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THE KASETSART JOURNALNatural Sciences

Volume 34, Number 1, 2000

ISSN 0075-5192

The Official Journal of Kasetsart UniversityPublished by the Kasetsart University, Bangkok 10900, Thailand

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ISSN 0075-5192

THEKASETSART JOURNALNatural Sciences http://www.rdi.ku.ac.th

January – March 2000Volume 34 Number 1

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Preliminary Report on Transfer Traits of Vegetative Propagation from Wild Rice Species to Oryza sativa via Distant

Hybridization and Embryo Rescue

............................................................................................................................Tao Dayun and Prapa Sripichitt 1

Effect of Mungbean Yellow Mosaic Virus (MYMV) on Yield and Yield Components of Mungbean

(Vigna radiata (L.) Wilczek)

........................................................................ G.S.S. Khattak, M.A. Haq, S.A. Rana, G. Abass and M. Irfag 12

Growth Period of Aquatic Plants for Birds Nesting at Bung Borapet, Nakhon Sawan

............................................... Suchada Sripen, Obhas Khobkhet, Sumon Masuthon and Sunanta Supanuchai 17

Study of Dropping Speed in Eggs of Oncomelania hupensis, a Snail Intermediate Host of Schistosomiasis

.......................... Xingjian Xu, Xianxiang Yang, Xiapin Li, Wei Zhang, Qingsang Pan and Zhengan Xiong 25

Selection for the Effective Species of Vesicular-Arbuscular Mycorrhizal Fungi on Soybean Root Infection

and Growth Enhancement

...................................................................................................................................................... Thongchai Mala 30

Residual Effects of 20 Annual Applications of Ammonium Sulfate and Triple Superphosphate for Corn

on Properties and Productivity of Oxic Paleustults

..........................................................................A. Suwanarit, I. Suwanchatri, J. Rungchuang and V. Verasan 40

Properties and Agricultural Potential of Skeletal Soils in Southern Thailand

.................................................................. Irb Kheoruenromne, Anchalee Suddhiprakarn and Sumitra Watana 52

Effect of Acetic Acid on Growth and Ethanol Fermentation of Xylose Fermenting Yeast and Saccharomyces cerevisiae

............................. Savitree Limtong, Tawatchai Sumpradit, Vichien Kitpreechavanich, Manee Tuntirungkij,

......................................................................................................................Tatsuji Seki and Toshiomi Yoshida 64

Cyanide Removal from Laboratory Wastewater Using Sodium Hypochlorite and Calcium Hypochlorite

...................................... Nusara Sinbuathong, Bussarin Kongseri, Panadda Plungklang and Roj Khun-anake 74

The Influence of Serum Concentration on tPA Production of CHO Cell

.............................................. Teerapatr Srinorakutara, Jin-Ho Jang, Mutsumi Takagi and Toshiomi Yoshida 79

Lectin Histochemistry of Glycoconjugates in Mandibular Gland of Chicken

.......................................................................... Apinun Suprasert, Surapong Arthitvong and Seri Koonjaenak 85

Prevalence of Antibody to Orientia tsutsugamushi in Dogs Along Thai - Myanmar Border

.............................................................................. Mongkol Chenchittikul, Decha Pangjai and Paijit Warachit 91

Process for Preparing Pre-fried and Frozen Sweetpotato French-fry Type Products

............................................................ Suparat Reungmaneepaitoon, Santi Tip-pyang and Sompoch Yai-eiam 98

Development of a Yogurt-type Product from Saccharified Rice

................................................................................ Chakamas Wongkhalaung and Malai Boonyaratanakornkit 107

Development of Instant High Fiber Processed Food

........ Plernchai Tangkanakul, Nednapis Vatanasuchart, Maradee Phongpipatpong and Patcharee Tungtrakul 117

Primary Productivity of the Pygmy Bamboo (Arundinaria pusilla) in the Dry Dipterocarp Forest at Sakaerat,

Nakhon Ratchasima

..................................................................................................................................................Niwat Ruangpanit 125

Electronic Knowledge Delivery: Developing a Web-based System for Computer Course

.................................................................................................................................................Anongnart Srivihok 139

Development of Water Allocation Strategy to Increase Water Use Efficiency of Irrigation Project

Varawoot Vudhivanich, Jesda Kaewkulaya, Ponsatorn Sopaphun, Watchara Suidee and Prapun Sopsathien 145

Superconducting Properties of (Bi,Pb)-Sr-Ca-Cu-O Ceramics

................................................................................................ Supreya Trivijitkasem and Wunchai Sratongluan 159

Removal of Naphthalene and 2, 4-Dinitrotoluene from Soils by Using Carboxymethyl-β-Cyclodextrin

................................................................................................................................................ Chatdanai Jiradecha 171

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The Kasetsart Journal

Home pages : http://www.ku.ac.th and http://www.rdi.ku.ac.th

Advisor : Supot Faungfupong Napavarn Noparatnaraporn

Suranant Subhadrabandhu Supamard Panichsakpatana

Editor-in-Chief : Wichien Yongmanitchai

Assistant Editors : Chakamas Wongkhalaung, Suparp Chatraphorn

Editorial Board : Natural Sciences Social SciencesPornsri Chairatanayuth Nuntana KapilakanchanaSuchint Deetae Nath BhanthumnavinEd Sarobol Nongnuch SriussadapornSaichol Ketsa Orasa SuksawangUthaiwan Sangwanit Suwanna ThuvachoteSutruedee Prathuangwong Pongpan TrimongkholkulPraparat Hormchan Suda PhiromkaiwAmara Tongpan Tarinee RodsonPornsawat WathanakulYupa MongkolsukApinun SuprasertKorchoke ChantawarangulSomnuk KerethoGunjana TheeragoolPhaisan WuttijumnongAree Thunyakijjanukij

Manager : Orawan Wongwanich

Assistant Managers : Wanpen Napativaumnauy

Business Office : Kasetsart University Research and Development Institute (KURDI)Kasetsart University, Chatuchak, Bangkok 10900.

The Kasetsart Journal is a publication of Kasetsart University intended to make available the resultsof technical work in the natural and the social sciences. Articles are contributed by Kasetsart University facultymembers as well as by those from other institutions. The Kasetsart Journal : Natural Sciences edition is issuedfour times per year in March, June, September and December while The Kasetsart Journal : Social Sciencesedition is issued twice a year in June and December.

Exchange publications should be addressed toThe Librarian,Main Library,

Kasetsart University,Bangkok 10900, Thailand.

Kasetsart J. (Nat. Sci.) 34 : 1 - 11 (2000)

1 Food Crop Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, The People’s Republic of China.2 Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.

INTRODUCTION

For Asian cultivated rice Oryza sativa, seedis the predominant way of propagation. Among 23

species of genus Oryza, there are different patterns

of vegetative propagation (Vaughan, 1994), whichcould be used to : 1) fix hybrid vigor (Xiu, 1995) 2)

breed for perennial rice (Schmit, 1996) 3) culture

ratooning rice (Krishnamurthy, 1988) and 4)multiply breeding lines and genetic stocks clonally

(Mahadevappa et al., 1989).

Tillering is a common trait of vegetative

Preliminary Report on Transfer Traits of Vegetative Propagationfrom Wild Rice Species to Oryza sativa via Distant Hybridization and

Embryo Rescue

Tao Dayun1 and Prapa Sripichitt2

ABSTRACT

There are diversified patterns of vegetative propagation in Oryza spp. If Oryza sativa is changedfrom annual type to perennial type via vegetative propagation, the perennial cultivar would be environmental

sound and economical viable. There would be a great potential to increase rice harvest area via ratoon

cropping or stubble cropping and some hope to break yield plateau via fixing heterosis by vegetativepropagation. Another advantage is that it would shorten the time interval from hybridization to form fixed

lines. A possible donor of the trait for ratoon or stubble cropping is O. rufipogon. The other species

possessing rhizome formation ability for breeding of perennial rice are O. longistaminata, O. officinalis,O. rhizomatis and O. australiensis. In this study perennial trait was transferred from wild species O.

longistaminata, O. rhizomatis and O. officinalis to cultivated rice (O. sativa) through distant hybridization.

Genotypes of O. sativa and wild species, pollen fertility of male species and environmental factors couldcontribute to crossability or germination rate of the resulting embryos. Finally “false” hybrid problem and

research on utilization of vegetative propagation in wild rice species were discussed.

Key words : vegetative propagation, wild rice, distant hybridization, embryo rescue, crossability

propagation of all species within genus Oryza,

which could be perennial (O. rufipogon, O.

glumaepatula, O. eichingeri, O. latifolia, O. alta,O. grandiglumis, O. longiglumis, O. meyeriana, O.

granulata) or annual (O. sativa, O. nivara, O.

meridionalis, O. glaberrima, O. barthii, O.punctata). Under certain environment, perennial

species O. glumaepatula has tillering ability as a

mean of vegetative propagation (Oka andMorishima, 1967), which could be used as a pattern

of vegetative propagation for all species of the

genus Oryza.

2 Kasetsart J. (Nat. Sci.) 34 (1)

Stem regeneration as indicated by ratooning,stubble planting (Mahadevappa et al., 1989) or

stoloniferous is also the common phenomenon

among all Oryza spp. especially perennial speciesof AA genome (Oka and Morishima, 1967) which

could also be used as a mean of vegetative

propagation for all species of the genus Oryza. O.australiensis, O. longistaminata, O. officinalis and

O. rhizomatis have a common trait of rhizome

(Vaughan, 1994), which usually could make thespecies perennial and adapt to temporal drought.

Thus it is the breeder’s ideal choice of breeding for

perennial ability in irrigated, upland or deepwaterrice.

Apomixis is the most promising way to fix

hybrid vigor (Hanna, 1995). However, the hope tofind obligate apomixis in Oryza spp. is dim. The

logical strategy is to transfer apomixis from other

remote species or to induce genetic mutation inOryza spp. In vitro propagation, especially artificial

seed, is another way to multiply rice or fix F1

heterosis (Juliano et al., 1993; Yoshida and Kata,1996).

From the viewpoint of breeding, strong

regeneration ability of stem is useful for breedingof rice ratooning. Rhizome is the most logical

pattern of propagation for breeding of perennial

rice especially upland rice (Schmit, 1996). Apomixisand in vitro propagation are the most promising

ways to fix heterosis of F1 hybrid.

In order to transfer rhizome character fromwild rice species to the cultivated species so as to

fix heterosis of rice, it is a prerequisite condition to

have diversified genetic resources. O.longistaminata, O. rhizomatis, O. officinalis and

O. australiensis should be the donors of perennial

character. This paper is to report preliminary resultsof transferring rhizome character from O.

longistaminata, O. officinalis, O. rhizomats to

cultivated rice so as to breed for perennial hybridrice via distant cross and embryo rescue.

MATERIALS AND METHODS

Cultivated riceKDML 105 (or Khao Dawk Mali 105, indica

rice)RD23 (indica rice)

CT6241-17-1-5-1 (japonica rice)

IR 42 (indica rice)

Wild speciesO. longistaminata (accession no. unknown)

was kindly supplied by Prof. Dr. Hiroshi Hyakutakewhich was derived from the Ministry of Agriculture

and Forestry, Japan.

O. rhizomatis (accession no. 20133) wassupplied by Pathumthani Rice Research Center,

Thailand and O. rhizomatis (accession no. W 95018

and accession no. unknown) were introduced fromInternational Rice Research Institute (IRRI) via

Yunnan Academy of Agricultural Sciences

(YAAS), China.O. officinalis (accession no. W9502 and

W9509) were introduced from IRRI via YAAS.

Planting methodAll materials were planted in pots and grown

in a greenhouse at the Department of Agronomy,

Kasetsart University. Each accession comprised 5-7 plants.

HybridizationHybridization was done between cultivated

rice and wild rice by emasculation of the female

parents. Upper one third of the glume of each

spikelet was cut off using scissors after 4 p.m..Then the anthers were removed using forceps. The

emasculated panicles were pollinated heavily from

the male parents one day afterwards for the durationof 3 days.

Kasetsart J. (Nat. Sci.) 34 (1) 3

Embryo rescueAfter pollination, ovaries were excised and

surface-sterilized by soaking in 75 % ethanol for 5

minutes followed by 25 % Chlorox contatining a

few drops of wetting agent, Tween 20, for 20minutes. After washing in sterilized distilled water

4 times, the lower part of the ovary was excised and

cultured on 1/4 MS (Murashige and Skoog, 1962)medium (3 % sucrose and 0.7 % agar, pH 5.8) in the

dark at 25°C (Guzman Emerita, 1983; Jena and

Khush, 1984). After germination, seedlings werekept in the light until they reached the three-leaf

stage. For acclimatization, the cultured bottles were

transferred to a room without direct sunlight forone week, then the seedlings were cultured in pipe

water with little NPK compound fertilizer for

another week before the plantlets were transplantedinto soil in pots and grown in a greenhouse.

Pollen fertilitySix spikelets were sampled from one panicle

of the wild species used as male parents before

anthesis. Pollen grains were crushed out and stained

with 2-KI solution. Dark, round and big pollengrains were considered as fertile ones, and about

1,000 pollen grains were counted for each material.

RESULTS

Observation on rhizome character of different

species of wild riceO. rhizomatis was collected from seasonally

dry grassland in Sri Lanka. The extensive thick rootsystem and rhizomes suggest its usefulness perhaps

as a source of drought tolerance (Vaughan, 1990).

It had vigorous tillers and the growth of rhizomedid not overlap with the main cropping. When

cutting the main cropping at maturation stage, all

rhizome grew and had tillers vigorously within 10days, and new ratooning tillers could head

simultaneously. However, the plant height and

panicle length of regenerated cropping were shorterthan the main cropping (Table 1). The cropping of

rhizome separating and planting was much better

than that of in situ plant.The perenniality of O. longistaminata was

due to permanent rhizome formation. It was easy to

form clonal population via rhizome propagation.However, tillering ability was weak and generation

overlapping was obvious. Thus, it was not easy to

induce panicle initiation.Both accessions of O. officinalis had rhizome

and strong tillering ability. Though they possessed

Table 1 Plant height and panicle length of the main cropping and ratoon cropping for different species

of wild rice.

Species Accession Plant height (cm) Panicle length (cm)

no. Main Ratoon Difference Main Ratoon Difference

O. officinalis W 9502 171 180.3 -9.3 29.8 31 -1.2

W 9509 150.2 174.3 -24.1 29.4 30.5 -1.1

O. rhizomatis W 95018 171.6 122.4 49.2** 31.4 22.4 9*O. rhizomatis 170.4 119 51.4** 27.9 22.4 5.5**

* ** indicate significance at the 0.05 and 0.01 level of probability, respectively.

4 Kasetsart J. (Nat. Sci.) 34 (1)

permanent rhizome formation and tillering, theplant usually showed synchronous flowers. When

cutting the plant at maturation stage, rhizome growth

and tiller formation were becoming vigorouslywithin 10 days, and there was no obvious change in

plant height, panicle length of main cropping and

those of regenerated cropping. This type ofvegetative propagation is promising for breeding

of perennial ability.

Crossability and germination rate of different

interspecific combinationsRD23/O. longistaminata could give the

highest percentage of seed set (42.86 %) (Table 2)since both parents have the same genome (AA),

followed by O. sativa/O. rhizomatis (0.26-19.3 %),

Table 2 Interspecific hybrids produced via embryo rescue.

Crosses No. florets Seed set No. embryos Germination No. hybrid

pollinated cultured rate plants

No. % (%)

RD 23/O. rhizomatis

(no. 20133) 172 14 8.14 8 50 1

(9 days old)

IR 42/O. rhizomatis

(no. 20133) 696 58 8.33 22 22.73 0

(5 days old)

KDML 105/O. rhizomatis

(no. 20133) 1,154 3 0.26 3 33.33 0

(8 days old)

RD 23/O. rhizomatis

490 83 16.94 45 0 0

(5-10 days old)

RD 23/O. rhizomatis

(W 95018) 1,011 196 19.39 27 0 0

(6-10 days old)

RD 23/O. officinalis

(W 9502) 479 8 1.67 3 0 0

(13 days old)

RD 23/O. officinalis

(W 9509) 388 27 6.96 2 0 0

(13 days old)

CT 6241/O. officinalis

(W 9502) 991 11 1.2 6 82.33 3

(15 days old)

RD 23/O. longistaminata 119 51 42.86 33 3.03 1(5-10 days old)

Kasetsart J. (Nat. Sci.) 34 (1) 5

and O. sativa/O. officinalis with the lowest seed set(1.2-6.96 %). O. rhizomatis (no. 20133) was crossed

with three different cultivars of O. sativa. Among

the three cultivars, KDML 105 gave the lowestseed set (0.26 %) while IR 42 exhibited the highest

seed set (8.33 %). The rate of seed set varied from

8.14 to 19.39 % when the cultivated variety RD23was used as a female parent and hybridized with

different accessions of O. rhizomatis, whereas the

percentage of seed set ranged from 1.67 to 6.96when RD23 was crossed with two different

accessions of O. officinalis. The results indicated

that there was crossability difference within thespecies of O. rhizomatis and O. officinalis.

In order to obtain interspecific hybrids,

embryo rescue is necessary. The embryos of O.sativa/O. rhizomatis began to degenerate about 8-

10 days after pollination and the embryo older than

10 day old was not sucessfully rescued. The hybridembryos of O. sativa/O. longistaminata began to

degenerate 6 days after pollination. The 5-10 day-

old embryos gave considerably low germinationrate (3.03 %). Development of the seeds of CT6241-

17-1-5-1/O. officinalis (no. W9502) was very poor,

but the degeneration rate was rather slow. Fifteen-day-old embryos could germinate quite well (82.33

%).

Relationship between crossability and pollen

fertility of male speciesPartial pollen sterility is a common

phenomenon for wild species of rice. The results of

Table 3 indicated that there was some relationship

between pollen fertility of male species andcrossability. The higher fertility of pollen caused

the higher percentage of seed set. To confirm the

concordance observation, five plants of O.rhizomatis (no. 20133) were crossed with RD23

separately and pollen fertility of each plant was

investigated. The results once again indicated thatthere was some relationship between pollen fertility

of male species (or different plant within accession)

and crossability (Table 4).

“False” hybrids problemFalse hybrid was an important problem for

distant cross breeding (Chen et al., 1989). From thereview of literatures, the average rate of false

hybrid for intraspecific hybridization was below 5

%. The self-fertilization seed set of emasculatedpanicles in this experiment was below 1 %.

However, the data in Table 2 shows that most

plantlets of embryo rescue were false hybrids. Itwas surprising that a few normal developmental

seeds were obtained when RD23 and IR42 were

Table 3 Pollen fertility of the male species and seed set of interspecific crosses.

No. florets Pollen fertility of Seed set

pollinated male species(%) No. %

RD 23/O. rhizomatis (no. 20133) 1,561 60.17 207 13.26

RD 23/O. rhizomatis 490 31.75 83 16.94

RD 23/O. rhizomatis (W 95018) 1,011 86.00 196 19.39RD 23/O. officinalis (W 9502) 497 16.08 8 1.67

RD 23/O. officinalis (W 9509) 388 51.31 27 6.96

CT 6241/O. officinalis (W 9502) 991 16.08 11 1.11RD 23/O. Longistaminata 199 64.49 51 42.86

6 Kasetsart J. (Nat. Sci.) 34 (1)

hybridized with O. rhizomatis, while the seedsgerminated normally. All plants of IR42/O.

rhizomatis (no. 20133) and 3 out of 7 plants of RD

23/O. rhizomatis (no. 20133) died after 6-7 days ofgermination, the remaining were all false hybrids.

RD23 was crossed with 5 separated plants

of O. rhizomatis (no. 20133). Each cross obtainedcould form very few normal seeds (Table 5).

However, all plants of these progenies were

apparently like the maternal parent in morphology.The rate of normal seed set was lower than 1 %,

which could be regard as true false hybrids of self

fertilization during the course of emasculation. The

normal seeds could be obtained for RD23/O.rhizomatis (no. 20133-3) as high as 10.67 %, which

could not explain the situation of self-fertilization.

Another possible explanation of false hybridis the pollen of wild species could induce

parthenogenesis, and the plant like the maternal

parent is presumed to be resulted from haploidgametes stimulated by the pollen of wild species.

This phenomenon is known as matromorphy

(Farooq et al., 1996). If it is true, there would beanother effective way to produce diploid plant of

rice from female gametophyte. To confirm the

hypothesis, CMS line V20A and Fl hybrid of

Table 4 Pollen fertility of the male plants of O. rhizomatis (no. 20133) and seed set of interspecificcrosses.

No. florets Pollen fertility of Seed setpollinated male plants

(%) No. %

RD 23/No. 20133-1 246 82.01 74 30.08

RD 23/No. 20133-2 451 50.39 37 8.20RD 23/No. 20133-3 300 54.46 72 24.00

RD 23/No. 20133-4 232 57.28 9 3.88

RD 23/No. 20133-5 332 39.55 15 4.52

Table 5 Normal seed set of certain interspecific hybrids.

No. florets Normal seeds obtained Germination

pollinated rateNo. % (%)

IR 23/O. rhizomatis (no. 20133) 362 2 0.55 100

RD 23/O. rhizomatis (no. 20133) 136 7 5.15 100

RD 23/O. rhizomatis (no. 20133-1) 246 2 0.81 100RD 23/O. rhizomatis (no. 20133-2) 451 9 1.96 100

RD 23/O. rhizomatis (no. 20133-3) 300 32 10.67 93.75

RD 23/O. rhizomatis (no. 20133-4) 232 2 0.86 100RD 23/O. rhizomatis (no. 20133-5) 332 2 0.60 100

Kasetsart J. (Nat. Sci.) 34 (1) 7

RD23/KDML 105 are suggested to be used asfemale parents to hybridize with O. rhizomatis (no.

20133 plant 3).

Preliminary observation on hybridsOne plant of RD23/O. rhizomatis, three

plants of CT6241-17-1-5-1/ O. officinalis (no.

W9502) and one plant of RD23/O. longistaminata

were morphological characterized. They were

intermediate in morphological characters between

the two parents, but much closer to their respectivewild parents (Figure 1-6). They headed much earlier

than their cultivated parents in winter. The spikelets

were awned, small and easy shattering. Somediscriminative traits such as ligule length of lower

leaves, presence of rhizome, presence of whorl of

branches at panicle base and spikelets inserted inthe lower half of lower panicle branches were

dominant or recessive traits in F1 generation

according to different interspecific hybrids (Table6).

Pollen fertility of RD23/O. rhizomatis (no.

20133) ranged from 0 to 1.54 %. No seed set wasobserved when the hybrid was backcrossed to

RD23; even 2,886 florets were pollinated by RD23.

RD23/O. longistaminata hybrid had rhizome andindehiscent anthers (Figure 7). Pollen fertility of

the hybrid was as low as 32.53 % while the male

parent O. longistaminata possessed 64.51 % pollenfertility before hybridization.

DISCUSSION

There are diversified pattern of vegetative

propagation in Oryza spp. If Oryza sativa is changedfrom annual habit to perennial type via vegetative

propagation, the perennial crop would have the

potential to provide environmental sound andeconomically viable alternatives for the use on

upland, irrigated, rainfed lowland and flood-prone

ecosystem (Schmit, 1996; Wagoner, 1990). There

might be an alternative to double or triple croppingof rice in tropical and subtropical area to expand

rice harvest area via ratooning (Krishnamurthy,

1988). Another advantage is to fix heterosis viavegetative propagation so as to make hybrid rice

become available to poor farmers and fragile

ecosystems (upland, flood-prone and rainfedlowland ecosystems) (Xiu, 1995). Subsequently,

rice yield per hectare could be increased on a large

scale. For breeding strategies, perennial charactercould shorten the time interval from hybridization

to form fixed lines. Thus, breeding rice for perennial

or vegetative character has environmental,economical, and theoretical preference.

Interspecific crossability is a complex

character which could be contributed by femalevariety of O. sativa, male species or accession of

wild rice, pollen fertility of male species or

accession, and environmental factors. The presentresults indicated that it is important to select

accession or plant of high pollen fertility as male

parent so as to get high seed set when interspecificcross is produced.

Nearly all hybrids between O. sativa and

wild species of the genus Oryza have beensuccessfully obtained (IRRI, 1993). Some useful

traits have been successfully transferred to O. sativa

from wild species of rice. However, very littleattention was paid on research and utilization of

propagation habit. There is a trend for food

production toward intensive and fragile area.Research and utilization of diversified vegetative

habit could partly meet today’s pressing global

concerns on agriculture, availability of food,conservation of resources, and sustainability of the

environment.

Finally, hybrid of RD23/O. longistaminata

might be useful for transferring rhizome character

from wild species to cultivated rice and to tag the

gene (s) responsible to rhizome formation becauseit has rhizome in Fl generation and relatively high

8 Kasetsart J. (Nat. Sci.) 34 (1)

Tab

le 6

Mor

phol

ogic

al c

hara

cter

s of

cer

tain

inte

rspe

cifi

c hy

brid

s.

Cro

sses

or

pare

nts

Res

cue

Tra

nspl

antin

gH

eadi

ngPl

ant

Pani

cle

Flag

Flag

Lig

ule

Lig

ule

Len

gth

Rhi

zom

eW

horl

of

Spik

elet

s in

sert

edda

teda

te (

m/d

)da

te (

m/d

)he

ight

leng

thle

afle

afle

ngth

of

leng

th o

fof

aw

npr

esen

cebr

anch

in th

e lo

wer

hal

f(m

/d)

(cm

)(c

m)

leng

thw

idth

flag

leaf

the

thir

d(c

m)

pres

ence

of lo

wer

pan

icle

(cm

)(c

m)

(mm

)le

af (

mm

)br

anch

es

RD

231

10/2

911

/41/

2796

24.5

461.

811

290

nono

yes

RD

23/

O. r

hizo

mat

is2

10/1

611

/10

12/1

780

21.5

331.

67

61.

0no

noye

s

O. r

hizo

mat

is (

no. 2

0133

)110

/29

11/4

1/20

6515

.523

.51.

12

50

yes

noye

sC

T 6

241-

17-1

-5-1

112

/18

1/13

3/28

6025

441.

74

170

nono

noC

T 6

241/

O. o

ffic

inal

is2

12/4

1/13

2/20

6519

282.

14

82.

5no

yes

no

O. o

ffic

inal

is (

W 9

502)

212

/18

1/13

3/17

120

3135

.52.

53

42

yes

yes

noR

D 2

3212

/28

1/27

4/9

102

2527

1.7

720

0.4

nono

yes

RD

23/

O. l

ongi

stam

inat

a212

/28

2/24

4/13

127

4169

2.3

3031

3.3

yes

noye

s

O. l

ongi

stam

inat

a3-

2/24

--

--

--

--

yes

--

1 D

irec

t ger

min

atio

n2

Em

bryo

res

cue

3 R

hizo

me

prop

agat

ion.

Kasetsart J. (Nat. Sci.) 34 (1) 9

Figure 1 Plants of RD 23 (left), RD 23/O.rhizomatis (middle) and O. rhizomatis

(no. 20133, right).

Figure 4 Panicles of CT 6241-17-1-5-1 (left),CT 6241/O. officinalis (middle) and

O. officinalis (no. W 9502, right).

Figure 2 Panicles of RD 23 (left), RD 23/O.rhizomatis (middle) and O. rhizomatis

(no. 20133, right).

Figure 5 Plants of RD 23 (left), RD 23/O.

longistaminata (middle) and O.longistaminata (right).

Figure 3 Plants of CT 6241-17-1-5-1 (left), CT

6241/O. officinalis (middle) and O.officinalis (no. W 9502, right).

Figure 6 Panicles of RD 23(lower) and RD 23/O.

longistaminata (upper).

10 Kasetsart J. (Nat. Sci.) 34 (1)

pollen fertility. It was different from other hybridsreported at least on rhizome expression (Ghesquiere,

1991).

ACKNOWLEDGEMENTS

This research was partly funded byTingthanathikul Foundation, Thailand. Our

profound gratitude also goes to Dr. Songkran

Chitrakorn, Pathumtani Rice Research Center,Thailand, Prof. Dr. Hiroshi Hyakutake, Japan

Science and Technology Cooperation and Dr. D.A.

Vaughan, IRRI, Philippines, for supplyingexperimental materials.

LITERATURE CITED

Chen, S. B., X. L. Duan and J. L. Fu. 1989. Genetic

variation in rice/sorghum hybrids and theirapplication in rice breeding, pp. 261-268. In

IRRI (ed.). Progress in Irrigated Rice Research.

IRRI, Manila.Farooq, S., N. Iqbal, T.M. Shah and M. Asghar.

1996. Problems and prospects for utilizing

Porteresia coarctata in rice breeding programs.Cereal Res. Comm. 24 (1) : 41-17.

Ghesquiere, A. 1991. Reexamination of genetic

control of the reproductive barrier betweenOryza longistaminata and O. sativa, and

relationship to rhizome expression, pp. 729-

730. In IRRI (ed.). Rice Genetics II, IRRI,Manila.

Guzman Emerita, V. de. 1983. Recent progress in

rice embryo culture at IRRI, pp. 215-228. InCell and Tissue Culture Techniques for Cereal

Crop Improvement. Proceedings of a

Workshop cosponsored by the Institute ofGenetics, Academia Sinica and IRRI. Science

Press, Beijing.

Hanna, W.W. 1995. Use of apomixis in cultivardevelopment. Adv. Agron. 54 : 333-350.

IRRI. 1993. Program Report for 1992. IRRI, Manila.

156 p.Jena, K. K. and G.S. Khush. 1984. Embryo rescue

of interspecific hybrids and its scope in rice

improvement. RGN 1 : 133-134.Juliano, A., D. A. Vaughan, C.Y. Wu and F.J.

Zapata. 1993. In vitro propagation of conserved

rice germplasm. IRRN 18(4) : 4-5.Krishnamurthy, K. 1988. Rice ratooning as an

alternative to double cropping in tropical Asia,

pp. 3-15. In W.H. Smith and V. Kumble (eds.).Rice Ratooning. IRRI, Manila.

Mahadevappa, M., N. D. Vishakantha, R. K. Sarma,

and K. G. Ovindaraj. 1989. Stubble planting-promising vegetative propagation method for

hybrid rice. IRRN 14 (4) : 9-10.

Murashige, T. and F. Skoog. 1962. A revisedmedium for rapid growth and bioassay with

Figure 7 Rhizome of RD 23/O. longistaminata

(F1 generation).

Kasetsart J. (Nat. Sci.) 34 (1) 11

tobacco tissue cultures. Physiol. Plant. 15 :474-497.

Oka, H. I. and H. Morishima. 1967. Variations in

the breeding systems of a wild rice, Oryza

perennis. Evolution 21 : 249-258.

Schmit, V. 1996. Improving sustainability in the

uplands through the development of a perennialupland rice, pp. 265-273. In C. Piggin, B.

Courtois, and V. Schmit (eds.). Upland Rice

Research in Partnership. Proceedings of theUpland Rice Consortium Workshop. 4-13

January 1996. Manila.

Vaughan, D. A. 1990. A new rhizomatous Oryza

species (Poaceae) from Sri Lanka. Bot. J.

Linnean Society 103 : 159-163.

Vaughan, D.A. 1994. The wild relatives of rice.IRRI, Manila, 137 p.

Wagoner, P. 1990 Perennial grain development :

Past efforts and potential for the future. CriticalRev. Plant Sci. 9 (5) : 381-408.

Xiu, L. Q. 1995. Breeding for Perennial Hybrid

Rice. Division Seminars of Plant Breeding,Genetics, and Biochemistry. IRRI, Manila. p.

Yoshida, T. and H. Kata. 1996. In vitro propagation

of hybrid rice (Oryza sativa L.). JARQ 30 : 9-14.

Received date : 14/10/98Aecepted date : 23/06/99

Kasetsart J. (Nat. Sci.) 34 : 12 - 16 (2000)

Effect of Mungbean Yellow Mosaic Virus (MYMV) on Yield andYield Components of Mungbean (Vigna radiata (L.) Wilczek)

G.S.S. Khattak1, M.A. Haq1, S.A. Rana2, G. Abass1 and M. Irfag1

ABSTRACT

Fourteen MYMV susceptible F3 progenies from a cross NM 92 x VC 1560D showed significantdifferences for MYMV disease infection, yield and yield components. These progenies suffered from 18.5

to 40.5 percent decrease in plant height, 11.7 to 64.0 percent reduction in number of pods per plant, 5.8 to

82.2 percent reduction in seeds per pod, 7.4 to 35.0 percent decrease in pod length, 10.6 to 53.3 percentreduction in 1000 seed weight and 32.2 to 78.6 percent decrease in grain yield per plant. The MYMV

incidence showed significant correlation (0.526) with the decrease in 1000 seed weight. The decrease in

yield and other yield components showed non significant positive correlation with MYMV incidence.Key words : mungbean yellow mosaic virus, yield, mungbean

1 Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan.2 Bahauddin Zakariya University, Multan, Pakistan.

INTRODUCTION

Mungbean Yellow Mosaic Virus (MYMV)

has been found widely distributed in India andPakistan causing enormous losses in the production

of several leguminous crops (Chenulu and Verma,

1988). The most seriously affected leguminouscrops by this disease are mungbean, blackgram and

soybean. It is the most destructive disease of

mungbean during summer season in Pakistan(Ahmad, 1975). MYMV disease is reported to be

transmitted by an insect vector, Bemisia tabaci and

not by seed, soil and mechanical inoculation (Nairand Nene, 1973; Ahmad and Harwood, 1973). The

effect of disease varies with cultivar to cultivar and

is subjected to the genetic make up of the cultivar.The present study reports the results on the

quantitative determination of the effect of MYMV

disease on the yield and yield components of the

susceptible F3 progenies from the cross of a localMYMV resistant variety NM 92 and an exotic

MYMV susceptible accession VC 1560D.

MATERIALS AND METHODS

Fourteen F3 MYMV susceptible progeniesresulting from a cross of NM 92 x VC 1560D along

with the MYMV susceptible parent VC 1560D

were planted in a randomized complete block designin three replications at Nuclear Institute for

Agriculture and Biology, Faisalabad – Pakistan

during summer 1997 in two sets of trial. Theprogenies were planted in 2 m long, 2 rows per plot

with 30 and 10 cm distances between rows and

pants respectively. Of the two sets of trials, one wasprotected from whiteflies invasion and hence from

MYMV infection, by spraying insecticide Polo at

the rate of 250 ml/ha at five days interval from 15th

day of sowing. The second trial was subjected tonatural invasion of white flies. The epiphytotic

conditions were created by planting mung kabuli

(MYMV susceptible check) around and in the trialafter each entry as a spreader for MYMV disease

inoculum to the vector (whitefly). Both the trials

were planted at a distance from each other tomaintain the ideal conditions of each trial.

The MYMV disease infection was recorded

in percent at the peak of the disease on the basis ofwhole plot in each replication from unprotected

(diseased) trial. At physiological maturity five plants

from each plot in each replication of protected(healthy) and unprotected (diseased) trial were

randomly selected and data were recorded for plant

height, pods per plant, pod length, seeds per pod,1000 seed weight and grain yield per plant. The

decrease over control in percent was calculated for

each character by the following formulla:

Decrease over control (%) =

Pr otected plot value – Unprotected plot value

Protected plot value×100

The analysis of variance of all the charactersand correlation coefficient between the MYMV

incidence and decrease in yield and yields

components were performed on microcomputerusing MSTATC Software.

RESULTS AND DISCUSSION

All the entries in both sets of trial were

found significantly different from each other for allthe characters (Table 1) Effect of MYMV infection

on yield and yield components of F3 susceptible

progenies of mungbean varied greatly (Table 2).All the entries were invariably affected by virus

infection and suffered 18.5 to 82.2 percent in seeds

per pod. Pod length, though much less affected,suffered from 7.4 to 35.0 percent decrease. The

seed size and grain yield per plant suffered from

10.6 to 52.3 percent and 32.2 to 78.6 percentreduction respectively. Singh (1981) reported the

disease to cause considerable reduction in growth

components and upto 38.2 percent reduction inplant height of mungbean. Chand and Verma (1983)

Table 1 Mean square values from ANOVA of yield and yield components of F3 progenies of mungbean

evaluated in two sets of trial.

SOV Disease Plant Pods per Seeds per Pod length 1000 seed Grain yield

incidence height plant pod (cm) wt (g) per plant(%) (cm) (g)

Protected

Rep. - 1.48NS 1.98* 0.02NS 0.31NS 3.56* 0.17

Ent. - 57.75** 23.22** 1.27** 1.31** 71.93** 5.27**

Error - 2.27 0.53 0.05 0.15 0.63 0.59

Unprotected

Rep. 1.63NS 0.91NS 0.46NS 0.17NS 0.08NS 0.15NS 0.05NS

Ent. 89.08** 78.44** 49.62** 0.94** 0.79** 67.98** 2.92**

Error 5.38 0.63 0.47 0.06 0.05 1.10 0.27

*,** = Significant at P<0.05 and 0.01 respectively.

NS = Non significant.

Kasetsart J. (Nat. Sci.) 34 (1) 13

14 Kasetsart J. (Nat. Sci.) 34 (1)

Tab

le 2

MY

MV

inci

denc

e (%

) an

d de

crea

se o

ver

cont

rol (

%)

of y

ield

and

yie

ld c

ompo

nent

s du

e to

MY

MV

in m

ungb

ean

F3 p

roge

nies

.

Ent

/D

isea

sePl

ant h

eigh

tPo

d le

ngth

Seed

s/Po

dPo

ds/P

lant

1000

see

d w

eigh

tG

rain

yie

ld/P

lant

Prog

eny

inci

denc

e(c

m)

(cm

)(g

)(g

)

(%)

H. p

lant

sD

. Pla

nts

Dec

reas

eH

. pla

ntD

. Pla

nts

Dec

reas

eH

. pla

ntD

. Pla

nts

Dec

reas

eH

. pla

nts

D. P

lant

sD

ecre

ase

H. p

lant

D. P

lant

sD

ecre

ase

H. p

lant

D. P

lant

sD

ecre

ase

over

over

over

over

over

over

cont

rol

cont

rol

cont

rol

cont

rol

cont

rol

cont

rol

(%)

(%)

(%)

(%)

(%)

(%)

1.58

.0fg

63.1

cde

50.8

a19

.58.

1f7.

0cde

13.6

6.9e

6.5e

fg5.

827

.9a

10.2

ef63

.446

.9g

40.5

cde

13.6

10.2

ef3.

3efg

67.6

2.68

.0de

61.8

de48

.6b

21.4

8.1f

7.5b

7.4

8.3d

7.0b

cd15

.722

.3de

19.0

a14

.842

.4h

37.9

fg10

.68.

7g5.

9a32

.2

3.65

.7e

56.8

f40

.9f

28.0

9.1d

e6.

8de

25.3

8.7c

d6.

6def

24.1

21.9

e17

.5b

20.1

52.7

e41

.4cd

21.4

11.5

abcd

e4.

7bcd

59.1

4.66

.3e

61.9

de36

.8h

40.5

9.0d

e7.

2bcd

20.0

8.7c

d6.

5efg

25.3

22.1

e10

.4ef

52.9

50.2

f37

.9fg

24.5

10.1

efg

3.1f

g69

.3

5.66

.0e

56.6

f39

.0g

31.1

8.4e

f7.

0cde

16.7

8.3d

6.3f

g24

.121

.7e

12.9

d40

.654

.2d

42.1

c22

.311

.0cd

ef3.

5ef

68.2

6.71

.3cd

56.9

f36

.4h

36.0

8.8d

e6.

8cde

22.7

8.6d

6.9c

d19

.818

.4g

11.4

e38

.054

.7cd

44.3

b19

.08.

8g3.

5ef

60.2

7.72

.0cd

67.9

a44

.5d

34.5

9.5b

cd6.

8cde

28.4

9.1b

6.1g

hi33

.027

.2a

9.8f

64.0

46.6

g37

.4gh

19.7

11.7

abcd

2.5g

78.6

8.75

.0bc

56.7

f39

.4g

30.5

8.8d

e6.

8cde

22.7

8.5d

6.3f

gh25

.925

.9b

10.3

ef60

.251

.7e

40.2

de22

.211

.5ab

cde

3.2f

g72

.29.

57.7

fg60

.6e

49.4

b18

.59.

4cd

7.2b

cd23

.48.

5d6.

8de

20.0

23.8

c18

.4ab

22.7

42.8

h35

.3i

17.5

9.7f

g3.

8def

60.8

10.

56.3

g62

.0de

39.8

fg35

.89.

1de

8.2a

9.9

9.3a

b7.

3abc

21.5

24.6

c18

.5ab

24.8

47.5

g39

.1ef

g17

.711

.2bc

de3.

8def

66.1

11.

61.4

f51

.8g

39.2

g24

.310

.1ab

8.2a

18.8

9.0b

c7.

4ab

82.2

23.5

cd18

.2ab

22.6

49.5

f35

.7hi

27.9

12.3

abc

5.2a

b57

.712

.71

.6cd

66.3

ab49

.9ab

24.7

8.9d

e6.

8cde

30.9

9.3b

5.9h

i36

.621

.4e

18.9

a11

.755

.6bc

39.5

ef29

.012

.9a

5.1a

bc60

.5

13.

77.1

ab65

.4ab

c46

.1c

29.5

10.3

a6.

7e35

.09.

7a5.

7i41

.220

.1f

14.9

c25

.956

.6ab

45.9

ab18

.912

.6ab

4.2c

de66

.7

14.

54.1

g62

.6de

49.1

b21

.69.

9abc

7.2b

c27

.39.

3ab

6.1g

hi34

.418

.7g

8.3g

55.6

55.9

bc46

.7a

16.5

10.7

def

2.5g

76.6

VC

1560

D80

.0a

64.2

bcd

42.8

e33

.39.

1de

7.9a

13.2

9.1b

7.6a

16.5

21.9

e18

.1ab

17.4

57.4

a27

.4i

52.3

12.7

a3.

7def

70.9

(Par

ent)

H-H

ealth

y D

-Dis

ease

d

Kasetsart J. (Nat. Sci.) 34 (1) 15

reported that mungbean cultivars might suffer 66.6percent decrease in plant yield and 25.7 percent

decrease in 1000 seed weight due to MYMV. Ayub

et al. (1989) reported reduction upto 91.6, 24.7,56.6 and 80.2 percent for pod number, pod size,

seed/pod and plant yield respectively in mungbean.

The variation in the effect of MYMV onyield and yield components among the F3 progenies

in the present study may be explained on the basis

of differences in the genetic make up of the progeniesresulting from new recombinations due to crossing

over of the genetic material during meiosis. The

variation may also be expected on the basis of earlyor late infection of the cultivars, as early and

severely infected plants usually bear much number

of pods while latter infection has been reported todelay plant maturity and yield (Singh, 1980, Singh

et al. 1982).

The decrease in yield and yield componentswas positively correlated with MYMV incidence

(Table 3) indicating that the MYMV disease affected

each component and decreased yield as well.Decrease in 1000 seed weight showed highest

significant correlation coefficient (0.526) with

MYMV disease amongst all the yield components.

This adverse effect of MYMV incidence on 1000seed weight might be the main cause of decrease in

yield. Yohe and Poehlman (1975) also reported

significant and negative correlation between virusscore and yield and yii $ components.

LITERATURE CITED

Ahmad, M. 1975. Screening of mungbean (Vigna

radiata) and urdbean (Vigna mungo)germplasm for resistance to yellow mosaic

virus. J. Agri. Res. 13(1): 349-354.

Ahmad, M. and R.F. Harwood. 1973. Studies onwhitefly-transmitted yellow mosaic disease of

cowpea (Vigna unguiculata). Plant Dis. Rep.

62 : 224-226.Ayub, M.A., M.B. Ilyas, and M.A.R.Bhatti. 1989.

Growth responses of mungbean cultivars to

mungbean yellow mosaic virus infection. Pak.J .Phytopath. 1 (1-2) : 38.

Chand, P. and J.P. Verma. 1983. Effect of yellow

mosaic on growth components and yield ofmungbean and urdbean. Haryana Agri. Univ.

J. Res. 13(1) : 98-102.

Chenulu, V.V. and A. Verma. 1988. Virus andvirus like diseases of pulse crops commonly

grown in India, pp. 338-370. In B. Baldev,

S.Ramajunam, and H.K. Jain (eds.). PulseCrops, New Delhi, Oxford and IBH.,

Nair, N.G. and Y.L. Nene. 1973. Studies on the

yellow mosaic of urdbean (Phaseolus mungo

L.) caused by mungbean yellow mosaic virus.

2. virus-vector relationships. Indian J. Farm

Science 1: 62-70.Singh, B.R., M. Singh, M.D. Yadav, and

S.M.Dinghra. 1982. Yield loss in mungbean

due to yellow mosaic. Sci. and Culture 48 (12): 435-436.

Singh, J.B. 1981. Effect of viruses on growth

components and yield of mungbean (Vigna

radiata) and urdbean (Vigna mungo). Indian

Table 3 Correlation between disease (MYMV)

incidence and decrease in yield and yield

components due to MYMV in mungbeanF3 progenies.

Character Disease (MYMV)

incidence

Plant height 0.434

Seeds per pod 0.024Pod length 0.254

Pods per plant 0.149

1000 seed wt. 0.526*

Grain yield per plant 0.033

* = Significant at P<0.05

16 Kasetsart J. (Nat. Sci.) 34 (1)

Phytopath. 33(7): 405-408.Singh, R.N. 1980. Natural infection of bean by

mungbean yellow mosaic virus. Indian J.

Mycol. and Pl. Pathol. 9(1) : 124-126.Yohe, J.M. and Poehlman. 1975. Regressions,

correlations, and combining ability in

mungbeans (Vigna radiata (L.) Wilczek). Trop.Agric. Trinidad. 52(4) : 343-352.

Received date : 25/11/98Accepted date : 28/06/99

Kasetsart J. (Nat. Sci.) 34 : 17 - 24 (2000)

Growth Period of Aquatic Plants for Birds Nestingat Bung Borapet, Nakhon Sawan

Suchada Sripen1, Obhas Khobkhet2, Sumon Masuthon1 and Sunanta Supanuchai3

ABSTRACT

Studies on the growth period of aquatic plants that were to be nested by birds was undertaken at BungBorapet, Changwat Nakorn Sawan. It was found that the minimum densities of various plants for bird

nesting were different and depended on nesting conditions. Salvinia cucullata Roxb., Potamogeton

malaianus Miq. and Pheudoraphis spinescens Vickery with density of 267, 203 and 229 g m-2were usedfor nesting by Hydrophasianus chirurgus, respectively. While Porphyrio porphyrio, Porzana cinerea and

Ixobrychus cinnamoneus preferred to nest over the water level, therefore, Eichhornia crassipes (Mart.)

Solms, Pheudoraphis spinescens Vickery, Typha angustifolia L. and Nelumbo nucifera Gaerth with thedensities of 1,050, 464, 4,133 an 886 g m-2 were utilized, respectively. Growth period requirements of

aquatic plants also differed accordingly to plant species and environmental factors. Pseudoraphis

spinescens required the longest period of 11-15 months, whilst Potamogeton malaianus required theshortest period of 5 months. Whereas, Eichhornia crassipes, Nelumbo nucifera and Typha angustifolia

required the period of 6 months, Salvinia cucullata required 7 months. The physical and chemical quality

of the water obtained from Bung Borapet were found suitably for all common aquatic plants in thefollowing ranges : depth of 135-345 cm, water transparency of 57-182 cm., temperature of 27.5-29.2 °C,

DO of 2.9-5.2 mg/l, B.O.D. of 2.1-3.1 mg/l, alkalinity of 95.2-108.9 mg/l, pH of 7.2-7.8. The average

amounts of base nutrient, nitrate, phosphate and potassium were 0.16, 0.01 and 3.9 mg/l, respectively. Theground-table soil was characterized as clay with the pH of 5.1 and 1% organic matter.

Key words : growth period, aquatic plant, bird nesting, Bung Borapet

INTRODUCTION

Bung Borapet is a big source of fresh water

of Nakhon Sawan province and also of the central

region of Thailand. This fruitful swamp is servedfor the fresh fishery development center and for the

source of fishery cultures. With the great density of

various aquatic plants, the swamp is characterized

to be the suitable habitat and nesting of several

birds, and has been notified as the animal forbiddenarea since 1975. Results from the great density of

various aquatic plants has led to the sedimentation

and more shallowness. To solve this problem, theDepartment of Fishery launched the project for

water drainage and area renovation during February

to October, 1992. This caused an ecology change in

1 Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.2 Department of Forest Biology, Faculty of Forestry, Kasetsart University, Bangkok 10900, Thailand.3 Kongtong School, Department of General Education, Ministry of Education, Bangkok, Thailand.

18 Kasetsart J. (Nat. Sci.) 34 (1)

the lost of a number of aquatic plants along with themore impact of severely weeds such as

Pseudoraphis spinescens. This ecological change

also had impact to several aquatic plants requiredfor bird nesting.

Recently, a number of aquatic plants in

Bung Borapet has declined progressively accordingto the report of 73 species identified (Sripen, 1979)

to 46 species (Plordprasop, 1982). After the swamp

drainage in 1992 and re-reservoir, the Division ofFresh Fishery reported for the only remaining 32

species. Suchada (1987) pointed out that factors

influencing the growth of aquatic plants including,the water depth level, light, water transparency,

temperature, gases content, and the chemical

component of minerals and compounds. Otherfactors affecting the distribution of aquatic plants

were wind, water level, rainfall, and the competition

among the plants (Amornrat, 1984). Study onnesting and laying behavior of birds in Bung Borapet

(Siriporn, 1983) revealed more than 10 species : 1

species in the area of water-level aquatic plants, i.e.

Hydrophasianus chirurgus ; 5 species in the area of

emerged aquatic plants, i.e. Hydrophasianus

chirurgus, Dendrocygna javanica, Metopidius

indicus, Ixobrychus cinnamoneus, and Ixobrychus

sinensis ; 6 species in the area of sedges, i.e.

Ixobrychus cinnamoneus, Ixobrychus sinensis,Dupetor flavicollis, Dendrocygna javanica, and

Gallicrex cineru ; 3 species in the area of aquatic

forest, i.e. Ixobrychus cinnamoneus, Dendrocygna

javanica, and Porzana cinerea. Wildlife

Conservation Division (1983) reported 11 species

of birds that required nesting materials from theaquatic plants, i.e. Salvinia cucullata, Nelumbo

nucifera, Scirgus grossus , Eichhornia crassipes,

Typha angustifolia and Pheudoraphis spinescens.Obhas (1991) found more than 107 species of birds

in the Bung Borapet, those that nested only around

the edge of the swamp were about 14 species. Mostof the birds mest in rainy season during July. This

indicated the significance of aquatic plants onnesting behaviors, particularly the water birds. The

objectives of this study were, therefore, to determine

the effects of growth period and density of particularaquatic plants on bird nesting behaviors in the

Bung Borapet, and to evaluate the factors

influencing the growth of water birds, i.e. physicaland chemical properties of water and ground-table

soil of the swamp. The information obtained,

particularly on the elementary biology, will be veryuseful and can be exploited for area management to

facilitate further development correspondingly with

the maximized preservation of resources andenvironment with less disadvantage.

MATERIALS AND METHODS

The aquatic plants nested by the birds at Bung

Borapet were utilized in the study were :1. Cuculate salvini, Salvinia cucullata Roxb.

2. Deepreenam, Patamogeton malaianus

Miq.

3. Narrow leaved Typha angustifolia L.

cattail,

4. Water hyacinth, Eichhornia crassipes (Mart)

Solms

5. Sacred lotus, Nelumbo nucifera Gaerth.

6. Yak preak nam, Pseudoraphis spinescens

Vickery.

The birds studied at Bung Boraphet were :-

1. Nok E Jaew Hydrophasianus chirurgus

2. Nok E Koang Porphyrio porphyrio

3. Nok Unchun Porzana cinerea

Kue Khao4. Nok Yang Fai Ixobrychus cinnamoneus

Thummada

5. Nok Yang Fai Ixobrychus sinensis

Hua Dum

6. Nik Yang Dum Dupetor flavicollis

7. Nok Ped Dang Dendrocygna javanica

8. Nok Prik Metopidius indicus

Kasetsart J. (Nat. Sci.) 34 (1) 19

Density of bird nesting aquatic plantsOn the area of the swamp where bird nesting

could be observed, the 1 × 1 quadrat was used for

the collecting of sampling aquatic plants, i.e. S.

cucullata, E. crassipes, P. malaianus, N. nucifera,

T. angustifolia, and P. spinescens. Sampling plants

were then determined for fresh and dry weight, anddensity (biomass) per m2. In all cases, maximum

number of practical samplings were suggested.

Growth period of bird nesting aquatic plantsThree plots of the size 2 × 2 m were assigned

for each of the investigated floating aquatic plants,i.e. S. cucullata and E. crassipes. For the submerged

and emergent aquatic plants such as P. malaianus,

N. nucifera, and T. angustifolia, a plot size of 5 × 3m was assigned for each plants. All aquatic plants

were allowed for their natural growth and then

samplings were executed by using a 1 × 1 mquadrat. Sampling plants were analyzed for fresh

and dry weight as well as the density at monthly

interval. For the P. spinescens, only the naturallywell growth plot of the size 5 × 3 m was selected to

determine for growth change at monthly interval

using the same sampling method.The data observed for plant density (per m2)

of each plant were compared for the growth period

that in turn was suitable for bird nesting accordingto the method indicated.

Water quality of the swampThe Van-dron water sampling tube was

used for the collecting of sampling water from 4

sites of the Bung Borapet. These sites were assignedfor the evaluation of the amount of water soluble

oxygen, B.O.D. value, pH, nitrate (NO3-N),

phosphate (PO4-P) and potassium (K) using theStandard Method (Swingle, 1969). The pH value

was measured wiith pH-meter. The values of water

transparency and depth were obtained through theSecchi dich. Temperature was measured with

thermometer. Data were collected at monthlyinterval for the period of 12 months from May 1993

to April 1994.

The study was conducted on the area ofinternal edge of the Bung Borapet, Nakhon Sawan

Province. Four sites were selected for sampling of

the water swamp. The first site was the outlet ofwaterway close to the Fresh Water Fisheries

Development Center, Amphur Muang, Changwat

Nakhon Sawan. The second site was at the center ofthe Bung Borapet, Ban Kloe Ta Seng. The third site

was at the first inlet of waterway, Klong Bon, Ban

Panomset while the fourth site was the second inletof waterway, Klong Huayhin, Amphur Tha Ta Ko.

In addition, plot layout for the growth study

of the aquatic plants was assigned at the center ofthe Bung closely to the Bung Borapet Conservation

Section, Ban Kloe Ta Seng.

RESULTS AND DISCUSSION

Density of bind nesting aquatic plantsThe results from the study on density of the

plants that birds can nest was calculated as the

relative density to the dry weight m-2 basis (Table1 and Table 2). It was found that density of each

plant differed greatly depending upon the condition

of nesting behavior of different birds. Those nestingat the water level, e.g. H. chirurgus, preferred not

much dense materials such as S. cucullata at the

lowest density of 267 g m-2, P. malaianus of 203g m-2 and P. spinescens of 229 g m-2. For birds

nesting over the water level, i.e. P. porphyrio, P.

cinerea, I. cinnamoneus, I. sinensis, and M. indicus,required the more dense materials such as E.

crassipes, P. spinescens, and T. angustifolia, at the

densities of 1,050, 454 and 4,133 g m-2, respectively.In case of N. nucifera, most of the birds did not

require to nest directly but preferred the leaves that

floated at the water level and over the water levelfor hiding or supplementing nesting materials. The

20 Kasetsart J. (Nat. Sci.) 34 (1)

Table 1 Species of aquatic plants and species of birds living on the plants at Bung Borapet.

Aquatic plants Bird species

1 2 3 4 5 6 7 8

S. cucullata + + +

E. crassipes + + + + +P. malaianus + + +

T. angustifolia + + + + +

N. nucifera +P. spinescens + + + + + +

1. H. chirirgus

2. D. flavicollis

3. P. porphyrio

4. P. cinerea

5. I. sinensis

6. I. cinnamoneus

7. D. javanica

8. M. indicus

Table 2 The biomass (gm/m2) of some aquatic plants that birds can nest at Bung Borapet, Nakhon Sawan.

Aquatic plant Sample 1* Sample 2 Sample 3 Average

Salvinia cucullata Roxb. 267* 320 389 325.3

Eichhornia crassipes (Mart.) Solms 1,050* 1,225 1,365 1,213.3Potamogeton malaianus Miq. 203* 240 285 242.7

Nelumbo nucifera Gaertn 886* 1,064 1,132 1,027.3

Typha angustifolia L. 4,133* 5,227 5,887 5,075.6Pheudoraphis spinescens Vickery1 229* 264 321 271.3

Pheudoraphis spinescens Vickery2 454* 543 678 561.7

1 = nesting at the water level

2 = nesting over the water level* = lowest density of aquatic plants that birds can nesting

clump of this plant that birds preferred wouldnormally have the emergent leaves at the density at

least 886 g m-2.

Moreover, the requirement of more suitablenesting materials was considered, e.g. for the ability

of supporting the weight of bird’ s egg and body.

For example, H. chirurgus, G. cinerea, andD.flavicollis preferred to nest a rough one on the

cluster of S. cucullata, P. malaianus, or the decayed

plants and laid their eggs over the water level. Bycontrast, P. porphyrio preferred to nest over the

water level in the area of dense aquatic plants and

Kasetsart J. (Nat. Sci.) 34 (1) 21

hided their nests in the clump of E. crassipes orbeneath the leaves of N. nucifera. This place of

nesting would be very secure in preventing of not

to be dispersed easily by wind and swamp water. Itwas also noticed that birds might use different

nesting materials, e.g. in the past, H. chirurgus use

S. cucullata to build up 2-3 layers of the nest(Division of Wildlife Conservation, 1983). But

today, the birds preferred more to nest on the dead

clump of P. malaianus and P. spinescens at thewater level.

Growth period of bind nesting aquatic plantsThe growth period of S. cucullata, E.

crassipes, P. malaianus, N. nucifera, and T.

angustifolia in Bung Borapet through theobservation of dry weight (g m-2) and the ecological

change of aquatic plants after the re-reservoir of the

swamp water since January 1993 to April 1994,indicated that growth period of each plant required

for bird nesting differed significantly depending on

plant species and environmental conditions. P.

malaianus required the shortest period of 5 months,

whilst, E. crassipes, N. nucifera and T. angustifolia

required 6 months. The other, S. cucullata required7 mouths and P. spinescen required 11-15 mouths.

It was then considered that at the time of study, the

swamp condition was probably most suitable forthe growth of P. malaianus.

The average of water level of 229 cm over

the year would be best suitable for the growth of P.

malaianus (Chanpen, 1983). The average of water

transparency was relatively high of 113 cm due to

rainfall and less removal of the alluvium causingfull light of this plant. On the contrary other aquatic

plants e.g. S. cucullata and E. crassipes that were

mixed with N. nucifera or algae had faster earlygrowth stage than those of the loose clumps. Yet

lower growth rate was noticed at the later stage of

growth due to shading of the above water levelleaves of N. nucifera. Besides the annually growth

habit, growth period of the aquatic plants in BungBorapet was influenced by the infestation of the

insect larvae in the family Noctuidae. The larvae

preferred to maintain feeding on the emergentleaves and the reproductive parts of the plants

causing death of the aquatic plants. This type of

ecosytem change occurs at leat once a year duringAugust to September.

In case of P. spinescens, the study was

emphazied on growth change of the plantspredominating over the swamp at the time of water

drainage and commencing of re-reservoir of the

water. Well growth of the plants at the early stagewas noticed correspondingly with plant elongation.

Thereafter, the growth declined due to no longer

adaptation to the very long period of submergence.At the time of this study, it was noticed that the

birds began to use P. spinescens for nesting in 2

types, the one at the water level and that over thewater level. And the birds, espectially H. chirurgus,

preferred to use more P. spinescens than in the past.

Water quality of the swampDetailed study in both physical and chemical

water qualities through the analyses of samplingwater from the 4 designated sites revealed that the

Bung Borapet water was characterized to be suitable

for normal growth and the living of most animalsand plants (table 3). The average of water level over

the year was 135-345 cm and none of any adverse

effects on the classified layers of water temperaturewas detected. The average of 113 cm of water

transparency was considered as relatively clear

water. The 28.5oC mean temperature was claimedto be optimal range for the growth of living

organisms in the swamp. Water soluble oxygen

(D.O.) content at the average of 4.4 mg/l was alsodenoted in the optimal range (Swigle, 1969). Except

that of the second inlet of the waterway where a

relatively lower value of 2.9 mg/l was observed dueto severe waste water release from houses located

22 Kasetsart J. (Nat. Sci.) 34 (1)

around the edge of the swamp correspondingly

with the high density of aquatic plants. B.O.D

value of the swamp averaged 2.6 mg/l eventhougha high value of 3.1 mg/l from the second inlet of the

waterway was detected. Hence, the swamp water

was considered acceptably and was not claimed tobe waste water according to the standard B.O.D.

value in the range of 1.5-4.0 mg/l given for the

ground–surface soil water quality (Division ofEnvironmental Quality Standard, 1991). The

average of pH was 7.5. The average of base nutrients

was 102.5 mg/l. The average amount of nitrate,phosphate and potassium were 0.16, 0.01 and 3.9

mg/l, respectively. The amount of swamp nutrients

was acceptably in the case of being natural waterresource, but was considered relatively low in the

sense of plant water quality causing poor growth of

the plants. The porn bottom soil of the swamp wasclassified as clay with relatively low organic matter

of 1%. In addition, poor growth of the submerged

plants was also denoted.According to the Bung Borapet

Development Project, the processes of water

drainage, the clean out of the pond bottom soil, andthe drying out of the ground-surface wamp would

result in the marked change of the ecosystem

towards the drought prone area. These would reflect

directly to the growth of a number of aquatic plants

in quantity and species. In addition, indirectinfluence was noticed for animals that required

those plants. The authors would suggest the

modified methods of water drainage that mighthave less adverse effects on the ecosystem of the

swamp, at the same time would serve the maximum

objectives of the project as follows :1. No fully water drainage at once is strongly

recommended to avoid the severe effects on

aquatic plants. At the same time, sequential zonesfor step further development should be designed.

For instance, the water drainage at certain zone

planned for the development will be use for aquaticanimal preserve or serve as the preservation area of

the living and nesting birds. This can be done

through the preserve of the area in the way whichcauses less ecosystem change along with other

developmental processes.

2. Long period of drying out of the ground-surface should be avoided to prevent the lost of

tuberous aquatic plants. At the same time some

inland weeds such as P. spinescens would disperseover the swamp. This weed was considered to be

one of the noxious weeds due to the tolerance to

Table 3 Chemical and physical properties of water at Bung Borapet.

Depth 135 – 345 cm 240 cm

Water Transparency 57 – 182 cm 113 cmTemperature 27.5 – 29.5 °C 28.5 °C

pH 7.2 – 7.8 7.5

D.O. 2.9 – 5.2 4.4B.O.D. 2.1 – 3.1 2.6

Base nutrient

- nitrate 0.16 mg/l- phosphate 0.01 mg/l

- potassium 3.9 mg/l

Kasetsart J. (Nat. Sci.) 34 (1) 23

either drought and submerge area in the swamp.Moreover, the weed grows and disperses quickly,

therefore is difficult to control. After the re-reservoir

of the swamp water, some of this weed would diecausing bad smell and the shallowness of the swamp.

It is suggested that water drainage should commence

in dry season from December when the water levelis certainly low. The clean up of the pond bottom

soil must complete prior to the coming rainy season.

Supply of water in the early stage of re-reservoir isessentially to allow the period that all aquatic plants

can grow well since June which is the time that bird

nesting could commence at least 3-4 months earlierthan usual.

CONCLUSION

The conclusion of the studies could be drawn as

follows :1. A study on density of the aquatic plants

that birds can nest revealed the lowest density of

each plant to be differed according to therequirement of different birds. All birds apparently

preferred N. nucifera as hiding place or for a

supplemented nesting materials at the lowest densityof 886 g m-2.

2. The growth periods of the aquatic plants

that birds can nest differed greatly depending onplant species and environmental conditions. Birds

could nest in 2 types : the one over the water level

at the period of 11 months and, that of the waterlevel at 15 months. For aquatic plants mixed with

N. nucifera or algae would grow well in the early

stage due to the better and firmly clumps of theplants. But later, poor growth was denoted due to

the shading effects of the leaves of N. nucifera.

3. Results from the analyses of physicaland chemical qualities revealed the swamp water to

be acceptable for normal standard of the living

organisms.4. The competition of soil at the ground-

surface level of the swamp was characterized asclay with pH 5.1 and 1% of organic matter.

LITERATURE CITED

Amornrat Sermwattanakul. 1984. Dispersal of the

aquatic plants and their related animals inBung Borapet, Nakhon Sawan. M.S. thesis,

Kasetsart University, Bangkok. (In Thai)

Chanpen Praklongwong. 1983. Study on thebotanical of Potamogeton malaianus Miq. at

Bung Borapet. M.S. thesis, Kasetsart

University, Bangkok. (In Thai)Division of Environmental Quality Standard. 1991.

The Water Quality Standard in Thailand. Office

of the Environmental Policy and PlanningCommittee, Bangkok. 135 p. (In Thai)

Division of Fresh Water Fisheries. 1992. The Survey

of Biofishery in Bung Borapet during thePeriod of Water Preservation. Department of

Fisheries, Ministry of Agriculture and Co-

operative, Bangkok. 79 p. (In Thai)Obhas Kobket. 1991. The White-eyed River Matin

and other birds in Bung Borapet. Royal Institute

Journal 17(1) : 19-35. (In Thai)Ploadprasop Suraswadi. 1983. The Improvement

and Development of Bung Borapet. Additional

study in 1982-1983. The Institute of SocialScience, Chulalongkorn University, Bangkok.

131 p. (In Thai)

Siriporn Tongaree. 1983. Study on bird nesting andlaying in the area of Bung Borapet. M.S.

Special Problem, Kasetsart University,

Bangkok. (In Thai)Suchada Sripen. 1987. Aquatic Plants. Department

of Botany, Faculty of Science, Kasetsart

University, Bangkok. 233 p. (In Thai)Sripen, S. 1979. Study on the Aquatic Weeds at

Borapet Lake, p. 385. In Proceeding 7th Asia

Pacific Weed Sci. Soc. Conf., Sydney,Australia.

24 Kasetsart J. (Nat. Sci.) 34 (1)

Swingle, H.S. 1969. Method of Analysis for WaterOrganic Matter and Pond Bottom Soils Used

in Fisheries Research. Auburn University,

Alabama. 119 p.Wildlife Conservation Division. 1983. Study on

the population and nesting laying of the birds

in the forbidden area of Bung Borapet, Nakhon

Sawan. Department of Forestry, Ministry ofAgriculture and Co-operative, Bangkok. 85 p.

(In Thai)

Received date : 18/10/99Accepted date : 29/12/99

Kasetsart J. (Nat. Sci.) 34 : 25 - 29 (2000)

Study of Dropping Speed in Eggs of Oncomelania hupensis,a Snail Intermediate Host of Schistosomiasis

Xingjian Xu,1 Xianxiang Yang,1 Xiapin Li,1 Wei Zhang,2

Qingsang Pan2 and Zhengan Xiong2

ABSTRACT

Oncomelana hupensis (Gredler, 1881) is an intermediate host of Schistosomiasis japonica in China.

In order to understand the factors controlling sedimentation and drifting of adult snails and snail eggs in

rivers, three aspects were investigated by experiment. Firstly, the specific gravity of the snail eggs whichwas found to be 2.29 g/cm3 ; secondly, the range of dropping speed of the snail eggs through a water column

which was found to be 1.19 to 3.77 cm/s; thirdly, a formula to determine dropping speed of the eggs was

established. The formula was statistically validated by comparison with observed values. The results arerelevant to both the development and design of irrigation schemes, and also to river management, where

dispersal of snails and their eggs needs to be controlled.

Key words : dropping speed, Oncomelania hupensis, snail eggs

1 Hubei Institute of Schistosomiasis Control, Wuhan 430070, The People’s Republic of China.2 Science Institute of Yangtze River, Water Conservancy Commission of Yangtze River, Wuhan, The People’s Republic of China.

INTRODUCTION

Schistosomiasis is now endemic in 74countries and territories of the world and

Schistosoma japonica is mainly distributed in

Southeast Asian and the Western Pacific Region(WHO, 1991). One of the most difficult problems

for schistosomiasis control is the dispersal of the

snail intermediate host along rivers and irrigationschemes. This dispersal expands the endemic areas

of schistosomiasis and increases the prevalence of

the disease (Hunter, 1993; Mott, 1990). It isimportant, therefore, to explore methods for

controlling the snail host. The ecology of the water

snails in relation to water flow has been well-researched and some effective models have been

proposed (Dussart, 1987; Yin et al., 1987; Bolton,

1988; Xu and Fang, 1989; Yang et al., 1992; Green

et al., 1992).

S. japonica is mainly distributed in eightprovinces in the southern part of China, with foci in

five provinces along the middle and lower reaches

of the Yangtze River (MPH, 1991; Mao, 1990).Oncomelania hupensis, the intermediate snail host

of S. japonica, is mainly distributed on banksides

of rivers, ditches and irrigation schemes throughoutthe flood plain and the snail habitats are increasing

year by year. The disease seriously threatens farming

and the daily life of local people, and also affectsthe socioeconomic development of these areas

(MPH, 1989; Chen, 1989; Xu and Fang, 1990).

Thus, snail control is one of the most importantfactors for disease control. To understand the

parameters which might control the drifting and

26 Kasetsart J. (Nat. Sci.) 34 (1)

dropping of snails and their eggs in rivers, theexperiments on specific gravity and dropping speed

for the adult snails have been carried out (Xu et al.

1996). The aim of present study was to carry outmeasurement on the specific gravity and dropping

speed for the eggs of O. hupensis, to contribute to

the development of a solid basis for the preventionof snail dispersal.

MATERIALS AND METHODS

Snail eggs were obtained from snail habitats

in Han Yang county in Hubei province at the end ofApril 1997. Adult snails were collected and put in

a bowl of mud at 25 °C for 15 days. After two

weeks, the snails were removed and snail eggswere washed from the surface of the mud in the

bowl. All eggs selected for measurement were in

the primitive gut embryonic stage. A 25 ml specificgravity flask was used to determine specific gravity.

Dropping speed was measured in a water-filled

glass tube of 160 cm in length and 4.5 cm indiameter, in which a water column of 130 cm

length was available to observe the eggs dropping.

Measurement of the specific gravity of the snaileggs was carried out in accordance with the routine

measurement method for the determination of

specific gravity of river silt in China of Sha (1963).The snail eggs washed from the mud bowl

were collected by suction tube in a tissue culture

dish 9 cm in diameter. The snail eggs were weighedand separated on Whatman’s No. 4 filter paper for

8 h to absorb excess surface water from the eggs.

The flask containing the eggs was put into adesiccator for 8 h to continue the drying process.

The snail eggs were weighed, placed in a graduated

flask and made up to 25 ml; the flask was then filledwith distilled water and shaken gently before being

weighed on a balance. Thus the weight included

graduated flask, the snail eggs and distilled water.The distilled water and snail eggs were then

discarded from the flask. The flask was dried insideand outside, distilled water was added to the level

of 25 ml and the flask was weighed again; thus the

weight included only graduated flask and distilledwater.

The following formula was used to calculate

the specific gravity of the snail eggs :SGe = ge / ge + g1 – g2 * SGH2Owhere,

SGe = Specific gravity of the snail eggs. (g/cm3)

ge = Weight of the snail eggs (g)

g1 = Weight of flask plus distilled water (g)g2 = Weight of the snail eggs plus flask and

distilled water (g)

SGH2O = Specific gravity of distilled waterat 4 °C (g/cm3)

Measurement of dropping speed of snaileggs in stable water. The diameter of each snailegg was measured under the microscope. The egg

was put into the 160 cm glass tube and dropping

speed was measured directly (MWE, 1965).Establishment and deduction of formula

for dropping speed of snail eggs. The formula for

snail eggs dropping speed in stable water was basedon the established principle for measuring the

dropping speed of a round object (Qian and Wan,

1983). The difference between the theoreticallypredicted and the measured values was statistically

investigated.

RESULTS

Specifix gravity of snail eggs. The meanspecific gravity was 2.29 ± 0.0162 g/cm3, ranging

from 2.25 to 2.33 g/cm3.

Dropping speed of the snail eggs. Thediameter of each of 84 snail eggs was measured; 74

eggs had shapes which resembled a spherical pill.

The range of diameter of the snail eggs was 0.43 to0.83 mm; the dropping speed of the snail eggs was

Kasetsart J. (Nat. Sci.) 34 (1) 27

2.48 ± 0.2614 cm/s ranging from 1.19 to 3.77cm/s.

Establishment and deduction of theformula for dropping speed of the snail eggs. Asnail egg drops with uniform acceleration when

water resistance and the specific gravity of the egg

become balanced. Because the shape of the snaileggs is similar to spherical pill, the force (G) acting

on the eggs in stable water is given by G = π/6*d3

(γ1-γ0)............(1) (Qian and Wan, 1983).where,

G = Gravitational force acting on the snail

eggs in water (g)F = Water resistance

W = Dropping speed of the snail eggs in

stable water (cm/s)Ca = Resistance coefficient

γ1 = Specific gravity of the snail eggs (g/

cm3)γ0 = Specific gravity of water (g/cm3)

g = Gravity plus speed (cm/s)

d = Diameter of the snail eggs (mm)Snail eggs which drop in water meet water

resistance. The resistance equation of the snail

eggs in stable water isF = Ca * γ1 * π/4 * d2 * w2 /2g............(2)

When G = F, the resistance snail eggs drop

with uniform acceleration. The equation is thereforeCa = 4/3 (γ1 - γ0) / γ0 * gd / w2............(3)

Based on a general principle of silt motion,

there is a resistance coefficient (Rd ........ alsocalled circumflow Rd). The function of the

relationship is given by :

Ca = f (Rd)............(4)In which Rd = wd / v , where w = dropping speed

of the snail eggs; d = diameter of the snail eggs; and

v = flow rate. When a correlation was made withthe experimental data, the result was

Ca = 250 / Rd............(5)

Therefore, the dropping speed formula ofthe snail eggs in stable water can be formed from

equation (3) and (5), and is :w = 4/750v * (γ1 - γ0) / γ0 * gd2............(6)

The unit of each symbol in the formula is: w

= cm/s; d = cm; γ1 = 2.29 g/cm3; γ0 = 1 g/cm3; g =98 cm/s2, which can change with the water

temperature.

A t test was used to investigate the differencebetween the practical measured value and the

predicted value of the dropping speed. With P >

50%, there was no significant difference betweenthe predicted and measured value.

DISCUSSION

Oncomelania hupensis is distributed mainly

in marshland and lakes in southern China. The peakegg laying period is spring and autumn of every

year. The snail eggs are light and small, so they drift

easily in the flood season. Once the eggs settle insuitable hatching sites, new snail populations

develop. Therefore, it is important to try to control

the dispersal of snails and their eggs in irrigationschemes in endemic areas.

The snails lay eggs at the edge of lakes and

rivers. When eggs are laid on moist mud, the eggsbecome covered with soft mud so that they have the

appearance of a small mud pill. It has been observed

that if the mud layer comes off at the firstdevelopment stage of the eggs, the eggs do not

develop into a juvenile snail (Guo, 1983). Thus, the

mud layer seems to plays an important role in thegrowth phase of the snail eggs. Microscopic

observation showed that the drying procedures

used here did not damage the internal or externalphysical and biological characteristics of the eggs.

The measured values would therefore appear to be

reliable.The specific gravity of the eggs of O.

hupensis is an indispensable basic physical

parameter in the calculation of dropping speed anddrift distance for snail eggs in still- and running-

28 Kasetsart J. (Nat. Sci.) 34 (1)

water. This information may contribute to atheoretical basis for the prevention of snail dispersal

in the field.

Based on the dropping speed of snail eggsmeasured in this study, the drifting style of the snail

eggs can be compared with the dropping speed of

silt in rivers. Usually, 0.5 mm diameter silt particlesare suspended in flowing water but drop at a rate of

5.67 cm/s in still water. Our results show the

dropping speed for snail eggs to be 1.19 to 3.77cm/s, which is lower than the result for silt that is

suspended as drift in rivers. Therefore, it can be

inferred that snail egg dispersal will at least conformwith the drift of silt in the rivers. These observations

may have practical value for developing engineering

measures to control and mitigate the dispersal ofsnails and their eggs.

CONCLUSIONS

Investigations were carried out into specific

gravity and dropping speed of snail eggs and anappropriate formula has been devised to link these

parameters. Statistical analysis showed no

significant difference between measured valuesand the values predicted from a formula for dropping

speed based on Newtonian principles. The result

could be used as a basic parameter in the control ofOncomelania hupensis when water conservation

facilities are designed or rebuilt in areas in which

Schistosomiasis is endemic.

ACKNOWLEDGEMENT

The study was supported by UNDP/

WORLD BANK/WHO Special Program for

Research and Training in Tropical Diseases (TDR);Ministry of Public Health and Chinese Scholarship

Council in China. The authors gratefully

acknowledge the help of Dr. Banpot Napompeth(National Biological Control Research Center,

Kasetsart University, Thailand) and Dr. GeorgesDussart (Canterbury Christ Church University

College, UK) in the revision of the manuscript.

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Chen, M.G. 198. Schistosomiasis control programin the People’s Republic of China : A review.

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and Public Health 20 : 511-517.Dussart, G.B. 1987. Effect of water flow on the

detachment of some aquatic pulmonate

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Gredler, V. 1881. Zur Conchylien-Fauna von China.

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Green, P., Dussart, G., and C. Gibson. 1992.

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(Muller), Biomphalaria glabrata (Say) and

Bulinus jousseaumei (Dautzenberg). Journalof Molluscan Studies 58 : 169-179.

Guo, Y.H. 1983. Scanning observation on

Oncomelania hupensis with electronmicroscope. Treatise compilation of Chinese

Molluscs Society. Science Press, Beijing,

China 1 : 81-83.Hunter, J.M. 1993. Parasitic Diseases in Water

Resources Development : the Need for

Intersectoral Negotiation. Geneva, WorldHealth Organisation. 12 p.

Mao, S.B. 1990. Schistosomiasis biology and

schistosomiasis control. People’s Health Press,Beijing, Chiina. p. 260-330.

Mott, K.E. 1990. Parasitic diseases and urban

development. Bulletin of World HealthOrganisation 68 : 691-698.

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Ministry of Public Health (MPH). 1989.Consultative Committee of Schistosomiasis

Control in China. National report on prevalence

of Schistosomiasis in 1988. Chinese Journal ofSchistosomiasis Control 1 : 5-7

Ministry of Public Health (MPH). 1991.

Consultative Committee of Schistosomiasis

Control in China. National report on prevalence

of Schistosomiasis in 1990. Chinese Journal of

Schistosomiasis Control 3 : 193-194.Ministry of Water Electricity (MWE). 1965. Rules

of operation on earthwork. Chinese Industry

Press. Beijing, China. p. 2-22Qian, L. and Z.H. Wan, 1983. Silt movement

kinetics. Chinese Science Press, Beijing, China.

p. 36-88.Sha, Y.Q. 1963. Silt motion theory. Chinese Science

Press, Beijing, China. p. 20-45.

WHO. 1991. Schistosomiasis control. Geneva,World Health Organisation.

Xu, X.J. and T.Q. Fang. 1989. Observation on

collecting Oncomelania hupensis when thesluices open during flood season in Yangtze

River. Kasetsart J. (Nat. Sci.) 22 : 251-260.Xu, X.J. and T.Q. Fang. 1990. Relationship between

sluicing for irrigation and spreading of

Oncomelania hupensis in Hubei province.Kasetsart Journal (Nat. Sci.) 23 : 281-286.

Xu, X.J., X.X. Yang, and X.P. Li 1996.

Establishment and deduction of a formula ofthe snail dropping speed in stable water. Hubei

Journal of Preventive Medicine 7 : 26-28.

Yang, X.X, X.J. Xu, and Z.H. Yu. 1992. Observationon preventing snail drift through an inverted

siphon. Chinese Journal of Zoology 27(4) : 12-

15.Yin, C.M., T.S. Wan, D..S. Mao, L.C, He and Z.,G.

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from spreading with double blocking nets atthe ditches. Chinese Journal of Zoology 33 :

194-196.

Received date : 16/09/99Accepted date : 20/12/99

Kasetsart J. (Nat. Sci.) 34 : 30 - 39 (2000)

INTRODUCTION

Vesicular-arbuscular mycorrhizal fungi(VAMF) is one of the most interesting soil

microorganisms which can solubilize fixed

phosphate and retentive phosphate in soil. Throughobservation, it was found that VAMF enhanced

plant growth and P uptake particularly in the soil

with low to moderate P level (Schenck, 1982;Bolan, 1991). In certain soils with high level of P,

positive relations between VAMF and plant growth

were also found (Kiernan et al; 1984., Mala et al.,

1997). However, many species of VAMF can notenhance plant growth and P uptake of plant even in

low fertile soils, mainly due to some limiting

conditions appearing in the soils. The quantity ofavailable P is one of the important factors which

limits the efficiency of VAMF. The mechanisms

for acquisition of P and other mineral nutrients intheir deficient soils, drought tolerance and protection

Selection for the Effective Speciesof Vesicular-Arbuscular Mycorrhizal Fungi

on Soybean Root Infection and Growth Enhancement

Thongchai Mala

ABSTRACT

The selection for the effective species of vesicular-arbuscular mycorrhizal fungi (VAMF) wasconducted in the greenhouse of Soil Science Department, Kasetsart University at Kamphaengsaen

Campus. Nine VAMF species, from pot production in Yangtalard soil, consisted of Acaulospora dilatata,

Acaulospora scrobiculata, Entrophospora colombiana, Gigaspora sp. No 2, Glomus aggregatum, G.

claroideum, G. geosporium, G. tenuis and Scutellospora sp. were inoculated to SJ5 soybeans grown in a

6–litre clay pots with sterilized sand medium. The results revealed that various VAMF had different

abilities to infect soybean roots. The VAMF species having fast infection rate and high intensities of rootcolonization were E. colombiana, A. scrobiculata, G. aggregatum, G. geosporium and Scutellospora sp.

The intensities of root colonization were 96.75, 95.25, 93.75, 90.50 and 89.25 %, respectively. The ability

of VAMF on hyphal development in soil were differed among species. G. aggregatum, G. geosporium,

Scutellospora sp. and E. colombiana had greater hyphal development than the rest while their lengths of

extraradical hyphae at harvest period were 168.33, 137.37, 134.85 and 128.90 cm, respectively. Finally,

high yield and high mycorrhizal dependency were found in plants inoculated with Scutellospora sp., E.

colombiana, G. aggregatum and G. geosporium. These VAMF species showed high potential to enhance

soybean growth and should be further examined with different levels and kinds of phosphorus fertilizer in

various conditions.Key words: extraradical hyphae, root infection, soybean, vesicular-arbuscular mycorrhizal fungi.

Department of Soil Science, Faculty of Agriculture, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom 73140, Thaialnd.

of plant root from destruction by pathogen ofmycorrhizal plants are described (Bolan, 1991).

Therefore, the compatibility among certain soil

environment, plants and VAMF species must berealized in order to acquire appropriate benefit

from VAMF-plant association. VAMF are differed

in their responses to phosphate application, producedifferent amounts of hyphae in the soil even at the

same phosphate treatment (De Miranda and Harris,

1994). Most of VAMF species decrease theirabilities on infecting plant roots with increasing

phosphate application but some species can tolerate

and show high activity even in high phosphateconditions (Marschner and Dell, 1994). The study

on various VAMF species and levels of rock

phosphate which are suitable for the application tosome soils in order to obtain maximum return from

individual plant cultivation is highly recommended.

The objective of this experiment is to compare theeffectiveness of selected VAMF species on soybean

growth enhancement.

MATERIALS AND METHODS

The experiment was conducted in thegreenhouse of Soil Science Department, Kasetsart

University at Kamphaengsaen Campus using

completely randomized design with 4 replications.Treatments consisted of soybean plants grown in

six-litre clay pots without VAMF(control) and

nine other species of VAMF; Acaulospora dilatata,

Acaulospora scrobiculata, Entrophospora

colombiana, Gigaspora sp. No 2., Glomus

aggregatum, G. clariodeum, G. geosporium, G.

tenuis and Scutellospora sp. Before starting the

experiment, the surface of every 6-litre clay pot

was cleaned and sterilized using 70 % ethyl alcohol.Seven kilograms of sterilized sand for individual

pot was prepared. Inoculums of VAMF were

prepared in sterilized Yangtalard soil as describedby Mala et al.(1997). Individual species of

mycorrhiza was inoculated into the soil by mixing1,000 ml of soil with 200 ml of inoculum. The

mixture of inoculum (inoculum layer) was spreaded

and pressed down into the pot, then, 600 ml of theremaining soil was topped over the inoculum layer,

leveled and pressed down. Sterilized inoculum was

used in the control treatment.Four-surface sterilized SJ5 soybean seeds

were sowed at 2 cm deep under the soil surface.

Water and nutrient solution were alternativelyapplied (Asher, 1975). One plant per pot was left

after 2-week emergence in the greenhouse until

harvest. Insecticide (monocrotophos) was sprayedtwice at 6 and 10 weeks after planting. The

percentage of mycorrhizal root infection using

Grideline Intersect Method (Shenck, 1982;Giovanetti and Mosse, 1980), length of extraradical

hyphae (Bethlenfalvay and Ames, 1987) and plant

height were determined both at 6 weeks afterplanting and at harvest time. Measurement of stem,

root dry weight, yield and yield component were

also conducted. Mycorrhizal dependency onsoybean yield (MDSY) were determined according

to Plenchette et al. (1983) as follows:

MDSY = Yi Yo

Yo

−

where Yi = yield of VAMF inoculated soybean;Yo = yield of control soybean.

Data were analyzed using analysis ofvariance procedure and the significance between

average means were compared using Duncan’s

new multiple range test at 95 % confidence.

RESULTS AND DISCUSSION

Colonization of VAMF on SJ5 soybean root

Colonization of VAMF on the root is the

first indication of relationships between the twosymbionts. The fungal structures in root and soil of

various VAMF were illustrated in Figure 1. In

Kasetsart J. (Nat. Sci.) 34 (1) 31

32 Kasetsart J. (Nat. Sci.) 34 (1)

contrast, there was not any structure of VAMFinfected to plant root in control plant (Figure 1-a).

There were many types of structures such as

intraradical hyphae, arbuscules, vesicles, restingspores and extraradical hyphae when plants were

inoculated with A. dilatata, A. scrobiculata, E.

colombiana, G. aggregatum, G. claroideum, G.

geosporium and G. tenuis (Figure 1-b, c, d, f, g, h,

and i). In plant inoculated with Gigaspora sp. No.

2 and Scutellospora sp., vesicle in plant roots werenot found. However, extraradical and intraradical

resting spores were found together with similar

fungal structures as found in other species (Figure1-e and j).

The main structures of various VAMF found

at harvest period were different (Table 1). Vesicleswere main structures found in roots inoculated with

E. colombiana and G. aggregatum, meanwhile,

arbuscules were prominent in Scutellospora sp.inoculation but hyphae were prominent in the

remaining species.

The intensity of colonization showed thetendency of VAMF ability on enhancing plant

growth. In this experiment, the abilities of various

VAMF on soybean root colonization wereillustrated in Table 2. Root infection percentages of

soybean caused by various VAMF at both stages,such as 6-week and harvest periods, were found to

be highly significant. Four species of VAMF, E.

colombiana, G. geosporium, Scutellospora sp. andG. aggregatum showed very high potential of

soybean root infection, meanwhile the rest of the

species showed rather low potential at 6 week-period. The most rapidly effective species at 6

weeks was E. colombiana (89.75 %) and the lowest

infection was found in soybean inoculated with A.

dilatata ( 7.75 %).

At harvest, the intensity of root infection of

various VAMF species were examined. Highpotential species (group a) which showed a very

high percentage of root infection were E.

colombiana, A. scrobiculata, G. aggregatum, G.

geosporium and Scutellospora sp. with the range of

infection were 89.25-96.75 %. The minimum

infection was found in A. dilatata inoculation as24.25 %.

The intensity of root infection of individual

VAMF species at harvest period was increased ascompared to its intensity at 6 weeks. In Some

species whose infections along the plant root

developed rapidly in the early stage (E. colombiana),the intensity of infection at this stage was further

Table 1 Main structure of VAMF found in the root of SJ5 soybean.

VAMF Main Quantity(%)

structure (compared to the whole VAMF structures)

A. dilatata Hyphae 95.95A. scrobiculata Hyphae 69.14

E. colombiana Vesicle 56.58

Gigaspora sp. No 2 Hyphae 87.39G. aggregatum Vesicle 46.15

G. claroideum Hyphae 68.00

G. geosporium Hyphae 55.96G. tenuis Hyphae 59.35

Scutellospora sp. Arbuscule 43.00

Kasetsart J. (Nat. Sci.) 34 (1) 33

Figure 1 Characteristics of VAMF root colonization on SJ5 soybean root ( a = non-inoculation, b = A.

dilatata, c = A. scrobiculata, d = E. colombiana, e = Gigaspora sp. No. 2, f = G. aggregatam,

g = G. claroideum, h = G. geosporium, i = G. tenuis and j = Scutellospora sp., bar = 0.2 mm).

progressed continuously and finally reached thepeak at 96.75 %. Certain species showed moderate

potential of infection at 6 weeks, such as G.

geosporium, Scutellospora sp. (71.00 and 69. 25%); however, the development of infection was

quite fast at harvest stage for these species up to

90.50 and 89.25 %, respectively. The species which

showed high ability for increasing infection from 6weeks to harvest period of plant growth was A.

scrobiculata. The root infection at 6 weeks was 31.

25 % and increased up 64 % to 95.25 % atharvest .

The difference of root infection between

two stages of plant growth explained how VAMF

34 Kasetsart J. (Nat. Sci.) 34 (1)

can enhance soybean successfully at differentgrowth stage. The species which infected plant root

intensively in early stage was also showed the

signal of high potential to enhance plant growth.The species which infected the plant root with

slower rate will be losing this advantage. Generally,

the infection rates of various VAMF are different.By determining the interval of root infection

intensity between two periods of plant growth, it

was found that the root infection of some VAMFspecies increased in a very high intensity, meanwhile

those of root infection caused by other species

increased in negligible amount. These roles maysuggest the categories that, the colonization on

plant root of those VAMF in early stage (6 weeks)

were highly intensive depending upon individualVAMF ability to infect and developing their

colonization through host root. VAMF in this group

were E. colombiana, G. geosporium andScutellospora sp. which initially infected host root

at a very fast rate and the intensity increased abruptly

to reach above 70 % within 6 weeks. These VAMF,however, still further developed colonies actively

and spreading their infection continuously to other

parts of plant root and reached their peak at harvestas shown in Table 2. The another category is that,

the intensity of root infection in early stage was low

due to the slowly development of VAMF on thehost root. This group of VAMF such as Gigaspora

sp. and A. dilatata, showed low root infectious

intensity at both stages of plant growth. Due to thelow efficiency on root infection at both stages, root

infectious intensity of both species were 21.50 and

7. 75 % at 6 weeks, and the development of rootinfection in the second stage was slowly occurred.

Their intensities of infection were 41.50 and 24.25

% which increased to 20.00 and 16.50 %,respectively.

In case of other species, the intensity of root

infection was at moderate level. But thedevelopment of VAMF occurred very fast in the

second stage which caused high intensity of root

Table 2 Colonization of various VAMF species on the root of soybean.

VAMF Root infection(%) Increased infectionAt 6 weeks At harvest after 6 weeks

Non inoculation(control) 0f 0f 0

A. dilatata 7.75f 24.25e 16.50

A. scrobiculata 31.25d-e 95.25a 64.00E. colombiana 89.75a 96.75a 7.00

Gigaspora sp. No 2 21.50e 41.50d 20.00

G. aggregatum 54.50c 93.75a 39.25G. claroideum 46.75c 79.25b 32.50

G. geosporium 71.00b 90.50a-b 19.50

G. tenuis 33.75d 62.25c 28.50Scutellospora sp. 69.25b 89.25a-b 20.00

CV.(%) 18.93 11.39

F-test ** **

Note: Means in each column followed by the same letter are not significant by DMRT at 95 % of confidence.

Kasetsart J. (Nat. Sci.) 34 (1) 35

infection at harvest. The VAMF in this groupincluding A. scrobiculata, G. aggregatum, G.

claroideum and G. tenuis showed the increasing of

colonization as 64.00, 39.25, 32.50 and 28.50 %,respectively.

The differences of VAMF manner, extent

and their abilities on infecting plant root wereattributed (Bolan, 1991; Abbott and Gazey, 1994).

Factors affecting VAMF infection may come from

the differences in inoculum quantity, types ofVAMF species, concentration of VAMF propagules

in inoculum, kinds of plant and concerned

environments. This experiment was carried out inthe same environment with the same amount of

inoculum. The quantity of spore of each VAMF

was above 40 spores/g. Various inoculums wereproduced and stored in the same condition.

Therefore, the difference in ability of VAMF on

infecting soybean root may be due to thecompatibility of individual VAMF on soybean

plant.

Extraradical hyphae of VAMFThe length of extraradical hyphae of

different VAMF species inoculated to SJ5 soybean

was illustrated in Table 3. The results of statistical

analysis of the extraradical hyphae of variousVAMF at both periods of plant growth were highly

significant. Three species of VAMF, G.

aggregatum, Scutellospora sp. and G. geosporium,showed prominent ability to produce extraradical

hyphae at 6 weeks as much as 47.65, 61.73 and

65.15 cm g-1of soil. Meanwhile, the other specieshad shorter extraradical hyphae.

After 6 weeks, the extraradical hyphae of

certain VAMF associating with SJ5 soybeandeveloped intensively through soil volume. Since

the rate of hyphal development of various VAMF

from the early stage to 6 weeks was different, theextraradical hyphae of 4 species, E. colombiana,

Scutellospora sp., G. geosporium and G.

aggregatum at harvest period were longer than 1 mg-1 meanwhile those of the remaining species were

Table 3 Length of extraradical hyphae of various VAMF associated with soybean(cm g-1).

VAMF At 6 weeks At harvest Increasing after 6 week

Non inoculation(control) 0d 0d 0

A. dilatata 37.53b-c 45.83c 8.30A. scrobiculata 27.38b-c 62.38c 35

E. colombiana 36.95b-c 128.90b 91.95

Gigaspora sp. No 2 20.58c-d 41.95c 21.37G. aggregatum 47.65a-b 168.33a 120.68

G. claroideum 26.98b-c 58.73c 31.75

G. geosporium 65.15a 137.37a-b 72.23G. tenuis 24.73b-c 28.75c-d 4.02

Scutellospora sp. 61.73a 134.85a-b 73.12

C.V.(%) 41.04 27.68

F-test ** **

Note: Means in each column followed by the same letter are not significant by DMRT at 95 % of confidence.

36 Kasetsart J. (Nat. Sci.) 34 (1)

shorter.Extraradical hyphae of G. geosporium,

Scutellospora sp. and G. aggregatum developed

thoroughly to the soil. The hyphal length of threespecies in soil at harvest was conspicuous which

increased more than 72 cm g-1. Markedly, the

hyphal length of E. colombiana at the first stage ofplant growth was moderate at 36.95 cm g-1, then,

the hyphae developed rapidly afterward and the

length was increased up to 91.95 cm at harvestperiod. This result revealed that 4 species of VAMF

are conspicuous having high potential on the

development of extraradical hyphae and enhancingplant growth through these special structures. The

result was coincided with the suggestion of De

Miranda and Harris (1994), who examined 3 speciesof VAMF using sorghum plant and found that

Scutellospora heterogama produced extraradical

hyphae more profusely than others. In contrast,

Abbott and Robson (1985) found that Glomus

calospora and Acaulospora laevis were prominent

on producing more hyphae in soil when associated

with subterranean clover. Finally, Abbott andRobson (1985) suggested that extraradical hyphae

are the important device for enhancing plant growth.

Normally, nutrients are taken up throughextraradical hyphae and then translocated to plant

root. Longer hyphae will have more advantages of

absorbing nutrients beyond the depletion zone ofnormal root.

The effects of VAMF on growth of SJ5 soybeanThe height of SJ5 soybean inoculated with

different VAMF at 6 weeks was significant at both

periods of measurement as shown in Table 4. The

group “a” of VAMF species, G. aggregatum, E.

colombiana and Scutellospora sp., exposed high

potential to stimulate the growth of soybean

Table 4 The effects of VAMF on growth of SJ5 soybean.

VAMF Plant height(cm) Dry weight(g plant-1)At 6 weeks At harvest Increased height Shoot Root

Non inoculation(control) 25.9c 37.0b 11.1 0.58c 0.50d

A. dilatata 35.1b-c 37.3b 2.2 0.53c 0.58d

A. scrobiculata 31.6b-c 34.7b 3.1 0.67b-c 0.84c-d

E. colombiana 43.1a-b 52.7a-b 9.6 1.68a 2.04a

Gigaspora sp. No 2 36.1b-c 38.0b 1.9 0.67b-c 0.96c-d

G. aggregatum 42.6a-b 58.5a 15.9 1.77a 1.68a-b

G. claroideum 34.9b-c 41.4a-b 6.5 0.82b-c 1.34b-c

G. geosporium 38.1b-c 42.5a-b 4.4 1.08b 1.54a-b

G. tenuis 35.3b-c 36.5b 1.2 0.59c 0.65d

Scutellospora sp. 52.5a 57.3a 4.8 1.90a 1.89a-b

C.V.(%) 20.81 27.24 – 29.23 29.45

F-test ** * – ** **

Note: Means in each column followed by the same letter are not significant by DMRT at 95 % of confidence.

Kasetsart J. (Nat. Sci.) 34 (1) 37

presented as measured by plant height at 6 weeks.The height of soybean inoculated with those three

species were 42.6, 43.1 and 52.3 cm, respectively.

Plants inoculated with other VAMF species wereshorter, while the height of non-inoculated plant in

control treatment was shortest.

The tallest group of soybeans at harvestperiod were those inoculated with G. aggregatum,

Scutellospora sp. and E. colombiana, while the

shortest plants were those inoculated with A.

scrobiculata at 34.7 cm. In soybeans inoculated

with G. aggregatum, plant height was up to 15.8 cm

at 6 weeks, reaching the peak of growth at harvestperiod at 58.5 cm. Some VAMF species exposed

rather low efficiency in stimulating plant height

during the period from 6 weeks to harvest. Thesegroups were A. scrobiculata, G. tenuis, A. dilatata

and Gigaspora sp. No. 2. Plant height inoculated

with those species was in the range of 34.7 to 38.0cm.

Although the height of soybean in every

treatment increased after 6 weeks, plant-height ofmost treatments increased negligibly.

Interestingly, the plant inoculated with

individual species of VAMF included Scutellospora

sp., E. colombiana and G. aggregatum grew very

fast and produced over 40 cm of plant height at 6

weeks. Consequently, plant height was increasedrather distinctively in most of the treatments except

those inoculated with Scutellospora sp. which the

increased height was only 4.75 cm. This resultsuggested that VAMF enhanced plant growth at 6

weeks stage and when the enhancement of VAMF

was prominent, the growth of soybean was furtherenhanced through harvest period by that particular

VAMF.

On the contrary, other group of VAMFcaused soybean plant height to develop slowly at

the first stage of growth. Later, plant height still

developed in the same manner excepted for non-inoculation treatment. The difference of plant height

between two stages of plant growth of this groupwere lower than 6.45 cm. These plants were those

inoculated by G. tenuis, G. claroideum, Gigaspora

sp. No. 2, G. geosporium, A. scrobiculata and A.

dilatata.

The dry weight of soybean was determined

in term of shoot and root dry weight as shown inTable 4. Different species of VAMF influenced the

shoot dry weight. The shoot dry weight of soybean

in treatments inoculated with Scutellospora sp., G.

aggregatum and E. colombiana were 1.90, 1.77

and 1.68 g plant-1, accomplished in the higher

quantity than the ability of the other species. Theminimal dry weight was found in soybean inoculated

with A. dilatata and in non-inoculated soybean as

0.53 and 0.58 g plant-1, respectively.Root dry weight of soybean inoculated with

different species of VAMF was highly significant.

The maximum root dry weight was found in soybeaninoculated with E. colombiana. In this treatment,

the root of plant developed intensively through the

volume of soil which was determined as root dryweight at 2.04 g plant-1, but the root distribution

declined sequentially to 1.89, 1.68, 1.54 and 1.34 g

plant-1 of soybeans inoculated with Scutellospora

sp., G. aggregatum, G. geosporium and G.

claroideum, respectively. The minimal root dry

weight was found in non-inoculated treatment at0.50 g plant-1.

Effects of VAMF on soybean yieldThe number of pod, seed and yield of

soybean inoculated with different VAMF species

were highly significant. But the size of seed

presented as 100-seed weight was significant (Table5). VAMF-inoculated soybean had more quantities

of pod, number of seed and seed size than those of

non-inoculated plant. The group of soybeaninoculated with Scutellospora sp., G. aggregatum,

E. colombiana and G. geosporium had more

quantities of pod than those of plant inoculated

38 Kasetsart J. (Nat. Sci.) 34 (1)

with the other species. The maximum quantity ofpod was found in soybean inoculated with

Scutellospora sp. at 28.28 pod plant -1.

The number of soybean seed affected byVAMF inoculation is similar in effect to the number

of pod. Four species of VAMF, Scutellospora sp.

G. aggregatum, G. geosporium and E. colombiana,had high potential to enhance soybean yield of

47.50, 41.50, 40.75 and 39.75 seed plant-1,

respectively. The remaining from the rest specieswas lower as compared to those one.

The larger seed size were found on plant

inoculated with G. claroideum, E. colombiana,Scutellospora sp. and G. aggregatum, compared to

the rest of the species. They showed the 100 -seed

weight of 15.95, 15.18, 13.81 and 13.65 g,respectively, with the smallest seed size found in

non-inoculated soybean or control plant.

Similar to the effect on yield component,four species of VAMF accomplished higher soybean

yield were Scutellospora sp., E. colombiama, G.

aggregetum and G. geosporium, gave the yield as

much as 6.42, 5.77, 5.37 and 4.34 g plant-1,

respectively (Table 5). Since non-inoculated plantgave the yield of 0.44 g plant-1, the yield above this

level will come from the encouragement of VAMF

affectability. Scutellospora sp. had highest abilityto accomplish the increased yield as much as 5.98

g plant-1. E. colombiana, G. aggregatum and G.

geosporium gave less encouragment to soybeanyield and their encouragement were 5.33, 4.97 and

3.90 g plant-1. Meanwhile, A. dilatata gave least

encouragement at 0.31 g plant-1.In term of mycorrhizal dependency on

soybean yield, five species of VAMF had high

potential to enhance soybean growth and raised thelevel of soybean yield as shown in Table 5. Those

species were Scutellospora sp., E. colombiana, G.

aggregatum, G. geosporium and G. claroideum.The mycorrhizal dependency of attributed-VAMF

Table 5 Effects of VAMF on SJ5 soybean yield, yield component and mycorrhizal dependency onsoybean yield (MDSY).

VAMF Number of Number of 100-seed Yield MDSYpod plant-1 seed plant-1 weight(g) (g plant-1)

Non inoculation(control) 3.75c 5.75c 7.63d 0.44e 0

A. dilatata 6.00b-c 8.25c 9.29c-d 0.75d-e 0.71

A. scrobiculata 12.25b 17.25b-c 11.28a-d 1.99d 3.52E. colombiana 23.75a 39.75a 15.18a-b 5.77a 12.11

Gigaspora sp. No 2 8.00b-c 12.75c 11.72a-d 1.55d-e 2.52

G. aggregatum 26.00a 41.50a 13.65a-c 5.37a-b 11.21G. claroideum 11.75b 26.75b 15.95a 3.90c 7.86

G. geosporium 22.25a 40.75a 10.83a-d 4.34b-c 8.86

G. tenuis 8.25b-c 15.00b-c 10.11b-d 1.55d-e 2.52Scutellospora sp. 28.25a 47.50a 13.81a-c 6.42a 13.59

C.V.(%) 27.04 34.07 27.02 26.96

F-test ** ** * **

Note: Means in each column followed by the same letter are not significant by DMRT at 95 % of confidence.

Kasetsart J. (Nat. Sci.) 34 (1) 39

on plant enhancement to increase the yield overcontrol were 13.59, 12.11, 11.21, 8.86 and 7.86,

respectively. These increased yields were obviously

encouraged by individual VAMF. The mycorrhizaldependency of the remaining VAMF were lower

than those species attributed above.

CONCLUSION

The effectiveness of various VAMF in thisexperiment is different. Certain species of VAMF

showed high potential on infecting soybean root

and consequently enhanced plant growth. The yieldof SJ5 soybean inoculated with Scutellospora sp.

was the highest at 6.24 g plant-1. Three species of

VAMF, E. colombiana, G. aggregatum and G.

geosporium were also high potential species of

high effectiveness on plant stimulation and obtain

high yield in the order of 5.77, 5.37 and 4.34 gplant-1, respectively. Therefore, these VAMF

should be further examined with soybean in different

phosphate conditions.

LITURATURE CITED

Abbott, L. K. and A. D. Robson. 1985. Formation

of external hyphae in soil by four species of

vesicular-arbuscular mycorrhizal fungi. NewPhytol. 99 : 245-255.

Abbott, L. K. and C. Gazey. 1994. An ecological

view of the formation of VA mycorrhizas.Plant and Soil 159 : 69-78.

Asher, C. J. 1975. Plant Nutrition I : Practical

Notes. Department of Agriculture, Universityof Queensland. 35 p.

Bethlenfalvay, G. I. and R. N. Ames. 1987.

Comparision of two methods for quantifyingextraradical mycelium of vesicular-arbuscular

mycorrhizal fungi. Soil Sci. Soc. Am. J., 51 :

834-837.Bolan, N. S. 1991. A critical review on the role of

mycorrhizal fungi in the uptake of phosphorusby plants. Plant and Soil 134 : 189-207.

Giovanetti, M. and B. Mosse. 1980. An evaluation

of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New

Phytol. 84 : 489-500.

Kiernan, J. M., J. W. Hendrix, L. P. Stoltz, and D.M. Maronex. 1984. Characterization of

strawbery plants produced by tissue culture

and infected with specific mycorrhizal fungi.Hort. Sci. 19 : 883-885.

Mala, T., S. Vangnai, P. Suwanarit, C. Suwanarat,

C. Hongprayoon, W. Phuengsaeng, and J.Phumpetch. 1997. The Utilization of Vesicular

Arbuscular Mycorrhizal Fungi Associated with

Cassava as a Mean to Improve PhosphateAvailability in Acid Soil. Final report submitted

to National Research Council of Thailand. 83

p.Marschner, H. and B. Dell. 1994. Nutrient uptake

in mycorrhizal symbiosis. Plant and Soil 159 :

89-102.De Miranda, J. C. C. and P. J. Harris. 1994. The

effect of soil phosphorus on the external

mycelium growth of arbuscular mycorrhizalfungi during the early stages of mycorrhizal

formation. Plant and Soil 166 : 271-280.

Plenchette, C., J. A. Fortin, and V. Furlan. 1983.Growth response of several plant species to

mycorrhiza in soil of moderate P-fertility. I.

Mycorrhizal dependency under fieldconditions. Plant and Soil 70 : 199-209.

Schenck, N. C. 1982. Methods and Principles of

Mycorrhizal Research. The AmericanPhytopathological Society, St. Paul, MN, USA.

244 p.

Received date : 16/09/99Accepted date : 20/12/99

Residual Effects of 20 Annual Applications of Ammonium Sulfateand Triple Superphosphate for Corn on Properties and Productivity

of Oxic Paleustults

A. Suwanarit1 , I. Suwanchatri2, J. Rungchuang3 and V. Verasan1

ABSTRACT

The residual effects of 20 successive annual applications of N and P fertilizer for corn productionon properties and productivity of an Oxic Paleustults were examined by field experiment. The experiment

consisted of 4 application rates of N and P fertilizer, i.e., 0-0, 60-60, 120-120, and 180-180 kg N-P2O5/ha/

year in the forms of ammonium sulfate and triple superphosphate.The pH of the surface soil and sub-soil was decreased with increased rates of the fertilizers with

more pronounced effects in the sub-soil. The EC of the surface soil was not affected by the fertilization

while that of the sub-soil was increased with increased rates of the fertilizer. The CEC of the surface soilwas not affected by the fertilizers while that of the sub-soil decreased with increased rates of the fertilizers.

The OM and total-N contents of the surface soil tended to increase with increased rates of the fertilizer while

those of the sub-soil were not affected by the fertilization. The available P of the surface soil wasdramatically increased with increased rates of the fertilizers while that of the sub-soil was less affected. The

exchangeable K, Ca and Mg either tended to decrease or significantly decreased with increased rates of the

fertilizer, with more pronounced effect in the sub-soil. The DTPA-extractable Fe of the surface soil wasincreased with increased rates of the fertilizers while that of the sub-soil was not affected by the

fertilization. The DTPA-extractable Mn and Zn of the top-layer soil was slightly increased with increased

rates of the fertilizer whereas those of the middle-layer soil were similarly decreased by the threeapplication rates of fertilizer and those of the bottom-layer soil were not affected. The DTPA-extractable

Cu of soils of the top and middle layers was slightly increased with increased rates of the fertilizer whereas

that of the bottom-layer soil was slightly decreased with increased rates of the fertilizer. The bulk densityof the surface soils showed slight trends to decrease with increased rates of the fertilizers while that of the

sub-soil showed no effect of the fertilizers. The aggregation of the soil was decreased with increased rates

of the fertilizers. The hydraulic conductivity of the saturated soils was not affected by the fertilization. Theinfection on corn roots of arbuscular mycorrhizal fungi (AMF) showed consistent trends to decrease with

increased rates of the fertilizer whereas AMF spore intensity in the soil was not affected by low rates of

the fertilizer but decreased with increased rates of the fertilizer at high rates of fertilizer. The populationof the free-living N2-fixing bacteria in the soil showed consistent trends to increase with increased rates

of the fertilizer. The productivity of the soil was increased with increased rates of the fertilizers.

Key words : ammonium, fertilizer, properties, long-term, phosphorus, productivity, soil

Kasetsart J. (Nat. Sci.) 34 : 40 - 51 (2000)

1 Department of Soil Science, Faculty of Agriculture, Kasetsart University, Chatuchak, Bangkok 10900, Thailand.2 Central Laboratory, Faculty of Natural Resources, Prince of Songkla University, Hadyai, Songkla 90110, Thailand.3 The National Corn and Sorghum Reasearch Center, Kasetsart University, Pakchong, Nakhon Ratchasima Province, Thailand.

INTRODUCTION

Though nitrogen and phosphorus fertilizers

have been widely used to increase crop yields, dataon a long-term basis on effects of these fertilizers

on properties and productivity of soil are scarce and

have been of continuing concern. Results of long-term application of fertilizers so far reported sug-

gested that the length of successive application of

the fertilizers and cropping conditions and/or crop-ping system were among factors influencing the

conclusions. The results of study by Owensby et al.

(1969), for example, led to a conclusion that annualapplication of N and P fertilizers at different rates

for 20 year for bromgrass did not affect organic

matter (OM) content of the soil whereas results ofstudy by Schwab et al. (1990), who worked with

the same plots as those of Owensby et al. (1969),

led to a conclusion that, after 40 annual applica-tions with the fertilizers, organic matter content of

the soil increased with increased rates of the nitro-

gen fertilizer. Moreover, Hasathon (1982), work-ing with lowland rice soils, reported that OM

contents of the soils increased with increased rates

of ammonium sulfate applied for rice in the previ-ous 5 years.

This paper presents results of a study on

effects of 20 annual applications of ammoniumsulfate and triple superphosphate for corn grown

on a Reddish Brown Lateritic soil in Thailand on

chemical, physical and biological properties andproductivity of the soil.

MATERIALS AND METHODS

Site and soilThe experiment was conducted on a

Pakchong series soil, clayey kaolinitic, Oxic

Paleustults at the National Corn and Sorghum

Research Center, Nakhon Ratchasima Province,Thailand.

Design and treatmentsA randomized complete block design with

6 treatments and 3 replications had been employed.

However, only four of the treatments were ac-counted for in this study. Each plot measured 7.5m

x 15.0m. Alleys of 175 cm and 75 cm had been

inserted between the two adjacent plots along theshorter and the longer sides of the plots, respec-

tively. The treatments studied were as follows.

F0: without fertilizer application.F60: application of 60 and 60 kg N and

P2O5/ha/year as ammonium sulfate and triple su-

perphosphate in annual cropping with corn duringthe previous 20 years.

F120: as F60 but the rates of fertilizers had

been 120 and 120 kg N and P2O5/ha/year.F180: as F60 but the rates of fertilizers had

been 180 and 180 kg N and P2O5/ha/year.

Throughout the previous 20 years, all of theplots were planted to corn during the late rainy

season (mid July to November) in each year. The N

fertilizer was applied by balanced split applicationat planting and one month after planting whereas

all of the P fertilizer was applied at planting. The

fertilizers at planting were mixed and applied bybanding under the soil surface on one side of each

corn row. The fertilizer at one month after planting

was applied by banding on the soil surface on oneside of each corn row. In the present cropping, all

of the plots were planted to corn and received no

fertilizer.

Basal fertilizer and crop residue managementZnSO4 at the rate of 62.5 kg/ha was applied

to all treatments in the second and third annual

cropping to ensure adequate supply of Zn. During

the 20 years prior to the present study, only cornears were removed from the plots. The stubble

from each cropping was left on the plots and then

chopped and plowed into the soil when land wasprepared at about one month before the following

Kasetsart J. (Nat. Sci.) 34 (1) 41

42 Kasetsart J. (Nat. Sci.) 34 (1)

cropping.

Soil sampling and sample preparationSoil samples for chemical analysis were

taken a few weeks before the crop residue incorpo-

ration into the soil. Within area of 25cm × 75cm

across the direction of the corn rows, the soil wasdug up in three separated layers, i.e., at 0-15cm, 15-

30cm, and 30-60cm depths. The soil from each

layer was crushed, well mixed and quartered. Onequarter of the soil was then taken and proceeded

with the preceding process. This process was re-

peated until a quarter of about 2 kg of soil wasobtained. The soil sample obtained was then air-

dried and crushed to pass a 2-mm sieve.

Soil samples for physical properties exami-nation were taken from the central area of 3m × 3m

of each plot before plowing for the present crop-

ping. Undisturbed soil samples for bulk densityand hydraulic conductivity examination were taken

with steel cylindrical (63.71 cc) soil samplers.

Samples from the centers of the surface (0-15cmdepths) and sub-surface (15-30cm depths) layers

were taken as representatives of the surface soil and

sub-soil, respectively. From each plot, two kinds ofsamples were taken, one from the center between

two adjacent hills of corn stumps and another from

the center between two adjacent rows of cornstumps. Each kind of sample was taken from two

sites in each plot. Soil clods for aggregate examina-

tion were also taken from the two kinds of sites butonly one sample of each kind was taken.

One composite soil sample for examination

of population of arbuscular mycorrhizal fungi(AMF) and free-living N2-fixing bacteria was taken

from the central area of 3m × 3m of each plot at one

week before harvest of the present cropping. Sur-face soil (0-15 cm depth) from three squares, each

measuring 50cm × 50cm and having two stumps of

corn plants (grown 75 cm between rows and 25 cmbetween hills of one plant) in the central area, was

dug out, put together and well mixed. An aliquot ofthe soil was then taken for the examination.

Analyses of soil and plant samplesChemical properties: pH of the soils were

measured with pH meter according to Peech (1965).

Saturation extracts of the soils for electrical con-ductivity (EC) measurement were obtained ac-

cording to Bower and Wilcox (1965). The cation

exchange capacity (CEC) was measured accordingto Chapman (1965), using neutral N NH4OAc and

10% NaCl as saturating and replacing solutions,

respectively. OM content of the soil was measuredaccording to Walkley and Black (1934). Total N

content of the soil was measured by Kjeldhal

method (Jackson, 1958), using the H2SO4 + Na2SO4+ Se digestion mixture. The available P was meas-

ured by Bray II method (Olsen and Dean, 1965).

Exchangeable K, Ca and Mg were measured asdescribed by Pratt (1965), using neutral N NH4OAc.

Extractrable Fe, Mn, Zn and Cu were measured

using DTPA according to Linsay and Norvell(1978).

Physical properties: The core soil samples

were ovened at 105°C to constant weights forgravimetric bulk density determination. Aggregate

analysis of the soils was done by wet seiving

according to Kemper and Chepil (1965). The hy-draulic conductivity of saturated soils was deter-

mined according to Klute (1965), respectively. The

state of aggregation was the proportion of the totalsample weight occurring in the aggregates of larger

than 0.25mm. The degree of aggregations was the

proportion (m1-m2)/(ms-m2), where m1 was theweight of aggregates larger than 0.25mm, m2 the

weight of primary particles larger than 0.25mm

and ms the total sample weight. The mean weight-diameter was the sum of products of (1) the mean

diameter of each size fraction and (2) the propor-

tion of the total sample weight occurring in thecorresponding size fraction.

Kasetsart J. (Nat. Sci.) 34 (1) 43

Biological properties: Infection in cornroot and spore intensity in the soil of AMF in the

soil was determined according to Danial and Skip-

per (1987). Number of the free-living N2-fixingbacteria was determined by the Total Plate Count

Technique.

Uptake of N, P and K: Grain and stubble ofcorn were analyzed for N, P and K by wet digestion

(Jackson, 1958). Total N in the digest was then

measured with micro Kjeldhal distillation method(Bremner, 1965). P content of the digest was meas-

ured with the vanadomolybdophosphoric yellow

calorimetric method (Jackson, 1958) using aSpectronic-20 colorimeter. K content of the digest

was measured by flame photometry.

RESULTS AND DISCUSSION

pH, EC, CEC and OMpH, EC, CEC and %OM of the soils are

shown in Figure 1.

In all of the three soil layers, pH decreasedwith increased rates of the fertilizers with more

pronounced changes in the lower soil layers. The

effects of fertilizers on pH were presumably dueprimarily to oxidation of the applied NH4-ions

which released H+ ions. More pronounced effects

in the lower layers were presumably results oflower buffering capacities of the soils due to lower

OM contents. The results were slightly different

from those obtained by Tattao (1987), who workedwith the same experimental plots after fertilization

for 10 years and found decreases in pH of the

surface soil (at 0-30cm depths) with increased ratesof the fertilizers but only trends of decreases in pH

of the sub-soils (at 30-100cm depths) with in-

creased rates of the fertilizers.EC of the top-layer soil was not affected by

the fertilizer application while those of the middle-

layer and bottom-layer soils generally increasedwith increased rates of the fertilizers. The effects of

0.81

0.98

0.870.951.

461.60

1.411.49

3.04

2.77

2.81

2.69

0

2

4

6

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

a a

30-60 cm depth

Treatments

% O

M

aaa a a a a

a a a

12.3

12.3

13.1

13.1

13.8

13.5

14.115

.0

17.3

18.7

17.1

17.4

0

7

14

21

28

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm deptha a

30-60 cm depth

Treatments

CE

C, c

mol

/100

g aaa a a a a a b b

1.29

1.30

0.84

0.48

1.08

1.06

1.0

1

0.70

1.19

1.27

1.15

1.22

0.0

0.5

1.0

1.5

2.0

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm deptha a

30-60 cm depth

Treatments

EC

, mS/

cm aa

ab b b

a

ab

b b

4.84

4.805.135.87

5.325.93

6.206.

81

6.27

6.697.

11

7.22

0

4

8

12

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

bc c

30-60 cm depth

Treatments

pH

aba aa a a a

b b b

Figure 1 pH, EC, CEC and %OM of soils asaffected by rates of N and P fertilizers

applied in the previous 20 years. Within

each group, bars with a common letterwere not significantly different by

DMRT.05. % CV: 3.2%, 8.4% and 3.3%;

17.1%, 13.0% and 23.4%; 14.0%,10.8% and 8.1%; 5.3%, 29.3% and

11.9% for pH, EC, CEC and %OM of

soils, each at 0-15cm, 15-30cm and 30-60cm depths, respectively. F0, F60, F120,

and F180 : application with 0 and 0, 60

and 60, 120 and 120, 180 and 180 kg Nand P2O5/ha/year in the previous 20

years.

44 Kasetsart J. (Nat. Sci.) 34 (1)

fertilizers were more pronounced in the bottomlayer. The results suggested that free salts from the

fertilizers were mostly leached down to the lower

layers.CEC of the top-layer soil was not affected

by the fertilization whereas those of soils of the

middle and bottom layers either showed consistenttrends to decrease or significantly increased with

increased rates of the fertilizers. The effects of

fertilizers on the lower-layer soils were presum-ably due to lower pH which resulted in more

dissolution and leaching from the profile of

sesquioxides which were sources of pH-dependentcharges. However, the CEC of the top-layer soil

was not affected by the fertilizers since the reduc-

tion in CEC as a result of reduction of sesquioxidescontent was compensated for by the CEC from the

increased OM contents. The results were not in

agreement with those of Tattao (1987) whichshowed trends of increases in CEC’s of the surface

soil and sub-soil with increased rates of the fertiliz-

ers.OM contents of the top-layer soils showed

consistent trends to increase with increased rates of

the fertilizers whereas those of soils of the middleand bottom layers showed no effect of the fertiliza-

tion. The increases in OM content of the top layer

soil were presumably due to increased root growthin response to increased rates of the fertilizers of

corn grown in the previous cropping. The results

were similar to those of Tattao (1987) which showedtrends of increases in %OM of the surface soil and

sub-soil with increased rates of the fertilizers up to

F120 but only a slight trend of increase from F180.

Total N, extractable P and exchangeable K, Caand Mg in soils

Amounts of total N, extractable P and ex-

changeable K, Ca and Mg in the soils are shown in

Figure 2.Total-N contents of the top-layer soils

1.39

1.29

1.29

1.15

1.01

0.77

0.74

0.63

0.71

0.49

0.51

0.42

0.0

0.5

1.0

1.5

2.0

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm deptha a

30-60 cm depth

Treatments

Exc

hang

eabl

e M

g, c

mol

/100

g

aa

ab b b a

b b b

4.6

4.6

3.75.

7

6.8

7.0

6.89.1

13.2

14.6

13.9

14.4

0

6

12

18

24

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

a a

30-60 cm depth

Treatments

Exc

hang

eabl

e C

a, c

mol

/100

g

aa

a a a a

ac b b

0.06

0.10

0.08

0.10

0.080.

15

0.14

0.17

0.370.580.55

0.58

0.0

0.2

0.4

0.6

0.8

1.0

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

a

a

30-60 cm depth

Treatments

Exc

hang

eabl

e K

, cm

ol/1

00g

aa

a a a a a a a a

1.84

1.74

1.851.21

5.63

4.05

3.10

1.60

58.5

9

45.5

9

18.9

3

4.62

0

30

60

90

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

cc

30-60 cm depth

Treatments

Ava

ilabl

e P

, mg/

kg

b

a a ab b c a a a a

0.05

4

0.06

2

0.05

6

0.06

1

0.10

6

0.08

2

0.09

3

0.09

0

0.16

1

0.14

8

0.14

5

0.13

5

0.0

0.1

0.2

0.3

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

a a

30-60 cm depth

Treatments

Tot

al N

, %

aa

a a a aa a a a

Figure 2 Amounts of total N, available P andexchangeable K, Ca and Mg of soils asaffected by rates of N and P fertilizerapplied in the previous 20 years. % CV:6.2%, 24.3% and 11.2%; 21.0%, 21.9%and 19.1%; 14.0%, 35.5% and 17.0%;5.5%, 18.6% and 6.7%; 6.3%, 14.3%and 15.5% for N, P, K, Ca and Mg ofsoils, each at 0-15cm, 15-30cm and 30-60cm depths, respectively. Refer toFigure 1 for captions.

Kasetsart J. (Nat. Sci.) 34 (1) 45

showed consistent trends to increase with increasedrates of the fertilizers whereas those of soils of the

middle and bottom layers showed no effect of the

fertilization. The increases in total-N content of thetop-layer soil were presumably due to the trends to

increase with increased rates of the fertilizers of

OM content of the soil. The results were similar tothose of Tattao (1987) which showed trends of

increases in total-N content with increased rates of

the fertilizer up to F120 but only a slight trend ofincrease from F180 in the case of surface soil and

trends to increase with increased rates of the ferti-

lizers in the case of sub-soil.Extractable P of the top-layer soils increased

markedly with increased rates of the fertilizers

whereas those of the middle-layer soils slightlyincreased with increased rates of the fertilizers and

those of the bottom-layer soils were not affected by

the fertilization. The results showed that very littleof the fertilizer P was moved down to the middle

and bottom layers. The results were in good agree-

ment with those of Tattao (1987) who worked withthe same experimental plots after fertilization for

10 years.

Exchangeable K of soils in the three layerswere not affected by F60 and F120 but showed

trends to be decreased by F180. This effect of

fertilization was presumably due to the increase insoil acidity as a result of the fertilization which was

largest in the case of F180. The results were slightly

different from those of Tattao (1987) which showedtrends of increases in exchangeable K by the ferti-

lization both in the cases of surface soil and sub-

soil.Exchangeable Ca of the top-layer soils

generally showed trends to decrease with increased

rates of the fertilizers whereas those of the middle-layer soils showed similar trends of decreases by

F60, F120 and F180. Exchangeable Ca of the bottom-

layer soils were similarly decreased by F120 andF180 and was most markedly decreased by the F60.

Figure 3 Amounts of extractable Fe, Mn, Zn and

Cu of soils as affected by rates of N andP fertilizers applied in the previous 20

years. % CV: 14.0%, 10.8% and 8.1%;

18.3%, 13.9% and 22.1%; 11.0%,22.8% and 12.7%; 7.2%. 12.5% and

19.3% for Fe, Mn, Zn and Cu of soils,

each at 0-15cm, 15-30cm and 30-60cmdepths, respectively. Refer to Figure 1

for captions.

0.94

0.90

1.01

1.101.

54

1.331.65

1.271.

69

1.55

1.51

1.48

0

1

2

3

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

a a

30-60 cm depth

Treatments

Ext

ract

able

Cu,

mg/

kg

aaa

a aa

a a a a

0.42

0.53

0.56

0.54

1.37

1.29

1.401.

91

3.25

3.11

2.442.67

0

2

4

6

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

a a

30-60 cm depth

Treatments

Ext

ract

able

Zn,

mg/

kg

aaa a a a

a a a a

15231916

43324153

152

134

116

98

0

100

200

300

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

aa

30-60 cm depth

Treatments

Ext

ract

able

Mn,

mg/

kg

aaa b b ab

a a a a

11.5

13.5

12.1

12.4

14.1

13.9

14.3

12.5

12.9

7.8

5.3

4.4

0

6

12

18

24

F0 F60

F120

F180 F0 F60

F120

F180 F0 F60

F120

F180

0-15 cm depth 15-30 cm depth

b

c

30-60 cm depth

Treatments

Ext

ract

able

Fe,

mg/

kg

aa

a a a aa a

aa

These effects of fertilization were presumably thenet results of the effects of fertilization on soil

acidity and the increased uptake of Ca. The ratio

between the increased uptake of Ca by the previouscorn crops in response to fertilization and the

46 Kasetsart J. (Nat. Sci.) 34 (1)

amounts of Ca carried by the applied triple super-phosphate for F60 was presumably higher than

those for F120 and F180 resulting in larger decrease

in the amount of exchangeable Ca relative to ratesof the fertilizers. The results for the top layer well

agreed with those of Tattao (1987) who worked

with the same experimental plots after fertilizationfor 10 years. However, Tattao (1987) found in-

creases in exchangeable Ca with increased rates of

the fertilizers in the case of sub-soil which wasregarded as soil at the 30-100cm depths.

Exchangeable Mg of soils of the three layers

showed effects of the fertilization that were similarto the effects on the exchangeable Ca but the effects

on Mg were more pronounced than those on Ca,presumably due to lower Mg content of the applied

triple superphosphate. Tattao (1987) obtained

results on exchangeable Mg similar to thosementioned above in the case of exchangeable Ca.

Extractable Fe, Mn, Zn and Cu in soilsExtractable Fe of the top-layer soils gener-

ally increased with increased rates of the fertilizers

whereas those of the middle-layer soils showedsimilar trends to be increased by the three treat-

ments and those of the bottom-layer soils showed

no effect of the fertilization (Figure 3). The resultssuggested that the amount extractable Fe in the top-

Figure 4 Mean weight diameter (MWD), state of aggregation, degree of aggregation, bulk density and

hydraulic conductivity of soils as affected by rates of N and P fertilizers applied in the previous20 years. % CV: 11.3% and 12.4%; 6.8% and 9.5%; 7.9% and 9.4%; 1.9% and 4.7%; and 29.2%

and 14.1% for MWD, state of aggregation, degree of aggregation, bulk density and hydraulic

conductivity at 0-15cm and 15-30cm depths, respectively. Refer to Figure 1 for captions.

1.16

1.10

1.15

1.08

1.16

1.15

1.09

0.74

0.0

0.5

1.0

1.5

2.0

F0

F60

F12

0

F18

0 F0

F60

F12

0

F18

0

Treatments

MW

D, m

m

0-15 cm depth 15-30 cm depth

a a a a a a aa

80.6

76.0

76.7

69.9 80

.9

81.5

75.9

67.9

0

40

80

120

F0

F60

F12

0

F18

0 F0

F60

F12

0

F18

0

Treatments

Stat

e of

Agg

rega

tion,

%

0-15 cm depth 15-30 cm deptha a a a

a a aa

79.0

74.0

75.3

68.4 79

.9

78.1

72.0

68.8

0

40

80

120

F0

F60

F12

0

F18

0 F0

F60

F12

0

F18

0

Treatments

Deg

ree

of A

ggre

gati

on,

%

0-15 cm depth 15-30 cm deptha a a a

a a a a

1.28

1.29

1.26

1.24 1.38

1.31

1.34

1.36

0

1

2

3

F0

F60

F12

0

F18

0 F0

F60

F12

0

F18

0

Treatments

Bul

k de

nsity

, gm

/cm

3

0-15 cm depth 15-30 cm depth

a a a a a a a a

0.73

2.53

1.39

1.20 0.

30

1.05

1.02 0.

19

0

1

2

3

4

F0

F60

F12

0

F18

0 F0

F60

F12

0

F18

0

Treatments

Hyd

raul

ic c

ondu

ctiv

ity,

cm/h

r

0-15 cm depth 15-30 cm depth

aa

a aa

a aa

Kasetsart J. (Nat. Sci.) 34 (1) 47

layer soil depended primarily on soil acidity. Greaterextractable Fe in soils of the middle and bottom

layers, in the case of the unfertilized plots, than that

of the top-layer soil suggested that amounts ofextractable Fe of the middle and bottom layers

were more dependent on soil aeration than on soil

acidity and thus showed no effect of the fertiliza-tion. The results for the top layer well agreed with

those of Tattao (1987) who worked with the same

experimental plots after fertilization for 10 years.However, Tattao (1987) found trends of increases

in extractable Fe with increased rates of the fertiliz-

ers in the sub-soil.Extractable Mn and Zn of the top-layer soils

showed consistent trends to increase with increased

rates of the fertilizers whereas those of the middle-layer soils showed comparable decreases by the

three fertilizer treatments and those of the bottom-

layer soils showed no effect of the fertilization. Theresults suggested that the amounts of extractable

Mn and Zn in the top-layer soil depended primarily

on pH of the soil. Similar decreases in extractableMn and Zn in the middle-layer soils by F60, F120and F180 were presumably net results of removal of

Mn and Zn by the corn crops which increased withincreased rates of the fertilizers and the solubility

of Mn and Zn which were higher in the soils

subjected to higher rates of fertilizers because oflower pH’s. The results for the top layer well

agreed with those of Tattao (1987). However, Tattao

(1987) found trends of increases in extractable Fewith increased rates of the fertilizers in the sub-soil.

Amounts of extractable Cu of soils of the

top and the middle layers showed trends of slightincreases with increased rates of the fertilizers

whereas those of the bottom-layer soils showed

trends of decreases with increased rates of thefertilizers. The results suggested that the amounts

of extractable Cu of soils of the top and middle

layers depended primarily on soil acidity. Thetrends to decrease with increased rates of the ferti-

lizers of the extractable Cu in the bottom-layer soilssuggested that acidity of the soil was not the main

factor affecting it. The results of Schwab et al.

(1990), which showed no effects of applicationwith ammonium nitrate for 40 years on DTPA-

extractable Cu in soil, supported this postulation.

Physical propertiesBulk density of the surface soils showed

slight trends to decrease with increased rates of thefertilizers while that of the sub-soil showed no

effect of the fertilizers (Figure 4). The results were

presumably due to the positive effects of thefertilization on OM contents of the soil. These

results were similar to those of Tattao (1987) who

worked with the same experimental plots afterfertilization for 10 years. State of aggregation and

degree of aggregation of both the surface soil and

sub-soil decreased with increased rates of thefertilizers (Figure 4). The effects of fertilization on

aggregation were presumably due primarily to the

increased acidity as a result of the fertilizationwhich in turn resulted in increased dispersion of

soil particles. The aggregate-size distribution

showed that proportion of the soil mass bound intosmaller aggregates increased with increased rates

of the fertilizers whereas that bound into larger

aggregates decreased with increased rates of thefertilizers resulting in increased aggregate-size

uniformity with increased rates of the fertilizers

(Figure 5). Mean weight diameters of aggregatesalso either tended to decrease or significantly

decreased with increased rates of the fertilizers

(Figure 4). The results thus showed that theaggregation decreased with increased rates of the

fertilizers and agreed well with results of Tattao

(1987) who worked with the same experimentalplots after fertilization for 10 years. Hydraulic

conductivity of the saturated soils was not effected

by the fertilization (Figure 4).

48 Kasetsart J. (Nat. Sci.) 34 (1)

F0 F60 F120 F180

10.8 a

16.7 a

23.7 a

25.5 a

14.8 a

14.5 a

16.9 a

23.0 a

21.4 ab

14.7 a

14.6 a

14.5 a

21.3 a

26.7 a

14.5 a

18.5 a

18.0 a

19.3 a

15.6 b

17.0 a

0%

20%

40%

60%

80%

100%

F0 F60 F120 F180

2.00-5.00

1.00-2.00

0.50-1.00

0.25-0.50

0.10-0.25

< 0.10

Agg

rega

tion

Treatments

Aggregate

diameter, mm

Aggregate-Size Distribution at 0-15cm Depth

8.6 a 9.5 a 8.7 a 11.6 a

F0 F60 F120 F180

8.2 ab

10.9 a

16.3 a

25.0 a

24.9 a

14.7 a

5.0 b

14.6 a

19.3 a

19.9 a

27.3 a

14.0 a

11.7 a

13.8 a

17.9 a

22.5 a

18.8 ab

15.4 a

11.9 a

20.2 a

23.5 b

24.6 a

13.4 b

6.4 b

0%

20%

40%

60%

80%

100%

F0 F60 F120 F180

2.00-5.00

1.00-2.00

0.50-1.00

0.25-0.50

0.10-0.25

< 0.10

Agg

rega

tion

Treatments

Aggregate

diameter, mm

Aggregate-Size Distribution at 15-30cm Depth

Figure 5 Aggregate size distribution of soils at 0-15cm and 15-30cm depths as affected by rate of NP

fertilizer applied in the previous 20 years. % CV: in case of 0-15 cm depth, 20.3%, 14.6%,

10.3%, 15.6%, 23.9%; and 21.0% for aggregate sizes 2.00-5.00mm, 1.00-2.00mm, 0.50-1.00mm, 0.25-0.50mm, 0.10-0.25mm and 0.10mm, respectively, and 20.1%, 20.8%, 12.4%,

9.6%, 30.0% and 28.3%for aggregate sizes 2.00-5.00mm, 1.00-2.00mm, 0.50-1.00mm, 0.25-

0.50mm, 0.10-0.25mm and 0.10mm, respectively, in the case of 15-30cm depth. Refer to Figure1 for captions.

Figure 6 AMF infection of corn roots, intensity of AMF spores in the soils and intensity of free-living

N2-fixing bacteria in the soils as affected by rate of NP fertilizer applied in the previous 20 years.

% CV: 5.9%, 12.3% and 13.3%, respectively. Refer to Figure 1 for captions.

86.7

83.3

78.9

73.3

0

40

80

120

F0

F60

F12

0

F18

0

Treatments

Infe

ctio

n, %

a a a a

AMF Infection

14.2

14.8

12.8

10.0

0

5

10

15

20

F0

F60

F12

0

F18

0

Treatments

No.

of

spor

es/g

soi

l

a aab

b

AMF Spores

1210

1279 13

14

1340

0

1000

2000

F0

F60

F12

0

F18

0

Treatments

Cel

ls/g

soi

l (x

104 )

a a a a

N2-Fixing Bacteria

Kasetsart J. (Nat. Sci.) 34 (1) 49

Biological propertiesThe infection on corn roots of AMF showed

consistent trends to decrease with increased rates

of the fertilizers whereas AMF spore intensity inthe soil was not affected by F60 but decreased with

increased rates of the fertilizers when the rates were

higher than that of F60 (Figure 6). The result onAMF spore intensity was slightly different from

that of Tattao (1987) who found trends of increase

in spore intensity with increased rates of the ferti-lizers up to F120 but a trend of decrease by F180.

The total plate counts of the free-living

nitrogen-fixing bacteria in the soil showed consist-ent trends to increase with increased rates of the

fertilizers. The results were different from those of

Tattao (1987) which showed no significant effectof the fertilization on Azotobacter spp. population

up to F120 and a trend of decrease in the population

by F180.

Soil productivityGrain yields of corn showed no effects of

F60, a trend to be increased by F120 and was

increased by F180 whereas stubble yield showed

consistent trends to increase with increased rates ofthe fertilizers (Figure 7). The uptake of N, P and K

either showed trends to increase or significantly

increased with increased rates of the fertilizers(Figure 7). The results therefore showed that pro-

ductivity of the soil increased with increased ratesof the fertilizers.

CONCLUSIONS

pH of the surface soil and sub-soil de-

creased with increased rates of the fertilizers withmore pronounced effects in the sub-soil. EC of the

surface soil was not affected by the fertilization

while that of the sub-soil increased with increasedrates of the fertilizers. CEC of the surface soil was

not affected by the fertilizers while that of the sub-

soil decreased with increased rates of the fertiliz-ers. OM and total N contents of the surface soil

tended to increase with increased rates of the ferti-

lizers while those of the sub-soil were not affectedby the fertilization. Available P of the surface soil

was dramatically increased with increased rates of

the fertilizers while that of the sub-soil was lessaffected. Exchangeable K, Ca and Mg either tended

to decrease or significantly decreased with in-

creased rates of the fertilizers, with more pro-nounced effect in the sub-soil. DTPA-extractable

Fe of the surface soil increased with increased rates

of the fertilizers while that of the sub-soil was notaffected by the fertilization. DTPA-extractable Mn

and Zn of the surface soil slightly increased with

increased rates of the fertilizers whereas those ofthe middle-layer soil were similarly decreased by

Figure 7 Grain (15% moisture) yields, dry stubble yields and total uptake of N, P and K of corn as affected

by rates of NP fertilizer applied in the previous 20 years. % CV: 14.4%, 10.1%, 9.9%, 15.7%

and 21.9% for grain, stubble, N, P and K, respectively. Refer to Figure 1 for captions.

2093

1991

2489 31

76

2849

3171

3477 37

51

0

2000

4000

6000

F0

F60

F12

0

F18

0 F0

F60

F12

0

F18

0

Treatments

Yie

ld, k

g/ha

Grain Stubble

a a ab

ba a a a

8.5 10.6

12.7

16.2

39.0

39.9

44.1

43.862

.1

48.2

42.4

41.7

0

30

60

90

F0

F60

F12

0

F18

0 F0

F60

F12

0

F18

0 F0

F60

F12

0

F18

0

Treatments

Tot

al u

ptak

e, k

g/ha

N P K

a aa

b

a ab bc c a a a a

50 Kasetsart J. (Nat. Sci.) 34 (1)

the three rates of fertilizers and those of the bottom-layer soil were not affected. DTPA-extractable Cu

of soils of the top and middle layers slightly in-

creased with increased rates of the fertilizerswhereas that of the bottom-layer soil slightly de-

creased with increased rates of the fertilizers.

Bulk density of the surface soils showedslight trends to decrease with increased rates of the

fertilizers while that of the sub-soil showed no

effect of the fertilizers. Aggregation of the soildecreased with increased rates of the fertilizers.

Hydraulic conductivity of the saturated soils was

not effected by the fertilization.Infection on corn roots of AMF showed

consistent trends to decrease with increased rates

of the fertilizers whereas AMF spore intensity inthe soil was not affected by low rates of the fertiliz-

ers but decreased with increased rates of the ferti-

lizers at high rates of fertilizers. Population of thefree-living nitrogen-fixing bacteria in the soils

showed consistent trends to increase with increased

rates of the fertilizers.Productivity of the soil increased with in-

creased rates of the fertilizers, with grain yield of

plot with the highest fertilizer rate being about151% relative to that of the non-fertilized plot.

ACKNOWLEDGMENT

The authors express their appreciation to

Kasetsart University for financial support on thiswork.

LITERATURE CITED

Bremner, J. M. 1965. Total nitrogen, pp. 1149-

1178. In C. A. Black, D. D. Evans, J. L. White,

L. E. Ensminger, and F. E. Clark (eds.). Meth-ods of Soil Analysis. Part 2: Chemical and

Microbiological Properties. Amer. Soc.

Agron., Inc., Madison, Wisconsin.

Bower, C. A. and L. V. Wilcox. 1965. Soluble salts,pp. 933-951. In C. A. Black, D. D. Evans, J. L.

White, L. E. Ensminger, and F. E. Clark (eds.).

Methods of Soil Analysis. Part 2: Chemicaland Microbiological Properties. Amer. Soc.

Agron., Inc., Madison, Wisconsin.

Chapman , H. D. 1965. Cation-exchang capacity,pp. 891-901. In C. A. Black, D. D. Evans, J. L.

White, L. E. Ensminger, and F. E. Clark (eds.).

Methods of Soil Analysis. Part 2: Chemicaland Microbiological Properties. Amer. Soc.

Agron., Inc., Madison, Wisconsin.

Danial, D. H. and H. D. Skipper. 1987. Method forthe recovery and quantitative estimation of

propagules from soil, pp. 29-36. In N. C.

Schenck (ed.). Method and Principle ofMycorrhizal research. Am. Phytopathol. Soc.,

St. Paul, Minnesota.

Hasathon, Y. 1982. Influences of long-term ammo-nium sulfate application to paddy soils of the

central plain on chemical properties and

growth, yields and chemical composition ofrice. M. S. thesis, Kasetsart University, Bang-

kok.

Jackson, M. L. 1958. Soil Chemical Analysis.Prentice-Hall, Inc. Engelwood Cliffs, N. J.

488 p.

Kemper, W. D. and W. S. Chepil. 1965. Sizedistribution of aggregates, pp. 499-519. In C.

A. Black, D. D. Evans, J. L. White, L. E.

Ensminger, and F. E. Clark (eds.). Methods ofSoil Analysis. Part I: Physical and Mineralogi-

cal Properties, Including Statistics of Meas-

urement and Sampling. Amer. Soc. Agron.,Inc., Madison, Wisconsin.

Klute, A. 1965. Laboratory measurement of hy-

draulic conductivity of saturated soil, pp. 210-221. In C. A. Black, D. D. Evans, J. L. White,

L. E. Ensminger, and F. E. Clark (eds.). Meth-

ods of Soil Analysis. Part I: Physical andMineralogical Properties, Including Statistics

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of Measurement and Sampling. Amer. Soc.Agron., Inc., Madison, Wisconsin.

Lindsay, W. L. and W. A. Norvel. 1978. Develop-

ment of a DTPA soil test for zinc, iron, manga-nese and copper. Soil Sci. Soc. Am. J. 42 : 421-

428.

Olsen, S. R. and L. A. Dean. 1965. Phosphorus, pp.1035-1049. In C. A. Black, D. D. Evans, J. L.

White, L. E. Ensminger, and F. E. Clark (eds.).

Methods of Soil Analysis. Part 2: Chemicaland Microbiological Properties. Amer. Soc.

Agron., Inc., Madison, Wisconsin.

Owensby, E. C., K. L. Anderson, and D. A. Whitney.1969. Some chemical properties of silt loan

soil after 20 years nitrogen and phosphorus

fertilization of smooth bromgrass (Bromus

inermis, Leyss). Soil Sci. 108 : 24-29.

Peech, M. 1965. Hydrogen-ion activity, pp. 914-

926. In C. A. Black, D. D. Evans, J. L. White,L. E. Ensminger, and F. E. Clark (eds.). Meth-

ods of Soil Analysis. Part 2: Chemical and

Microbiological Properties. Amer. Soc.Agron., Inc., Madison, Wisconsin.

Pratt, P. F. 1965. Potassium, pp. 1022-1034. In C.A. Black, D. D. Evans, J. L. White, L. E.

Ensminger, and F. E. Clark (eds.). Methods of

Soil Analysis. Part 2: Chemical and Microbio-logical Properties. Amer. Soc. Agron., Inc.,

Madison, Wisconsin.

Schwab, A. P., C. E. Owensby, and S. Kulyingyong1990. Changes in soil chemical properties due

to 40 years of fertilization. Soil Sci. 149 : 35-

43.Tattao, D. 1987. Influence of Long-Term N-P

Fertilizers, Cropping Systems and Rainfall on

Corn Yields and on Soil Properties. Ph.D.thesis. Kasetsart University, Bangkok.

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Received date : 9/01/98Accepted date : 8/09/98

Kasetsart J. (Nat. Sci.) 34 : 52 - 63 (2000)

Properties and Agricultural Potential of Skeletal Soilsin Southern Thailand

Irb Kheoruenromne, Anchalee Suddhiprakarn and Sumitra Watana

ABSTRACT

A study on properties and agricultural potential of skeletal soils in Southern Thailand placed a majoremphasis on representative skeletal soils that can be found in large extent, moderate extent and limited

extent in the region. Study method included field investigation and pedon analysis of the representative

soils and collecting soil samples from their genetic horizons, and laboratory analysis on their physical andchemical properties to evaluate their vital properties and their agricultural potential. Eight representative

soil series included Chumphon series, Khao Khat series, Nong Khla series, Phato series, Ranong series,

Phayom Ngam series, Sawi series and Yi-ngo series.Results of the study revealed that most of them are upland soils mainly occupying undulating and

rolling terrains with a major slope range of 3-8 percent. Most of them are deep to very deep, highly leached,

well developed Ultisols. The skeletal materials in these soils vary but the ones that can be found frequentlyare ironstone nodules. Their basic fertility status is generally poor and problem on available phosphorus

in most soils exists. Their high extractable acidity is also a vital adversary property that can affect their

exchange properties markedly whereas the presence of skeletal materials in the soils does not appear to poseany serious problem on their uses and management. These soils are not suited for paddy rice but they are

moderately suited for upland field crops and livestock pasture, and well suited for tree crops including fruit

trees with different degrees of problem on topography and the presence of skeletal materials. Since thesesoils have been used mainly for rubber and fruit tree production, land use problem on them can be coped

with quite well by the use of existing agricultural technologies. Therefore, sustainability in their use, at

present, is somewhat achieved.Key words : skeletal soils, ironstone nodules, Ultisols, agricultural potential, Southern Thailand

Department of Soil Science, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.

INTRODUCTION

Skeletal soils are those having substantial

amount of coarse materials present within 50centimeters from the soil surface (Soil Survey

Staff, 1975). They were considered problem soils

in Thailand (Panichapong, 1982). It was reportedthat skeletal soils can be found extensively in upper

Northeast and substantial area of these soils are inSouthern Thailand (Vijarnsorn, 1984). Basic

properties of these skeletal soils vary in many

aspects, i.e., nature of skeletal materials, depth ofsoils and their specific ecology (Dee-saeng, 1993).

Their agricultural uses and potential, in general, are

poor to very poor (Chintaskul, 1989). The uses ofthese soils for crop practices in Thailand become

more common nowadays due to the need of land forcropping practices. In Southern Thailand, these

soils have been used generally for tree crop

production such as for para rubber, tropical fruittrees and oil palm and problems on their uses, so

far, have been least reported. It appears that the

soils can support the uses in this aspect sufficiently.This study was conducted on skeletal soils

in Southern Thailand with three objectives including

1) to determine the vital properties of the skeletalsoils, 2) to evaluate their agricultural potential, and

3) to analyze problems on their uses.

MATERIALS AND METHODS

Method of the study consisted of two parts:field investigation and laboratory analysis. The

field study included identification, pedon analysis

and sampling of skeletal soils distributed in SouthernThailand by standard field study method

(Kheoruenromne, 1987; Soil Survey Division Staff,

1993) using several soil maps published by theLand Development Department, Ministry of

Agriculture and Cooperatives as guides to locations

of the soils. The Laboratory analysis was on thephysical and chemical properties of the soil samples

collected from the field using standard methods of

soil analysis (Soil Survey Laboratory Staff, 1992).Parameters involved in the laboratory analysis of

the soil samples include particle size distribution,

chemical properties related to major plant nutrientspresent in the soils and chemical parameters

indicating their exchange properties.

RESULTS AND DISCUSSION

Ecology and profile models of skeletal soilsResults of field investigation indicated that

many skeletal soils can be found in Southern

Thailand. However, some of them are quite similar

or can be grouped together. From the groups of

these skeletal soils, seven soil series representingmost of them were chosen to be studied in details.

These are Chumphon series which can be found

extensively in Southern Thailand, Khao Khat series,Nong Khla series and Phato series which can be

found in a moderate extent, Phayom Nyam series,

Sawi series and Yi-ngo series which can be foundin a limited extent in Southern Thailand (Vijarnsorn,

1985).

Some data on ecology of skeletal soils inSouthern Thailand are summarized in Table 1. It is

clear that most of skeletal soils have been formed

on old alluvial deposits and residuum and colluviumderived from clastic sedimentary and

metasedimentary rocks such as shale, phyllite and

quartzitic sandstone on undulating to rolling andhilly terrains. Most of them are well drained soils

and their uses are mainly for tree crop production

such as para rubber and tropical fruit trees. Theecology of these skeletal soils appears to be different

from what can be seen for skeletal soils in the North

and Northeast of Thailand (Kheoruenromne, 1991).Profile models of these skeletal soils are

shown in Figures 1, 2 and 3. Chumphon series, a

skeletal soil that can be found rather extensively inSouthern Thailand (Figure 1) is a very deep soil and

the coarse fragments are ironstone nodules. This is

a highly developed soil and have a distinct profilefeature. The profile models of Khao Khat series,

Nong Khla series, Phato series and Ranong series

(Figure 2) illustrate well of skeletal soils havingdifferent nature of the coarse fragment and profile

arrangement relating to the presence of coarse

materials. Khao Khat and Nong Khla series areboth deep and well developed soils and plinthite

layer in the Khao Khat profile does not limit root

penetration seriously. Both profiles indicate a welldeveloped soils. For Phato and Ranong series, their

profile features indicate a similar nature of the

coarse materials as being rock fragments. However,the arrangement of soil horizons in their profile is

Kasetsart J. (Nat. Sci.) 34 (1) 53

54 Kasetsart J. (Nat. Sci.) 34 (1)

Tab

le 1

Eco

logy

of

skel

etal

soi

ls in

Sou

ther

n T

haila

nd.

1/ D

rain

age

: WD

= w

ell d

rain

ed, S

PD =

som

ewha

t poo

rly

drai

ned

2/ R

= a

nnua

l rai

nfal

l, T

= r

ange

of

mea

n an

nual

tem

pera

ture

Kasetsart J. (Nat. Sci.) 34 (1) 55

different. Based on their profile models, Phatoseries can be considered a deep soil whereas Ranong

series is generally a shallow soil and its quartzitic

sandstone parent material can limit penetration ofplant roots. Within this group of skeletal soils

found in a moderate extent in southern Thailand,

Ranong series portrays the soil of poorest profile

Texture: C = Clay, GC = Gravelly clay; GSL =Gravelly sandy loam, L = Loam,

SL = Sandy loam, SGC = Slightly gravelly

clay, VGC = Very gravelly clayVGCL = Very gravelly clay loam;

VGSCL = Very gravelly sandy clay

loamStructure: SBK = Subangular blocky, 1 = Weak,

2 = Moderate

Figure 2 Profile models of some skeletal soilsfound in a moderate extent in Southern

Thailand.

feature to support the use of land for cropping

practices.

Phayom Ngam series, Sawi series and Yi-ngo series are skeletal soils found in a limited

extent in Southern Thailand. Skeletal materials in

Phayom Ngam and Sawi series are ironstonenodules, and somewhat broken plinthite layer exists

in Phayom Ngam series. This layer does not appear

to restrict penetration of plant roots either. Both of

Texture: GSL = Gravelly sandy loam,SGSL = Slightly gravelly sandy loam,

VGC =Very gravelly clay,

VGSC = Very gravelly sandy clayStructure: SBK =Subangular blocky, 1 = Weak,

2 = Moderate

Figure 1 Profile model of a skeletal soil foundrather extensively in Southern

Thailand.

56 Kasetsart J. (Nat. Sci.) 34 (1)

Texture: GC = Gravelly clay, GCL = Gravelly

clay loam, GSCL = Gravelly sandy clay

loam,GSL = Gravelly sandy loam, SL = Sandy

loam SiC = Silty clay,

VGCL = Very gravelly clay loamStructure: ABK = Angular blocky, SBK =

Subangular blocky,

1 = Weak, 2 = Moderate

Figure 3 Profile models of some skeletal soils

found in a limited extent in Southern

Thailand.

However, the condition would also depend on theirspecific physico-chemical properties.

Physical properties and status of basic fertility

of skeletal soilsTable 2 summarizes data on particle size

distribution, organic matter and available

phosphorus and potassium of a skeletal soil found

rather extensively in Southern Thailand (Chumphonseries). A vital parameter found in this soil as

indicated in Table 2 is the relatively sandy nature in

the upper part of its profile and the sharp break inthe amount of clay from the depth of 30 centimeters

downwards. This condition when added with the

low organic matter content, very low availablephosphorus and low available potassium making

its basic fertility status rather poor.

Four skeletal soils found in a moderatelyextent in Southern Thailand have been studied.

They are Khao Khat series, Nong Khla series,

Phato series and Ranong series. Table 3 summarizesdata on particle size distribution, organic matter

content and available phosphorus and potassium of

these soils. Khao Khat and Nong Khla series haverelatively finer texture comparing to that of Phato

and Ranong series. Considering data on organic

matter available phosphorus and potassium of thesesoils it appears that all the more clayey soils tend to

have a better basic fertility status than the sandy

ones. It should be noted here, that organic mattercontent values in the surface layer of these soils

show no problem for crop practices. Available

phosphorus data is too low for general cropproduction in these soils. Available potassium

values however, are varied but most of them except

those in Ranong series appear to be within a readilymanageable range. Therefore, another vital property

of these skeletal soils is the available phosphorus in

their fine earths.Data on particle size distribution, organic

matter and available nutrients of Phayom Ngam

these soils illustrate a generally deep and highlydeveloped soils. Yi-ngo series is a very deep, well

drained and highly developed soils having quartz

gravels as skeletal materials. This differs from theother soils within this group substantially. Another

characteristic deviating from other soils is the thick

surface layer in this soil making it more supportivefor crops with a shallow rooting zone.

Both data on the ecology of these soils and

their profile models suggested quite clearly thatthese soils are readily manageable for crop practices.

Kasetsart J. (Nat. Sci.) 34 (1) 57

Table 2 Particle size distribution in fine earths (USDA grading), organic matter (O.M.) and availablenutrients of a skeletal soil found rather extensively in Southern Thailand.

Depth Horizon Particle size distribution (g kg-1) Textural 1/ O.M. Avail.P Avail.K(cm) Sand Silt Clay class (g kg-1) ← (mg kg-1 ) →

Chumphon series0-10 A 710 210 80 SL 9.1 1.2 5410-20 Bw 700 190 110 SL 3.3 0.8 2420-30 Btc1 690 190 120 SL 3.6 1.0 1830-75 Btc2 460 150 390 SC 3.6 0.7 4575-115 Btc3 420 130 450 C 2.1 1.0 39115-185+ Btc4 470 190 340 C 4.6 1.2 36

1/ C = Clay, SC = Sandy clay, SL = Sandy loam.

Table 3 Particle size distribution in fine earths (USDA grading), organic matter (O.M.) and available

nutrients of some skeletal soils found in a moderate extent in Southern Thailand.

Depth Horizon Particle size distribution (g kg-1) Textural 1/ O.M. Avail.P Avail.K

(cm) Sand Silt Clay class (g kg-1) ← (mg kg-1 ) →

Khao Khat series0-10 A 270 470 260 SiL 39.0 4.1 14310-22 Bc 235 385 380 CL 23.4 1.7 6222-65 Btc 140 335 525 C 7.1 1.3 4165-122+ Bv 185 395 620 C 8.1 1.9 35Nong Khla series0-8 Ap 330 250 420 C 23.4 2.0 678-18 Bt1 205 190 605 C 24.1 3.7 4718-35 Bt2 170 170 660 C 20.8 1.9 3235-75 Btc1 155 110 735 C 21.1 1.4 2975-110+ Btc2 150 110 740 C 21.1 1.4 29Phato series0-12/17 Ap 735 165 100 SL 16.2 2.5 5017-35 Bt1 690 200 110 SL 8.4 1.7 3835-50/55 Bt2 670 180 150 SL 4.8 1.2 4155-110+ Bc 595 175 230 SL 3.6 0.9 59Ranong series0-10 A 680 260 60 SL 17.2 4.3 3910-20 Bc1 680 270 50 SL 6.9 2.9 3020-47 Bc2 690 250 60 SL 1.9 1.6 2147-100+ C ---------------------- Saprolite of quartzitic sandstone ----------------------

1/ C = Clay, CL = Clay loam, SiL = Silt loam, SL = Sandy loam.

58 Kasetsart J. (Nat. Sci.) 34 (1)

Table 4 Particle size distribution in fine earths (USDA grading), organic matter (O.M.) and available

nutrients of some skeletal soils found in a limited extent in Southern Thailand.

Depth Horizon Particle size distribution (g kg-1) Textural 1/ O.M. Avail.P Avail.K

(cm) Sand Silt Clay class (g kg-1) ← (mg kg-1 ) →

Phayom Ngam series

0-15 A 445 315 240 SCL 35.4 3.1 6415-29 Bw 390 270 340 SCL 30.7 3.9 38

29-65 Btc 330 215 450 C 18.1 3.3 29

65-155+ Bvg 323 205 470 C 7.5 1.1 29Sawi series

0-9 Ap 710 235 55 SL 13.8 1.9 39

9-25 Bw 705 230 65 SL 7.6 1.8 2125-52/58 Bt 700 235 65 SL 2.4 1.4 26

58-110+ Btc 580 270 150 SL 1.5 1.9 36

Yi-ngo series0-22 Ap 521 354 125 SL 17.2 64.2 62

22-50 Btc1 472 312 216 SL 8.8 60.8 48

50-135 Btc2 421 338 241 SL 3.8 58.2 36135-160+ C 434 366 200 SL 1.7 59.1 33

1/ C = Clay, SCL = Sandy clay loam, SL = Sandy loam.

series, Sawi series and Yi-ngo series, skeletal soilsthat occupy a limited extent in Southern Thailand

are summarized in Table 4. Phayom Ngam series is

only soil having clayey texture where clay contentdetermines the activities and chemical properties

of the soil. Also, this soil differs from other two soil

series in this group in its more poorly drainedenvironment. The trend for their basic fertility

relating to the amount of clay present in the fine

earths follows what can be found in other groups ofskeletal soils. The soils do not have any problem on

organic matter content and their available potassium

values are also within a readily manageable range.Available phosphorus in general is the problem for

Phayom Ngam and Sawi series but very high

values of available phosphorus were found in theanalysis of soil samples collected from the profile

of Yi-ngo series. This makes basic fertility status of

Yi-ngo series relatively better than the other twosoils for a general cropping practices (Sanchez,

1976).

Data on physical properties and basic fertilityof these skeletal soils found in Southern Thailand

indicate mildly that most of them have a generally

light texture in their fine earths. Most of them havemanageable level of organic matter content and

available potassium. Available phosphorus in these

soils can vary markedly and most of them have toolow available phosphorus. This aspect should be

looked into in using them for crop practices.

Exchange properties of skeletal soils in SouthernThailand

Table 5 summarizes laboratory analyticaldata related to exchange properties in the fine

earths of Chumphon series, a skeletal soil found

Kasetsart J. (Nat. Sci.) 34 (1) 59

rather extensively category being highly leached,

well developed soil poor exchange properties of

this soil can be expected. Though this poor soil stillfavour cation exchange in the soil system since its

delta pH (∆ pH) is negative, other chemical

parameters such as the low extractable bases,relatively high extractable acidity, relatively low

cation exchange capacity and low base saturation

make them have poor exchange properties in theirfine earths. With at least 35 percent by volume of

coarse fragments in the soils the overall exchange

properties of soil mass can be quite poor.Table 6 summarizes laboratory data related

to exchange properties in the fine earths of four soil

series found in a moderate extent in SouthernThailand. Normally, the pH level of these soils do

not pose any serious condition and they also favour

cation exchange judging from their negative deltapH values. The problem of their exchange properties

generally lies in their low extractable basic cations

and their markedly high extractable acidity. Thisimplies the excessive acidic cations (H+, Al3+) in

the soil system affecting balance of plant nutrient

status. The cation exchange capacity values of

these soils range widely but depending on their

extractable acidity mainly since their base saturationcan be considered quite low. This condition in the

soils can be quite serious for crop practices and

their soil-fertilizer management. The condition inKhao Khat and Nong Khla series is more serious

than the one in Phato and Ranong series.

Laboratory analytical data related toexchange properties in the fine earths of three

skeletal soils found in a limited extent in Southern

Thailand are summarized in Table 7. Pattern of thedata on their exchange properties appear to strictly

follow those of the ones shown in Table 6. Though

it is obvious that their soil system still favour cationexchange, acidic cations in the soils adversely

affect their cation exchange capacity and base

saturation. The excessive extractable acidic cationsin Phayom Ngam series makes its cation exchange

poorest. A moderate condition on cation exchange

properties of these soils can be found in Sawiseries. Among these three soils the exchange

properties of Yi-ngo series are the best one.

Table 5 Laboratory analytical data related to exchange properties in the fine earths of a skeletal soil foundrather extensively in Southern Thailand.

Depth Horizon pH (1:1) Extractable bases Sum E.A.1/ C.E.C.2/ B.S.3/

(cm) H2O KCl Ca Mg Na K bases (sum) %

← cmol kg-1→

Chumphon series0-10 A 6.0 4.8 1.9 0.3 0.1 0.1 2.4 3.7 6.1 5610-20 Bw 5.5 3.8 0.8 0.2 0.1 0.1 1.2 3.7 4.9 22

20-30 Btc1 5.3 3.9 0.9 0.2 0.1 0.1 1.3 4.4 5.7 23

30-75 Btc2 5.7 4.1 4.0 0.9 0.2 0.1 5.2 7.6 12.8 3875-115 Btc3 5.7 4.2 4.0 1.1 0.2 0.1 5.4 7.3 12.7 37

115-185+ Btc4 5.6 4.1 3.4 1.3 0.2 0.1 5.0 5.6 10.6 34

1/ E.A. = Extractable acidity

2/ C.E.C. (sum) = Cation exchange capacity by the sum of E.A. and sum of bases

3/ B.S. = Base saturation

60 Kasetsart J. (Nat. Sci.) 34 (1)

Table 6 Laboratory analytical data related to exchange properties in the fine earths ofsome skeletal soils found in a moderate extent in Southern Thailand.

Depth Horizon pH (1:1) Extractable bases Sum E.A.1/ C.E.C.2/ B.S.3/

(cm) H2O KCl Ca Mg Na K bases (sum) %

← cmol kg-1→

Khao Khat series

0-10 A 5.1 4.2 0.7 1.0 0.1 0.1 1.9 19.8 21.7 910-22 Bc 5.0 4.0 0.1 1.1 0.1 0.1 1.4 15.8 17.2 8

22-65 Btc 5.5 4.2 0.1 0.2 0.1 0.1 0.5 13.4 13.9 4

65-122+ Bv 5.7 4.1 0.1 0.1 0.2 0.1 0.5 13.0 13.5 4Nong Khla series

0-8 Ap 4.6 3.7 0.3 0.3 0.2 0.1 0.9 12.5 13.4 7

8-18 Bt1 4.9 3.8 0.3 0.2 0.3 0.1 0.9 12.7 13.6 718-35 Bt2 5.0 3.7 0.2 0.1 0.2 0.1 0.6 12.8 13.4 4

35-75 Btc1 5.4 3.8 0.2 0.1 0.2 0.1 0.6 12.5 13.1 5

75-110+ Btc2 5.5 3.8 0.2 0.1 0.2 0.1 0.6 12.5 13.1 5Phato series

0-12/17 Ap 4.8 4.0 0.5 0.2 0.1 0.1 0.9 4.9 5.8 15

17-35 Bt1 4.8 3.9 0.2 0.1 0.1 0.1 0.5 3.9 4.4 1135-50/55 Bt2 4.8 3.7 0.2 0.1 0.1 0.1 0.5 3.9 4.4 11

55-110+ Bc 4.9 3.7 0.2 0.1 0.1 0.1 0.5 5.2 5.7 9

Ranong series0-10 A 4.6 4.0 0.9 0.1 0.2 0.1 1.3 3.4 4.7 28

10-20 Bc1 4.5 4.0 0.5 0.1 0.2 0.1 0.9 3.3 4.2 21

20-47 Bc2 5.0 4.0 0.2 0.1 0.1 0.1 0.4 1.4 1.8 2247-100+ C --------------------------Saprolite of quartzitic sandstone--------------------------

1/ E.A. = Extractable acidity

2/ C.E.C. (sum) = Cation exchange capacity by the sum of E.A. and sum of bases3/ B.S. = Base saturation

Data related to exchange properties of theseskeletal soils indicate few of their common vital

properties. Firstly, these skeletal soils possess a

system favouring cation exchange. Secondly, dueto their highly leached and well developed nature

their exchange complex is generally affected

strongly by the acidic cations since the extractableacidity values are generally higher than their total

extractable basic cations. Thirdly, the high amount

of clay in the fine earths of these soils does not reallygive positive effect on their exchange properties.

Some of the soils with high clay content in the fine

earths appear to show more adverse effect of theacidic cations. These conditions of their exchange

properties seem to suggest that soil-fertilizer

management in crop practices for these soils shouldnot emphasize the cation form of fertilizer elements

too strongly. Anionic forms of nutrients should

Kasetsart J. (Nat. Sci.) 34 (1) 61

also be considered more seriously for a moreefficient fertilizer management. The presence of

skeletal materials in these soils would not have

strong negative effect in soil-crop practices.Based on their morphology and data on

their properties these soils can be classed mainly as

Ultisols. Chumphon series and Yi-ngo series areHapludults; Khao Khat series is Plinthudult; Nong

khla series, Phato series and Sawi series are

Paleudults, Phayom Ngam series is a Plinthaquult.However, Ranong series is a Udorthent (Soil Survey

Staff, 1998).

Table 7 Laboratory analytical data related to exchange properties in the fine earths of some skeletal soilsfound in a limited extent in Southern Thailand.

Depth Horizon pH (1:1) Extractable bases Sum E.A.1/ C.E.C.2/ B.S.3/

(cm) H2O KCl Ca Mg Na K bases (sum) %

← cmol kg-1→

Phayom Ngam series

0-15 A 4.8 3.8 0.3 0.2 0.3 0.1 0.9 15.0 15.9 615-29 Bw 4.8 3.7 0.2 0.1 0.4 0.1 0.8 10.6 11.4 8

29-65 Btc 5.1 3.8 0.2 0.1 0.4 0.1 0.8 10.1 10.9 7

65-155+ Bvg 5.1 3.7 0.1 0.1 0.3 0.1 0.6 12.0 12.6 5Sawi series

0-9 Ap 5.3 4.4 0.9 0.2 0.1 0.1 1.3 2.6 3.9 33

9-25 Bw 5.5 4.3 0.9 0.2 0.1 0.1 1.3 2.3 3.6 3625-52/58 Bt 6.3 4.6 0.1 0.2 0.1 0.1 0.5 1.7 2.2 23

58-110+ Btc 6.1 3.8 0.1 0.3 0.1 0.1 0.5 2.5 3.0 17

Yi-ngo series0-22 Ap 4.7 3.7 0.3 0.1 7.8 0.6 8.8 5.3 14.1 62

22-50 Btc1 4.5 3.7 0.3 0.2 7.7 1.3 9.5 5.1 14.6 65

50-135 Btc2 4.7 3.9 0.4 0.2 7.2 0.8 8.6 7.5 16.1 53135-160+ C 5.1 3.9 0.2 0.2 0.6 0.6 1.6 4.5 6.1 26

1/ E.A. = Extractable acidity

2/ C.E.C. (sum) = Cation exchange capacity by the sum of E.A. and sum of bases

3/ B.S. = Base saturation

Agricultural potential of skeletal soils inSouthern Thailand

Data on ecology, profile models and physicaland chemical properties of these skeletal soils

illustrate that these soils are generally deep soils

having skeletal materials of different nature but themost common one is the ironstone nodule.

Generally, the soils are well developed, highly

leached Ultisols and their fine earths have poorexchange properties. Though their surface layers

would appear to possess a better quality for crop

practices, the condition within the whole soils inmost of them are poor. This is a typical condition of

Ultisols in the humid tropics. Based on their

properties and morphology most of these soils can

62 Kasetsart J. (Nat. Sci.) 34 (1)

be classed into U-IVct according to the landcapability classification system and P-Vt, N-IIIct,

F-IIct (T-IIct), L-IItc according to land suitability

classification (FAO, 1976; Land ClassificationDivision and FAO Project Staff, 1973) where U

indicates upland cropping, P = paddy rice, N = non-

flooded annual crops (upland field crops), F = fruittrees, T = tree crops, L = livestock pasture, IV =

marginally suited (land capability classification) V

= not suited, III = moderately suited, II = well suited(for suitability classess of paddy rice, upland field

crops and fruit trees or tree crops) and II = moderately

suited (for suitability class of livestock pasture).Two limiting parameters can be envisioned for

these soils include topography (t) and gravels or

coarse materials (c) in the soils.In a strict sense these skeletal soils do not

have potential for lowland crop production in

agriculture. They are moderately suitable for uplandfield crop and livestock pasture, well suited for

fruit trees and tree crop production with two inherent

problems, slope and coarse materials in the soils.From these analyses it should be noted that these

skeletal soils in Southern Thailand can be used

readily in crop practices but attention on types ofcrops is needed.

Sustainable uses of skeletal soils in SouthernThailand

Development of crop production and

practices in Southern Thailand has been quiteadaptive to climatic and soil conditions. For the

upland region including areas occupied by skeletal

soils, para rubber and fruit tree have beenemphasized. The general condition on agricultural

technology is also directed to perennial tree crop

production. These include both soil-fertilizermanagement and integrated pest management. As

indicated in Table 1 on the ecology of the skeletal

soils, most of them are now supporting para rubberplanting with some limited part under fruit trees.

Stands of these crops are quite acceptable and theiryields have been quite satisfactory with common

plantation and orchard management basing on

existing technologies. Therefore, the overallagricultural use of upland soils including the skeletal

ones in most parts of Southern Thailand can be

considered stable and productive.A factor that can affect the stability of land

use on these soils is strictly economic. Marketing

of the products, at time, becomes problem, but withthe relatively fast development in agro-industry in

the country the situation can be sustained for a good

period of time. Though it appears to be somewhataccidental the use of skeletal soils in Southern

Thailand at present is well adapted and generally

sustainable.

CONCLUSION

Results of the study on vital properties and

agricultural potential of skeletal soils in Southern

Thailand revealed that most of them are uplandsoils mainly occupying undulating and rolling

terrains with a major slope range of 3-8 percent.

Most of them are deep to very deep, highly leachedand well developed Ultisols. The skeletal materials

in these soils vary but ones that can be found

frequently are ironstone nodules. Their basic fertilitystatus is generally poor and available phosphorus

problem in most soils exists. Their high extractable

acidity can affect their exchange propertiesmarkedly whereas the presence of skeletal materials

does not appear to pose serious problem on their

uses and management.Evaluation of their suitability for cropping

reveals that they are not suited for paddy rice, but

they are moderately suited for upland field cropsand livestock pasture, and well suited for tree crops

including fruit trees with different degrees of

problem on topography and the presence of skeletalmaterials. Since they have been used mainly for

Kasetsart J. (Nat. Sci.) 34 (1) 63

rubber and fruit tree production, land use problemon them can be coped with quite well by the use of

existing agricultural technologies. Therefore,

sustainability in their use, at present, is somewhatachieved.

ACKNOWLEDGEMENT

This study was a part of the Project on

Properties and Fertility of Skeletal Soils in Thailandsupported by research fund granted through the

Kasetsart University Research and Development

Institute (KURDI), Kasetsart University, Bangkok.

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interpreting soil survey. United StatesDepartment of Agriculture. U.S. Government

Printing Office, Washington D.C. 754 p.

Soil Survey Staff. 1998. Keys to Soil Taxonomy.8th ed., United States Department of

Agriculture. U.S. Government Printing Office,

Washington D.C. 326 p.Vijarnsorn, P. 1984. Skeletal soils of Thailand, pp.

F2.1-F2.14. In Proceedings of the Fifth ASEAN

Soil Conference, Vol. 1. Department of LandDevelopment, Ministry of Agriculture and

Cooperatives. Bangkok, Thailand.

Vijarnsorn, P. 1985. General Soil Map of theSouthern Region, Thailand (scale 1:500,000).

Department of Land Development, Ministry

of Agriculture and Cooperatives. Bangkok (2map sheets).

Received date : 1/06/99Accepted date : 19/10/99

Kasetsart J. (Nat. Sci.) 34 : 64 - 73 (2000)

indicated that xylose fermenting yeasts did not

ferment well in undetoxified hydrolysate. Ferrari et

al. (1992) reported that acetic acid in the eucalyptuswood hemicellulose acid hydrolysate caused low

pH, reduced the ethanol production rate and yield

of Pichia stipitis fermentation. Its toxicity dependednot only on the concentration but also the pH of

hydrolysate. Some investigators reported the

inhibitory effects of acetic acid on activities of

Effect of Acetic Acid on Growth and Ethanol Fermentationof Xylose Fermenting Yeast and Saccharomyces cerevisiae

Savitree Limtong1 , Tawatchai Sumpradit1, Vichien Kitpreechavanich1, Manee Tuntirungkij2 , Tatsuji Seki3 and Toshiomi Yoshida3

ABSTRACT

Growth of some xylose fermenting yeasts, Candida shehatae, Pichia stipitis CBS5773, fusant F101

and fusant F198, was completely inhibited in xylose medium added with 0.5% v/v acetic acid which caused

the reduction of pH to 4.1. Only one xylose fermenting strain, Pachysolen tannophilus NRRL-Y2460,showed relatively low growth and ethanol fermentation. However, in the medium added with 1.0% v/v

acetic acid (pH 3.7) all of these strains were completely inhibited. When the medium was adjusted by

hydrochloric acid to pH 4.1 and 3.7, all xylose fermenting strains showed almost the same growth as in themedium without pH adjustment (pH 6.2). In glucose medium added with 0.5% v/v acetic acid, various

strains of Saccharomyces cerevisiae, M30, Sc90, N1, G/3, G/5, G/2, TJ3 and SH1089, grew with lower

specific growth rate and provided lower maximal cell concentration rate than in medium without addingacetic acid (pH 6.2). All strains, except N1, produced slightly higher maximal ethanol concentration.

However, all of them yielded lower ethanol production rate. Among S. cerevisiae, strain B120 was more

sensitive to acetic acid than the others since its growth was completely inhibited by 0.5% v/v acetic acid.In glucose medium, 0.5% v/v acetic acid did the same role as in xylose medium to xylose fermenting strains.

Hence, the xylose fermenting yeasts revealed higher sensitivity to acetic acid than S. cerevisiae.

Key words : acetic acid, xylose fermenting yeast, ethanol fermentation, xylose fermentation,Saccharomyces cerevisiae

INTRODUCTION

Hydrolysate of lignocellulose contains not

only a mixture of sugars, mainly glucose from

cellulose and xylose from hemicellulose, but alsosome substances that exert inhibitory effects on

yeast such as acetic acid, furfural and lignin

derivatives (Tsao et al., 1982; Olsson and Hahn-Hagerdal, 1993). Linden and Hahn-Hagerdal (1989)

1 Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.2 Central Laboratory and Greenhouse Complex, Kasetsart University Research Development Institute, Kasetsart University,

Kamphaengsaen Campus, Nakhon Pathom 73140, Thailand.

3 International Center for Biotechnology, Osaka University, Yamadaoka, Suita-shi, Osaka 565, Japan.

several yeasts. For examples; Mariorella et al.

(1983) showed the inhibitory effects of different

metabolic products of yeast including acetic acid.

Pampulha and Loureioro (1989) and Ferrari, et al.(1992) indicated the important role of undissociate

form of acetic acid which diffuse into yeast cells,

causing decreased pH of cytoplasm and inhibitingthe activity of some enzymes, especially endolase,

phosphoglyceromutase, aldolase and

triosephosphate isomerase. Phowchinda et al.

(1995) showed the inhibitory effect of acetic on

growth and fermentation activity of Saccharomyces

cerevisiae and indicated that it was more effectiveon the biomass synthesis than on ethanol production.

However, there were no comparative study on

sensitivity to acetic acid between xylose fermentingyeast and S. cerevisiae. Therefore, in this study the

inhibitory effect of acetic acid on growth and

ethanol fermentation of some xylose fermentingyeasts and some strains of S. cerevisiae was

demonstrated, as well as comparison on sensitivity

of both yeasts to acetic acid.

MATERIALS AND METHODS

Yeast strainsYeast used in this study included

Saccharomyces cerevisiae strain Sc90, M30, N1,

TJ3, B120, G/2, G/3, G/5 and SH1089 and wildstrains of xylose fermenting yeast, Candida

shehatae, Pichia stipitis CBS5773, Pachysolen

tannophilus NRRL-Y2460. Fusant F101, a fusantfrom intergeneric protoplast fusion of P. stipitis

CBS5773 and S. cerevisiae AM12, and F198, a

fusant from intraspecific protoplast fusion of P.

stipitis CBS5773 (Chomtong, 1995) were also

included.

Growth and ethanol fermentationInoculum was prepared by inoculation of

24 h culture of yeast cultivated on a slant YPD agar

(1% yeast extract, 2% peptone, 2% D-glucose and1.2% agar) or YPX agar (same ingredient as YPD

except D-xylose was used instead of D-glucose),

depending on yeast strains, into 50 ml of YPD brothor YPX broth in 250 ml flask and incubated on a

rotary shaker, 200 rpm, at room temperature for 24

h. Cells were harvested, washed twice andresuspended in distilled water.

Fermentation was carried out in 100 ml of

YPD18 broth (YPD broth containing 18% D-glucose) or YPX4 broth (YPX broth containing 4%

D-xylose) in 250 ml Erlenmeyer flask. In acetic

acid treatment, pre-calculated volume ofconcentrated acetic acid was added prior to

sterilization. For comparative study on effect of pH

1 N hydrochloric acid was used. Inoculum wasadded into the medium to obtain the initial cell

concentration, as optical density (OD) at 660 nm, at

1.0. Incubation was performed at room temperatureon a rotary shaker at 180 rpm.

AnalysesEthanol was determined by gas

chromatography (Shimadzu GC-9A, Japan) and

propanol was used as internal standard. Cell

concentration was quickly determined as OD at660 nm by Spectrophotometer (Shimadzu model

UV-240, Japan).

RESULTS AND DISCUSSIONS

Effect of acetic acid on growth and ethanolfermentation in xylose medium of xylosefermenting yeast

In xylose medium (4% xylose and pH 6.2),P. stipitis CBS5773 showed the highest specific

growth rate (µ), 0.21 h-1 though its maximal cell

concentration measured as OD at 660 nm, was thelowest, 33.6, at 96 h (Figure 1 A). This yeast also

produced the highest ethanol concentration, 1.51%

w/v after 36 h, with the highest ethanol production

Kasetsart J. (Nat. Sci.) 34 (1) 65

66 Kasetsart J. (Nat. Sci.) 34 (1)

Figure 1 Growth of C. shehatae (◆ ), P. stipitis

CBS5773 (▲), Pa. tannophilus NRRL-

Y2460 ( ), F101 (■ ) and F198 (● ) in

xylose medium without (A) and with pHadjustment by HCl to pH 4.1 (B) and 3.7

(C).

Figure 2 Ethanol fermentation of C. shehatae (◆ ),

P. stipitis CBS5773 (▲), Pa. tannophilus

NRRL-Y2460 ( ), F101 (■ ) and F198(● ) in xylose medium without (A) and

with pH adjustment by HCl to pH 4.1

(B) and 3.7 (C).

Kasetsart J. (Nat. Sci.) 34 (1) 67

rate of 0.41 g/l/h (Figure 2A). Likewise, the twofusants, F198 and F101, produced ethanol at 1.44

and 1.41% w/v after 36 h with the production rate

of 0.40 and 0.39 g/l/h, respectively. C. shehatae

accumulated 1.40 % w/v of ethanol at 48 h with low

production rate of 0.29 g/l/h, while Pa. tannophilus

NRRL-Y2460 produced only 0.77% w/v of ethanolwith the lowest production rate of 0.10 g/l/h.

Accordingly, Pa. tannophilus NRRL-Y2460

revealed the lowest specific growth rate, 0.158 h-1,though its maximal OD was the highest, 47.0.

Addition of 0.5% v/v acetic acid into xylose

medium resulted in reduction of pH to 4.1.Cultivation of xylose fermenting yeasts in this

medium showed that only Pa. tannophilus NRRL-

Y2460 could grow with low specific growth rate,0.076 h-1, and yielded low maximal cell

concentration as OD at 29.30 after 96 h. This yeast

produced maximal ethanol concentration, 0.56%w/v at 120 h resulted in production rate of 0.046 g/

l/h. In 1.0% v/v acetic acid treatment causing pH

reduction to 3.7, no xylose fermenting yeasts wereobserved in the medium. The results obviously

indicated the inhibitory effect of acetic acid to

growth and fermentation of xylose fermentingyeasts. These findings agreed with the report of

Ferrari et al. (1992) on inhibitory effect of acetic

acid on ethanol fermentation by P. stipitis in acidhydrolysate of hemicellulose in eucalyptus wood

which contained various products including 3%

xylose and 1% acetic acid. In addition they reportedflocculation of yeast and lost of viability after

inoculation, however, growth was resumed after a

period of time.To exhibit the effect of acetic acid on

reduction of growth and ethanol fermentation and

not from low pH caused by adding the acid,experiments were carried out in xylose medium

where pH was adjusted by 1 N hydrochloric acid to

the same pH level as obtained by adding aceticacid. Results showed that growth and ethanol

production by each xylose fermenting yeast inxylose medium with pH adjusted to 4.1 and 3.7

were not much different as compared to the control

(Figure 1B, 1C, 2A and 2B). On the contrary, theproduction rate of each xylose fermenting yeast in

the medium with hydrochloric acid treatment was

slightly higher than in the normal xylose medium(pH 6.2), except C. shehatae (Figure 3B) where

ethanol production rate was markedly improved

from 0.29 g/l/h in normal xylose medium to 0.45 g/l/h, in both treated media. Subsequently, the specific

growth rate of most xylose fermenting yeasts in

hydrochloric treated media was slightly higher ascompared to the control (Figure3A). These indicated

Figure 3 Specific growth rate (A) and ethanol

production rate (B) of C. shehatae, P.

stipitis CBS5773, Pa. tannophilus

NRRL-Y2460, F101 and F198 in xylose

medium without (■ ) and with pH adjust-ment by HCl to pH 4.1 ( ), and 3.7 ( ).

68 Kasetsart J. (Nat. Sci.) 34 (1)

Effect of acetic acid on growth and ethanol

fermentation of S. cerevisiae in glucose mediumIn glucose medium (pH 6.2), S. cerevisiae

N1 and Sc90 produced relatively high cell

concentration as OD at 72.20 and 66.90 after 36

and 24 h, respectively (Figure 4A). Consequently,the specific growth rates of all strains of S.

cerevisiae, except SH1089, were relatively high

and were not much different (Figure 4B).All strains of S. cerevisiae, namely M30,

Sc90, N1, G/3 and TJ3, except SH1089, produced

high ethanol concentrations at 7.16, 7.42, 7.53,

that low pH (pH 4.1 and 3.7) did not inhibit growthand ethanol fermentation of xylose fermenting

yeasts, except in the case of C. shehatae which

lower pH promoted better growth and ethanolproduction.

In conclusion, the results revealed that

growth and ethanol fermentation of all xylosefermenting yeasts used in this study was inhibited

by acetic acid not by lower pH.

Figure 4 Maximal cell concentration (A) and specific growth rate (B) of S. cerevisiae M30, Sc90, N1, G/

3, G/5, G/2, TJ3, B120 and SH1089 in glucose medium without (■ ) and with addition of acetic

acid ( ) and with pH adjustment by HCl to 4.1 ( ) and 3.7 ( ).

Kasetsart J. (Nat. Sci.) 34 (1) 69

Figure 5 Maximal ethanol concentration (A) and production rate (B) of S. cerevisiae M30, Sc90, N1,

G/3, G/5, G/2, TJ3, B120 and SH1089 in glucose medium without (■ ) and with addition of aceticacid ( ) and with pH adjustment by HCl to 4.1 ( ) and 3.7 ( ).

7.32 and 7.48% w/v, respectively, at fairly shortfermentation time of 24 h with relatively high

production rates of 2.98, 3.09, 3.13, 3.05 and 3.11

g/l/h, respectively (Figure 5B). On the other hand,ethanol produced by strains G/5, G/2 and B120

were similar at 36 h, 7.63, 7.42 and 7.07% w/v,

respectively, with slightly longer incubation timeof 36 h and hence their production rates were lower.

For S. cerevisiae SH1089 lowest concentration of

ethanol of 5.97% w/v was obtained after 48 h withthe lowest production rate of 1.24 g/l/h.

By addition of 0.5 and 1.0% v/v acetic acid

into glucose medium, pH changes of the mediumwere similar to those observed in xylose medium.

Likewise no growth of all S. cerevisiae strains were

observed in medium added with 1.0% v/v. Withaddition of 0.5% v/v acetic acid, only S. cerevisiae

B120 could not grow while the other strains

produced comparable maximal cell concentrationto those in basal glucose. However, the specific

growth rates were slightly lower (Figure 4A and

4B). Though all strains, except S. cerevisiae N1,yielded slightly higher maximal ethanol

concentration, their production rates were lower

70 Kasetsart J. (Nat. Sci.) 34 (1)

than in normal glucose medium (Figure 5B).The results revealed that acetic acid at 0.5%

v/v reduced maximal cell concentration but had no

effect on maximal ethanol concentration of S.

cerevisiae. However, both specific growth rate and

ethanol production rate decreased. These findings

confirmed the report of Phowchinda et al. (1995)which concluded that acetic acid inhibited the

activities of S. cerevisiae and the inhibition was

more effective on the biomass synthesis than ethanolsynthesis.

Comparison on the effect of acetic acid on

ethanol production rate and specific growth rate ofS. cerevisiae in the medium added with 0.5% v/v

acetic acid showed that the inhibition on production

rate ranged from 67.7% to 27.5% and on specificgrowth rate were 72.9 to 35.6% (Figure 6). The

results indicated stronger inhibition of acetic acid

on specific growth rate than on production rate.In the medium where pH was adjusted to 4.1

by hydrochloric acid specific growth rate of N1was the highest, while G/2 showed the highest

specific growth rate in medium with pH was at 3.7

(Figure 4B). As far as ethanol fermentation wasconcerned, all strains of S. cerevisiae demonstrated

high performance in pH adjusted medium. At pH

4.1, N1 produced high ethanol concentration, 8.44%w/v at 12 h, with the highest production rate, 7.03

g/l/h (Figure 5), while G/3 produced the highest

ethanol concentration, 8.58% w/v at 24 h, withproduction rate of only 3.57 g/l/h. On the contrary,

B120 produced the lowest ethanol concentration,

7.75% w/v, with the lowest production rate, 1.61 g/l/h. At pH 3.7, Sc90 fermented ethanol with the

highest production rate, 6.85 g/l/h, while ethanol

produced was 8.23% w/v at 12 h.Comparison on the effect of both acids

revealed that growth and ethanol fermentation of S.

cerevisiae were inhibited by acetic acid not by lowpH as reported by Phowchinda et al. (1995).

Figure 6 The percentage of inhibition on specific growth rate (A) and ethanol production rate (B) ofvarious strains of S. cerevisiae M30, Sc90, N1, G/3, G/5, G/2, TJ3, B120 and SH1089 in glucose

medium added with 0.5% v/v acetic acid comparing with the rates in medium without addition

of acetic acid.

Kasetsart J. (Nat. Sci.) 34 (1) 71

Comparison on effect of acetic acid on growthand ethanol fermentation of xylose fermentingyeast and S. cerevisiae in glucose medium

Ethanol fermentation in glucose medium

without adding acetic acid (pH 6.2) by xylose

fermenting yeasts was compared to two strains ofS. cerevisiae, M30 and Sc90. The result showed

that both strains of S. cerevisiae produced higher

ethanol concentration and at the higher rate than allxylose fermenting strains (Figure 7C). Among

xylose fermenting strains tested in this work, Pa.

tannophilus NRRL-Y2460 produced the highestethanol concentration of 6.45% w/v at 48 h, with

the highest production rate of 1.34 g/l/h. In contrast,

P. stipitis CBS 5773 produced only 3.39% byweight at 72 h with production rate of 0.47 g/l/h. In

addition the specific growth rate of both strains of

S. cerevisiae was higher than those of all xylosefermenting strains (Figure 7A).

In the medium added with 0.5% v/v acetic

acid, no growth and ethanol fermentation of C.

shehatae, P. stipitis CBS5773, F101 and F198

Figure 7 Growth and ethanol fermentation of C. shehatae (◆ ), P. stipitis CBS5773 (▲), Pa. tannophilus

NRRL-Y2460 ( ), F101 (■ ), F198 (● ), S. cerevisiae M30 (✲ ) and S. cerevisiae Sc 90 (▼) in

glucose medium without (A, C) and with addition of 0.5% v/v acetic acid (B, D).

72 Kasetsart J. (Nat. Sci.) 34 (1)

were observed. P. tannophilus NRRL-Y2460 wasthe only xylose fermenting yeast that could grow

and ferment ethanol (Figure 7B). However, it grew

at lower specific growth rate, 0.059 h-1, andproduced lower ethanol concentration, 4.44% by

weight at 96 h, with lower production rate, 0.46 g/

l/h. For S. cerevisiae, strain M30 produced nearlythe same cell concentration as in glucose medium

without adding acetic acid, while Sc90 provided

much lower cell concentration (Figure 7B).However, the specific growth rates of both strains

of S. cerevisiae were much lower than in medium

without adding acetic acid.

CONCLUSION

In conclusion, acetic acid at the

concentration of 0.5% v/v completely inhibited

growth of most xylose fermenting yeasts, C.

shaehatae, P. stipitis CBS5773 and the two xylose

fermenting fusants, F101 and F198, in medium

containing glucose and xylose as a sole source ofcarbon. However, Pa. tannophilus NRRL-Y2460

relatively tolerated to acetic acid since it could

grow and ferment ethanol in both media added with0.5% v/v acetic acid. This acid plays the same role

on growth and ethanol fermentation of various

strains of glucose fermenting yeast, S. cerevisiae.Despite most strains of S. cerevisiae investigated in

this study showed higher tolerance to acetic acid

than xylose fermenting yeasts. The inhibition ongrowth and ethanol fermentation was proved to be

the result from acetic acid and not from low pH as

shown by hydrochloric acid.

ACKNOWLEDGMENTS

This research was supported by the National

Research Council of Thailand and Kasetsart

University Research and Development Institute,Kasetsart University, Thailand. The authors thank

Assistant Professor Dr.Sarote Sirisansaneeyakulfor his helpful discussion. We also thank Dr.Wichien

Yongmanitchai for English editing this manuscript.

LITERATURE CITED

Chomtong, S. 1995. Construction of new yeasthybrid for ethanol production from xylose by

protoplast fusion technique. M.S. Thesis,

Kasetsart University. Bangkok.Fanta, G.F., T.P. Abbott, A.I. Herman, R.C. Burr,

and W.M. Doane. 1984. Hydrolysis of wheat

straw hemicellulose with trifluoroacetic acid:Fermentation of xylose with Pachysolen

tannophilus. Biotechnology Bioengineering

26 : 1122-1125.Ferrari, M.E., E. Neirotti, C. Albornoz, and E.

Saucedo. 1992. Ethanol production from

eucalyptus wood hemicellulose hydrolysateby Pichia stipitis. Biotechnology

Bioengineering 40 : 753-759.

Linden, T. and B. Hahn-Hagerdal. 1989.Fermentation of lignocellulose hydrolysates

with yeasts and xylose isomerase. Enzyme

Microbiology 1: 583-589.Maiorella, B., H.W., Blanch, and C.R. Wilke,

1983. By-product inhibition effects on

ethanolic fermentation by Saccharomyces

cerevisiae. Biotechnology Bioengineering

25 : 103-121.

Olsson, L. and B. Hahn-Hagerdal. 1993.Fermentative performance of bacterial and

yeasts in lignocellulose hydrolysate. Process

Biochemistry 28 : 249-257.Pampulha, M.E. and M.C. Loureiro-Dias. 1989.

Interaction of the effect of acetic acid and

ethanol on inhibition of fermentation inSaccharomyces cerevisiae. Biotechnology

Letters 11 : 269-274.

Pampulha, M.E. and M.C. Loureiro-Dias. 1990.Activity of glycolytic enzymes of

Kasetsart J. (Nat. Sci.) 34 (1) 73

Saccharomyces cerevisiae in the presence ofacetic acid. Applied Microbiology

Biotechnology 34 : 375-380.

Phowchinda, O., M.L Delia-Dupuy, and P.Strehaiano. 1995. Effects of acetic acid on

growth and fermenting activity of

Saccharomyces cerevisiae. BiotechnologyLetters 17(2) : 237-242.

Tsao, G.T., M.R. Ladisch, M. Voloch, and P.Bienkowski. 1982. Production of ethanol and

chemicals for cellulosic materials. Process

Biochemistry Sep/Oct : 34-38.

Received date : 16/04/99Accepted date : 8/11/99

Kasetsart J. (Nat. Sci.) 34 : 74 - 78 (2000)

INTRODUCTION

The high toxicity of cyanide to life is welldefined. Seventy mg KCN has a 50% chance of

killing a 70 kg person within 15 minutes (Lygre,

1994). Standard regulation for the maximumcontaminant level (MCL) of cyanide in wastewater,

set by the Ministry of Science Technology and

Environment, is 0.2 mg/l as HCN, i.e., 0.182 mg/las CN-. Cyanide concentrations up to 30 mg/l have

been successfully treated in biological process

(Eckenfelder, 1966). Cyanide concentration was

Cyanide Removal from Laboratory Wastewater UsingSodium Hypochlorite and Calcium Hypochlorite

Nusara Sinbuathong1, Bussarin Kongseri2, Panadda Plungklang3

and Roj Khun-anake2

ABSTRACT

Removal of cyanide (CN-) from laboratory wastewater using sodium hypochlorite (NaOCl) andcalcium hypochlorite (Ca(OCl)2) were performed at the reaction time of 30 minutes. The product of

chlorination at an alkaline pH of 12.3 was CNO - which could be oxidized further to N2. Colorimetric

method was used to determine the amount of CN- before and after chemical treatments. The optimum dosesof chemicals used were determined. It was found that 100% removal of this contaminant could be achieved.

The optimum doses and chemical costs of NaOCl and Ca(OCl)2 varied depending on the initial cyanide

concentration. The optimum doses of NaOCl and Ca(OCl) 2 for 100% CN- removal were Y = 17.3X andY = 3.32 X, respectively (where X = initial CN- concentration in mg/l, and Y = chemical dose in mg/l). The

chemical costs of NaOCl and Ca(OCl) 2 were Y = 0.69 X and Y = 0.50X, respectively (where X = initial

CN- concentration in mg/l, and Y = cost, baht/m3 of wastewater). Ca(OCl)2 is more effective than NaOClconsidering the cost and dosage used.

Key words: cyanide removal , laboratory wastewater, chlorination

1 Scientific Equipment Centre, Kasetsart University Research and Deveolpment Institute (KURDI), Kasetsart University, Bangkok

10900, Thailand.2 Department of Environmental Science, Thammasart University, Bangkok 12121, Thailand.3 Water Quality Control Division, Provincial Water Works Authority, Bangkok 10210, Thailand.

reduced from 75,000 mg/l to 0.2 mg/l after 18 daysof treatment using electrolytic oxidation under

optimum operating condition (Easton, 1967).

Electrolytic oxidation is economical and efficientfor use with concentrated waste. However, treating

dilute solution has some restriction because low

conductivity results in poor current efficiency (Larryet al., 1982). Alkali chlorination is another technique

used for cyanide treatment. The first reaction product

is cyanogen chloride(CNCl), a highly toxic gas. Atan alkali pH, CNCl hydrolyzes to cyanate ion

(CNO-), which has low toxicity. The CNO- can be

oxidized further by chlorine at a nearly neutral pHto CO2 and N2 (APHA and AWWA, 1992).

Cyanide generated from most laboratory

wastewater is usually in CN- form as used incalibration curve for determination of cyanide.

These standards must be freshly prepared because

they gradually lose strength. Moreover, thedissolved KCN salt used as standards are in alkali

solutions. Thus, pH of standard solutions are above

12 which is an optimum condition for cyanideremoval. In the case of concentration and volume

of cyanide from laboratory wastewater is not high,

chlorination may be the appropriate technique touse. In this study, chlorine compounds were used

instead of chlorine gas (Cl2). The method was

focused on the chemical optimum doses and costsof chlorine compounds for removal at various

cyanide concentrations.

MATERIALS AND METHODS

The wastewater with CN- concentrationsranging from 1-100 mg/l were prepared in NaOH

solutions. The pH of these solutions were already at

12.3 which needed not be adjusted further. Differentamount of NaOCl or Ca(OCl)2 was added to each

cyanide concentration. The reaction time was set at

30 minutes. The residual CN- left at any dose wasdetermined by colorimetric method at maximum

absorption of 587 nm (APHA and AWWA, 1992).

The graphs were plotted to show the relationship ofresidual cyanide and the amount of NaOCl and

Ca(OCl)2 added to determine the optimum doses

for each initial CN-concentration. The chemicalcosts were calculated from the optimum doses.

After that, the relationships showing the different

initial cyanide concentrations and the optimumdoses of chemical for CN- removal and costs were

expressed in the form of graphs and equations. The

calculated doses were also tested with the laboratorywastewater containing high CN- concentration.

RESULTS AND DISCUSSION

The optimum doses of chemical were

defined as the amount of NaOCl or Ca(OCl)2 usedto achieve MCL and 100% removal. At 10 mg/l

cyanide removal, the optimum doses of NaOCl

were 86.8 mg/l and 90 mg/l to get MCL and 100 %CN-removal, respectively (Figure 1). At this initial

cyanide concentration, the optimum doses of

Ca(OCl)2 were 20 mg/l and 22 mg/l to achieveMCL and 100% CN- removal, respectively (Figure

2). The optimum doses of chemical varied

depending on the initial cyanide concentrations.These results were determined and summarized in

Table1 and 2 and the graphs were plotted

accordingly as shown in Figure 3 and 4.Chemicals used in CN- removal varied

depending on the initial cyanide concentrations of

0-100 mg/l (Figure 3 and 4). In case of usingNaOCl, the relationships were:-

Y = 17.3 X (for 100%CN- removal) (1)

Y = 15.2 X (for MCL) (2)

Figure 1 The optimum doses of NaOCl for 10

mg/l initial CN- removal.

0.182

2

4

6

8

10

50 86.8 150

90

Residual CN- (mg/l)

NaOCl dose (mg/l)

0

Kasetsart J. (Nat. Sci.) 34 (1) 75

76 Kasetsart J. (Nat. Sci.) 34 (1)

Figure 2 The optimum doses of Ca(OCl)2 for 10

mg/l initial CN- removal.

22

0.00

4.00

8.00

12.00

0 10 3020 40

Residual CN- (mg/l)

Ca(OCl)2 dose(mg/l)

0.1820

500

Y = 15.2 X

Y = 17.3 X

Initial CN- conc (mg/l)

0 10 40 60 80 100

2000

1500

1000

(for 100% removal)

(for MCL)

NaOCl (mg/l)

Figure 3 The optimum doses of NaOCl for cya-nide removal at various initial concen-

trations.

As for Ca(OCl)2 , the relationships were:-

Y = 3.32 X (for 100% CN-removal) (3)Y = 2.93 X (for MCL) (4)

where

X = initial CN- concentration (mg/l)Y = chemical dose (mg/l)

In chlorination using chlorine gas, 2.7mg/l of Cl2 was required for 1 mg/l of CN- (Larry

et al., 1982). Compare to this study, 17.3 mg/l of

NaOCl or 3.32 mg/l of Ca(OCl)2 was required for1 mg/l of CN-.

The 0.67 mg/l CN- laboratory wastewater

Table 1 The optimum doses of NaOCl for cyanide

removal at various initial concentrations.

Initial CN- NaOCl dose (mg/l) forconcentration MCL 100%

(mg/l) removal

0.20 n.a. 5

1.00 8.7 133.75 6.3 20

5.90 25.8 30

10.00 86.8 9050.00 823.1 850

100.00 1502.6 1750

n.a. = not analyzed

Table 2 The optimum doses of Ca(OCl)2 forcyanide removal at various initial

concentrations.

Initial CN- Ca(OCl)2 dose (mg/l) for

concentration MCL 100 %

(mg/l) removal

0.20 n.a. 2.21.00 2.68 3.0

3.75 9.13 15.0

5.90 13.85 30.010.00 20.00 22.0

50.00 126.49 130.0

100.00 303.85 350.0

n.a. = not analyzed

Kasetsart J. (Nat. Sci.) 34 (1) 77

was treated with NaOCl and Ca(OCl)2 at the doses

obtained from equations (1) and (3), respectively.The experiments were also done with the 88.0

mg/l CN- laboratory wastewater. There was no

cyanide detected after treatment.Moreover, test was also done on high CN-

concentration of laboratory wastewater (270 mg/l)

using Ca(OCl)2 at the dose calculated from equation(3). It was found that 98.89 % of CN- was removed.

The remaining cyanide of 2.99 mg/l was gradually

removed and meet the standard regulation of 0.182mg/l as CN- (MCL) after 47 hours .

Considering the oxidation reaction of

chlorine compound which has been changing fromCN- to CNO-, hypochlorite ion (OCl-) is the active

chlorine group in the oxidation process. The

Ca(OCl)2 has 2 groups of OCl- presenting moreeffective in oxidation than NaOCl. Therefore, the

lower dosage of Ca(OCl)2 was used in cyanide

removal.The chemical cost was calculated from the

optimum dose of NaOCl and Ca(OCl)2 (commercial

grade NaOCl costs 40 baht/kg and Ca(OCl)2 costs150 baht/kg). The chemical costs depended on the

initial cyanide concentrations were summarized in

Table 3.For 100% CN- removal, the relationships

were:-

Y = 0.69 X (for NaOCl) (5)Y = 0.50 X (for Ca(OCl)2) (6)

where

X = initial CN- concentration (mg/l)Y = chemical cost (baht/m3)

0.08 baht was required to remove 1 cubicmetre of 1 mg/l CN- wastewater using chlorine gas

( Cl2 costs 30 baht/kg) whereas 0.69 , 0.50 baht was

required to remove 1 cubic metre of 1 mg/l CN-

using NaOCl and Ca(OCl)2, respectively.

Although the costs of using chlorine

compounds are higher than using chlorine gas inremoving the same amount of CN- and wastewater

volume, but chlorine compounds are recommended

as the practical alternative for CN- removal fromlaboratory wastewater which contains less volume

than industrial wastewater. The chlorine compounds

are readily available and not dangerous to use whilethe handling process is much more convenient

Figure 4 The optimum doses of Ca(OCl)2 for

cyanide removal at various initial con-

centrations.

0

50

100

150

200

250

300

350

400

0 100 12020 40 60 80

Initial CN- conc. (mg/l)

Ca(OCl)2 (mg /l)

Y = 2.93 X (for MCL)

Y = 3.32 X(for 100% removal)

Table 3 Chemical costs for 100% CN-removal at

various initial cyanide concentrations.

Initial CN- NaOCl cost Ca(OCl)2cost

conc.(mg/l) (baht/m3) (baht/m3)

0.20 0.2 0.33

1.00 0.5 0.45

3.75 0.8 2.255.90 1.2 4.50

10.00 3.6 3.30

50.00 34.0 19.50100.00 70.0 52.50

78 Kasetsart J. (Nat. Sci.) 34 (1)

ACKNOWLEDGEMENTS

We would like to express our sincere thanks

to KURDI for grant support and also to Dr. AmaraThongpan for reviewing this manuscript.

LITERATURE CITED

Lygre, D.G. 1994. General, Organic and Biological

Chemistry. Brooks/Cole Publishing Company,California. 670 p.

Eckenfelder, W.W. 1966. Industrial Water

Pollution Control. McGraw-Hill Book Company,New York . 400 p.

Easton, J.K. 1967. Electrolytic Decomposition of

Concentrated Cyanide Plating Wastes, JournalWater Pollution Control Federation. 39:1621.

Larry, D. B. , F. Joseph , Judkins, Jr. and L.W.

Barron. 1982. Process Chemistry for Waterand Wastewater Treatment. Prentice-Hall

INC., Englewood Cliffs, New Jersey. 510p.

American Public Health Association (APHA) andAmerican Water Work Association (AWWA).

1992. Standard Method for the Examination

of Water and Wastewater, 19 th ed. VictorGraphics INC. , Maryland . 1268 p.

Received date : 15/10/99Accepted date : 29/12/99

Figure 5 The chemical costs for 100% CN- re-

moval at various initial cyanide concen-

trations.

0

20

40

60

80

0 100 12020 40 60 80Initial CN- conc. (mg/l)

Chemical cost ( baht / m3)

NaOCl

Y = 0.69 X

Y = 0.50 X

Ca(OCl)2

compared to chlorine gas. Using equations (1),

(3), (5) and (6), to remove an equal amount of

CN-, Ca(OCl)2 was 5.2 and 1.4 times more effectivethan NaOCl considering the dosage and cost.

CONCLUSION

The removal of CN- from laboratory

wastewater using either NaOCl or Ca(OCl)2 couldbe achieved. The optimum dose and cost of each

chemical have been reported. Ca(OCl)2 was more

effective than NaOCl considering the cost anddosage. We, therefore, have an alternative of using

these 2 chemicals instead of chlorine gas.

Kasetsart J. (Nat. Sci.) 34 : 79 - 84 (2000)

INTRODUCTION

Tissue culture medium is always

supplemented with serum to provide the cell with

necessary growth factors and trace mineral. Thecomposition of serum is complex and ill-defined.

More importantly, serum is the most expensive raw

material in cell culture medium. In addition, serumproteins contaminate the product and subsequently

increase purification costs. Therefore, it is desirable

to reduce the amount of serum or other growthfactors used in the production of the desired product

while maximizing volumetric productivity.

Reduction of the percentage of NCS in theculture medium is an important aim in animal cell

The Influence of Serum Concentrationon tPA Production of CHO Cell

Teerapatr Srinorakutara1 , Jin-Ho Jang2 , Mutsumi Takagi2 and Toshiomi Yoshida2

ABSTRACT

The effect of initial serum concentration on cell growth, and the production of tissue plasminogen

activator (tPA) is investigated across batch fermentation of a Chinese hamster ovary (CHO) growing in

newborn calf serum (NCS) medium. It was found that initial serum concentration influenced on CHO cellgrowth rate and cell yield. At high-serum content, the uptake rate of glucose decreased although the specific

growth rate was higher, indicating a serum component becoming growth limiting. An average glucose

uptake rate in 10% (v/v) NCS medium was 13.84 mg/106 cells/h as in 1% (v/v) NCS medium was 18.17mg/106 cells/h. It was also found that the tPA or the maximum tPA productions increased with initial serum

concentration. At 7.12% NCS, the maximum tPA production gave the highest value (5.55 mg/l). The effect

of initial serum concentration on the main product was similar to that of major by-product (e.g. lactate andammonia).

Key words: serum medium, tPA, CHO cell, animal fermentation

culture, because of improved economy, easierpurification of products (e.g. tPA) and a better

defined production process (van der Pol et al.,

1990).While these media are highly optimized, a

lower cost alternative is desirable for large-scale

protein production, which is also serum-free andsupports reasonably good cell growth and

recombinant protein production. Also, when

specific formulas are desired, it is useful to knowwhat the necessary components are for designing a

simple yet effective medium without a painstaking

optimization process. Other investigators havesought to replace the serum to reduce cost, reduce

lot-to-lot variability in serum, reduce potential risk

1 Thailand Institute of Scientific and Technological Research, 196 Phahonyothin Road, Chatuchak, Bangkok 10900, Thailand.2 ICBiotech, Osaka University, 2-1, Yamada-oka, Suita, Osaka 565, Japan.

80 Kasetsart J. (Nat. Sci.) 34 (1)

of contamination by mycoplasma, and reduce theprotein load in the supernatant to facilitate

downstream processing of recombinant proteins

(Donaldson and Shuler, 1998).In this study, the effect of initial serum

concentration on growth rate, maximum cell yield

and tPA production rate on the production of tPAwas studied on CHO cultured in spinner bottles.

MATERIALS AND METHODS

Cell Line, medium and cultivation methodsCHO ATCC CRL-9606, a Chinese hamster

ovary (CHO) cells producing tPA used in this studywas kindly provided by International Center of

Biotechnology (ICBiotech), Osaka University,

Japan. The original culture medium consisted of10.64 g/l Ham’s F-12 (ICN Biomedicals Inc., Ohio,

USA) supplement with 10% (v/v) NCS (GIBGO

Laboratories Life Technologies Inc., USA), 500nM methotrexate (MTX, Sigma), 1.18 g/l NaHCO3(Wako Pure Chemical Industries Ltd., Japan), and

200 µl of 1,000 units penicillin G or 1 mgstreptomycin (Sigma). The inoculumn was prepared

from stock culture of cells scaled to 10 ml of 10%

(v/v) NCS medium in 55 cm2 petridishes. Thepetridishes were then incubated in CO2 SANYO

MCO-345 incubator (SANYO, Japan) at 37oC, 5%

CO2 concentration for 3 days. Cells in petridishesused as seed were fully-grown to 8.8 × 105 viable

cells/ml before introduction to the four 100-ml

spinner bottles (HARIO Co., Ltd., Tokyo, Japan).Four 100-ml spinner bottles with working volume

of 30 ml of serum free medium contained the NCS

at concentration of 1, 2, 5, and 10%(v/v) respectivelyand were then put into a CuSO4 solution bath with

magnetic stirrer SW-600S (NISSIN Scientific

Corp., Japan). The CuSO4 solution bath wascontrolled at 37°C and 70 rpm. In order to control

pH, the CuSO4 solution bath was surface-aerated

with humidified air containing 5% CO2 through a

0.2 mm membrane airfilter (Millipore). CO2concentration was daily checked using “FYRITE”

Bacharach (Bacharach Inc., Pittsburgh, USA). The

initial cell concentration in spinner bottles wasapproximately 2 × 105 cells/ml.

Analytical MethodsSample consisting of 750 µl suspended cells

was taken daily, and was divided into 2 parts. The

1st part, 250 µl suspended cells, was used for

determination of cell concentration and viabilityby tryphan blue exclusion on a hemacytometer.

The 2nd part, 500 µl suspended cells, was centrifuged

using TOMY high-speed refrigeratedmicrocentrifuge MX-150 (TOMY TECH USA Inc.)

at 4°C, 1000 rpm for 4 min. The supernatant was

then used for determination of glucose and lactateconcentrations using biochemistry analyzer YSI

2700-D SELECT (YSI Incorporated, Yellow

Springs Instrument Co., Ltd., Yellow Springs,OHIO 45384-0279, USA). The remaining

supernatant was aliquoted and stored at –20°C for

later analysis of ammonia, glutamine, and tPAcontents.

An enzymatic method was used to determine

ammonium concentration using a NH3 kit (WakoPure Chemical Industries Ltd., Japan).

Glutamine and glutamate concentrations

were determined using biosensor BF-4 (OjiScientific Instruments, Hyogo, Japan).

The Biopool Imulyse tPA (Biopool

international, CA, USA), an immunoassay (ELISA)for the quantitative determination of single-chain

and two-chain tPA antigen in human plasma or in

other biological fluids, such as cell culturesupernatants, was used to determine tPA

concentration.

Determination of specific growth rate and

metabolic quotientsThe specific growth rate, µ, assuming

Kasetsart J. (Nat. Sci.) 34 (1) 81

negligible cell lysis, was calculated from datacollected during the exponential growth phase and

is defined as follows:

µ = 1

x

dx

dtv

t ..................................... (1)

Where xv denotes the concentration of viablecells and t denotes the cultivation time. The specific

metabolic quotient calculations for glucose

consumption and lactate formation (qGlu and qLac,respectively) were also based on data collected

during the exponential phase of growth. They are

defined as follows:

–qGlu = 1

x

d Glu

dtv

[ ] ............................ (2)

qLac = 1

x

d Lac

dtv

[ ] ............................ (3)

where [Glu] and [Lac] are glucose and lactateconcentrations respectively.

Glutamine spontaneously decomposes

following first-order kinetics to pyrroilidonecarboxylate and ammonia (Tritsch and Moore,

1962). The specific glutamine composition and

ammonia production rates (qGlu and qNH4+,respectively) were determined by accounting for

the degradation of glutamine at 37°C (Glacken et

al., 1988; Ozturk and Palsson, 1990).

− [ ]d G In

dt= k[G In] + qG InXv ............. (4)

d NH4+[ ]

dt= k G In q X

NH v [ ] + +4

....... (5)

where [Gln] is the glutamine concentration (mM ormg); [NH4

+] is the ammonia ion concentration

(mM or mg); k is the first-order rate constant for

glutamine decomposition (h-1). Since the first-order decomposition rate varied with serum and

medium components (Miller et al., 1988; Ozturk

and Palsson, 1990) the values of k were measured

experimentally.The yield coefficients of glucose consumed

to lactate produced and of glutamine consumed to

ammonia produced (YLac/Glu and YNH4+

/Gln,respectively) are defined as follows:

YLac / Glu = q

qLac

Glu

........................................ (6)

YNH4

+ =q

q

NH

G In

4+

...................................... (7)

The specific tPA productivity, qtPA was

based on the data obtained from each batch cycle:

qtPA =tPA tPA

x dtvtc

[ ] − [ ]

∫0

0

........................ (8)

where [tPA] and [tPA]0 denote the concentration oftPA and the initial concentration of tPA for each

batch cycle, respectively.

RESULTS AND DISCUSSION

Culture Time (h)

0 20 40 60 80 100 120 140 160 180

Viable Cell concentration (105 cells/ml)

2

3

4

5

6

789

1

10

Culture Time v 1% NCS Culture Time v 2% NCS Culture Time v 5% NCS Culture Time v 10% NCS

Figure 1 The growth curves for CHO ATCC

CRL-9606 cultivated in four spinner

bottles containing 1, 2, 5, and 10% (v/v)initial NCS concentrations.

82 Kasetsart J. (Nat. Sci.) 34 (1)

The growth curve for each spinner bottle ispresented in Figure 1. Clearly, the concentration of

serum component(s) plays an important role in

both CHO cell growth rate and cell yield. Only aslight reduction in viable cell number occurred

when the serum content was reduced from 10 to

2%. The cell concentration rapidly decreased,however, from 2% to 1% FCS, indicating a serum

component becoming growth limiting. The

maximum cell yields were due to initial serumlevel, indicating stoichiometric as well as kinetic

limitation by serum component (s) (Dalili and

Ollis, 1989).Glucose and glutamine, major carbon and

energy sources in most cell culture media, required

for cell growth, were measured during thecultivation. The major byproducts, lactate and

ammonia, were also measured. The glucose and

lactate concentrations during the cultivation areshown in Figure 2. The glucose levels decreased

markedly during the exponential growth, and

glucose utilization was accompanied by a

Figure 2 Glucose consumption (dotted lines) and

lactate production (solid lines) of CHOATCC CRL-9606 cultivated in four spin-

ners bottles containing 1, 2, 5, and 10%

(v/v) initial NCS concentrations.

Figure 4 tPA production of CHO ATCC CRL-9606 cultivated in four spinner bottles

containing 1, 2, 5, and 10% (v/v) initial

NCS concentrations.

Figure 3 Glucose uptake rate of CHO ATCC

CRL-9606 cultivated in four spinner

bottles containing 1, 2, 5, and 10% (v/v)initial NCS concentrations.

Culture Time (h)

0 20 40 60 80 100 120 140 160 180

Lactate concentration (mg/L)

0

200

400

600

800

1000

1200

Glucose concentration (mg/l)

200

400

600

800

1000

1200

1400

1600

1800

2000

1% NCS

2% NCS

5% NCS

10% NCS

Culture Time (h)

0 20 40 60 80 100 120 140 160 180

Glucose consumption rate (mg/106 cells/h)

0

10

20

30

40

50

1% NCS 2% NCS 5% NCS 10% NCS regression lines

Culture Time (h)

0 20 40 60 80 100 120 140 160 180

tPA production (mg/L)

0

1

2

3

4

5

6

Culture Time v 1% NCS Culture Time v 2% NCS Culture Time v 5% NCS Culture Time v 10% NCS

Kasetsart J. (Nat. Sci.) 34 (1) 83

corresponding accumulation of lactate. Glucosewas not a limiting nutrient for cell growth in all

culture conditions tested.

With high-serum medium, the uptake rateof glucose decreased (Figure 3), although the

specific growth rate was higher (Fig. 1). This result

indicated that the cells utilize glucose moreefficiently in high-serum medium. An average

glucose uptake rate of the cells in 10% (v/v) NCS

medium was 13.84 mg/106 cells/h, while the averageglucose uptake rate increased to 18.17 mg/106

cells/h in 1% (v/v) NCS medium.

Although tPA production (Figure 4) ormaximum tPA production (Figure 5) increased

with initial serum concentration and reached the

highest maximum tPA production (5.5498 mg/l) at7.12 % (v/v) NCS, at higher serum content some

factor(s) might become inhibitory for tPAproduction. The effect of initial serum concentration

on the main product (tPA) was similar to that of the

major by-products (e.g. lactate and ammonia).Concentration of tPA and lactate gave the highest

values at 5% (v/v) NCS (Figure 4) as the highest

ammonia production was at 10% (v/v) NCS(Figure 6). Concentration of serum at 5% (v/v)

NCS may be a more suitable value for producing

the tPA and lactate.Next to sugar, glutamine is the most

abundant constituent of tissue culture media (Eagle,

1955). It has been long established that themetabolism of glutamine can provide significant

quantities of energy in mammalian cells (Reitzer et

al., 1979). This cellular energy is produced via totalor partial oxidation of glutamine. The complete

oxidation of glutamine to CO2 occurs via the TCA

cycle, whereas its partial oxidation is accomplishedby a linear pathway, which involves several

intermediates of the TCA cycle. The end product of

Figure 6 Ammonia (NH3) production of CHO

ATCC CRL-9606 cultivated in four spin-

ner bottles containing 1, 2, 5, and 10%(v/v) initial NCS concentrations.

Figure 5 Relationship between initial serum con-

centrations and maximum tPA concen-

trations for cultivation of CHO ATCCCRL-9606 grown in spinner bottles. The

line shown is regression of initial serum

concentration against maximum tPAconcentration, with a regression coeffi-

cient of r2 = 0.98.

Initial serum concentration (% v/v)

0 2 4 6 8 10 12

Maximum

tPA concentration (mg/l)

2

3

4

5

6

7

regression coefficient (r2) = 0.98

Culture Time (h)

0 20 40 60 80 100 120 140 160 180

NH

3 concentration (mg/L)

0

5

10

15

20

25

30

Culture Time v 1% NCS Culture Time v 2% NCS Culture Time v 5% NCS Culture Time v 10% NCS

84 Kasetsart J. (Nat. Sci.) 34 (1)

this pathway is pyruvate and /or lactate. By analogyto naming the oxidation of glucose to lactate as

glycolysis, McKeehan (1982) has coined the term

glutaminolysis for the partial oxidation of glutamine.Data for concentration of glutamine and glutamate

in spinner bottles containing 1, 2, 5, and 10% (v/v)

NCS determined as a function of time were notshown in this report because some data was lost.

However, from the remaining data it showed that

the concentration of glutamine decreased with time.Unfortunately, amino acid was not analyzed

in this study because of limitation of research time.

Therefore, no amino acids data lead to lacking ofinformation concerning available effect of amino

acid components on such as tPA production and so

on.

ACKNOWLEDGEMENT

The authors wish to thank the UNESCO, for

financial support throughout this work. The authors

express their sincere thanks to the technical staff ofthe international center of biotechnology

(ICBiotech) for providing the facilities and technical

assistance throughout this work. Finally, the authorswould like to thank Ms Toshiko Nagai and Ms

Kumiko Gojo for financial arrangement.

LITERATURE CITED

Dalili, M. and D.F. Ollis. 1989. Transient kinetic ofhybridoma growth and monoclonal antibody

production in serum-limited cultures.

Biotechnology and Bioengineering 33 : 984-990.

Donaldson, M.S. and M. L. Shuler. 1998. Low-cost

serum medium for the BTI-Tn5B1-4 insectcell line. Biotechnology Progress 14 : 573-

579.

Eagle, H. 1955. Nutritional needs of mammaliancells in tissue culture. J. Biol. Chem. 214 : 839.

Glacken, M.W., R.J. Fleischaker, and A.J. Sinskey.

1988. Reduction of waste product excretionvia nutrient control: possible strategies for

maximizing product and cell yields on serum

in cultures for mammalian cells. Biotechnologyand Bioengineering 28 : 1376-1389.

Lee, G.M., A. Varma, and B.O. Palsson. 1991.

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells:

effect of serum. Biotechnology and

Bioengineering 38 : 821-830.McKeehan, W.L. 1982. Glutaminolysis in animal

cells. Cell Biol. Int. Rep. 6 : 635.

Miller, W.M., C.R. Wilke, and H.W. Blanch. 1988.Transient responses of hybridoma metabolism

to changes in oxygen supply rate in continuous

culture. Bioprocess Eng. 3 : 103-111.Ozturk, S.S. and B.O. Palsson. 1990. Chemical

decomposition of glutamine in cell culture

media type, pH, and serum concentration.Biotechnol. Prog. 6 : 121-128.

Reitzer, L.J., B.M. Wice, and D. Kennel. 1979.

Evidence that glutamine, not sugar, is themajor energy source for culture HeLa cells. J.

Biol. Chem. 254 : 2669-2674.

Tritsch, G.L. and G.E. Moore. 1962. Spontaneousdecomposition of glutamine in cell culture

media. Exp. Cell Res. 28 : 360-364.

van der Pol, L., G. Zijlstra, M. Thalen, and J.Tramper. 1990. Effect of serum concentration

on production of monoclonal antibodies and

shear sensitivity of a hybridoma. BioprocessEngineering 5 : 241-245.

Received date : 12/05/99Accepted date : 8/11/99

Kasetsart J. (Nat. Sci.) 34 : 85 - 90 (2000)

INTRODUCTION

A number of glycoconjugates are present inanimal tissue, particularly on the secretory granules

and cell surface. These molecules contain

carbohydrate chains with a specific sequence. Inthe submandibular gland of different mammals,

numerous histochemical studies have been made

on glycoconjugates elaborated by their secretorycells (Shackleford and Wilborn 1968; Laden et al.,

1984). However, little information is available as

to the detail histochemical properties ofglycoconjugates in the comparable mandibular

gland of chicken. Recently, various kinds of lectins

were employed to detect sugar residues inglycoconjugates (Goldstein and Hayes, 1978; Roth

1978; Schulte et al., 1984). In view of the

Lectin Histochemistry of Glycoconjugatesin Mandibular Gland of Chicken

Apinun Suprasert, Surapong Arthitvong and Seri Koonjaenak

ABSTRACT

The distribution of glycoconjugates in the chicken mandibular gland was studied by means of lightmicroscopic histochemical methods. The staining procedures employed were horseradish peroxidase

conjugated lectin, Alcian blue pH 2.5-periodic acid-Schiff (AB pH 2.5-PAS), and high iron diamine-alcian

blue pH 2.5 (HID-AB pH 2.5). The lectins used in the present study were Concanavalin A (Con A), Ricinus

communis agglutinin-I (RCA-I), wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ulex

europeues agglutinin - I (UEA-I), Limax flavus agglutinin (LFA), Lotus tetragonolobus agglutinin (LTA)

and peanut agglutinin (PNA). According to the results obtained, acidic and neutral glycoconjugates withα-D-mannose, α- D-glucase, β-D-galactose, N-acetyl-D-glucasamine, α-L-fucose and sialic acid residues

were visualized in the secretory cells of chicken mandibular gland.

Key words : lectin, glycoconjugates, mandibular gland, chicken

circumstance mentioned above, attempts were made

to analyze glycoconjugates involved in the

mandibular gland of chicken, employing thecurrently available light microscopic methods of

peroxidase - conjugated lectins.

MATERIALS AND METHODS

Mandibular glands from both male andfemale adult Brown Leghorn chicken were fixed

by immersion with 10% formalin containing 2%

calcium acetate for 12 h at 4°C. or in Carnoy’s fluidfor 6 hours at room temperature. The tissue

specimens were then processed to embed in

paraplast. Sections were made at 3 µm thick andstained with the following histological and

histochemical procedures. Hematoxylin and Eosin

Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.

86 Kasetsart J. (Nat. Sci.) 34 (1)

(H&E), alcian blue pH 2.5-periodic acid-Schiff(AB pH 2.5-PAS), and high iron diamine alcian

blue pH 2.5 (HID-AB pH 2.5). To access the

saccharide residues further, the peroxidase-conjugated lectin-diaminobenzidine procedure was

performed to the paraplast sections. Following

lectins were employed : Concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I), wheat

germ agglutinin (WGA), Dolichos biflorus

agglutinin (DBA), Ulex europeus agglutinin-I(UEA-I), Limax flavus agglutinin (LFA), Lotus

tetragonolobus agglutinin ( LTA) and peanut

agglutinin (PNA). All these lectin preparationsconjugated with peroxidase were purchased from

E.Y. Laboratory (San Mateo, California, U.S.A.).

To detect sialic acid residues, sections were treatedwith neuraminidase (from Arthrobacter ureafaciens

Marukinshoyu Co. Ltd., Japan) 1 unit/ml in acetate

buffer pH 5.3 containing CaCl2 at 39-41°C for 12-

16 hours prior to staining with LFA or PNA, or ABpH 2.5-PAS.

RESULTS AND DISCUSSION

The secretory epithelium of the chicken

mandibular gland was found to consist exclusivelyof mucous cells (Figure 1). Which agreed with the

previous report (Suprasert et al., 1986). The staining

results of the mucous cells are listed in Table 1. Inthe mucous cells, the AB pH 2.5-PAS stained cells

were reddish blue (Figure 2). However, they

changed into red with AB pH 2.5-PAS after enzymedigestion with neuraminidase. In the mucous cells

of mandibular gland, furthermore, the dual staining

with HID-AB pH 2.5 resulted in blue colorationtogether with some pin point staining of black color

around certain area (Figure 3). The binding patterns

of Con A (Figure 4), RCA-I, PNA (Figure 5), LFA

Table 1 Histochemical staining of mucous cells in mandibular gland of the chicken.

Lectins References Intensity1 Binding specificities

Con A Kiernan 1975 2 Br α-D-glucase,α-D-mannoseRCA-I Yamada and Shimizu ,1977 1-2 Br β-D-galactose

WGA Goldstein and Hayes, 1978 1 Br β-D-Glc NAC

DBA Goldstein and Hayes, 1978 O α-D-Gal NACUEA-I Goldstein and Hayes, 1978 O α-L-fucose

LFA Schulte et al., 1984 2 Br Neu AC.

LTA Goldstein and Hayes ,1978 2 Br α-L-fucosePNA Stoward et al., 1980 1-3 Br Gal β 1-3-Gal NAC

N2)-LFA Schulte et al., 1984 O Neu AC lost to stain

AB pH 2.5-PAS Spicer et al., 1967 3 BM Acidic and neutral glycoconjugateHID-AB pH 2.5 Spicer et al., 1967 2 B Bl* Sulfated and nonsulfated

glycoconjugates

N2-AB pH 2.5-PAS Spicer et al., 1967 2-3 M Neu AC lost to stain with AB pH 2.5

1) B = Blue, Bl = Black, Br = Brown, M = MagentaO = negative staining. Number indicates intensity of staining reaction.

2) Neuraminidase

* All mucous cells stain blue. However, there are some pin point staining of black coloration around certain area.

Kasetsart J. (Nat. Sci.) 34 (1) 87

Figure 1 The secretory portions of chicken mandibular gland consist of a single layer of high columnar

cells, which contain a flattened nucleus in the basal cytoplasm. The cytoplasm stain lightly witheosin. Hematoxylin and eosin. X 260.

Figure 2 In the mucous cells of chicken mandibular glands, the dual staining with AB pH 2.5-PAS result

in deep blue coloration. X 260.Figure 3 The dual staining with HID-AB pH 2.5 results in blue coloration with some pin point staining

of black color around certain areas.

Figure 4 The mucous cells exhibit moderate positive reaction. Con A. X 260Figure 5 As in Figure 4. PNA. X 260

88 Kasetsart J. (Nat. Sci.) 34 (1)

Figure 6 All mucous cells are moderately reactive. LFA. X 260Figure 7 The mucous cells are stained negatively with LFA after neuraminidase digestion. X 260

Figure 8 The mucous cells exhibit variable intensities of positive reaction. LTA. X 260

Figure 9 The mucous cells are weakly stained with WGA. X 260

Kasetsart J. (Nat. Sci.) 34 (1) 89

(Figure 6) and LTA (Figure 8), was moderately tointensely stained the stored secretion products of

mucous cells. However, they were weakly stained

with WGA (Figure 9) but negatively stained withDBA and UEA-I.

The staining patterns with PNA, LFA, and

LTA corresponded to the presence of terminalgalactose-(1-3) N-acetylgalactosamine, terminal

sialic acid and α -L-fucose residues respectively.

The negative staining with LFA (Figure 7) and thechange in color from deep blue to magenta with AB

pH 2.5-PAS of mucous granules of mucous cells

after neuraminidase digestion further confirmedthe presence of acidic glycoconjugates with terminal

sialic acid residues (Spicer et al.,1967, Schulte et

al., 1984). Furthermore, glycoconjugates with α-D-mannose, α-D-glucose and β-D-galactose were

also confirmed the previous study (Suprasert et al.,

1986) as judged from their positive staining withCon A and RCA-I. The weakly WGA and

negativiely DBA reactions are taken to be an

evidence that the mucous cells contain small amountof N-acetylglucosamine residues but they are devoid

of N-acetylgalactosamine residues.

Comparison the staining specificities of theUEA-I and LTA in the mucous cells of the chicken

mandibular gland, each having a nominal binding

specificity for α-L-fucose residues, revealed someinteresting findings. The UEA-I reactivity could be

attributed to the presence of O-glycosidically-linked

secretory glycoprotein whereas the LTA bindingwas restricted to the presence of N-linked

glycoprotein (Schulte and Spicer ,1983). The

positive LTA but negative UEA-I reactionsconfirmed the different binding affinity for the

same terminal sugar having different glycosidic

linkage.The main function of the mandibular gland,

through its salivary secretion is known to be

lubrication of food boli and prevention ofmicroorganism and chemicals in oral cavity. Sialic

acid is believed to play an essential role of thelubrication and protection in the digestive tract

(Werner et al., 1982). The physiological role of

terminal galactose and terminal fucose residuesfound in the chicken mandibular gland must await

further investigation.

LITERATURE CITED

Goldstein, I.J. and C.E. Hayes 1978. The lectins :Carbohydrate-binding proteins of plant and

animals. Adv. Carbohyd. Chem. Biochem. 35

: 127-340.Kiernan, J.A. 1975. Localization of α-D-glucosyl

and αD-glucasyl and -D mannosyl groups s.

Concanavalin A and horseradish peroxidase.Histochemistry 44 : 39-45.

Laden, S.A., B.A. Schulte, and S.S. Spicer 1984.

Histochemical evaluation of secretoryglycoproteins in human salivary glands with

lectin-horseradish peroxidase conjugates. J.

Histochem. Cytochem. 32 : 965-972.Roth, J. 1978. The lectins : Molecular probes in cell

biology and membrane research. Exp. Path.

Suppl. 3 : 186Schulte, B.A. and S.S. Spicer 1983. Light

microscopic detection of sugar residues in

glycoconjugates in salivary glands and thepancreas with lectin -horseradish peroxidase.

Histochem. J. 15 : 1217-1238.

Schulte, B.A., S.S. Spicer, and R.L. Miller 1984.Histochemical localization of sialoglyco-

conjugates with sialic acid-specific lectin from

slug Limax flavus. Histochem. J. 18 : 1125-1132.

Shackleford, J.M. and W.H. Wilborn 1968.

Structural and histochemical diversity inmammalian salivary glands. Alabama J. Med.

Sci. 5 : 180-203.

Spicer, S.S., R.G. Horn, and T.J. Leppi. 1967.Histochemistry of connective tissue

90 Kasetsart J. (Nat. Sci.) 34 (1)

mucopolysaccharides,pp.251-303. In B.M.Wagner and D.E. Smith (eds). The Connective

Tissue. Williams and Wilkins, Baltimore

Stoward, P.J., S.S. Spicer, and R.T. Miller. 1980.Histochemical reactivity of peanut lectin-

horseradish peroxidase conjugate. J.

Histochem. Cytochem. 28 : 979-990.Suprasert, A., T. Fujioka, and K. Yamada. 1986.

Glycoconjugates in the secretory epithelium

of the chicken mandibular gland. Histochem.J. 18 : 115-121.

Werner, R., K. Eckart, B. Christian, and G.

Wolfgang. 1982. Biological significance of

sialic acid, pp 263-305. In R. Schauer (ed.)Sialic acid : Chemistry, Metabolism and

Function. Cell Biology Monographs vol. 10,

Springer New York.Yamada, K. and S. Shimizu. 1977. The

histochemistry of galactose residues of

complex carbohydrates as studied byperoxidase-labeled Ricinus Communis

agglutinin. Histochem. 53 : 143-156.

Received date : 22/03/99Accepted date : 27/08/99

Kasetsart J. (Nat. Sci.) 34 : 91 - 97 (2000)

Prevalence of Antibody to Orientia tsutsugamushi inDogs Along Thai - Myanmar Border

Mongkol Chenchittikul, Decha Pangjai and Paijit Warachit

ABSTRACT

Three hundred and ninety six dog sera collected from ten provinces along Thai-Myanmar borderin 1997 were tested for antibody to Orientia tsutsugamushi by Indirect Fluorescent Antibody test (IFA).

The average percentage of positive antibody (Ab) to scrub typhus in all provinces was 39.65%. The Ab

prevalence to scrub typhus in each province ranged from 21.95 to 62.16%, northern provinces had thehighest prevalence (54.00%) whereas southern provinces had the lowest prevalence (24.39%). Geometric

mean titer (GMT) of the Ab in dog sera were 1:191 (range from 1:100 to 1:594). GMT of the Ab in Chiengrai

province was the highest titer (1:594) and Chumphon province was the lowest titer (1:100).Key words : dog, IFA, Orientia tsutsugamushi,prevalence, scrub typhus

National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nontaburi 11100, Thailand.

INTRODUCTION

Scrub typhus, caused by Orientia

tsutsugamushi (Rickettsia tsutsugamushi), is afebrile disease (Frances et al., 1997), transmitted to

man by the bite of an infected, larval mite (chigger)

of the family Trombiculidae, order Acarina.Numerous cases were reported from Kwe Noi

Valley and Phuket in 1943 (Oaks et al., 1983).

However, scrub typhus in Thai patient was firstconfirmed by serology in 1952 (Thainua, 1952).

Since then, it has been recognized as endemic in

Thailand. Since 1973, every cases had to reportedas the surveillance disease to the Division of

Epidemiology, Ministry of Public Health. Recently,

the reported cases in 1995-1997 were 1,203, 1,210,and 2,064 respectively (Division of Epidemiology,

1995; 1996; 1997).The highest prevalence of scrub

typhus was in the north and the lowest prevalencewas in the south.

Rodents have been recorded as being naturalhost of scrub typhus rickettsia (Azad and Beard,

1998). Dogs infected with O. tsutsugamushi were

found in the endemic area of Vietnam and PeninsularMalaysia (Alexander et al., 1972; Huxsoll et al.

1977). In Thailand, isolation of O. tsutsugamushi

from rodents had been reported (Traub et al., 1954).Seroprevalence of scrub typhus in human also had

been carried out, demonstrated that human was

exposed to O. tsutsugamushi (Johnson et al., 1982),but serosurveys on dogs had not been conducted.

This report presents the serosurvey of scrub typhus

in dogs along Thai-Myanmar border.

MATERIALS AND METHODS

SeraSerum samples of 396 Dogs were collected

from ten provinces in November 1997 along Thai-Myanmar border, Thailand for an epidemiological

92 Kasetsart J. (Nat. Sci.) 34 (1)

survey of plague (Figure 1). Two villages fromeach province were selected randomly for collecting

the samples. Dogs were privately owned and belong

to inhabitants of the respective study areas. Bloodsamples were collected from cephalic vein and

allowed to clot. The serum samples were maintained

at -20°C prior to testing.

Serological testThe sera were tested for antibodies specific

to the three prototype (Gilliam, Karp and Kato)

strains of O. tsutsugamushi, Rickettsia typhi

Wilmington strian and Thai tick typhus TT118

strain. An indirect fluorescent antibody test (IFA)

using fluorescein conjugated goat anti-dog IgG(Kirkegaard Perry Laboratories Inc., America)

were employed in the IFA. The IFA was performed

as described previously (Tamura et al., 1984).Serum dilution, beginning at 1:25 were test for

screening. Positive sera were further diluted two

fold serially, titer ≥1:50 was considered as positiveserodiagnosis (Huxsoll et al., 1977).

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Figure 1 Map of Thailand showing ten provinces along Thai-Myanmar border from which specimens

were collected.

Kasetsart J. (Nat. Sci.) 34 (1) 93

(Northern : Chiang Mai, Chiang Rai, Tak, MaeHong Son, Central : Phetchaburi, Ratchaburi,

Kanchanaburi, Prachuap Khiri Khan, Southern :

Chumphon, Ranong) are 56,404,166,23,1,11,2,5,1and 0 respectively (Division of Epidemiology,

1997). The sum of reported cases in three parts of

Thailand were presented in Figure 2. This figurealso showed the GMT of dog antibodies in each

part.

DISCUSSION

The prevalence of antibodies to scrub typhusin dogs along Thai-Myanmar border, Thailand

were 39.65 %. The data presenting here have

demonstrated that natural infections of scrub typhusoccur in dogs in every province along Thai-

Myanmar border, Thailand. All villages in this

study were rural areas. This correlated to the formerstudy by Johnson et al. (1982) which showed 77%

of seroprevalence in rural Thai village. And also,

O. tsutsugamushi could be isolated from patientsand rodents in rural areas (Trishnananda et al.,

1964).

The prevalence of scrub typhus antibodiesin three parts of Thailand was correlated well with

the number of reported patient cases. Northern part

was the high risk areas than central and southernparts, respectively.

The prevalence of antibodies to scrub typhus

were not only varied by province but also byvillage. The highest prevalence of antibodies was

84.21% at Muang Na village, Chiang Mai where as

Klong Loi village in Prachuap Khiri Khan showedthe lowest with a prevalence of 4.76% . For GMT,

the highest was 1:635 at Rong village, Chiang Rai,

but the lowest was 1:71 at Wang Ta Kian village,Tak.

The present study correlated well with the

previous studies. Alexander et al. (1972) showed45% of antibodies against O. tsutsugamushi in

RESULTS

Of the 396 dog serum samples, the number

of antibody positive against O. tsutsugamushi was157 (39.65%). All sera were negative for R. typhi

and TT118. The samples collected from 10

provinces ranged from 37-42 cases/province(average 39.6). The prevalence of antibodies to

scrub typhus were varied (Table 1) by provinces

(range from 21.95 to 62.16%). Chiang Mai had thehighest prevalence with 62.16% where as Prachuap

Khiri Khan and Chumphon had the lowest with

21.95%. Geometric mean titer (GMT) of the Ab indog sera were 1:191 (range from 1:100 to 1:594).

GMT of the Ab in Chiengrai province was the

highest titer (1:594) and Chumphon province wasthe lowest titer (1:100).

Table 2 showed the prevalence of antibodies

to scrub typhus in each village (range from 4.76 to84.21). The highest prevalence of antibodies was

84.21% at Muang Na village, Chiang Mai where as

Klong Loi village in Prachuap Khiri Khan showedthe lowest with a prevalence of 4.76%. For GMT,

the highest titer was 1:635 at Rong village, Chiang

Rai. The lowest titer was 1:71 at Wang Ta Kianvillage, Tak.

Table 3 showed the prevalence of antibodies

against scrub typhus rickettsia in dog in threedifferent geographical parts of Thailand. Northern

provinces were the highest, with a prevalence of

54.00% and southern provinces were the lowest,with a prevalence of 24.39%. GMT of the Ab in dog

sera in three parts were highly significant difference

(p<0.01). However, it was not different betweencentral and northern parts (p>0.05). The GMT in

northern part (1:220) was highly significant

difference (p<0.01) from southern part (1:111).But GMT in central part (1:190) was only significant

difference from southern part (p<0.05).

From the Annual Epidemiological report,in 1997 the reported cases in the studied provinces

94 Kasetsart J. (Nat. Sci.) 34 (1)

Tab

le 1

The

pre

vale

nce

of a

ntib

odie

s to

scr

ub ty

phus

in d

ogs

in 1

0 pr

ovin

ces.

Prov

ince

Num

ber

Ant

ibod

ies

tite

rG

MT

a)%

Pos

itive

exam

ined

<1:

251:

251:

501:

100

1:20

01:

400

1:80

01:

1600

1:32

00

Chi

ang

Rai

3715

10

12

77

31

594

21/3

7 (5

6.76

)

Chi

ang

Mai

3714

01

57

62

02

279

23/3

7 (6

2.16

)M

ae H

ong

Son

378

77

93

30

00

107

22/3

7 (5

9.46

)

Tak

3924

06

26

10

00

110

15/3

9 (3

8.46

)

Rat

chab

uri

4125

11

41

34

11

348

15/4

1 (3

6.59

)Ph

etch

abur

i42

282

21

62

10

018

912

/42

(28.

57)

Prac

huap

Khi

ri

K

han

4132

01

42

11

00

159

9/41

(21

.95)

Kan

chan

abur

i40

173

74

61

11

013

220

/40

(50.

00)

Chu

mph

on41

257

23

31

00

012

69/

41 (

21.9

5)

Ran

ong

4122

84

34

00

00

100

11/4

1 (2

6.83

)T

otal

396

210

2931

3640

2516

54

191

157/

396

(39.

65)

a) G

MT

, Geo

met

ric

Mea

n tit

er

Kasetsart J. (Nat. Sci.) 34 (1) 95

Table 2 The prevalence of antibodies to scrub typhus in dogs in 20 villages.

Provinces Villages No. examined No. positive (%) GMTa)

Chiang Rai Viang Hom 20 12 (60.00) 566

Rong 17 9 (52.94) 635Chiang Mai Aruno Tai 18 7 (38.89) 135

Muang Na 19 16 (84.21) 383

Mae Hong Son Mae Sa Riang 20 12 (60.00) 100Mae Ja Ton 17 10 (58.82) 115

Tak Rim Mei 19 7 (36.84) 181

Wang Ta Kian 20 8 (40.00) 71Ratchaburi Bo Moo 20 7 (35.00) 400

Tago Lang 21 8 (38.09) 308

Phetchaburi Teng Nuea 21 4 (19.05) 200Suan Yai Pattana 21 8 (38.10) 183

Prachuap Khiri Khan Dan Sing Khon 20 8 (40.00) 168

Klong Loi 21 1 (4.76) 100Kanchanaburi Tai Mhuang 20 9 (45.00) 147

Pra Chum Mai 20 11 (55.00) 121

Chumphon Ran Tad Phom 19 3 (15.79) 79Santi Nimitr 22 6 (27.27) 159

Ranong Nai Krung 20 5 (25.00) 115

Had Tun 21 6 (28.57) 89Total 396 157 (39.65) 191

a) GMT, Geometric Mean titer

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Figure 2 Number of reported cases and geometric mean titer of dogs in three studied parts.

96 Kasetsart J. (Nat. Sci.) 34 (1)

military scout and tracker dogs worked in ruralareas in Vietnam. Huxsoll et al. (1977) also showed

the seroprevalence antibodies in dog in Malaysian

rural areas.Dogs usually live in the same habitat as

human and are frequently exposed to the same

disease organisms. But their movements in thescrub areas are more frequently than man. So, they

are more risk of infection by scrub typhus rickettsia

than man. It is recommended to use dogs as sentinelsfor scrub typhus and also in determining areas of

potential risk to man.

Antibodies against R. typhi were not detectedin this study. This supported that murine typhus

was a disease of urban areas where man and rats

shared the same habitat in Southeast Asia but not inrural areas (Brown et al., 1988; Sankasuwan et al.,

1969). Okabayashi et al. (1996) also showed that

wild rats in rural areas of Thailand were not foundantibodies against R. typhi. However, it was contrast

to 38% of sero-prevalence to R. typhi in dogs in

rural areas in Malaysia and 0.4% of prevalence inthe Suez Canal area in Egypt (Huxsoll et al., 1977;

Soliman et al., 1989).

Antibodies against TT118 were also notfound in this study. This disease may be more

prevalence in the areas near the forest. Two of three

reported cases were working and living near theforest areas (Sirisanthana et al., 1994). However,

Okabayashi et al., (1996) showed a 64.71%

prevalence of antibody to Thai tick typhus (TT118)in wild rats in rural areas of Thailand.

LITERATURE CITED

Alexander A.D., L.N. Binn, B. Elisberg, P. Husted,

D.L. Huxsoll, J.D. Marshall,Jr., C.F. Needy,and A.D. White.1972. Zoonotic infections in

military scout and tracker dogs in Vietnam.

Infection and Immunity 5 : 745-749.Azad A.F. and C.B. Beard. 1998. RickettsialT

able

3T

he p

reva

lenc

e of

ant

ibod

ies

to s

crub

typh

us in

dog

s in

3 p

arts

.

Part

Num

ber

Ant

ibod

y ti

ter

%

GM

Ta)

exam

ined

<1:

251:

251:

501:

100

1:20

01:

400

1:80

01:

1600

1:32

00Po

ssiti

ve

Nor

th15

061

814

1718

179

33

81/1

50 (

54.0

0)22

0

Cen

tral

164

102

611

1315

77

21

56/1

64 (

34.1

5)19

0P

<0.

01

P<

0.05

Sout

h82

4715

66

71

00

020

/82

(24.

39)

111

Tot

al39

621

029

3136

4025

165

415

7/39

6 (3

9.65

)

a) G

MT

, G

eom

etri

c M

ean

titer

Kasetsart J. (Nat. Sci.) 34 (1) 97

pathogens and their arthropod vector. EmergingInfectious Diseases 4 : 179-186.

Brown A.E., S.R. Meek, N. Maneechai, and G.E.

Lewis. 1988. Murine typhus among Khmersliving at an evacuation site on the Thai-

Kampuchean border. Am. J. Trop. Med. Hyg.

38 : 168-171.Division of Epidemiology. 1995. Annual

Epidemiological Surveillance Report, Ministry

of Public Health, Bangkok. 310 p.Division of Epidemiology. 1996. Annual

Epidemiological Surveillance Report, Ministry

of Public Health, Bangkok. 307 p.Division of Epidemiology. 1997. Annual

Epidemiological Surveillance Report, Ministry

of Public Health, Bangkok. 304 p.Frances S.P., C. Eamsila, and D. Strickman. 1997.

Antibodies to Orientia tsutsugamush in soldiers

in northern Thailand. Southeast Asian J. Trop.Med. Public Health 28 : 666-668.

Huxsoll D.L., A. Shirai, D.M. Robinson, L.F. Yap,

and B.L. Lim. 1977. Presence of antibodies toscrub typhus and murine typhus in dogs from

Selangor, Peninsular Malaysia. Southeast

Asian J. Trop. Med. Pub. Hlth. 8 : 232-235.Johnson D.E., J.W. Crum, S. Hanchalay, and C.

Saengruchi. 1982. Sero-epidemiological

survey of Rickettsia tsutsugamushi in a ruralThai village. Trans. R. Soc. Trop. Med. Hyg.

76 : 1-3.

Oaks S.C. Jr, R.L. Ridgway, A. Shirai, and J.C.Twartz. 1983. Scrub typhus. Institute for

Medical Research Bulletin No.21, Malaysia. :

1-4.Okabayashi T., K. Tsutiya, Y. Muramatsu, H.

Ueno, and C. Morita. 1996. Serological survey

of spotted fever group rickettsia in wild rats inThailand in the 1970s. Microbiol. Immunol.

40 : 895-898.

Sankasuwan V., P. Pongpradit, P. Bodhidatta, K.Thonglongya, and P. E. Winter. 1969. Murine

typhus in Thailand. Trans. Roy. Soc. Trop.

Med. Hyg. 63 : 639-643.Sirisanthana T., V. Pinyopornpanit, V. Sirisanthana,

D. Strickman, D. J. Kelly, and G. A. Dasch.

1994. First cases of spotted fever grouprickettsiosis in Thailand. Am. J. Trop. Med.

Hyg. 50 : 682-686.

Soliman A.K., A.M. Botros, T.G. Ksiazck, H.Hoogstraal, I. Helmy, and J. C. Morrill. 1989.

Seroprevalence of Rickettsia typhi and

Rickettsia conorii infection among rodentsand dogs in Egypt. Ann. Trop. Med. Hyg. 92 :

345-349.

Tamura A., K. Takahashi, T. Tsuruhara, H.Urakami, S. Miyamura, H. Sekikawa, M.

Kenmotsu, M. Shibata, S. Abe, and H. Nezu.

1984. Isolation of Rickettsia tsutsugamushi

antigenically different from Kato, Karp and

Gilliam strains from patients. Microbiol.

Immunol. 28 : 873-882.Thainua M.A. 1952. Case report of scrub typhus. J.

Med. Assoc. Thai. 35 : 9-27.

Traub R., P.T. Johnson, M.L. Miesse, and R. E.Elbel. 1954. Isolation of Rickettsia

tsutsugamushi from rodents from Thailand.

Am. J. Trop. Med. Hyg. 3 : 356-359.

Received date : 10/09/99Accepted date : 29/12/99

Kasetsart J. (Nat. Sci.) 34 : 98 - 106 (2000)

Process for Preparing Pre-fried and FrozenSweetpotato French-fry Type Products

Suparat Reungmaneepaitoon1, Santi Tip-pyang2 and Sompoch Yai-eiam1

ABSTRACT

Three processes for producing pre-fried and frozen sweetpotato French-fry type products wereinvestigated. The best process was obtained by blanching sweetpotato strips in 0.5% of disodium acid

pyrophosphate (SAPP) solution for 1-2 min, followed by partially frying in hot oil at 176°C for 20 sec.

These sweetpotato strips were partially dehydrated with hot air oven at 176°C for 4 min, packaged in LDPEbags, and then stored at –20°C. Chemical, physical and sensory properties of a sweetpotato French-fry type

product were also determined. Analysis included measurements of yields, moisture loss during process,

moisture content, reducing sugar, oil content, vitamin C, color, texture and sensory panel scores for color,odor, flavor and texture. The best process produced a pre-fried sweetpotato product contained reducing

sugar and oil content of about 2-3% and 7-10% respectively. The pre-fried products had a good stability

in frozen storage for one year period and were acceptable in the sensory scores for color, odor, flavor andtexture.

Key words : sweetpotato, french-fry, pre-fried, frozen

1 Institute of Food Research and Product Development, Kasetsart University Bangkok 10900, Thailand.2 Department of Chemistry, Faculty of Science, Chulalongkorn University., Bangkok 10500, Thailand.

INTRODUCTION

Sweetpotatoes (Ipomoea batatas L) havebeen grown for domestic consumption in Thailand

for many years. Commercial plantations are very

few. Home and cottage-scale processing units havesupplied products to local markets on a daily basis.

These processed products such as boiled

sweetpotato in heavy syrup etc. must be consumedwithin one to two days, due to short shelf-life.

Calories contribution by sweetpotato in the diet is

quite small because people consume sweetpotatoas dessert and snackfood product.

Since 1987 the Government of Thailand has

had a National Policy Plan (1987-1991) to promote

commercial production through activities led by

the Department of Agriculture and Department of

Agriculture Extension. In this regard, food industryis required extensive research to develop an

appropriate technology for processing a product

with a longer shelf-life and to meet consumerpreferences.

One prospective product is the frozen

French-fry product. Recently, the rate of French-fries consumption from white potatoes have

increased rapidly in fast-food shops. If a similar

product prepared from sweetpotatoes are available,it might be acceptable to a larger number of

consumers and would be a value-added product

from sweetpotatoes.

The sweetpotato French-fry type productwas judged of good quality and acceptability by

consumer panels. (Walter and Hoover, 1986) Since

roots are harvested seasonally and storage costs arehigh, roots should be processed into a finished

product.

The objectives of this study were to providean improved process to produce a pre-fried and

frozen French-fry type product from sweetpotato

having a low oil content, and the moisture contentabout 30 to 32% and to evaluate chemical, physical

and sensory changes of the sweetpotato French-fry

type product from various processes.

MATERIALS AND METHODS

Raw materialsApproximately 250 kg sweetpotatoes (Kaset

cultivar) were obtained from a producer in Suphan

Buri province. The roots were kept for one week at30°C and 65% relative humidity before processing

into strips and frozen.

Processing conditionsRoots were processed into two sizes of the

French-fry type strips. The roots were washed,

hand-peeled, rinsed and sliced into so-calledshoestring strips with an average length about 9-10

cm and cross-sectional side dimension of 6 mm ×6 mm and 8 mm × 8 mm with vegetable slicer(Robot Coupe, France). Small or uneven strips

were discarded to improve product uniformity.

These sweetpotato strips were then processed intoa French-fry type following the flowchart in Figure

1. Three processes were done for producing the

frozen French-fry type product. The deep fat fryerused in this study was NSF Testing Laboratory

having a capacity of 15 kg of frying oil.

All strips were packaged in low densitypolyethylene bags and stored in a freezer at –20°C.

After one week and one year storage time, theywere thawed and analyzed for yields, weight loss,

physical and chemical properties. They were fried

in palm oil at 176oC at different periods (Table 1)for sensory panel test.

Chemical analysesThe analyses were performed on samples of

products handled and processed under various

processing treatments. All chemical analyses wereperformed in duplication. Dry matter was

determined by weighing duplicated samples before

and after drying in oven at 102 ± 3°C for 2 hr. Oilcontent of fried strips was determined by Soxhlet

Extraction with petroleum ether for 16 hr, followed

by evaporation of the solvent and weighing theresidues. Reducing sugar was determined using

Fehling’s solution described by Lane and Eynon

(AOAC. 1990). Ascorbic acid was determinedusing 2, 6-Dichloroindophenol titration method

(AOAC, 1990).

Physical measurementsYields, weight losses and size of pre-fried

sweetpotato strips were determined.Color CIELAB L* a* b* C* and h were

determined from reflectance measurement with a

ACS Spectro-sensor II (Applied Color System,inc. Princeton, NJ 08543). Four rows of strips were

placed side by side in 5 cm × 4 cm cell. Color data

were determined with daylight illuminant induplicate.

Shear measurements were made using an

Instron Universal Testing Machine. (Model 1140).Samples of 15 g pre-fried product and 2.5 g finish-

fried product were placed perpendicular to the

blades of a Kramer shear cell with a slotted bottomand kg shearing force measured. The cross-head

speed was set at 200 mm/min. The shear force for

each treatment was determined 5 times.

Kasetsart J. (Nat. Sci.) 34 (1) 99

100 Kasetsart J. (Nat. Sci.) 34 (1)

Sweetpotato

Washing

Peeling

Cutting

Soaking in 0.5% SAPPSolution for 5 min.

Process (BF1) Process (SF2) Process (SF3)

Blanching in 0.5%SAPP solutionfor 1-2 min.

1. Frying in hot oil at 176oCfor 20 sec.

1. Frying in hot oil at176oC for 20 sec.

Steaming for 1-2 min. Steaming for 1-2 min.

1. Frying in hot oil at176o C for 20 sec.

2. Frying in hot oil at 176oCfor 20 sec.

2. Frying in hot oil at176oC for 20 sec

Drying in oven at176o C for 4 min.

Drying in oven at 176oCfor 4 min.

Steaming for 1-2 min.

Freezing at ï20oC Freezing at -20 oC 3. Frying in hot oil at176oC for 20 sec

Drying in oven at176oC for 4 min

Freezing at ï20oC.

Figure 1 Process of preparing of French-fry type products from sweetpotato.

Sensory evaluationSensory evaluations were performed on all

finish-fried products. For panel evaluation, thefrozen strips were fried at 176°C for 50-150 seconds.

The finish-fried products were presented to 10

members of research staff of Institute of FoodResearch and Product Development. Panels were

served coded samples on white plate and were

asked to evaluate color, odor, flavor, texture andoverall acceptability. Sensory scores were judged

with a score range from 1 to 9 on the following

scoring system. 1 dislike extremely, 2 dislike very

much, 3 dislike moderately, 4 dislike slightly, 5neither like nor dislike, 6 like slightly, 7 like

moderately, 8 like very much, 9 like extremely.

Statistical analysesThe data collected were analyzed by the

analysis of variance (ANOVA) using the statistically

analysis system (Duncan’s Multiples range test).

Kasetsart J. (Nat. Sci.) 34 (1) 101

RESULTS AND DISCUSSION

Process for pre-fried productSweetpotato pre-fried products were

prepared from various processing treatments. The

first process (BF1) comprised only one partial

frying steps, under controlled time and temperatureconditions, the second process (SF2) had two partial

frying steps, the third process (SF3) had three

partial frying steps after steaming or blanching step(Figure 1). There were differences in products for

dry matter, oil content, reducing sugar, vitamin C,

yield, size, weight loss, shear force measurements.These data are presented in Table 1-3. The data

from this study indicated that processing treatments

affected these properties differently. The overalleffect was to reduce the weight of sweetpotato

French-fry type product from about 30% to about

47% of the weight of the original strips, and tocause a concentration of solid on the surface for

improved surface texture.

Chemical compositional analysisThe data of the composition of pre-fried

prodcuts indicated that both samples of pre-fried

products produced by steaming and frying in oil 2and 3 times were higher in reducing sugar than the

blanched sample (Table 1). Blanching process used

in this study extracted more sugar from the samples.Blanching the sweetpotato strips caused decrease

Table 1 Effect of various processing treatments on dry matter, fat, reducing sugar, vitamin C of

sweetpotato French-fry type products.

Processing Dry matter Fat Reducing sugar Vitamin C

treatments (g/100 g) (g/100 g) (g/100g) (g/100 g d.b) (mg/100 g d.b)

Fresh sweetpotato 30.34 - 6.31 20.79 63.75

Pre-fried product

BF 1-66 44.35 9.82 2.10 4.73 50.90SF 2-66 49.36 13.98 6.00 12.15 40.06

SF 3-66 59.46 18.22 5.30 8.91 30.75

BF 1-88 42.12 7.54 2.14 5.08 52.14

SF 2-88 48.89 11.08 6.10 12.47 36.32

SF 3-88 56.62 14.48 5.60 9.89 29.00

Finish-fried product Frying time (sec)

BF1-F66 72.72 26.16 90 9.15SF2-F66 75.91 24.26 60 8.30

SF3-F66 81.22 25.37 50 6.13

BF1-F88 72.47 31.34 150 10.42SF2-F88 73.78 24.88 120 8.26

SF3-F88 81.88 23.22 105 6.90

d.b - dry weight basis

102 Kasetsart J. (Nat. Sci.) 34 (1)

Table 2 Effect of various processing treatments on frying time, cross sectional size, yield and weight loss,of pre-fried sweetpotato French-fry type product.

Processing Size Yield (n = 4) Weight loss (n=4)

treatments (mm) (g/100 g w.b.) (g/100 g d.b.) (g/100 g w.b)

Pre-fried productBF1-66 5.5 × 5.5 69.26 28.81 30.73

SF2-66 4.5 × 5.5 55.94 29.21 44.06

SF3-66 4.0 × 4.0 52.73 32.99 47.27

BF1-88 7.6 × 7.6 67.44 26.38 32.56

SF2-88 7.4 × 7.2 62.27 29.77 37.73SF3-88 6.4 × 6.2 58.50 33.12 41.50

w.b. - wet weight basis, d.b - dry weight basis

Table 3 Effect of various processing treatments on color values and shear measurements of sweetpotato

French-fry type product.

Processing CIELAB SYSTEM Shear force

Treatments L* a* b* C* h (Kg.)

Pre-fried productBF 1-66 75.38 -1.45 29.86 29.89 92.79 23.37

SF 2-66 68.60 -0.87 24.65 24.67 92.02 26.87

SF 3-66 67.80 -0.13 27.80 27.80 90.27 28.16

Finish-fried productBF1-F66 62.69 1.29 28.53 28.56 87.35 23.32

SF2-F66 59.87 1.22 29.64 29.66 87.65 22.66

SF3-F66 58.57 5.43 24.86 25.44 77.67 24.32

Pre-fried productBF1 -88 70.75 -1.24 30.56 30.58 92.32 23.32

SF2 -88 65.74 -1.28 25.65 25.68 92.86 25.12

SF3 -88 63.11 -2.30 24.69 24.80 95.33 26.82

Finish-fried productBF1-F88 62.35 -0.95 24.46 24.48 92.22 20.62

SF2-F88 60.15 3.21 25.46 25.66 82.82 22.50

SF3-F88 60.18 2.84 24.85 25.01 83.49 26.25

L* is the lightness a* is the red-green color component b* is the yellow-blue color componentC* is the chroma (saturation) = (a*2 + b*2)1/2 h is the hue angle = arctan (b*/a*)

Kasetsart J. (Nat. Sci.) 34 (1) 103

in dry matter because part of sugars and otherwater-soluble materials were extracted by the hot

water. Pre-fried sweetpotato products of BF1

process had reducing sugar content of 2-3% andalso had the lowest oil content in average 7-10 %.

Vitamin C content in this product was higher

compared to those of the other products (50.90-52.14 mg/100 g dry weight basis), which indicated

high nutritious value for BF1 processing product.

Sensory evaluationThe sensory scores for sweetpotato French-

fry type product odor, flavor showed no significantdifference among types of processing treatments

(Table 5 and 6). Panelists scored the odor and

flavor of products between slightly like andmoderately like. Most panelists score the odor and

flavor of products in both sizes processed in BF1 as

like moderately, due to the lower content of flavorand reducing sugar (2-3 %). It is likely that when a

blanching or water extraction step is used, some of

the flavor components will also be extracted.Blanching was effective in lowering the reducing

sugar content of sweetpotato strips. The scores for

sweetpotato French-fry type product color showedsignificant difference (P< 0.05) among types of

process. Panelists scored the color of products

between slightly like and very much like. The meanscores of sweetpotato French-fry type product of

BF1 process was the highest in color, texture,

overall acceptability among other products as shownin Table 5, 6. Toma et al (1986) reported that darker

french fried product were produced from potatoes

of lower specific gravity because of the higherreducing sugar content. The gray discoloration was

thought to be caused by the reaction between o-

dihydroxyphenols and iron III (Hoover, 1963) anddiscoloration is nonenzymatic browning, which

results when reducing sugars condense with amino

groups (Spark, 1969). The rate of this reactionincreased at high temperatures attained in oil frying.

The discoloration could be reduced if the o-dihydroxyphenol-Fe III and sugar-amino reaction

were minimized.

The L*, a*, b*, C*, h color values arepresented in Table 3. Three partial frying steps

decreased L* value as indicating a change to a

darker product. The L*, a*, b* color values weresimilar in all pre-fried products of SF2 and SF3

processes. The color of sweetpotato pre-fried

products of BF1 process were lighter (higher L*value) than product of SF2 process and SF3 process

(Table 4). Frying decreased L* and caused an

increase in a* values as anticipated indicating achange to a darker, more red product.

Mean scores for texture and overall

acceptability also showed trend toward a maximumat BF1 process. Mean scores for texture and overall

acceptability of the best product were rated between

moderately like and very much like. The quality ofbest product maintained crispiness on the exterior,

moist mealy interior and golden yellow in color.

Product of twice partial frying process (SF2) hadcrisp slight tough exterior, dry mealy interior and

sweet taste. Product of SF3 process had very crisp

exterior, darker color, dry tough interior, sweettaste, smallest size under standard size among

other products. (Table 2).

Processing treatments affected the shearforce of strips (Table 3). Pre-fried and finish-fried

strips of the SF3 process were tougher than strips of

SF2 process due to surface hardening and moistureloss during processing so it required greater force

to shear relative to others.

Pre-fried product stability during storageOverall acceptability of sweetpotato French-

fry type products throughout the one year frozen

storage period, were rated between slightly likeand very much like in all sensory categories by the

panelists. (Table 7).

Mean scores of sensory evalution of

104 Kasetsart J. (Nat. Sci.) 34 (1)

Tab

le 4

Col

or d

iffe

renc

e of

sw

eetp

otat

o Fr

ench

-fry

type

pro

duct

s by

com

pari

ng to

BF1

pro

cess

.

Proc

essi

ngC

IEL

AB

DIF

FER

EN

CE

Exp

lana

tion

of c

olor

trea

tmen

tD

E*

DL

*D

a*D

b*D

C*

DH

*

SF2-

668.

41-6

.58

0.58

-5.2

1-5

.23

-0.3

6D

arke

r, m

ore

red,

less

sat

urat

ed th

an B

F1-6

6

SF3-

667.

97-7

.58

1.32

-2.0

6-2

.10

-1.2

7D

arke

r, m

ore

red,

less

sat

urat

ed th

an B

F1-6

6

SF2-

F66

3.03

-2.8

2-0

.10

1.11

1.11

0.15

Dar

ker,

mor

e gr

een,

mor

e sa

tura

ted

than

BF1

-F66

SF3-

F66

6.89

-4.1

34.

11-3

.67

-3.1

1-4

.55

Dar

ker,

mor

e re

d, le

ss s

atur

ated

than

BF1

-F66

SF2-

887.

01-5

.01

-0.0

5-4

.91

-4.9

00.

27D

arke

r, m

ore

gree

n, le

ss s

atur

ated

than

BF1

-88

SF3-

889.

69-7

.64

-1.0

7-5

.07

-5.7

91.

45D

arke

r, m

ore

gree

n, le

ss s

atur

ated

than

BF1

-88

SF2-

F88

4.81

-2.2

14.

151.

001.

08-4

.11

Dar

ker,

mor

e re

d, m

ore

satu

rate

d th

an B

F1-F

88

SF3-

F88

4.38

-2.1

83.

780.

390.

53-3

.77

Dar

ker,

mor

e re

d, m

ore

satu

rate

d th

an B

F1-F

88

CIE

LA

B D

IFFE

RE

NC

ED

L*

is th

e lig

htne

ss d

iffe

renc

e

Da*

is th

e re

d-gr

een

colo

r di

ffer

ence

Db*

is th

e ye

llow

-blu

e co

lor

diff

eren

ceD

E*

= [

(DL

*)2

+ (

Da*

)2 +

(D

b*)2

] 1/

2

DC

*=

[(D

a*)2

+ (

Db*

)2]1

/2

DH

*=

[(D

E*)

2 -

(DL

*)2

- (D

C*)

2 ] 1

/2

Kasetsart J. (Nat. Sci.) 34 (1) 105

Table 5 Mean sensory scores* of French-fry type product (size 6 × 6) of sweetpotatoes subjected tovarious processing treatments.

Processing Color Odor Flavor Texture Overalltreatments acceptability

BF1-F66 7.80a 7.20a 7.20a 7.40a 7.50a

SF2-F66 6.90b 7.00a 6.70a 6.60b 6.50b

SF3-F66 6.80b 7.10a 7.00a 7.00ab 6.95ab

* - Hedonic scale where 9-like extremely; 1-dislike extremely (n-10)

- Means followed by the same letter are not significantly different within each column at P<0.05 level.

Table 6 Mean sensory scores* of French-fry type product (size 8 × 8) of sweetpotato subjected to various

processing treatments.

Processing Color Odor Flavor Texture Overall

treatments acceptability

BF1-F88 8.00a 7.20a 7.40a 7.20a 7.50a

SF2-F88 6.80b 6.70a 6.80a 6.50b 6.60b

SF3-F88 6.65b 6.70a 6.70a 6.30b 6.60b

* - Hedonic scale where 9-like extremely; 1-dislike extremely (n-10)

- Means followed by the same letter are not significantly different within each column at P<0.05 level.

Table 7 Effect of different processes on mean sensory scores* of French-fry type product after one yearfrozen storage period.

Processing Color Odor Flavor Texture Overall

treatments acceptability

BF1-F66 7.40a 7.40a 7.33a 7.36a 7.50a

SF2-F66 7.13a 6.93a 7.06a 7.06a 7.10a

SF3-F66 6.83a 6.93a 6.86a 6.66a 6.86a

* - Hedonic scale where 9-like extremely; 1-dislike extremely (n-15)- Means followed by the same letter are not significantly different within each column at P<0.05 level.

sweetpotato french-fry type product were not

statistically different (P<0.05). Peroxide values ofoil extracted from the pre-fried products were below

10 mEq/kg, indicating not to be affected by the

process treatment, and the products had no rancid

taste during storage time (Table 8). A rancid tasteoften begins to be noticeable when PV values is

between 20 and 40 mEq/kg (Pearson, 1976).

106 Kasetsart J. (Nat. Sci.) 34 (1)

Table 8 Moisture, oil content, and the peroxide value (mEq/Kg) of oil extraction from sweetpotato pre-fried prodcut after one year frozen storage period.

Pre-fried product Moisture Oil Peroxide value% % (mEq/Kg Fat)

BF1-66 59.36 9.81 7.76

SF2-66 47.33 13.45 7.92

SF3-66 43.99 16.34 7.30

CONCLUSION

Improved process to produce a pre-friedsweetpotato French-fry type product having a low

oil content was to blanch sweetpotato strips in hot

water at 100°C containing 0.5% SAPP and partiallyfried in hot oil at 176°C for 1-2 min to give an oil

content 7-10%. The sweetpotato strips were frozen

and packaged for later finish frying. The quality ofproduct had a crisp, palatable outer surface, golden

yellow uniform color, a fluffy interior, enhance

flavor and relative low oil perception.

ACKNOWLEDGEMENT

The authors are grateful to International

Potato Center (CIP) for funding this work.

LITERATURE CITED

AOAC. 1990. Official Method of Analysis (15thed). The Association of Official Analytical

Chemists, Washington, D.C. 1298 p.

Hoover, M.W. 1963. Preservation of the natural

color in processed sweetpotato products. Food

Technol. 17 : 128.Pearson, D. 1976. The chemical analysis of food.

7th ed. Churchill Livingstone, Edinburg

London and New York. 575 p.Spark, A.A. 1969. Role of amino acids in

nonenzymatic browning. J. Science Food

Agric. 20 : 308.Toma, R.B., H.K. Leung, J. Augustin and W.M.

Iritani. 1986. Quality of French fried potatoes

as affected by surface freezing and specificgravity of raw potatoes. J. Food Sci. 51 (5) :

1213.

Walter, W.M. Jr. and M.W. Hoover. 1986.Preparation, evaluation and analysis of a

French-fry-type product from sweetpotatoes.

J. Food Sci. 51 (5) : 967.

Received date : 26/03/99Accepted date : 4/11/99

Kasetsart J. (Nat. Sci.) 34 : 107 - 116 (2000)

Development of a Yogurt-type Product from Saccharified Rice

Chakamas Wongkhalaung and Malai Boonyaratanakornkit

ABSTRACT

A yogurt-type product from saccharified rice was developed. Jasmine rice was saccharified withamylolytic enzymes at 55 °C. Changes of reducing sugars, sugar components and compositions during

hydrolysis were studied to obtain optimum condition and yield of glucose. Rice milk was prepared by

fortification of saccharified rice with 3 % casein, 3% soybean oil and 0.4 % calcium lactate to improve bothnutritive quality and organoleptic properties.

Mixed cultures of Lactobacillus acidophilus and L. casei subsp. rhamnosus were used for lactic

fermentation of rice milk. The effect of beta glycerophosphate, inoculum size and incubation period wereinvestigated. Improvement for a more palatable yogurt was achieved by blending rice yogurt with pectin

and strawberry preserve. Resulted rice-based yogurt with strawberry contained 3.05 % protein, 2.67 % fat,

24.4 % glucose, 1.5 % sucrose, 47 mg/100g of calcium, 0.86 % acidity as lactic, pH 3.48 and lactic bacteriacount of 7.6 × 107 CFU/g. Sensory evaluation revealed that rice-based yogurt with strawberry was well

accepted by panelists when compared with commercial strawberry yogurt. Shelflife of rice-based yogurt

stored at 4 °C was at least 20 days.Key words : rice based yogurt, rice milk, lactic acid fermentation, saccharified rice

Institute of Food Research and Product Development, Kasetsart University, Bangkok 10900, Thailand.

INTRODUCTION

Lactic acid fermentation of cereals has been

studied extensively in the past few decades. Yogurt-

like products have been produced from variouskinds of cereals such as liquefied starch (Shin,

1989); prefermented and extruded rice flour (Lee et

al., 1992); and cooked maize meal mixture (Zulu et

al., 1997). A product, socalled Risogurt, was

produced from mixture of fermented rice and soy

protein isolate (Mok et al., 1991). Method forproducing a highly concentrated lactic product

from rice with improved quality by a secondary

enzymatic treatment during fermentation wasfurther developed by Mok et al. (1993).

Development of lactic beverages from mixture of

rice and soybean were also reported by Lee et al.

(1988).

The cultures starter for lactic fermentation

of yogurt-like products from cereals and other non-dairy raw materials were often Lactobacillus,

Streptococcus and Leuconostoc spp. A mixed

culture of Streptococcus thermophilus,

Lactobacillus bulgaricus and L. plantarum were

used by Shin (1989). Lee et al. (1992) compared

different kinds of lactic acid bacteria, namely,Leuconostoc mesenteroides, L.plantarum, L. casei

and L. lactis subsp. diacetylactis in fermentation of

pre-fermented and extruded rice flour. S.

thermophilus and L. mesenteroides have been used

108 Kasetsart J. (Nat. Sci.) 34 (1)

as starter for a yogurt-like product from rice flourand soymilk mixture (Collado et al., 1994).

Tominaga and Sato (1996) reported the production

of fermented beverage from rice flour using enzymehydrolysis followed by lactic fermentation by L.

mesenteroides. Other lactic bacteria used for

developing a fermented rice product was amylolyticBifidobacterium spp. (Park et al., 1997).

This paper reported a method to develop a

new yogurt-type product by lactic fermentation ofsaccharified rice using Lactobacillus spp. as starter

cultures.

MATERIALS AND METHODS

Rice Jasmine rice (Khao Dok Mali 105)was used in this experiment. It contains 8.17 %

protein, 1.1 % fat and 79.5 % starch, of which 17.3

% are amylose (P. Tungtrakool, unpublished data).Enzyme Heat stable alpha amylase from

Bacillus licheniformis, Termamyl 120L, and

amyloglucosidase from Aspergillus niger, AMG300L, were obtained from Novo (Novo Nordisk

Co., Bagsvaerd, Denmark). According to Novo

Nordisk ’s Standard Method, one KiloNovo alphaamylase Unit (1 KNU) is the amount of enzyme

which breaks down 5.26 g starch per hour at pH 5.6,

temperature 37 °C and reaction time 7-20 min. OneNovo Amyloglucosidase Unit (AGU) is defined as

the amount of enzyme which hydrolyzes 1

micromole maltose per unit at pH 4.3, temperature25 °C and reaction time 30 min. Both enzymes are

food grade products complied with FAO/WHO

JECFA and FCC recommendations for food gradeenzymes.

Microorganisms Lactic acid bacteria

(LAB) used in this study were Lactobacillus

acidophilus (IFRPD 2013) and L. casei subsp.

rhamnosus (IFRPD 2020) from Culture Collections

of Institute of Food Research and ProductDevelopment, Kasetsart University, Thailand. The

strains were cultured and maintained on MRSmedium (de Man et al., 1960).

Analytical methods Reducing sugars were

determined by DNS method (Chaplin and Kennedy,1986). Sugar composition was analyzed by HPLC

(Sugars Analyzer I, Waters Associates, Milford,

MA, USA) using Sugar Pak column. Thetemperature of column was 90 °C with 50 mM

EDTA-deionized water at flow rate of 0.2 ml/min

and 20 µl injection volume. DifferentialRefractometer Detector, Waters 410, was used.

Titratable acidity as lactic acid was determined by

the method of Frazier et al. (1968). Proximatecomposition and calcium content were determined

by the methods of AOAC (1990). Color was

determined by Spectraflash SF 600 Plus (DataColor International, USA) and viscosity by

Brookfield Viscometer (Model LV, Brookfield

Engineering Laboratories, USA).Total viable count, yeast, mold and coliforms

were determined according to AOAC (1990). LAB

was detected by plating method on MRS Mediumsupplemented with 0.5 % CaCO3 and counting the

colonies surrounding with clear zones.

Saccharification process Rice was washedand soaked in water at 1:2.5 ratio, wt by wt, for

about 30 min. It was adjusted to pH 6.5, heated up

to 90-95 ° and 0.2 % Termamyl was added. Therice mixture was held at this temperature for 2 hr.

Saccharification process was carried out using 0.2

% of AMG at 55 °C, pH 5.0 for about 24 hr.Suspension obtained was filtered through 350-

mesh nylon (Monyl 140T) to remove coarse solids.

It was then heated at 90 °C for 30 min to sterilizeand inactivate the enzymes.

Preparation of rice milk Saccharified rices

was adjusted to 18 °Brix before blending with 3 %casein, 3 % soybean oil and 0.4 % calcium lactate.

The mixture was homogenized for 3 min at high

speed (Ultra-Turax, Janke u. Kunkel KG, Staufen,I Breisgar) and pasteurized at 90 °C for 15 min. The

Kasetsart J. (Nat. Sci.) 34 (1) 109

rice milk was instantaneously subjected to lacticacid fermentation.

Lactic acid fermentation Two strains of

Lactobacillus, L. acidophilus 2013 and L. casei

2020, were used as starter in this study. They were

propagated separately in MRS medium for 24 hr at

37 °C to get viable cell counts in the range of 108 to109 CFU/g. Beta glycerophosphate was added at

different concentrations to enhance growth of LAB.

Various inoculum sized at 2, 3 and 4 % as well asincubation periods of 18, 24 and 36 hr were

investigated.

Preparation of rice-based yogurtImprovement of texture and flavor of rice-based

yogurt obtained after lactic fermentation was made

by addition of pectin and strawberry preserve. Themixture was blended with 1 % pectin and 20 % fruit

preserve in a mixer (Bamix, Model M 133,

Switzerland). Strawberry flavor (F 11720 UniversalFlavors, 0.1 %) and color (Winner’s Ponceur 4 R ,

0.03 %) were also added to disguise the fermented

rice odor and simulate strawberry dairy yogurt.The product was kept chilling at 4 °C for 1-2 hr

before sensory testing.

Sensory evaluation Sensory evaluation ofrice-based yogurt was performed in comparison

with commercial dairy yogurt of the same flavor.

They were scored for color, odor, flavor, texture/consistency and overall acceptability by 20

expereinced panels, using 9-point hedonic scale

rating from dislike very much (1) to like extremely(9).

Keeping quality and safety Strawberry

rice-based yogurt was kept at 4 °C for 20 days.Samples were taken during storage to examine for

titratable acidity, pH, LAB and non-LAB counts,

yeast, mold and coliforms.

RESULTS AND DISCUSSIONS

Saccharified riceRice was saccharified in closed container at

55 °C for 24 hr, during which samples were drawn

for analysis of reducing sugars and sugar

composition. Reducing sugar at 0 hr in cooked,liquefied solution before adding AMG was 9.36 %.

During the first few hours of saccharification, the

rate of hydrolysis was markedly high and sugarproduced was about double (18.9%) of the original

amount as shown in Figure 1. After that the rate was

gradually increased until about 20-21 hr when itreached the maximum content (23.8 %). Prolonged

incubation to 24 hr did not give higher yield of

reducing sugars.Sugar composition by HPLC revealed that

at 0 hr, major sugars in the solution were higher

oligosaccharides (Degree of polymerization [DP]= 3 and DP > 3) and maltose (DP =2) with only

small amount of glucose i.e. 15.74, 6.5 and 2.86 %,

respectively. However, after 30 min, glucoseincreased to 8.67 while higher oligosaccharides

decreased to 5.72 % (Figure 2). As saccharification

proceeded, glucose increased somewhat linearlyafter 1, 2, 3 and 4 hrs of incubation. Maltose

liberated was eventually broken down to glucose

and remained at low concentration in the solution.At 18th hr, 16.92 % glucose was obtained with

higher oligosaccharides and maltose comprised for

about 2 %. Saccharification process was consideredcompleted after 20-21 hr when maximum amount

of glucose (23.8 %) was produced with only 0.5 %

higher oligosaccharides and 0 % maltose left . Theyield of hydrolysis (100 x g glucose produced /

expected glucose in starch, dry basis) was 81 % and

the yield of glucose was 89.1 g per 100-g ricestarch. Glucose yield was calculated from the real

amount obtained in the saccharified solution

excluding glucose that remained in the spent solidremoved by filtration.

110 Kasetsart J. (Nat. Sci.) 34 (1)

0

5

1 0

1 5

2 0

2 5

Red

ucin

g s

ugar

(%

)

0 0 . 5 1 2 3 4 1 8 2 1 2 4

T i m e ( h r )

Figure 1 Changes of reducing sugars during saccharification of rice by amylolytic enzymes at 55 °C.

0

5

1 0

1 5

2 0

2 5

0 0 . 5 1 2 3 4 1 8 2 1 2 4

T i m e ( h r )

Sug

ar c

once

ntra

tion

(%)

G l u c o s e

M a l t o s e

D P 3

H i g h e r o l i g o s a c c h a r i d e s

Figure 2 Changes of sugars compositions during saccharification process of rice by amylolytic enzymesat 55 °C.

Preparation of rice milkSaccharified rice when adjusted to 18 °

Brix, contained 17.25 % reducing sugars. After

fortification with 3 % casein, 3 % soybean oil and

0.4 % calcium lactate and homogenized, the resultedsuspension obtained was called rice milk due to its

milky color similar to that of cow’s milk (Table 1).

The lightness was a little lower but it was less

greenish and yellowish than that of cow’s milk.Casein comprised for higher protein content and

soybean oil increased fat content as well as provided

smooth texture of the product. Calcium lactate is asalt of lactic acid which is very soluble in water and

has a high percentage of calcium and the lactate ion

is the L(+) isomer, the same as the naturally presentisomer in human. Fortification of calcium lactate to

Kasetsart J. (Nat. Sci.) 34 (1) 111

other products such as orange juice, calcium-enriched beverages and drinking yogurt was

recommended at 0.3 - 1.5 % level resulting in

calcium enrichment of 40-200 mg per 100 g product(Anonymous, 1993). Concentration of calcium

lactate higher than 0.4 % resulted in precipitation

of calcium salt in the rice milk.

Lactic fermentation of rice milkFrom preliminary study of Chakamas et al.

(1992), L casei 2020 and L acidophilus 2013 wereselected for lactic fermentation of rice hydrolysed

by koji prepared from Amylomyces rouxii. Since

both strains proved to grow well and produced upto 0.6 % lactic acid in hydrolyzed rice, they were

also used in this experiment as single inoculum andas the mixed cultures for lactic fermentation. The

effect of beta glycerophosphate(BG) is shown in

Figure 3. BG seemed to enhance acid production,especially when mixed culture (1 % each) was used

as starter. BG at 0.5 % level helped increasing the

acidity up to 0.8 % compared to 0.67 % of thecontrol. Sodium salt of beta glycerophosphate was

widely used in fermented dairy products,

particularly yogurt, drinking yogurt and cheesesince it was effective as buffering agent suitable for

growth of LAB and increased lactic acid production

(Rebecchi et al., 1993).The incubation period of 24 hr gave the

product with 0.8 % acidity and flavor and odor was

Table 1 Color measurement of rice milk in comparison with fresh cow’s milk.

Factors Rice milk Cow’s milk

Lightness (L*) 90.25 93.50a* (+ : red; - : green) -0.89 -1.93

b* (+ :yellow; - : blue) 2.86 16.25

C* (Chroma) 3.00 6.54H* (arctan) 107.28 107.13

0

2

4

6

8

10

12

0 0.25% 0.50%

Beta-glycerophosphate concentration

Aci

dity

as

lact

ic a

cid

(g

/l) L. acidophilus 2013

L. casei 2020

LA 2013 + LC 2020

Figure 3 Effect of beta-glycerophosphate on acidity of rice milk fermented with 2 % LAB at 37 °C for24 hr.

112 Kasetsart J. (Nat. Sci.) 34 (1)

0

2

4

6

8

1 0

1 2

18 hr 24 h r 30 h r

Incubat ion per iod

Aci

dity

as

lact

ic a

cid

(g

/l)

L . ac idophi lus 2013

L. casei 2020

LA 2013 + LC 2020

0

2

4

6

8

10

12

2% 3% 4%

Inoculum size

Aci

dity

as

lact

ic a

cid

(g

/l)

L. acidophilus 2013

L. casei 2020

LA 2013 + LC 2020

Figure 5 Effect of inoculum size on acidity of rice milk fermented at 37 °C for 24 hr.

found to be more palatable than the longer period.After 36-hr incubation, either using as single or

mixed cultures, the sour taste of the saccharified

rices was too profound (Figure 4). Inoculum sizehigher than 2 % gave no advantage for lactic

fermentation both when the strains were used

separately and as mixed culture (Figure 5). L.

acidophilus 2013 could grow well and produce

high lactic acid during the first stage of incubation

but it also gave strong acidic smell. L. casei 2020produced slower rate of lactic acid but the odor was

much more favorable. Combination of the twostrains, at 1 % each, resulted in the product with

appropriate acidity and improved flavor than when

separately used. Therefore, it has been chosen forthe fermentation of saccharified rice fortified with

0.5 % beta glycerophosphate for 24 hr at 37 °C.

Resulted rice-based yogurt contained 0.8% lacticacid at pH 3.58 and lactic bacteria count of 1.0 × 108

CFU/g.

L. acidophilus and L. casei are consideredto be probiotics and they are natural inhabitants of

Figure 4 Effect of incubation period on acidity of rice milk fermented with 2% LAB at 37 °C.

Kasetsart J. (Nat. Sci.) 34 (1) 113

the intestinal tracts (Saloff-Coste, 1997). L. casei,

Shirota has been used in Yakult, which is a well-

recognized drinking yogurt-like product. L. casei

subsp. rhamnosus, a human origin strain, wasreported to be used in fruit-flavored drink and

yogurt from whey (Salminen et al., 1991), and as a

direct-to-vat inoculum for fermented milk (Russell,1996).

Rice-based yogurt preparationRice-based yogurt obtained after 24-hr lactic

fermentation contained 0.8 % as lactic acid, pH

3.48 and viable cell count of LAB of 1.0 × 108 CFU/

g. The curd was well set, yet relatively soft butwithout separation of the liquid on the surface of

curd. Texture and consistency of the yogurt could

be improved by addition of 1 % pectin. Moreover,in order to enhance acceptability, 20 % strawberry

preserve was employed instead of sugar. The

preserve used which contains 65 % total sugar,consists of of 57 % glucose and 8 % sucrose. This

would result in about 11 % glucose and 1.6 %

sucrose in the resulted rice yogurt. The rice curdwas blended with the fruit and pectin to make

custard-type or swiss-style yogurt (Sellars andBabel, 1970). Strawberry flavor and color were

also added to disguise the fermented rice odor and

simulate strawberry dairy yogurt. After chilling at4 °C for 1-2 hr, texture of the curd became smooth

and firmer with a light pink color and strawberry

odor. Some physicochemical properties of rice-based yogurt in comparison with commercial dairy

yogurt are shown in Table 2. Despite protein content

of rice-based yogurt was a little lower than dairyyogurt, fat content was also lower and can be

furtherly adjusted to give a low-fat and cholesterol-

free product. The major component of sugars wasglucose with small amount of sucrose derived from

the preserve. Acidity was 0.8 % as lactic acid and

0.14 % as citric acid from the preserve.

Sensory evaluationThe overall acceptability of strawberry rice-

based yogurt with 0.86 % acidity as lactic, pH 3.58and LAB count of 7.6 × 107 was rated by the

panelists as like moderately (6.85) which was

comparable with commercial strawberry dairyyogurt (6.98) as shown in Table 3. Commercial

Table 2 Physico-chemical properties of strawberry added rice-based yogurt in comparison with commercialdairy yogurt with strawberry.

Rice-based yogurt Commercial dairy yogurt

Protein (%) 3.05 4.0Fat (%) 2.67 2.8

Ash (%) 0.26

Carbohydrate 25.1 27.0Glucose (%) 24.4 16.8

Sucrose (%) 1.5 9.9

Calcium (mg/100 g) 47 117Acidity (% as lactic acid) 0.86 1.02

pH 3.48 4.2

Viscosity (cps) 3,900 4,200Viable LAB count (CFU/g) 7.6 × 107 3.4 × 109

114 Kasetsart J. (Nat. Sci.) 34 (1)

yogurt was slightly preferred in all categories testedbut the differences were not significant at 95 %

confidential level.

Shelf-life study of Rice-based yogurtShelf-life study revealed that during 6 day-

storage at 4 °C, pH of rice-based yogurt decreased

slightly from 3.58 to 3.40 and remained unchanged.Acidity gradually increased throughout 20-day

storage while number of LAB increased from 7.6 ×107 CFU/g (day 1) to 4.5 × 108 at day 6 anddecreased thereafter to 103 at day 20 (Table 4).

Coliforms, yeasts, mold and bacteria other than

LAB were not detected during storage up to 20days. LAB counts of two commercial dairy yogurts

after 10 day-storage were none and 8 × 104 CFU/g

even though both contained about 1 % acidity.

CONCLUSION

Saccharification of Jasmine rice with

amylolytic enzymes at 55 °C yielded the highestamount of reducing sugar after 21 hr. Sugar

composition of saccharified rice by HPLC revealed

that 98 % was glucose and only 2 % were higheroligosaccharides (DP>3). Saccharified rice at 18 °Brix when fortified with 3% casein, 3% soybean oil

and 0.4% calcium lactate, homogenized andpasteurized, resulted in rice milk for lactic acid

fermentation.

Mixed culture of L. acidophilus and L. casei

subsp. rhamnosus, 1 % each, were selected for

lactic fermentation of rice milk. Optimum

conditions for lactic fermentation were as follows:rice milk added with 0.5 % beta glycerophosphate

Table 4 Shelf-life study of Rice-based yogurt with strawberry.

Day LAB count Lactic acid

(CFU/g) (%) pH

1 7.6 × 107 0.86 3.58

2 9.8 × 107 0.94 3.524 1.3 × 108 1.10 3.45

6 4.5 × 108 1.13 3.40

8 1.5 × 107 1.20 3.4010 5.6 × 106 1.18 3.40

13 1.9 × 104 1.25 3.40

16 1.0 × 104 1.26 3.4020 9.8 × 103 1.26 3.50

Table 3 Sensory evaluation of rice-based yogurt and commercial dairy yogurt with strawberry.

Color Odor Flavor Texture Acceptability

Commercial dairy yogurt 7.35a 6.87 a 7.10 a 7.18 a 6.98 a

Rice-based yogurt 6.70 a 6.73 a 6.75 a 7.05 a 6.85 a

* Values of each column with the same letter are not significantly different at 0.05 level.

Kasetsart J. (Nat. Sci.) 34 (1) 115

as substrate; 2% inoculum and 37°C incubation for24 hr. Resulted product contained 0.8% lactic acid,

pH 3.58 and lactic bacteria count of 1.0 × 108 CFU/

g. This rice yogurt was used as base to prepareswiss-style type yogurt by blending with 20%

strawberry preserve, 1 % pectin and flavoring and

coloring agents.Rice-based yogurt with strawberry consisted

of 3.05 % protein, 2.68 % fat, 24.4 % glucose, 1.5

% sucrose, 47 mg/100g of calcium, 0.86 % acidityas lactic, pH 3.58 and lactic bacteria count of 7.6 ×107 CFU/g. The viscosity was 3,900 centipoises

and could be kept at 4 °C for at least 20 days withoutdeterioration and separation of liquid. No

contamination from other microflora was dectected

throughout the storage period and at day 20, 9.8 ×103 CFU/g of viable lactic bacteria remained in the

product. Sensory evaluation indicated that rice-

based yogurt with strawberry was accepted bypanelists when compared with commercial

strawberry yogurt with no significant difference at

0.05 % level.

ACKNOWLEDGEMENT

The research was partially supported by

National Research Council of Thailand. The authors

acknowledged the technical assistance of Ms.Suparat Reungmaneepaitoon for Spectroflash SF

600 color measurement and Brookfield viscosity

determination.

LITERATURE CITED

A.O.A.C. 1990. Official Method of Analysis, 15th

ed. Association of Official Analytical Chemists

Washington D. C. 1298 p.Anonymous. 1993. Lactic Acid, pp. 56-60. In Asia

Pacific Food Industry. A P Food Industry

Publication Pte Ltd. Singapore.Chakamas Wongkhalaung, M. Boonyaratana-

kornkit, P. Chimanage and P Thammarate.1992. Process Development of Rice-based

Product through Lactic Acid Fermentation. A

Thai-ASEAN Project Report submitted toNational Research Council of Thailand. 47 p.

Chaplin, M.F. and J.F. Kennedy. 1986.

Carbohydrate Analysis. IRL Press. Oxford,Washington D.C. 450 p.

Collado, L.S., R.C. Mabesa, M.J.V. Sumague and

C.K. Mok. 1994. Yogurt-like products fromriceflour and soymilk. Philippine Agriculturist

77 (3) : 307-319.

deMan, J.C., M.C. Rogasa and M.E. Sharpe. 1960.A medium for the cultivation of lactobacilli. J.

Appl. Bacteriol. 23 : 130-135.

Frazier, W. C., E.H. Marth and R.H. Diebel. 1968.Laboratory Manual for Food Microbiology.

4th ed. Burgess Publishing Company,

Minneapolis, Minn., USA. 122 p.Lee, C.H., K.C.Min, M. Souane, M.J. Chung, T.E.

Mathiasen and J. Adler-Nissen. 1992.

Fermentation of prefermented and extrudedrice flour by the lactic acid bacteria from

Sikhae. Food Biotechnology, 6(3) : 239-255.

Lee, Cherl Ho, M. Souane and Ki-Hyung Rhu.1988. Effects of prefermentation and extrusion

cooking on the lactic fermentation of rice-

soybean based beverage. Korean J. of FoodSc. Technol. 20 (5) : 666-673.

Mok Chulkyoon, Jinsuk Han, Young Jin Kim,

Namsoo Kim, Dae Young Kwon andYoung June Nam. 1991. Risogurt, a mixture of

lactic acid fermented rice and soybean protein

: development and properties. Korean J. ofFood Sc. Technol. 23(6) : 745-749.

Mok Chulkyoon, Young Jung Nam and Young Jin

Kim. 1993. Method for producing highlyconcentrated, lactic-acid fermented product

utilizing unground grainy rice and improving

qualities thereof by the secondary, enzymatictreatment at fermentation. United States Patent

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No. US 5219597. USA.Park, J.H., H.K. Song, J.B. Ahn, G.E. Ji and C.

Mok. 1997. Rice fermentation by Korean

amylolytic Bifidobacterium spp. Korean J. ofFood Sci. and Technol. 29 (3) : 581-587.

Rebecchi, A., S. Bertuzzi, F. Lcchini, V. Bottazzi

and E. Brambilla. 1993. Lactic acid bacteriafor Grana Cheese production. 5. Lactic

microflora activity in whey buffered with

sodium beta-glicerophosphate. Scienza eTecnica Lattiero Casearia (Italy) 44(6) : 377-

388.

Russell, P. 1996. Yogurt and fermented milks – aChanging area. International Milk Industry.

98(11) : 5-6, 8-9.

Salminen, S., S. Gorbach and K. Salminen. 1991.Fermented whey drink and yogurt-type product

manufactured using Lactobacillus strain. Food

Technology (USA), June 1991. 45(6) : 112.Saloff-Coste, C.J. 1997. Lactobacillus acidophilus.

Danone World Newsletter 13 : 1-7.Sellars, R.L. and F.J. Babel. 1970. Cultures for the

Manufacture of Dairy Product. CHR. Hansen’s

Laboratory, Inc. West Maple Street, Wisconsin.Shin Dong Hwa. 1989. A yogurt like product

development from rice by lactic acid bacteria.

Korean J. Food Sci. Technol. 21 : 686-690.Tominaga, M. and K. Sato. 1996. Lactic acid

fermentation of saccharified solution from rice

flour. Journal of Food Science 61(3) : 627-631.

Zulu, R.M., V.M. Dillon and J.D. Owens. 1997.

Munkoyo beverage, a traditional Zambianfermented maize gruel using Rhynchosia root

as amylase source. International Journal of

Food Microbiology 34(3) : 249-258.

Received date : 15/10/99Accepted date : 17/01/00

Kasetsart J. (Nat. Sci.) 34 : 117 - 124 (2000)

INTRODUCTION

In recent years, epidemiological evidence

has suggested that a reduction in dietary fiber is

related to an increase in certain diseases such asdiverticulosis (Painter and Burkitt, 1971) and

colonic cancer (Burkitt, 1971). Dietary fiber acted

as a bulking agent that increased intestinal motilityand moisture content of feces (Forsythe et al.,

1976). It was postulated that those effects were

important in preventing disease of the colon(Trowell, 1973). Other studies showed evidence

that plant fiber could decrease serum cholesterol

level (Forsythe et al., 1976; Tsia et al., 1976) and

Development of Instant High Fiber Processed Food

Plernchai Tangkanakul, Nednapis Vatanasuchart,Maradee Phongpipatpong and Patcharee Tungtrakul

ABSTRACT

Five high fiber processed foods were formulated by using high fiber sources such as beans,

unpolished rice, sesame. The product formulas were : I) kidney bean : unpolished rice : white sesame

(70:20:10), II) kidney bean : sweet potato : job’s tears seed (45:35:20), III) mungbean : pineapple : pumpkin(50:30:20), IV) corn : mungbean : unpolished rice (60:30:10), and V) banana : pumpkin : corn : unpolished

rice (30:25:25:20). These products were prepared in powder form by drum dryer and then ground with pin

mill. The particle size, bulk density and viscosity of the plain products ranged from 141.6 - 186.5 µm, 0.75- 0.82 g/ml and 1,750-7,208 cps, respectively. After flavoring with sugar, skimmed milk, cocoa or vanilla,

the Water Absorption Index (WAI) and water activity (aw) of flavored products ranged from 2.81-5.59 and

0.27-0.31, respectively. Flavored products prepared for sensory evaluation were conducted by addingwarm water in four different ratio, products to water as 1:3, 1:4, 1:5 and 1:6 by weight. The results showed

that the ratio 1:4 of most formulas had scores of acceptance ranged from 6.29-7.35 which was higher than

the other ratios. Protein, fat and total dietary fiber of all flavored products ranged from 14.54-20.50 , 1.02-4.25, and 5.89-11.88 g/100 g , respectively.

Key words: high fiber, instant food, dietary fiber, drum dryer

improved oral glucose tolerance in humans (Kay,

1982).In view of the recently proposed

physiological role and medical advantage of dietary

fiber, along with the increasing interestdemonstrated in the scientific and consumer world,

it was the interest of food research and product

development to examine more closely theapplication of fiber ingredients in commercial

formulations. It led to the purpose of this study to

develop a profile of high-fiber processed food byusing several potential sources of dietary fiber as

the ingredients.

Institute of Food Research and Product Development, Kasetsart University, Bangkok 10900, Thailand.

118 Kasetsart J. (Nat. Sci.) 34 (1)

MATERIALS AND METHODS

1. Products preparationPulses, cereals, sesame and seeds were used

as potential sources of dietary fiber in the products.

Five product formulas are shown in as follows.

Formula Fiber source Ratio

I kidney bean : unpolished

rice : white sesame 70:20:10II kidney bean : sweet

potato : job’s tears seed 45:35:20III mungbean : pineapple :

pumpkin 50:30:20IV corn : mungbean :

unpolished rice 60:30:10V banana : pumpkin : corn :

unpolished rice 30:25:25:20

Products were formulated to meet

Recommended Daily Dietary Allowances forhealthy Thais which should contain protein about

20% of total energy and 5 g/100 g of dietary fiber

(RDA,1989). Process of high fiber food productionis shown as flow chart.

2. Physical propertiesParticle size Twenty five grams of milled

product were placed on the largest of a descending

60 , 80 , 100 mesh stainless steel U.S. Standard

Sieves that were fitted with a pan and cover. The“nested” sieves were shaken for 10 min ,

disassembled and contents were stirred lightly ,

then shaked for an additional 5 min. The residue oneach sieve was carefully removed with the aid of a

brush and weighed. Each residue was expressed as

percent by weight of the original sample.Density determinations For density

determination , a calibrated graduate cylinder was

Weighed raw materials

↓Cooked

↓Mixed

↓ ← added water

Ground

↓Drum dryer (Surface Temp. 135

o C , 4 rpm , clearance 0.1 mm.)

↓Pin mill

↓Plain product

↓ ← added sugar , skimmed milk powder , cocoa, vanilla

Flavored product

Kasetsart J. (Nat. Sci.) 34 (1) 119

filled, with each milled product and slightly shaked.The contents of milled product in the cylinder were

weighed and the average of triplicate determinations

was expressed as g/ml.Bulk density Bulk density was measured

with a calibrated graduate syringe (open and packed

with cotton). The syringe was filled with a knownamount of sample , which varied somewhat

depending on particle size and density. Pressure

was applied manually until additional pressurewould not furtherly reduce the volume.

Viscosity Viscosity was measured by using

a Brookfield model RVT viscometer with no. 3, 4,5 spindle at 50 rpm (Synchro-Lectric Viscometer

model RVT Brookfield engineering Laboratories,

INC).Water Absorption Index (WAI) Water

Absorption Index was measured by the procedure

of Anderson et al., 1969.Water activity (aw) Water activity was

measured by Novasina EEJA –3 at 25°C.

3. Chemical compositionMoisture, ash, protein and fat were analysed

by following AOAC procedure (1990). Dietary

fiber was determined by enzymatic gravimetricmethod (AOAC, 1990).

4. Preparation of flavored productsFlavoring agents and carboxy methyl

cellulose (CMC) were added into each formula

accordingly as follows.

Ingredients High fiber processed food formula number

(g) I II III IV V

Plain product 50 50 50 50 50Sugar 25 25 25 25 25

Skimmed milk 20 20 24.8 24.95 24.95

powder

Cocoa powder 4.95 4.95 - - -

Vanilla - - 0.15 - -

CMC 0.05 0.05 0.05 0.05 0.05

5. Sensory evaluation of flavored productsSensory evaluation of flavored products

was tested by adding warm water in the ratio of

instant powder to water, 1:3 1:4 1:5 and 1:6. Color,

odor , flavor , thickness , texture and acceptancewere evaluated by 20 experience panelists. The

acceptance was done using the 9-point hedonic

scale (max. value 9 = like extremely , min. value 1= dislike extremely) , whereas color , odour ,

flavor , thickness and texture were determined by

5-point scale for perceived intensity scores (max.value = 5, min. value = 1) (ASTM, 1968). Color

tone , strength of smell , sweetness and viscosity of

the tested products were comparatively justified.The hedonic and scoring rating were

analysed by Analysis of variance (Randomized

Complete Block Design) and Duncan’s MultipleRange Test at 95% confidential level.

RESULTS AND DISCUSSION

Materials selected to develop instant high

fiber processed foods were red kidney bean,mungbean, white sesame seeds, unpolished rice,

corn, sweet potato, pumpkin, pineapple, job’s tear

and banana. These food sources provided eithernutritional value, dietary fiber, fruity flavor and/or

color to the developing products. For nutritional

value point of view, protein content was the mostconcern. Red kidney bean, mungbean, white sesame

seeds and job’s tear contained a considered amount

of protein. The amounts of dietary fiber were highin red kidney beans , mungbean and sesame which

were 30.2 , 28.9 and 22.3 g/100 g (dry weigh basis),

respectively (Puwastien, 1990), whereas , dry matterof pumpkin, corn, sweet potato, pineapple, banana,

job’s tear and unpolished rice contained lower fiber

of 16.4, 13.4, 8.6, 8.0, 6.6, 4.15 and 2.4 g/100 g,respectively (Puwastien, 1990).

120 Kasetsart J. (Nat. Sci.) 34 (1)

Physical propertiesThe results showed that most of the particle

size (> 50%) of the formula I II III and IV ranged

from 180 - 150 µm (Table 1) which were smaller

than the particle size of the formula V (average186.5 µm). Mix of different particle sizes in one

formula implied existing of various fiber sizes.

Kimura (1977) reported that mix of various particlesize of fiber increased rate of water absorption in

human. Therefore, the formula I and II should be

superior to the formula III, IV and V.WAI of formula I was the lowest, 2.81,

while that of formula V was the highest, 5.59. The

result indicated that formula V imbibed more waterthan formula I as demonstrated by Chen et al.

(1988). Sieve size as well influenced on the water

absorption. Water absorption could be decreasedby the reduction of powder particle size (Cadden,

1987) , suggesting that small size of materials

might be less porous and would be unable to imbibeas much water as large size. This study displayed

WAI trend similar to Cadden (1987) except formulaI (Table 1). From calculating carbohydrate content,

formula I, II, III, IV and V contained 48.3, 59.1,

66.9, 70.7 and 82.4 g/ 100g (dry weight basis),respectively. Amount of carbohydrate might affect

water absorption, illustrated in formula I. Besides

that type of carbohydrate, starch or cellulose, alsoinfluenced water absorption.

Sensory evaluationDeveloped products displayed their

characteristic in color depend upon the ingredients.

Formula I and II contained red kidney beans,

therefore, both formulas had red-purplish tone.Mungbean provided greenish color and pineapple,

pumpkin and corn gave yellowish color to the

formula III and IV. Color of formula V was similarto formula III and IV, but brighter in yellow caused

by banana.

Each formula was designed to haveindividual characteristic in odor. Formula I expected

Table 1 Physical properties of plain and flavored products.

Property High fiber processed food formula number

I II III IV V

Plain product

Particle size*60-80 mesh (250-180 µm) 39.4 25.2 4.2 5.6 56.7

80-100 mesh (180-150 µm) 50.1 62.1 67.5 78.0 35.7

>100 mesh (<150 µm) 10.7 12.7 28.3 16.7 7.6Particle size (µm) 175.0 166.2 141.6 153.0 186.5

Direct density (g/ml) 0.66 0.62 0.66 0.60 0.54

Bulk density (g/ml) 0.82 0.75 0.82 0.80 0.79Viscosity (cps) 1750 6040 3820 7208 6440

Flavored product

Water Absorption Index (WAI) 2.81 3.75 3.80 3.80 5.59Water activity (aw) 0.27 0.31 0.30 0.31 0.27

* Percent of sample retained on U.S. standard sieves

Kasetsart J. (Nat. Sci.) 34 (1) 121

kidney bean flavor mix with cocoa. Job’s tear andcocoa powder contributed odor to the formula II.

Formula III was dressed with vanilla. Corn and

banana were natural odor in formula IV and V,respectively.

Sensory evaluation of flavored products is

shown in Table 2. The ratio of 1:4 in most formulas

had higher acceptance scores than the others. Inparticularly, the formula IV obtained the highest

scores. The reason was formula IV contained corn

up to 60%. This amount of corn provide good odorand flavor to the product. Panelists’ commendation

revealed that odor was the most important factor

for them to decide their preference.

Table 2 Sensory evaluation of 5 formulas of high fiber processed food.

Ratio Mean(Instant powder :

water) Color Odor Flavor Thickness Texture acceptance

Formula I

1 : 3 4.19a 3.44a 3.63a 4.44a 2.56a 6.25ab

1 : 4 3.00b 2.88ab 2.69b 3.06b 2.06b 7.00a

1 : 5 2.25c 2.63b 2.00c 2.25c 1.81b 6.13ab

1 : 6 1.38d 2.69ab 1.38d 1.31d 1.63b 5.50b

Formula II

1 : 3 4.07a 2.57a 3.43a 4.64a 2.36a 5.86a

1 : 4 3.21b 3.07a 2.71b 3.50b 2.29a 6.29a

1 : 5 2.14c 2.86a 2.21c 2.43c 2.07a 6.36a

1 : 6 1.43d 2.93a 1.50d 1.79d 1.86a 5.79a

Formula III 1 : 3 3.88a 2.41a 3.82a 4.41a 1.94a 5.94b

1 : 4 2.88b 2.47a 2.94b 3.18b 1.71ab 7.24a

1 : 5 2.18c 2.29a 2.41c 2.12c 1.47bc 7.18a

1 : 6 1.65d 2.18a 1.53d 1.24d 1.29c 5.71b

Formula IV

1 : 3 3.70a 3.00ab 3.40a 4.45a 2.50a 5.35b

1 : 4 2.95b 3.10a 2.90b 3.25b 1.95b 7.35a

1 : 5 2.30c 2.60b 2.10c 2.35c 1.90b 6.95a

1 : 6 1.75d 2.10c 1.50d 1.40d 1.60b 5.70b

Formula V

1 : 3 4.15a 3.54a 3.92a 4.62a 1.38a 5.15b

1 : 4 3.23b 2.77b 2.92b 3.38b 1.38a 7.15a

1 : 5 2.77c 2.38b 2.23c 2.62c 1.31a 6.92a

1 : 6 2.23d 1.77c 1.62d 1.77d 1.31a 6.69a

*In each formula, mean in the same column having different superscripts were significantly different

according to DMRT (P < 0.05)

122 Kasetsart J. (Nat. Sci.) 34 (1)

In all developed products, there was anunsatisfied characteristic of gritty texture. This

gritty texture may be caused by bean hull, corn seed

pericarp, sesame seed coat and cellulose inpineapple. Those coarse particles distributed

irritating effect to the throat. The result of this

experiment agreed to the previous report that If afiber with low water holding capacity was added to

a beverage system , the beverage may have a gritty

texture (Schmidl et al., 1985). However, throatirritated feeling could be minimized by increasing

water content (Table 2). The bigger number on

texture represented the more irritating feeling.

Chemical compositionThe protein contents of plain formula I to IV

ranged from 18.59 - 21.99 g per 100 g whichaccounted as 24.17% , 23.10% , 27.56% and 23.12%

of total calories, respectively (Table 3). These were

higher than the contents in the flavored products,which were 20.71% 20.08% 22.35% and 21.84%

of total calories, respectively. Among 5 formulas,the protein contents of plain (10.75 g/100g) and

flavored (14.54 g/100g) of formula V were the

lowest, 12.0% and 16.11% of total calories,respectively. The reason was main ingredients were

banana, pumpkin, corn and unpolished rice which

were naturally low in protein contents.Fat contents of all plain products, formula I

to V, accounted as 22.83%, 6.18%, 3.61%, 9.43%

and 7.83% of total calories, respectively. FormulaI provided highest fat content according to one of

the components was sesame which contained fat

56.2 g/100 g. (Nutrition Division, 1992).The results showed that the plain formula I

had a very high dietary fiber contents of 20.59 g/

100g. The reason was that 70% of total weight of allingredients were red kidney beans which were rich

in dietary fiber, 27.7 g/100g (Puwastien, 1990).

The plain formula II and III also provided a highlevel of dietary fiber of 15.38 - 15.85 g/100g which

was found to be associated with the selected

Table 3 Chemical composition of 5 formulas of plain and flavored high fiber processed food (per 100 g).

Product Moisture Protein Fat Ash Dietary fiber CHO Energy(g) (g) (g) (g) (g) (g) (Kcal)

Plain

Formula I 3.91 20.11 8.44 2.86 20.59 (5.84)* 44.09 332.76

Formula II 3.71 18.59 2.21 2.72 15.85 (4.49) 56.92 321.93Formula III 2.95 21.99 1.28 3.47 15.38 (4.36) 54.93 319.20

Formula IV 3.77 19.74 3.58 2.96 12.35 (3.50) 57.60 341.58

Formula V 2.82 10.75 3.12 2.85 8.62 (2.44) 71.84 358.44

Flavored

Formula I 3.08 18.07 4.25 3.10 11.88 (3.37)* 59.62 349.01Formula II 2.78 17.62 1.02 3.14 7.63 (2.16) 67.81 350.90

Formula III 2.48 20.50 3.21 3.56 6.26 (1.77) 63.99 366.85

Formula IV 2.90 19.57 1.46 3.43 5.89 (1.67) 66.75 358.42Formula V 2.35 14.54 1.46 3.28 5.96 (1.69) 72.41 360.94

* The numbers in the parenthesis referred to the contents of dietary fiber in g/1 - 0Z serving

Kasetsart J. (Nat. Sci.) 34 (1) 123

ingredients, kidney bean and mungbean The plainformula V possessed the lowest of dietary fiber of

8.62 g/100g.

The amount of dietary fiber in flavoredproducts ranged from 5.96 to 11.88 g/100 g which

were substantially higher than some commercial

products. It was reported that dietary fiber contentsof commercial breakfast cereal ranged from 0.2 -

34.06 g/100g (Jwuang, 1979; Baker, 1981; Douglass

et al., 1982; Mongeau and Brassard, 1982; Marlett,1992).

The flavored powder of formula I, II, III, IV

and V providing energy ranged from 319.20 -358.44 Kcal equaling 4.88% 9.00% 14.93% 4.93%

and 0.70%, respectively,which were greater than

the plained formulas. The increasing calorie wasdue to sugar and skimmed milk, 36.7 Kcal/100g,

(Whitney and Hamilton, 1981).

CONCLUSION

All of the flavored products provided dietaryfiber ranged from 5.89 - 11.88 g/100 g which were

substantially higher than thore of some comercial

products. The most preference one was Formula IVdue to accustomed and adored fragrance of corn. In

this study, the production cost of Formula IV

calculated only on raw materials was 40 baht / kg.The cost could certainly be reduced if industrial

scale was conducted. Producing these higher

nutritional products would provide another optionfor consumers who concern of their health.

ACKNOWLEDGEMENT

We would like to acknowledge Kasetsart

University Research and Development Institute(KURDI) for a grant to support this study.

LITERATURE CITED

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E.L. Griffin Jr. 1969. Gelatinization of corngrits by roll and extrusion cooking. Cereal Sci.

Today. 14 : 4.

AOAC. Official methods of analysis. 1990 15 thed. Association of Official Analytical

Chemists, Arlington, Virginia. 1298 p.

ASTM, 1968. Manual on Sensory Testing Methods.American Society for Testing and Materials,

Philadelphia, 77p.

Baker D. 1981. Fiber in breakfast cereal. J FoodSci. 46 : 396.

Burkitt, D.P. 1971. Epidemiology of cancer of the

colon and rectum. Cancer. 28 : 3.Cadden, A.M. 1987. Comparative effects of particle

size reduction on physical structure and water

binding properties of several plant fiber. J.Food Sci. 52 : 1595.

Chen, H., C.L. Rubenthaler, H.K. Leung, and J.D.

Baranowski. 1988. Chemical, physical , andbaking properties of apple fiber compared

with wheat and oat bran. Cereal Chem. 65 :

244.Douglass, J., R. Mathews, and F. Hepburn. 1982.

Composition of Food : Breakfast Cereals -

Raw , Processed, Prepared. Rev USDAAgriculture Handbook No. 8 – 8.

Forsythe, W.A., Chenoweth, and M.R. Bennink.

1976. The effect of various dietary fibers onserum cholesterol and laxation in the rat. J.

Nutr. 106 : 26.

Jwuang, W.J., M.E. Zabik. 1979. Enzyme neutraldetergent fiber analysis of selected commercial

and home - prepared foods. J Food Sci. 44 :

924.Kay, R.M. 1982 Dietary fiber. J. Lip Res. 23 : 221.

Kimura, K.K. 1977. High fiber diet - who need it?

Cereal Food World 22 : 16.Marlett,. J.A. 1992. Content and composition of

124 Kasetsart J. (Nat. Sci.) 34 (1)

dietary fiber in 117 frequently consumed foods.J. Am Diet Assoc. 92 : 175.

Mongeau, R., R. Brassard. 1982. Determination of

neutral detergent fiber in breakfast cereals :Pentose , hemicellulose , cellulose , and lignin.

J. Food Sci. 47 : 550.

Nutrition Division , Department of health , Ministryof public health. 1992. Nutritive Values of

Thai Foods. 48 p.

Painter, N.S. and D.P. Burkitt. 1971. Diverticulardisease of the colon : A deficiency disease of

western civilization. BMJ. 2 : 450.

Puwastien, P., U. Valaipatchara, and R.Kongkachuichai. 1990. Dietary fiber content

in common Thai food. J. Nutr. Assoc.

(Thailand) 24 : 43.RDA, 1989. The Committee on Recommended

Daily Dietary Allowances, Dept. of Health,

Ministry of Public Health. 161p.Schmidl, M.K., T.P. Labuza. 1985. Low calorie

formulations cutting calories , keeping quality.

Prepared Foods. 154 : 118.Trowell, H.1973. Dietary fiber , ischaemic heart

disease and diabetes mellitus. J. Nutr Soc. 32

: 151.Tsia, A.C., J. Elias, J.J. Kelly, R.C. Lin, and J.R.K.

Robson. 1976. Influence of certain dietary

fibers on serum and tissue cholesterol in rats.J. Nutr. 106 : 118.

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of food composition. Understanding nutrition,2 nd ed. (St. Paul , Minn ; West) : A 43.

Received date : 19/03/99Accepted date : 25/11/99

Kasetsart J. (Nat. Sci.) 34 : 125 - 138 (2000)

Primary Productivity of the Pygmy Bamboo (Arundinaria pusilla)in the Dry Dipterocarp Forest at Sakaerat, Nakhon Ratchasima

Niwat Ruangpanit

ABSTRACT

The aboveground and belowground biomass of Arundinaria pusilla in two strata of the drydipterocarp forest were monthly harvested and net primary production, accumulation and disappearance

rates by compartment were estimated. These data were further utilized for determination of system transfer

functions and the efficiency of energy capture. The results indicated that the aboveground standing cropof standing live, standing dead and litter varied considerably through different sampling intervals within

and between each stratum. More belowground biomass was concentrated in the upper soil layer and the

amount declined with an increase in depth. The community annual aboveground net primary productionwas estimated on ash-free basis to be 210 gm-2 on stratum 1 and 329 gm-2 on stratum 2 and the belowground

net production was 2,844 gm-2 on stratum 1 and 2,884 gm-2 on stratum 2. The accumulation of standing

dead (256 gm-2) and litter (214 gm-2) on stratum 1 was greater than the standing dead (198 gm-2) and litter(137 gm-2) on stratum 2. Annual disappearance of litter was also higher on stratum 1 (149 gm-2) than that

of stratum 2 (119 gm-2). But the belowground disappearance was much greater on stratum 2 (2,794

gm-2) than that of stratum 1 (2,573 gm-2). Annual efficiency of energy capture was estimated to be 1.77percent on stratum 2 and 1.86 percent on stratum 1based on 50 percent usable solar insolation. It was shown

that the belowground portion of Arundinaria pusilla played an important role in the dynamics of the system

as a whole. This study confirmed that the belowground biomass in this kind of plant community shouldbe considered as being very important in the study of the ecosystem functions.

Key words : biomass, primary production, system transfer function, energy flow

Department of Forest Biology, Faculty of Forestry, Kasetsart University, Bangkok 10900, Thailand.

INTRODUCTION

With the rising demand for food in the

world and increasing competition for the use of

natural and agricultural land, the efforts of manyscientists concerned with food production have

been directed towards raising production and

improving the efficiency of productivity fromexisting areas of cultivated land. Hence, primary

productivity of natural communities remains a

major area for ecological research.

Optimal use of primary producers dependson accurate understanding of the amount and

dynamics of herbage biomass. Analyses of

ecosystem processes are also dependent on theaccuracy of biomass estimates and the seasonal

patterns of biomass dynamics. The most important

fact, however, is that, without a knowledge of

126 Kasetsart J. (Nat. Sci.) 34 (1)

primary productivity, the ecosystem functioning isnot known as well as the calculation of an energy

budget for any ecosystem as a whole is impossible.

The present study was concerned with theestimation of the primary productivity of

Arundinaria pusilla the predominant perennial grass

community (known in Thai as Yaaphet) in the drydipterocarp forest at Sakaerat Environmental

Research Station (SERS), Pak Thong Chai, Nakhon

Ratchasima.The objective of the study was to determine:

1) seasonal variation in the aboveground and

belowground plant biomass; 2) aboveground andbelowground net primary producton and turnover;

3) net accumulation and disappearance rates; 4)

system transfer functions; 5) the calorific contentof biomass compartments; 6) efficiency of energy

capture; and 7) annual energy flow.

MATERIALS AND METHODS

According to the differences in size, heightand density of the stands, the study area was divided

into two strata. Stratum 2 is normally located on

soil which displays greater prominence of sandstoneboulders or rock out crop than that of stratum 1.

Three replications of 25 × 30 m were randomly

selected on each stratum. The aboveground andbelowground biomass were harvested monthly.

On each sampling date, ten sample plots of the

aboveground biomass were randomly selected andclipped at ground level by using 0.25 × 1.00 m

rectangular quadrats on each replication. Three

samples of belowground biomass were alsocollected in each plot by using a soil core method

to a depth of 40 cm, and divided into 0-10, 10-20,

20-30 and 30-40 cm sections. All plant materialswere oven-dried at 70°C for 48 hours and then

weighed to the nearest 0.01 g. The energy content

of each compartment was determined by using Parradiabatic bomb calorimeter.

The aboveground net primary production(ANP) was estimated using three methods, the

community peak standing crop (Odum, 1960;

Hadley and Kieckhefer, 1963), the sum of positiveincrease in biomass (Kelly et al., 1974) and the sum

of positive change in biomass plus mortality.

According to Singh and Yadava (1974) the sum ofpositive increases in standing dead for only those

sampling intervals which correspond to a positive

change in biomass is called mortality. Thebelowground net primary production (BNP) was

determined by a summation of the positive changes

in the belowground biomass on successive samplingdates (Sims and Singh, 1971; Singh and Yadava,

1974). The turnover rate of belowground biomass

was calculated by using the ratio betweenbelowground net production and maximum

belowground biomass which poposed by Dahlman

and Kucera (1965).Total net primary production (TNP) was

determined simply by adding the aboveground net

production (ANP) and belowground net production(BNP). System transfer functions, net accumulation

and disappearance rates were determined following

the method described by Grodins (1963), Golley(1965), and Sims and Singh (1971). The poduction

of standing dead (SD) was calculated by the

summation of positive changes in the standing cropof standing dead on successive sampling dates,

which represents the transfer of standing live to the

standing dead compartment. Litter production (L)was estimated from the sum of positive differences

in litter through the successive sampling dates.

Litter disappearance (LD) has been derived byadding the negative differences in the litter values

between different sampling intervals. Root or

belowground disappearance (RD) was representedby the summation of significant negative changes

in the belowground biomass on successive sampling

dates. Total disappearance (TD) was the sum oflitter disappearance (LD) and root disappearance

Kasetsart J. (Nat. Sci.) 34 (1) 127

(RD). In order to estimate the efficiency of energycapture and the flow of energy in the system, all

biomass values in gm-2 were converted to energy

values by multiplying those values with theappropriate calorific content of the harvested

samples and expresses in kcal m-2.

RESULTS AND DISCUSSION

Seasonal variation in the aboveground biomassThe aboveground standing crop of live,

dead, and litter, both on stratum 1 and stratum 2,

varied considerably through different samplingintervals (Tables 1 and 2).

The standing live standing crop on both

strata peaked in November at 364 gm-2 on stratum1 and 313 gm-2 on stratum 2. The average standing

live vegetation was 259 gm-2 on stratum 1 and 196

gm-2 on stratum 2.Ruangpanit (1981) estimated that the live

herbage of Arundinaria pusilla was 275 gm-2,

divided into 128 gm-2 stem biomass and 147 gm-2

leaf biomass. The standing live vegetation

decreased on all sampling dates after the peak with

minor fluctuations through to the last samplingdate in March.

Standing dead material was evaluated in the

same manner as standing live on both strata. Thestanding dead on stratum 1 peaked in March at 202

gm-2 and at 194 gm-2in February on stratum 2. The

rapid increase of this component in the dry seasonwas probably caused by the transfer of standing

live vegetation into this compartment after life

cycles were completed. The maximum rate ofincrease of standing dead material on stratum 1 and

stratum 2 was 3.01 and 3.77 gm-2 day-1, respectively,

both being recorded in January.The standing crop of ground litter on both

strata peaked in May at 237 gm-2 on stratum 1 and

199 gm-2 on stratum 2. The amount of litter on bothstrata then declined steadily through the wet season

starting from May and increased again during thedry season in February. Fluctuations in the amount

of litter were the net result of litter production and

disappearance by decomposition. The increase inthe amount of litter in the early part of the growing

season was probably caused by the addition of

material from the standing dead crop and the deathof green vegetation. It is also possible that part of

the litter could have been carried over from the

previous year. The gain in the amount of litterduring the dry season was indicative of the transfer

from standing dead to litter. The decline during the

growing season represents the decomposition oflitter. Lewis (1969) pointed out that litter fall was

most rapid during the dormant season, but

decomposition proceed more rapidly during warm,moist periods.

Seasonal variation in the belowground biomassThe belowground biomass in the upper 40

cm of soil profile on both strata exhibited the same

seasonal trend (Tables 3 and 4); only the maximumand minimum amounts of biomass occurred on

different dates. The seasonal peak on both strata

was recorded in February; with 3,623 gm-2 onstratum 1 and 3,481 gm-2 on stratum 2. On the

average, there were about 2,668 gm-2 of

belowground biomass in the upper 40 cm of soilprofile on stratum 1 and about 2,589 gm-2 on

stratum 2.

Both strata had a greater belowgroundbiomass during the early wet season followed by a

decline and increase immediately following the

decline again thereafter. Generally, the mid-seasondip in belowground biomass and subsequent

recovery was probably the result of stored

carbohydrates being utilized for growth and thenthe carbohydrates were restored later in the same

season.

The variation of belowground biomass onboth strata occurred primarily in the top 10 cm of

128 Kasetsart J. (Nat. Sci.) 34 (1)

Table 1 Seasonal variation in standing live, standing dead and litter standing crop (gm-2 dry mater ± SE1)(ash-free dry wt in parenthesis) of Arundinaria pusilla in the dry dipterocarp forest stratum 1.

Sampling date Standing live Standing dead Litter Total

Apr. 155.24 ± 14.26 80.51 ± 7.32 113.61 ± 9.11 349.36(143.43) (73.83) (100.09) (317.35)

May 307.56 ± 28.27 135.01 ± 17.40 236.65 ± 19.03 679.22

(287.57) (126.06) (183.14) (569.77)Jun. 290.90 ± 38.98 138.44 ± 21.44 215.02 ± 21.44 644.36

(267.69) (131.02) (174.60) (573.31)

Jul. 290.13 ± 2.31 87.55 ± 13.09 147.07 ± 18.70 524.75(265.50) (83.25) (118.92) (467.67)

Aug. 343.40 ± 38.09 79.84 ± 9.44 166.95 ± 16.37 590.19

(319.08) (73.39) (149.52) (541.99)Sep. 315.89 ± 29.81 142.65 ± 13.59 160.32 ± 11.43 618.86

(292.64) (132.75) (136.98) (562.37)

Oct. 282.46 ± 28.58 123.95 ± 12.04 141.75 ± 14.38 548.16(256.93) (113.72) (118.57) (498.22)

Nov. 364.27 ± 30.03 69.61 ± 5.76 90.84 ± 6.30 524.72

(335.82) (65.48) (81.62) (482.92)Dec. 320.38 ± 28.54 50.93 ± 6.81 71.42 ± 8.72 442.73

(290.62) (48.10) (64.37) (403.09)

Jan. 268.01 ± 25.21 103.83 ± 13.99 92.40 ± 8.24 464.24(244.53) (97.20) (82.32) (424.05)

Feb. 107.61 ± 23.53 197.12 ± 15.63 116.90 ± 6.84 421.63

(100.53) (182.22) (103.69) (386.44)Mar. 58.87 ± 8.21 202.02 ± 13.85 184.73 ± 10.02 445.62

(55.56) (187.90) (164.65) (408.21)

Average 258.73 117.62 144.81 521.15

(238.33) (109.58) (132.21) (471.12)

1 Standard error of the mean.

the soil profile and decreased with an increase in

depth. It was possible that the size and amount of

rhizome of Arundinaria pusilla which dominatedthe belowground biomass caused more variation.

Bartos and Sims (1974) believed that the variation

in the amount of root mass in the grassland rangecould come from the fluctuation in the amount of

plant crowns. The dynamics of belowground

biomass, however, may be interpreted either using

concepts of growth and decomposition, or theconcept of translocation of photosynthate material

down to or up from the root, or the combination of

both.

Kasetsart J. (Nat. Sci.) 34 (1) 129

Table 2 Seasonal variation in standing live, standing dead and litter standing crop (gm-2 dry mater ± SE1)

(ash-free dry wt in parenthesis) of Arundinaria pusilla in the dry dipterocarp forest stratum 2.

Sampling date Standing live Standing dead Litter Total

Apr. 209.54 ± 18.39 90.59 ± 7.80 101.41 ± 11.24 401.54

(191.06) (82.07) (91.23) (364.36)May 191.23 ± 21.84 120.01 ± 19.66 199.44 ± 23.99 510.68

(178.15) (113.17) (155.74) (447.06)

Jun. 184.78 ± 26.18 125.11 ± 20.59 188.09 ± 19.94 497.98(170.04) (117.49) (143.66) (431.19)

Jul. 227.24 ± 29.40 92.04 ± 14.30 164.36 ± 20.10 438.64

(212.08) (86.51) (133.30) (431.89)Aug. 286.30 ± 25.54 112.24 ± 17.35 141.56 ± 18.35 540.10

(267.52) (103.03) (124.88) (495.43)

Sep. 206.71 ± 20.04 139.03 ± 16.32 113.49 ± 10.23 459.23(191.27) (127.91) (92.72) (411.90)

Oct. 228.76 ± 31.23 100.82 ± 15.46 123.17 ± 12.40 452.75

(210.19) (94.22) (106.73) (411.14)Nov. 312.74 ± 30.86 91.42 ± 17.94 116.68 ± 11.28 520.84

(287.60) (84.94) (105.40) (477.94)

Dec. 255.63 ± 24.52 58.27 ± 6.96 71.17 ± 8.59 385.07(232.39) (54.64) (64.67) (351.70)

Jan. 147.84 ± 23.03 76.93 ± 11.01 75.16 ± 9.26 299.93

(133.25) (70.76) (66.40)) (270.41))Feb. 55.79 ± 5.79 193.86 ± 20.63 143.49 ± 8.44 393.14

(51.94) (175.99) (122.97) (350.90)

Mar. 50.08 ± 9.56 115.50 ± 13.59 123.92 ± 8.19 289.50(46.92) (105.94) (108.33) (261.19)

Average 196.38 109.65 130.16 436.19

(181.03) (101.39) (109.67) (392.09)

1 Standard error of the mean

Aboveground net primary productionThe annual net production based on the

community peak standing crop, sum of positivechanges in biomass plus mortality and summation

of positive biomass increase on dry matter basis

were 364, 342 and 287 gm-2, respectively, onstratum 1 and 313, 228 and 208 gm-2 on statum 2

(Table 5). In every case the estimate of annual

aboveground net production on stratum 1 was more

productive than that on stratum 2, probably becausethe larger number of rock outcrops on stratum 2

retarded the growth and distribution of Arundinaria

pusilla. It should be pointed out that the communitypeak standing crop gave maximum estimates of net

130 Kasetsart J. (Nat. Sci.) 34 (1)

Table 3 Belowground biomass (gm-2 dry mater ± SE1) (ash-free dry wt in parenthesis) of Arundinaria

pusilla in various soil depth in the dry dipterocarp forest stratum 1.

Sampling Soil depth (cm) Total

date 1 - 10 10 – 20 20 – 30 30 - 40

Apr. 1306.84 ± 181.08 354.95 ± 32.03 140.52 ± 10.35 74.24 ± 12.00 1876.55(1108.72) (275.62) (108.79) (55.72) (1548.85)

May 1376.57 ± 111.08 1055.36 ± 100.93 155.77 ± 12.62 108.48 ± 10.61 2696.18

(1101.53) (818.33) (119.72) (85.32) (2124.90)Jun. 1602.60 ± 181.45 1331.37 ± 181.45 125.28 ± 11.90 66.50 ± 11.88 3125.75

(1290.41) (1041.93) (96.29) (52.30) (2480.93)

Jul. 1511.66 ± 132.04 674.76 ± 83.33 163.06 ± 10.13 89.04 ± 9.92 2438.52(1262.39) (554.45) (125.33) (70.03) (2012.20)

Aug. 1877.28 ± 210.27 824.70 ± 120.17 180.70 ± 6.33 118.87 ± 7.53 3001.58

(1619.53) (710.00) (137.77) (93.55) (2551.85)Sep. 1393.01 ± 136.13 760.53 ± 106.53 181.62 ± 4.97 114.45 ± 13.19 2449.61

(1204.26) (623.41) (138.49) (90.07) (2056.23)

Oct. 1793.69 ± 166.93 649.91 ± 85.35 176.53 ± 17.29 98.10 ± 8.20 2718.23(1535.94) (543.78) (134.57) (77.20) (2291.49)

Nov. 1846.65 ± 204.39 569.64 ± 95.75 142.51 ± 16.38 92.58 ± 14.55 2651.38

(1624.68) (493.42) (106.55) (72.80) (2297.45)Dec. 1446.77 ± 181.60 594.01 ± 72.17 151.79 ± 20.01 85.95 ± 13.69 2278.52

(1192.76) (491.37) (113.49) (63.29) (1861.01)

Jan. 1986.78 ± 213.36 718.91 ± 111.49 174.55 ± 17.71 70.48 ± 11.99 2950.72(1660.95) (582.46) (130.51)) (55.43)) (2429.35)

Feb. 2516.78 ± 322.57 842.26 ± 140.86 159.30 ± 8.66 104.95 ± 8.28 3623.29

(2195.39) (634.14) (123.33) (78.78) (3031.64)Mar. 1338.66 ± 183.66 616.70 ± 69.88 158.64 ± 7.83 94.34 ± 9.58 2202.34

(1174.67) (491.39) (122.82) (70.81) (1859.69)

Average 1666.44 749.43 159.19 93.17 2668.22

(1414.28) (604.28) (121.47) (72.11) (2212.13)

1 Standard error of the mean

production over the other two methods. On stratum

1, net production based on the community peakwas 6 percent more than the sum of positive change

in biomass plus mortality and 21 percent more than

the summation of positive biomass increase. A

similar trend occurred on stratum 2, where net

production based on the community peak was 17and 34 percent more than estimated by the sum of

positive changes in biomass plus mortality and

summation of positive biomass increase

Kasetsart J. (Nat. Sci.) 34 (1) 131

respectively. However, each method of calculation

has its own application and the difference in netproduction varied depending on the method of

estimation and the sampling interval utilized.

For further discussion in aboveground net

production, however, the estimates obtained by

sum of positive changes in biomass plus mortalityhas been used primarily because this method is well

adapted for determining the primary productivity.

Singh and Yadava (1974) also found this method

Table 4 Belowground biomass (gm-2 dry mater ± SE1) (ash-free dry wt in parenthesis) of Arundinaria

pusilla in various soil depth in the dry dipterocarp forest stratum 2.

Sampling Soil depth (cm) Total

date 1 - 10 10 – 20 20 – 30 30 - 40

Apr. 1692.41 ± 191.10 655.75 ± 105.00 149.80 ± 16.94 74.46 ± 16.01 2582.42

(1402.67) (513.49) (115.98) (55.89) (2088.03)

May 1624.87 ± 146.98 1299.42 ± 163.92 156.87 ± 13.18 82.85 ± 21.04 3164.01(1263.01) (960.53) (120.57) (65.16) (2409.27)

Jun. 1353.51 ± 158.80 684.31 ± 84.05 104.06 ± 23.71 26.07 ± 9.74 2167.95

(1127.20) (560.04) (79.98) (20.50) (1787.72)Jul. 1498.93 ± 157.73 705.59 ± 93.30 142.07± 8.10 83.30 ± 6.57 2429.89

(1285.78) (586.91) (109.20) (65.52) (2047.41)

Aug. 1589.14 ± 136.67 997.70 ± 219.67 175.43 ± 16.24 97.22 ± 11.28 2859.49(1325.50) (845.35) (133.73) (76.51) (2381.09)

Sep. 1516.36 ± 162.50 608.88 ± 95.58 170.35 ± 15.75 85.51 ± 16.46 2381.10

(1325.50) (503.94) (129.86) (67.30) (2004.90)Oct. 1291.40 ± 129.04 574.61 ± 146.30 188.24 ± 19.08 95.23± 10.72 2149.48

(1104.15) (480.03) (143.49) (74.95) (1802.62)

Nov. 1946.21 ± 176.01 530.47 ± 101.24 116.00 ± 14.78 75.72 ± 11.01 2668.40(1761.51) (462.04) (86.73) (59.55) (2369.83)

Dec. 1287.35 ± 119.09 478.67 ± 63.66 86.83 ± 19.95 37.12 ± 15.64 1889.97

(1042.11) (395.76) (64.92) (29.19) (1531.98)Jan. 1760.75 ± 275.88 637.65 ± 134.14 144.72 ± 17.61 90.59 ± 8.90 2633.71

(1458.43) (527.97) (108.21)) (71.24) (2165.85)

Feb. 2579.15 ± 254.65 605.50 ± 69.39 186.04 ± 8.04 109.81 ± 7.24 3480.50(2221.42) (446.62) (144.03) (82.42) (2894.49)

Mar. 1994.53 ± 242.17 433.63 ± 47.48 157.31 ± 8.13 80.20 ± 7.12 2665.67

(1618.16) (337.84) (121.79) (60.20) (2137.99)

Average 1677.88 685.18 148.14 78.17 2589.38(1409.48) (551.71) (113.20) (60.70) (2135.10)

1 Standard error of the mean

132 Kasetsart J. (Nat. Sci.) 34 (1)

appears to be the best estimate compared to theothers. Since this method was the only method in

the study that took mortality into account and gave

the estimates in between the other two methods, itis reasonable to utilize this method for further

discussion.

Belowground net primary production andturnover

Estimates of annual belowground netproduction on strata 1 and 2 were 3,426 g m-2 (9.39

g m-2 day-1) and 3,383 g m-2 (9.27 m-2 day-1),

respectively (Table 6). The turnover rate ofbelowground biomass was calculated by the method

proposed by Dahlman and Kucera (1965). The

ratio between belowground net production andmaximum belowground biomass gave a turnover

value. It was estimated that approximately 95

percent of the belowground biomass in stratum 1and 97 percent in stratum 2 of Arundinaria pusilla

would be replaced each year (Table 6). The figures

were rather high compared to the grassland range intemperate zones. Nilsson (1970) found that about

50 percent of the root would be replaced each year

or turnover every two years. Dahlman and Kucera(1965) indicated that 25 percent of the root system

in grassland range would be replaced each year,

producing turnover rate of roots every four years.In the ungrazed shortgrass prairie, Sims and Singh

(1971) reported the turnover rate was 36 percent

per year.

Net primary production, accumulation anddisappearance rates

Net primary production, accumulation and

disappearance rates by compartments for stratum 1

and 2 are presented in Table 7.These data showed that total net production

(3,212 g m-2), aboveground net production (329 g

m-2), and belowground net production (2,883g m-2) as well as their rates of production on

stratum 1 were greater than those on stratum 2. The

Table 5 Comparison of estimates of aboveground net primary production of Arundinaria pusilla basedon three methods of estimation in the dry dipterocarp forest strata 1 and 2.

Method of estimating production Stratum 1 Stratum 2

ANP 1/ Rate of ANP 1/ Rate of (g m-2) production (g m-2) production

(g m-2 day-1) (g m-2 day-1)

1. Based on the community 364.27 ± 30.032/ 0.99 312.74 ± 30.86 0.86peak standing crop (335.82)3/ (0.92) (287.60) (0.79)

2. Based on the sum of 341.90 0.93 227.75 0.62

positive change in biomass (328.84) (0.90) (210.33) (0.58)plus mortality

3. Based on the sum of positive 287.40 0.79 207.55 0.57

increase in biomass (276.61) (0.76) (193.81) (0.53)

1/ ANP = annual aboveground net production2/ Standard error of the mean3/ Ash-free dry weight in parenthesis.

Kasetsart J. (Nat. Sci.) 34 (1) 133

Table 6 Annual belowground net production and turnover rate of belowground biomass in the drydipterocarp forest stratum 1 and 2. (ash-free dry weight in (g m-2) parenthesis)

Stratum Belowground net production1/ Rate of production Maximum biomass Turnover(g m-2) (g m-2 day -1) (g m-2) %

1 3425.65 9.39 3623.29 94.5

(2883.58) (7.90) (3031.64) (95.1)

2 3382.58 9.27 3480.50 97.2(2844.33) (7.79) (2894.49) (98.2)

1/ Based on the sum of positive changes in biomass on successive sampling dates.

Table 7 Annual net primary production accumulation and disappearance rates (ash-free basis) by

compartments of Arundinaria pusilla in the dry dipterocarp forest strata 1 and 2.

Compartment Stratum 1 Stratum 2

gm-2 gm-2day-1 gm-2 gm-2day1

Total net primary production (TNP) 3212.42 8.80 3054.66 8.37

Aboveground net primary production (ANP) 328.84 0.90 210.33 0.58

Standing dead (SD) 256.35 0.70 198.17 0.54Litter (L) 213.93 0.59 136.82 0.37

Litter disappearance (LD) 149.37 0.41 119.72 0.33

Belowground net primary production (BNP) 2883.58 7.90 2844.33 7.79Belowground biomass disappearance (RD) 2572.74 7.05 2794.37 7.66

Total disappearance (TD) 2722.11 7.46 2914.09 7.98

rate of accumulation of organic matter in thestanding dead compartment was also greater on

stratum 1 than on stratum 2. Moreover,

accumulation of organic material in the littercompartment was higher on stratum 1 (214 g m-2)

than on stratum 2 (137 g m-2). Annual diappearance

of litter was also higher on stratum 1 than onstratum 2. But the belowground disappearance as

well as the total disappearance, was much greater

on stratum 2 than on stratum 1, although thebelowground net production on both strata were

only slightly different.

There was an annual surplus of 490 g m-2 ofdry matter on stratum 1, much higher than that on

stratum 2 (140 g m-2). This is because the rate of

belowground biomass disappearance on stratum 2(7.66 g m-2 day-1) was greater than that on stratum

1 (7.05 g m-2 day-1) although there was a higher rate

of litter disappearance, 0.41 g m-2 day-1 on stratum1 compared to 0.33 g m-2 day-1 on stratum 2. It was

clear that the belowground portion of this kind of

grass plays an important role in the dynamic of thesystem as a whole. This study confirmed that the

belowground biomass in this kind of plant

134 Kasetsart J. (Nat. Sci.) 34 (1)

community should be considered as being very

important in the study of the ecosystem functions.

System transfer functionThe system transfer function is the quantity

by which the system block multiplied the input to

generate the output (Grodin, 1963) or it is the ratio

of output to input (Golley, 1965). The systemtransfer function for the whole year on strata 1 and

2 is presented in Table 8. The functions were

calculated using the compartment values in Table 7.On the annual basis, the transfer functions

on stratum 1 had 10 percent aboveground and 90

percent belowground production campared to 7percent aboveground and 93 percent belowground

production on stratum 2. There was 78 percent of

aboveground net production which found its wayinto the standing dead compartment on stratum 1

and about 94 percent on stratum 2. The annual

transfer of standing dead to litter was 84 percent onstratum 1 and 42 percent on stratum 2. This

amounted to 65 percent of the aboveground net

production on stratum 1 and 39 percent on stratum2. It is evident that some direct transfer may also

occur from the live vegetation to the litter

compartment. Galley (1965) stated that a relativeamount of litter might be contributed by current

live vegetation. Uresk et al. (1975) noted that littermay increase from live herbage because of rain,

hail, wind and insects. Of the total litter produced,

70 percent on stratum 1 and 88 percent on stratum2 were decomposed within the same year.

The annual transfer from belowground net

production to belowground disappearance was 89percent on stratum 1 and 98 percent on stratum 2.

Within the year, about 85 percent of total net

production disappeared from the system on stratum1 and 95 percent on stratum 2. This gave on annual

net gain of total net production of about 15 percent

on stratum 1 and 5 percent on stratum 2.

Calorific content of biomass compartmentsCalorific content of Arundinaria pusilla

was determined by a composite sample from both

strata. The average calorific values of standing

live, standing dead, litter and belowground biomasswere 4.312, 4.375, 4.237 and 4.462 kcal g-1 ash-

free dry weight, respectively. Biomass in g m-2

may be converted to energy by multiplying thesevalues by the appropriate calorific equivalents to

estimate the standing crop of energy in the

community. On an ash-free basis, the energystored aboveground was 2,029 kcal m-2 on stratum

1 and about 1,689 kcal m-2 on stratum 2. The

Table 8 System transfer functions of Arundinaria pusilla in the dry dipterocarp forest.

Compartments Stratum 1 Stratum 2

TNP to ANP 0.102 0.069

TNP to BNP 0.898 0.931ANP to SD 0.780 0.942

SD to L 0.835 0.690

ANP to L 0.650 0.651L to LD 0.698 0.875

BNP to RD 0.892 0.892

TNP to TD 0.847 0.954

Note : For notation see table 7.

Kasetsart J. (Nat. Sci.) 34 (1) 135

average energy stored belowground fluctuated

around 9,900 to 9,588 kcal m-2 on stratum 1 and 2,respectively.

Efficiency of energy captureIn order to determine the energy capture in

aboveground and belowground production in kcalm-2, the appropriate mean calorific value mentioned

above has been used to convert the aboveground

and belowground net production on both stratafrom g m-2 to kcal m-2 (Table 9). The efficiency of

energy capture was expressed as percentages of

energy within the visible portion of the spectrumwhich was assumed to be 50 percent of the total

solar insolation (Golley, 1960; Odum, 1971). The

efficiency values have been calculated and presentedin Table 9, which includes the aboveground,

belowground and total net production on both

strata.The efficiency of annual energy capture for

aboveground net production on stratum 1 (0.18%)

was more efficient than that on stratum 2 (0.12%).The efficiency of energy capture of aboveground

net production was less than that of belowground

net production. The efficiency of energy capture inbelowground (1.68%) and total net production

(1.86%) on stratum 1 was more than that in

Table 9 Usable solar insolation, annual net primary production and efficiency of annual energy capture(ash-free dry weight) of Arundinaria pusilla in the dry dipterocarp forest stratum 1 and 2.

Energy used by compartments Annual net primary 2/ Efficiency (%)production (kcal m-2)

Stratum 1 Stratum 2 Stratum 1 Stratum 2

Usable solar insolation1/ 767,845 767,845 - -

Abeveground net production 1,418 907 0.18 0.12Belowground net production 12,867 12,691 1.68 1.65

Total net production 14,285 13,598 1.86 1.77

1/ 50% of total solar insolation.2/ Net production/usable solar insolation.

belowground (1.65%) and total net production

(1.77%) on stratum 2.In the shortgrass ecosystem Sims and Singh

(1971) reported that the efficiency of total net

production based on 50 percent of solar insolationwas 0.57 at the Pawnee and 0.19 percent at the

Pantex site. Klipple and Costello (1960) found theefficiency based on 45 percent of solar insolation

was 1.3 percent. However, for most ecosystems

the annual efficiency of solar energy conversion inthe visible spectrum into net production potentially

was near 1 percent or less (Woodwell and Whittaker,

1968).These data indicated that the variation of the

efficiency values depended on the time intervals

and the estimation of solar insolation used in thedetermination, as well as the community types of

vegetation under study.

Annual energy flowBased on the system transfer functions in

table 8, the estimate of the annual energy flowthrough the primary producer compartments on

stratum 1 and 2 of Arundinaria pusilla in the dry

dipterocarp ecosystem is depicted in Figures 1 and2, respectively. The usable solar insolation was

767, 845 kcal m-2, based on 50 percent of the

136 Kasetsart J. (Nat. Sci.) 34 (1)

incident solar energy. Golley (1965) estimated therespiration by analyzing the CO2 content of the air

in a chamber around the plant and found that about

47 percent of gross primary production was lostthrough respiration. Values of gross primary

production and respiation in the present study were

based on the assumption that, in general, 50 percentof the gross primary production was dissipated by

respiration and the remaining 50 percent was total

net primary production (Odum, 1971).There was a surplus of energy on both sites,

15 percent on stratum 1 and 5 percent on stratum 2.

More surplus energy was stored in belowground

than aboveground on stratum 1 but on the contraryfor stratum 2. Arundinaria pusilla on stratum 1

stored 36.5 percent surplus energy in aboveground

production and 63.5 percent in belowgroundproduction. But Arundinaria pusilla on stratum 2

stored 63.9 percent surplus energy in aboveground

production and 36.1 percent in belowgroundproduction.

Therefore, if these two strata remain

Figure 1 Annual energy flow (kcal m-2) through primary producer compartment of Arundinaria pusilla

in the dry dipterocarp forest stratum 1.

Figure 2 Annual energy flow (kcal m-2) through primary producer compartment of Arundinaria pusilla

in the dry dipterocarp forest stratum 2.

Kasetsart J. (Nat. Sci.) 34 (1) 137

unburned or ungrazed for a long period of time, thesurplus of energy in the form of organic matter

would probably accumulate and increase soil

organic matter and eventually improve soil moisturestorage. The excess production, therefore, may

change the environmental conditions of the

ecosystem in the longrun. But in reality, plants andlitter in this dry dipterocarp forest were burned out

every year during the dry season. In order to reduce

the ground fuel and utilize the surplus energy of thesystem efficiently, the introducing of cattle raising

in this forest at the early of the growing season

should be carried out every year. However, a studyof the nutrient cycling of the undergrowth in this

ecosystem is recommended.

ACKNOWLEDGEMENTS

This research study was sponsored by theNational Research Council of Thailand and under

the close supervision of the Faculty of Forestry,

Kasetsart University. The author is extremelygrateful to the Agriculture Chemistry Division for

their valuable assistance in chemical analyses.

Special appreciation must be expressed to the staffof SERS for their assistance in the field and use of

their facilities.

LITERATURE CITED

Bartos, D.L. and P.L. Sims. 1974. Root dynamicsof a shortgrass ecosystem. Journal of Range

Management. 27 : 33-36.

Dahlman, R.C. and C.L. Kucera. 1965. Rootproductivity and turnover in native prairie.

Ecology 46 : 84-89.

Golley, F.B. 1960. Energy dynamics of a foodchain in an old field community. Ecological

Monographs 30 : 187-206.

. 1965. Structure and function of anold-field broomsedge community. Ecological

monographs 35 : 113-137.Grodins, F.S. 1963. Control theory and biological

systems. Columbia University Press, New

York. 205 p.Hadley, E.B. and B.J. Kieckhefer. 1963.

Productivity of two prairie grasses in relation

to fire frequency. Ecology 44 : 389-395.Kelly, J.M., G.M. van Dyne and W.F. Harris.

1974. Comparison of three methods of assesing

grassland productivity and biomass dynamics.The American Middland Naturalist 92 :

357-367.

Klipple, G.E. and D.F. Costello. 1960. Vegetationand cattle responses to different intensities of

grazing on shortgrass ranges on the central

Great Plains. United State Department ofAgriculture. Technical Bulletin number 1216.

82 p.

Lewis, J.K. 1969. Primary producers in grasslandecosystem, pp. 241-1 to 241-87. In R.L. Dix

and R.G. Beidleman (eds.). The grassland

ecosystem : A preliminary synthesis. RangeScience Department. Science Series. No.2

supplement. Colorado State University, Fort

Collins, Colorado.Nilsson, J. 1970. Notes on the biomass and

productivity of belowground organs of a south-

Swedish hay-meadow. Botanist Notiser 123 :183-194.

Odum, E.P. 1960. Organic production and turnover

in old-field succession. Ecology 41 : 34-49. . 1971. Fundamentals of Ecology. 3rd

ed. W.B. Saunders Co. 574 p.

Ruangpanit, N. 1981. Some ecologicalcharacteristics of Arundinaria pusilla. Forest

Research Bulletin Number 80, Faculty of

Forestry, Kasetsart University. Bangkok. 21 p.Sims, P.L. and J.S. Singh. 1971. Herbage dynamics

and net primary production in certain ungrazed

and grazed grassland in North America, pp.59-124 In N.R. Frech (ed.). Preliminary

138 Kasetsart J. (Nat. Sci.) 34 (1)

analysis of structure and function in grassland.Range Science Department. Science Series

Number 10, Colorado State University, Fort

Collins, Colorado.Singh, J.S. and P.S. Yadava. 1974. Seasonal

variation in composition, plant biomass, and

net primary productivity of a tropical grasslandat Kurushetra, India. Ecological Monographs

44 : 351-356.

Uresk, D.W., P.L. Sims, and D.A. Jameson. 1975.

Dynamics of blue grama within a shortgrassecosystem. Journal of Range Management 28

: 205-208.

Woodwell, G.M. and R.H. Whittaker. 1968. Primaryproduction in terrestrial ecosystems. American

Zoologist 8 : 19-30.

Received date : 9/08/99Accepted date : 30/12/99

Kasetsart J. (Nat. Sci.) 34 : 139 - 144 (2000)

Electronic Knowledge Delivery: Developing a Web-based Systemfor Computer Course

Anongnart Srivihok

ABSTRACT

The universe of information provided on the World Wide Web (WWW) has been delivered to userstremendously. These millions of Web documents induce the oversupply of information. The study which

improved a Web-based system development might increase the successful experience and satisfaction of

users. This present study identifies the WWW publishing and highlights methods and tools for developinga Web-based system. Further, this paper describes the design and the development of a Web-based system

for a computer course. The system allows a personalised user interface and a presentation of course

materials through the WWW.Key words: Web-based system, electronic knowledge, WWW, development.

Department of Computer Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

INTRODUCTION

The WWW is a wide area hypermediainformation system providing a global access to

large amounts of documents (Foo and Lim, 1997).

The multitude of Web sites has created muchcomment on the existence of information overload.

Hyper Text Markup Language (HTML) is a

hypermedia language used to build WWWdocuments. This language allows the user to define

the logical organizational and various formats

including ordinary text documents, graphics,multimedia, hyperlinks to other HTML documents

and Internet resources. Hyperlinks are powerful

utilities for navigating and cross referencinginformation. The WWW resource has a unique

address namely Uniform Resource Locator (URL).

Components of web publishingThe essential elements required for

publishing and access information on the WWWare as follows:

1. Web server. It is compulsory that an

individual or enterprise subscribes to an informationsystem service providing access to the internet.

Organizations which have substantial computing

resources can set up their own HTTP server andconnect to the internet.

2. HTML authoring tool. To compose

the Web pages an authoring tool is needed. Webpages are made of HTML which is an ASCII text

file consisting of HTML tags. Common text editors

can be used for this task. To create commercial ormore complicated HTML, such as multimedia Web

pages, a higher functionality tool such as Perl

(O’Rielley and Associates Inc., 1998), HoTMetal(SoftQuad, 1998) and FrontPage (Microsoft

Corporation, 1998) are used for PC based editors.

Alternatively, Pheonix (University of Chicago,1998) , EMAChhm (Minar, 1998) and HoTMetal

140 Kasetsart J. (Nat. Sci.) 34 (1)

(SoftQuad, 1998) are Unix based editors.3. Web browser. Web browsers are client

software which can be used to navigate and access

the large volume of Web pages stored in differentWeb servers on the internet. The browsers are text

and graphics-based, however the later is more

preferred because they can display graphics andicons which are more user friendly. The most

commonly used Web browsers are Netscape

Communicator (Netscape Communications 1998)and Microsoft Internet Explorer (Microsoft

Corporation 1998).

Development of web-based systemsPrevious studies have suggested different

factors involved in the design and development of

the Web-based systems. These factors include stylesof Web sites, objectives of designers, communities

of users and spectrum of tasks (Schneiderman,

1997) and domains of user works (Erskni, David,Carter-Tod and Burton, 1997; Picciano, 1998).

Interestingly, no study has introduced the

framework or the relationships between the factorsof the system development.

As in any development process of

Information Systems, it requires the developers toundertake a number of related activities. Benyon

(1997)’s study proposes six essential processes for

developing Web-based systems. They includesystem specification, instructional design,

multimedia design, integration, implementation

and evolution. However, Benyon (1997) suggeststhat new tools and approaches are needed to enhance

the effective development of a Web-based system.

Thus, a study of the comprehensive process ofdesign and development of Web-based system is

required to improve the efficiency and the

effectiveness of the system.This paper aims to present a model of Web-

based system development and to provide tools and

methods to guide Web designers. Besides,

difficulties of the Web publication and somerecommendations for improving communication

through the WWW will be presented.

MATERIALS AND METHODS

A web-based system for computer courseCPI is a Web-based teaching system

designed for an undergraduate course named

418211 Computer Programming I. It is a 3 credit

course which is offered at the Department ofComputer Science, Faculty of Science, Kasetsart

University.

Various Web-publishing tools had been usedfor developing CPI. These tools include a Web

server, a Web browser and HTML authoring tools.

First, the Web server (HTTP) is a Sun Sparc 1000Eresiding at the Department of Computer Science,

Faculty of Science, Kasetsart University. This server

is connected to the Internet via Kasetsart Universitynetwork. Second, the two commonly used Web

browsers, Netscape Navigator (Netscape

Communications, 1998) and Microsoft InternetExplorer (Microsoft Corporation, 1998), can be

used for CPI. The former browser is preferred

because it is more popular in Thailand. Third,HTML authoring tools are text editor and Web

authorizer. The text editor, Microsoft Word 97,

was used to compose large text documents for theWeb. Additionally, Perl (O’Reilley and Associates

Inc., 1998) and Java (Sun Microsystems Inc., 1998)

were selected to build more attractive features onWeb sites and allow users to interact with the

system easily.

The Computer Programming I course wasused as the information profile designed to be

published on the WWW. The information profile

was broken systematically and logically to treestructures revealed in Figure 1. The hierarchical

tree structure can be used as a model to define and

cross-link the components of the Web-based system.

Kasetsart J. (Nat. Sci.) 34 (1) 141

The hyperlinks connect different informationtogether, further cross-hierarchical tree structures

can be connected by linking these structures. For

this purpose, these structures are used for making amodel to publish Web documents.

The Web home page referring to the first

document intended to be viewed is placed at the topof the tree. This home page includes an introduction

and a main menu of documents within the

publication. Generally, it is associated with aparticular web site, person, named collection, or

private or public enterprise. The main menu consists

of the hyperlinks to other home pages or documents.With the homepage, the main menu of CPI

contains the principal focus and is differentiated

into different common components as depicted inFigure 2.

The components of CPI1. Chapters. The important parts of each

chapter are selected and published on the Web

pages. The reasons are the limitations of the space

on the Web site and a large graphic Web pagerequires overly long response time. Users can

download full text of all chapters from the server by

using the given password. The materials in thesechapters are comprehensive and fulfilled the

requirements provided in the course outline.

2. Exercises. The exercises of each chapterare provided in both HTML and text.

3. Help. It offers the details of hardware

and software configuration required to work withthe system. Furthermore, it includes the

methodology in download document files provided

by the lecturer.4. Course Syllabus. It provides the

objectives of the course, course outline, course

description, text books, contact address, gradingsystem and schedule.

5. News. It reveals an announcement which

the information from the lecturer to users such asassignments and current news.

6. General Information. It provides the

objectives of the current Web-based system.

Figure 1 Information profile of 418211 Computer Programming I course on Web sites.

418211

News General Chapters Exercises Syllabus Help Information

Announcement

I/O commands Data types Control Structure Array Records

Files Pointers Sorting Searching

142 Kasetsart J. (Nat. Sci.) 34 (1)

By using HTML tools, the Web documentsare coupled and cross-linked with the detailed

information provided for each component. As the

information comes from various sources ofdocuments, it is noted that the published information

should be uniform and homogenous. Consequently,

a schedule of regular maintenance to manageproposed changes and to present information is

also required to enhance overall effectiveness and

efficiency of the system.

RESULTS AND DISCUSSIONS

Since June 1998, CPI was published on the

Web. Students who enroll in 418211 Computer

Programming I class can access the course materialson Web sites and communicate with the lecturer via

email through the links provided on the Web page.

However, there are still problems in utilisation ofthe system. These problems are presented in the

following paragraphs.

Difficulties of web-based system implemen-

tationSome problems of CPI implementation have

been raised by the users after the system was

published on the WWW.

1. Inappropriate search engine. There aremany search engines provided in the WWW

includes Lycos, Infoseek, Excite and Yahoo.

Interestingly, these engines often provide verylong lists of matching documents if the index is

provided. Then, the documents have to be navigated

one at a time. The home page of the document isrevealed in order to access the hypermedia areas

via hyperlinks. This is performed until the relevant

information is delivered. That is appropriate forsearching information from several resources. Still

this is time consuming and unsuitable for cases

when a specific and detailed query is known.Additionally, not many search engines in other

languages from English are provided. Applying

English-based search engine to a foreign language

Figure 2 CPI home page using Netscape Communication, the URL address is http://www.sci.ku. ac.th/~fsciang2/index.html.

Kasetsart J. (Nat. Sci.) 34 (1) 143

database such as Thai creates some difficulties indata presentation on the Web. In this case,

developers need to build their own search engine

for specific purposes and tasks.2. Degradation of performance. The large

number of WWW users decrease the system

performance, the network is overloaded due to theextremely heavy traffic. This situation always occurs

with the popular Web sites. It leads to the frustration

of the users and loss of interest in utilization of theWWW.

3. Maintainability of publication. As the

time passes, more information is always added onthe WWW. This can result in a high growth rate of

Web documents. As a result this will lead to the

point which is difficult to maintain and ensure theaccuracy of information. It is suggested that the

management of Web documents is needed.

4. Data transmission. In developingcountries such as Thailand, there are various

limitations of data transmission. These factors

include poor communication infrastructure, low-cost devices, and low-bandwidth wireless access.

Since the data transmission is very slow, multimedia

which require high speed of transmission cannot beapplied on the Web effectively. The text version of

Web-based system is recommended for this case.

CONCLUSIONS

The major contribution of this study is anattempt to present tools and methods in developing

a Web-based system. CPI has been implemented to

demonstrate the research idea and allow the structureand contents to publish on the Web. The design is

practical and might be used in developing a

prototype for Web-based systems, specifically acourseware for internal or for distance learning.

Future research on the development of Web

publishing might be done to improve theeffectiveness and efficiency of the system. The

study of the new designing techniques or theframework of Web-based system is recommended

for further research.

ACKNOWLEDGEMENT

The author would like to acknowledge theKasetsart University Research and Development

Institute for providing funding for this study.

LITERATURE CITED

Benyon, D., D. Stone, and M. Woodroffe. 1997.Experience with developing multimedia

courseware for the World Wide Web: the need

for better tools and clear pedagogy. Int. J.Human-Computer Studies. 47 : 197-218.

Erskni, L. E., R. David, N. Carter-Tod and J. K.

Burton. 1997. Dialogical techniques for thedesign of web sites. Int. J. Human-Computer

Studies. 47 : 169-195.

Exite Inc. 1998. Exite. Available http://www.exite.com/home/, July 1998.

Foo, S. and E. P. Lim. 1997. A hypermedia database

to manage World-Wide-Web documents.Information & Management. 31 : 235-250.

Infoseek Corporation. 1998. Infoseek Guide.

Available http://www.infoseek.com/ home,July 1998.

Lycos Inc. and Carnegie Mellon University. 1998.

Lycos. Available http://lycos.com/, July 1998.Microsoft Corporation. 1998. Microsoft FrontPage.

Available http://www.microsoft. com/

frontpage/, July 1998.Microsoft Corporation. 1998. Internet Explorer.

Available http://www.microsoft.com/

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Received date : 13/10/98Aecepted date : 23/06/99

Kasetsart J. (Nat. Sci.) 34 : 145 - 158 (2000)

INTRODUCTION

Whenever the water available is less than

the expected amount or the irrigation water

requirements exceed the availability, the watershortage takes place even in the irrigated area. The

issues of high irrigation water requirements is

usually related to the efficiency in water delivery

Development of Water Allocation Strategy to IncreaseWater Use Efficiency of Irrigation Project

Varawoot Vudhivanich1, Jesda Kaewkulaya1, Ponsatorn Sopaphun1,Watchara Suidee2 and Prapun Sopsathien3

ABSTRACT

In development of water allocation strategy to meet crop water requirement and to utilize rainfall

effectively, a computerized water allocation scheduling and monitoring system named WASAM 2 wasdeveloped. Song Phi Nong irrigation project was selected as a case study. WASAM 2 was used for water

allocation scheduling in Song Phi Nong project for 3 cultivation seasons of 1994 dry and wet seasons and

1995 dry season. The discharge at 5 key regulators which are the entrance to 5 water masters weremonitored on daily basis. The actual delivery performance of the 3 studied seasons indicated that the actual

measured discharge was about 20-30% higher than the recommended WASAM 2 discharge because Song

Phi Nong project was the downstream project and had to take all the remaining water from the upstreamPhanom Tuan project. It is recommended that the water allocation scheduling system of Song Phi Nong

and Phanom Tuan projects will be combined into one system to increase the efficiency in water allocation.

The average irrigation efficiency of Song Phi Nong was 37.6% in dry season and 28.5% in wet season.WASAM 2 increased the wet season efficiency by 3-5%. The irrigation efficiency in the dry season was

on the average 7.7% higher than that in the wet season. Some water master has the efficiency up to 46.9%

in dry season. The irrigation efficiency of the project is still low by two reasons : (1) being unable tocompletely control the discharge, and (2) high seepage loss in the canal system.

Key words : water allocation, water management, irrigation efficiency, Mae Klong

and application. As commonly known, the projectirrigation efficiency in Thailand is still low at only

30-50%. If the project irrigation efficiency is

increased, it can improve water shortage problemat more cost effectively than developing the new

water resources project.

In order to improve the water deliverly,application efficiency and the equity of water

1 Department of Irrigation Engineering, Kasetsart University, Kamphaengsaen, Nakhon Pathom 73140, Thailand.2 College of Irrigation, Royal Irrigation Department, Nonthaburi 11120, Thailand.3 Regional Office 10, Royal Irrigation Department, Kanchanaburi 71110, Thailand.

146 Kasetsart J. (Nat. Sci.) 34 (1)

distribution among the head-and-end users, RoyalIrrigation Department (RID) has developed various

water allocation system including Water

Management Systems (WMS) of the Greater ChaoPhraya Irrigation Project having an irrigated area

of over 7.0 million rai. The main feature of WMS

is the prediction of planting area for 1-2 weeks inadvance and the utilization of return flow in the

downstream irrigation project. The computer

program was developed for calculation of waterrequirements on block basis; each block

representing an area of approximately 50,000 rai

(Chalong, 1984). On the same purpose, WaterAllocation Scheduling and Monitoring system or

WASAM was developed for the Greater Mae Klong

Irrigation Project (GMKIP). The main purpose ofWASAM is to determine the required discharge of

the canal section, 3,000-5,000 rai each, from the

long term average potential evapotranspiration(ETo), crop coefficient (Kc) and the cropping pattern

surveyed at the beginning of the season. The

irrigation water requirements are adjusted accordingto the field wetness and the deviation of the actual

rainfall from the anticipated value. The main

improvement of WASAM is to incorporate thehydraulic properties of the canal in water allocation

planning. Besides, the actual water distribution in

the canal system (at several key regulators) ismonitored on weekly basis in order to check field

water delivery performance (Ilaco/Empire M&T,

1984; 1985; 1986(a); 1986(b); 1986(c)). Later onWASAM has been applied to Lam Pao (RID,

1988(a); 1988(c)) and Nong Wai (RID,

1988(d))irrigation projects in the Northeast ofThailand. Water Use Analysis (WUA) program

was also developed to assist the irrigation engineer

in evaluation of water use efficiency of the LamPao project at zone, water master and main canal

levels (RID, 1988 (b)).

Although WMS, WASAM and WUA aregood tools for project water allocation, they require

a lot of field data including planting area, rainfalland flow measuring data. Good trained field staff

and additional expenditures are needed to implement

these water allocation system. This prohibits theapplication of these systems except in the irrigation

project having water management expert and special

funding system.As mentioned above, the project water

allocation techniques which responses to the crop

water requirements, reduces the water losses andincreases an effective use of rainfall in such way

that the irrigation efficiency of an irrigation project

increases, need to be developed. The objective ofthis research is to develop an effective water

allocation scheduling and monitoring system of an

irrigation project.

MATERIALS AND METHODS

Song Phi Nong irrigation project in

Suphanburi province which is a typical irrigation

project in Mae Klong basin is selected for the study.The methods adopted for this research are :

(1) to study the existing method of water

allocation in Song Phi Nong irrigation project,identify the problems and constraints,

(2) to evaluate irrigation efficiency of the

present condition and use as a benchmark forevaluating the effectiveness of the improved water

allocation method,

(3) to analyze the basic data and informationrequired for effective water allocation and design

the data collection and processing system which

are appropriated to the project,(4) to develop water allocation method

which takes into consideration of crop water

requirement, canal delivering capacity and effectiveutilization of rainfall.

(5) to Develop WASAM 2 computer

program to be a tool for project water allocation.

Kasetsart J. (Nat. Sci.) 34 (1) 147

Present water allocation practices of Song Phi

Nong Irrigation ProjectSong Phi Nong irrigation project is one of

the 3 irrigation projects on the upper left bank of

GMKIP, having the command area of 416,106 rai.

Its upstream and downstream projects receivingirrigation water from the same 2L canal are Phanom

Tuan and Banglane. The project divides the

operation and maintenance responsibility into 5water masters (43 zones) as shown in Table 1.

There are totally 39 operation and maintenance

staffs where 20 are zoneman. Each zoneman isresponsible for O&M in an approximated area of

20,000 rai which is exceeding the normal criteria of

RID, 5,000-10,000 rai per zoneman. Therefore, theproject is in shortage of field O&M staff which may

effect the water delivery control practices.

The principle of water allocation of GMKIPand Song Phi Nong irrigation project is to allocate

water on weekly basis according to crop water

requirements. Thursday (6.00 am.) is the first dayof the irrigation week. Upstream control with

continuous delivery is practiced in the main and

lateral canals while rotation delivery is practiced atthe tertiary level of paddy growing area (almost no

on-farm water distribution system in sugarcane

area). Each farm ditch has a fixed duration-varieddischarge rotation schedule.

At the beginning of irrigation week

(Thursday at 6:00 pm), all the regulators are adjustedby zoneman according to the new water allocation

schedule. The adjustment normally starts in

sequence from the most upstream to the downstreamregulators according to the upstream control

concept. In order to check the actual water

distribution, Song Phi Nong project has set the flowmonitoring points at the head regulator of 5L-2L,

the main intakes to water master section 2, 3, 4 and

5, and at the cross regulator of 2L at km 22.700, themain headworks of the project. However, the project

is lack of field staff to do the routine daily

measurement and most of the regulators areuncalibrated.

At the present, the management of Song Phi

Nong project faces the following problems :(1) unreliable water supply since Song Phi

Nong is located on the downstream of Phanom

Tuan project,(2) too few number of rain gages, only 4

stations (Uthong, Song Phi Nong, Kamphaengsaen

and Phanom Tuan), are used in water allocationscheduling,

(3) most of the key regulators are not

calibrated, assuming 0.7 of the discharge coefficient,resulting in uncorrected delivery and distribution

of water to the farm land,

(4) many farmers in water master 2 and 3pump water directly from the main canal because

Table 1 Division of responsibility for O&M in Song Phi Nong Irrigation Project.

Water Responsible area Zone Command area Main crop

master (rai)

1 Bor Suphan 1-8 92,734 Sugarcane

2 Nong Phaw 9-18 106,421 Sugarcane3 Talad Khet 19-25 59,904 Sugarcane

4 Jorrakhe Sarmpun 26-32 55,375 Paddy (2 crops a year)

5 Sra Punglarn 33-43 101,672 Paddy (2 crops a year)Total 416,106

148 Kasetsart J. (Nat. Sci.) 34 (1)

of lack of farm turn outs, making difficulty in thewater delivery control and oftenly unfair water

distribution to tail-end users.

The existing WASAM computer programwhich was originally developed by Ilaco/Empire

M&T for GMKIP in 1985 had some drawback in

real practices. It requires a large number of fielddata exceeding the capability of field staff to handle

the daily data collection effectively. WASAM has

no capability for evaluation of managementperformance of an irrigation project. Lastly the

WASAM computer program is not user friendly

making it difficult to use. Therefore, the existingWASAM needs improvement.

RESULTS AND DISCUSSIONS

Development of WASAM 2Due to the shortcoming of the existing

WASAM as mentioned earlier, WASAM 2 wasdeveloped to help the irrigation engineer in charge

of an irrigation project in calculation of the required

discharge of the regulators, in evaluation of thewater management performance and in monitoring

analysis of the actual water distribution. The

differences between WASAM 2 and the existingWASAM are shown in Figure 1.

In calculation of the required discharge, the

Song Phi Nong Irrigation Project is divided into 57canal sections as shown in Figure 2. Each canal

section is the canal reach between two regulators.

The irrigation water requirements of each canalsection is first calculated using the following

equations :

R(N) =

EV ES CF w I LP w I COR RE RS A I NI

NC( ) * ( , ) ( , ) ( ) * ( , )

, *

1 1

378 0001

+ + −[ ]=∑

SUE(w1, I)

..........(1)

R(N) = Irrigation water requirement of canalsection N in week W (cms)

EV(ES) = Weekly potential evapotranspiration

of station ES (mm)CF(w1,I) = Crop factor of crop I at the age of w1

weeks

NC = No. of crop types (there are 6 typesof crop ; 1 = dry season paddy ; 2 =

wet season paddy ; 3 = sugarcane ; 4

= upland crop ; 5 = fruit tree and 6 =fish pond)

LP(w1,I) = Weekly land preparation and

percolation of crop I at the age of w1weeks (mm)

COR = Correction factor due to rainfall or

field wetness conditionsRE(RS) = Expected rainfall of station RS (mm)

A(I,N) = Area of crop I in canal section N (rai)

SUE(w1,I) = Service unit irrigation efficiency ofcrop I at the age of w1 weeks

The required discharge of any canal section(N) is calculated as follow :

Q(N) = R(N)+ Q I LOSS NI

M( ) ( )+

=∑

1

............ (2)

WhereQ(N) = Required discharge of canal section

N (cms)

R(N) = Irrigation water requirements ofcanal section N (cms)

Q(I) = Required discharge of the

downstream canal section I (cms)LOSS(N) = Conveyance losses from the source

of water supply to canal section N

(cms)The calculated Q(N) is thus adjusted

according to the following water distribution

criteria :(1) If Q (N) < 0.4 Qmin(N) (the discharge

is too small)

Kasetsart J. (Nat. Sci.) 34 (1) 149

Figure 1 Comparison of the structure of the WASAM 2 and WASAM (Vudhivanich et al.,1998).

150 Kasetsart J. (Nat. Sci.) 34 (1)

Figure 2 Schematic diagram showing the structure of canal sections of Song Phi Nong Irrigation Project.

Kasetsart J. (Nat. Sci.) 34 (1) 151

Q (N) = 0and D (N) > 0 (special counter for shortage)

(2) If 0.4 Qmin(N) < Q (N) < Qmin(N)

Q (N) = Qmin (N) (to maintain the minimumsupply level in section N)

Except R (N) = 0 and the canal sections

branching off directly from section N do not requireto maintain the minimum supply level in N.

If Q (N) has to increase to Qmin (N), the

excessive allocation water, V = Q (N)-Qmin (N),will be distributed to the M downstream canal

sections according to the following rules :

- allocate V to the canal section with D(I)>0by the amount of D(I),

- allocate the remainder V to increase the

discharge of the M downstream sections to Qmin,- allocate the remainder proportionally to

the allocated amount.

(3) If Qmin (N) < Q (N) < Qmax (N)No adjustment is needed.

(4) If Q (N) > Qmax (N)

Q (N) = Qmax (N)Water deficit will take place.

P (deficit) = Q (N) - Qmax (N)

The deficit P is allocated to the Mdownstream canal sections proportionally to the

allocated amount.

The irrigation efficiency is calculated bythe following equation :

IE(L) =R N

QI

J

ply

' ( )

sup

=∑

1 .................................... (3)

WhereIE(L) = Irrigation efficiency of water master L

(%)

R’(N) = Net irrigation water requirements ofcanal section N (cms)

R’(N) =

EV ES CF w I LP w I ER RSI

NC( ) * ( , ) ( , ) ( ) *

,

1 1

378 0001

+ −[ ]=∑ A(I, N)

.............(4)

J = No. of canal section in water master LER(RS) = Effective rainfall of station RS (mm)

QSupply = (Qin–Qout) of water master L (cms)

Required basic data for water allocation by

WASAM 2As Shown in Figure 1, the basic data required

for water allocation are

- the potential evapotranspiration (ETo)- the expected rainfall and effective

rainfall

- the crop coefficients- the land preparation water requirements

- the percolation in paddy field

- the canal system data- the discharge monitoring points

The monthly ETo of 2 stations ; (1)

Kamphaengsaen located on the south of the projectand (2) Uthong on the north, was calculated via the

CROPWAT 5.7 computer program (Smith, 1992).

The monthly ETo calculated from the long termaverage agroclimatological data (more than 20

years) is shown in Table 2.

The simple average rainfall is used as theexpected rainfall in WASAM 2. The effective

rainfall was derived by the simulation model via

Water Use Study Model Version 4 (Kamnertmanee,1989). In the simulation, it is assumed that the

maximum, normal and minimum water levels are

130, 100 and 70 mm respectively for paddy and 0,0, and –65 mm for non-paddy. The daily rainfall of

4 stations having the reconds of more than 40 years

(1953-1992) was used to derive the relationshipbetween rainfall and effective rainfall on weekly

basis. The formula is shown below :

If R < R* ; RE = RIf R > R* ; RE = AxR+B........................(5)

152 Kasetsart J. (Nat. Sci.) 34 (1)

crops including dry season paddy, wet season paddy,sugarcane, upland crops, fruit tree. The weighted

crop coefficients are shown in Table 4.

The land preparation water requirement is250 mm for paddy and 50 mm for sugarcane. The

land preparation is progressed at decreasing rate of

25.2, 22, 18.8, 15.2, 12 and 6.8 % of the total areafrom week 1 to week 6 respectively for paddy and

at uniform rate during Jan.-Apr. for sugarcane. The

percolation in paddy field is 0.5 mm/day in wetseason and 1 mm/day in dry season (Ilaco/Empire

M&T, 1984).

The canal conveyance loss measured byInflow-Outflow Method from 3 different locations

on 3 types of canal showed that the loss rate was

very high as shown in Table 5.The canal system of Song Phi Nong

Irrigation Project is divided into 57 sections for

water allocation calculation by WASAM 2. Thedetail of the canal sections are the name, location,

canal section number, number of the upstream

section, maximum and minimum discharges,conveyance loss rate, zone, water master section,

ETo station number, rainfall station number,

command area, etc, (Vudhivanich et al., 1998).For monitoring and evaluation purposes,

the discharge of 5 main regulators as shown in

Table 6 were measured on daily basis. The zonemen

Table 2 Monthly potential evapotranspiration(ETo) in mm at Kamphaengsaen and

Uthong stations.

Month Station %

Kamphaengsaen Uthong Difference

Jan. 116.8 118.2 1.19

Feb. 123.2 125.9 2.13Mar. 164.6 171.6 4.07

Apr. 168.3 177.6 5.23

May. 152.6 166.7 8.41Jun. 128.1 138.6 7.53

Jul. 134.5 142.9 5.88

Aug. 126.2 136 7.2Sep. 116.4 119.5 2.59

Oct. 116.3 118.3 1.7

Nor. 111.6 117.9 5.35Dec. 111.3 117.8 5.51

Table 3 R*, A and B in effective rainfall formulas.

Month For paddy For non-paddy

R* A B R* A B

Nov.-Apr. 59 0.55 26.10 29 0.78 6.38

May. 53 0.44 29.68 25 0.63 9.25Jun. 55 0.46 29.70 27 0.70 8.10

Jul. 60 0.75 15.00 26 0.65 9.10

Aug. 50 0.56 22.00 25 0.64 9.00Sep. 42 0.39 25.62 22 0.42 12.76

Oct. 30 0.25 22.50 18 0.27 13.14

Where

R = the weekly rainfall (mm)RE = the weekly effective rainfall (mm)

R*,A,B = the constants which depend on the crop

types and the month of year, see Table3.

WASAM 2 was designed to calculate the

water requirements of fish pond and 5 different

Kasetsart J. (Nat. Sci.) 34 (1) 153

Table 4 Weighted crop coefficients.

Types of CropWeek Dry season Wet season Sugarcane Upland Fruit tree Fish pond

paddy paddy 5 years 3 years crops

1 0.13 0.13 0.47 0.48 0.3 0.5 1.0

2 0.36 0.36 0.47 0.48 0.3 0.5 1.0

3 0.57 0.57 0.46 0.47 0.3 0.5 1.04 0.76 0.76 0.44 0.43 0.3 0.5 1.0

5 0.91 0.91 0.44 0.43 0.7 0.5 1.0

6 1.03 1.03 0.44 0.43 0.7 0.5 1.07 1.07 1.07 0.44 0.43 0.7 0.5 1.0

8 1.09 1.09 0.60 0.58 0.9 0.5 1.0

9 1.10 1.10 0.70 0.64 1.2 0.5 1.010 1.10 1.10 0.70 0.64 1.0 0.5 1.0

11 1.10 1.10 0.70 0.64 1.0 0.5 1.0

12 1.11 1.09 0.80 0.74 0.7 0.5 1.013 1.14 1.08 0.91 0.87 0.5 0.5 1.0

14 1.17 1.07 0.91 0.87 0.5 0.5 1.015 1.20 1.06 0.91 0.87 0.5 0.5 1.0

16 1.21 1.04 0.91 0.87 0.5 1.0

17 1.17 1.02 1.06 1.03 0.5 1.018 1.02 0.9 1.06 1.03 0.5 1.0

19 0.77 0.69 1.06 1.03 0.5 1.0

20 0.52 0.48 1.06 1.03 0.5 1.021 0.3 0.28 1.12 1.1 0.5 1.0

22 0.11 0.09 1.15 1.13 0.5 1.0

23 1.15 1.13 0.5 1.024 1.15 1.13 0.5 1.0

25 1.16 1.14 0.5 1.0

26-29 1.19 1.18 0.5 1.030-37 0 0 0.5 1.0

38-41 1.18 1.17 0.5 1.0

42 1.04 11.00 0.5 1.043-45 1.02 0.98 0.5 1.0

46-52 0.5 1.0

154 Kasetsart J. (Nat. Sci.) 34 (1)

Table 5 Canal conveyance loss rate.

Types of canal Conveyance loss rate

(% of Qmax/km)

Lined main canal 0.44Lined lateral 0.91

Unlined lateral 2.35

Table 6 Discharge monitoring points.

Monitoring point Canal section Location Main regulators of

no. no. water master section

1 2 2L cross regulator at km 22.70 12 14 5L-2L head regulator at km 0.020 2,3,4 and 5

3 26 5L-2L cross regulator at km 9.813 3,4 and 5

4 34 5L-2L cross regulator at km 26.401 45 42 3R-5L-2L head regulator at km 0.020 5

2. The water management performance of the pastweek, in terms of irrigation efficiency, of the 5

water master sections and of the project was

evaluated.The weekly allocated discharge or “adviced

discharge” of the 5 main regulators was compared

with the actual discharge in Figure 3. The projectand water master irrigation efficiency of the 3

seasons was compared with the values during 1991-

1993, the period before WASAM 2 was used, asshown in Figure 4.

Dry season 1994Figure 3(1) – Figure 3(5) show the actual

discharges of water master section 1 to be very

close to the WASAM 2 adviced discharge. The

actual discharges of water master sections 2-5 wereabout 10-50% higher than the adviced values,

particularly during the last few weeks of the dry

season. The analysis of the actual water distributionfound that although the 2L cross regulator at km

22.700 (Figure 3(1)) was one of the two head

regulators for the project; it was the tail end structureof Phanom Thuan Irrigation Project. By the present

water management practice of Song Phi Nong, this

regulator controlled water distribution to the mostupstream area of the project (zone 1 of water master

section 1). Zoneman usually tried to control the

discharge as WASAM 2 adviced. On the opposite,the 5L-2L head regulator at km 0.020 (Figure 3(2))

is responsible for the measurement and report to thewater management officer of Song Phi Nong

Irrigation Project by Wednesday. All the five

regulators were calibrated in field for the accuratecontrol of the discharge.

Performance analysis of water allocation with

WASAM 2WASAM 2 was used for water allocation of

Song Phi Nong Irrigation Project for 3 consecutive

seasons of ;

- dry season 1994 (Feb.-Jun.)- wet season 1994 (Jul.-Nov.)

- dry season 1995 (Feb.-Jun.)

On weekly basis, the ETo, rainfall, plantingarea, canal system data and field wetness conditions

were analyzed by WASAM 2 on Tuesday. The

irrigation water requirements of each canal sectionwere calculated using Equation 1. Consequently,

the discharge allocated to the 5 main regulators and

each canal section were calculated using Equation

Kasetsart J. (Nat. Sci.) 34 (1) 155

Figure 3 Comparison of WASAM 2 adviced discharge and actual discharge at 5 main regulators in dry

and wet season 1994 and dry season 1995.

which was the entrance to water master section 2-5, was recognized as the tail part of the system.

Whenever there was the excess water from Phanom

Thuan, it would distribute to this regulator and itwould do so for the water deficit. The degree of the

problem was higher for the further downstream

regulators. This could be seen by the delivery

performance of 3R-5L-2L head regulator at km

0.020 (water master section 5) where the actual andthe adviced discharges were most different.

This study indicated that the water

management of the large scale irrigation projectsuch as GMKIP was a lot more difficult than a

small irrigation project. The water management

156 Kasetsart J. (Nat. Sci.) 34 (1)

practices of the upstream project such as PhanomThuan had a direct effect to the downstream project

such as Song Phi Nong.

The irrigation efficiency of dry season 1994was 39.8 % which was about the average value for

an irrigation project. However, it was about 2-3 %

higher than that of the dry season irrigationefficiency of 1991-1993 which had the efficiency

between 36.7-38.3 %.

The irrigation efficiency of Song Phi Nongwas not high because of 2 reasons. First, the seepage

loss in the canal system was high. Second, the

actual discharge was higher than the adviced onebecause the water management practices of Phanom

Thuan had some effects on Song Phi Nong as

mentioned before.

Wet season 1994Figure 3(6) – Figure 3(10) show the actual

discharges delivered to the 5 water master sectionsto be 20-30 % higher than the WASAM 2 adviced

discharges in most of the wet season (week 29-48).

This was caused by the excess water from PhanomThuan Project . Song Phi Nong was unable to

completely control the flow. The operation of

Phanom Thuan Project could affect the water supplyto Song Phi Nong. However, the excess water

affected the water distribution of the 5 water master

sections almost equally.The irrigation efficiency was 32.1 %. It was

3-5 % higher than the efficiency during 1991-1993

because WASAM 2 used the rainfall moreeffectively.

Dry season 1995As shown in Figure 3(11) - 3(15), the actual

discharge during week 7-16 (Feb.-Apr.) was veryclose to the adviced discharge, except the case of

Figure 4 Irrigation efficiency of Song Phi Nong Irrigation Project and 5 water master sections during

1991-1995.

WMS = Water Master Section

Kasetsart J. (Nat. Sci.) 34 (1) 157

5L-2L cross regulator at km 26.401 (the downstreamregulator). The actual discharge of this regulator

was 20-30 % higher than the adviced one, However

during the later period of the season (week 17-22),all 5 water master sections got water more than the

WASAM 2 adviced value similar to the situation of

the previous 2 seasons.

CONCLUSION

The performance of WASAM 2 for water

allocation analyzed from the comparison of the

actual and the adviced discharges and the irrigationefficiency indicated that WASAM 2 could help

improing the irrigation efficiency in wet season

more than dry season. However the irrigationefficiency in the dry season was on the average

7.7% higher than that of the wet season.

The difficulty in discharge control wasfound. The actual discharge was about 20-30 %

higher than the WASAM 2 adviced value because

of the operation effect of the upstream PhanomThuan Project. This reduced the irrigation efficiency

of Song Phi Nong Project. It is recommended that

the water allocation system of the two projects is tobe integrated in one system in order to improve the

water management of Song Phi Nong Project.

ACKNOWLEDGEMENTS

This research was financially supported bythe National Research Council of Thailand.

LITERATURE CITED

Ilaco/Empire M&T. 1984. Water Management and

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Ilaco/Empire M&T. 1985. Water Management andO&M Report No.5 : Water Allocation

Scheduling and Monitoring at Project Level,

Supporting Document 5.3 - Rainfall Estimate.Mae Klong Irrigation Project. Royal Irrigation

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Ilaco/Empire M&T. 1986(a). Water Managementand O&M Report No.5 : Water Allocation

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Hydrometeorological Data Recording LAMPAO. Chi Basin Water Management

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Coop. Bangkok. 64 p.RID. 1988 (d). Maintenance of Irrigation Projects

158 Kasetsart J. (Nat. Sci.) 34 (1)

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Received date : 7/07/99Accepted date : 29/10/99

Kasetsart J. (Nat. Sci.) 34 : 159 - 170 (2000)

INTRODUCTION

High critical temperature ,TC, copper oxide

superconductors are based on perovskite-likestructure. The Bi-based superconducting oxide is

well known to have mixed structure of Bi-2212 low

TC and Bi-2223 high TC phases, which involvesseveral layers that insulate the CuO2 layer, the

main superconducting zone.

It was found that substituting lead for part ofbismuth increased easily volume fraction of high-

TC phase, and a number of mechanisms wereproposed to explain the reaction route in forming

this phase. Yet, the formation mechanisms have

not been well understood. Rhee et al. (1989) studiedthe effects of Pb on the formation of the high TCphase with various Bi/Pb ratios and found that

substitution of 30% Pb for Bi was the most preferablefor forming high TC phase.

Partial substitution of bismuth by lead in Bi

system shows three types of superconductingcompounds : (Bi,Pb)2Sr2CuOx semiconducting

Superconducting Properties of (Bi,Pb)-Sr-Ca-Cu-O Ceramics

Supreya Trivijitkasem and Wunchai Sratongluan

ABSTRACT

Sixteen specimens of four initial nominal composition ceramics were synthesized by solid statereaction for a given initial cation ratio (Bi:Pb)/Sr = (1.5:0.5)/2.0, and the varied cation ratio Ca/Cu = 2.5/

4.0 , 2.5/4.5 , 3.0/4.0 and 3.5/4.5.

SEM images of each specimen showed different grain sizes with porous structure. The grain sizeincreased with sintering time. The specimen synthesized by reground-repressed process and sintered for

50 h , possessed smallest grain size. Three different grain structures were observed. The platelike dark gray

grain , flake-pellet bright grain and sphere-like bright grain corresponded to high TC phase, low TC phaseand Ca2PbO4, respectively. The platelike grain changed to granular after the second grinding.

XRD spectra showed high TC phase coexisting with low TC phase and Ca2PbO4 in each specimen.

The ratio of lattice parameter of high TC and low TC phases , c/a , were 6.84-6.88 and 5.70-6.02,respectively. Magnetic-susceptibility measurement showed that TC was significantly affected by the

composition changed. Each specimen revealed nearly the same onset critical temperature, TC onset , at

107.0-108.5 K. TC and HC2 increased when the sintering time was extended from 50 h to 100 h. Anintermediate ground-pressed process enhanced TC but depressed HC2 at 90 K. The optimum atomic ratio

leading to the formation of the Bi-2223 phase was Ca/Cu = 0.75. The highest TC=106.0 K belonged to the

specimen which was sintered for 100 h by an intermediate ground-pressed process, the critical field at 90K was 427 A/m. The highest HC2= 539 A/m at 90 K belonged to the specimen which was sintered for 100

h by reground-repressed process,and TC=105.2 K.

Key words : fixed (Bi:Pb)/Sr , varied Ca/Cu , superconductivity

Department of Physics, Faculty of Science, Kasetsart University, Bangkok,10900,Thailand.

160 Kasetsart J. (Nat. Sci.) 34 (1)

phase,(Bi,Pb)2Sr2CaCu2Oy low TC phase and(Bi,Pb)2Sr2Ca2Cu3Oz high TC phase with

superconducting transition temperatures of ~7 K ,

~ 80 K and ~110 K, respectively. It is obvious thatthe 110 K superconducting phase is likely the most

interesting one for practical applications; and the

most efficient way to synthesize this phase is tostart with initial composition close to the nominal

composition (Bi,Pb)2 Sr2Ca2Cu3Oz.

Kikuchi et al. (1989) reported characteristicsof Bi-Pb-Sr-Ca-Cu oxide superconductor closely

related to the preparatory conditions such as the

sintering temperature and cooling rate. Xu et al.(1990) studied the superconductivity and

microstructure of Bi1.92Pb0.48Sb0.1Sr2Ca2Cu3.2Oxand Bi1.8Pb0.3Sb0.1Sr2Ca2Cu3Oy samples, theyfound that different superconducting behavior due

to different sintering conditions and were controlled

by powder grain size and chemical activity.Kusano et al. (1994) annealed the Pb-

substituted Bi ceramics at 500 °C to 850 °C and

subsequently reheating at 850 °C, they reportedthat the composition of high TC phase changed

while the superconducting properties remained

unaffected. Toledano et al. (1995) examined theeffect of varying the ratio (Bi:Pb)/Cu as well as the

ratio Sr/Ca, and found that the kinetics of forming

high TC phase greatly enhanced by an excess ofcalcium , while an excess of strontium prevented its

formation.

Sung and Hellstrom (1995) found thatCa2PbO4 had both positive and negative roles in

forming (Bi,Pb)-2223. The positive role was Ca

and Pb which released from Ca2PbO4 reacted with(Bi,Pb)-2212 and CuO to form (Bi,Pb)-2223. The

negative role was Ca which could also react with

CuO to form (Sr,Ca)2CuO3, since forming(Sr,Ca)2CuO3 added an extra reaction path to form

(Bi,Pb)-2223.

The extensive studies for the equilibrium ofthe lead-substitute system as a function of

composition and temperature has been performedby us recently. The phase equilibrium of Bi1.5Pb0.5Sr2.0Ca2.0Cu3.5Os and Bi1.5Pb0.5Sr2.0Ca2.5Cu3.5Oβwere determined (1999), the second compositionwith atomic ratio Ca/Cu = 0.71 formed more high

TC phase. Prunglag et al. (1997) determined the

phase equilibrium of (Bi0.85Pb0.15)2.0Sr2.0CaxCuyOα , 2.0 ≤ x ≤ 3.5 and 3.0 ≤ y ≤ 4.5, and found

that high TC phase increased when the atomic ratio

of Ca to Cu was 0.75.In order to complete the knowledge of the

range of the 2223 phase, this study was extended to

nominal compositions of (Bi : Pb): Sr : Ca : Cu=(1.5 : 0.5) : 2.0 : x : y , here x=2.5,2.5,3.0,3.5 and

y= 4.0,4.5,4.0,4.5, respectively. The procedure of

determination of the phase equilibrium wasdescribed in another paper (1999). In the present

work the influence of calcium and copper contents

for the formation of the 2223 phase with varioussintering conditions are reported. The results of x-

ray diffraction (XRD) spectra and scanning electron

microscope (SEM) observations are specified.Superconductivity determined from AC

susceptibility as a function of temperature are

discussed. The high critical field will becharacterized in a separate study.

MATERIALS AND METHODS

The starting materials were Bi2O3 (99.5% ,

purity) , PbO (99.0%), SrCO3 (98.0%), CaCO3(99.0%) and CuO (99.0%), powders were weighed

in the cation ratio of (Bi:Pb):Sr:Ca:Cu = (1.5:0.5):

2.0:2.5:4.0 , (1.5:0.5):2.0: 2.5:4.5 , (1.5: 0.5): 2.0:3.0:4.0 and (1.5:0.5):2.0:3.5:4.5. The powders were

mixed and ground in a mortar and pestle for 1 h , put

the mixed powders in an alumina crucible andcalcined in air at 800 °C for 12 h at a heating rate of

10 °C/min , then ground and cold pressed at 107

MPa into pellet with dimension of 13.1 mm diameterand ~4.7 mm thickness, sintered (first time) in air

Kasetsart J. (Nat. Sci.) 34 (1) 161

Table 1 Initial atomic ratio, mass change ∆m/m0and mass density ρ of specimens, here I,

II denotes number of pressing and 50,

100 denotes 50, 100 h in sintering.

Specimen (Bi:Pb):Sr:Ca:Cu ∆m/mo ρ(%) (g/cm3)

IA50 (1.5:0.5):2.0:2.5:4.0 1.72 3.63IA100 3.55 3.04IIA50 1.65 3.98IIA100 3.32 3.76IB50 (1.5:0.5):2.0:2.5:4.5 1.81 3.90IB100 3.45 3.82IIB50 1.59 4.05IIB100 3.07 3.92IC50 (1.5:0.5):2.0:3.0:4.0 3.29 3.08IC100 3.69 3.02IIC50 2.22 3.44IIC100 3.57 3.27ID50 (1.5:0.5):2.0:3.5:4.5 2.35 3.47ID100 2.64 3.43IID50 1.78 3.61IID100 2.48 3.47

at 840 °C for 20 h, the heating rate from 800-840 °C was 5 °C/min. The specimen was cooled down in

the furnace until 300 °C and quenched in air. Then

the process was repeated by not reground-repressed(process I) or reground-repressed (process II), and

sintered (second time) the specimen at 845 °C in air

for 50 h or 100 h. Weight and diameter of specimenswere measured before and after sintering process.

Mass density of the bulk specimens were

determined by density determination (Sartorius).The identification of the superconducting phases

was deduced from x-ray powder diffraction spectra

at room temperature with Ni-filtered Cukα radiationat 30 kV and 25 mA, on a Philips model PW 3710

wide angle goniometer. Microstructure was taken

by SEM , JEOL, model JSM-220A.AC susceptibility measurement was

performed by means of an AC susceptometer ,

Lake Shore model 7130. The pellet specimen wascut in a rectangular shape of dimensions

~1.5x1.6x9.1 mm3 and mass ~0.1 g , cooled the

specimen down to 45-55 K in the AC susceptometerby zero field cooling (ZFC) , then applied AC

magnetic field Hac(t)=H sin ωt at the fundamental

frequency f= 125 Hz and various field amplitude H.The specimen was heated at a rate of 0.86 °C/min

and fundamental susceptibility χ was recorded as a

function of increasing temperature.

RESULTS AND DISCUSSION

Four initial nominal compositions , Bi1.5Pb0.5 Sr2.0 Ca2.5 Cu4.0 Oa (A) , Bi1.5 Pb0.5 Sr2.0Ca2.5 Cu4.5 Ob (B) , Bi1.5 Pb0.5 Sr2.0 Ca3.0 Cu4.0 Oc(C) and Bi1.5 Pb0.5 Sr2.0 Ca3.5 Cu4.5 Od (D) , were

prepared by the conventional solid state reaction.

Initial atomic ratio , mass density and mass change,∆m/m0, of the specimens are shown in Table 1;

here ∆m= m0-m , m0 was mass of pellet specimen

before the first sintering in process I or before thesecond sintering in process II, and m was mass of

pellet specimen after the second sintering.

Mass change of the specimen is shown inFigure 1 (a). Here m0 of process I and process II

were not at the same condition. It was found that

the mass of specimens decreased significantly whensintering time ts increased ; the mass lost was due

to evaporation of specific chemical substance, such

as PbO, of specimen.Mass density of the specimen depended on

the process of synthesis as shown in Figure1(b).

The specimen sintered for 50 h by intermediatereground-repressed process, revealed highest mass

density and least mass lost. Specimen IA100 and

IC100 exhibited lowest mass density ρ=3.0 g/cm3

, while the mass density of specimen IIA50 increased

significantly as shown in Figure 1 (b).

The powder XRD patterns of 16 pellet

162 Kasetsart J. (Nat. Sci.) 34 (1)

specimens were recorded from 2θ = 3°-70° as

shown in Figure 2. The 2θ ranging from 20°-40°was a specifically region for pointing out of themain superconducting phases of (Bi,Pb)-2212 and

(Bi,Pb)-2223. Peaks corresponding to different

phases were marked by various patterns for clarity.XRD pattern showed peaks belonging to

the high TC (Bi,Pb)-2223 phase (H) as a majorcomponent together with peaks due to the low TC(Bi,Pb)-2212 phase (L). The high TC phase was

labeled in the form of (00P) and (11Q); here P andQ were even and odd number, respectively. There

was no peak of semiconducting phase (Bi,Pb)-

2201 at 2θ =21.9°. Each specimen showed a peakat 2θ =17.8° which belonged to Ca2PbO4. The

present of Ca2PbO4 was known to result in a liquid

phase which accelerated the anisotropic growth of(Bi,Pb)-2223 phase.

The diffraction peaks of (002)H, (002)L,

(115)L and (0012)H appeared at 2θ=4.7°,4.8°,27.7° and 28.8°, respectively. It could be seen from

Figure 2 that more high TC phase appeared in

specimen C and D than specimen A and B. Onlystrong diffraction peaks of (002)H appeared in

specimen A, B and D, while specimen C exhibited

both diffraction peaks of (002)H and (002)L.The peak intensity of (002)H of specimen

IC100 was stronger than specimen IC50, partly

due to more Ca2PbO4 occurred in specimen IC50.

The peak intensity of (0012)H and (119)H of

specimen C became stronger, while (115)L becameweaker with increasing sintering time ts. More high

TC phase was formed in specimen C than the others

and the formation of the high TC phase in eachspecimen enhanced when the sintering time

increased.Lattice parameter, a,b,c,c/a; and cell volume

v of low and high TC phases are shown in Table 2.

The crystal structure of low TC phase was tetragonal;the ratio of c/a was between 5.70-6.02 and cell

volume v was between 867-967(A°)3. The crystal

structure of high TC phase was orthorhombic; theratio of c/a was between 6.84-6.88, and cell volume

v was 1036-1120 (A°)3.

Since the Bi(Pb)-Sr-Ca-Cu-O ceramicscontains five cations, the phase relations for forming

2223 phase are more complicated. In order to

characterized the formation of phase in thespecimen, microstructure of all specimens were

carried out by SEM.

Figure 3 showed grain orientation ofspecimens. Each specimen was polycrystal and the

grains were anisotropic. Three different grain

structures were observed; the platelike dark graygrain, flake-pellet bright grain and sphere-like bright

grain corresponded to high TC phase, low TC phase

Figure 1 (a) Mass change and (b) mass density vs. atomic ratio Ca/Cu of specimens.

Kasetsart J. (Nat. Sci.) 34 (1) 163

Figure 2 Powder x-ray diffraction patterns of specimen (a) A , (b) B , (c) C and (d) D ; ■ denotes peakdue to Ca2PbO4, ▲ denotes low TC (Bi,Pb)-2212 phase and ◆ denotes high TC (Bi,Pb)-2223

phase.

164 Kasetsart J. (Nat. Sci.) 34 (1)

Table 2 Lattice parameter (a,b,c,c/a) and cell volume (v) of low TC and high TC phases.

Specimen Tetragonal Orthorhombic

a(A°) c(A°) c/a v(A°)3 a(A°) b(A°) c(A°) c/a v(A°)3

IA50 5.3109 31.0600 5.8483 876.1000 5.4692 5.4555 37.5438 6.8646 1120.2026

IA100 5.3084 31.0851 5.8558 875.9505 5.4205 5.4153 37.1394 6.8517 1090.1763

IIA50 5.2952 31.1107 5.8752 872.3289 5.3924 5.3829 36.9718 6.8563 1073.1712

IIA100 5.3126 31.0773 5.8497 877.1243 5.3894 5.3672 36.8428 6.8362 1065.7144

IB50 5.2831 31.1214 5.8907 868.6419 5.4477 5.4421 37.3661 6.8591 1107.7901

IB100 5.2824 31.0711 5.8820 866.9973 5.3938 5.3842 36.9295 6.8467 1072.4806

IIB50 5.2831 31.1214 5.8907 868.6419 5.3841 5.3614 36.8423 6.8428 1036.5014

IIB100 5.3021 31.0928 5.8642 874.0890 5.3821 5.3661 36.8256 6.8422 1063.5559

IC50 5.3964 30.7791 5.7036 896.3223 5.4285 5.4024 37.1896 6.8508 1090.6568

IC100 5.5296 31.6138 5.7172 966.6386 5.4313 5.4239 37.2398 6.8565 1097.0409

IIC50 5.4411 30.9978 5.6970 917.7075 5.4826 5.4043 37.7136 6.8788 1117.4395

IIC100 5.3160 31.0968 5.8497 878.7911 5.4044 5.3851 36.9888 6.8442 1076.4937

ID50 5.2984 31.2026 5.8891 875.9519 5.4133 5.4054 37.0981 6.8531 1085.5294

ID100 5.2575 31.6374 6.0175 874.4991 5.3739 5.3771 36.8418 6.8557 1064.9768

IID50 5.2608 31.6409 6.0144 875.6941 5.4423 5.4211 37.3058 6.8548 1100.6424

IID100 5.2618 31.6085 6.0072 875.1299 5.3868 5.3743 36.9556 6.8604 1069.8749

and Ca2PbO4, respectively. The areas with black

contrast corresponded to pores. The grain sizeincreased with sintering time. The platelike grain

changed to granular after second grinding. The

broad face of grain corresponded to the ab plane.The specimen sintered for 50 h by reground-

repressed process, exhibited the smallest grain size

and relatively dense, while the intermediate ground-pressed specimen showed more porous structure.

The grain size of specimen IIC100 was smaller and

more ordering than specimen IC100. The SEMmicrograph of specimen IIC100 showed that the

grains were oriented more dense than specimen

IC100,which was agree well with the mass densityof the specimen.

Zero-field-cooled measurement of the

superconducting transition was determined fromheating curve of fundamental susceptibility χ=χ/-

iχ//. The applied field amplitude for each running

were H=0.1,1,10,50,100,200,300,400 and 500 A/m. Figure 4 shows temperature dependence of

external susceptibility at field amplitude H=0.1 A/

m of specimen A and B. Figure 5 shows χ(T) of

specimen C and D at various fields.AC susceptibility was separated into 2

components : real part or dispersive component χ/

and imaginary part or dissipative component χ//. χ/

and χ// showed two steps of superconducting

transition. According to Mazaki et al. (1988), the

upper component of heating curve of χ/

corresponded to high TC phase, which was regarded

as the superconductivity of the grain or the bulk

phase; and the lower component of χ/ correspondedto low TC phase, which was regarded as the

diamagnetism due to the Josephson-like coupling

or the coupling phase. Two maxima of χ//

corresponded to intrinsic and coupling losses,

respectively.

It was obvious from Figure 5 that χ/ of lowTC phase had stronger amplitude dependence than

χ/ of high TC phase. The second maximum of

heating curve χ// (T) in process II of all specimens

Kasetsart J. (Nat. Sci.) 34 (1) 165

Figure 3 SEM micrographs of powder specimen showing microstructural characteristics.

was smaller than that in process I, i.e. , there wasonly small intrinsic loss in reground–repressed

process.

According to Sumiyama et al.(1989), thediamagnetism attributed to the Josephson-like weak

coupling was sensitive to the synthetic conditions

and grain contact-structure.When the applied field H was lower than

low critical field HC1 , χ/ would show perfect

diamagnetism, i.e. , χ/ = –1, exactly in SI unit; andχ// =0 , since the magnetization was linear reversible

and lossless. When H>HC1, χ/ increased from -1

and χ// ≠ 0. But the data in Figure 4 and Figure 5were not corrected for demagnetization factor,

hence χ/ deviated from –1.Critical temperature was determined from

χ/ at applied external field amplitude H=0.1 A/m

and f = 125 Hz. The onset of the coupling betweengrains in χ/ (T) was defined as critical temperature

TC of the specimen, and Tc onset was defined as the

temperature at which χ/ and χ// separated into 2parts. Table 3 shows atomic ratio of Ca/Cu, critical

temperature and onset of critical temperature of

specimens. Each specimen revealed TC onset at107.0-108.5 K.

Figure 6(a) shows critical temperature TCas a function of atomic ratio Ca/Cu, which increaseswith increasing sintering time; reground-repressed

IA50 IA100 IIA50 IIA100

IB50 IB100 IIB50 IIB100

IC50 IC100 IIC50 IIC100

ID50 ID100 IID50 IID100

166 Kasetsart J. (Nat. Sci.) 34 (1)

Table 3 Atomic ratio, critical temperature TC ,onset of critical temperature TC onset and high critical fieldHc2 at 90 K of specimens.

Specimen Ca/Cu Tc (K) Tc onset (K) Hc2 (A/m)

IA50 2.5/4.0=0.62 100.2 107.2 175IA100 102.8 107.5 195

IIA50 98.2 107.0 145

IIA100 101.3 107.3 266IB50 2.5/4.5=0.56 94.0 107.1 7

IB100 96.8 107.4 24

IIB50 92.0 107.0 5IIB100 94.6 107.2 38

IC50 3.0/4.0=0.75 104.2 108.1 352

IC100 106.0 108.5 427IIC50 104.0 108.0 408

IIC100 105.2 108.2 539

ID50 3.5/4.5=0.78 104.8 108.0 300ID100 105.7 108.3 416

IID50 103.0 107.8 335

IID100 105.1 108.1 527

process depresses slightly the critical temperature.

The synthetic process that provided higher critical

temperature was to sinter specimen at 845°C for100 h by an intermediate ground-pressed process.

TC of all specimens were significantly

affected by the composition changed. The highestcritical temperature was 106.0 K which belonged

to specimen IC100, atomic ratio of Ca/Cu=0.75.

The next higher TC =105.7 K belonged to specimenID100. The other 2 compositions , IA100 and

IB100 revealed much lower critical temperature ,

TC=102.8K and 96.8 K, respectively. It could beseen from Figure 1(b) and Figure 6(a) that lower

mass density pellet specimen revealed higher TC.

Low and high critical field of the specimencould be determined from the AC χ measurement

at various magnetic field. The onset of the first

maximum of χ// ≠ 0 from heating curve was definedas low critical field HC1(T), and the onset of the

coupling between grain in χ/(T) at various field wasdefined as high critical field HC2 (T) of the specimen

(Dodrill and Krause. 1996).

The temperature dependence of HC2 at 90 Kare shown in Table 3 and Figure 6(b). HC2 at 90 K

of process II was higher than that of process I, and

HC2 increased with sintering time. It was obviousfrom Figure 6(b) that HC2 of specimen C was

slightly higher than specimen D. The highest

HC2=539 A/m at 90 K belonged to specimen IIC100,while specimen IID100 showed HC2= 527 A/m at

90 K. The high critical field of the other 2

compositions, IIA100 and IIB100 were much lower.HC2 at 90 K of specimen II-50 was lower

than that of specimen I-100. Specimen IC100 which

exhibited highest TC revealed rather low HC2 = 427A/m at 90 K. Hence the optimum atomic ratio that

formed more volume fraction of Bi-2223 phase

was Ca/Cu = 0.75. The best synthetic condition that

Kasetsart J. (Nat. Sci.) 34 (1) 167

Figure 4 Volume susceptibility of specimen A and B as a function of increasing temperature T, at

H=0.1A/m and f=125 Hz.

168 Kasetsart J. (Nat. Sci.) 34 (1)

Figure 5 Volume susceptibility of specimen C and D as a function of increasing temperature T, at f=125Hz and various field amplitude H, here a,b,c,d,e,f,g,h and i denotes H=0.1,1,10,

50,100,200,300,400 and 500 A/m, respectively.

Kasetsart J. (Nat. Sci.) 34 (1) 169

lead to higher TC and highest HC2 at 90 K was to

sinter the specimen at 845°C for 100 h by reground-repressed process.

CONCLUSION

Sixteen specimens of four initial nominalcomposition ceramics were synthesized by solid

state reaction. The fixed initial cation ratio was

(Bi:Pb)/Sr = (1.5:0.5)/2.0 and the varied ratioswere Ca/Cu = 2.5/4.0, 2.5/4.5, 3.0/4.0 and 3.5/4.5.

Mass density, critical temperature and high

critical field of the specimen depended on theprocess of synthesis.

More mass loss was found when the sintering

time was prolonged. The mass lost,∆m/ m0 , of thespecimens was 1.59-3.69% .

Mass density decreased with increasing

sintering time. Specimen sintered for 50 h byintermediate reground-repressed process revealed

the highest mass density and least mass lost.

Powder XRD pattern taken from the pelletspecimen showed that each specimen contained

Bi-2223, Bi-2212 and Ca2PbO4 phases. More high

TC phase was formed in the specimen than low TCphase, when the duration of the sintering was

prolonged. The ratio of lattice parameter of high TCand low TC phases were c/a=6.84-6.88 ,and 5.70-

6.02, respectively.

SEM micrograph revealed different grainsizes in each specimen. Three different grain

structures were found; platelike dark gray grain ,

flake-pellet bright grain and sphere-like bright graincorresponded to high TC phase, low TC phase and

Ca2PbO4, respectively. The areas with black

contrast corresponded to pores. The grain size ofspecimen I-100 was larger than that of specimen II-

100, which was sintered by reground-repressed

process; that is the platelike grain changed togranular after the second grinding.

The AC susceptibility χ =χ/-iχ// depended

on temperature T, and was separated into 2 parts :real susceptibility χ/ and imaginary susceptibility

χ// at T<TC onset. Magnetic-susceptibility

measurements showed that TC was significantlyaffected by the composition changed, and TCincreased while mass density decreased with

increasing sintering time. Each specimen revealednearly the same onset critical temperature, TC onset= 107.0-108.5 K. Critical temperature TC and high

critical field HC2 were determined from χ/. Eachspecimen showed increasing TC and HC2 at 90 K

when the sintering time was extended from 50 h to

100 h. Reground-repressed process depressed TCbut enhanced HC2 at 90 K.

The highest TC and HC2 belonged to the

Figure 6 (a) Critical temperature and (b) high critical field at 90 K vs. atomic ratio Ca/Cu of specimens.

170 Kasetsart J. (Nat. Sci.) 34 (1)

initial composition Bi1.5 Pb0.5 Sr2.0 Ca3.0 Cu4.0 OCwhose initial atomic ratio of Ca/Cu was 0.75. The

specimen IIC100, which was sintered for 100 h by

reground-repressed process, exhibited asuperconducting transition at 105.2 K and highest

critical field HC2=539 A/m at 90 K, while the

specimen IC100 exhibited the highest TC=106.0 Kand much lower critical field, HC2= 427 A/m at 90

K. Critical temperature and HC2 at 90 K of specimen

IID100 were slightly lower than that of specimenIIC100.

ACKNOWLEDGEMENT

The authors are grateful to the Kasetsart

University Research and Development Institutefor the grant support of this paper under project No.

7.41 .

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conductor in (Bi0.85Pb0.15)2.0Sr2.0Cax

CuyO10-s when 2.0≤x≤3.5 and 3.0≤y≤4.5, pp.310-316. In The 35th Kasetsart University

Annual Conference, Science section. Bangkok.

Rhee, C. K. , C. J. Kim, H. G. Lee, I. H. Kuk, J. M.Lee, I. S. Chang, C. S. Rim, P. S. Han , S. I.

Pyun, and D. Y. Won. 1989. Effects of Pb

content on the formation of the high-TC phasein the (Bi,Pb)-Sr-Ca-Cu-O system. Jpn. J. Appl.

Phys. 28 (7) : L1137-L1139.

Sumiyama, A. , H. Endo, J. Tsuchiya, N. Kijima,M. Mizuno, and Y. Oguri. 1989. A.C.

susceptibility of superconducting Bi- (Pb)-Sr-

Ca-Cu-O system. Jpn. J. Appl. Phys. 28 (3) :L373-L376.

Sung, Y. S. and E. E. Hellstrom. 1995. A s t u d y

of reactions amongst (Bi,Pb)Sr2Ca2Cu3Ox andphases on the line between CaO and CuO to

form (Bi,Pb)2Sr2Ca2Cu3Ox. Phys. C. 252 :

155-159.Toledano, J. C. , D. Morin, J. Schneck, H. Fagir, O.

Monnereau, G. Vacquier, P. Strobel, and V.

Barnole. 1995. Stability of the 2223 phase inthe lead-substituted bismuth cuprates. Phys.

C. 253 : 53-62.

Trivijitkasem, S. and W. Sratongluan. 1999.Microstructural evolution in forming Bi 2223

superconductor from Bi-Pb-Sr-Ca-Cu-O

system. The Kasetsart J. (Nat. Science) 33 (4): 554-563.

Trivijitkasem, S. and W. Sratongluan. 1999. Grain

orientation and growth of high TC Bi(Pb)-Sr-Ca-Cu-O superconductor, pp. 160-168. In The

37th Kasetsart University Annual Conference,

Science section. Bangkok.Xu, Q., Z. Chen, G. Meng, and D. Peng. 1990.

Microstructure and superconductivity in Bi-

Sr-Ca-Cu-O system doped with Pb and Sb.Jpn. J. Appl. Phys. 29 (10) : L1918-L1923.

Received date : 17/08/99Accepted date : 27/12/99

Kasetsart J. (Nat. Sci.) 34 : 171 - 178 (2000)

INTRODUCTION

The remediation of subsurface (both soils

and groundwater) has been the most costly andtime consuming part of site cleanups. After the

source of contamination has been removed and

treated, contaminated soils and groundwater maystill remain and require treatment. Nowadays in-

situ technologies have become very attractive for

treating contaminated soils and groundwaterbecause of lower costs, less disruption to the

environment, and reduced worker exposure to

Removal of Naphthalene and 2, 4-Dinitrotoluene from Soilsby Using Carboxymethyl-βββββ-Cyclodextrin

Chatdanai Jiradecha

ABSTRACT

Spillage of petroleum and petroleum products from the underground storage tanks has caused

significant contamination of groundwater aquifers by organic contaminants. These compounds are

generally difficult to remove since the chemicals are hydrophobic, having a low solubility and thereforeprefer to adsorb onto the soil. The ability of carboxymethyl-β-cyclodextrin (CMCD) to increase the

solubilities of contaminants in soils was studied. Furthermore, studied partitioning of soil-contaminants

and CMCD-contaminants were also included. Soils consisting of 7.4% clay content and 2.2% organiccarbon content were used as contaminated soils. Naphthalene and 2, 4-dinitrotoluene (2, 4-DNT) were

selected as the representative low polarity organic contaminants. In the batch studies, the partition

coefficient between soil-naphthalene and soil-2, 4-DNT were 51.3 and 5.6 L/kg. In addition, 269.2 and 22.4L/kg were determined from the partitioning between CMCD-naphthalene and CMCD-2, 4-DNT respectively.

The results from column experiments showed that CMCD greatly enhanced the removal of naphthalene

and 2, 4-DNT from soils. Using 0.01 N NaNO3 as the flushing agent, 32 and 40% of naphthalene and 2,4-DNT were removed. Meanwhile, 70 and 72% naphthalene and 2, 4-DNT were removed after 2 g/L

CMCD solution was flushed through the soil columns.

Key words : CMCD, naphthalene, 2, 4-dinitrotoluene, solubility

hazardous materials. Addition of agents such as

organic cosolvents and surfactants are known toincrease the transport of low polarity organics from

the soils (Laha and Luthy, 1992; Edward et al.,

1994; Thangamani and Gina, 1994). However, ithas been found that both cosolvents and surfactants

have some disadvantages for soil remediation

applications. Cosolvents are not effective insolubilizing the organics unless their volume-

fraction concentrations are above 10%, and

surfactants form high-viscosity emulsions that aredifficult to remove from the soils (Wang and

Department of Environment Engineering, Faculty of Engineering, Kasetsart University, Bangkok 10900, Thailand.

172 Kasetsart J. (Nat. Sci.) 34 (1)

Brusseau, 1993). Furthermore, synthetic surfactantsinhibit microbial activity in soils thereby reducing

their natural bioremediation capability. Hence,

contaminant transport-enhancing additives that donot adsorb or retain in the soil, and also do not

inhibit natural soil microbial activity are desired.

One such type of additives is the microbially-produced compounds, known as biosurfactants

which can enhance low-polarity and nonpolar

organics contaminant removal from soils withoutaccumulating in the soil, or affecting the natural

microbial activity.

Another class of microbially-producedcompounds that is known to increase the aqueous

solubilities of low polarity organics by forming

water-soluble 1:1 inclusion complexes are calledcyclodextrins. Cyclodextrins are produced from

starch by bacteria, and are widely used in the

pharmaceutical industry to improve the dissolutionof non-polar drugs. These are cyclic oligosaccarides

containing 6-8 glucose units arranged to form a

hydrophobic shell, and a 5-8 °A hydrophobic cavity.It is demonstrated that carboxymethyl-β-

cyclodextrin (CMCD) is a highly water soluble,

anionic, non-emulsifying and non-toxiccyclodextrin. Biodegradable cyclodextrins can form

complex with cationic heavy metals, significantly

enhance the aqueous solubility of low polarityorganic compounds, and hence enhance their

removal from soils (Brusseau et al., 1997). The

hydrophobic cavity of β-cyclodextrin is reported to

be 0.346 nm3. Hence, low polarity organiccontaminants that have molecular volume (MV)

less than 0.346 nm3 are likely to form inclusion

compounds with β-cyclodextrin.

MATERIALS AND METHODS

Materials. Naphthalene and 2, 4-

dinitrotoluene (2, 4-DNT) were selected as the test

compounds. The physical and chemical propertiesof these compounds are shown in Table 1.

Carboxymethyl-β-cyclodextrin (CMCD) was used

as additive agent to increase the solubility of thetest compounds. The soil samples were collected

up to a depth of 12 inches after clearing the top 2

inches of debris and grass. The samples werebrought to the laboratory for analysis in polyethylene

bottles. The soils were sieved, and the fraction

passing a No. 10 (2 mm) sieve was air dried andstored at room temperature in polyethylene bottles.

The physical and chemical properties of these soils

are shown in Table 2.Adsorption tests. Samples for the

determination of adsorption isotherms were

prepared by adding a known amount of adsorbentinto a series of 40 mL vials (Teflon-lined vial caps)

that contained 25 mL of 0.01 N NaNO3 and different

amounts of contaminant. The solution was adjustedto the desired pH (4.0, 6.5 and 9.0) by adding 0.01

N HNO3 or 0.01 N NaOH prior to adding the

adsorbate. These vials were rotated in the tumbler

Table 1 Physical and chemical properties of naphthalene and 2, 4-dinitrotoluene.

Properties Naphthalene 2,4-dinitrotoluene

Formula C10H8 CH3C6H3(NO2)2Molecular weight 128.17 182.14

Density (g/cm3) 1.145 1.321

Molecular volume (nm3) 0.186 0.229Solubility (mg/L) 35.5 300

Kasetsart J. (Nat. Sci.) 34 (1) 173

for 3 days. The samples were filtered through 0.45mm nylon membrane filters and analyzed by the

UV spectrophotometer (Perkin Elmer Lambda 3A)

at wavelengths 276 and 250 nm for naphthaleneand 2, 4-DNT at the end of the tests.

Contaminants-CMCD partition tests.These tests were prepared by adding a known

amount of contaminant into a series of 40 mL vials

(Teflon-lined vial caps) that contained 25 mL of0.01 N NaNO3 and different amounts of CMCD.

The amount of contaminant added was

approximately 10 times higher than the solubilitylimit. The solution was adjusted to the desired pH

(4.0, 6.5 and 9.0) by adding 0.01 N HNO3 or 0.01

N NaOH prior to adding the adsorbate. These vialswere rotated in the tumbler for 3 days. The samples

were filtered through 0.45 mm nylon membrane

filter and the filtrate was analyzed by the UVspectrophotometer at the end of the test.

Column tests. Polycarbonate columns 2.54

cm in diameter and 4 cm long were used throughoutthe experiments. The columns were packed in

incremental steps with dry soils to establish uniform

bulk density. Soil was prevented from leaching outof the columns by nylon membrane filter paper

supported on stainless steel screen. After packing,

the columns were slowly wetted from the bottom

with electrolyte (0.01 N NaNO3) and contaminantsolution. During this procedure the effluent from

the columns was collected and determined for the

contaminant. Elution experiments were conductedafter the contaminant solution was pumped through

the soil columns for 14 days. The effluent solution

was analyzed by the UV spectrophotometer atvarious times to determine the amount of

naphthalene and 2, 4-DNT. After 14 days of elution,

the contaminated soils were extracted by placingthe contaminated soils in glass containers containing

methanol for 1 day. Two types of elution

experiments were conducted: (1) elution with 0.01N NaNO3 solution, (2) elution with 2 and 5 g/L of

CMCD and 0.01 N NaNO3 solution.

RESULTS

Adsorption isotherm. Adsorption isothermequations such as Freundlich and Langmuir

equations were used to simulate the experimental

data. The plot between the solid phase and aqueousphase naphthalene concentrations is shown in Figure

1. The relationship between solid phase and aqueous

phase 2, 4-DNT concentrations is shown in Figure2.

Naphthalene oncentration, (mg/L

1 2 3 4 5 6 7 8 9 1

Naphthalene dsorption, (m

300

400

500

600

700

800

Freundlich isotherLangmuir Isotherm

c cc

a

Figure 1 Adsorption isotherm of naphthalene in

soil.

Table 2 Characteristic parameters of test soil.

Soil type I

pH 7.2

Organic matter content 4.9 %Organic carbon content 2.2 %

Specific gravity 2.46

Clay content 7.4 %Silt content 17.4 %

Sand content 75.2 %

pHzpc 7.0Soil type Sandy loam

174 Kasetsart J. (Nat. Sci.) 34 (1)

2, 4 Dinitrotoluene oncentration,

0 2 4 6 8 10 12 14 16 18

2, 4initrotoluene

orptio

150

200

250

300

350

Freundlich isothermLangmuir Isotherm

c

ads

d

Naphthalene oncentration, (mg/L)

0 2 4 6 8 10 12 1

Naphthalene dsorption, (

0

200

400

600

800

1000

1200

pH 6.5 pH 4

pH 9

a

c

2, 4 initrotoluen oncentration, mg/L

0 2 4 6 8 10 12 14 16 18

2, 4 initrotoluendsorption, mg/kg

150

200

250

300

350

400

pH 6.5 pH 4 pH 9

ad

d e c

Figure 2 Adsorption isotherm of 2, 4-dinitroto-

luene in soil.

Figure 3 Adsorption isotherm for naphthalene atpH 4, 6.5 and 9.

Figure 4 Adsorption isotherm for 2, 4-dinitroto-

luene at pH 4, 6.5 and 9.Figure 5 Plot of relative aqueous-phase naphtha-

lene concentration versus the CMCD

concentration.

Figure 6 Plot of relative aqueous-phase 2, 4-dinitrotoluene concentration versus the

CMCD concentration.

Figure 7 Plot of the relative aqueous-phase naph-

thalene concentration versus the CMCD

concentration at pH 4, 6.5 and 9.

Concentration of CMCD, (kg/L)

0.00 0.02 0.04 0.06 0.08

Relative que

ous-hase oncentra

tion

0

5

10

15

20

25c

ap

Concentration of CMCD, (kg/L)

0.00 0.02 0.04 0.06 0.080

1

2

3

4

ap

cRelative que

ous-hase oncentra

tion

Concentration of CMCD, (kg/L)

0.00 0.02 0.04 0.06 0.08

0

5

10

15

20

25

30

pH 4 pH 6.5 pH 9

ap

cRelative que

ous-hase oncentra

tion

Kasetsart J. (Nat. Sci.) 34 (1) 175

To understand the effect of pH on thesorption capacity of soils, three adsorption isotherms

with different pH were conducted at the same time

using the exact procedure. Figure 3 and 4 show theadsorption data obtained for naphthalene and 2, 4-

DNT at different pH values.

Partition between contaminants andCMCD. The relative aqueous-phase concentrations

(St/S0) of naphthalene and 2, 4-DNT were plotted

against the CMCD concentration in Figures 5 and

Figure 8 Plot of relative aqueous-phase 2, 4-

dinitrotoluene concentration versus the

CMCD concentration at pH 4.5, 6 and 9.

Figure 9 Elution of soil contaminated with naph-

thalene using CMCD and NaNO3 solu-tion.

Figure 10 Elution of soil contaminated with 2, 4-

dinitrotoluene using CMCD and NaNO3

solution.

Concentration of CMCD, (kg/L)

0.00 0.02 0.04 0.06 0.080

1

2

3

4

pH 4 pH 6.5 pH 9

ap

cRelative que

ous-hase oncentra

tion

1.0

0.8

0.6

0.4

0.2

0.0

Nap

htha

lene

frac

tion

rem

oval

0 20 40 60 80 100 120 140 160 180

Number of pore volume

0.01 N NaNO3 SolutionCMCD (2 g/L)CMCD (5 g/L)

1.0

0.8

0.6

0.4

0.2

0.0

2,4

Din

itrot

olue

ne fr

actio

n re

mov

al

0 20 40 60 80 100 120 140 160 180

Number of pore volume

0.01 N NaNO3 SolutionCMCD (2 g/L)

6 respectively. Furthermore, to determine the effect

of pH on the partition of naphthalene and 2, 4-DNT

to the soil, three different pH solutions were preparedsimultaneously by the same procedure. The plots

between the aqueous-phase concentration versus

the CMCD concentration at three different pHvalues are shown in Figures 7 and 8 for naphthalene

and 2, 4-DNT respectively.

Column tests. A comparison between the

log Kd and log Kcw values from the above

experiments reveals that both naphthalene and 2, 4-DNT have log Kcw values higher than log Kdvalues. These results suggest that CMCD has the

ability to extract naphthalene and 2, 4-DNT fromthe soils. Column tests were conducted to confirm

these results. The results from column experiments

were plotted between number of pore volumes ofelutant versus contaminant fraction removed.

Elution curves for naphthalene and 2, 4-DNT

removal from the soil are shown in Figures 9 and 10respectively.

176 Kasetsart J. (Nat. Sci.) 34 (1)

DISCUSSION

Adsorption isotherm. Relative to the

Langmuir isotherm, the results show that theFreundlich isotherm is a better fit to the experimental

data according to the sum of square error (SSE)

between the experimental and model results.Freundlich partition coefficients were determined

for each system by plotting the sorption capacity

versus the aqueous concentrations for naphthaleneand 2, 4-DNT. It has been determined for

hydrophobic compounds that, if the equilibrium

aqueous phase concentration is less than one half ofthe solute water solubility, sorption isotherms to

natural sediments are linear (Karickhoff et. al.,

1979). Assuming that the constant 1/n = 1, andusing only the linear portion of the curve, the

partition coefficient is then the slope of the line

(Roger, 1989). The log Kd values for naphthaleneand 2, 4-DNT were found to be 1.71 and 0.75

respectively. Relative to the naphthalene system,

the capacity of the soil for 2, 4-DNT was muchsmaller. This result demonstrated that naphthalene

has a stronger affinity to the soil surface than 2, 4-

DNT. Due to the lower solubility of naphthalene,water can cause naphthalene to sorb onto the

hydrophobic surface of the soils more than 2, 4-

DNT since naphthalene attempts to minimize itscontact with water and migrates to the relatively

hydrophobic soil organic matter.

The effect of pH on the sorption capacity ofsoils indicates that the log Kd values between the

soils and naphthalene were 1.78, 1.76, and 1.71 at

pH 4, 6.5, and 9 respectively. The log Kd values for2, 4-DNT at pH 4, 6.5, and 9 were 0.86, 0.75, and

0.71. Using statistical t-test, there was no statistically

significant difference in the log Kd values ofnaphthalene and 2, 4- DNT at the three different pH

at the 95 percent confidence interval. The results

indicate that the solution pH has no effect on thesorption capacity of naphthalene and 2, 4-DNT in

soils. However, a charge-induced dipole interactioncould occur between a positively charged surface

and the electron-rich π system of the PAHs (Mader

et al.,1997). At pH lower than pHzpc, the soilsurface becomes positively charged and tends to

interact with the negative charges in the electron-

rich π system of naphthalene and 2, 4-DNT. Thisinteraction causes the affinity of the soils to increase

at lower pH. From this study, it is obvious that the

surface charge density does not affect sorptioncapacity since the dominant force is hydrophobic

interaction. Meanwhile, the surface charge tends to

affect the affinity of the soils by electrostaticinteraction.

Partition between contaminants andCMCD. Linear regression was performed for thesedata following the contaminant/CMCD partition

model. The linear equation fitted the observed data

closely for both contaminants. The CMCD/waterpartition coefficient (Kcw) for naphthalene and 2,

4-DNT were obtained from linear regression of St/

S0 versus the concentration of CMCD. The log Kcwfor naphthalene and 2, 4-DNT were found to be

2.43 and 1.35 respectively. The log Kcw reported

by Wang and Brusseau (1993) for naphthalene/hydroxypropyl-β-cyclodextrin (HPCD) was about

2.72, which is higher than naphthalene/CMCD

measured in this research. Since HPCD and CMCDare both β-cyclodextrin having 0.346 nm3 cavity

(Szejtli, 1982), the difference in the partition of the

contaminants may be due to the charge of thecarboxylic functional groups and the polarity of the

CMCD molecules (Brusseau, 1997). Hence, HPCD

having less polarity and no charge functional groupsmay cause larger sorption of contaminants. The

results from this study indicate that CMCD can

enhance the solubility of naphthalene and 2, 4-DNT due to the hydrophobic cavity of the CMCD.

The log Kcw for naphthalene is higher than

2, 4-DNT as naphthalene is more hydrophobic andhas a smaller molecular volume. This causes the

Kasetsart J. (Nat. Sci.) 34 (1) 177

arrangement of naphthalene into the CMCDhydrophobic cavity to be easier and stronger

compared to 2, 4-DNT. The molecular volumes of

naphthalene and 2, 4-DNT were calculated fromthe density and were found to be 0.186 and 0.229

nm3 respectively. The results from this study

indicate that CMCD produces a significant increasein the apparent aqueous solubilities of naphthalene

and 2, 4-DNT due to its hydrophobic cavity.

The log Kcw for naphthalene at pH 4, 6.5,and 9.0 were 2.46, 2.43, and 2.42 while the log Kcwfor 2, 4-DNT were 1.34, 1.35, and 1.34 at pH 4, 6.5,

and 9.0. Using statistical t-test, there was nostatistically significant difference in the log Kcwvalues of naphthalene and 2, 4-DNT at the three

different pH values at the 95 percent confidenceinterval. The results confirmed that the solution pH

has no effect on the partition between the

contaminants and CMCD and has little effect onthe naphthalene and 2, 4-DNT solubilities. So the

increase in the solubilization of naphthalene and 2,

4-DNT is strongly dependent upon the complexationbetween the hydrophobic cavity of CMCD and low

polarity organic compounds.

The formation ratio for the naphthalene/CMCD and 2, 4-DNT/CMCD inclusion complexes

were determined from the amount of CMCD added.

At pH 4 and 6.5, the formation of the naphthalene/CMCD inclusion complexes was 1 to 0.07 (mole

CMCD : mole naphthalene) while at pH 9 it was 1

to 0.06. The 1 to 0.03 (mole CMCD : mole 2, 4-DNT) 2, 4-DNT/CMCD inclusion complexes were

found at pH 4, 6.5, and 9. The formation value of

naphthalene inclusion complexes is higher thanthat of 2, 4-DNT as naphthalene has a smaller

molecular size that enables easier arrangement into

the CMCD cavity. However, the naphthalene and2, 4-DNT inclusion complexes values were less

than the assumed 1 to 1 inclusion complexes value

as described by Blyshak et al., 1989.Column tests. Clearly, CMCD greatly

enhanced the removal of naphthalene and 2, 4-DNT from soils as compared to the NaNO3 solution.

For example, 70 percent of the initial naphthalene

was removed by 2 g/L CMCD solution and 72percent was removed by 5 g/L of CMCD solution

after 160 pore volumes of flushing as compared to

32 percent using NaNO3 solution. Furthermore, 73percent of the initial 2, 4-DNT was removed after

140 pore volumes of 2g/L CMCD solution was

pumped through the column while only 40 percentwas removed by the NaNO3 solution. Adding more

CMCD did not affect the total naphthalene removal.

It may be that the diffusion of the contaminantsfrom the soils to the bulk liquid was rate limited.

Furthermore, naphthalene and 2, 4-DNT could not

be completely removed from the soils as thedesorption of these contaminants by the hard soil

organic matter domain is slow and only partially

reversible (Huang, 1997). However, it can increasethe removal rate of naphthalene. At 50 pore volumes

of CMCD flushing, the percent naphthalene

removed increased from 38 to 49 percent whenCMCD solution concentration increased from 2 to

5 g/L. Since the Kcw value is higher than the Kdvalue, naphthalene and 2, 4-DNT tend to leave thesoils and complex with CMCD in the aqueous

solution. The total removal efficiencies for

naphthalene and 2, 4-DNT are approximately equalsince the Kd and Kcw values for naphthalene and 2,

4-DNT differs slightly.

The study of low polarity organiccompounds in enhancing solubility show that

CMCD greatly enhances the desorption and elution

of naphthalene and 2, 4-DNT, which were used asrepresentative low polarity organic contaminants.

The partition coefficients between contaminants

and CMCD (Kcw) were determined by using thecontaminants/CMCD model. The results show that

this linear equation fitted the experimental data

extremely well for both naphthalene and 2, 4-DNT.Furthermore, there was no difference in Kcw values

178 Kasetsart J. (Nat. Sci.) 34 (1)

for naphthalene and 2, 4-DNT when the pH waschanged. Further research will be focused on the

removal of low polarity organic contaminants from

low hydraulic conductivity soils based oncyclodextrin.

LITERATURE CITED

Brusseau, M.L., X.J. Wang, and W.Z. Wang. 1997.

Simultaneous elution of heavy metals andorganic compounds from soil by cyclodextrin.

Environ. Sci. Technol. 31(4) : 1087-1092.

Edwards, D.A., Z. Liu, and R.G. Luthy. 1994.Surfactant solubilization of organic compounds

in soil/aqueous systems. J. Envir. Engrg. 24(1)

: 5.Huang, W. 1997. Sorption and desorption by soils

and sediments: effects of sorbent heterogeneity.

Ph.D. Dissertation, University of Michigan,Michigan.

Karickhoff, S.W., D.S. Brown, and T.A. Scott.

1979. Sorption of hydrophobic pollutants onnatural sediments. Water Research 11 (3) :

241.

Laha, S. and R.G. Luthy. 1992. Effects of nonionic

surfactants on the solubilization andmineralization of phenanthrene in soil-water

systems. Biotechnology and Bioengineering

40 : 1367.Mader, B.T., K.U. Goss and S.J. Eisenreich. 1997.

Sorption of nonionic, hydrophobic organic

chemicals to mineral surfaces. Environ. Sci.Technol. 31(4) : 1079-1086.

Rogers, J.A. 1989. Humidity effects on low

temperature thermal desorption of adsorbedvolatile organic compounds from soil. Ph.D.

Dissertation, Illinois Institute of Technology,

Illinois.Thangamani, S. and S.S Gina. 1994. Effect of

anionic biosurfactant on hexadecane

partitioning in multiphase systems. Environ.Sci. Technol. 28(12) : 1993.

Wang, X.J. and M.L. Brusseau. 1993. Solubilization

of some low-polarity organic compounds byhydroxypropyl-b-cyclodextrin. Environ. Sci.

Technol. 27(13) : 2021-2025.

Received date : 18/10/99Accepted date : 30/12/99

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the research, and state objectives of your work.

Materials and Methods. Provide sufficient detail so work can be repeated. Describe new methods in

detail; accepted methods briefly with references. Use subheadings as needed for clarity.

Use of Trade names. Trade names are to be avoided in defining products whenever possible. Ifnaming a product trade name cannot be avoided, the trade names of other like products also should be

mentioned, and first use should be accompanied by the superscript symbol TM followed in parentheses by

the owner’s name. If a product trade name is used, it is imperative that the product be described in sufficient

detail so the nature of the product will be understood by professionally trained readers. Do not use tradenames in titles.

Use of abbreviations and acronyms. At fist text use, define in parentheses. Do not use

abbreviations and acronyms in titles.Statistical analysis. If variation within a treatment (coefficient of variation, the standard

deviation divided by the mean) is small (less than 10%) and difference among treatment means is large

(greater than 3 standard deviations), it is not necessary to conduct a statistical analysis. If the data do notmeet these criteria, appropriate statistical analysis must be conducted and reported.

Results and Discussion. Present and discuss results concisely using figures and tables as needed. Donot present the same information in figures and tables. Compare results to those previously reported, and

clearly indicate what new information is contributed by the present study.

Conclusion. State conclusions (not a summary) briefly.

Literature Cited. List only those references cited in the text. Required format of references is describedbelow.

Acknowledgments. List sources of financial or material support and the names of individuals whosecontributions were significant but not deserving of authorship. Acknowledgment of an employer’s

permission to publish will not be printed.

Appendix. This section is rarely needed in a research paper but can be added if deemed necessary (e.g.,

complicated calculations, detailed nomenclature).

Tables. Number each table with Arabic numerals. Place a descriptive caption at the top of each table.

Print one table per page. Columns and their headings are usually (but not always) used to display the

dependent variable(s) being presented in the table. Foot-notes should be identified by lower case lettersappearing as superscripts in the body of the table and preceding the footnote below the table. The same data

should not appear in both tables and figures.

Figures (graphs, charts, line drawings, photographs). Supply one illustration per page with the figurenumber indicated in the lower comer of page; figure and figure numbers should appear on the same page.

Figure captions should be double-spaced and listed consecutively on page(s) separate from figures: use

Arabic numerals. Include one original set of illustrations with an original manuscript, marked,“ORIGINAL.” Illustrations for other copies of the manuscript may be photocopies, provided they are clear.

Exceptions are photomicrographs, gel electrophoretic patterns, etc.; furnish four glossy photographs for

each of these.Authors are responsible for obtaining permission to reproduce previously copyrighted illustrations.

Proof or certification of permission to reproduce is required.

Lettering, data lines, and symbols must be sufficiently large so as to be clearly visible when the figureis reduced to a size commonly used in the journal. When a color presentation is deemed necessary, please

note this in the cover letter of the submission.

REFERENCE FORMAT

Manuscripts should follow the name-year reference format of the Council of Science Editors(formerly Council of Biology Editors). Cite only necessary publications. Primary rather than secondary

references should be cited, when possible. It is acceptable to cite work that is “in press” (i.e., accepted but

not yet published) with the pertinent year and volume number of the reference. Work that is “submitted”but not yet accepted should not be cited.

In text. Cite publications in text with author name and year. Three or more authors use “et al.”

In parenthetical citations, separate author and year with a comma. Use suffixes a, b and c to separatepublications in same year by the same author. Semi-colons separate citations of different authors. Cite two

or more publications of different authors in chronological sequence, from earliest to latest. For example:

• The starch granules are normally elongated in the milk stage (Brown, 1956).• Smith et al. (1994) reported growth on vinasse.

• …and work (Dawson and Briggs 1984,1987) has shown that . . .

• …and work (Dawson 1984; Briggs 1999) has shown that . . .

In Literature Cited section. List only those references cited in the text. References should be

listed alphabetically by the first author’s last name. Single author precedes same author with Co-authors.Type references flush left as separate paragraphs. Do not indent manually. Let the text wrap with first line

hanging indented. Use the following format.

• Journal articles: Author(s). Year. Article title. Journal title, volume number: inclusive pages.Example: Citation in text: (Smith et al., 1999)

Smith J.B., L.B. Jones and K.R. Rackly. 1999. Maillard browning in apples. J. Food Sci. 64: 512-518.

• Books: Author(s) or editor(s). Year. Title. Publisher name. Place of publication. Number of pages.Example: Citation in text: (Spally and Morgan, 1989)

Spally M.R. and S.S. Morgan. 1989. Methods of Food Analysis. 2nd ed. Elsevier. New York. 682 p.

• Chapter: Author(s) of the chapter. Year. Title of the chapter, pages of the chapter. In author(s) oreditor(s). Title of the book. Publisher. Place of publication.

Example: Citation in text: (Rich and Ellis, 1998)

Rich R.Q. and Ellis M.T. 1998. Lipid oxidation in fish muscle, pp. 832-855. In Moody J.J. and W. Lasky,(eds). Lipid Oxidation in Food. 6th ed. Pergamon. New York.

For journal abbreviations and other examples of reference formats please refer to articles in a2000 issue of this journal or contact the editorial office at KURDI.

EDITORIAL REVIEW AND PROCESSING

Peer Review. All submitted manuscripts are screened by the Scientific Editor for importance,substance, appropriateness for the journal, general scientific quality, and amount of new information

provided. Those failing to meet current standards are rejected without further review. Those meetings these

initial standards are sent to expert referees for peer review. Referees identities are not disclosed to the

author. Author identities are also not disclosed to the referees. Referee comments are reviewed by anAssociate Editor and he/she, often after allowing the author to make changes in response to the referee’s

comments, advises the Scientific Editor to either accept or reject the manuscript. The Scientific Editor

informs the author of the final decision.Rejected manuscripts. Rejected manuscripts may, in some instances, be appropriate for

publication in other journals. Rejected manuscripts including original illustrations and photographs will

be returned to authors.Accepted manuscripts. The author(s) will be asked to review a copyedited pageproof. The

author(s) is responsible for all statements appearing in the galley proofs. The author will be informed of

the estimated date of publication.Inquiries regarding status of the manuscript. Direct inquiries to: Orawan Wongwanich,

Manager, Kasetsart University Research and Development Institute (KURDI), Kasetsart University, 50

Pahonyothin Road, Chatuchak, Bangkok 10900, Tel. 66-2-579-0032, 579-5548, 561-1474, Fax. 66-2-561-1474, E-mail: rdi@nontri.ku.ac.th.

INSTRUCTIONS FOR SUBMITTING A MANUSCRIPT

Submit the following items.Cover letter. Identify the corresponding author and provide his/her full name, address, numbers

for telephone and fax, and e-mail address.

Manuscript. Double-space all components of the manuscript except tables. Type on one sideof A4 paper. Use one inch margins. Number all pages.

Send an original manuscript (with original figures, marked “ORIGINAL”) and 3 photocopies.

Hard copies are required. Staple each manuscript copy, including the original manuscript in the upper leftcomer. Include no paper clips or binders.

Disk. Include an IBM-formatted, 3-l/2" disk, containing the manuscript in Microsoft Word

(version 97 preferred).

MAIL MANUSCRIPT TO:

Orawan Wongwanich, Manager,

Kasetsart University Research and Development Institute (KURDI)Kasetsart University,

50 Pahonyothin Road, Chatuchak,

Bangkok 10900, ThailandTel. 66-2-579-0032, 579-5548, 561-1474

Fax. 66-2-561-1474

E-mail: rdi@nontri.ku.ac.th

PRE-SUBMISSION CHECKLIST

(See “Instructions for Authors” for additional information.)

Cover letter & form❏ Full contact information for the corresponding author (full name, address, phone,

fax, e-mail).

Manuscript❏ Original manuscript (with original figures, marked çORIGINALé)❏ Three photocopies

❏ Page numbering

❏ Each manuscript stapled in upper left corner (no Paper clips or binders)❏ Abstract of less than 200 words

❏ 5 key words

❏ Figure captions listed consecutively on a page separate from figuresDISK

❏ 3-l/2 in IBM-formatted disk with full manuscript Microsoft Word (version 97 preferred) file.

(Submission of the title page and abstract by electronic means is an acceptable alternative.)

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