UNIVERSITI PUTRA MALAYSIA

MOHD YUSOFF BIN ABD SAMAD

FBSB 2011 52

BIOFERTILIZER FORMULATION COMPRISING PHOSPHATE SOLUBILIZING BACTERIA TO ENHANCE PHOSPHATE

AVAILABILITY FOR COCOA GROWTH

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BIOFERTILIZER FORMULATION COMPRISING PHOSPHATE

SOLUBILIZING BACTERIA TO ENHANCE PHOSPHATE AVAILABILITY FOR COCOA GROWTH

By

MOHD YUSOFF BIN ABD SAMAD

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

January 2012

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DEDICATION

I dedicate this precious effort, the fruit of my thoughts and study to my

affectionate Mother, brothers and sisters, wife Ameza and children Nur Aina,

Nur Farhana and Muhammad Firdaus who inspired me to the

higher destiny of life.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in Fulfillment of the requirement for the degree of Doctor of Philosophy

BIOFERTILIZER FORMULATION COMPRISING PHOSPHATE SOLUBILIZING BACTERIA TO ENHANCE PHOSPHATE

AVAILABILITY FOR COCOA GROWTH

By

MOHD YUSOFF ABD SAMAD

January 2012

Chairman : Professor Raja Noor Zaliha Raja Abd Rahman, D.Eng.

Faculty : Biotechnology and Biomolecular Sciences Production of cocoa can be enhanced by good fertilizer application which

normally using inorganic fertilizers. However, prolonged use of inorganic

fertilizers poses many environmental problems. The use of microorganisms

as biofertilizer with specific functional abilities in the mobilization of

nutrients is an alternative to mineral fertilizer for enhancement of soil

fertility and crop production in sustainable agriculture. The general

objectives of this study were to screen phosphate-solubilizing bacteria (PSB),

to formulate the formulation of the bacteria inoculants using cocoa wastes

and to study the shelf life of the formulations. Screening of all bacterial

isolates from various sources of agricultural soils and wastes were carried

out. Twenty-two isolates showed the capability of solubilizing insoluble

phosphate by both plate assay and liquid medium techniques. The top three

PSB namely Pseudomonas aeruginosa strain AGKT1, Serratia marcescens strain

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AGKT4 and Bacillus amyloliquefaciens were chosen for subsequent studies. In

preparation of seed culture for use in dry and liquid formulations, optimum

concentrations of carbon source, nitrogen source, and density of inoculum

were determined using response surface methodology. Carrier materials

from cocoa pod husk and cocoa bean shell were found to be superiors as

organic carrier-based materials for the selected PSB as compared to peat

moss. The shelf life of bioinoculants in the dry and liquid formulations was

found to be good as they had a high number of viable cells for up to 6

months with the cfu above 5 log10 cfu/g when stored at both temperatures

of 10 oC and 28 oC. Application of the dry and liquid formulations on cocoa

seedlings showed the good responses to the growth, biomass and

chlorophyll content of cocoa seedlings. All the dry formulations significantly

increased (P<0.05) fresh and dry plant biomass as well as vegetative growth

of cocoa seedlings. The three formulations using cocoa bean shell with

Pseudomonas aeruginosa strain AGKT1, Serratia marcescens strain AGKT4 and

Bacillus amyloliquefaciens resulted in an increase of 86.74%, 114.31%, and

84.35% of total fresh weight of cocoa seedlings, respectively. The dry matter

weight of cocoa seedlings also significantly increased at 30.05%, 48.90%, and

19.69% in the formulation using cocoa bean shell with these three PSB as

compared to the control. Enhancement of nutrient uptake by the cocoa

seedlings and the increase in nutrients in the soil, in particular to phosphorus

(P) availability (> 15 ppm) by most dry formulations indicated that the

formulations were superiors as bioinoculant preparation.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan Ijazah Doktor Falsafah.

FORMULASI BAJA BIO MENGANDUNGI BAKTERIA PELARUT FOSFAT BAGI MENINGKATKAN KEDAPATAN FOSFAT UNTUK

PERTUMBUHAN KOKO

Oleh

MOHD YUSOFF ABD SAMAD

Januari 2012

Pengerusi : Profesor Raja Noor Zaliha Raja Abd Rahman, D.Eng.

Fakulti : Bioteknologi dan Sains Biomolekul

Pengeluaran koko dapat ditingkatkan dengan pemberian baja yang baik

yang biasanya mengunnakan baja bukan organic. Namun, penggunaan

jangka panjang baja bukan organik menimbulkan pelbagai masalah kepada

persekitaran. Penggunaan mikroorganisma sebagai baja biologi dengan

kemampuan fungsi-fungsi tertentu dalam mobilisasi nutrien adalah satu

alternatif bagi baja mineral bagi meningkatkan kesuburan tanah dan

pengeluaran tanaman dalam pertanian yang mampan. Tujuan umum kajian

ini adalah untuk memencilkan bakteria pelarut fosfat, untuk merumuskan

formulasi inokulan bakteria menggunakan hasil buangan koko dan untuk

mengkaji kemampuan hidup bakteria dari formulasi-formula tersebut.

Bakteria dipencilkan dari pelbagai sumber tanah pertanian dan bahan

buangan. Pemencilan 22 bakteria mampu melarutkan fosfat tidak larut oleh

kedua teknik ujian plat asai dan media cecair. Tiga bakteria terbaik dari

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Pseudomonas aeruginosa strain AGKT1, Serratia marcescens strain AGKT4 dan

Bacillus amyloliquefaciens telah dipilih dalam kajian selanjutnya. Dalam

penyediaan kultura asas untuk digunakan dalam penyediaan formulasi

cecair dan kering, kepekatan sumber karbon, nitrogen dan ketumpatan

inokulum telah ditentukan dengan mengunakan kaedah tindakbalas

permukaan. Bahan pembawa dari kulit buah koko dan kulit biji koko telah

didapati sebagai pembawa terbaik sebagai bahan pembawa berasas organik

untuk bakteria pelarut fosfat terpilih jika dibandingkan dengan tanah

gambut. Daya hidup inokulan dalam formulasi kering dan cecair telah

didapati baik kerana mempunyai bilangan sel bernas yang tinggi sehingga

6 bulan dengan unit pembentukan koloni adalah melebihi 5 log10 cfu/g

apabila disimpan pada suhu 10 oC dan 28 oC. Applikasi formulasi kering

dan cecair ke atas anakbenih-anakbenih koko menunjukkan tindakbalas

kepada pertumbuhan, biojisim dan kandungan klorofil anakbenih koko.

Semua formulasi kering meningkat secara bererti (P<0.05) bagi biojisim

basah dan kering dan pertumbuhan tampang anakbenih koko. Tiga

formulasi menggunakan kulit biji koko dengan penggunaan Pseudomonas

aeruginosa strain AGKT1, Serratia marcescens strain AGKT4 dan Bacillus

amyloliquefaciens memberikan peningkatan 86.74%, 114.31% dan 84.35% dari

jumlah berat basah anakbenih koko, masing-masing. Jumlah berat kering

anakbenih koko juga meningkat dengan ketara iaitu sebanyak 30.05%,

48.90% dan 19.69% dalam formulasi menggunakan kulit biji koko dengan

tiga bakteria pelarut fosfat ini jika dibanding dengan kawalan. Peningkatan

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pengambilan nutrien-nutrien oleh anakbenih koko dan nutrien-nutrien

dalam tanah terutamanya kesediaan unsur fosforus (P) (>15 ppm) oleh

kebanyakan formulasi kering menunjukkan formulasi-formulasi tersebut

adalah yang terbaik sebagai penyediaan bioinokulan.

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ACKNOWLEDGEMENTS

My sincere appreciation is extended to Prof. Dr. Raja Noor Zaliha Raja Abd

Rahman, the chairman of the Supervisory Committee, for her kind interest,

invaluable guidance, diligent advice and helpfulness provided during the

duration of my study and preparation of this thesis. Her valuable help

patience, encouragement and generosity are highly appreciated.

I am also very grateful to Prof. Dato’ Dr. Abu Bakar Salleh and Prof. Dr.

Mahiran Basri, members of the supervisory committee, for their invaluable

assistance and guidance at the various stages of my research and preparation

of this thesis.

The support of Malaysian Cocoa Board through the Division of

Administration and the Division of Transfer of Technology for awarding me

all fees and funding is gratefully acknowledged.

I wish to thank Dato’ Dr. Azhar Ismail, the Director General of Malaysian

Cocoa Board, Hajah Sapiyah Subali, the Director of Transfer of Technology

and Mr. Kelvin Lamin, the Director of Biology for their dedicated support

and encouragement and faith in me for pursuing this study.

I am greatly indebted to all technical staffs of the Biology Division of

Malaysian Cocoa Board, especially to Mr. Ali Bin Gadong and

Mr. Mohamad Yusof Mohd Yunus as well as Mr. Sainudin Ali, the staff of

the Division of Transfer of Technology for assisting in the running of

various stages of experiments.

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Last but not least, to all my family members, especially my wife (Ameza

Talib), my daughters (Nur Aina, Nurfarhana), my son (Muhammad Firdaus)

and my mother (Siti Zainab Man), thanks for their prayers, patience, love,

care, sacrifices, endless emotional and physical support, and motivation.

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I certify that a Thesis Examination Committee has met on 17 January 2012 to conduct the final examination of Mohd Yusoff Abd Samad on his thesis entitled "Biofertilizer Formulation Comprising Phosphate Solubilizing Bacteria to Enhance Phosphate Availability for Cocoa Growth” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Thesis Examination Committee were as follows: Mohd Puad Abdullah, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Janna Ong Abdullah, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Umi Kalsom Md Shah, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Praveen K. Saxena, Phd Professor Department of Plant Agriculture University of Guelph Canada (External Examiner) ___________________________ SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies, Universiti Putra Malaysia

Date: 23 April 2012

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Raja Noor Zaliha Raja Abd Rahman, D.Eng. Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Abu Bakar Salleh, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Mahiran Basri, PhD 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 citation 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 other institutions. _____________________________________ MOHD YUSOFF ABD SAMAD Date: 17 January 2012

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TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL x DECLARATION xii LIST OF TABLES xvii LIST OF FIGURES xxii LIST OF ABBREVIATIONS xxvii CHAPTER

1 INTRODUCTION 1 2 LITERATURE REVIEW 7 2.1 Cocoa Industry in Malaysia 7 2.2 The Importance of Phosphorus (P) in Cocoa and Its

Availability in Malaysian Soils

9 2.3 Phosphate-Solubilizing Bacteria (PSB) 12 2.4 Mechanism of Phosphate Solubilization 20 2.5 Medium Composition for Phosphate Solubilization by

PSB and Medium Optimization

25 2.6 Formulation and Production of Bioinoculant 32 2.7 Critical Assessment of Research on PSB in Sustainable

Agriculture 40

3 SCREENING OF PHOSPHATE-SOLUBILIZING

BACTERIA

43 3.1 Introduction 43 3.2 Materials and methods 47 3.2.1 Isolation of Bacteria 47 3.2.2 Screening for PSB Using Plate Assay 47 3.2.3 Screening of PSB in Liquid Culture Medium 48 3.2.4 Ability of Selected PSB to Solubilize Rock

Phosphates

48 3.2.5 Quantitative Measurement of Phosphate

Solubilization in Liquid Medium

50 3.2.6 Morphological Observation of Selected PSB 51

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3.2.7 Identification of the Selected PSB Using 16s rDNA Sequencing

52

3.2.8 Statistical Analysis 54 3.3 Results and Discussion 55 3.3.1 Isolation of Bacteria 55 3.3.2 Screening of PSB by Plate Assay 57 3.3.3 Solubilization of Phosphate by PSB in Liquid

Medium

61 3.3.4 Changes in pH by PSB in Liquid Medium 64 3.3.5 Ability of Selected PSB to Solubilize Poorly

Soluble Rock Phosphates

68 3.3.6 Morphological Observation of Selected PSB 72 3.3.7 Identification of the Selected PSB Using 16s

rDNA Sequencing

76 3.4 Conclusion 79 4 MEDIA SELECTION AND OPTIMIZING PHOSPHATE

SOLUBILIZATION

81 4.1 Introduction 81 4.2 Materials and Methods 86 4.2.1 Bacteria Isolates and Inoculum 86 4.2.2 Effect of Carbon Sources 86 4.2.3 Effect of Nitrogen Sources 87 4.2.4 Effect of Grinded Cocoa Bean Shell and Cocoa

Pod Husk as Organic Sources

87 4.2.5 Effect of pH 87 4.2.6 Effect of Temperature 88 4.2.7 Optimizing Phosphate Solubilization 88 4.2.8 Statistical Analysis 90 4.3 Results and Discussion 94 4.3.1 Effect of Carbon Sources 94 4.3.2 Effect of Nitrogen Sources 97 4.3.3 Effect of Grinded Cocoa Bean Shell and Grinded

Cocoa Pod Pod Husk as Organic Sources

101 4.3.4 Effect of pH 103 4.3.5 Effect of Temperature 107 4.3.6 Optimizing Phosphate Solubilization by

Response Surface Methodology

110 4.4 Conclusion 130

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5 FORMULATION OF SELECTED PHOSPHATE-SOLUBILIZING BACTERIA

133

5.1 Introduction 133 5.2 Materials and Methods 135 5.2.1 Preparation of Carrier Materials for Dry

Formulation

135 5.2.2 Determination of Physical Properties of Carrier

Materials

136 5.2.3 Determination of the Chemical Properties of

Carrier Materials

138 5.2.4 Bacterial Strain 147 5.2.5 Preparation of Bacterial Inoculum 148 5.2.6 Dry Formulation of Selected PSB 148 5.2.7 Liquid Formulation of Selected PSB 150 5.2.8 Statistical Analysis 154 5.3 Results and Discussion 155 5.3.1 Physical Properties of Carrier Materials 155 5.3.2 Chemical properties of Carrier Materials 156 5.3.3 Survivability of Bioinoculants in the Dry

Formulations

158 5.3.4 Effect of Storage Duration on the Moisture

Content of the Dry Formulations

167 5.3.5 Effect of Storage Duration on the pH of the Dry

Formulations

170 5.3.6 Survivability of Bioinoculant in the Liquid

Formulations

173 5.3.7 Effect of Storage Duration on the pH of the

Liquid Formulations

178 5.3.8 Effect of Storage Duration on the Color Change of

the Liquid Formulations

181 5.3.9 Effect of Storage Duration on the Reducing Sugar

Content in the Liquid Formulations

184 5.4 Conclusion 186 6 EVALUATION OF BACTERIAL FORMULATIONS ON

THE GROWTH OF COCOA

188 6.1 Introduction 188 6.2 Materials and Methods 191 6.2.1 Experimental Site 191 6.2.2 Preparation of Soil for Potting and Seedlings 191 6.2.3 Experimental Design and Treatment 192 6.2.4 Measurement of Plant Height 195 6.2.5 Measurement of Stem Diameter 196

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6.2.6 Measurement of Number of Leaves 196 6.2.7 Measurement of Chlorophyll Content (SPAD

Value)

196 6.2.8 Determination of Fresh and Dry Weight of Plant

Components 196

6.2.9 Soil and Leaf Sample Preparations 197 6.2.10 Physical and Chemical Analyses of Soil Samples 198 6.2.11 Plant Leaf Tissue Analysis 211 6.2.12 Determination of PSB Population in the Potting

Soil

212 6.2.13 Statistical Analysis 212 6.3 Results and Discussion 213 6.3.1 The Effects of Treatments on Growth Parameters 213 6.3.2 The Effects of Treatments on Fresh Weight of

Plant Components

216 6.3.3 The Effects of Treatments on Dry Weight of Plant

Components

219 6.3.4 The Effects of Treatments on Chlorophyll Content

(SPAD Value)

222 6.3.5 The Effects of Treatments on Leaf Nutrient Status 224 6.3.6 The Effect s of Treatments on Total Leaf Nutrient

Uptake

230 6.3.7 The Effects of Some Chemical Properties of

Potting Soil Before and After Treatments

234 6.3.8 The Effects of Treatments on the Population of

PSB in the Potting Soil

245 6.4 Conclusion 250 7 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATION FOR FUTURE RESEARCH

253 REFERENCES 264 APPENDICES 285 BIODATA OF STUDENT 321