ORIGINAL PAPER

Genetic and metabolic diversity of streptomycetes in pulpand paper mill effluent treated crop fields

Binu Mani Tripathi • Rajeev Kaushik •

Priyanka Kumari • Anil Kumar Saxena •

Dilip Kumar Arora

Received: 8 May 2010 / Accepted: 15 November 2010 / Published online: 27 November 2010

� Springer Science+Business Media B.V. 2010

Abstract Irrigation of farm field with water mixed

with pulp and paper mill effluent from Century pulp and

paper mill in Uttrakhand state of India for over last

25 years in succession increased streptomycetes population

(120 9 105) compared to the fresh water irrigated fields

(48 9 103 in WIF). Denaturing gradient gel electrophore-

sis, amplified ribosomal DNA restriction analysis, 16S

rRNA gene sequencing, BIOLOGTM substrate usage, pro-

duction of extracellular enzymes (xylanase and cellulase)

and plant growth promoting attributes were applied to

monitor changes in genetic and metabolic diversity of

streptomycetes. Significant variation was observed for

production of extracellular enzymes, Indolic compounds,

siderophore and P-solubilisation among isolates. Metabolic

substrate usage of Streptomyces isolates was evaluated

using the BIOLOGTM GP2 plates and unique carbon sub-

strate usage profiles were observed. Based on 16S rRNA

gene sequencing, the isolates were identified as Strepto-

myces variabilis, Streptomyces spp. S. glaucescens,

S. viridochromogenes, S. cinnabarinus, S. aburaviensis,

S. viridis, S. xylophagus, S. macrosporeus, S. thermocarb-

oxydus, and S. albogriseolus. The diversity index parame-

ters like Shannon index, reciprocal of Simpson’s index

(1/D), and Pielou index of evenness based on ARDRA

revealed that streptomycetes community in effluent

irrigated field (EIF) was more diverse. DGGE profiles

of Streptomyces specific 16S rRNA gene fragments

(16S-DGGE) amplified directly from soil samples were

highly similar in both soils.

Keywords ARDRA � BIOLOGTM � DGGE � PGP �Pulp and paper mill effluent � Streptomyces

Introduction

Pulp and paper mill is the major industry in India. The

heavy demand for the paper helps in steady expansion of

paper industries. Since early fifties, the number of paper

pulp mills in India has increased from 17 to more than

406 in 2008, with simultaneous increase in paper pro-

duction from 0.13 to 1.9 million tons per annum (Singh

and Thakur 2006). Pulp and paper mills are utilizing huge

amount of lignocellulosic plant components along with

various chemicals and thus regarded as major polluting

industries. Irrigating crops with pulp and paper mill

effluent is a cheap and attractive alternative for discharge

of effluent to natural waterways. Pulp and paper mill

effluent contains several elements including important

plant nutrients such as nitrogen (N), phosphorus (P) and

potassium (K), which contribute to higher crop yields

when applied to nutrient deficient soils (Udayasoorian

et al. 2004). Other elements (magnesium, sodium, chlo-

rides, sulfur) and organic compounds (chlorinated lignins,

phenolic derivatives) that are common in pulp and paper

mill effluent can cause toxicities and nutrient imbalance

in plants. The tendency of certain elements (especially

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11274-010-0614-1) contains supplementarymaterial, which is available to authorized users.

B. M. Tripathi � R. Kaushik (&) � P. Kumari � D. K. Arora

National Bureau of Agriculturally Important Microorganisms,

Mau, Uttar Pradesh 275101, India

e-mail: rknbaim@gmail.com

A. K. Saxena

Division of Microbiology, Indian Agricultural Research

Institute, New Delhi, India

123

World J Microbiol Biotechnol (2011) 27:1603–1613

DOI 10.1007/s11274-010-0614-1

Na) to accumulate in pulp and paper mill effluent irri-

gated soils affects soil structure, increases soil salinity,

resistance to root expansion and reduces water percolation

and soil aeration (Howe and Wagner 1996). Furthermore,

the addition of such a ‘‘mixed bag’’ of compounds may

induce changes in physiochemical properties of the soil

and also the significant shifts in structure and function of

the associated microbial community, which in turn may

ultimately affect the soil viability for agriculture purposes

(Oved et al. 2001).

Pulp and paper mill effluent application to soil is known

to increase the population of bacteria, actinomycetes, fungi

and yeast (Kannan and Oblisami 1990a). Gram-positive,

filamentous bacteria of the genus Streptomyces within the

class Actinobacteria (Stackebrandt et al. 1997) are regarded

as common saprophytic soil bacteria with a complex life

cycle. They typically occur in soil as spores, which germi-

nate and produce substrate mycelium under favourable

nutritional conditions. Streptomyces appears to be most vital

ecologically and accounts for approximately 90% of the soil

actinomycetes community. It is also well known that

Streptomyces play an important role in the transformation of

lignocelluloses (Ramachandra et al. 1987). These microor-

ganisms are able to degrade cellulose and hemicelluloses as

they oxidize and solubilize the lignin component. Several

works reported dominance of streptomycetes population in

wetlands constructed for industrial waste water treatment

(Shatoury et al. 2004), however no systematic study has been

carried out to account for the diversity of Streptomyces

specifically in arable soils facing paper mill effluents over a

long period. Therefore, the aim of present study was to

determine the effect of irrigating sugarcane crop with the

water from local channel mixed with paper mill effluent

from Century paper mill (CPM), Lal Kuan, Uttrakhand,

India, on culturable as well as unculturable streptomycetes

community.

Materials and methods

Field sites and collection of soil samples

The effluent from the factory Century Pulp and Paper mill

(CPM) at Lal Kuan, Uttrakhand, India (79�100E longitude

and 29�30N latitude), which is discharged in local water-

way containing fresh water is being used as source of

irrigation to the sugarcane fields since last 25 years. A field

was selected from this site for sampling and was designated

as Effluent irrigated field (EIF). Samples were also col-

lected from a tube well-water irrigated field (WIF) from the

same area for comparison. The chemical composition of

effluent from CPM, effluent mixed with water and tube

well water is given in Table 1. Samples were collected

during March (2008) using a soil auger along zigzag paths

(Zigzag sampling) to account for the randomness from a

depth of approximately 15 cm. The samples were trans-

ported to the laboratory in the insulated container at 4�C.

The soil was sandy loam texture with pH 8.16; electrical

conductivity (EC), 0.73 dSm-1; organic carbon (OC),

0.95%; available N, 64.85 kg ha-1; Olsen P, 16.86 kg ha-1

and extractable K, 130.62 kg ha-1. The soil of control site

also had sandy loam texture, pH, 7.30; electrical conduc-

tivity (EC), 0.30 dSm-1; organic carbon (OC), 0.81%;

available N, 63.09 kg ha-1; Olsen P, 12.57 kg ha-1 and

extractable K, 168.56 kg ha-1.

Selective isolation and enumeration of presumptive

streptomycetes

One gram of each composite sample was suspended in

9 ml of � Ringer’s solution (Oxoid) in a universal bottle.

The resultant 10-1 preparations were agitated on a shaker

at 150 revolutions per minute (rpm) for 10 min at room

temperature, heated at 55�C for 6 min in a water bath, and

Table 1 Chemical nature of effluent from CPM, effluent used for irrigation (EUI), ground water from effluent irrigated field (GWE) and fresh

water from tube well (FWTW) used for irrigating control fields

Sample detail pH EC (d sm-1) Na (me l-1) K (ppm) SAR TDS BOD COD

CPM effluent 8.4 1.6 4.37 11.8 2.1 955 86.9 269

EUI (mixed with drain water) 7.75 1.20 3.82 10.48 1.91 722 64.3 205.7

GWE 7.78 0.74 1.02 2.48 0.57 572 3.9 14.5

FWTW 7.29 0.59 0.55 3.52 0.33 435 3.1 12.3

CPM century pulp and paper mill

EC electrical conductivity

SAR sodium absorption ratio

TDS total dissolved solids

BOD biological oxygen demand

COD chemical oxygen demand

Source: Joshi 2008

1604 World J Microbiol Biotechnol (2011) 27:1603–1613

123

cooled at room temperature. The resultant preparations

were serially diluted down to 10-6, using � strength

Ringer’s solution, and aliquots of each dilution (100 ll)

spread over the surfaces of dried starch casein agar plates

amended with cycloheximide (50 lg ml-1). The pre-

sumptive streptomycetes were ascertained from the leath-

ery colonies and an aerial spore mass, counted and

expressed as the number of colony forming units (CFUs)

per gram dry soil. The isolates were examined for the

presence of isomers of diaminopimelic acid (A2pm) in

whole organism hydrolysates using the procedure descri-

bed by (Staneck and Roberts 1974). A standard solution

(10 lM) of A2pm (Sigma) containing a mixture of DL-,

LL- and meso-A2pm isomers was used as a reference.

Purified isolates were maintained on oatmeal agar slopes

while spores and hyphal fragments stored in glycerol (20%,

v/v) at -20�C. Isolates obtained from EIF and WIF are

designated by prefixing NBE and NBC before isolate

number, respectively.

Assay of xylanase and cellulase and plant growth

promoting activity

All the isolates were tested qualitatively for production of

xylanase and cellulase. Based on initial screening, selected

isolates were also screened on quantitative basis. The

production of xylanase and cellulase was estimated fol-

lowing the method as described by Sanghi et al. (2007) and

Zvereva et al. (2006). Indolic compounds production was

estimated colorimetrically (Gordon and Weber 1951). All

the isolates were also screened for phosphate solubilization

on Pikovskaya’s agar plates (Mehta and Nautiyal 2001).

Siderophore production was examined on chrome-Azurol-

S agar medium as described (Schwyn and Neilands 1987).

Carbon substrate usage

Carbon substrate usage by isolates was measured using

the BIOLOGTM GP2 automated identification system

(Hayward, California). The strains were grown on oatmeal

agar plates for 7 days at 32�C when spores were harvested

by scrapping them from agar surfaces. The resultant spore

preparations were suspended in sterile distilled water and

washed by centrifugation at 14,000 rpm for 10 min and the

supernatants discarded (Kieser et al. 2000). This procedure

was repeated five times and the resultant washed spore

suspensions (20 ll) pipetted into two 1.5 ml microfuge

tubes each of which contained 500 ll of peptone-yeast

extract broth (Kieser et al. 2000) and incubated at 32�C in

an horizontal shaker at 220 rpm. Tubes were removed from

the shaker after 72 h and resultant preparations were cen-

trifuged, and the pelleted cells suspended in a sterile

solution. Cell density was adjusted to an OD590 of

between 0.34 and 0.39. The BIOLOGTM GP2 microplates

were immediately inoculated with 125 ll of cell suspen-

sion per well. Triplicate plates were used for each isolate

and were incubated at 32�C in sealed plastic bags for 96 h.

Substrate oxidation was measured with a microplate reader

at 590 nm. Clustering was based on binary data (usage/

nonusage) for each of the 95 substrates. Data were sub-

mitted to cluster analysis using a simple matching coeffi-

cient (SSM) (Sokal and Michener 1958) and clustering was

achieved by the unweighted-pair-group method of associ-

ation (UPGMA) (Sneath and Sokal 1973).

Genomic DNA extraction from isolates and soil

samples

Genomic DNA extraction from all the isolates was carried

out as described by Pospiech and Neumann (1995). The

total microbial community DNA was extracted directly

from WIF and EIF soil samples (0.25 g of each sample in

duplicate) using power soil DNA kit (MO BIO, USA). Soil

DNA was further purified by using Wizard DNA clean up

system (Promega, USA) to remove humic acid contamina-

tion. DNA preparations were visualized after electropho-

resis in a 0.8% agarose gel in 19 TBE buffer to assess their

integrity and then stored at 4�C prior to PCR amplification.

Amplified ribosomal DNA restriction analysis

(ARDRA)

The gene encoding 16S rRNA from selected isolates was

amplified by PCR using the pair of universal primers pA

and pH and the conditions described in Massol-Deya et al.

(1995). The PCR products were monitored through gel

electrophoresis (1% agarose, w/v), followed by ethidium

bromide staining and UV transillumination. Approximately

1 lg of PCR-amplified 16S rRNA gene fragments were

restricted with endonucleases Dde I, Mbo I and Taq I

(Fermentas, USA) separately at 37�C for overnight and

resolved by electrophoresis in 2.5% agarose. Banding

pattern was visualized by ethidium bromide staining and

documented in gel documentation and analysis system

(Alphaimager, USA). Strong and clear bands were scored

for similarity and clustering analysis using the software,

NTSYS-PC2 package (Numerical taxonomy analysis pro-

gram package, Exeter software, USA). Similarity among

the strains was calculated by Jaccard’s coefficient, and

dendrogram constructed using UPGMA method (Nei and

Li 1979).

16S rRNA gene sequencing and phylogenetic analysis

Purified 16S rRNA gene of representative isolates from

each cluster was used as a template in cycle sequencing

World J Microbiol Biotechnol (2011) 27:1603–1613 1605

123

reactions with fluorescent dye-labeled terminators (Big

Dye, Applied Biosystems). Both primers pA and pH were

used for sequencing and run in 3130 9 l ABI prism auto-

mated DNA sequencer. All the sequences were compared

with 16S rRNA gene sequences available in the GenBank

databases by BLASTn search. Identification to the species

level was determined based on 16S rRNA gene sequence

similarity ([97%) with that of a prototype strain sequence.

Multiple sequence alignment of approx 1,500-bp sequences

was performed using CLUSTAL W, version 1.8 (Thomp-

son et al. 1994). A phylogenetic tree was constructed using

the neighbor-joining method (Saitou and Nei 1987). Tree

topologies were evaluated through bootstrap analysis of

1,000 data sets by MEGA 4.0 package (Tamura et al.

2007). The 16S rRNA gene sequences were submitted to

NCBI GenBank database under accession numbers

GQ268015 to GQ268026 and GU136398 to GU136399.

Diversity indices

The Shannon index (H) (Shannon and Weaver 1949),

reciprocal of Simpson’s index (1/D) (Magurran 1988) and

Pielou index (E) (Pielou 1969) were chosen to characterize

the Streptomyces communities based on the ARDRA pro-

file in WIF and EIF soils. The use of 1/D instead of the

original formulation of Simpson’s index ensures that an

increase in the reciprocal index reflects an increase in

diversity (Magurran 1988).

PCR amplification of 16S rRNA coding genes

for DGGE

Fragments of 16S rRNA genes were amplified using DNA

from both WIF and EIF soils by first PCR with primers

specific to streptomycetes StrepB 50-ACA AGC CCT GGA

AAC GGG GT-30 and StrepF 50-ACG TGT GCA GCC

CAA GAC A-30 described by Rintala et al. (2001). Second

PCR was performed by using first PCR product as template

with primers F243 50-GGA TGA GCC CGC GGC CTA

and R513-GC 50-CGC CCG GGG CGC GCC CCG GGC

GGG GCG GGG GCA CGG GGG GCG GCC GCG GCT

GCT GGC ACG TA-30 Heuer et al. (1997). The PCR

products were visualized by agarose gel electrophoresis

(1.4% gel) followed by staining with ethidium bromide.

Amplicons were stored at -20�C until DGGE analysis.

DGGEs were carried out using a Bio-Rad DCode Universal

Mutation Detection System (Bio-Rad Laboratories,

Germany). PCR products (approximately 150 ng) were

applied directly onto 8% (w/v) polyacrylamide gels in

19 TAE buffer (40 mM Tris-acetate; pH 7.4, 20 mM

sodium acetate, 1 mM disodium EDTA) containing a

denaturing gradient of urea and formamide varying from

40 to 60%. The gels were run for 6 h at 60�C and 150 V.

Statistical analysis

Data was subjected to analysis of variance (ANOVA) using

software SPSS ver. 10 for enzyme assays and Indolic

compounds production in the respective isolates and least

significant difference (LSD) at P = 0.01 among means

compared using standard error.

Results

Selective isolation and enumeration

The streptomycetes population was significantly higher

(120 9 105 cfu g-1 dw soil) in EIF soils compared to WIF

counterpart (48 9 103 cfu g-1 dw soil) (Table 2). The 55

isolates (29 from WIF and 26 from EIF soils) gave whole-

organism hydrolysates rich in LL-A2pm thereby confirming

that they were members of the genus Streptomyces, were

selected for further analysis.

Enzyme assays and PGP attributes

Significant variation was observed among the isolates

(from WIF or EIF soils) with respect to production of

xylanase and cellulase. Significant variation was rule with

regard to numerical dominance of producer of these

Table 2 CFU count and percentage of Streptomyces isolates from WIF and EIF with production of extracellular enzymes and plant growth

promoting activity

Site CFU gm-1 soil Morphotypes % of positive isolates

Xylanase Cellulase Siderophore IAA P-solubilizers Siderophore

WIF 48 9 103 29 13.79 10.34 13.79 13.79 24.13 13.79

EIF 120 9 105 26 46.15 42.30 38.46 50.00 7.69 38.46

WIF water irrigated field

EIF effluent irrigated field

1606 World J Microbiol Biotechnol (2011) 27:1603–1613

123

enzymes among the two soil sources (EIF and WIF).

Xylanase producers were more in EIF soils (12 from EIF

and 4 from WIF) with maximum production by isolate

NBE43 from EIF soils. Likewise, producers of cellulose,

more in EIF soils (11 from EIF and 3 from WIF) with

maximum production by NBE51 from EIF (Table 3).

The percentage of isolates exhibiting plant growth pro-

moting attributes among the EIF and WIF soils also

showed significant variation. Indolic compounds and sid-

erophore producers were more in EIF soils whereas higher

phosphate solubilization was observed in WIF soils

(Table 3).

Carbon substrate usage

The result of clustering analysis is shown in Fig. 1 based

on use/non-use of 95 substrates studied by the BIOLOGTM

system. At a 58% similarity level all Streptomyces isolates

were grouped into two major groups (A and B) (Fig. 1).

D-melezitose, D-melibiose, b-methyl-D-galactoside, 3-me-

thyl glucose, a-methyl-D-glucoside, b-methyl-D-glucoside,

a-methyl-D-mannoside, D-raffinose, salicin, 20-deoxy ade-

nosine and L-fucose were not utilized by any of the isolates

of group A whereas D-fructose-6-phosphate and a-D-glu-

cose-1-phosphate were not utilized by the isolates of group

Table 3 Production of

extracellular enzymes (IU) by

Streptomyces isolates from WIF

and EIF

ND not detected

?: weak reaction; ??:

intermediate reaction; ???:

strong reactiona Isolates designated NBC are

from WIF and NBE from EIF

Isolatea Xylanase Cellulase Indolic compounds production

(lg mg-1 protein)

Siderophore

production

P-solubilization

NBE3 193.53 ND 128.17 ND ND

NBE4 ND ND ND ? ND

NBE6 168.59 ND 121.52 ?? ND

NBE7 ND 33.81 399.63 ??? ND

NBE9 201.20 ND ND ND ND

NBE12 194.24 ND 55.50 ND ND

NBE13 77.46 ND 369.74 ?? ??

NBE14 214.39 54.68 496.72 ?? ND

NBE23 142.45 ND 627.10 ??? ??

NBE24 ND 28.78 ND ND ND

NBE27 ND ND 408.49 ?? ND

NBE28 139.57 44.12 ND ND ND

NBE35 ND 43.17 186.67 ND ND

NBE40 ND 24.70 603.94 ??? ND

NBE43 283.69 66.43 ND ND ND

NBE45 ND 49.40 423.61 ?? ND

NBE50 220.62 ND 283.54 ND ND

NBE51 ND 74.58 ND ? ND

NBE55 202.88 ND ND ND ND

NBE56 ND 73.38 362.92 ND ND

NBE57 213.43 53.96 ND ND ND

NBC5 ND ND 84.88 ND ??

NBC11 ND 28.78 114.27 ND ???

NBC15 ND ND ND ND ??

NBC25 ND ND 24.08 ND ??

NBC29 82.49 ND ND ? ND

NBC32 45.08 36.45 ND ?? ???

NBC33 ND ND ND ND ND

NBC38 ND 40.77 ND ND ???

NBC39 ND ND 226.01 ?? ND

NBC41 201.20 ND ND ND ND

NBC46 100.48 ND ND ND ND

NBC49 ND ND ND ND ??

NBC54 ND ND ND ?? ND

LSD(P20.01) 13.40 9.62 24.11 – –

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123

B. Only one carbon source, D-ribose, was used universally

by all the isolates and sedoheptulosan was the only sub-

strate not utilized by any isolate from both groups.

The substrate usage patterns for the isolates tested

revealed a broad variability, isolate NBE13 from group A

used the fewest substrates, 10/95, about 9.5% of the total

no. of substrates tested. Whereas two isolates NBE7 and

NBE35 from group B used the most, 78/95, about 78.94%

of the substrates.

Amplified ribosomal DNA restriction

Restriction digestion of 16S rRNA gene using three

endonucleases (Dde I, Mbo I and Taq I) yielded 7–9 dis-

tinct restriction patterns for each enzyme. About 2–8

restricted fragments of varying sizes were common to each

of the restriction patterns. Cluster analysis of combined

16S rRNA gene restriction pattern based on Jaccard’s

similarity index, grouped all the 55 isolates under 14 dis-

tinct groups with similarity percentage ranging from 17 to

100% (Fig. 2). Majority of the isolates were under group I

(43% of the total number), while the remaining 57% iso-

lates shared rest of groups. ARDRA cluster I, III, VI and

VIII had isolates from both WIF and EIF. ARDRA cluster

II, IV, VII, X, XI, XIII and XIV had isolates only from EIF,

where as cluster V, IX and XII had isolates only from WIF.

16S rRNA gene sequence

For sequence analysis, a total 14 isolates was chosen in

such a way that each of the isolates represents a group with

similar ARDRA pattern as generated by three different

restriction enzymes. The representative isolates from all 14

clusters were identified as S. variabilis, Streptomyces spp.

S. glaucescens, Streptomyces spp., S. viridochromogenes,

S. cinnabarinus, S. aburaviensis, S. viridis, Streptomyces

spp., S. xylophagus, S. macrosporeus, S. thermocarboxy-

dus, Streptomyces spp., and S. albogriseolus (Fig. 3;

Table 4).

Diversity index of streptomycetes

It is evident from different diversity index that EIF

harboured high streptomycetes diversity (H = 2.098 and

1/D = 6.259) relative to WIF (H = 1.419 and 1/D =

2.870).The evenness of the community structure was

higher in EIF (E = 0.874) compared to WIF (E = 0.729)

soils (Table 5).

Analysis of streptomycetes DGGE fingerprints

DNA was recovered from both WIF and EIF soil samples.

Streptomycetes community fingerprints were generated

from two replications of each of the sample, and repro-

ducible DGGE profiles of 16S rRNA gene were obtained.

The DGGE profiles were found to be very similar among

the both soils (WIF and EIF). No difference (presence or

absence of bands) could be observed visually when the two

patterns were compared (Fig. 4), indicating that the pre-

dominant unculturable streptomycetes community found in

these soils does not vary regarding the effluent treatment.

Discussion

Pulp and paper mill effluent is usually discharged in local

channel in many parts of India. Sugarcane, rice and wheat

growing regions of Northern Indo-Gangetic plains often

face water scarcity during critical stages of crop growth. In

Lal Kuan, Uttrakhand, India, farmers use water from

channel containing effluent of CPM for irrigating their

sugarcane fields since last 25 years. It is a common practice

near many other paper mills in irrigated agro-ecosystems of

Northern Indo-Gangetic plains, in terms of being the

readily available and inexpensive option to water. How-

ever, irrigation with such type of effluent mixed water also

has the possible environmental side effects; this transports

a wide variety of elements into the environment (Juwarkar

and Subrahmanyam 1987) and in long run this practice

has led to significant changes in soil physicochemical

Fig. 1 Dendrogram showing the clustering of 14 Streptomyces on the

basis of use/non-use of 95 substrates studied by the BIOLOGTM

system). The simple matching (SSM) coefficient and unweighted-pair

group method with average (UPGMA) clustering were used

1608 World J Microbiol Biotechnol (2011) 27:1603–1613

123

properties i.e. increase in soil pH, electrical conductivity

and organic C, N, P more particularly in EIF over WIF

soils (Juwarkar and Subrahmanyam 1987). Earlier, we

observed the dominance of Gram positive bacteria over

Gram negative in pulp and paper mill effluent mixed water

irrigated fields (EIF) as compared to freshwater irrigated

fields (WIF) (Unpublished data). The present study reports

significant increase in the streptomycetes population in EIF

(120 9 105 gm-1 soil) over WIF soils (48 9 103). This

could be due to the increased availability of residual,

complex structural compounds in the effluents used for

irrigation (Pokhrel and Viraraghavan 2004). Actinomy-

cetes are the active decomposers of organic matter in soils,

including lignin and other recalcitrant polymers, and can

degrade agricultural and urban wastes (McCarthy 1987).

Also, it is the proven fact that streptomycetes prefer alka-

line pH for growth (Goodfellow 1983) and the EIF soils

had alkaline pH (8.16) compared to WIF (7.13). Build up

of population of bacteria, actinomycetes, fungi, yeasts and

rhizobia was already reported in soils irrigated with pulp

and paper mill effluent for 15 years (Kannan and Oblisami

1990a).

The most appropriate measures to determine the effect

of the contamination on ‘‘soil health’’ is to assess the

activity rather than the presence or absence of different cell

types (Lawlor et al. 2000). The percentage of isolates

Fig. 2 Dendrogram with

clustering of 55 isolates of

Streptomyces from WIF and EIF

generated from restriction of

1,500 bp 16S rRNA gene

amplicon by three different

restriction enzymes

World J Microbiol Biotechnol (2011) 27:1603–1613 1609

123

NBE57 (GU136398)

Streptomyces xylophagus AB184526

Streptomyces thermocarboxydus EU593727

NBE34 (GQ268015)

NBC47 (GQ268021)

Streptomyces spp. EU368776

NBE6 (GQ268025)

Streptomyces viridochromogenes EU812168

NBE9 (GQ268020)

Streptomyces spp. FJ842682

NBC5 (GQ268022)

Streptomyces variabilis EU841659

NBE40 (GQ268024)

Streptomyces albogriseolus FJ486402

NBC8 (GQ268026)

Streptomyces viridis AB184361

NBE12 (GQ268016)

Streptomyces macrosporeus EF063494

NBC54 (GU136399)

Streptomyces spp. DQ663155

NBC18 (GQ268017)

Streptomyces glaucescens X79322

NBE7 (GQ268018)

Streptomyces spp. EF063473

NBE13 (GQ268019)

Streptomyces cinnabarinus AJ399487

NBE35 (GQ268023)

Streptomyces aburaviensis AB184178

Escherichia coli U00096

100

100

62

100

99

97

67

67

99

96

80

9399

49

81

92

99

63

72

56

33

66

4453

74

71

0.02

Fig. 3 Phylogenetic tree based

on the 16S rRNA gene

sequences of Streptomycesisolates and their closest

phylogenetic relatives. The tree

was created by the neighbour-

joining method. The numbers on

the tree indicate the percentage

of bootstrap sampling derived

from 1,000 replicates

Table 4 Phylogenetic

affiliations of Streptomycesisolates from WIF and EIF

a Species identified based on

16S rRNA gene sequence

similarity ([97%) of

representative isolateb Percent similarity of the

sequence in BLAST result

Site Representative isolate 16S rRNA sequence homology

Species identified NCBI accession numbera % homologyb

WIF NBC5 Streptomyces variabilis GQ 268022 99

NBC8 Streptomyces viridis GQ 268026 99

NBC18 Streptomyces glaucescens GQ 268017 99

NBC47 Streptomyces spp. GQ 268021 99

NBC54 Streptomyces spp. GU 136399 99

EIF NBE6 Streptomyces viridochromogenes GQ 268025 97

NBE7 Streptomyces spp. GQ 268018 97

NBE9 Streptomyces spp. GQ 268020 100

NBE12 Streptomyces macrosporeus GQ 268016 99

NBE13 Streptomyces cinnabarinus GQ 268019 97

NBE34 Streptomyces thermocarboxydus GQ 268015 99

NBE35 Streptomyces aburaviensis GQ 268023 98

NBE40 Streptomyces albogriseolus GQ 268024 99

NBE57 Streptomyces xylophagus GU 136398 99

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exhibiting xylanase and cellulase production was signifi-

cantly higher in EIF as compared to WIF soils. The results

are thus suggesting that continuous application of pulp and

paper mill effluents containing water has led to enrichment

of Streptomyces species with high xylanase and cellulase

activity. It was observed earlier that the application of such

effluents to sugarcane crops increased soil enzyme activi-

ties (Kannan and Oblisami 1990b). The percentage and

quantitative level of Indolic compounds and percentage of

isolates with siderophore production was more in EIF soils.

This very well correlates with earlier reports that sidero-

phores promote auxin synthesis by chelating metals

(Dimkpa et al. 2008). Chelation makes the metals less

inhibitory to synthesis of auxins, which in turn enhances

growth of indolic compound producing bacteria over non-

producing ones (Manulis et al. 1994). Pulp and paper mill

effluents have been reported to contain various trace metals

(Skipperud et al. 1998). The establishment and perfor-

mance of P-solubilizing microorganisms is severely

affected by environmental factors, especially the stressful

conditions (Mehta and Nautiyal 2001). Streptomycetes are

earlier reported to solubilize phosphate (Hamdali et al.

2008). In the present study, a decrease in the number of

isolates showing P-solubilisation activity (from EIF) indi-

cates that effluent irrigation negatively affected the P-solu-

bilisation capacity in streptomycetes. Relating BIOLOGTM

experimental findings to the in situ ecology of streptomy-

cetes is challenging, but because some of the substrate usage

was exclusive to isolates cultured from a specific collection

site, one could hypothesize that metabolic activity is a

reflection not just of the species, but is also a reflection of the

specific environmental conditions.

The Shannon index (H), reciprocal of Simpson’s index

(1/D) and Pielou index (E) revealed that sterptomycetes

community in EIF was more diverse compared to those

from WIF soils. A possible explanation for this difference

could be the wider range of organic substrates and

increased nutrient status of the soil (Øvreas and Torsvik

1998).

ARDRA was used in this study to assist in distin-

guishing among taxonomic groups. This technique has

been shown to be a useful tool for screening environmental

bacterial isolates and/or clone libraries (Sjoling and Cowan

2003). Among the presently described 55 isolates, 44%

closely related to Streptomyces variabilis, previously

reported to be soil inhabitants (Preobrazhenskaya et al.

1957), 13% to S. viridochromogenes, as colonizers

of sugarcane roots (Fernandez and Szabo 1982), 7% to

S. viridis, that alleviate soil contaminations (Wyszkowska

et al. 2008), and 7% to S. cinnabarinus from hot climate

Table 5 Diversity index of Streptomyces isolates based on ARDRA profile and carbon-substrate pattern

Sites Diversity indicesa

Shannon index of diversity (H0) Pielou index (E) Reciprocal of Simpson’s index (1/D)

WIF 1.419 0.729 2.870

EIF 2.098 0.874 6.259

a The number of isolates showing similar ARDRA profile are grouped and used for estimating diversity indices

Fig. 4 DGGE fingerprints of Streptomyces specific 16S rRNA gene

fragments amplified from soil DNA templates obtained from WIF and

EIF: (lanes C1, C2)—WIF, (lanes E1, E2)—EIF

World J Microbiol Biotechnol (2011) 27:1603–1613 1611

123

soils (Preobrazhenskaya et al. 1957). Streptomyces ther-

mocarboxydus, S. aburaviensis, S. macrosporeus, S. xyl-

ophagus and S. albogriseolus were specifically prevalent in

EIF soils. Earlier workers reported S. thermocarboxydus

from composts, soils and sewage (O’Donnell et al. 1993)

whereas S. xylophagus is a well studied xylanase producing

actinomycetes (Tangnu et al. 1981). One isolate was found

similar to S. glaucescens from WIF, reported to be the soil

inhabitant by Preobrazhenskaya et al. (1957). These

observations collectively suggest that irrigation of agri-

cultural fields with pulp and paper mill effluent exceeding

20 years in succession has altered the genetic and meta-

bolic diversity of streptomycetes. Oved et al. (2001) also

observed the significant and consistent shift in population

composition of ammonia oxidizing bacteria as a result of

effluent irrigation with the enrichment of Nitorosmonas-

like population. The DGGE fingerprints obtained for the

streptomycetes communities analyzed here hardly varied,

regardless of effluent treatment (Fig. 4). It indicates that

the effect of effluent irrigation on the dominant bands

observed in DGGE analysis of DNA extracted directly

from soil was minimal compared to the effects on the

culturable portion of the community. This observation

supports the previous studies that readily culturable bac-

teria are probably the largest, most active prokaryotes in a

given sample (Bakken 1997) and so provide a useful, rapid

assessment of biological responses to determining the

impact of anthropogenic activity.

This study indicated that pulp and paper mill effluent

contamination did not have a significant effect on the total

genetic diversity of streptomycetes but affected physio-

logical and metabolic status, so that the number of strep-

tomycetes isolates capable of responding to laboratory

culture and their taxonomic distribution were altered. Thus,

it appears that plate counts may be a more appropriate

method for determining the effect of effluent contamination

on streptomycetes than culture-independent approaches.

Streptomyces isolates in this study could be further

exploited for commercial production of enzymes of bio-

mass degradation as well as promoting plant growth in

such soils.

Acknowledgments This research was conducted with funds pro-

vided by the Indian Council of Agricultural Research under Network

Project on Application of Microorganisms in Agriculture and Allied

Sectors.

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