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PERFORMANCE CHARACTERISTICS of FUNGAL DEGRADED RICE BRAN and PALM KERNEL CAKE SUBSTITUTED in Clarias Gariepinus FEEDS by JULIANA

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PERFORMANCE CHARACTERISTICS of FUNGAL DEGRADED RICE BRAN and PALM KERNEL CAKE SUBSTITUTED in Clarias Gariepinus FEEDS by JULIANA PERFORMANCE CHARACTERISTICS OF FUNGAL DEGRADED RICE BRAN AND PALM KERNEL CAKE SUBSTITUTED IN Clarias gariepinus FEEDS BY JULIANAH FOLARIN ALUKO B.Sc. (HONS); M.Sc. Microbiology (Ibadan) MATRIC. NO.: 33005 A Thesis in the Department of MICROBIOLOGY submitted to the Faculty of Science in partial fulfilment of the requirement for the award of the degree of DOCTOR OF PHILOSOPHY (Ph. D) of the UNIVERSITY OF IBADAN, NIGERIA. FEBRUARY, 2014 ABSTRACT Fish farming enterprise has increased in recent times due to public demand for fish protein. However, rising costs of fish feed ingredients have greatly reduced the profit margin of local fish farmers. Cheap, readily available and locally sourced ingredients may be better substitutes to the costly fish feeds. The aim of this study was to evaluate the performance of fermented Palm Kernel Cake (PKC) and Rice Bran (RB) as substitutes in the diet of Clarias gariepinus. Fungi were isolated from Unfermented PKC (UPKC), RB (URB) and from Naturally-Fermented PKC (NFPKC) and RB (NFRB) ligno-cellulosic substrates using Potato Dextrose Agar (PDA). Isolates were identified and screened for enzyme production (amylase and cellulase) using standard methods. They were then subjected to physico-chemical (pH, temperature, incubation period, carbon and nitrogen sources) analysis to obtain optimal conditions for enzyme production. Proximate analysis of fungi-fermented samples was carried out to select the starters used in degrading the ligno-cellulosic substrates. Starters that gave the highest protein contents were selected. Starter-Degraded PKC (SDPKC) and RB (SDRB) were analysed for proximate and mineral compositions, amino acid profile, anti-nutrition factors, vitamins and heavy metals. The degraded and undegraded ligno-cellulosic bulks were substituted in the diets of fingerling and compared with a standard fish feed as control for three months. Fish performance characteristics [(Mean Weight Gain (MWG), Specific Growth Rate (SGR)] and Feed Conversion Ratio (FCR)) were evaluated. Data were analysed using ANOVA at p=0.05. Ten fungi isolates obtained were Aspergillus fumigatus, Aspergillus niger, Aspergillus clavatus, Aspergillus tamarii, Aspergillus terreus, Aspergillus versicolor, Rhizopus stolonifer, Rhizopus oryzae, Rhizopus sp. and Trichoderma sp. Highest amylase (63.2units/mg) at pH 6.0, 50.0oC, Vmax 3.9 (UI/mL) and Km 1.3 (g/mL) was produced by Aspergillus niger which grew best on wheat bran (8.0 mg/g) as carbon source and ammonium nitrate (4.6 mg/g) as nitrogen source. o Highest cellulase (3.1Units/mg) at pH 8.0, 20.0 C, Vmax 0.9 (UI/mL) and Km 0.8(g/mL) was produced by Rhizopus oryzae which grew best on RB (7.3 mg/g) as carbon source and potassium nitrate (1.6 mg/g) as nitrogen source. Selected starters, Aspergillus clavatus and Aspergillus tamarii, gave protein contents of 6.5% and 17.9%. Protein contents of NFPKC, NFRB, UPKC and URB were 5.7, 22.0, 5.2 and 16.0% respectively. Highest zinc composition (520.0µg/g) was recorded in SDPKC and SDRB with reduction in amino acid profile (16.7 – 0.03µg/g). Fermentation decreased oxalate (24.1-18.2 µg/g) among the anti-nutritional factors and had no effect on the vitamin contents, while no heavy metal was detected in the feeds. The MWG of fish fed with URB and UPKC ranged from 18.9g - 35.8g and that of SDRB and SDPKC ranged from 12.8 - 43.3g while that of the control feed was 31.5g. Fish fed SDPKC gave the highest SGR of 1.3g/day and the lowest FCR of 1.4. Significant difference in SGR was observed between fish fed with SDPKC and UPKC. Fungi degraded palm kernel cake and rice bran supported the growth of Clarias gariepinus better than the control feed and could be good sources of fish feed components. Keywords: Palm kernel cake, Rice bran, Fungal degradation, Clarias gariepinus feed. Word count: 497 ii ACKNOWLEDGEMENT I acknowledge with a heartfelt gratitude and appreciation the God Almighty, the Alpha and Omega for His presence, love, mercies, assistance, provision and protection for me in life and particularly during the period of this research work. I must express my unreserved gratitude to my Supervisor, Prof. A.A. Onilude for his concern, contribution, guidance, assistance and patience from the beginning of this work to the end. I appreciate your concern and patience without which this work would not have been completed. I wish to acknowledge Prof. O. E. Fagade the Head of Department of Microbiology also for his concern and encouragement for me to successfully complete the programme. I acknowledge the opportunity I was given to undertake this academic programme by the management of the National Institute of Freshwater Fisheries Research, New-Bussa under the leadership of the Executive Director Dr. A. N. Okaeme, past and present staff of Central Service Laboratory, staff of Nutrition Unit, Pastor Adebayo Aremu of Genetics Laboratory, brother Olanrewaju Ajayi at the time of the fish feeding trial and all the staff of the headquarters office for their care in one way or the other. My appreciation goes to Dr. A. A. Ogunjobi for his involvement and willingness to assist me at any stage of this work. I appreciate you. I am indeed grateful to Dr. B. C. Adebayo - Tayo for her readiness to accept and assist me in this work. I will ever be grateful to all the members of staff and students of Microbiology Botany Departments of the University of Ibadan, my colleagues under the supervision of Prof. A. A. Onilude and many others whose names I may not be able to mention. I bless the name of God for Pastors D.I. Olowe, Paul Olumide, Rev. J. D. Oni and all the men and women of God and church members of All Christian Evangestic Ministry (A.C.E.M), New- Bussa who took it as duties to pray for me. I cannot but appreciate my children for their endurance, understanding and prayers, my mother, sisters and brothers for their prayers and as many as have contributed in one way or the other to my success. I thank you all. Julianah F. Aluko, February, 2014. iii CERTIFICATION I certify that the research work reported in this thesis for the degree of Doctor of Philosophy (Microbiology) was carried out under my supervision in the Department of Microbiology, University of Ibadan, Ibadan. --------------------------------------------------------------- Supervisor Prof. A. A. Onilude B.Sc., M.Sc., (Ife), Ph.D (Ibadan) Professor of Microbiology. Supervisor iv DEDICATION I dedicate this work to my late husband, Dr. P. O. Aluko, my late father, Pa Benjamen Kolawole, my mother, Beatrice Aduke Kolawole, my sisters, brothers and my children – David Opeyemi, Deborah Oluwafunke and Daniel Oluwaseyi Aluko. v TABLE OF CONTENTS Content Page Title i Abstract ii Acknowledgement iii Certification iv Dedication v Table of Contents vi List of Tables viii List of Figures x List of Abbreviations xiii Chapter One Introduction 1 1.1 Feedstuff and animal protein 1 1.2 Statement of problem 2 1.3 Justification 4 1.4 Aims and Objectives of the project 4 Chapter Two Literature Review 6 2.1 Fish feeds 6 2.2 Natural and Artificial Fish Feeds 6 2.3 Conventional and Unconventional Feeds 7 2.4 Agro-Industrial by-products 9 2.5 Constraints in the use of AIBs 13 2.6 Improvement in AIBs Utilization 15 2.7 Anti-nutritive Factors in Animal Feedstuffs 22 vi Content Page 2.8 Biodegradation of Materials 24 2.9 Performance characteristitics 30 2.10 Fish 31 Chapter Three Materials and Methods 39 3.1 Sampling and Culture Methods 39 3.1.1 Collection of Samples 39 3.1.2 Spontaneous fermentation 40 3.1.3 Isolation of Fungi 40 3.1.4 Collection of pure cultures 42 3.1.5 Maintenance of Stock Cultures 42 3.1.6 Identification Procedure 42 3.1.7 Physiological characterization and enzymatic 43 studies of fungal isolates. 3.1.8 In-vitro Biodegradation of Lignocellulosic 57 Substrates Samples 3.1.9 Evaluation of the performance and other 68 characteristics of fungi-degraded bulk of these lignocellulosic substrates substituted in the diets of Clarias gariepinus 3.1.10 Water quality Analysis 75 Chapter Four Results 76 Chapter Five Discussion 174 Chapter Six Conclusion 192 References 194 Appendix 225 vii LIST OF TABLES Table Page 3.1 Nature and Sources of Lignocellulosic Samples utilized in the work 41 3.2 The seven (7) treatments used in the fish feeding trials1 and 2 70 3.3 Feed samples used in the fish feeding trial 3 71 3.4 Feed samples used in the fish feeding trial 4 72 4.1 Morphological Characteristics of the fungi isolated from categorized fermented 77 and unfermented lignocellulosic substrates. 4.2 Frequency of occurrence of fungi isolated from categorized fermented and 78 unfermented lignocellulosic substrates. 4.3 Temperature (oC) and pH changes during spontaneous fermentation of the 81 lignocellulosic substrates 4.4 Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF), Acid Detergent 83 Lignin (ADL), Cellulose and Metabolizable energy profile of the lignocellulosic samples. 4.5 Proximate Composition (%) of Biodegraded lignocellulosic samples under 84 spontaneous fermentation 4.6 Amino acid profiles analysis of unfermented and spontaneously fermented 87 lignocellulosic substrates (Rice bran and Palm kernel cake). 4.7a Performance characteristics of Local and Dutch Clarias gariepinus fed with 153 seven (7) different feed samples for 90 days 4.7b Performance characteristics of Dutch Clarias gariepinus fed for 90 days with 155 feed substituted with unfermented and spontaneously fermented substrates 4.7c Performance characteristics of Dutch Clarias gariepinus fed for 90 days with 156 feed substituted with fungi degraded
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