UNIVERSITY OF THE PHILIPPINES Doctor of Philosophy in Environmental Engineering NGUYEN KHAC KIEM MICROBIAL POPULATION DYNAMICS IN A SEMI CONTINUOUS ANAEROBIC SYSTEM TREATING LIPID RICH WASTEWATER Diss.
UNIVERSITY OF THE PHILIPPINES Doctor of Philosophy in Environmental Engineering NGUYEN KHAC KIEM MICROBIAL POPULATION DYNAMICS IN A SEMI-CONTINUOUS ANAEROBIC SYSTEM TREATING LIPID-RICH WASTEWATER Dissertation Adviser Florencio Jr Ballesteros, PhD Environmental Engineering Programs Chemical Engineering University of the Philippines Diliman Date of Submission November 2017 Dissertation Classification F This dissertation is available access to the general public MICROBIAL POPULATION DYNAMICS IN A SEMI-CONTINUOUS ANAEROBIC SYSTEM TREATING LIPID-RICH WASTEWATER Dissertation by NGUYEN KHAC KIEM Nationality: Vietnamese BS Chemical and Food Technology MS Food Science Submitted to the National Graduate School of Engineering College of Engineering University of the Philippines In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In Environmental Engineering National Graduate School of Engineering College of Engineering University of the Philippines Diliman, Quezon City November 2017 University Permission Page “I hereby grant the University of the Philippines a non-exclusive, worldwide, royalty-free license to reproduce, publish and publicly distribute copies of this thesis or dissertation in whatever form subject to the provisions of applicable laws, the provisions of the UP IPR policy and any contractual obligations, as well as more specific permission marking on the Title Page.” “Specifically I grant the following rights to the University: a) To upload a copy of the work in the theses database of the college/school/institute/department and in any other databases available on the public internet; b) To publish the work in the college/school/institute/department journal, both in print and electronic or digital format and online; and c) To give open access to above-mentioned work, thus allowing “fair use” of the work in accordance with the provisions of the Intellectual Property Code of the Philippines (Republic Act No 8293), especially for teaching, scholarly and research purposes.” Nguyen Khac Kiem Student-author ii Curriculum vitae Born in Hau Giang, a South-Western province of Mekong Delta, 250 km from Ho Chi Minh City, Vietnam, Mr Nguyen Khac Kiem spent his childhood for studies at the hometown After finishing his high school at Ly Tu Trong High School for the Grifted in Can Tho City where he specialized in Mathematics, in 2000 he went to Ho Chi Minh City to enroll into Department of Chemical Technology, Ho Chi Minh City University of Technology (HCMUT) Then he received his Bachelor in Chemical and Food Technology in 2005 After that, he continued to study Food Science and obtained his master degree at Department of Chemical Technology, Ho Chi Minh City University of Technology (HCMUT) in the year 2008 After graduation, he joined Industrial University of Ho Chi Minh City and worked as a lecturer at Institute of Biotechnology and Food Technology In 2014, he started to study PhD at The University of the Philippines, Diliman A part of his PhD work was done at the Department of International Development Engineering, Tokyo Institute of Technology, Japan under AUN-SEED Net – JICA Project The results of his PhD research are presented in this manuscript iii Acknowledgements I would like to express my sincere gratitude to my adviser Prof Dr Florencio C Ballesteros and Prof Dr Kiyohiko Nakasaki for the continuous support of my PhD study and research His expertise in the field and for his patience, motivation, enthusiasm, and immense knowledge are much appreciated His guidance helped me a lot with the research and writing of this thesis Thank you for the panel members for their valuable comments and suggestions during the oral defense Also, I would like to thank the JICA – ASEAN University Network – Southeast Asia Engineering Education Development Network (AUN-SEED-Net) scholarship for all the financial assistance that helped me finished with my studies and experiments on time I would like to offer my thanks to the directors, coordinator, and staffs of JICA office at Bangkok, Manila, and Tokyo My special thanks to Mrs Sirin for closely supporting me during my study period I would like to thank Ate Lynn, Ma’am Baby and all staffs of the Environmental Engineering Graduate Program, for their help during the study period at Department of Chemical Engineering, University of the Philippines, Diliman I also would like to thank all Professors who taught me courses in Environmental Engineering Finally, for my family who always there and believe in everything I do, thank you so much iv Abstract of Thesis Lipid-rich wastewaters are an ever-growing environmental concern These wastewaters, which can be traditionally characterized as fats, oils, and greases (FOG), come from domestic sewage and industrial effluents of restaurants and food services, foodprocessing facilities, vegetable oil plants, dairy industries, livestock farms, wool scouring facilities FOG reduce operational performance and they are the main reasons of process failure in traditional anaerobically biological wastewater treatment plants because of flotation, waste discharges, unpleasant odors, blockages, and inhibiting the contact between soluble substrates and biomass The main objective of this work is to find out the microbial groups responsible for the anaerobic digestion of lipid-rich wastewaters To accomplish the main objective, first we characterized sludge and substrate properties; then we revealed microbial groups responsible for each step of oil anaerobic degradation; finally, we investigated how the key oil degraders change when disturbance applied The results from our first research revealed that granular sludge became active and was able to produce a stable amount of methane after two weeks of acclimation to GAL Moreover, anaerobic digestion of GAL was inhibited by LCFA at the concentration of g L-1 LCFA The anaerobic digestion of glycerol, LCFA and soybean oil was able to accomplish using one-month-old acclimated sludge For LCFA and oil, although they were v ready for anaerobic digestion, lag phases were much longer than glycerol The outcomes from this research are helpful for understanding anaerobic digestion of lipid-rich wastewater, and especially guideline for experimental design in next research In the second research, semi-continuously fed reactors with three related substrates glycerol, long-chain fatty acids (LCFA) and oil, and stepwise increase in organic loading rate (OLR) were conducted for 90 days Microbial community was analyzed using NextGeneration Sequencing (NGS) with Miseq Illumina platform By comparing changes in microbial community in three types of operations we found that Clostridiales, Bacteroidales, and Spirochaetales orders were important for glycerol degradation while Syntrophobacterales and Thermobaculales orders including Leptospirales were shown to have played a crucial role in the β-oxidation step of LCFA mineralization This is a significant finding as the participation of Leptospirales has not been reported in the literature The results also suggest that feeding single related substrate while gradually increasing OLR and using NGS provides a clearer picture of the population dynamics occurring at given conditions In the final research, an artificial disturbance designed by a ten days’ period of starvation was applied to anaerobic systems to evaluate the stability of microbial groups involved in degradation of soybean oil and its components The dynamics of microbial community was traced by NGS, compared to before the disturbing event and linked to reactor performance The results showed that the microbial community was shifted profoundly, leading to declining methane production after 40 days of re-feed in reactors vi treated LCFA and soybean oil The abundance of orders Clostridiales, Spirochaetales and Desulfovibrionales was essential to overcome the difficult period in the reactors fed with glycerol More interestingly, a strong correlation was found between LCFA degrading orders, namely Thermobaculales, Syntrophobacterales, and Leptospirales and performance of reactors fed with LCFA and soybean oil The results also revealed that orders Actinomycetales, Synergistales, and Thermotogales as background organisms which did not contribute to glycerol, LCFA nor oil degradation, were able to surpass other useful bacteria and increased their relative abundance when methane production was ceased vii Table of contents Approval sheet i Curriculum vitae ii Acknowledgements iii Abstract of Thesis iv ACRONYMS xvi Chapter INTRODUCTION 1.1 General introduction 1.2 Research objectives 1.3 Scope of the study 1.4 Thesis outline Chapter LITERATURE REVIEW 2.1 Lipid-rich wastewaters 2.2 Anaerobic treatment of wastewater 13 2.3 Microbial communities degrading lipids-metabolic steps 16 2.3.1 Hydrolysis 17 2.3.2 Acidogenesis 17 2.3.3 Acetogenesis 24 2.3.4 Methanogenesis 24 2.3.5 Syntrophic association 35 66 the relationships between AD performance and the microbial community structure and function There are several reviews seeking to highlight the value and potential of applicable knowledge on the microbial communities involved in AD to achieve process optimization Based on current knowledge one does not know exactly the detail indices of the microbial community that will contribute a dominant role in AD (Goux et al 2015) They, however, assume that ecological features of reactor’s ecosystem such as syntrophic relationships (Kato & Watanabe 2010), diversity indicators, and the evenness of community as well as the capacity of key species are the main factors contributing the outcomes By applying ecological theory and adjusting these factors to anaerobic microbiology one can obtain better insight into the community and therefore propose more appropriate measures to estimate the outputs, control the process parameters and optimize the overall AD performance (Allison & Martiny 2008; 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