universiti putra malaysia enzyme activities …kitaran semula nutrien dilihat berkesan untuk...
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UNIVERSITI PUTRA MALAYSIA
YAN YI WEI
FS 2013 1
ENZYME ACTIVITIES AND ENHANCEMENT OF PLANT NUTRIENT CONTENT IN VERMICOMPOST
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ENZYME ACTIVITIES AND ENHANCEMENT
OF PLANT NUTRIENT CONTENT IN
VERMICOMPOST
YAN YI WEI
DOCTOR OF PHILOSOPHY
UNIVERSITI PUTRA MALAYSIA
YEAR
January 2013
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirement for the degree of Doctor of Philosophy
ENZYME ACTIVITIES AND ENHANCEMENT OF PLANT NUTRIENT
CONTENT IN VERMICOMPOST
By
YAN YI WEI
January 2013
Chairman: Nor Azwady Abd Aziz, PhD
Faculty: Science
Nutrient recycling has been seen as a viable way to return nutrient both to the soil
and plant. Recycling of organic wastes such as plant residues reduces the amount of
wastes that enter the landfills. Vermicomposting has been widely accepted as an
efficient, rapid and cost effective way in managing organic waste. The current study
investigated the efficiency of earthworms Eudrilus eugeniae and Perionyx excavatus
in vermicomposting rice straw. Vermicompost derived by P. excavatus was found to
contain higher concentrations of total N, P, K and Mg. Available P, K and Mg was
also higher in P. excavatus vermicompost. However E. eugeniae vermicompost was
found to contain higher total and available Ca. Humic acid content was also found to
be higher in E. eugeniae vermicompost. The experiment showed E. eugeniae took
134 days while P. excavatus took 171 days to complete vermicomposting. Though
Perionyx excavatus vermicompost contains higher total and available plant nutrient,
the rate of vermicompost generation by this species was relatively low compared to
E. eugeniae. Subsequent experiment was conducted by comparing plant nutrient
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availabilities and humic acids content in vermicompost generated from different plant
residues; grass clippings (GC), sago waste (SW) and rice straw (RS) using E.
eugeniae as vermicomposting agent. Total P was lower in vermicomposts as
compared to the controls, however, highest extractable P was found in RS
vermicompost (0.33 ± 0.00 g/kg). SW vermicompost contained highest total Ca
(22.79 ± 0.01 g/kg) compared to the GC (1.39 ± 0.01 g/kg) and RS (8.22 ± 0.06 g/kg)
vermicompost. Significant positive correlations between nutrient contents in raw
plant residues and vermicompost were observed for total N (r = 0.779), K (r = 0.998)
and Ca (r = 0.997). The study showed that nutrient in initial wastes material affects
the nutrient contents of vermicompost. This suggests that among the plant residues
studied, vermicomposting of rice straw produced vermicompost with the highest
plant extractable nutrient contents. The effect of storage on the enzymatic action and
microbial activity in the vermicompost was also determined. RS vermicompost was
stored for 6 months and samples were taken every month to analyse the microbial
and enzymatic activity in the vermicompost. Microbial and enzyme activities
(protease and phosphatase) in vermicompost were found to be relatively active
indicating that application of vermicompost after 6 months post harvest may still
stimulate N and P nutrient cycling in agricultural land. RS vermicompost was found
to contain high amount of plant extractable nutrients which could be easily taken up
by plants. Nevertheless when compared to inorganic fertilizers the nutrient content in
vermicompost is still relatively lower. Therefore, an attempt was made to further
improve the extractable nutrient in RS vermicompost by adding rock phosphate (RP)
during vermicomposting. The extractable P was 24% higher in vermicompost with
the addition of rock phosphate. In addition, extractable macronutrients N and K were
found to be significantly higher in the final product of vermicomposting with the
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addition of rock phosphate. The research studied the various aspects that influence
the quality of vermicompost. Based on the results obtained, an improvement in the
macronutrient content in vermicompost has also successfully achieved. As an overall,
the evidences raised for the current study proved that vermicomposting can be a
viable technology in recycling organic wastes such as rice straw in Malaysia. This
will benefit the farmers economically and contribute significantly in reducing open
burning activity, thus creating a cleaner environment.
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Abstrak thesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
AKTIVITI ENZIM DAN PENAMBAHANBAIKAN KANDUNGAN NUTRIEN
TUMBUHAN DALAM VERMI KOMPOS
Oleh
YAN YI WEI
Januari 2013
Pengerusi: Nor Azwady Abd Aziz, Ph.D.
Fakulti: Sains
Kitaran semula nutrien dilihat berkesan untuk mengembalikan nutrien ke dalam
tanah dan tumbuhan. Penggunaan semula sisa organik seperti sisa tumbuhan dapat
mengurangkan jumlah bahan buangan ke tapak pelupusan. Pengkomposan-vermi
(pengkomposan dengan menggunakan cacing tanah) telah diterima sebagai cara yang
efisien, pantas dan murah dalam pengurusan sisa organik. Kajian ini telah dilakukan
untuk menilai keberkesanan cacing tanah Eudrilus eugeniae dan Perionyx excavatus
dalam pengkomposan-vermi jerami padi. Vermikompos yang dihasilkan oleh P.
excavatus menunjukkan komposisi nutrien N, P, K dan Mg yang lebih tinggi. Ia juga
mengandungi P, K dan Mg tersedia dalam kepekatan yang lebih tinggi. Tetapi,
vermikompos E. eugeniae menunjukkan komposisi Ca, Ca tersedia dan asid humik
dalam kepekatan yang lebih tinggi. Kajian juga menunjukkan E. eugeniae
mengambil masa 134 hari bagi melengkapkan proses pengkomposan jerami padi
manakala P. excavatus pula mengambil 171 hari. Walaupun vermikompos P.
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excavatus menunjukkan kepekatan nutrien yang lebih tinggi namun, peratus
penjanaan vermikompos oleh spesies ini adalah lebih rendah berbanding dengan
vermikompos E. eugeniae. Seterusnya, kajian ini turut membandingkan kandungan
nutrien dan asid humik dalam vermikompos keratan rumput (GC), sisa sagu (SW)
dan jerami padi (RS). Kandungan nutrien tumbuhan dan asid humik dalam
vermikompos menggunakan E. eugeniae sebagai agen pengkomposan telah
dianalisa. Kandungan P menunjukkan kepekatan yang lebih rendah dalam
vermikompos berbanding dengan kawalan, walau bagaimanapun, vermikompos RS
menunjukkan kepekatan P tersedia paling tinggi (0.33 + 0.00 g/kg). Vermikompos
SW mengandungi kepekatan Ca yang paling tinggi (22.79 + 0.01 g/kg) berbanding
dengan vermikompos GC (1.39 + 0.01 g/kg) dan RS (8.22 + 0.06 g/kg). Kandungan
N (r=0.779), K (r=0.998) dan Ca (r=0.997) menunjukkan korelasi positif yang bererti
antara bahan asas (sisa organik) dan kandungan nutrien dalam vermikompos.
Vermikompos jerami padi mempunyai kandungan nutrien tersedia yang paling
tinggi. Kesan penyimpanan vermikompos ke atas aktiviti enzim dan mikroorganisma
dalam vermikompos turut dikaji. Vermikompos RS telah disimpan selama 6 bulan
dan sampel diambil setiap bulan bagi analisa aktiviti enzim dan mikroorganisma.
Aktiviti mikroorganisma dan enzim (protease dan phosphatase) dalam vermikompos
didapati masih aktif sepanjang tempoh kajian. Justeru, aplikasi vermikompos walau
setelah 6 bulan dituai adalah masih efektif serta dapat merangsangkan kitaran nutrien
N dan P dalam tanah pertanian. Vermikompos RS mengandungi nutrien tersedia
yang paling tinggi dan mudah diserap oleh tumbuhan. Namun begitu, kandungan
nutrien dalam vermikompos secara relatif masih rendah jika dibandingkan dengan
baja bukan organik. Oleh yang demikian, kajian telah dilakukan dengan
menambahkan ‘rock phosphate’ (RP) dalam proses pengkomposan-vermi. Hasil
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daripada penambahan ‘rock phosphate’, kandungan P tersedia didapati meningkat
sebanyak 24% berbanding dengan vermikompos yang dihasilkan tanpa penambahan
‘rock phosphate’. Kandungan makronutrien N dan K tersedia vermikompos turut
menunjukkan peningkatan dengan penambahan ‘rock phosphate’. Kajian ini
menyimpulkan pengkomposan-vermi merupakan teknologi yang berkesan dalam
mengitar semula sisa organik seperti jerami padi di Malaysia kepada produk
vermikompos yang berkualiti tinggi. Ini secara tidak langsung membawa faedah
ekonomi kepada para petani di samping menurunkan pembakaran terbuka seterusnya
mewujudkan persekitaran yang lebih bersih.
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ACKNOWLEDGEMENTS
This dissertation would not be able to be completed without the guidance, support
and help of several individuals who in one way or another have contributed or
extended their valuable assistance in the preparation and completion of this research.
First and foremost, my utmost gratitude to my supervisor Dr. Nor Azwady Abd Aziz
for his patience and steadfast encouragement as I hurdle all the obstacles in
completing the research. I would have been lost without him. Special thanks to Prof
Zulkifli Hj. Shamsuddin, his perpetual energy and enthusiasm has motivated many of
his advisees including me. My sincere thanks also goes to Assoc. Prof. Dr.
Muskhazli Mustafa and Prof. Dr. Suraini Abd Aziz for their inputs especially by
providing invaluable insights and sharing their experiences with me.
All my lab-mates and friends that have make both working in the lab and thesis
writing a more enjoyable experience. I warmly thank Suk Kuan, Rainbow, Jun, Izyan
and all other postgraduates (the ‘exhaustive’ list includes those in the Postgraduate
Room and Aquatic Lab) for their friendship and companion.
I owe my loving thanks to my husband for his continual support financially and
emotionally throughout the entire study. Special gratitude goes to my family and
extended family members for their loving support. Thank you all for giving me the
strength to plod through good and bad times. The financial support through Graduate
Research Fellowship (GRF) by Universiti Putra Malaysia is also gratefully
acknowledged.
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Nor Azwady Abd Aziz, PhD
Senior Lecturer
Faculty of Science
Universiti Putra Malaysia
(Chairman)
Suraini Abd Aziz, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Zulkifli Shamsuddin, PhD
Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Member)
Muskhazli Mustafa, PhD
Associate Professor
Faculty of Science
Universiti Putra Malaysia
(Member)
_____________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which
have been duly acknowledged. I also declare that it has not been previously, and is
not concurrently, submitted for any other degree at Universiti Putra Malaysia or at
any other institution.
_________________________
YAN YI WEI
Date: 14th
January 2013
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LIST OF TABLES
Table Page
2.1 Moisture requirements of earthworm species commonly used as
vermicomposting agents
52
4.1 Biological performances of earthworms Eudrilus eugeniae (EE)
and Perionyx excavatus (PE)
67
4.2 Amount of rice straw consumed and vermicompost harvested for
EE and PE when 70% of rice straw has been decomposed (EE at
D134 (day 134) and EE at D171 (day 172))
68
4.3 Plant nutrient concentrations, pH and humic acids of
vermicompost (EE and PE) and control harvested at 70%
decomposition of rice straw
69
4.4 Total and available plant nutrient contents generated per day by
earthworm EE and PE (weight of vermicompost generated per
day multiply nutrient concentrations in vermicompost)
70
5.1 Physicochemical and elemental analysis of raw materials used as
initial substrates (g/kg) on dry weight basis
80
5.2 Physicochemical and elemental analysis of vermicompost and
controls dry weight basis
81
5.3 Correlation of plant nutrient in vermicompost and controls with
the different raw materials
84
6.1 pH, humic acid and total plant macronutrient contents in fresh
(day 0) vermicompost and control (mean ± standard error, n=4)
101
6.2 Plant extractable macronutrients in fresh (day 0) vermicompost
and control (mean ± standard error, n=4)
102
7.1 Total plant macronutrients (N, P, K, Ca and Mg) in raw material
rice straw (mean ± standard error, n=4)
121
7.2 Total plant macronutrients (N, P, K, Ca and Mg) in vermicompost
and control (mean ± standard error, n=4)
122
7.3 Extractable plant macronutrients (N, P, K, Ca and Mg) in
vermicompost and control (mean ± standard error, n=4)
123
7.4 Population of phosphate solubilising bacteria (mean ± standard
error, n=4)
124
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LIST OF FIGURES
Figure Page
3.1 Plant residues used as vermicomposting substrates (A) grass
clippings (B) sago wastes (C) rice straw
43
3.2 Dark and shiny humic acid extracted from the vermicompost 53
5.1 Average (mean ± standard error) of plant nutrient contents in
vermicompost and control (A) total and extractable phosphorus
(B) total and extractable potassium (C) total and extractable
calcium
83
6.1 Amount of carbon dioxide liberated per gram of sample by
vermicompost and control depicting microbial activity at different
storage time (mean ± standard error, n=4)
98
6.2 Concentration of tyrosine produced from activities of protease at
in vermicompost and control at different storage time (mean ±
standard error, n=4)
99
6.3 Concentration of p-nitrophenol (PNP) produced from activities of
alkaline phosphatase at in vermicompost and control at different
storage time (mean ± standard error, n=4)
100
6.4 Concentration of p-nitrophenol produced from activities of acid
phosphatase at in vermicompost and control at different storage
time (mean ± standard error, n=4)
101
7.1 NBRIP media (A) NBRIP plate before inoculation of bacteria (B)
halozones formed by phosphate solubilising bacteria
117
7.3 Amount of p-nitrophenol liberated per hour as an estimation of
protease activity in the treatments (mean ± standard error, n=4)
125
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LIST OF ABBREVIATIONS
ANOVA Analysis of Variance
C Carbon
Ca Calcium
CD Cow dung
CFU Colony forming unit
CIRP Christmas island rock phosphate
D Diameter
EE Eudrilus eugeniae
GC Grass clippings
H Height
ICP Induction coupled plasma
K Potassium
Mg Magnesium
N Nitrogen
NBRIP National botanical research phosphate growth media
P Phosphorus
PE Perionyx excavatus
PSB Phosphate solubilising bacteria
RP Rock phosphate
RS Rice straw
SPSS Statistical package for social package
SW Sago waste
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TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK v
ACKNOWLEDGEMENTS viii
APPROVAL ix
DECLARATION x
LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF ABBREVIATIONS xiv
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 8
2.1 Sustainable agriculture practice 8
2.2 Common organic wastes 9
2.2.1 Rice straw 10
2.2.2 Sago residues 12
2.2.3 Grass clippings 13
2.3 Vermicomposting versus conventional composting 14
2.4 Earthworm 15
2.5 Vermicomposting – the process 17
2.6 Requirements for vermicomposting 19
2.6.1 Moisture 20
2.6.2 Temperature 22
2.6.3 pH 23
2.7 C:N ratio 24
2.8 Cow dung 25
2.9 Vermicomposting scenario in Malaysia 26
2.10 Quality of vermicompost 28
2.10.1 Nitrogen 29
2.10.2 Phosphorus 30
2.10.3 Humic acids 31
2.10.4 Microbial activity 33
2.10.5 Enzymes activities in vermicompost 35
2.10.5.1 Protease 36
2.10.5.2 Phosphatase 37
2.11 Stability of vermicompost 39
3 GENERAL MATERIAL AND METHODS 42
3.1 Experimental settings 42
3.2 Media preparation 42
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3.3 Moisture control and watering technique 43
3.4 Vermicompost harvesting technique 44
3.5 pH measurement 44
3.6 Moisture content 45
3.7 Total carbon 46
3.8 Total macronutrient analysis 46
3.8.1 Total nitrogen 46
3.8.2 Total phosphorus, potassium and calcium (dry
ashing)
47
3.9 Extractable nitrogen 49
3.10 Extractable phosphorus, potassium and calcium (Merlich
3)
49
3.10.1 EDTA stock reagent 49
3.10.2 Extraction reagent solution 50
3.10.3 Extraction of phosphorus, potassium and calcium 50
3.11 Humic acids 51
3.11.1 Extraction 51
3.11.2 Fractionation 52
3.11.3 Purification 52
3.11.4 Drying 53
3.12 Microbial activity (Bekku et al., 1997) 54
3.13 Enzymes activity 55
3.13.1 Acid and alkaline phosphatase (Tabatabai and
Bremner, 1969)
55
3.13.1.1 Modified universal buffer 55
3.13.1.2 Chemical solutions 56
3.13.1.3 Sample solutions 56
3.13.1.4 Quenching solutions 57
3.13.1.5 Standards solutions 57
3.13.1.6 Calculations for acid and alkaline
phosphatase
58
3.13.2 Protease (Ladd and Butler, 1972) 59
3.13.2.1 Sample solutions 59
3.13.2.2 Quenching solutions 60
3.13.2.3 Standard solutions (Cupp-Enyard, 2008) 60
3.13.2.4 Calculation for protease activity 61
4 VERMICOMPOSTING POTENTIAL AND PLANT
NUTRIENT CONTENTS IN RICE STRAW
VERMICOMPOST OF Perionyx excavatus AND Eudrilus
eugeniae
63
4.1 Introduction 63
4.2 Material and methods 65
4.3 Statistical analysis 66
4.2.2 Experimental setup 62
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4.2.3 Chemical analysis 63
4.2.4 Statistical analysis 63
4.4 Results 66
4.5 Discussion 70
5 COMPARISON ON PLANT NUTRIENT CONTENTS IN
VERMICOMPOST OF SELECTED PLANT RESIDUES
77
5.1 Introduction 77
5.2 Material and methods 78
5.2.1 Experimental setup 78
5.2.2 Chemical analysis 79
5.2.3 Statistical analysis 79
5.3 Results 79
5.4 Discussion 85
6 STABILITY OF ENZYMES (PROTEASE AND
PHOSPHATASE) AND MICROBIAL ACTIVITY
DURING SIX MONTHS STORAGE OF
VERMICOMPOST
94
6.1 Introduction 94
6.2 Material and methods 96
6.2.1 Experimental setup 96
6.2.2 Microbial and enzymes activity 96
6.2.3 Statistical analysis 97
6.3 Results 97
6.4 Discussion 102
7 ENHANCEMENT OF PLANT NUTRIENT
AVAILABILITIES IN RICE STRAW VERMICOMPOST
THROUGH THE ADDITION OF ROCK PHOSPHATE
111
7.1 Introduction 111
7.2 Material and methods 113
7.2.1 Preparation of feed materials 113
7.2.2 Selection of earthworms 114
7.2.3 Experimental setup 114
7.2.4 Analysis of vermicompost 115
7.2.5 National Botanical Research Institute phosphate
growth media (NBRIP)
115
7.2.5.1 Serial dilution 116
7.2.5.2 Phosphate Solubilizing Bacteria (PSB) spread
plates
116
7.2.6 Statistical analysis 118
7.3 Results 118
7.4 Discussion 125
8 CONCLUDING REMARKS AND FUTURE
RECOMMENDATIONS
135
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REFERENCES
APPENDICES
BIODATA OF STUDENT
LIST OF PUBLICATIONS
142
163
164
165