Konstanz, Univ., Diplomarb., 2000.

Hohenheim, Univ., Diss., 2006.

Benzene is chemically stable as it has no substituents which can be biochemically attacked and a well-known toxic contaminant whose anaerobic degradation pathway is still not fully resolved. As only a very few anaerobic benzene-mineralizing pure cultures have been described yet, research was usually done with enrichment cultures dominated by specific organisms capable of benzene degradation under different electron acceptor conditions. Remarkable progress has been made in recent years with regard to the initial mechanism of benzene transformation especially on the putative genes that are involved in anaerobic carboxylation of benzene and the benzoyl-CoA central pathway. Many phylotypes described to be primary benzene degraders in anaerobic enrichment cultures at various electron acceptor conditions belong to the Peptococcaceae. Here, the thesis focused on characterizing the structure and function of anaerobic benzene-mineralizing microbial communities enriched from two hydrocarbon-contaminated sites: hydrocarbon-contaminated sediment from Ogoni in Niger Delta of Nigeria and a benzene-contaminated aquifer in Zeitz (Germany). The Niger Delta is one of the world’s most damaged ecosystem mainly due to hydrocarbon exploration accidents. The natural attenuation potential of Niger Delta subsurface sediment for anaerobic hydrocarbon degradation was investigated using benzene as a model compound under iron-reducing, sulfate-reducing, and methanogenic conditions. Benzene was slowly mineralized under iron-reducing conditions using Fe(III) chelated with nitrilotriacetic acid, or poorly crystalline Fe(III) oxyhydroxides as electron acceptors, analyzed by measurement of 13CO2 produced from added 13C-labelled benzene. The highest mineralization rates were observed in microcosms amended with Fe(III) oxyhydroxides while microcosms amended with Fe(III) nitrilotriacetic acid produced methane. Abundant phylotypes were affiliated to Betaproteobacteriales, Ignavibacteriales, Desulfuromonadales, and Methanosarcinales of the genera Methanosarcina and Methanothrix, illustrating that the enriched benzene mineralizing communities were diverse and may contain more than a single benzene degrader. The study underpins the importance of microbial ecosystem services in contaminant degradation as a sustainable environmental means of mitigating harmful chemicals. Benzene degradation pathways in a benzene-mineralizing, nitrate-reducing enrichment culture from Zeitz was investigated. Benzene mineralization was dependent on the presence of nitrate and correlated to enrichment of a Peptococcaceae phylotype only distantly related to known anaerobic benzene degraders of this family. Its relative abundance decreased after benzene mineralization had terminated, while other abundant taxa - Ignavibacteriaceae, Rhodanobacteraceae and Brocadiaceae - slightly increased. Generally, the microbial community remained diverse despite amendment of benzene as single organic carbon source, suggesting complex trophic interactions between different functional groups. A subunit of the putative anaerobic benzene carboxylase (AbcA) previously detected in Peptococcaceae was identified by metaproteomic analysis suggesting that benzene was activated by carboxylation. Detection of proteins involved in anaerobic ammonium oxidation (Anammox) indicates that benzene mineralization was accompanied by Anammox, facilitated by nitrite accumulation and the presence of ammonium in the growth medium. The results suggest that benzene was activated by carboxylation and further assimilated by a novel Peptococcaceae phylotype and confirm the hypothesis that Peptococcaceae are important anaerobic benzene degraders. Only a few benzene mineralizing anaerobes have been isolated to date. In an attempt using classical isolation techniques to isolate benzene-mineralizing pure cultures from a benzene-mineralizing nitrate-reducing microbial community, two consortia were gained under nitrate-reducing conditions spiked separately with acetate and benzene as sole sources of carbon and energy with media containing ammonium or without ammonium. Both consortia – Bz4 (with ammonium) and Bz7 (without ammonium) - mineralized 13C-labelled acetate under anoxic conditions at 3.3 and 2.7 µM day-1, respectively, revealed by analysis of evolved 13CO2. However, only Bz4 mineralized 13C-labelled benzene (0.298 µM benzene mineralized day-1) generated up to 960.2 ± 0.3 ‰ ẟ13C-CO2 during 184 days while producing only slight amounts of nitrite (4.60 ± 0.004 µM). By 16S rRNA gene amplicon sequencing was determined that the isolated cultures were not pure cultures but still contained several different phylotypes. The gained Bz4 consortium that mineralized benzene under anoxic conditions can be further purified and explored for their metabolic potentials.:Acknowledgments ………………………………………………………................. ii Table of Contents …………………………………………………………………… iii Dissertation Summary ……………………………………………………………… vi Dissertation Zusammenfassung …………………………………………………… viii List of Tables ………………………………………………………………………… x List of Figures ……………………………………………………………………….. xi List of Appendices ………………………………………………………………….. xiii Abbreviations .………………………………………………………….................... xv Chapter 1: Introduction and Research Objectives ……………………………… 1 1.1 Introduction ……………………………………………………………… 2 1.2 Aims and Objectives ………………………………………………….... 4 Chapter 2: Anaerobic Benzene Degradation by Microbial Communities and Pure Cultures …… 6 2.1 Anaerobic benzene degradation – a brief introduction ...…………… 7 2.2 Anaerobic benzene degradation under different electron acceptor conditions … 9 2.2.1 Benzene degradation under methanogenic conditions ……… 9 2.2.2 Benzene degradation under sulfate-reducing conditions …… 14 2.2.3 Benzene degradation under nitrate-reducing conditions …… 20 2.2.4 Benzene degradation under iron-reducing conditions ……… 25 2.3 Anaerobic benzene degradation by pure cultures ………………… 26 2.4 Anaerobic benzene activation mechanisms and associated genes……………… 28 2.4.1 Hydroxylation of benzene …………………………………….… 30 2.4.2 Methylation of benzene ………………………………..………… 34 2.4.3 Carboxylation of benzene ……………………………....………. 34 2.5 Benzoyl-CoA central metabolic pathways ………………………… 37 2.6 Syntrophic interactions in benzene-degrading communities ……… 42 2.7 Prospects for the future ……..……………………………………………… 43 Chapter 3: Anaerobic Benzene Mineralization by Natural Microbial Communities from Niger Delta …………………………………………………………………........... 44 3.1 Introduction …………………………………………………………..... 45 3.2 Materials and Methods ……………………………………………….. 46 3.2.1 Chemicals ………………………………………………………... 46 3.2.2 Site description and sampling procedure ……………………… 47 3.2.3 Setup of enrichment cultures …………………………………… 47 3.2.4 Chemical and microscopic analysis …………………………… 48 3.2.5 Microbial community analysis …………………………………… 49 3.3 Results and Discussion …………………………………………………. 50 3.3.1 Mineralization of benzene at different electron-acceptor conditions …………... 50 3.3.2 Microbial community structure at different electron-acceptor conditions ……... 53 3.4 Conclusion ………………………………………….…………………… 61 Chapter 4: Structure and Functional Capacity of a Benzene-mineralizing, and Nitrate-reducing Microbial Community ……………………………………………......... 62 4.1 Introduction …………………………………………………………..... 63 4.2 Materials and methods ……………………………………………..... 64 4.2.1 Chemicals ………………………………………………………... 64 4.2.2 Microcosm setup and sampling ………………………………… 64 4.2.3 Chemical and physiochemical analyses ……………………… 66 4.2.4 Amplicon and metagenome sequencing ……………………… 67 4.2.5 Protein mass spectrometry ……………………………………. 67 4.2.6 Metaproteome analysis ………………………………………… 68 4.2.7 Cloning and sequencing of putative nitric oxide dismutase (nod) genes ………. 68 4.2.8 Data availability …………………………………………………… 69 4.3 Results …………………………………………………………………………. 70 4.3.1 Benzene mineralization under nitrate-reducing conditions …… 70 4.3.2 Changes in microbial diversity during benzene mineralization . 71 4.3.3 Metaproteome composition ……………………………………… 74 4.3.4 Presence of putative nitric oxide dismutase genes (nod) ……. 76 4.4 Discussion ……………………………………………………………... 76 4.4.1 Putative pathways for nitrate reduction coupled with benzene mineralization … 76 4.4.2 Elucidation of the benzene activation step ……………………… 78 4.4.3 Benzoyl-CoA central pathway ……………………………………. 79 4.4.4 Peptococcacea as putative primary benzene degraders ……… 80 4.4.5 Metabolic function of Anammox bacteria in the community …… 81 Chapter 5: Consortia Dominated by Gammaproteobacteria Isolated from a Denitrifying Benzene-degrading Enrichment Culture and their Capacity to Mineralize Benzene...................... 83 5.1 Introduction ……………………………………………………………… 84 5.2 Materials and methods ………………………………………………… 85 5.2.1 Chemicals ………………………………………………………… 85 5.2.2 Isolation procedure …………………………………………………… 85 5.2.3 Mineralization and nitrite analyses ……………………..……… 86 5.2.4 Genomic DNA extraction and 16S rRNA gene sequencing … 87 5.3 Results …………………………………………………………………. 87 5.4 Discussions …………………………………………………………… 91 Chapter 6: General Conclusions and Outlook …..……………………………… 95 6.1 Conclusions and novelty of the research …………………………… 96 6.2 Ignavibacteriales as benzene degrading consortia under iron-reducing conditions 96 6.3 Insights into benzene activation via carboxylation by Peptococcaceae … 97 6.4 Unraveling growth of Anammox bacteria during benzene mineralization … 98 6.5 Study significance ……………………………………………………… 99 6.6 General outlook ………………………………………………………… 100 References ………………………………………………………………………… 101 Appendices ………………………………………………………………………… 120 Contributions of other Authors …………………………………………………… 160