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. Ballesteros and Prof.
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, food- processing 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 1 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 Next- Generation 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. iii Abstract of Thesis. xvi Chapter 1 INTRODUCTION .3 Scope of the study.
7 Chapter 2 LITERATURE REVIEW .1 Lipid-rich wastewaters .2 Anaerobic treatment of wastewater .3 Microbial communities degrading lipids-metabolic steps .4 Common factors affect anaerobic digestion .2 Main carbon source .3 OLR and HRT .4 Volatile fatty acid .5 Substrate feeding regime.5 Microbial communities in anaerobic reactors .6 Methods to study on microbial community .1 Molecular techniques for studying microbial community .2 Next generation sequencing (NGS) .3 Statistical tools for microbial ecology .7 Measures to enhance biogas production. 67 Chapter 3 ACTIVATION OF SLUDGE AND EVALUATION OF SUBSTRATE PROPERTIES FOR ANAEROBIC TREATMENT OF LIPID-RICH WASTEWATER .2 Materials and methods .1 Activate stored anaerobic sludge with GAL.2 Inhibitory effect of LCFA on GAL digestion .3 Biochemical methane potential of glycerol, LCFA and soybean oil .3 Results and discussions .1 Activate stored anaerobic sludge with GAL.2 Inhibitory effect of LCFA.3 Biochemical methane potential of glycerol, LCFA and soybean oil. 100 Chapter 4 CHARACTERIZATION OF MICROBIAL COMMUNITY INVOLVED IN ANAEROBIC SYSTEM TREATING LIPID-RICH WASTEWATER .2 Methods and materials .1 Experimental set-up .3 Sampling and analysis of physical-chemical parameters.3 Results and discussion.2 The overall results of microbial community .3 Dynamics of microbial communities. 137 Chapter 5 THE EFFECT OF A FEED INTERRUPTION ON MICROBIAL COMMUNITY IN ANAEROBIC SYSTEM TREATING LIPID-RICH WASTEWATER .2 Materials and Methods .1 Experimental set-up and physical parameters analysis .3 Results and discussion.
Performance of the reactors. Overall dynamics of microbial community. Dynamics of glycerol degrading bacteria. Dynamics of LCFA degrading bacteria.
Dynamics of intermediate degrading bacteria. Dynamics of archaeal groups. Dynamics of non-growth with the soybean oil bacteria. Dynamics of background bacteria.
163 Chapter 6 CONCLUSIONS AND RECOMMENDATIONS. Recommendations for future works. 169 xii Table of tables Table 2.1 Biochemical methane potential from main substrates .2 Percentage of common LCFA found in lipid-rich waste water .3 Common fatty acids in nature .4 Characteristics of some methanogenic Archaea .5 Energetics of intracellular redox reactions .6 Organic acids degradation with possitive Gibbs energy .7 Primers used in PCR amplification .2 Trace element solution .4 Substrates and inoculum for inhibitory effect of LCFA on GAL digestion experiment.5 Substrates and inoculum for the biochemical methane potential of glycerol, LCFA and soybean oil experiment .6 LCFA used as substrate in LCFA reactors .1 Primer of PCR. 112 xiii Table of figures Figure 2.1 Four steps of anaerobic digestion .2 Metabolic pathways of the fermentation of glycerol .3 Beta-oxidation of fatty acids .4 Methanogenesis from CO2 plus H2.5 Effect of temperature and hydrogen partial pressure on the Gribbs free energy change (G’) for the oxidation of acetate to H2/CO2 or methanogenesis from H2/CO2.6 Methanogenesis from methanol and acetate.7 Thermodynamic thresholds for propionate and palmitate oxidation, and hydrogenotrophic methanogenesis.8 Typical procedure for microbial community analysis .9 Organization of data within OTU-base .10 Summarize steps in microbial community analysis in anaerobic digesters .1 Biogas production rate of the activated process of anaerobic sludge with GAL.2 Cumulative biogas production of GAL fermentation receiving LCFA at various concentrations.3 pH values of GAL fermentation receiving LCFA at various concentrations at the final day.4 Cumulative biogas production of culture receiving glycerol, LCFA, and oil.1 Performance of reactors .2 Time courses of the relative abundance of characteristic microorganisms at phylum level.3 Time courses of the relative abundance of characteristic microorganisms at order level .4 Time courses of the relative abundance of microorganisms #6, #12, and #21.5 Time courses of the relative abundance of microorganism #11, #19, and #22.6 Time courses of the relative abundance of microorganism #7.7 Time courses of the relative abundance of microorganisms #1 and #3.8 Time courses of the relative abundance of microorganism #17.9 Time courses of the relative abundance of microorganism #4, #23, and #24.10 Pathway of anaerobic digestion of lipid.1 Performance of the reactors .2 Time courses of the relative abundance of characteristic microorganisms at order level .3 Time course of the relative abundance glycerol degrading microorganisms 154 Figure 5.4 Time courses of the relative abundance of LCFA degrading bacteria .5 Time courses of the relative abundance of oil and intermediates degrading organisms .6 Time courses of the relative abundance of producing archaea.7 Time courses of the relative abundance of non-growth with the oil organisms .8 Time courses of the relative abundance of background bacteria .