MARINE FISH WASTE DECOMPOSITION

CULTIVABLE BACTERIAL AND FUNGAL COMMUNITIES ASSOCIATED WITH A SELECTED

MARINE FISH WASTE DECOMPOSITION

BY

NURUL SYAKIRA BINTI ZAINUDDIN

A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology)

Kulliyyah of Science

International Islamic University Malaysia

AUGUST 2019

i

ABSTRACT

This study was conducted to identify the fungi and bacteria associated with fish waste decomposition. The rotten fish was ground and setup in blue tank approximately 15 kg for each tank closed with net and left in the tank for 30 days to decompose.

Physicochemical parameters including pH, temperature, moisture content and carbon to nitrogen (C:N) ratio were monitored throughout the process. Culture-dependent methods were used to identify the diversity of bacteria and fungi in this study. Fungi were isolated using eight different media with various compositions; potato dextrose agar, Rose Bengal agar, peptone water agar, Saboroud agar, yeast extract agar, oatmeal agar, malt extract agar and leachate medium agar while bacteria were isolated on yeast extract agar, diluted nutrient agar, leachate agar and tryptone agar. Fungal isolates were extracted using CTAB followed by 18S rRNA amplification using primers set FF390/FR1 meanwhile, PCR amplification of the 16S rRNA gene using primer sets 27F/1492R was performed directly from the bacterial colonies. Results from DNA sequencing were analyzed using BLASTn, and phylogenetic tree were constructed using ARB software. During the decomposition process, temperature of waste was in the range between 20-50°C, pH 6-8, moisture content started at 80% and reduced to 50% and C:N ratio 3-7. A total of 41 fungal isolates and 25 bacterial isolates were identified based on 18S and 16S rRNA gene respectively. For fungal isolates, the Ascomycota (91% of all sequences recovered) were the dominant phyla followed by Basidiomycota (9%). The 41 randomly isolated fungi were belonged to 22 OTUs of the genus Exophiala, Penicillium, Aspergillus, Monascus, Parengyodontium, Fusarium, Trichoderma, Chaetomium, Hypoxylon, Candida, Curvularia, Cladosporium, Paraconiothyrium, Cymatoderma and Trichosporon. From 16S gene rRNA identification and comparison, the 24 bacteria belonged to Pseudomonas, Bacillus, Enterobacter, Klebsiella, Stenotrophomonas, Alcaligenes, Acinetobacter, Streptomyces, Micrococcus, Brevundimonas, and Proteus. The utilization of different agar medium increase the possibility of isolating the diverse of fungal and bacterial community involved in fish waste decomposition.

ii

ثحبلا ةصلاخ

ةطبترملا ايريتكبلاو تايرطفلا ديدحتل ةساردلا هذه تيرجأ ب

للحت تلاضف .كامسلأا

نازخ يف دسافلا كمسلا عضو مت تا

قرز

،ءا ب يلاوح 51

نازخ لكل مجك

، تيطغو

بشلاب ا كرتو ك ت

نازخلا يف تا

ةدمل ملا دصر مت .للحتتل اًموي 03

تارشؤ ةيئايزيفلا

ةيئايميكلاو يهو ،

ةضومحلا ةجرد

، ةرارحلا ةجردو

، ةبوطرلا ىوتحمو

، ةبسنو

( نيجورتينلا ىلإ نوبركلا تمدختسا .ةيلمعلا لاوط ) C:N

قرطلا ىلع ةدمتعملا

تاتبنتسملا تايرطفلا لزع مت .ةساردلا هذه يف تايرطفلاو ايريتكبلا عونت ديدحتل

ةينامث مادختساب تاتبنتسم

ةفلتخم تابيكرتب يهو ،

،زورتسكدلا سطاطب راجأ و

راجأ

زور لا ،لاغنب و

،نوتببلا ءاملا راجأ و

،دووروباس راجأ و

راجأ تاصلختسم ،ةريمخلا

و ،نافوشلا راجأ و

راجأ تاصلختسم ريعشلا

، راجأو

،تاشتيللا ايرتكبلا لزع مت امنيب

راجأ ىلع تاصلختسم

،ةريمخلا و

،ةففخملا تايذغملا راجأ و

راجأ تاشتيللا ،

و راجأ

نوتبيرتلا جارختسا مت .

تلاوزعملا مادختساب ةيرطفلا

ميخضتب اًعوبتم CTAB

يموسوبيرلا يزوبيرلا يوونلا ضمحلا ــل

51 مادختساب S

لا ةعومجم ةئدابلا

FF390/FR1 امنيب ،

ءارجإ مت لا

ميخضت ب

تاريميلوبلا ةلسلس لعافت لل

يوونلا ضمح

يموسوبيرلا يزوبيرلا 51

مادختساب S لا

ةعومجم ةئدابلا

27F/1492R نم ةرشابم

جئاتن ليلحت مت .ةيريتكبلا تارمعتسملا مادختساب يوونلا ضمحلا لسلست

جمانرب

BLASTn و ،

ءانب مت روطت ةرجش

تلالاسلا جمانرب مادختساب

ةيلمع ءانثأ . ARB

للحتلا تحوارت

نيب تايافنلا ةرارح ةجرد 03

- 13 ةضومحلا ةجردو ،ةيوئم ةجرد

نيب 1 - 1 ةبوطرلا ىوتحم أدبو ، نم

13

٪ ىلإ ضفخو 13

٪

، و تحاورت ةبسن

C:N

نيب 0 - 7 . مت ىلع فرعتلا 15

لوزعم و يرطف

01 م زع و ىلع ءانب يريتكب ل

يموسوبيرلا يزوبيرلا يوونلا ضمحلا 51

و S 51 امهنم لكل S لل ةبسنلاب .

م زع و تلا

تناك ،ةيرطفلا ةيقزلا تايرطفلا

( 15

٪ ملا تلاسلستملا عيمج نم ةلوصح

يه )

ةبعشلا ةنميهملا

نمو اهدعب ةيماعدلا تايرطفلا

( 1

٪ .) ا متن ت ـلا تايرطفلا 15

ىلإ ًايئاوشع ةلوزعملا 00

ةدحو ةيليغشت فينصت سنج نم

لاايفوسكلإا لاو ،

مويليسينب ،

و ،مويتنودويجنيرابلاو ،سوكسانوملاو ،تايشاشرلاو ةيولازغملا

،امريدوكيرتلاو ،

،ايرلاوفركلاو ،تاضيبملاو ،نوليسكوبيهلاو ،مويموتيشتلاو و

ةيغوبلا تايقوطلا ،

نوروبسوكيرتلاو ،امريدوتاميكلاو ،مورياتوينوكارابلاو نم .

ةنراقملاو فرعتلا للاخ

ب يموسوبيرلا يزوبيرلا يوونلا ضمحلا ، 16S

تمتنا ايريتكبلا

ــلا 01 بعش ىلإ

،تايلقملاو ،سانوموفورتونيتسلاو ،تلايسبلكلاو ،تايئاعملأا ،تايوصعلا ،تافئازلا .تابلقتملاو ،سانوميدنوفيربلاو ،تاريكملاو ،تلاسلستملاو ،تادكارلاو مادختسا

تاتبنتسم لأا

راج ةفلتخملا داز

لزع ةيناكمإ نم تاعمجملا

يرطفلا ة يريتكبلاو ة

طروتملا ة

للحت يف تلاضف

سلأا

.كام

iii

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master in Science (Biotechnology)

………..

Mohd Faez bin Sharif Supervisor

………..

Suhaila binti Omar Co-Supervisor

………..

Mohd Hafiz bin Jamaluddin Co-supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master in Science (Biotechnology)

………..

Mardiana Mohd Ashaari Internal Examiner

………..

Mohd Ali Hassan External Examiner

This thesis was submitted to the Department of Biotechnology and is accepted as a fulfilment of the requirement for the degree of Master in Science (Biotechnology)

………..

Mardiana Mohd Ashaari Head, Department of Biotechnology

This thesis was submitted to the Kulliyyah of Science and is accepted as a fulfilment of the requirement for the degree of Master in Science (Biotechnology)

………..

Shahbudin Saad

Dean, Kulliyyah of Science

iv

DECLARATION

I hereby declare that this thesis is the result of my own investigation, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Nurul Syakira binti Zainuddin

Signature………. Date …...

vii

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA DECLARATION OF COPYRIGHT AND AFFIRMATION OF

FAIR USE OF UNPUBLISHED RESEARCH

CULTIVABLE BACTERIAL AND FUNGAL COMMUNITIES ASSOCIATED WITH SELECTED MARINE FISH WASTE

DECOMPOSITION

I declare that the copyright holder of this thesis are jointly owned by the student and IIUM

Copyright ©2019 by Nurul Syakira binti Zainuddin and International Islamic University Malaysia. All rights reserved.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below.

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by Nurul Syakira binti Zainuddin

……..……..……… ………..

Signature Date

viii

ACKNOWLEDGEMENTS

First and foremost, I express my deep grateful, thanks to the Almighty Allah SWT, for his blessings throughout my study year to complete the master’s thesis complete.

I would like to express sincere thanks to my great supervisor, Dr Mohd Faez bin Sharif for the continuous support of my master study. His motivation and guidance helped me in all the time of study and writing of this thesis.

Beside my supervisor, I would like to express gratitude the rest of my study and thesis committee: my co-supervisor, Dr Suhaila binti Omar and Dr Mohd Hafiz bin Jamaluddin for their valuable comments and encouragement.

I also like to acknowledge Dr Mohd Naim as the third reader of this thesis and I am thankful to his very precious and valuable comments on this thesis.

Not forget to my fish waste groupmates, Sr Syuhada, Sr Shahira Illiyin, Sr Atisha, Sr Fatin, Br Izzat, Br Zakwan and Br Najib who were involved in this research study. Without their passionate participation, this study could not have been successfully conducted.

Next, I must express my very deep appreciation to my beloved parents, Mr Zainuddin bin Mohd Zawawi and Mrs Siti Zubaidah bt Awang Ahmad and beloved family for continuous encouragement and giving support throughout my years of study.

Finally, my thanks to all the people who have supported me to complete my work directly and indirectly.

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TABLE OF CONTENTS

Abstract ... i

Abstract in Arabic ... ii

Approval Page ... iii

Declaration ... iv

Copyright ... vii

Acknowledgements ... viii

Table of Contents ... ix

List of Tables ... xii

List of Figures ... xiii

List of Symbols ... xiv

CHAPTER ONE: INTRODUCTION ... 1

Background Study ... 1

Problem Statement ... 2

Objectives ... 3

Research Hypothesis... 3

Research Questions... 4

CHAPTER TWO: LITERATURE REVIEW ... 5

Marine Fish Diversity ... 5

2.1.1 Composition Of Marine Fish ... 5

2.1.2 Sardines Diversity And Their Compositions ... 6

Marine Fish Waste ... 7

2.2.1 Uses Of Fish Waste ... 7

Decomposition Process... 9

2.3.1 Physico-Chemicals Parameters Of Decomposition ... 10

2.3.1.1 Temperature ... 10

2.3.1.2 Moisture Content ... 11

2.3.1.3 pH ... 12

2.3.1.4 CN Ratio ... 12

Microbial Community In Waste Decomposition ... 13

2.4.1 Molecular Identification Of Microbial Community ... 13

2.4.2 Fungal Community In Waste Decomposition ... 16

2.4.3 Bacterial Community In Waste ... 17

CHAPTER THREE: PHYSICO-CHEMICAL ANALYSIS DURING FISH WASTE DECOMPOSITION ... 19

Introduction ... 19

Methods And Materials ... 20

3.2.1 Sample Collection And Preparations ... 20

3.2.2 Measurement Of Physico-Chemical Parameters ... 21

3.2.2.1 Measurement Of Temperature ... 21

x

3.2.2.2 Measurement Of pH ... 21

3.2.2.3 Measurement Of Moisture Content ... 21

3.2.2.4 Measurement Of CN Ratio ... 22

3.2.3 Statistical Analyses ... 22

Results ... 22

3.3.1 Measurement Of Physico-Chemical Parameters ... 22

3.3.1.1 Temperature ... 23

3.3.1.2 Moisture Content ... 24

3.3.1.3 Ph ... 24

3.3.1.4 Total Nitrogen, Total Carbon And Cn Ratio ... 25

Discussions ... 26

Conclusion ... 30

CHAPTER FOUR: CULTIVABLE OF FUNGI COMMUNITY IN FISH WASTE DECOMPOSITION ... 31

Introduction ... 31

Method And Materials ... 32

4.2.1 Isolation And Identification Of Fungal Cultures From Fish Waste ... 32

4.2.1.1 Preparation Of Media ... 32

4.2.1.2 Culture And Isolation Of Fungal Community ... 35

4.2.1.3 Morphological Observation Of Fungal Colonies ... 35

4.2.2 Molecular Identification Of Selected Fungal Colonies... 35

4.2.2.1 Preparation Stock Of CTAB/NaCl Solution ... 35

4.2.2.2 Genomic Dna Extraction ... 36

4.2.2.3 Measurement Of Dna Concentration And Gel Electrophoresis ... 37

4.2.2.4 Polymerase Chain Reaction (Pcr) Amplification ... 38

4.2.3 Sequence Assembly, Editing And Data Analysis ... 38

4.2.4 Phylogenetic Analysis Of 18s Rrna Sequences ... 39

4.2.5 MDS Analysis ... 40

4.2.6 Nucleotide Sequence Accession Numbers ... 40

Results ... 40

4.3.1 Isolation, Identification And Characterization Of Fungal Cultures From Fish Waste Compost ... 40

4.3.1.1 The Selected Fungal Isolates In Different Medium Growth . 40 4.3.1.2 Colony Morphology Of The Selected Fungal Colonies ... 41

4.3.1.3 Identification And Characterization Of Fungal Isolates ... 44

4.3.1.4 The Diversity Of Cultivable Of Fungal ... 45

4.3.1.5 MDS Analysis ... 49

Discussions ... 49

Conclusion ... 52

CHAPTER FIVE: CULTIVABLE OF BACTERIA COMMUNITY FROM FISH WASTE DECOMPOSITION ... 54

Introduction ... 54

Methodology ... 55

5.2.1 Sequence Assembly, Editing And Data Analysis ... 55

5.2.2 Computational Of 16s Rrna Sequences ... 55

xi

5.2.3 MDS Analysis ... 56

Results ... 56

5.3.1 Isolation And Cultivation Of Bacteria From Fish Waste ... 56

5.3.1.1 The Selected Bacterial Isolates In Different Type Of Growth Media ... 56

5.3.2 Identification And Characterization Of Bacteria Community ... 57

5.3.2.1 16s Rrna Gene Analysis Using Ncbi Blast Database ... 57

5.3.2.2 Cultivable Of Bacterial Diversity From Fish Waste Decomposition ... 58

5.3.3 Growth Medium And The Associated Taxa ... 62

Discussions ... 63

Conclusion ... 66

CHAPTER SIX: GENERAL DISCUSSION AND CONCLUSION ... 67

6.1 Future Outlook And Conclusion... 71

REFERENCES ... 72

APPENDIX ... 83

xii

LIST OF TABLES

Table 3.1 The Total Nitrogen and Carbon Over Decomposition Days 25

Table 4.1 Trace Salt Solution 33

Table 4.2 The Colony Morphological of Selected Fungal and Their Successfully PCR Amplification

42

Table 4.3 Operational Taxonomic Unit (OTUs) of Fungal Isolates Derived from Fish Waste Decomposition Clustered 97% Similarity. Nearest Neighbours are Obtained from NCBI and SILVA Database

44

Table 5.1 Operational Taxonomic Unit (OTUs) of Bacterial Isolates Derived from Fish Waste Decomposition Clustered 97% Similarity. Nearest Neighbours are Obtained from NCBI Database

58

xiii

LIST OF FIGURES

Figure 3.1 The Changes of Temperature for Waste Over 30 Days 23 Figure 3.2 The Changes of Moisture Content for Waste Over 30 Days 24

Figure 3.3 The Changes of pH For Waste Over 30 Days 25

Figure 3.4 The Changes of C:N Ratio Over 30 Days 26

Figure 4.1 The Number of Selected Isolates Identified by Different Medium 41 Figure 4.2 Proportion Of Fungal Isolated From Fish Waste Decomposition at

Phylum Level (A), Order Level (B) And Genus Level (C).

46

Figure 4.3 Bayesian Phylogram Based On 18S rRNA Gene Sequences of 22 Selected OTUs and Their Nearest Neighbours. OTUs From This Study are in bold.

48

Figure 4.4 The Nonmetric Multidimensional Scaling (NMDS) Plot Obtained from Bray-Curtis Similarity Analysis Based on Abundance of Fungal Isolates and Type of Medium Used.

49

Figure 5.1 The Number of Bacterial Isolates from Different Types of Growth Media

57

Figure 5.2 The Composition of Bacteria Isolated from Fish Waste Decomposition at Classes Level (A), Order Level (B) and Genus Level (C).

59

Figure 5.3 Bayesian Phylogram Based on 16S rRNA Gene Sequences of 18 Selected OTUs and Their Nearest Neighbours. OTUs from This Study are in Bold

61

Figure 5.4 Nonmetric Multidimensional Scaling (NMDS) Plot Derived from Bray-Curtis Similarity Analysis Based on Abundance of Bacterial Genus and Medium Used

62

xiv

LIST OF SYMBOLS

°C Degree Celsius

% Percentage

1

CHAPTER ONE INTRODUCTION

BACKGROUND STUDY

The fishery sector is an important sub-sector for Malaysian economy. It serves as a major food chain supply for Malaysian population. According to the Food and Agriculture Organisation (FAO), Malaysia is a main fish-consuming country in Asia with approximately above 40kg/capita/year. Fish industries produce huge quantities of daily solid and liquid waste. Approximately 75% of fish production is used for human consumption while the 25% of the residues were left as fish waste (Ben Rebah &

Miled, 2012) which has potential to be valorized as functional products. The fish waste and its by-product is known as the best source for microbial growth to produce many metabolites such as enzymes and lysine (Coello, Montiel, Concepcion, &

Christen, 2002). The viscera, heads, and chitinous material of fish are the best growth substrates for production of enzyme such as protease, lipase, chitinolytic and ligninolytic enzymes.

The increasing volume of waste and lack of proper management will leads to environmental problem such as pollution and health problem to human and animals.

The waste usually ends up at the landfill and contributing to the global warming.

Since decomposition is an effective method to recycle the organic matters, it can divert the solid waste disposal into landfill. Product from the decomposition process can be applied in agriculture as compost.

2

Decomposition is defined as the natural biological process by organic matter is broken down and through activities of microorganisms involved. The final products of decomposition are simple molecules like water and carbon dioxide. It is a natural process and the productivity of the decomposition process was influenced by some factors such as temperature, pH, moisture content, CN ratio, substrate used and the microbial community involved. Decomposition involves microorganism to convert mixture of organic matter to humus-like product. According to Kreith &

Tchobanoglous (2002) stated organisms that play role in decomposition process based on their frequency are fungi, bacteria, and actinomycetes.

Nevertheless, little is known on the microbial community associated with fish waste decomposition. Hence, this study aimed to identify fungal and bacteria involved in the fish waste decomposition process via culture dependent method.

PROBLEM STATEMENT

Landfilling is the method used to dispose various waste in Malaysia. Improper management of wastes from various sources (including fish industries) that being send directly to landfill for disposal can leads to environmental and community health problem. Potential effects on health resulted from exposure of toxic gas such as methane and carbon dioxide. Contamination of groundwater by leachate may interrupt domestic water supply and other beneficial uses. However, some fish waste was discarded directly to river and ocean can interrupt the ecology and cause water pollution. Hence, decomposition process that can be used to decrease the volume of fish waste that generated from fish processing industry and reducing the volume of

3

waste to be landfilled and thrown away to ocean and river and also produce valuable product such as compost.

There are many studied review on uses and application of fish waste such as production of collagen, enzymes and proteins. However, less study had been done to explore the uses of fish processing or by-product as composting materials and the valuable microorganism during decomposition process. The knowledge on microbial community including bacteria and fungi in fish waste decomposition is limited as well as the microorganisms are responsible in decomposition process.

OBJECTIVES

i. To evaluate the temperature, pH, moisture content and CN ratio during fish waste decomposition process

ii. To identify fungi during fish waste decomposition through cultivation on various growth media

iii. To determine fungal and bacteria present in fish waste decomposition by using molecular identification

RESEARCH HYPOTHESIS

Cultivation on various growth media coupled with 16S and 18S rRNA gene analysis will identify fungi and bacteria community that involved in the fish waste decomposition.

4

RESEARCH QUESTIONS

1. What are the observed physico-chemical parameters patterns during the fish waste decomposition?

2. What are the phyla and genus of bacteria and fungi that obtained from marine fish waste decomposition?

3. Does the type of medium used in the cultivation process determine the microorganism isolated?

4. What is the novelty of the isolated microorganisms in term of taxonomy?

5

CHAPTER TWO LITERATURE REVIEW

MARINE FISH DIVERSITY

Fish are an important community of marine habitats. Approximatively 11,300 species are found in coastal and littoral waters depth to 200 m. The diversity of fish is higher in the Indo-West Pacific, between Papua-New Guinea and Australia. Malaysia is located in the Indo-pacific region that included sea surrounding The South China Sea is well-known for its extreme productivity and rich diversity of animals and plants (Matsunuma, Motomura, Matsuura, Shazili, & Ambak, 2011). Malaysia is located in the Indo-Pacific region that includes sea surrounding. Survey of fishes done in Terengganu (Matsunuma et al., 2011) stated that 441 marine and estuarine fish species of 108 families was reported to be found here.

2.1.1 Composition of Marine Fish

The composition of fish is different depending on the type of species, age, sex, nutritional status, health and time of year. Main constituents in a portion of fish are protein, lipid (fat and oil), water and minerals. Most of the fish comprises 15-30% of protein, 0-25% of fat and 50-80% moisture. The three types of fish proteins that originate from all parts of fish are structural proteins, sarcoplasmic proteins and connective tissues proteins which can be extracted by chemical and enzymatic process. Fish contains well balanced amino acid compositions that produced from fish proteins and contain eight amino acids and eight non-essential amino acids. Marine

6

lipid consists of long-chain n-3(omega-3) polyunsaturated fatty acid (PUFAs), mostly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is essential fatty acids to human health, nutrition and disease prevention. The presence of proteins and unsaturated essential fatty acids, minerals and vitamins give health beneficial when fish is consumed in our meals. This has led to high fish stock request for human consumption.

2.1.2 Sardines Diversity and Their Compositions

Sardine is small, elongated and silvery fishes with short dorsal fin, no lateral fine and no scales on head. It has been categorized to phylum Chordata, family Clupeidae, and members of genera Sardina, Sardinella and Sardinops. Sardines are also recognized as a small pelagic fishes which belong to diverse group of planktivorous fishes that share same habitats which not exceed to 200m in water depth.

Sardines is well known as food fishes and mostly consumed by humans in their daily life. Fish belonging to family Clupeidae have higher fatty acid content and could become a source of polyunsaturated fatty acids omega 3 especially DHA and EPA. These fatty acids had been proved to give advantages for human health in cardiovascular disease, inflammation, immunity and cancer (Alasalvar & Taylor, 2002). Chemical composition in two types of Sardinella sp (spotted and goldstrip) was studied by (Suseno et al., 2014) and showed this two Sardinella sp have higher fat content and proteins. Higher fatty acid in this study presented in spotted Sardinella was palmitic acid, oleic acid and DHA while in goldstrip Sardinella was palmitic acid, palmitoleic acid and DHA. ⁠

7

MARINE FISH WASTE

The catching and processing of marine fish can cause large amount of waste includes fins, scales, viscera and bones. The 75% of fish production is used for human consumption and residue approximately 25% were left as fish waste (Ben Rebah &

Miled, 2012). These wastes can trigger serious environmental problem if not disposed properly. Currently, most of fish by-product was discarded directly in soil as waste materials thus will cause odor problem and may attract animals. Furthermore, the wastes are discarded directly to water; interrupting the marine ecology.

The fish wastes incorporate the head, liver, skin, bones and intestine that contains 58% of proteins, 19% of ether extract or fat, minerals, 22% abundant of mono-saturated acid, palmitic acid and oleic acids in fish waste (Ghaly, Ramakrishnan, Brooks, Budge, & Dave, 2013). Substantial amount of lipid can be extracted from the wastes. Khoddami, Ariffin, Bakar, & Ghazali (2009) study in Sardinella lemuru waste discovered with 5.8% of extracted oils found in the fish’s liver and the predominant fatty acids are palmitic, stearic, oleic and docosahexaenoic acid.

2.2.1 Uses of Fish Waste

Fish is nutritious and rich with micronutrient, minerals, proteins and essential fatty acids. Aquatic animals like fish are good source of raw material for functional food due to their easily digested proteins, minerals and vitamins. Treated fish waste has many uses such as animal feed, biodiesel or biogas, dietic product, natural pigments, food-packaging application, cosmetics, enzyme isolation, soil fertilizer and moisture maintenance in foods (Arvanitoyannis & Kassaveti, 2008). Fish processing industry generated 20-80% amount of wastes depending on the level of processing and type of

8

fish used that can be utilized as fish silage, fish sauce, fishmeal and produce various values added products such as proteins, amino acids, oils, minerals, enzymes, collagen, gelatin and bioactive products (Ghaly et al., 2013).

The fish proteins can be used as functional ingredients in foods items because of their properties such as water holding capacity, oil absorption, gelling activity, foaming capacity and emulsifying properties. Nutrient and minerals such as 6% of nitrogen, 5% of phosphorus and 4% of potassium are present in fish waste that contribute to plant growth study in commercial plants (Lycoperscon esculantum, Hibiscus esculenta and Solanum melongena) (Ramalingam, Thirunavukkarasu, Chandy, & Rajaram, 2014). Fish oil can be converted to non-toxic, biodegradable, environment friendly biodiesel using chemical and enzymatic transesterification.

Husin, Mazlina, Kamal, Chuan, & Muhammad (2015) studied that peptones generated from sardines and mackerel waste by enzymatic hydrolysis are possibly to replace current commercial peptones for microbial growth media. Fish waste and fish wastewater are valuable used as substrates were processed by different ways for microbial enzymes production.

Fish bone that generated from fish processing industries contains higher amount of calcium, carbonate and phosphorus. Fish bones contains of hydroxyapatite (HAP) that have potential as UV absorbance. Zul Helmi Rozaini et al., (2017) claimed in his study that HAP existing in Sardinella fimbriata or Tamban fish bone waste was synthesized, utilized and exhibits as sunscreen in cosmeceutical with addition of FeCl2. In addition, leftover fish bone produce hydroxyapatite (HAP) which have potential to reduce pollution, used as bone replacement implants, heart valves and other implants in human body systems (Bin, Dara, Sontang, Zuha, & Nina, 2013).

Moreover, treating fish waste and drift seaweed with an effective ways such as

9

decomposition process can produce organic soil fertilizers for agriculture (Illera- Vives, Seoane Labandeira, Brito, López-Fabal, & López-Mosquera., 2015).

DECOMPOSITION PROCESS

Decomposition is the process of breakdown the raw organic matter into more simple organic matter. Three succession phases that involve in decomposition are mesophilic phase (~45°C), thermophilic phase (~70°C) and curing phase (cooling to ambient temperature) (J Ryckeboer et al., 2003). The productivity of the decomposition process is determined by several factors such as the number and type of microorganisms presence, temperature, pH, electrical conductivity, moisture content and nutrient balance (Gebeyehu & Kibret, 2013). Microbial have to degrade or oxidize the organic matters and produce carbon dioxide, water, heat and stabilized end of organic product. The rate of microbe’s activity in the decomposition influences by several physical and chemical factors. In initial stages of decomposition, mesophilic microorganisms speedily break down the soluble and readily degradable substances and compost temperature increase due to these microbes produced heat. After temperature increase over 40 °C which is thermophilic stages, mesophilic microbes are less competitive and switched by thermophilic microbes. During thermophilic stages when temperature was high, the proteins, fats, cellulose and hemicellulose were rapidly breakdown. Most of pathogens are destroyed when temperature exceed than 55 °C. Lastly, the temperature slowly decrease and mesophilic microbes take over again for the maturation of organic matter.

Martins et al.(2013) stated that various biomass decomposed enzymes can be found during decomposition process. Furthermore fish waste has been used as fertilizers because it is rich in nitrogen and phosphorus (Arvanitoyannis & Kassaveti,

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2008). As natural bio-fertilizer, decomposition can improve soil fertility and health thus increasing agricultural productivity, enhanced soil biodiversity, diminished ecological risks and provide healthier environment.

The narrative of the microorganisms that involved in decomposition process is complicated since the community’s change continuously of microorganisms, pattern of temperature, pH, nutrient obtainability, oxygen concentration and water content.

2.3.1 Physico-Chemicals Parameters of Decomposition

Temperature, moisture content, pH, aeration, CN ratio are major factors that affecting decomposition process. These parameters are regulated by changing of aeration, ingredient mix ratios, moisture and turning frequency (Makan, Assobhei, &

Mountadar, 2013).

2.3.1.1 Temperature

Temperature is one of the factors that contribute for decomposition efficiency. The rising of temperature over decomposition indicates a microbial activity (Zein, 2015).

There were two ranges of temperature during the decomposition process which are mesophilic and thermophilic. Temperature for mesophilic range is between 10°C and 40°C and was characterized by the proliferation of the microbiota. During initial phase of decomposition, a mesophilic microbe is swiftly broken down to degrade the compounds. The heat will produce thus increase the waste temperature and changes to thermophilic phase. The temperature during thermophilic phase ranged around 45- 70°C (C Sundberg, Smårs, & Jönsson, 2004). Known as an active stage, the higher rate of biodegradation, thermophilic microbe’s presences and the inhibition of non-

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thermotolerant organisms were commonly observed. High temperature promotes the growth of microbial activity, maximizing biodegradation and killed the pathogens.

Decomposition process is obligatory through thermophilic phase to guarantee the safety of waste from pathogens (Bernal, Alburquerque, & Moral, 2009). Later, the temperature drops to last phase which is cooling phase, stabilization and maturation, characterized by growth of mesophilic microbes and the humidification of the compost. High temperature promotes the growth of microbial activity, maximizing biodegradation and killed the pathogens. The variations of temperature during decomposition showed the changes of microbes in the waste.

2.3.1.2 Moisture Content

Moisture content affords a medium for transport of dissolved nutrients that needed for microbes metabolic and physiological activity and greatly affect the rate of decomposition process (Liang, Das, & McClendon, 2003). Generally, optimum moisture ranged between 25% to 80% on a wet basis however recommended values of moisture content is 60% to 80% (Ahn, Richard, & Glanville, 2008). Initial moisture content of 75% can be used as the right parameter for successful decomposition process municipal solid waste (Makan et al., 2013). Moisture content during decomposition process was influenced by microbial activity and biodegradation of organic materials. This parameter regularly aids substitution for other factors such as water availability where microbial activity active in low percentage of moisture content.