Acknowledgements I would like to express my sincere thanks and gratitude to my research supervisor, Associate Processor Wen-Tso LIU who provided high quality intellectual support and constructive supervision throughout this research. My sincere appreciation also goes to my co-supervisor, Professor Say Leong ONG for his valuable comments and thoughtful inspiration. Further appreciation is given to all my classmates, friends and staffs in the Environmental Molecular Biotechnology Laboratory and Water Science Technology Laboratory for their kindly support and assistance throughout these years. My gratitude is also extended to Dr Jer-Horng WU in National Cheng Kung University (Taiwan) and Dr Hervé MACARIE in Institut de recherche pour le développement (France) for their kind advices; and to Dr Serena TEO in Tropical Marine Science Institute and Associate Processor I-Cheng TSENG in National Cheng Kung University (Taiwan) for their pertinent suggestions and great encouragements. Last but not least, I wish to express my deepest thanks to my family for their love, encouragement and moral support. i Table of Contents Title page Acknowledgements i Table of contents ii Summary vii List of tables xii List of figures xiii Abbreviations xviii Introduction 1.1 Background 1.2 Problem statements 1.3 Objectives 1.4 Organization of the thesis Literature review 11 2.1 12 Anaerobic treatment of terephthalate- and phenol-containing wastewaters 2.1.1 PTA wastewater 13 2.1.1.1 PTA wastewater production 13 2.1.1.2 Treatment of PTA wastewater 14 2.1.1.3 Anaerobic treatment under mesophilic conditions 14 2.1.1.4 Anaerobic treatment under thermophilic conditions 18 2.1.2 Phenolic wastewater 2.2 2.3 19 2.1.2.1 Phenolic wastewater production 19 2.1.2.2 Treatment of phenol-containing wastewater 20 2.1.2.3 Anaerobic treatment under mesophilic conditions 21 2.1.2.4 Anaerobic treatment under thermophilic conditions 23 Methanogenic degradation of terephthalate and phenol 23 2.2.1 Syntrophic association 23 2.2.2 Proposed terephthalate degradation pathway 24 2.2.3 Proposed phenol degradation pathway 26 Microbial communities for terephthalate and phenol degradation 27 ii 2.3.1 2.3.2 Microorganisms involved in terephthalate degradation 27 2.3.1.1 Culture-dependent studies 27 2.3.1.2 Culture-independent studies 29 Microorganisms involved in phenol degradation 30 2.3.2.1 Culture-dependent study 30 2.3.2.2 Culture-independent studies 31 Materials and methods 33 3.1 Terephthalate- and phenol-degrading microbial consortia 34 3.1.1 Thermophilic anaerobic terephthalate-degrading reactor 34 3.1.2 Anaerobic terephthalate-degrading reactor operated at 35 46−50°C 3.2 3.3 3.4 3.1.3 Mesophilic and thermophilic phenol-degrading enrichments 37 3.1.4 Full-scale phenol-degrading anaerobic sludge sample 38 Anaerobic degradation batch test 39 3.2.1 Anaerobic culture medium 39 3.2.2 Batch substrate degradation 40 3.2.3 Chemical analyses 40 16S rRNA gene-based molecular analysis 41 3.3.1 DNA extraction 41 3.3.2 PCR amplification 43 3.3.3 T-RFLP 44 3.3.4 45 Clone library construction 3.3.5 DGGE 45 3.3.6 RFLP 46 3.3.7 Sequencing 46 3.3.8 Phylogenetic analysis and probe design 46 3.3.9 Nucleotide sequence accession numbers 47 Microscopic observation 47 3.4.1 47 Scanning electron microscopy (SEM) 3.4.2 FISH 48 iii Microbial community structure in a thermophilic anaerobic hybrid 51 reactor degrading terephthalate 4.1 Introduction 52 4.2 Results and discussion 54 4.2.1 Reactor performance 54 4.2.2 SEM-based morphological observations 59 4.2.3 Effect of operational conditions on microbial population 60 dynamics as revealed by 16S rRNA gene-based T-RFLP 4.2.4 Thermophilic terephthalate-degrading consortium as 63 revealed by 16S rRNA gene clone libraries 4.2.5 Phylogenetic identity of T-RFs observed in community 69 T-RFLP profiles 4.2.6 Thermophilic terephthalate-degrading syntrophic 69 consortium as revealed by FISH 4.3 Summary Microbial community 73 structure in a terephthalate-degrading 74 anaerobic hybrid bioreactor operated at 46−50°C 5.1 Introduction 75 5.2 Results 77 5.2.1 Reactor performance 77 5.2.2 Microbial compositions as revealed by T-RFLP of amplified 79 16S rRNA genes 5.2.3 Microbial populations revealed by FISH 81 5.2.4 Microbial compositions as revealed by 16S rRNA gene 84 clone library 5.2.5 Batch degradation of terephthalate at different temperatures 88 5.3 Discussion 89 5.4 Summary 91 Identification of important microbial populations in the mesophilic 93 and thermophilic phenol-degrading methanogenic consortia 6.1 Introduction 94 iv 6.2 Results 96 6.2.1 Enrichment of phenol-degrading consortia 96 6.2.2 Microbial compositions as revealed by 16S rRNA gene 97 clone library 6.2.3 Predominant microbial populations as revealed by FISH 104 6.2.4 Population changes of phenol-degrading MP and TP 106 enrichments associated with terephthalte and benzoate degradation 6.3 Discussion 106 6.4 Summary 112 Diversity and localization of microbial consortium in a full-scale 114 phenol-degrading granular activated carbon anaerobic reactor 7.1 Introduction 115 7.2 Results 117 7.2.1 Batch degradation 117 7.2.2 SEM observation of GAC sludge 118 7.2.3 Microbial compositions as revealed by 16S rRNA gene 119 T-RFLP 7.2.4 Microbial compositions as revealed by 16S rRNA gene 120 clone library 7.2.5 Important microbial populations in the GAC sludge 125 revealed by FISH 7.3 Discussion 128 7.4 Summary 133 Conclusions and recommendations 134 8.1 135 Conclusions 8.1.1 Operation of anaerobic terephthalate-degrading reactors 135 under 46−50°C and 55°C conditions 8.1.2 Anaerobic degradation of phenol under mesophilic (37°C) 135 and thermophilic (55°C) conditions v 8.1.3 Terephthalate- and phenol-degrading microbial consortia 137 under different temperatures 8.1.4 Deltaproteobacteria group TA and Pelotomaculum-related 140 populations 8.2 Recommendations 141 8.2.1 Linking the laboratory-study to full-scale processes 141 8.2.2 Clarification of phylogenetic positions 142 8.2.3 Cultivation of yet-to-be cultured microorganisms 142 8.2.4 Functional importance of yet-to-be cultured microorganisms 143 8.2.5 Metagenomics of yet-to-be cultured microorganisms 144 References 145 Publications 159 vi Summary The demand for raw materials such as terephthalate and phenol for the production of various petrochemical-related products has increased over the years. As a result, wastewaters containing high concentrations of these anthropogenic compounds are generated and needed to be properly treated. Although a number of anaerobic (methanogenic) biological processes have been applied to treat terephthalate- and phenolcontaining wastewaters, these processes were mainly focused under mesophilic (30−37ºC) conditions. These mesophilic processes usually require long start-up time and sometimes are sensitive to the environmental changes, which are caused by unsuitable seeding biomass or inadequate growth conditions for the microbial consortia. Until now, the feasibility of treating terephthalate- and phenol-containing wastewaters above 37ºC is poorly known and warrants further study. To better select seeding sludge and operate these anaerobic processes in full-scale at temperature of 37ºC and beyond 37ºC, this study is carried out to address the microbial community structures in terephthalate- and phenol-degrading consortia enriched in laboratory-scale and full-scale reactors under different temperature ranges. To assess the feasibility of treating terephthalate-containing wastewaters above 37ºC, two laboratory-scale anaerobic hybrid reactors under 55°C and 46−50°C conditions were successfully started-up and operated for 272 and 547 days, respectively. Overall performance results suggested that these two reactors could effectively degrade terephthalate, and could be potentially scaled up to treat purified terephthalate (PTA) wastewater. These two anaerobic hybrid reactors showed extremely high resistance to vii the environmental perturbations (i.e., heat shock and pump failure), and their performances recovered in 1−2 weeks after normal operation conditions were restored. In the thermophilic (55°C) terephthalate-degrading reactor, a significant shift in bacterial population structure in the sludge bed but not on the biofilm attached on the surface of packing materials was observed during severe environmental perturbations. The finding suggested that the attached growth in the hybrid reactor therefore could serve as a good seeding source and helped the reactor to rapidly recover from process perturbations. The microbial structures in theses two reactors were characterized using 16S rRNA genebased terminal restriction fragment length polymorphism (T-RFLP), clone library and fluorescence in situ hybridization (FISH) with specific oligonucleotide probes. In the thermophilic (55ºC) terephthalate-degrading hybrid reactor, Methanothrix thermophilarelated methanogens, Pelotomaculum-related fat rod-shaped bacterial populations (171bp T-RF) were the key members responsible for terephthalate degradation under thermophilic methanogenic conditions throughout the reactor operation except during periods when the reactor experienced heat shock and pump failure. After system recovery, many other bacterial populations emerged, which belonged mainly to the Gram positive low G+C group (LGC) and Cytophaga-Flexibacter-Bacteroides (CFB) as well as Betaproteobacteria, Planctomycetes, and Nitrospira. These newly emerged populations were probably capable of degrading terephthalate in the hybrid system, but were outcompeted by those bacterial populations before perturbations. viii Under 46−50°C conditions, T-RFLP showed a significant shift in microbial community structure during the start-up period. A T-RF with a length of 100-bp was predominant in both sludge bed and the biomass taken from the surface of the packing materials on Day 346. The 100-bp T-RF was further identified to affiliate with 16S rRNA gene sequences representing the members of Pelotomaculum spp. FISH and cloning results further suggested that Methanosaeta- and Pelotomaculum-related populations were the key members responsible for terephthalate degradation under 46−50°C conditions. Phylogenetic analysis further suggested that these microbial populations were likely to be different from those found in mesophilic (37°C) and thermophilic (55°C) consortia. The experimental results suggested different microorganisms are responsible for terephthalate degradation in reactors operated under different temperature ranges, and batch degradation results further suggested the microbial consortia in the 46−50°C reactor were neither mesophiles nor thermophilies. It implies that using the seeding sludge from different temperature conditions may require a long start-up time to enrich the different specific microbial populations. To study the phenol-degrading microbial community, active mesophilic and thermophilic phenol-degrading methanogenic consortia were obtained after 18 months of acclimation process, and characterized using rRNA-based molecular approaches. As revealed by cloning, FISH and T-RFLP, these two enrichment cultures differed greatly in the community structures. Results strongly suggested that group TA in the Deltaproteobacteria (88.0% of EUBmix FISH-detectable bacterial cell area) and Pelotomaculum spp. in the Desulfotomaculum family (81.2%) were the predominant ix fermentative bacteria under mesophilic and thermophilic conditions, respectively. These populations closely associated with mesophilic and thermophilic members of Methanosaetaceae, Methanobacteriaceae and Methanomicrobiales to mineralize phenol as the sole carbon substrate to carbon dioxide and methane. These findings from the enrichment cultures were subsequently examined in a mesophilic full-scale granular activated carbon anaerobic fluidized bed (GAC-AFB) reactor treating the phenol-containing wastewaters from the phenolic resin manufacturing processes. TRFLP analysis revealed that the bacterial fingerprinting pattern was similar to that of the mesophilic phenol-degrading enrichment. Cloning and FISH analyses further suggested that Deltaproteobacteria group TA-, Syntrophus- and Methanosaeta-related microbes were the key populations in the phenol-degrading GAC-AFB reactor. FISH results further suggested that thin-filaments related to Chloroflexi-like populations may play an important role in the phenol-degrading GAC sludge. FISH analysis of the thin-sectioned GAC sludge showed that a non-layered structure was observed among those bacterial and archaeal (i.e., methanogens) populations. These findings greatly improve our understanding on the diversity and distribution of microbial populations in a full-scale mesophilic bioreactor treating actual phenol-containing waste stream. By combining the findings between the laboratory-scale enrichment and a full-scale reactor, the microbial populations involved in mesophilic phenol degradation were successfully identified. The overall results showed that the methanogenic microbial communities involved in terephthalate and phenol degradation were temperature-dependent, and the most x predominant microbial populations found in terephthalate- and phenol-degrading consortia were phylogenetically closely related. The experimental results further demonstrated that the two phenol-degrading enrichment cultures could mineralize terephthalate and benzoate, suggesting that these phenol-degrading consortia could be used as the seeding sludge for a bioreactor degrading terephthalate, or it is possible to use phenol as a co-substrate to enrich terephthalate-degrading microbial consortia, shortening the start-up time. Scanning electron microscopy (SEM) and FISH with oligonucleotide probes clearly revealed the cell morphology of those predominant yet-to-be cultured Deltaproteobacteria group TA-related cells (oval-shaped), Pelotomaculum-related cells (fat-rod, some of which may contain spherical spores at the central of cells), and Methanosaeta-related cells (filamentous bamboo-shaped or rods with flat-ends). These findings could serve as the basis to facilitate the identification and isolation in the future. Findings in this thesis further improved our understanding on the diversity, distribution, abundance, and dynamics of the microbial populations in the anaerobic degradation of terephthalate and phenol under different temperatures conditions. Keywords: anaerobic; methanogenetic; terephthalate; phenol; microbial, phylogenetic; clone library; terminal restriction fragment length polymorphism, T-RFLP; fluorescence in situ hybridization, FISH. xi List of Tables Table 2.1 Number of full-scale anaerobic bioreactors constructed for treating 12 petrochemical wastewaters. Table 2.2 Full-scale anaerobic bioreactors treating PTA wastewaters. Table 2.3 Performance of UASB reactors treating 17 phenol-containing 22 wastewaters under mesophilic and methanogenic conditions. Table 2.4 Syntrophic degradation of organic matters and the standard Gibbs 24 free-energy. Table 3.1 Trace elements and vitamins composition in anaerobic medium. 40 Table 3.2 16S rRNA genes primers used in this study. 43 Table 3.3 Oligonucleotide probes used in FISH analysis. 50 Table 4.1 Performances of the thermophilic reactor and specific degrading 57 activity of its sludge. Table 6.1 Degradation of benzoate and terephthalate in phenol-degrading 106 enrichments within 28 days. Table 8.1 Summary of microbial community structures in anaerobic 139 degradation of terephthalate and phenol under different temperatures conditions based on analyses of 16S rRNA gene clone libraries. xii List of Figures Figure 1.1 Proposed terephthalate and phenol degradation under methanogenic conditions. Figure 2.1 Proposed anaerobic degradation of terephthalate (A). Theoretical 25 overall reaction ofor terephthalate fermentation with hydrogentrophic methanogensis (B). Figure 2.2 The phenol degradation pathway, based on phenol transformation to 26 benzoate and acetate in BES-amended cultures (A) (Karlsson et al., 1999). Overall phenol degradation equation under methanogenic condition (B). Figure 3.1 Sketch of the laboratory-scale thermophilic anaerobic terephthalate- 35 degrading hybrid reactor. Figure 3.2 Sketch of the laboratory-scale anaerobic terephthalate-degrading 36 hybrid reactor operated at 46−50°C, and the packing materials on Day 346. Figure 3.3 Full-scale phenol-degrading anaerobic granular sludge sample. 38 Figure 4.1 Terephthalate degradation and concomitant methane production 59 during a thermophilic anaerobic batch experiment (■, terephthalate; ○, methane). Figure 4.2 SEM of dominant microorganisms observed in thermophilic 60 terephthalate-degrading anaerobic sludge sample taken at day 200. Figure 4.3 Electropherograms of MspI-digested T-RFLP fingerprints obtained 63 form thermophilic terephthalate-degrading sludge bed samples at days 103 (A), 172 (B), 200 (C), 259 (D), and packing materials (E). Figure 4.4 Phylogenetic affiliations of the 16S rRNA gene sequences retrieved 67 from the cloning analyses of domain Archaea. The phylogenetic tree was constructed using the neighbor-joining algorithm with JukesCantor parameter (bootstrapping number = 1000). The 16S rRNA gene sequence of Methanobrevibacter smithii (U55233) was selected as the out group. Only bootstrap values greater than 50% of the xiii replicates were shown. The abundance for individual clones was indicated at the end of individual sequences in parentheses. Scale bar = substitution per 50. Figure 4.5 Phylogenetic affiliations of the 16S rRNA gene sequences retrieved 68 from the cloning analyses of domain Bacteria. Calculation was based on the neighbor-joining algorithm with 1000 bootstrapping. The tree was rooted with 16S rRNA gene sequence of Methanococcus vannielii (M36507). Numbers in nodes and parentheses are identical as in Fig. 4.4. Scale bar = substitution per 10 nucleotides. In addition, the corresponding MspI-digested T-RF sizes for those bacterial clones were determined and shown. Those T-RFs in common to both clone libraries were highlighted with grey background. Figure 4.6 FISH of thermophilic terephthalate-degrading anaerobic sludge. 72 Microbial distribution was revealed by simultaneous hybridization with domain and group specific probes labeled with Cy3 (green) and Cy5 (red), respectively. (A) Hybridized with bacterial domain probe (EUB338mix; green) and archaeal domain probe (ARC915, red) (B) Hybridization with MX825 probe (red) specific for Methanosaeta and DFMI227a (green) probe specific for Desulfotomaculum. (C) Hybridized with MB1174 probe specific for Methanobacteriaceae and Methanosaeta specific probe (MX825, red) (D) Hybridization with TA55_OP5 probe (red) specific for clones in OP5 clade and bacterial domain probe (EUB338mix; green) (E) Hybridization with Delta_402 probe (red) specific for most of Deltaproteobacteria and bacterial domain probe (EUB338mix; green). (F) Hybridization with CF319a probe (red) specific for the members of the CFB group and bacterial domain probe (EUB338mix; green). Figure 5.1 Performance of the 46−50°C terephthalate (TA)-degrading hybrid 79 reactor over 547 days operation. xiv Figure 5.2 T-RFLP electropherograms of 16S rRNA gene from the 46-50°C 80 terephthalate- degrading hybrid reactor on Day (A) 0, (B) 42, (C) 104 and (D) 168 in the start-up period. Figure 5.3 T-RFLP electropherograms of 16S rRNA gene from the 46-50°C 81 terephthalate-degrading hybrid reactor on Day 346 in the stable period. (A) Biomass from packing materials. (B) Sludge bed. Figure 5.4 FISH analyses of bioflim (A and C) and sludge (B and D) samples 83 from the 46−50°C terephthalate-degrading hybrid reactor on Day 346 in the stable period. In panels (A) and (B), samples were hybridized with Cy3-labeled EUBmix probe specific for the domain Bacteria (green), and the Cy5-labeled ARC915 probe specific for the domain Archaea (red). (C and D) Samples were hybridized with FITC- labeled EUBmix probe (green), Cy3-labeled Ih820 probe specific for Desulfotomaculum subcluster Ih (red; combined with EUBmix was yellow), and Cy5-labeled MX825 probe specific for Methanosaetaceae (blue). Bar = 10 µm. Figure 5.5 Phylogenetic tree of 16S rRNA gene sequences constructed for 85 archaeal clones obtained from the 46−50°C terephthalate-degrading hybrid reactor on day 346 in the stable period. It was constructed using the neighbor-joining algorithm with Jukes-Cantor parameter (bootstrapping number = 1000). Only bootstrap values greater than 50% of the replicates are indicated at branch points. The scale bar represents the estimated number of nucleotide changes per sequence position. Figure 5.6 Phylogenetic tree of 16S rRNA gene sequences constructed for 87 bacterial clones obtained from the 46−50°C terephthalate-degrading hybrid reactor on Day 346 in the stable period. Figure 5.7 Degradation of terephthalate at 37, 46, 50 and 55°C. (A) Bioflm on 88 packing materials from upper region of reactor. (B) Biomass from sludge bed. xv Figure 6.1 Degradation of phenol in the (A) mesophilic and (B) thermophilic 97 enrichments. Figure 6.2 Phylogenetic tree of 16S rRNA gene sequences constructed for 99 archaeal clones obtained from mesophilic and thermophilic phenoldegrading enrichments. The relevant sequences were aligned using ClustalW program provided in MEGA3 package. The tree was constructed using the neighbor-joining algorithm with Jukes-Cantor model (bootstrapping number = 1000). The 16S rRNA gene sequence of Methanopyrus kandleri (M59932) was used as the outgroup. Only bootstrap values greater than 50 % of the replicates are indicated at branch points. The scale bar represents the estimated number of nucleotide changes per sequence position. Figure 6.3 Phylogenetic trees of bacterial clones obtained from mesophilic and 101 thermophilic phenol-degrading enrichments for domain Bacteria. Figure 6.4 Phylogenetic trees of bacterial clones obtained from mesophilic 102 phenol-degrading enrichment for Deltaproteobacteria group TA. Figure 6.5 Phylogenetic trees of bacterial clones obtained from thermophilic 103 phenol-degrading enrichment for Desulfotomaculum subcluster Ih. Figure 6.6 FISH analyses of mesophilic (A and C) and thermophilic (B and D) 105 phenol-degrading enrichments. In panels (A) and (B), samples were hybridized with Cy3-labeled EUBmix probe specific for the domain Bacteria (green), and the Cy5-labeled ARC915 probe specific for the domain Archaea (red). (C) Samples were hybridized with Cy3- labeled EUBmix probe (green) and Cy5-labeled DETLA-TA664 probe specific for Deltaproteobacteria group TA (red). (D) Samples were hybridized with Cy3-labeled EUBmix probe (green) and Cy5labeled Ih820 probe specific for Desulfotomaculum subcluster Ih (red). Arrow indicates a spore-forming cell. Bar = 10 µm. Figure 6.7 T-RFLP fingerprints of 16S rRNA genes from (A) mesophilic phenol- 108 degrading, (B) mesophilic terephthalate-degrading, (C) mesophilic benzoate-degrading, (D) thermophilic phenol-degrading, (E) xvi thermophilic terephthalate-degrading, and (F) thermophilic benzoatedegrading batch cultures. Figure 7.1 Batch degradation of phenol using GAC sludge sample. (A) Phenol 118 degradation. (B) SMA of phenol degradation. Figure 7.2 SEM images of a whole sludge granule (A) and the surface (B) of 119 granule from the full-scale phenol-degrading GAC-AFB reactor. Figure 7.3 T-RFLP fingerprints of 16S rRNA genes PCR-amplified from the 120 full-scale phenol-degrading GAC-AFB reactor. Figure 7.4 Phylogenetic tree of 16S rRNA gene sequences constructed for 122 archaeal clones obtained from a full-scale phenol-degrading GACAFB reactor. It was constructed using the neighbor-joining algorithm with Jukes-Cantor parameter (bootstrapping number = 1000). Only bootstrap values greater than 50% of the replicates are indicated at branch points. The scale bar represents the estimated number of nucleotide changes per sequence position. Figure 7.5 Phylogenetic tree of bacterial clones obtained from a full-scale 124 phenol-degrading GAC-AFB reactor. Figure 7.6 FISH analyses of the full-scale phenol-degrading GAC-AFB sludge. 127 In panels (A and B), samples were hybridized with Cy3-labeled EUBmix probe specific for the domain Bacteria (green), and the Cy5labeled ARC915 probe specific for the domain Archaea (red). Samples were hybridized with Cy3-labeled ARC915 probe (green) and Cy5-labeled MX825 (red) specific for Methanosaetaceae (C); and Cy3-labeled EUBmix probe and Cy5-labeled Delta-TA664, Syn424, and GNSB941 probes specific for Deltaproteobacteria group TA (D), Syntrophus (E) and Chloroflexi (F) (red), respectively. xvii Abbreviations AC Anaerobic contact AF Anaerobic filter AFB Anaerobic fluidized bed BES Bromoethanesulfonate BLAST Basic local alignment search tool bp Base pair Bv Volumetric organic loading rate CFB Cytophaga-Flexibacter-Bacteroides of the Bacteroidetes CLSM confocal laser scanning microscopy COD chemical oxygen demand CSTR continuous stirred tank reactor CTA crude terephthalic acid Cy3 Cyanine (fluorescent dye) Cy5 Cyanine (fluorescent dye) D Day DAF downflow anaerobic filter DAPI 4’,6-diamidino-2-phenylindole DGGE denaturing gradient gel electrophoresis dNTP Deoxyribonucleotide triphosphate DSFF Downflow stationary fixed film EDTA Ethylenediaminetetraacetic acid EGSB expanded granular sludge bed EPA Environmental Protection Agency EUBmix EUB338_I, EUB338_II and EUB338_III mixed FA Formamide FISH Fluorescence in situ hybridization FITC Fluorescein isothiocyanate (fluorescent dye) g Gram xviii GAC Granular activated carbon ∆Go' Gibbs free energy hr Hour HRT Hydraulic retention time IC Internal circulation ITS Internally transcribed spacers L Liter Lab Laboratory LGC Gram positive low G+C group M Molar MAR-FISH Microautoradiograph-fluorescence in situ hybridization m-cresol meta-cresol mg Milligram µg Microgram Minute mL Milliliter µL Microliter mM Millimolar µM Micromolar mm Millimeter µm Micrometer mmole Millimole MP Mesophilic phenol-degrading enrichment µsmax maximum specific growth rate nm Nanometer OTU Operational taxonomy unit PBS Phosphate buffered saline PCR Polymerase chain reaction p-cresol para-cresol PET Polyethylene terephthalate xix PTA Purified terephthalate p-toluate para-toluate RDP Ribosomal database project RFLP Restriction fragment length polymorphism rpm Rate per minute of centrifuge rRNA Ribosomal RNA s Second SEM Scanning electron microscopy SDS Sodium dodecyl sulfate SIP Stable-isotope probing SMA Specific methanogenic activity TA Terephthalate T-RF Terminal restriction fragment T-RFLP Terminal restriction fragment length polymorphism TP Thermophilic phenol-degrading enrichment UAF Upflow anaerobic filter UASB Upflow anaerobic sludge bed UV Ultraviolet v Volume VFA Volatile fatty acid VSS Volatile suspended solid Yxtots biomass yields xx [...]... Performances of the thermophilic reactor and specific degrading 57 activity of its sludge Table 6.1 Degradation of benzoate and terephthalate in phenol- degrading 106 enrichments within 28 days Table 8.1 Summary of microbial community structures in anaerobic 139 degradation of terephthalate and phenol under different temperatures conditions based on analyses of 16S rRNA gene clone libraries xii List of Figures... Table 2. 3 Performance of UASB reactors treating 17 phenol- containing 22 wastewaters under mesophilic and methanogenic conditions Table 2. 4 Syntrophic degradation of organic matters and the standard Gibbs 24 free-energy Table 3.1 Trace elements and vitamins composition in anaerobic medium 40 Table 3 .2 16S rRNA genes primers used in this study 43 Table 3.3 Oligonucleotide probes used in FISH analysis 50... central of cells), and Methanosaeta-related cells (filamentous bamboo-shaped or rods with flat-ends) These findings could serve as the basis to facilitate the identification and isolation in the future Findings in this thesis further improved our understanding on the diversity, distribution, abundance, and dynamics of the microbial populations in the anaerobic degradation of terephthalate and phenol. .. Proposed terephthalate and phenol degradation under methanogenic 4 conditions Figure 2. 1 Proposed anaerobic degradation of terephthalate (A) Theoretical 25 overall reaction ofor terephthalate fermentation with hydrogentrophic methanogensis (B) Figure 2. 2 The phenol degradation pathway, based on phenol transformation to 26 benzoate and acetate in BES-amended cultures (A) (Karlsson et al., 1999) Overall phenol. .. terephthalate- degrading, (C) mesophilic benzoate-degrading, (D) thermophilic phenol- degrading, (E) xvi thermophilic terephthalate- degrading, and (F) thermophilic benzoatedegrading batch cultures Figure 7.1 Batch degradation of phenol using GAC sludge sample (A) Phenol 118 degradation (B) SMA of phenol degradation Figure 7 .2 SEM images of a whole sludge granule (A) and the surface (B) of 119 granule from... Terephthalate degradation and concomitant methane production 59 during a thermophilic anaerobic batch experiment (■, terephthalate; ○, methane) Figure 4 .2 SEM of dominant microorganisms observed in thermophilic 60 terephthalate- degrading anaerobic sludge sample taken at day 20 0 Figure 4.3 Electropherograms of MspI-digested T-RFLP fingerprints obtained 63 form thermophilic terephthalate- degrading sludge... bacterial clones obtained from the 46−50°C terephthalate- degrading hybrid reactor on Day 346 in the stable period Figure 5.7 Degradation of terephthalate at 37, 46, 50 and 55°C (A) Bioflm on 88 packing materials from upper region of reactor (B) Biomass from sludge bed xv Figure 6.1 Degradation of phenol in the (A) mesophilic and (B) thermophilic 97 enrichments Figure 6 .2 Phylogenetic tree of 16S rRNA gene...predominant microbial populations found in terephthalate- and phenol- degrading consortia were phylogenetically closely related The experimental results further demonstrated that the two phenol- degrading enrichment cultures could mineralize terephthalate and benzoate, suggesting that these phenol- degrading consortia could be used as the seeding sludge for a bioreactor degrading terephthalate, ... full-scale phenol- degrading GAC-AFB reactor Figure 7.3 T-RFLP fingerprints of 16S rRNA genes PCR-amplified from the 120 full-scale phenol- degrading GAC-AFB reactor Figure 7.4 Phylogenetic tree of 16S rRNA gene sequences constructed for 122 archaeal clones obtained from a full-scale phenol- degrading GACAFB reactor It was constructed using the neighbor-joining algorithm with Jukes-Cantor parameter (bootstrapping... Keywords: anaerobic; methanogenetic; terephthalate; phenol; microbial, phylogenetic; clone library; terminal restriction fragment length polymorphism, T-RFLP; fluorescence in situ hybridization, FISH xi List of Tables Table 2. 1 Number of full-scale anaerobic bioreactors constructed for treating 12 petrochemical wastewaters Table 2. 2 Full-scale anaerobic bioreactors treating PTA wastewaters Table 2. 3 Performance . 24 2. 2.3 Proposed phenol degradation pathway 26 2. 3 Microbial communities for terephthalate and phenol degradation 27 iii 2. 3.1 Microorganisms involved in terephthalate degradation 27 2. 3.1.1. 2. 1 .2. 4 Anaerobic treatment under thermophilic conditions 23 2. 2 Methanogenic degradation of terephthalate and phenol 23 2. 2.1 Syntrophic association 23 2. 2 .2 Proposed terephthalate degradation. 18 2. 1 .2 Phenolic wastewater 19 2. 1 .2. 1 Phenolic wastewater production 19 2. 1 .2. 2 Treatment of phenol- containing wastewater 20 2. 1 .2. 3 Anaerobic treatment under mesophilic conditions 21 2. 1 .2. 4