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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


(Member)

Zulkifli Shamsuddin, PhD

Professor

Faculty of Agriculture


(Member)

Muskhazli Mustafa, PhD

Associate Professor

Faculty of Science


(Member)

_____________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of graduate Studies


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



5.2.2 Chemical 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



6.2.2 Microbial and enzymes activity 96


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


7.2.1 Preparation of feed materials 113

7.2.2 Selection of earthworms 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


7.3 Results 118

7.4 Discussion 125

8 CONCLUDING REMARKS AND FUTURE

RECOMMENDATIONS

135

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