UNIVERSITI PUTRA MALAYSIA ENZYME ACTIVITIES AND ENHANCEMENT OF PLANT NUTRIENT CONTENT IN VERMICOMPOST YAN YI WEI

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1 UNIVERSITI PUTRA MALAYSIA ENZYME ACTIVITIES AND ENHANCEMENT OF PLANT NUTRIENT CONTENT IN VERMICOMPOST YAN YI WEI FS

2 (UPM LOGO) ENZYME ACTIVITIES AND ENHANCEMENT OF PLANT NUTRIENT CONTENT IN VERMICOMPOST YAN YI WEI DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA YEAR January 2013

3 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 Chairman: Nor Azwady Abd Aziz, PhD Faculty: Science By YAN YI WEI January 2013 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 ii

4 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 iii

5 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. iv

6 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. v

7 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 ( g/kg). Vermikompos SW mengandungi kepekatan Ca yang paling tinggi ( g/kg) berbanding dengan vermikompos GC ( g/kg) dan RS ( 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 vi

8 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. vii

9 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. viii

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11 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: x

12 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: 14 th January 2013 xi

13 LIST OF TABLES Table Page 2.1 Moisture requirements of earthworm species commonly used as vermicomposting agents 4.1 Biological performances of earthworms Eudrilus eugeniae (EE) and Perionyx excavatus (PE) 4.2 Amount of rice straw consumed and vermicompost harvested for EE and PE when 70% of rice straw has been decomposed (EE at D 134 (day 134) and EE at D 171 (day 172)) 4.3 Plant nutrient concentrations, ph and humic acids of vermicompost (EE and PE) and control harvested at 70% decomposition of rice straw 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) 5.1 Physicochemical and elemental analysis of raw materials used as initial substrates (g/kg) on dry weight basis 5.2 Physicochemical and elemental analysis of vermicompost and controls dry weight basis 5.3 Correlation of plant nutrient in vermicompost and controls with the different raw materials 6.1 ph, humic acid and total plant macronutrient contents in fresh (day 0) vermicompost and control (mean ± standard error, n=4) 6.2 Plant extractable macronutrients in fresh (day 0) vermicompost and control (mean ± standard error, n=4) 7.1 Total plant macronutrients (N, P, K, Ca and Mg) in raw material rice straw (mean ± standard error, n=4) 7.2 Total plant macronutrients (N, P, K, Ca and Mg) in vermicompost and control (mean ± standard error, n=4) 7.3 Extractable plant macronutrients (N, P, K, Ca and Mg) in vermicompost and control (mean ± standard error, n=4) Population of phosphate solubilising bacteria (mean ± standard error, n=4) 124 xii

14 LIST OF FIGURES Figure 3.1 Plant residues used as vermicomposting substrates (A) grass clippings (B) sago wastes (C) rice straw 3.2 Dark and shiny humic acid extracted from the vermicompost 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 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) 6.2 Concentration of tyrosine produced from activities of protease at in vermicompost and control at different storage time (mean ± standard error, n=4) 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) 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) 7.1 NBRIP media (A) NBRIP plate before inoculation of bacteria (B) halozones formed by phosphate solubilising bacteria 7.3 Amount of p-nitrophenol liberated per hour as an estimation of protease activity in the treatments (mean ± standard error, n=4) Page xiii

15 LIST OF ABBREVIATIONS ANOVA Analysis of Variance C Carbon Ca CD CFU CIRP D EE GC H ICP K Mg N NBRIP P PE PSB RP Calcium Cow dung Colony forming unit Christmas island rock phosphate Diameter Eudrilus eugeniae Grass clippings Height Induction coupled plasma Potassium Magnesium Nitrogen National botanical research phosphate growth media Phosphorus Perionyx excavatus Phosphate solubilising bacteria RS SPSS Rock phosphate Rice straw Statistical package for social package SW Sago waste xiv

16 TABLE OF CONTENTS ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS CHAPTER 1 INTRODUCTION 1 Page ii v viii ix x xii xiii xiv 2 LITERATURE REVIEW Sustainable agriculture practice Common organic wastes Rice straw Sago residues Grass clippings Vermicomposting versus conventional composting Earthworm Vermicomposting the process Requirements for vermicomposting Moisture Temperature ph C:N ratio Cow dung Vermicomposting scenario in Malaysia Quality of vermicompost Nitrogen Phosphorus Humic acids Microbial activity Enzymes activities in vermicompost Protease Phosphatase Stability of vermicompost 39 3 GENERAL MATERIAL AND METHODS Experimental settings Media preparation 42

17 3.3 Moisture control and watering technique Vermicompost harvesting technique ph measurement Moisture content Total carbon Total macronutrient analysis Total nitrogen Total phosphorus, potassium and calcium (dry 47 ashing) 3.9 Extractable nitrogen Extractable phosphorus, potassium and calcium (Merlich 49 3) EDTA stock reagent Extraction reagent solution Extraction of phosphorus, potassium and calcium Humic acids Extraction Fractionation Purification Drying Microbial activity (Bekku et al., 1997) Enzymes activity Acid and alkaline phosphatase (Tabatabai and 55 Bremner, 1969) Modified universal buffer Chemical solutions Sample solutions Quenching solutions Standards solutions Calculations for acid and alkaline 58 phosphatase Protease (Ladd and Butler, 1972) Sample solutions Quenching solutions Standard solutions (Cupp-Enyard, 2008) Calculation for protease activity 61 4 VERMICOMPOSTING POTENTIAL AND PLANT 63 NUTRIENT CONTENTS IN RICE STRAW VERMICOMPOST OF Perionyx excavatus AND Eudrilus eugeniae 4.1 Introduction Material and methods Statistical analysis Experimental setup 62

18 4.2.3 Chemical analysis Statistical analysis Results Discussion 70 5 COMPARISON ON PLANT NUTRIENT CONTENTS IN 77 VERMICOMPOST OF SELECTED PLANT RESIDUES 5.1 Introduction Material and methods Experimental setup Chemical analysis Statistical analysis Results Discussion 85 6 STABILITY OF ENZYMES (PROTEASE AND 94 PHOSPHATASE) AND MICROBIAL ACTIVITY DURING SIX MONTHS STORAGE OF VERMICOMPOST 6.1 Introduction Material and methods Experimental setup Microbial and enzymes activity Statistical analysis Results Discussion ENHANCEMENT OF PLANT NUTRIENT 111 AVAILABILITIES IN RICE STRAW VERMICOMPOST THROUGH THE ADDITION OF ROCK PHOSPHATE 7.1 Introduction Material and methods Preparation of feed materials Selection of earthworms Experimental setup Analysis of vermicompost National Botanical Research Institute phosphate 115 growth media (NBRIP) Serial dilution Phosphate Solubilizing Bacteria (PSB) spread 116 plates Statistical analysis Results Discussion CONCLUDING REMARKS AND FUTURE RECOMMENDATIONS 135

19 REFERENCES APPENDICES BIODATA OF STUDENT LIST OF PUBLICATIONS