This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Analysis of microbial components of two‑liquid phase bioreactors for improved volatile organic compund biofiltration Ng, Chow Goon 2014 Ng, C. G. (2014). Analysis of microbial components of two‑liquid phase bioreactors for improved volatile organic compund biofiltration. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/61656 https://doi.org/10.32657/10356/61656 Downloaded on 24 Sep 2021 17:27:32 SGT ANALYSIS OF MICROBIAL COMPONENTS OF TWO-LIQUID PHASE BIOREACTORS FOR IMPROVED VOLATILE ORGANIC COMPOUND BIOFILTRATION NG CHOW GOON SCHOOL OF BIOLOGICAL SCIENCES 2014 ANALYSIS OF MICROBIAL COMPONENTS OF TWO-LIQUID PHASE BIOREACTORS FOR IMPROVED VOLATILE ORGANIC COMPOUND BIOFILTRATION NG CHOW GOON School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfilment of the requirement for the degree of Doctor of Philosophy 2014 ACKNOWLEDGEMENTS The journey to complete this thesis was an experience that was challenging, yet illuminating and enjoyable! It would not have been possible without the help and the support of many people in both my professional and private life. To my professor, Assistant Professor Sze Chun Chau, a big Thank you! In the past four years of research, she had been the source of positive and genuine guidance, not just in the scientific aspects, but also in my social life. It had been a privilege and an honour to be part of her team to learn and become more comfortable with sharing of scientific ideas. I am thankful to my lab members, Miao Huang, Shalini, Yang Nan, Sock Hoai, Zhang Rui, Yulan, Ley Byan, Yu Ling and Kian Wee for providing a friendly and conducive atmosphere to work in. Not just in the working aspect, thank you for the moments of joy and leisure we shared together in our outings. I am also grateful to my scientific graduate committee members, Professor Christiane Ruedl and Associate Professor Ravi Kambadur for giving their time to listen and advice on this project and progress. Lastly, to my family, thank you for your kind understanding and support! To my dearest mum, thank you for the numerous late night suppers and tonic soups you prepared for me. This work was supported by Academic Research Fund Tier 1 (AcRF1) from Ministry of Education, Singapore. TABLE OF CONTENTS LIST OF PUBLICATIONS VII LIST OF TABLES VIII LIST OF FIGURES X LIST OF ABBREVIATIONS XVI ABSTRACT S XIX Chapter 1: General Introduction 1 1.1 Volatile organic compounds (VOCs) 1 1.1.1 Current treatment methods for control of VOC release 2 1.2 Bioavailability and toxicity of VOCs to microorganisms 5 1.3 Two-liquid-phase partitioning bioreactor (TPPB) system 6 1.3.1 Use of single strain versus microbial consortium in TPPB 9 1.4 Hexane 11 1.4.1 Impact on environment and health 12 1.4.2 Biodegradation/mineralization of hexane 13 1.5 Silicone oil as NAL phase in TPPB for hexane removal 14 1.6 Stirred tank TPPB configuration 15 1.7 Objectives of this study 16 1.8 Thesis organization 17 Chapter 2: Development of Microbial Biomass in Aqueous and Interfacial Fractions of TPPB 19 2.1 Introduction 19 2.1.1 Use of acclimated microbial consortia as bioreactor inocula 19 2.1.2 Biological manifestation of hexane removal/utilization 20 2.1.3 Microbial association at the aqueous-NAL interface 21 2.2 Materials and methods 23 2.2.1 Culture media 23 I 2.2.2 Culturable cell count of microbial consortia from soil samples before and after enrichment by hexane 24 2.2.3 Collection and acclimatization of microbial consortium from petroleum-contaminated soil 24 2.2.4 Bioreactor operating conditions 27 2.2.5 Monitoring to biomass and biological activities 28 2.2.5.1 Cell turbidity at OD600 28 2.2.5.2 Quantification of total cellular protein 29 2.2.5.3 Quantification of adenosine triphosphate (ATP) 29 2.2.5.4 Culturable cell count 30 2.2.6 Microscopy 30 2.2.6.1 Brightfield and fluorescence microscopy 30 2.2.6.2 Confocal Laser Scanning Microscope (CLSM) 31 2.2.7 Hexane degradation kinetic study 31 2.3 Results 32 2.3.1 Assessment of soil microbial consortia for suitability as bioreactor inocula 32 2.3.2 Acclimatization of the car park soil microbial consortia 33 2.3.3 Development of biomass and biological activities in the bioreactors 37 2.3.3.1 Microbial biomass in aqueous phase of TPPBs versus MPBs 37 2.3.3.1.1 Cell density(OD600) of aqueous phase cultures 37 2.3.3.1.2 Total cellular protein content of aqueous phase culture 39 2.3.3.1.3 ATP quantification of aqueous phase cultures 40 2.3.3.1.4 Culturable cell count of aqueous phase cultures 42 2.3.3.1.5 Hexane degradation efficiency 43 2.3.3.2 Microbial biomass associated with TPPB interfacial fractions 46 2.3.3.2.1 Microscopic examination of microorganisms within IF 47 2.3.3.2.2 Cell density(OD600) of TPPB IFs 50 2.3.3.2.3 Culturable cell count of TPPB IFs 51 2.3.3.2.4 Hexane degradation efficiency of TPPB IFs 53 2.4 Discussion and Conclusions 55 2.4.1 Enhanced microbial growth and metabolic activities in TPPBs 55 II 2.4.2 Localization of microbial subpopulations at aqueous/NAL interface 56 2.4.3 Development of higher hexane degradation efficiency by microbial consortia in TPPB over time 57 Chapter 3: Bioreactor Microbial Community Analysis 59 3.1 Introduction 59 3.1.1 Microbial community analysis 59 3.1.2 Culture-based approaches for analysis of microbial community 60 3.1.3 Culture- independent approaches based on DNA sequences 62 3.1.3.1 Choice of ribosomal RNA gene as candidate for phylogenetic classification 62 3.1.3.2 Real time (quantitative) PCR 63 3.1.3.3 Sequencing of full-length rRNA genes 64 3.1.3.4 Use of 16S rRNA (rDNA) library in community analysis 65 3.1.3.4.1 Amplified ribosomal DNA restriction analysis (ARDRA) 66 3.1.3.5 Denaturing gradient gel electrophoresis (DGGE) 67 3.1.3.6 Metagenomic and high throughput approaches 69 3.1.4 Culture-independent approaches based on other cellular components 69 3.1.5 Strategies applied in this thesis to identify the microorganisms and its dynamics changes in the population 70 3.2 Materials and Methods 72 3.2.1 Extraction of genomic DNA from bioreactor samples 72 3.2.2 Polymerase-chain reaction (PCR) 73 3.2.2.1 Primers 73 3.2.2.2 Real time PCR (qPCR) 75 3.2.2.3 End-point PCR 76 3.2.3 Agarose gel electrophoresis 77 3.2.4 Distribution of colony morphotypes in microbial communities 77 3.2.5 Construction of 16S rDNA clone library 78 3.2.6 Molecular analysis of bacterial isolates and clones 79 3.2.6.1 PCR-ARDRA 79 3.2.6.2 16S rDNA sequencing and analysis 80 3.2.6.3 Sequence analysis and dendrogram construction 80 III 3.2.7 Denaturing Gradient Gel Electrophoresis (DGGE) 81 3.2.7.1 Gel Electrophoresis and visualization 81 3.2.7.2 DGGE data analysis 81 3.2.7.3 Sequencing of excised bands 83 3.3 Results 84 3.3.1 Determination of bacterial/fungal/archeal abundance by qPCR 84 3.3.1.1 Specificity of universal primers for bacterial, fungal and archaeal rRNA genes 84 3.3.1.2 Standard curves and detection limits 86 3.3.1.3 Proportion of bacterial, fungal and archaeal population in the microbial consortium 87 3.3.2 Dynamics of bacterial communities in bioreactors 90 3.3.2.1 Community dynamics based on colony morphotype distribution 91 3.3.2.2 Community dynamics based on DGGE of 16S rRNA V3 region 94 3.3.2.2.1 Community dynamics based on moving window analysis 97 3.3.2.2.2 Community structures in comparison to initial microbial consortium 99 3.3.2.2.3 Diversity richness of bacterial communities in bioreactors 100 3.3.2.2.4 Functional organization of bacterial communities in bioreactors 102 3.3.2.2.5 Cluster analysis, multi-dimensional scaling (MDS) and principle component analysis (PCA) of DGGE profiles 104 3.3.2.2.6 Similarity comparison between sets of bioreactors 108 3.3.2.2.6.1 Between aqueous phase of TPPBs and MPBs 108 3.3.2.2.6.2 Between aqueous phase and IF of TPPBs 110 3.3.3 Identification of bacterial taxa present in bioreactors 112 3.3.3.1 16S rDNA clone libraryof bioreactor inoculum (0th week sample) 112 3.3.3.2 Culturable isolates sampled from bioreactors 113 3.3.3.3 Molecular identification of clones/isolates based on strategy combining PCR-ARDRA and 16S rDNA sequencing 114 3.3.3.3.1 Identification of selected clones and isolates by 16S rDNA sequencing 115 IV 3.3.3.3.2 ARDRA profiles and 16S rDNA-based taxonomic identification 116 3.3.3.3.3 Association of colony morphotypes to taxonomic groups 122 3.3.3.4 Phylogenetic relationships of bacteria in initial inoculum and bioreactor communities 124 3.3.3.5 Intra-genus diversity of clones/isolates 126 3.3.4 Association of DGGE profiles to MOTU identities 130 3.3.4.1 Construction of reference DGGE ladder using MOTU-designated clones/isolates 130 3.3.4.1.1 Coverage of bioreactor consortia by reference DGGE ladder 136 3.3.4.2 Identification of MOTU corresponding to excised DGGE bands138 3.3.5 Analysis of community dynamics at genus level resolution 138 3.3.5.1 Selection of Prominent Bands (PB) 141 3.3.5.2 Dynamics of key genera in bacterial communities of bioreactors 142 3.4 Discussion and Conclusions 151 3.4.1 Dynamics of the key genera distribution as analyzed by MDS and PCA 151 3.4.2 Dynamics of key subpopulations during exponential growth phase 152 3.4.3 Dynamics of key subpopulation during stationary growth phase 153 3.4.4 Probable key bacterial genera involved in the hexane degradation 155 3.4.5 Conclusions 156 Chapter 4: Characterization of Hexane Degraders Isolated From Bioreactors 160 4.1 Introduction 160 4.1.1 Alkane