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Biodegradation of Aromatic Hydrocarbons in Oxygen-Limited Groundwater

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Biodegradation of Aromatic Hydrocarbons in Oxygen-Limited Groundwater TECHNISCHE UNIVERSITÄT MÜNCHEN Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl für Mikrobologie Biodegradation of aromatic hydrocarbons in oxygen-limited groundwater Lauren Bradford Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzende(r): Hon.-Prof. Dr. Michael Schloter Prüfer der Dissertation: 1. Prof. Dr. Tillmann Lüders 2. Prof. Dr. Wolfgang Liebl Die Dissertation wurde am 27.01.2020 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 08.06.2020 angenommen. 1 Abstract Hydrocarbon contamination of groundwater is a global problem, negatively impacting human health and ecosystem functioning. One of the main ways to mitigate this issue is bioremediation, the use of microorganisms to sequester or break down pollutants. Bacteria capable of metabolizing hydrocarbons are widespread in the environment, including groundwater. They rely on a variety of metabolic and respiratory strategies, distinguished primarily by the availability of oxygen. Oxygen can act as a terminal electron acceptor (TEA) during respiration, but also as a metabolic co-substrate during the breakdown of stable compounds like aromatic hydrocarbons. Anaerobic degradation relies on alternative TEAs and mechanisms of aromatic destabilization. Strictly aerobic and anaerobic hydrocarbon degradation have been well studied in recent decades, but hot spots of degradation occur at the fringes of contaminant plumes, where mixing of oxygen and other TEAs create dynamic, TEA- limited conditions. Microaerobes and other microbes with specialized strategies that could offer advantages in such conditions have been poorly investigated, despite their probable contribution to pollutant degradation in environmentally-relevant situations. In this thesis, I focus on two such strategies: oxygenesis via nitric oxide dismutation, and oxygen-activation, nitrate-respiration whereby an organism respires an alternative TEA and reserves limited oxygen for aromatic destabilization. To unambiguously connect the identities and functional expression patterns of these degraders, I improved upon the method of RNA stable isotope probing (RNA-SIP) by developing semi-quantitative total-RNA-SIP. In the first part of this doctoral work, I developed PCR assays for the putative nitric oxide dismutase gene (nod) and probed for its abundance in contaminated aquifers and other aquatic systems. Dismutation of nitric oxide into N2 and O2 has been linked to oxygen-dependent metabolism of methane and alkanes in anoxic enrichment cultures. The nod gene is believed to code for the dismutase enzyme catalyzing this process. We hypothesize that it could also provide oxygen for aromatic hydrocarbon degradation, even in anoxic and low-oxygen conditions. Following the design and testing of nod-targeted degenerate primers, I found evidence of nod genes in contaminated aquifers in Siklós, Hungary and Düsseldorf, Germany, as well as in experimental wastewater treatment systems. This shows that oxygenesis may be more widespread and relevant to bioremediation and the global nitrogen cycle than previously thought, although the respective organisms still resist cultivation and more detailed characterisation. 2 I followed this with the development and a proof-of-principle experiment for total-RNA-SIP. RNA-SIP is a well-established technique used to identify microbes in a complex community actually metabolizing a substrate of interest by means of 13C-labelling. Most RNA-SIP studies to date have focused solely on identification via sequencing of PCR-amplified “heavy” small subunit (SSU) rRNA. By instead combining SIP with sequencing of total heavy RNA, I was able to analyze the identity (via SSU rRNA) and function (via mRNA) of degraders from SIP gradient fractions. In combination with enrichment factor calculations, this is a powerful tool for identifying not only process-relevant microbes, but also the biochemical pathways used. I performed a proof-of-principle experiment on microcosms containing sediment from a hydrocarbon-contaminated microoxic aquifer and provided with 13C-labelled toluene. Members of the Rhodocyclaceae family were overwhelmingly the most abundant and highly labelled members of the sediment community, consistent with previous work on this site. The functional transcript data showed interesting patterns that would have been missed using other methods, such as the importance of cell motility, prevalence of catechol-2,3-dioxygenase over catechol-1,2-dioxygenase for aromatic ring opening, and use of phenol hydroxylase for aromatic activation. Moreover, I applied this new method to a series of microcosms designed to unravel the catabolic and respiratory adaptations of toluene degraders to simultaneous but limited availability of oxygen and nitrate as TEAs. I successfully resolved the involvement of distinct microaerobic vs. aerobic and anaerobic populations in pollutant degradation. In particular, differential labelling of catabolic and respiratory transcripts including phenol-hydroxylase (dmpN) revealed the adaptation of degraders within the Commamonadaceae under microoxic conditions with nitrate amendment. This demonstrates, for the first time, the previously hypothesized oxygen-activating, nitrate-respiring degradation strategy for a complex aquifer microbiota, explaining how degraders can increase catabolic efficiency under limited electron acceptor supply. Overall, this thesis provides important insights into the largely unrecognized contributions to hydrocarbon degradation made by microbes employing unconventional metabolic and respiratory adaptations. This has implications for future bioremediation efforts, for example by encouraging treatment with low oxygen concentrations combined with nitrate, rather than more expensive oxygen sparging. It also represents a novel and valuable method for overcoming the notorious limitations of environmental non-target transcriptomics, by unambiguously linking substrate metabolism to the identity and gene expression of process-relevant microbes. 3 Zusammenfassung Die Verunreinigung von Grundwasser mit Kohlenwasserstoffen ist ein globales Problem, das sich negativ auf die menschliche Gesundheit und auf Ökosysteme auswirkt. Ein grundlegender Ansatz zur Lösung dieses Problems ist die Bioremediation, d.h. der Abbau von Schadstoffen durch Mikroorganismen. Bakterien, die in der Lage sind, Kohlenwasserstoffe zu metabolisieren, sind in der Umwelt, einschließlich des Grundwassers, weit verbreitet. Sie sind auf eine Vielzahl von Stoffwechsel- und Atmungsaktivitäten spezialisiert, die sich vor allem durch die Verwendung von Sauerstoff unterscheiden. Sauerstoff kann als terminaler Elektronenakzeptor (TEA) bei der Atmung, aber auch als metabolisches Co-Substrat im Abbau von chemisch stabilen Schadstoffen wie z.B. aromatischen Kohlenwasserstoffen wirken. Der anaerobe Abbau basiert auf alternativen Atmungsprozessen und Aktivierungsmechanismen zur Destabilisierung der Aromaten. Der sowohl rein aerobe oder anaerobe Abbau von Kohlenwasserstoffen wurde in den letzten Jahrzehnten bereits intensiv untersucht. Am Rand von Schadstofffahnen gibt es in natürlichen Grundwasserleitern allerdings wichtige «Hot- Spots» des Abbaus, in denen die Durchmischung von Sauerstoff und anderen TEAs dynamische und teils auch limitierende Bedingungen für verschiedene Atmungen schafft. Mikroaerobier und andere Mikroben mit spezifischen Strategien, die unter solchen Bedingungen physiologische Vorteile bieten können, sind trotz ihres möglichen wichtigen Beitrags zum Schadstoffabbau in umweltrelevanten Situationen bisher nur unzureichend untersucht worden. In dieser Dissertation konzentriere ich mich auf zwei Strategien: Oxygenese durch Stickoxid-Disproportionierung und Sauerstoff-Aktivierung bei Nitrat- Atmung, in welcher ein Organismus Nitrat veratmet, während limitierender Sauerstoff nur zur Aktivierung aromatischer Substrate verwendet wird. Um die Identitäten und prozessrelevante Genexpression solcher Abbauer gezielt zu untersuchen, habe ich die Methodik des RNA «Stable Isotope Probings» (RNA-SIP) einen neuen, semi-quantitativen Ansatzes für Transkriptom-SIP wesentlich erweitert. Im ersten Teil dieser Doktorarbeit habe ich PCR-Assays für das Gen der postulierten Stickoxid- Dismutase (nod) entwickelt, um dessen Häufigkeit in kontaminierten Aquiferen und anderen aquatischen Systemen zu untersuchen. Die Disproportionierung von Stickstoffmonoxid zu N2 und O2 wurde bereits mit der Oxidation von Methan und Alkanen in anoxischen Anreicherungskulturen in Verbindung gebracht. Es wird angenommen, dass das Nod-Gen für ein Dismutase-Enzym kodiert, welches diesen Prozess katalysiert. Wir vermuten, dass es auch Sauerstoff für den Abbau von aromatischen Kohlenwasserstoffen bereitstellen könnte, sowohl 4 unter anoxischen oder sauerstoffarmen Bedingungen. Nach der Entwicklung geeigneter PCR Primer, fand ich Hinweise auf eine unerwartete Diversität und Abundanz von Nod-Genen in kontaminierten Grundwasserleitern und in Kläranlagen. Dies zeigt, dass die Oxygenese möglicherweise weiter verbreitet und relevanter ist, als bisher angenommen, sowohl in der Bioremediation als auch im globalen Stickstoffkreislauf. Die entsprechenden Mikroorganismen entziehen sich jedoch weiterhin einer Kultivierung
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