Microbial population dynamics in a semi-continuous anaerobic system treating lipid-rich wastewater :Doctor of philosophy - Major: Environmental engineering

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

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Nguyen Khac Kiem

Student-author

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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.

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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.

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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

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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

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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

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Table of contents

Approval sheet.................................................................................................................. i

Curriculum vitae.............................................................................................................. ii

Acknowledgements........................................................................................................iii

Abstract of Thesis........................................................................................................... iv

ACRONYMS ............................................................................................................... xvi

Chapter 1 INTRODUCTION .......................................................................................... 1

1.1 General introduction............................................................................................. 1

1.2 Research objectives.............................................................................................. 5

1.3 Scope of the study ................................................................................................ 6

1.4 Thesis outline ....................................................................................................... 7

Chapter 2 LITERATURE REVIEW .............................................................................. 8

2.1 Lipid-rich wastewaters......................................................................................... 8

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