UNIVERSITI PUTRA MALAYSIA

MAYA ERNA BINTI NATNAN

IB 2012 19

INDIGENOUS IMMOBILIZED NITRIFYING BACTERIA FOR REDUCTION OF AMMONIA IN SHRIMP LARVICULTURE SYSTEM

© COPYRIG

HT UPM

INDIGENOUS IMMOBILIZED NITRIFYING BACTERIA FOR

REDUCTION OF AMMONIA IN SHRIMP LARVICULTURE SYSTEM

By

MAYA ERNA BINTI NATNAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Master of Science

November 2012

© COPYRIG

HT UPM

i

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science

INDIGENOUS IMMOBILIZED NITRIFYING BACTERIA FOR

REDUCTION OF AMMONIA IN SHRIMP LARVICULTURE SYSTEM

By

MAYA ERNA BINTI NATNAN

November 2012

Chairperson: Prof. Dato’ Mohamed Shariff Mohamed Din, PhD

Faculty/Institute: Institute of Bioscience

The shrimp industry has grown rapidly due to the increase in market demand, which

in turn resulted in intensive shrimp postlarvae (PL) cultivation practices. The

ultrahigh stocking densities and over loading of feeds cause serious water quality

problems in PL hatchery system. Exposure of shrimp PLs to toxic compounds like

ammonia leads to stress and diseases that eventually causes PL mortality. The

present study investigated the ability of local indigenous immobilized nitrifying

bacteria, isolated from the marine environment, to reduce total ammonia nitrogen

(TAN) in a shrimp larviculture system. For isolation of nitrifying bacteria, soils and

water samples were collected from the west coast of Peninsular Malaysia such as

Kuala Juru, Kuala Gula, Morib and shrimp farms in Kuala Langat. Bacteria were

grown in Skinner and Walker medium and then screened for their ability to reduce

ammonia. Since nitrifying bacteria cannot be isolated in a pure culture condition,

they were cultured and maintained as consortia. The nitrifying bacteria consortia

were then tested for their ability to reduce ammonia and the consortium that showed

the lowest ammonia reading was selected for further experiments. Consortium M1,

isolated from the mangrove area of Morib, showed the best reduction rate (0.154

mg/l/day) of ammonia from 2.0 ± 0.1 mg/l to 0.06 ± 0.01 mg/l in 14 days, when

tested in the laboratory. Using 16S rDNA primers, three species of bacteria were

identified from the consortium M1, namely Pseudomonas aeruginosa with 99%

© COPYRIG

HT UPM

ii

homology, Pseudomonas stutzeri with 98% homology and Nocardioides albus with

98% homology after BLAST analyses in Genebank. Further experiments were

conducted to test the efficacy of the consortium M1 for its ability to reduce ammonia

in a shrimp larviculture system. The bacteria consortium M1 was immobilized using

alginate solution. Tanks treated with immobilized bacteria were able to achieve high

ammonia reduction proficiency to 76.52%, followed by tanks that were replaced with

50% fresh seawater on alternate days (68.10 %), tanks treated with non-immobilized

bacteria (36.84%) and tanks treated with alginate beads without bacteria (18.37%).

However, in control tanks (without treatment) ammonia increased to 40.79% at 14

days. Tanks treated with immobilized bacteria has significantly (P < 0.05) increased

survival rate (72.44%) and specific growth rate (SGR) (12.86%) of shrimp PL

compared to other treatments.

© COPYRIG

HT UPM

iii

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

BAKTERIA NITRIFIKASI TEMPATAN YANG DIIMMOBILISASIKAN

BAGI PENGURANGAN AMMONIA DARI SISTEM LARVIKULTUR

UDANG

Oleh

MAYA ERNA BINTI NATNAN

November 2012

Pengerusi: Prof. Dato’ Mohamed Shariff Mohamed Din, PhD

Fakulti/ Institut: Institut Biosains

Industri udang berkembang pantas menyebabkan kultur pascalarva (PL) udang

menjadi intensif dan seterusnya peningkatan permintaan pasaran. Kepadatan stok

yang tinggi dan makanan berlebihan boleh menyebabkan masalah kualiti air di pusat

penetasan pascalarva. Pendedahan PL udang kepada sebatian toksik seperti ammonia

menjadi punca tekanan dan penyakit yang seterusnya menyebabkan punca kematian

PL. Kajian ini menyiasat kemampuan pencilan bakteria nitrifikasi tempatan dari

persekitaran marin yang telah diimmobilisasi, dalam mengurangkan jumlah ammonia

nitrogen (TAN) dari sistem larvikultur udang. Sampel tanah dan air dari persekitaran

marin diambil di sepanjang pantai barat Semenanjung Malaysia seperti Kuala Juru,

Kuala Gula, Morib dan ladang kolam udang di Banting, Kuala Langat. Bakteria

dibiakkan dalam media Skinner dan Walker, kemudian disaring untuk mengenalpasti

bakteria yang boleh mengurangkan ammonia. Disebabkan bakteria tidak dapat

dipelihara dalam keadaan kultur tulen, ia dikultur dan dipelihara dalam keadaan

perkongsian. Perkongsian bakteria nitrifikasi kemudian diuji untuk keupayaan

mengurangkan ammonia, dan perkongsian yang menunjukkan bacaan ammonia

paling rendah telah dipilih untuk kajian lanjutan. Kultur perkongsian M1 yang

diambil dari kawasan bakau di Morib menunjukkan tahap pengurangan ammonia

(0.154 mg/l /hari) yang paling baik iaitu dari 2.0 ± 0.01 mg/l kepada 0.06 ± 0.01 mg/l

© COPYRIG

HT UPM

iv

dalam masa 14 hari apabila diuji di makmal. Dengan menggunakan primer 16S

rDNA, tiga spesies bakteria telah dikenalpasti dari perkongsian M1, iaitu

Pseudomonas aeruginosa dengan homologi 99%, Pseudomonas stutzeri dengan

homologi 98% dan Nocardioides albus dengan homologi 98% selepas analisis

BLAST di Genbank. Eksperimen selanjutnya dilakukan bagi menguji keberkesanan

perkongsian M1 dalam kebolehan mengurangkan ammonia di dalam sistem tangki

larvikultur udang. Tangki yang dirawat dengan bakteria immobilisasi menunjukkan

keberkesanan paling baik dalam pengurangan ammonia (76.52%), diikuti oleh tangki

yang telah digantikan dengan 50% air laut selang sehari (68.84%), tangki yang

dirawat dengan bakteria yang tidak diimmobilisasi (36.84%) dan tangki yang dirawat

dengan manik alginat tanpa bakteria (18.37%). Bagaimanapun, air di dalam tangki -

kawalan (tanpa rawatan), menunjukkan penambahan ammonia kepada 40.79%

selepas 14 hari. Tangki yang dirawat dengan bakteria immobilisasi menunjukkan

signifikasi (P < 0.05) lebih tinggi kadar hidup (72.44%) dan kadar pertumbuhan tentu

(SGR) (12.86%) bagi PL udang jika dibandingkan dengan rawatan lain.

© COPYRIG

HT UPM

v

ACKNOWLEGEMENT

Assalammualaikum. Praise to Allah (S.W.T) the all powerful, the most wise and the

most merciful, and greetings to His Messenger, the Saviour of mankind, Prophet

Muhammad (S.A.W), his family, Moslems present and past. To Allah the Almighty,

belong all praise and glory. Without His will and blessing, I would not have

succeeded in completing this thesis.

I would like to express my heartfelt thanks and gratitude to my supervisor Prof.

Dato’ Dr. Mohamed Shariff Mohamed Din, Faculty of Veterinary Medicine, UPM

for his valuable guidance and supervision towards completing the course of research

and this thesis. His meticulous nature in evaluated my thesis has made this thesis a

valuable document. Not to forget Prof. Dr. Fatimah Md Yusoff, Faculty of

Agriculture, UPM, member of my advisory committee for her guidance and

encouragement throughout the course of study.

I am also thankful to Dr. Sanjoy Banerjee and Dr. Helena Khatoon, who have been

very helpful in contributing the necessary information and needs during the course of

my study. Last but not least, I wish to thank my beloved family, labmates and

colleagues for their patience, opinion, motivation, support and understanding

throughout the process in finishing this research course and preparing this thesis.

Dearest beloved, all the experiences we have been through together and your

encouragement have inspired me to develop myself personally and professionally.

My thanks to Universiti Putra Malaysia for giving me the opportunity for pursue my

Master’s degree.

© COPYRIG

HT UPM

vi

APPROVAL SHEET

I certify that an Examination Committee has met on 6th

November 2012 to conduct

the final examination of Maya Erna binti Natnan, on her Master thesis entitled

“Indigenous Immobilized Nitrifying Bacteria for the Reduction of Ammonia in

Shrimp Larviculture System” in accordance with Universities and University

Colleges Act 1971 and the Constitution of the Universiti Pertanian Malaysia [P.U.

(A) 106 15 March 1998. The Committee recommends that the student be awarded

the Master of Science.

Members of the Examination Committee were as follows:

Mohd Hair Bejo, PhD

Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Chairman)

Aziz bin Arshad, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Internal Examiner)

Muta Harah binti Zakaria, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Internal Examiner)

Jiann Chu Chen, PhD

Professor

Department of Aquaculture

National Taiwan Ocean University

Taiwan

(External Examiner)

______________________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

© COPYRIG

HT UPM

vii

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Master in Science. The

members of the Supervisory Committee were as follows:

Mohamed Shariff Mohamed Din, PhD

Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Chairman)

Fatimah Md. Yusoff, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Member)

_____________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

© COPYRIG

HT UPM

viii

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 or

concurrently submitted for any degree at Universiti Putra Malaysia or other

institutions.

_________________________

MAYA ERNA BT NATNAN

Date: 6 November 2012

© COPYRIG

HT UPM

ix

TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION viii

TABLE OF CONTENTS ix

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF ABBREVIATIONS xiv

CHAPTER

1 INTRODUCTION 1

1.1 Background of Study 1

2 LITERATURE REVIEW 5

2.1 General Introduction to Aquaculture 5

2.1.1 Aquaculture in Malaysia 6

2.1.2 Shrimp Production 7

2.1.3 Problems in Shrimp Aquaculture 8

2.1.3.1 Total Ammonia Nitrogen 9

2.1.3.2 Nitrite Waste 9

2.1.3.3 Diseases 10

2.2 Strategy to Overcome Pollution Problems 11

2.2.1 Bioremediation 12

2.3 Nitrification 14

2.3.1 Nitrifying Bacteria 15

2.3.1.1 Autotrophic Nitrifying Bacteria 16

2.3.1.2 Heterotrophic Nitrifying Bacteria 17

2.4 Source of Nitrifying Bacteria 18

2.5 Requirement in Nitrifying Bacteria Growth 19

2.6 Role of Nitrifying Bacteria 20

2.7 Identification using 16S rDNA 20

2.8 Immobilization 21

3 SCREENING, ISOLATION AND IDENTIFICATION OF

NITRIFYING BACTERIA 23

3.1 Introduction 23

3.2 Methodology

3.2.1 Isolation and Screening of Nitrifying Bacteria 24

3.2.2 Effect of Selected Nitrifying Bacteria Consortia on Ammonia 26

3.2.2.1 Total Ammonia Nitrogen Analysis (TAN) 26

3.2.3 Bacteria Count 27

3.2.4 Genomic DNA Extraction (Boil Method) 27

© COPYRIG

HT UPM

x

3.2.5 Polymerase Chain Reaction (PCR) 27

3.2.6 Stock Maintenance 28

3.3 Results 29

3.4 Discussion 36

4 IMMOBILIZATION OF AMMONIA OXIDIZING BACTERIA 40

4.1 Introduction 40

4.2 Methodology

4.2.1 Immobilizing Nitrifying Bacteria 41

4.2.1.1 Bacteria Count in Beads 42

4.2.2 Effect of Immobilized Bacteria on ammonia 42

4.2.2.1 Total Ammonia Nitrogen Analysis (TAN) 42

4.2.2.2 Nitrite-N Analysis 43

4.3 Results 44

4.4 Discussion 51

5 GROWTH AND SURVIVAL OF Penaeus monodon POSTLARVAE

IN SHRIMP LARVICULTURE SYSTEM TREATED WITH

IMMOBILIZED BACTERIA 55

5.1 Introduction 55

5.2 Methodology

5.2.1 Source of PL 56

5.2.2 Tank Preparation 56

5.2.3 Stocking of PL 56

5.2.4 Preparation of Immobilized Nitrifying Bacteria, Non-immobilized

Bacteria and Beads without Bacteria 56

5.2.5 Preparation of Different Treatments in Shrimp Larviculture System 57

5.2.6 Growth and Survival Study of PL in Shrimp Larviculture System

Treated with Immobilized Bacterial 57

5.2.7 Data Analysis 58

5.3 Results 58

5.4 Discussion 67

6 SUMMARY, GENERAL CONCLUSION AND RECOMMENDATION

FOR FUTURE RESEARCH 72

REFERENCES 75

APPENDICES 92

BIODATA OF STUDENT 100