Microbial community structure in anaerobic degradation of terephthalate and phenol 2

Table of Contents Title page Acknowledgements i Summary vii Abbreviations xviii 2.1 Anaerobic treatment of terephthalate- and phenol-containing wastewaters 12 2.1.1.1 PTA wastewater pro

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

Table of Contents

Title page

Acknowledgements i

Summary vii

Abbreviations xviii

2.1 Anaerobic treatment of terephthalate- and phenol-containing

wastewaters

12

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

2.1.2.1 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

2.2 Methanogenic degradation of terephthalate and phenol 23

2.2.2 Proposed terephthalate degradation pathway 24

2.2.3 Proposed phenol degradation pathway 26 2.3 Microbial communities for terephthalate and phenol degradation 27

2.3.1 Microorganisms involved in terephthalate degradation 27

2.3.1.1 Culture-dependent studies 27

2.3.1.2 Culture-independent studies 29 2.3.2 Microorganisms involved in phenol degradation 30

2.3.2.2 Culture-independent studies 31

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

46−50°C

35

3.1.3 Mesophilic and thermophilic phenol-degrading enrichments 37 3.1.4 Full-scale phenol-degrading anaerobic sludge sample 38

3.2.2 Batch substrate degradation 40

3.3 16S rRNA gene-based molecular analysis 41

3.3.8 Phylogenetic analysis and probe design 46 3.3.9 Nucleotide sequence accession numbers 47

3.4.1 Scanning electron microscopy (SEM) 47

4 Microbial community structure in a thermophilic anaerobic hybrid

reactor degrading terephthalate

51

4.2.2 SEM-based morphological observations 59 4.2.3 Effect of operational conditions on microbial population

dynamics as revealed by 16S rRNA gene-based T-RFLP

60

4.2.4 Thermophilic terephthalate-degrading consortium as

revealed by 16S rRNA gene clone libraries

63

4.2.5 Phylogenetic identity of T-RFs observed in community

T-RFLP profiles

69

4.2.6 Thermophilic terephthalate-degrading syntrophic

consortium as revealed by FISH

69

5 Microbial community structure in a terephthalate-degrading

anaerobic hybrid bioreactor operated at 46−50°C

74

5.2.2 Microbial compositions as revealed by T-RFLP of amplified

16S rRNA genes

79

5.2.3 Microbial populations revealed by FISH 81 5.2.4 Microbial compositions as revealed by 16S rRNA gene

clone library

84

5.2.5 Batch degradation of terephthalate at different temperatures 88

6 Identification of important microbial populations in the mesophilic

and thermophilic phenol-degrading methanogenic consortia

93

6.2.1 Enrichment of phenol-degrading consortia 96 6.2.2 Microbial compositions as revealed by 16S rRNA gene

clone library

97

6.2.3 Predominant microbial populations as revealed by FISH 104 6.2.4 Population changes of phenol-degrading MP and TP

enrichments associated with terephthalte and benzoate degradation

106

7 Diversity and localization of microbial consortium in a full-scale

phenol-degrading granular activated carbon anaerobic reactor

114

7.2.2 SEM observation of GAC sludge 118 7.2.3 Microbial compositions as revealed by 16S rRNA gene

T-RFLP

119

7.2.4 Microbial compositions as revealed by 16S rRNA gene

clone library

120

7.2.5 Important microbial populations in the GAC sludge

revealed by FISH

125

8 Conclusions and recommendations 134

8.1.1 Operation of anaerobic terephthalate-degrading reactors

under 46−50°C and 55°C conditions

135

8.1.2 Anaerobic degradation of phenol under mesophilic (37°C)

and thermophilic (55°C) conditions

135

8.1.3 Terephthalate- and phenol-degrading microbial consortia

under different temperatures

137

8.1.4 Deltaproteobacteria group TA and Pelotomaculum-related

populations

140

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

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 phenol-containing 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

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 gene-based 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 thermophila-related methanogens, Pelotomaculum-thermophila-related fat rod-shaped bacterial populations

(171-bp 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 out-competed by those bacterial populations before perturbations

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

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 T-RFLP 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

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

List of Tables Table 2.1 Number of full-scale anaerobic bioreactors constructed for treating

petrochemical wastewaters

12

Table 2.2 Full-scale anaerobic bioreactors treating PTA wastewaters 17

Table 2.3 Performance of UASB reactors treating phenol-containing

wastewaters under mesophilic and methanogenic conditions

22

Table 2.4 Syntrophic degradation of organic matters and the standard Gibbs

free-energy

24

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

activity of its sludge

57

Table 6.1 Degradation of benzoate and terephthalate in phenol-degrading

enrichments within 28 days

106

Table 8.1 Summary of microbial community structures in anaerobic

degradation of terephthalate and phenol under different temperatures conditions based on analyses of 16S rRNA gene clone libraries

139