Anaerobic digestion technologies have been utilized in Vietnam for more than 30 years with thousands of domestic small scale plants, mostly for agricultural and livestock wastes. For municipal solid waste (MSW) the development of biogas plants is far below the current high waste generation rates. The aim of this paper is to present the results of a feasibility assessment of implementing AD to treat the organic fraction of municipal solid waste (OFMSW) in Vietnam. For this purpose, an environmental analysis was performed comparing three treatment scenarios: two hypothetical AD technologies (a wet and a dry fermentation system) and the existing industrial composting facility at Nam Binh Duong Waste Treatment Complex in South Vietnam. This study sought for the technology to recover the most possible resources and energy from the OFMSW, and reduce greenhouse gas (GHG) emissions. The results were then combined with a policy review to support a holistic approach on the feasibility of these technologies in Vietnam. The outcome indicates that by implementing the dry AD system, up to 16.7 GWh of power and 14.4 GWh of heat energy can be generated annually and it can potentially save up to 5,400 Mg of CO2 equivalent per year, presenting the highest resource/energy benefits. The performance of the wet system and composting facility present some advantages particularly if there is a previous segregation of the organic material from the rest of the household wastes. Moreover, current reforms in Vietnam demonstrate the government’s interest in AD technologies, translated into the development of fiscal and financial revenues which incentivize participation from the public and private sector. Finally, these technologies are constantly under development and have the potential to be further improved, which gives hopes that waste treatment systems can be optimized to meet the waste and energy challenges of the future generations. / Phương pháp lên men kị khí đã được áp dụng tại Việt Nam từ hơn 30 năm nay với hàng nghìn các công trình nhỏ chủ yếu xử lý chất thải nông nghiệp và chăn nuôi. Sự phát triển hiện nay của các nhà máy sinh khí biogas còn quá ít cho xử lý lượng phát thải cao rác thải đô thị. Bài báo này trình bày các kết quả việc đánh giá tính khả thi khi áp dụng công nghệ lên men kị khí xử lý phần hữu cơ của chất thải rắn đô thị tại Việt Nam. Với mục đích này, phân tích môi trường được thực hiện để so sánh ba kịch bản xử lý: hai công nghệ lên men kị khí giả định (một cho công nghệ lên men ướt và một cho công nghệ lên men khô) và nhà máy hiện hữu lên men hiếu khí làm phân bón compost tại khu liên hợp xử lý chất thải Nam Bình Dương ở miền Nam Việt Nam. Nghiên cứu này tìm kiếm giải pháp công nghệ để thu hồi nhiều nhất có thể các tài nguyên và năng lượng từ rác thải đô thị và và giảm phát thải khí nhà kính. Các kết quả sau đó được kết hợp với đánh giá chính sách để hỗ trợ cách tiếp cận toàn diện về tính khả thi của các công nghệ này vào Việt Nam. Kết quả cho thấy áp dụng công nghệ lên men kị khí khô có thể tạo ra đến 16,7 GWh điện năng và 14,4 GWh nhiệt năng hàng năm và có khả năng làm giảm đến 8,000 Mg CO2 tương đương mỗi năm, thể hiện lợi ích cao nhất giữa tài nguyên và năng lượng. Hiệu suất của hệ thống lên men kị khí ướt và lên men hiếu khí thể hiện một số lợi thế đặc biệt khi nguyên liệu hữu cơ cho quá trình lên men được tiền phân loại ra khỏi hỗn hợp rác sinh hoạt. Hơn nữa, các đổi mới hiện nay ở Việt Nam thể hiện sự quan tâm của Chính phủ đến các công nghệ lên men kị khí, thể hiện qua sự tăng trưởng tài chính và doanh thu để khuyến khích sự tham gia của khu vực công và tư nhân. Chắc chắn rằng các công nghệ sẽ liên tục được phát triển và có khả năng được cải tiến tốt hơn, mang đến cho chúng ta những hy vọng rằng các hệ thống xử lý chất thải được tối ưu hóa để đáp ứng được các thách thức về chất thải và năng lượng của các thế hệ tương lai.

Die Acetyl-CoA Synthase (ACS) beinhaltet ein einzigartiges Metallcluster (Cluster A) in seinem aktiven Zentrum, welches wichtig für ein autotrophes Wachstum von Bakterien und Archaeen ist, die den reduktiven Acetyl-CoA-Weg nutzen. Der letzte Schritt dieses Syntheseweges wird von der ACS am proximal zum [4Fe-4S] Cluster liegenden Ni ion katalysiert (Nip). In meiner Arbeit wurde die ACS von Carboxydothermus hydrogenoformans (ACSCh) auf seine Ligandenbindung untersucht und in verschiedenen Konformationen kristallisiert. Zuerst wurde die ACSCh in einer neuen Konformation (ACSCh-closed) kristallisiert und deren Struktur mit einer Auflösung von 2.1 Å bestimmt. Diese wies einen geringeren Kontakt zur Solventumgebung auf als die vorher bekannte Struktur der ACSCh (ACSCh-open, PDB-ID: 1RU3). Das Cluster A der ACSCh-closed unterscheidet sich von der ACSCh-open in der Koordination des Nip, welches verzerrt tetraedrisch koordiniert in ACSCh-closed vorliegt und quadratisch planar in ACSCh-open. Eine Analyse des Modells wies einen molekularen Tunnel auf, der nur in ACSCh-closed vorhanden ist, welcher als CO-Kanal zur Substratversorgung dienen könnte. Zweitens wurde die Kristallstruktur einer CO gebunden ACSCh-closed mit einer Auflösung von 2.0 Å gelöst. Darin wurde eine Elektronendichte am Nip identifiziert, die als CO überzeugend modelliert werden konnte. CO bindet am ebenfalls verzerrt tetraedrisch koordinierten Nip. Die Konformationsänderung von ACSCh-open zu ACSCh-closed scheint der entscheidende Schritt zu sein, um CO zur Bindungsstelle zu leiten. Die dritte Struktur zeigt eine CoA gebundene ACSCh Struktur mit einer Auflösung von 2.3 Å. CoA bindet am Nip, wobei Nip quadratisch planar koordiniert wird. Die Gegenwart von CoA wurde mit Berechnungen verschiedener Elektronendichte-Karten für CoA validiert. Die Bindung von CO und CoA an ACSCh wurde zudem mittels isothermaler Titrationskalorimetrie weiter charakterisiert. Dabei bindet CoA enthalpisch getrieben mit einem KD von 3.1 µM und CO entropisch getrieben mit einem KD von 9.4 µM an ACSCh. / The acetyl-CoA synthase (ACS) harbors a unique metal cluster (cluster A) in its active site, which is important for bacteria and archaea to survive in autotrophic growth using the reductive acetyl-CoA. The last step of this pathway is catalyzed by ACS at the Nip, the Ni ion proximal to the [4Fe-4S] cluster. In my study, the monomeric ACS of Carboxydothermus hydrogenoformans (ACSCh) was studied in its ligand binding and crystallized in different forms. At first, an ACSCh structure was solved in a new conformation at dmin of 2.1 Å and less solvent exposed (called ACSCh-closed) than the known structure of ACSCh (called ACSCh-open, PDB-ID:1RU3). The cluster A of ACSCh-closed differs to that of ACSCh-open in the coordination of the proximal Ni, which is distorted tetrahedrally coordinated in ACSCh-closed and square planar coordinated in ACSCh-open. Analysis of the model revealed a molecular tunnel that is only present in ACSCh-closed, which might act as CO channel for substrate delivery. Secondly, a CO-bound ACSCh-closed crystal was obtained and solved at a resolution of 2.0 Å. An electron density fitting with a diatomic ligand at the Nip site was clearly identified and modeled as a CO molecule. CO binds at the Nip site completing the tetrahedral coordination geometry of Nip. The conformational switch between open and closed is responsible for CO migration and binding to the catalytic site. A third crystal structure depicts a CoA bound ACSCh structure at 2.3 Å resolution. CoA binds at the Nip which shows a square planar geometry. This was further supported by calculating different electron density maps for CoA. The binding of CO and CoA to ACSCh has been characterized by isothermal calorimetry experiments. While CoA binding is enthalpically driven with a KD of 3.1 µM, CO binds to ACSCh by entropic contribution with a KD of 9.4 µM.

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

The characteristics of anaerobic batch co-digestion of water hyacinth (WH) with pig manure (PM) under seven mixing ratio 100%WH; 80%WH : 20%PM; 60%WH : 40%PM; 50%WH : 50%PM; 40%WH : 60%PM; 20%WH : 80%PM and 100%PM were investigated, each treatment was conducted in five replications with daily loading rate at 1 gVS.L-1.day-1. During the anaerobic digestion process of 60 days, maximum biogas production occurred in two periods, the first stage from 12- 22 days and second stage from 30 - 35 days. The maximum daily biogas productions from each stage were 17.2 L.day-1 and 15.1 L.day-1, respectively. The cumulative biogas production varied between 60 L (100%PM) and 360 L (60%WH : 40%PM). The results showed that the biogas yields of co-digestion 40- 80%WH were higher from 34.6 to 56.1% in comparison with 100%PM and from 109 to 143% in comparison with 100%WH. When mixing with WH, treatments were received more methane and the methane contents were higher than 45% (v/v) that good for energy using purposes. / Nghiên cứu được thực hiện nhằm khảo sát khả năng gia tăng lượng khí sinh học khi tiến hành đồng phân hủy yếm khí lục bình (WH) và phân heo (PM) ở các tỉ lệ phối trộn khác nhau gồm 100%WH; 80%WH : 20%PM; 60%WH : 40%PM; 50%WH : 50%PM; 40%WH : 60%PM; 20%WH : 80%PM và 100%PM. Các nghiệm thức được nạp lượng nguyên liệu là 1 gVS.L-1.ngày-1 và bố trí lặp lại 5 lần. Theo dõi quá trình phân hủy của các nghiệm thức trong 60 ngày ghi nhận có 2 khoảng thời gian lượng khí sản sinh nhiều nhất - giai đoạn 1 từ ngày 12 đến 22, giai đoạn 2 từ ngày 30 đến 35. Lượng khí sản sinh cao nhất tương ứng trong mỗi giai đoạn là 17.2 L.ngày-1 và 15.1 L.ngày-1. Lượng khí tích lũy trong suốt thời gian thí nghiệm ghi nhận thấp nhất ở nghiệm thức 100%PM đạt 60 L, và cao nhất ở nghiệm thức 60%WH : 40%PM đạt 360 L. Năng suất khí sinh ra của các nghiệm thức phối trộn lục bình từ 40 đến 80% cao hơn từ 34,6 đến 56,1% so với nghiệm thức 100%PM và cao hơn từ 109% đến 143% so với nghiệm thức 100%WH. Hàm lượng mê-tan sinh ra từ các nghiệm thức có phối trộn lục bình ổn định trong khoảng > 45% đảm bảo nhiệt lượng cho nhu cầu sử dụng năng lượng.

Anaerobic digestion technologies have been utilized in Vietnam for more than 30 years with thousands of domestic small scale plants, mostly for agricultural and livestock wastes. For municipal solid waste (MSW) the development of biogas plants is far below the current high waste generation rates. The aim of this paper is to present the results of a feasibility assessment of implementing AD to treat the organic fraction of municipal solid waste (OFMSW) in Vietnam. For this purpose, an environmental analysis was performed comparing three treatment scenarios: two hypothetical AD technologies (a wet and a dry fermentation system) and the existing industrial composting facility at Nam Binh Duong Waste Treatment Complex in South Vietnam. This study sought for the technology to recover the most possible resources and energy from the OFMSW, and reduce greenhouse gas (GHG) emissions. The results were then combined with a policy review to support a holistic approach on the feasibility of these technologies in Vietnam. The outcome indicates that by implementing the dry AD system, up to 16.7 GWh of power and 14.4 GWh of heat energy can be generated annually and it can potentially save up to 5,400 Mg of CO2 equivalent per year, presenting the highest resource/energy benefits. The performance of the wet system and composting facility present some advantages particularly if there is a previous segregation of the organic material from the rest of the household wastes. Moreover, current reforms in Vietnam demonstrate the government’s interest in AD technologies, translated into the development of fiscal and financial revenues which incentivize participation from the public and private sector. Finally, these technologies are constantly under development and have the potential to be further improved, which gives hopes that waste treatment systems can be optimized to meet the waste and energy challenges of the future generations. / Phương pháp lên men kị khí đã được áp dụng tại Việt Nam từ hơn 30 năm nay với hàng nghìn các công trình nhỏ chủ yếu xử lý chất thải nông nghiệp và chăn nuôi. Sự phát triển hiện nay của các nhà máy sinh khí biogas còn quá ít cho xử lý lượng phát thải cao rác thải đô thị. Bài báo này trình bày các kết quả việc đánh giá tính khả thi khi áp dụng công nghệ lên men kị khí xử lý phần hữu cơ của chất thải rắn đô thị tại Việt Nam. Với mục đích này, phân tích môi trường được thực hiện để so sánh ba kịch bản xử lý: hai công nghệ lên men kị khí giả định (một cho công nghệ lên men ướt và một cho công nghệ lên men khô) và nhà máy hiện hữu lên men hiếu khí làm phân bón compost tại khu liên hợp xử lý chất thải Nam Bình Dương ở miền Nam Việt Nam. Nghiên cứu này tìm kiếm giải pháp công nghệ để thu hồi nhiều nhất có thể các tài nguyên và năng lượng từ rác thải đô thị và và giảm phát thải khí nhà kính. Các kết quả sau đó được kết hợp với đánh giá chính sách để hỗ trợ cách tiếp cận toàn diện về tính khả thi của các công nghệ này vào Việt Nam. Kết quả cho thấy áp dụng công nghệ lên men kị khí khô có thể tạo ra đến 16,7 GWh điện năng và 14,4 GWh nhiệt năng hàng năm và có khả năng làm giảm đến 8,000 Mg CO2 tương đương mỗi năm, thể hiện lợi ích cao nhất giữa tài nguyên và năng lượng. Hiệu suất của hệ thống lên men kị khí ướt và lên men hiếu khí thể hiện một số lợi thế đặc biệt khi nguyên liệu hữu cơ cho quá trình lên men được tiền phân loại ra khỏi hỗn hợp rác sinh hoạt. Hơn nữa, các đổi mới hiện nay ở Việt Nam thể hiện sự quan tâm của Chính phủ đến các công nghệ lên men kị khí, thể hiện qua sự tăng trưởng tài chính và doanh thu để khuyến khích sự tham gia của khu vực công và tư nhân. Chắc chắn rằng các công nghệ sẽ liên tục được phát triển và có khả năng được cải tiến tốt hơn, mang đến cho chúng ta những hy vọng rằng các hệ thống xử lý chất thải được tối ưu hóa để đáp ứng được các thách thức về chất thải và năng lượng của các thế hệ tương lai.

Chlorbenzole sind aufgrund ihrer weiten Verbreitung in Industrie und Landwirtschaft und ihrer geringen chemischen Reaktivität sowie guten Fettlöslichkeit ubiquitäre Umweltkontaminanten, die sich in der Nahrungskette anreichern. Eine natürliche, mikrobielle Dechlorierung dieser Verbindungen ist von besonderem Interesse, da die Toxizität chlorierter Benzole mit der Anzahl der Chlorsubstituenten steigt. Im Gegensatz zu Mono- und Dichlorbenzol, die durch aerobe Laborreinkulturen dechlorierbar sind, werden höher chlorierte Benzole bevorzugt unter anaeroben Bedingungen in Mischkulturen mit unbekannter Spezieszusammensetzung dechloriert. Bioreaktoren, in denen solche undefinierten Mischkulturen kontinuierlich kultiviert werden, sind eine vielversprechende Technik bei der Behandlung Chlorbenzol-kontaminierter Abwässer. Aufgrund der unbekannten Zusammensetzung der Population muß die mikrobielle Aktivität jedoch als "black box" betrachtet werden, was eine direkte Steuerung und Optimierung solcher Bioreaktoren erschwert. Zur Bestimmung der mikrobiellen Diversität einer in ihrer Zusammensetzung unbekannten, Trichlorbenzol(TCB)-dechlorierenden Bioreaktorkultur wurde aufgrund der bekannten Limitierungen kulturabhängiger Methoden die kulturunabhängige, vergleichende Sequenzanalyse direkt amplifizierter und klonierter 16S-rDNA gewählt. Die durch eine neuentwickelte Hybridisierungsmethode wesentlich vereinfachte Analyse der 16S-rDNA-Genbibliotheken erlaubte eine phylogenetische Klassifizierung der in der TCB-dechlorierenden Mischkultur abundanten Mikroorganismen (Bakterien und Archaea) und zeigte das Auftreten von 51 bakteriellen 16S-rDNA-Klonfamilien, mit einem weiten phylogenetischen Spektrum und teilweise enge Verwandtschaften zu anaerob dechlorierenden Dehalobacter spp. oder zu unkultivierten Bakterien eines vergleichbaren Biotops. Dieser Diversität stand eine dominierende Methanosaeta-ähnliche Klonfamilie innerhalb der Archaea gegenüber. Die aus der phylogenetischen Klassifizierung abgeleiteten metabolischen Eigenschaften einiger Bakterien und Archaea der TCB-dechlorierenden Mischkultur konnten durch gezielte Anreicherung und in-vitro Kultivierung bzw. die kulturunabhängige Sequenzanalyse funktioneller Gene bestätigt werden. Die dargestellten Ergebnisse lassen eine spezifische Zusammensetzung der TCB-dechlorierenden Mischkultur vermuten und geben Hinweise auf Indikatororganismen, die ein Monitoring und eine damit verbundene Effizienzsteigerung der anaeroben TCB-Dechlorierung im Bioreaktor ermöglichen könnten. / Due to their widespread application in industry and agriculture and their chemical stability chlorobenzenes (CB) are ubiquitous pollutants in soil, sediments, and aquifers. Since toxicity of CBs increases with the number of chlorine substituents, microbial dechlorination of CBs is of major interest. In contrast to di- and monochlorobenzenes (DCB and MCB) higher chlorinated benzenes are more resistant to aerobic dechlorination. However, for these compounds reductive dechlorination by different anaerobic microbial communities is well known. Bioreactors inoculated with complex dechlorinating anaerobic microbiota seem to be promising technologies for bioremediation of CB-contaminated aquifers. Several studies showed the efficiency of such bioreactors in treating chloroaromatic contaminated wastewaters. However, due to the unknown species diversity microbial activity had to be treated as a "black box" and direct optimization was hampered. To determine the microbial diversity of an anaerobic consortium in a fluidized bed reactor used for dechlorination of trichlorobenzene (TCB) I employed comparative sequence analysis of 16S rRNA genes after direct PCR-amplification and cloning from community DNA since culture-based methods have shown to be strongly biased. The application of a new hybridization based screening approach for bacterial 16S rDNA clone libraries drastically simplified the analysis and allowed the phylogenetic classification of the abundant bacteria and archaea. A total of 51 bacterial 16S rDNA clone families were found, which showed a wide distribution among the main bacterial phyla. Several bacterial 16S rDNA clone families were closely related to anaerobic, dechlorinating Dehalobacter spp. and to yet-uncultured bacteria of a similar habitat. In contrast to the high bacterial diversity archaeal 16S rDNA clone libraries were clearly dominated by a Methanosaeta concilii-like clone family. Some yet-uncultured bacteria and archaea of the TCB-dechlorinating consortium were sufficiently closely related to studied organsims that reasonable physiological hypotheses could be formulated. These hypotheses were confirmed by either cultivation of the respective organism or by culture-independent sequence analysis of specific functional genes. The results suggest a specific community structure of the TCB-dechlorinating consortium and give evidence for indiator organisms. Moleculargenetic monitoring of these indicator organisms might allow the optimization of the continous TCB-dechlorination in the fluidized bed reactor.

A new technology for biotechnological treatment of mine waters with both high concentrations of heavy metals and sulphate was developed. The technology is based on the technical coupling of microbially mediated hydrolysis, fermentation and microbial sulphate reduction in a self-stabilising process. Electron donor for sulphate reduction is supplied by degradation of a solid substrate (silage). Elimination of metals is primarily achieved by sulphide precipitation within the sulphate reduction zone. The organic compounds are either supplied by elution or by hydrolysis of polymeric compounds which was named active elution. The concept was realised as a two-phase process with (active) elution in the first phase (R1) and sulphate reduction and metal elimination in the second phase (R2). With this process setup the supply of sufficient amounts of electron donor in R1, a stable and effective sulphate reduction yield as the basis of metal elimination in R2 and a stable separation of microbial processes in R1 and R2 was achieved at hydraulic retention times of 69 h in R1 and 40 h in R2. Almost complete elimination of heavy metals was achieved from wastewaters with 0.2 mM Ni2+, Cu2+, Zn2+, Fe2+ and Mn2. A structurised mathematical model describing the two-phase process was developed on the basis of literature values and tested with data from continuous experiments. Microbial processes were significantly influenced in the presence of precipitated heavy metal sulfides. The effect was dependent on both the bound metal (Ni2+ or Fe2+) and the relative distance between sediment and biomass. / Es wurde eine neuartige Technologie zur biotechnologischen Reinigung von schwermetallbelasteten, sulfathaltigen Bergbauwässern entwickelt. Die Technologie basiert auf der technischen Kopplung von mikrobiell vermittelter Hydrolyse, Fermentation und mikrobieller Sulfatreduktion in einem selbststabilisierenden Prozess, wobei aus Abbau eines festen Substanzgemisches (Silage) Elektronendonor zur Sulfatreduktion bereitgestellt wird. Die Schwermetallelimination erfolgt vorrangig durch sulfidische Fällung, die technisch einstufig mit der mikrobiellen Sulfatreduktion realisiert wurde. Die organischen Verbindungen wurden durch Elution bereitgestellt bzw. durch hydrolytischen Abbau von polymeren Verbindungen. Hierfür wurde der Begriff der ?Aktiven Elution? geprägt. Die Konzeption wurde technisch zweistufig umgesetzt. In der ersten Stufe (R1) erfolgt die (Aktive) Elution, in der zweiten Stufe (R2) erfolgen Sulfatreduktion und Schwermetallelimination. Mit der verfahrenstechnischen Umsetzung wurde die Bereitstellung einer ausreichenden Menge an Elektronendonor in R1, eine effektive und stabile Sulfatreduktionsausbeute als Bedingung der Schwermetallelimination in R2 und eine weitgehende Trennung der mikrobiellen Prozesse in R1 und R2 bei Verweilzeiten von 69 h in R1 und 40 h in R2 erreicht. Bei Behandlung von wässrigen Lösungen mit 0,2 mM Ni2+, Cu2+, Zn2+, Fe2+ und Mn2+ konnte eine nahezu vollständige Elimination der Schwermetalle aus der Lösung erreicht werden. Es wurde ein strukturiertes mathematisches Modell für den zweistufigen Prozess auf der Basis von Literaturangaben entwickelt und anhand der kontinuierlichen Laborversuche überprüft. Es wurde ein erheblicher Einfluss schwermetallsulfidischer Präzipitate auf die mikrobiellen Prozesse festgestellt. Dabei wurde dieser Einfluss in Abhängigkeit von der Art der gebundenen Metallionen (Ni2+ oder/und Fe2+) und in Abhängigkeit der relativen räumlichen Anordnung von Sediment und Biomasse festgestellt.

A new technology for biotechnological treatment of mine waters with both high concentrations of heavy metals and sulphate was developed. The technology is based on the technical coupling of microbially mediated hydrolysis, fermentation and microbial sulphate reduction in a self-stabilising process. Electron donor for sulphate reduction is supplied by degradation of a solid substrate (silage). Elimination of metals is primarily achieved by sulphide precipitation within the sulphate reduction zone. The organic compounds are either supplied by elution or by hydrolysis of polymeric compounds which was named active elution. The concept was realised as a two-phase process with (active) elution in the first phase (R1) and sulphate reduction and metal elimination in the second phase (R2). With this process setup the supply of sufficient amounts of electron donor in R1, a stable and effective sulphate reduction yield as the basis of metal elimination in R2 and a stable separation of microbial processes in R1 and R2 was achieved at hydraulic retention times of 69 h in R1 and 40 h in R2. Almost complete elimination of heavy metals was achieved from wastewaters with 0.2 mM Ni2+, Cu2+, Zn2+, Fe2+ and Mn2. A structurised mathematical model describing the two-phase process was developed on the basis of literature values and tested with data from continuous experiments. Microbial processes were significantly influenced in the presence of precipitated heavy metal sulfides. The effect was dependent on both the bound metal (Ni2+ or Fe2+) and the relative distance between sediment and biomass. / Es wurde eine neuartige Technologie zur biotechnologischen Reinigung von schwermetallbelasteten, sulfathaltigen Bergbauwässern entwickelt. Die Technologie basiert auf der technischen Kopplung von mikrobiell vermittelter Hydrolyse, Fermentation und mikrobieller Sulfatreduktion in einem selbststabilisierenden Prozess, wobei aus Abbau eines festen Substanzgemisches (Silage) Elektronendonor zur Sulfatreduktion bereitgestellt wird. Die Schwermetallelimination erfolgt vorrangig durch sulfidische Fällung, die technisch einstufig mit der mikrobiellen Sulfatreduktion realisiert wurde. Die organischen Verbindungen wurden durch Elution bereitgestellt bzw. durch hydrolytischen Abbau von polymeren Verbindungen. Hierfür wurde der Begriff der ?Aktiven Elution? geprägt. Die Konzeption wurde technisch zweistufig umgesetzt. In der ersten Stufe (R1) erfolgt die (Aktive) Elution, in der zweiten Stufe (R2) erfolgen Sulfatreduktion und Schwermetallelimination. Mit der verfahrenstechnischen Umsetzung wurde die Bereitstellung einer ausreichenden Menge an Elektronendonor in R1, eine effektive und stabile Sulfatreduktionsausbeute als Bedingung der Schwermetallelimination in R2 und eine weitgehende Trennung der mikrobiellen Prozesse in R1 und R2 bei Verweilzeiten von 69 h in R1 und 40 h in R2 erreicht. Bei Behandlung von wässrigen Lösungen mit 0,2 mM Ni2+, Cu2+, Zn2+, Fe2+ und Mn2+ konnte eine nahezu vollständige Elimination der Schwermetalle aus der Lösung erreicht werden. Es wurde ein strukturiertes mathematisches Modell für den zweistufigen Prozess auf der Basis von Literaturangaben entwickelt und anhand der kontinuierlichen Laborversuche überprüft. Es wurde ein erheblicher Einfluss schwermetallsulfidischer Präzipitate auf die mikrobiellen Prozesse festgestellt. Dabei wurde dieser Einfluss in Abhängigkeit von der Art der gebundenen Metallionen (Ni2+ oder/und Fe2+) und in Abhängigkeit der relativen räumlichen Anordnung von Sediment und Biomasse festgestellt.

Im Rahmen dieser Arbeit wurden verschiedene zweiphasige Vergärungssysteme hinsichtlich der mikrobiellen Biomasse und Abbauaktivität charakterisiert, mit dem Ziel, eine mikrobiologische Prozessüberwachung zu ermöglichen. Bei der analytischen Begleitung des Biogasprozesses stellte sich heraus, dass viele Biomasse- und Aktivitätsparameter anlagen- bzw. substratspezifische Werte aufweisen und Prozessstörungen bzw. Verfahrensmodifikationen über diese mikrobiologischen Kenngrößen detektiert und verifiziert werden können. Im Normalbetrieb der Vergärung von Maissilage konnte in der Hydrolysestufe eine starke Vermehrung der mikrobiellen Gesamtzellzahl sowie eine Zunahme des Archaea:Bacteria-Verhältnisses verzeichnet werden. Die Aktivitätsprofile von Hydrolasen (unspezifische Esterase, Polysaccharasen und Proteasen) erlaubten eine Visualisierung des Hydrolysefortschritts. Hierbei erwiesen sich die Esterase-, Cellulase- und Xylanaseaktivität als besonders aussagekräftig. Ähnlich ermöglichte dies die Analyse der Atmungsaktivität, die die mikrobielle Abbauaktivität des Eingangs- bzw. das Restgaspotential des Ausgangsmaterials der Hydrolysestufe wiedergibt. Die phylogenetischen Analysen der ersten Prozessstufe zeigten eine klare Dominanz von cellulolytischen Bakterien der Gattung Clostridium (Ø 35%). Der Anteil der hydrogenotrophen Methanogenen lag in den untersuchten Systemen etwa 50% über dem der Acetoklastischen, was darauf schließen lässt, dass der hydrogenotrophe Methanbildungsweg favorisiert wird. Es wurde aber auch eine erhöhte Abundanz der nicht-methanogenen Crenarchaeota festgestellt, deren Rolle im Biogasprozess noch ungeklärt ist. Im Zuge dieser Arbeit wurde weiterhin die Vergärbarkeit von HCH-belasteter Grassilage bzw. ein potentieller mikrobieller β-HCH-Abbau untersucht. Hier konnte gezeigt werden, dass die Aktivität der Mikroorganismen durch die Schadstoffbelastung des Substrates nicht inhibiert war. Darüber hinaus konnte ein Abbau des β-HCH nach der Hydrolyse der Grassilage nachgewiesen werden. Durch das Auftreten von Prozessstörungen bzw. Verfahrensmodifikationen konnten die Auswirkungen auf die untersuchten mikrobiologischen Kenngrößen näher untersucht und u. a. auch statistisch abgesichert verifiziert werden. Korrelationsanalysen verdeutlichten die mikrobiellen bzw. biochemischen Zusammenhänge im System. Besonders interessant sind dabei die signifikanten Korrelationen zwischen der Gesamtzellzahl, der Esteraseaktivität und dem Chemischen Sauerstoffbedarf, die für jede Verfahrensstufe nachgewiesen werden konnten. Diese Analysen zeigten erstmalig, dass die unspezifische Esteraseaktivität als allgemeiner mikrobieller Aktivitätsparameter in verschiedenen Prozessstufen von Biogasanlagen als Indikator zur Prozesseffizienz und -stabilität einsetzbar ist. Daher wurde die Methodik als Schnelltest weiterentwickelt, der auch vor Ort Anwendung finden kann. Dies ermöglicht eine direkte Analyse des Biogassubstrates, der Effizienz der Substratumsetzung sowie die Detektion von Störungen und eine entsprechende Steuerung und Regelung des Prozesses.

Nesse trabalho utilizou-se o reator anaeróbio horizontal de leito fixo (RAHLF) para produção de ácidos graxos voláteis (AGVs). O reator, com volume total de 2,38 litros, foi preenchido com partículas de argila expandida e alimentado com substrato sintético à base de glicose. Com concentração de glicose de 2000 mg/L, sem adição de alcalinizante e com tempo de detenção hidráulica (TDH) de 2 horas, houve produção de ácidos acético, butírico e propiônico. Com a variação do (TDH) de 2 horas para 0,5 hora, em condições idênticas, houve redução da produção do ácido butírico. Na segunda condição testada, em que a concentração da glicose foi 4000 mg/L, a concentração dos três ácidos aumentou, além de ser produzido o ácido capróico. Nas três últimas experimentações, com a glicose a 2000 mg/L e alcalinizada com bicarbonato de sódio, nas concentrações de 0,1000 e 2000 mg/L e TDH em 0,5 hora, as produções do ácido acético e propiônico aumentaram, enquanto que houve diminuição da produção dos ácidos butírico e capróico. A extração dos AGVs foi realizada em coluna de 320 mL de leito, com resina de troca iônica Dowex Marathon A - tipo aniônica base forte de amina quaternária em gel de divinil benzeno-estireno como matriz. O primeiro sistema extratante testado, foi com a resina condicionada na forma \'CL POT. -\' e a eluição foi feita com solução de \'NA\'\'CL\' em várias concentrações. Os percolados apresentaram alto teor de cloreto, inconveniente para as recuperações dos AGVs ou para o seu descarte no ambiente. O sistema testado com a resina condicionada na forma \'OH POT.-\' e a eluição com solução de \'NA\'\'OH\', em pH 13,5, demonstrou ser viável para a extração e concentração dos AGVs, com os fatores de concentração acima de 25, além de 80% do volume do percolado da fixação ser adequado para a devolução ao ambiente. No último teste o sistema utilizado foi o mesmo, com a alimentação apresentando a relação AVTs : sulfato de 1:1,27 e procurou-se fixar a máxima quantidade de AVTs. A eluição foi com solução de \'NA\'\'OH\' em três diferentes valores de pH: 12, 12,5 e 13. A presença do sulfato, nessa relação, dificultou a fixação dos AVs e, na eluição, competiu com os AVs, resultando em baixos fatores de concentração entre 2 e 3. Um volume de 47% do percolado da fixação e outro, de 29% da eluição, apresentou qualidade para o descarte. Na extração com solvente foram testados dois extratantes, a Alamina 336 (tri-octil/decil amina) e a Alamina 304-1 (tri-n-dodecilamina). O melhor sistema extratante, de solução de AVs impurificada com alto teor de sulfato, foi com a Alamina 336 condicionada com ácido nítrico. Entretanto, o consumo do ácido é alto o suficiente para inviabilizar economicamente a extração. Extração em sistema com baixo teor em sulfato e pH igual a 4,0 demonstrou ser técnicamente viável. Todavia, os valores da DQO residual nos refinados aquosos de todos os sistemas impossibilitam a devolução direta ao ambiente. / This work used the horizontal-flow anaerobic immobilized biomass (HAIB) reactor in order to produce volatile fatty acids (VFAs). The reactor, filled up with expanded clay particles, had a total volume of 2,38 liters and was fed with glucose based synthetic substrate. Production of acetic, butyric, and propionic acids occurred at an influent glucose concentration of 2000 mg/L without any addition of buffer agent and at a hydraulic retention time (HRT) of 2 hours. A reduction in butyric acid production occurred with the decrease of the HRT from 2 to 0,5 hours, with the reactor operating in identical conditions. In a second test condition, where the influent glucose concentration was increased to 4000 mg/L, the production of the three acids increased, beside the production of caproic acid. In the last three experiments, with influent glucose concentration at 2000 mg/L and buffered with sodium bicarbonate at concentrations of 0,1000 e 2000 mg/L and HRT of 0,5 hours, the production of acetic and propionic acids increased, whereas the production of butyric and caproic acids decreased. VFA extraction was accomplished in a 320 mL bed containing the ionic exchange resin Dowex Marathon A - strong base anionic type of quaternary amine in gel of benzene-styrene divinyl as matrix. The first extractant system was tested with the resin conditioned as \'C L POT.-\' and the elution was done with a solution of \'NA\'\'CL\' at different concentrations. The percolates presented high chloride concentration, which is inconvenient for the recovery of VFA or their release to the environment. On the other hand, the system with resin conditioned as \'OH POT.-\' and the elution with a solution of \'NA\'\'OH\' at pH 13,5 showed to be a viable option for the extraction and concentration of VFA with concentration factors above 25. In addition, the 80% of the volume of the fixation percolate is suitable for the devolution into the environment. The same system was used in the last test, however with the feeding at total volatile acids (TVA): sulfate ratios of 1:1,27 trying to fix the maximal amount of TVAs. The elution was done with \'NA\'\'OH\' at three different pH values: 12, 12,5 and 13. The presence of sulfate at this ration hampered the fixation of the volatile acids (VA) and during the elution competed with the VA, resulting in low concentration factors between 2 and 3. The volume of 47% of the fixation percolate and the volume of 29% of the elution presented good quality for discharge. Two extractants were tested in the extraction tests with solvents: Alamine 336 (tri-octyl/decyl amine) and the Alamine 304-1 (tri-n-dodecylamine). The best extractant system for the VA contaminated with high concentrations of sulfate was the Alamine 336 conditioned with nitric acid. The consumption of acid, however, was high enough to make the extraction economically unfeasible. On the other hand, extractions in systems with low sulfate concentrations and pH of 4 showed to be technically feasible, even though the residual chemical oxygen demand (COD) in the aqueous refined of all systems was too high for their direct devolution to the environment.