Widdel, Friedrich
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Widdel, Friedrich
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Widdel, Friedrich
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Item-typ:Veröffentlichung, Mikrobiologische Studien zur anaeroben Oxidation von Methan (AOM)(2003-11-26); ; ; Biogeochemical and molecular biological studies revealed new insights into the anaerobic oxidation of methane (AOM), but physiological aspects remained mostly unclear. Here incubation-experiments on AOM were performed, which gave insights into the physiology of the organisms. With sediment samples from Hydrate Ridge the dependence of sulphate-reduction on methane was shown, as well as the 1:1 stoichiometry. Vertical activity profiles confirmed that higher methane fluxes lead to higher activity. AOM showed a dependence on temperature, Hydrate Ridge samples were psychrophilic while samples from the Black Sea were mesophilic. Elevated methane partial pressure had a strong effect and increased the activity 2 to 5fold. Since AOM is most probably a syntrophic process by a sulphate-reducer and a methane-oxidizer we tried to find the intermediate that is exchanged between them. But non of the discussed and tested ones (H2, acetate, formate, methanol) seems to be the one in question. BES however stopped the AOM activity, indicating, that one of the partners is a methanogen operating in reverse. Molybdate only stopped the activity in Hydrate Ridge samples. In Black Sea samples the activity was only reduced. Antibiotics only active against bacteria did not effect the process of AOM. It was possible to increase the activity of the samples by continuously incubating them under elevated methane partial pressure (1,37MPa) and a regular exchange of the medium to supply the organisms with sulphate and methane and to remove sulphide. In 20 month the activity increased 6,5fold and also the amount of consortia increased significantly. The assumed doubling time of a consortium is 4.7 month. The classical isolation of sulphate-reducing-bacteria and methanogens seperatly lead to a number of strains, non of which however was closely elated to the members in the consortia. Also co-culture experiments did not reveal their ability to oxidize methane.Dissertation162 486 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Photosythesis and sulfur oxidation in microbial mats: Unravelling the role of versatile cyanobacteria in ancient ocean analogues(2015-12-14); ; ; The capability to perform oxygenic photosynthesis likely evolved in a cyanobacterial ancestor, probably in microbial mats. The cyanobacterial photosynthetic repertoire is not limited to oxygenic photosynthesis. In fact some specialized cyanobacteria can switch to using H2S as an electron donor instead of H2O in a process termed anoxygenic photosynthesis. Such photosynthetic versatility of cyanobacteria might have been an important adaptation strategy to sulfidic conditions in ancient microbial mats. Furthermore, cyanobacterial anoxygenic photosynthesis might have contributed to sustaining ocean euxinia in Proterozoic oceans. Therefore, studying the activity of modern day sulfide-adapted cyanobacteria and the competitiveness of oxygenic with anoxygenic photosynthesis has broad implications. The aim of this thesis was to gain insights into the activity of sulfide-adapted cyanobacteria in microbial mats that represent ancient Earth analogues. Overall, this thesis highlights the wide spectrum of adaptations to sulfidic conditions among cyanobacteria. A crucial factor determining success in the environment is the specific effect of the local dynamics of light and H2S on activity. The most successful cyanobacteria ancient Earth analogues are photosynthetically versatile. As exposure to sulphidic conditions is like a red line through the history of cyanobacteria, it seems intuitive that cyanobacterial anoxygenic photosynthesis might be an ancient trait. However, there is currently no robust evidence supporting this hypothesis. This thesis highlights that photosynthetically versatile cyanobacteria might, however, have had an important impact on the ancient biogeochemical cycling. Deeper knowledge concerning the timeline of the emergence of anoxygenic photosynthesis among cyanobacteria has the potential to explain major shifts on the global oxygen budgets and redox state of Earth through history.Dissertation407 581 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Microbiological study of the anaerobic corrosion of iron(2003-06-27); ; ; Anaerobic corrosion causes economically significant damages. The industrial branches that suffer from MIC most severely include the nuclear and fuel electric power-generating sectors as well as the oil industry. Despite of numerous investigations of anaerobic corrosion, the underlying mechanisms are insufficiently understood. In the present study, the anaerobic corrosion of iron was investigated in seawater under conditions of sulfate reduction and methanogenesis. Enrichment cultures of sulfate-reducing bacteria (SRB) and methanogenic archaea using metallic iron (Fe) as the only source of electrons were established with marine sediment samples as inocula. Further aspects that were subsequently investigated were the composition of the enriched microbial community, the key organisms involved in corrosion, and mechanistic aspects of the process at the iron surface.Dissertation387 168 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Die Gene der (1-Methylalkyl)succinat-Synthase im anaeroben n-Alkanabbau des Betaproteobakteriums Stamm HxN1(2012-03-16); ; ; The betaproteobacterial strain HxN1 completely oxidizes the n-alkanes pentane to octane under denitrifying conditions. The activation of n-alkanes is catalyzed by the glycyl radical enzyme (1-methylalkyl)succinate synthase, whose encoding mas genes are organized in an operon in strain HxN1. In this study, a genetic system for strain HxN1 was developed. The deletion of masD, encoding the catalytic subunit of the enzyme, revealed the presence of a second identical mas operon in strain HxN1. The physiological characterization of the mutant confirmed in vivo the anaerobic activation of n-alkanes by (1-methylalkyl)succinate synthase. The phenotype was restored by complementation with the entire mas operon. Regarding regulation, the mas operon was induced by several hydrocarbons and expression was not inhibited in the presence of a carboxylic acid or a sugar as second carbon source. Furthermore, it was attempted to crystallize the (1-methylalkyl)succinate synthase of strain HxN1.Dissertation333 114 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Untersuchungen zum Stoffwechsel des anaeroben Alkanabbaus(2009-12-15); ; ; Strain HxN1, an Azoarcus-like denitrifying bacterium, degrades the saturated hydrocarbon n-hexane under anoxic conditions. Former studies on metabolites formed during the anaerobic degradation of n-alkanes suggested an activation mechanism analogous to that in the anaerobic degradation of toluene. The postulated enzyme (Mas, methylpentylsuccinate-synthase) is expected to belong to the class of glycyl radical enzymes. It was biochemically characterized by an enzymatic study using cell-free extract of strain HxN1. Furthermore, the enzyme was successfully purified under strictly anoxic conditions proofing the predicted function of the enzyme. A fourth by then unknown subunit of the enzyme was identified. Homologues of this subunit could be found in other n-alkane degrading organisms as well. Another part focused on the relationship of different mas-genes. In three strains tentative n-alkane activating genes could be identified strongly related to methylpentylsuccinate-synthase.Dissertation278 105 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Dissimilatory sulfur metabolism coupled to anaerobic oxidation of methane(2011-02-18); ; ; The seafloor and its microbial inhabitants play an important role in the biogeochemical cycling of elements. These environments are generally anoxic but contain high concentrations of sulfate penetrating from the overlying seawater. The main carbon mineralization processes such as the anaerobic oxidation of methane (AOM; Eq. 1) are therefore generally coupled to sulfate reduction. CH4 SO42 → HCO3 HS H2O (Eq. 1) AOM plays a crucial role in both carbon and sulfur cycling. It oxidizes the majority of the methane a potent greenhouse gas diffusing from the seafloor and prevents its escape to the atmosphere. Methane oxidation also returns the carbon trapped in the form of recalcitrant methane back to the carbon cycle as carbon dioxide. The AOM-coupled sulfate reduction consumes a large portion of the downwards sulfate flux and forms sulfide, which diffuses upwards towards the seafloor where it supports free-living sulfide- and sulfur-oxidizers but also gutless worms, clams and mussels that rely for their nutrition on the thiotrophic symbionts. Despite the pronounced effect of AOM on the sediment geochemistry little is known about its biology. The organisms responsible for AOM a consortium of methanotrophic archaea and Deltaproteobacteria have been identified in situ but their slow metabolism complicates growing them in pure cultures and renders the physiological investigations challenging. So far, AOM research has predominantly focused on the C1 metabolism of the methanotrophic archaea. The investigations presented in this thesis address the dissimilatory sulfur metabolism of the organisms involved in AOM and the mechanisms of its coupling to methane oxidation. Chapters 2 and 3 describe the purification and characterization of the three known enzymes involved in dissimilatory sulfate reduction (SR enzymes; ATP sulfurylase, APS reductase, sulfite reductase). The enzymes were purified from a naturally enriched microbial mat using liquid chromatography. The identity of the SR enzymes was confirmed by N-terminal amino acid sequencing and their activity in total cell extracts as well as in individual chromatography fractions was quantified by corresponding enzyme essays. Our aim was to assign these enzymes to a particular organism in the mat sample. For this purpose, polyclonal antibodies against the purified ATP sulfurylase and sulfite reductase were used APS reductase could not be sufficiently purified for antibody generation in situ in the original environmental sample as well as in our other enrichment cultures. This combination of environmental proteomics and immunolocalization allowed us to unambiguously assign the isolated SR enzymes exclusively to the bacterial partner. The archaea did not express detectable amounts of the identified SR enzymes themselves and therefore likely depend on their bacterial partners to perform the sulfate reduction. These results are presented as manuscripts in revision (Manuscript 1) and in preparation (Manuscript 2). The following Chapter 4 introduces experiments that were performed in order to elucidate sulfur transfer and speciation in AOM consortia. We used stable and radioactive sulfur isotopes to follow sulfur exchange between the medium and biomass and on a single cell level among individual cells. Based on our results and thermodynamic consideration we propose a model, in which DSS bacteria reduce sulfate to a zerovalent sulfur compound (probably polysulfide) that might be utilized by ANME as an electron acceptor for methane oxidation. Thus, unexpectedly, ANME participate in the dissimilatory sulfur metabolism coupled to AOM. Our combined data suggest that ANME obtain this compound from the associated bacteria. Such sulfur shuttling between two organisms not only represents a unique mechanism for a syntrophic relationship but also has significant implications for our understanding of sulfur transformations in the AOM zones in marine sediments. These results are presented as a manuscript in preparation (Manuscript 3).Dissertation337 125 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Die dissimilatorische APS-Reduktase -Vorkommen, Phylogenie, Struktur und Anwendung in der funktionellen Genanalyse(2009-03-02); ; ; Within this doctoral thesis, the distribution, phylogeny and structure of the dissimilatory APS reductase (AprBA) of sulfate-reducing and sulfur-oxidizing prokaryotes (SRP and SOP) has been analyzed. An assay for aprA-based functional gene analysis was developed for determination of the SRP and SOP diversity in environmental samples. In addition, the distribution of the sulfate thiol hydrolase (SoxB, a component of the thiosulfate-oxidizing multi-enzyme system Sox) in SOP was investigated.Dissertation436 508 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Biochemie und Physiologie der Sulfatreduktion in der anaeroben Oxidation von Methan(2009-12-11); ; ; Anaerobic oxidation of methane coupled to sulfate reduction (AOM) is the major sink for methane produced in anoxic marine sediments. AOM is catalyzed by consortia of anaerobic methanotrophic archaea (ANME) and deltaproteobacteria closely related to known sulfate-reducing bacteria (SRB). The knowledge about the biochemical reactions involved in AOM is incomplete. The most common view is that the archaea are responsible for methane oxidation, while the deltaproteobacteria are SRB scavenging reducing equivalents and producing sulfide. There is biochemical evidence that ANME catalyze the initial step of methane activation by the enzyme methyl-coenzyme M reductase as a reversal of the last step of methanogenesis. However, the pathway of sulfate reduction coupled to methane oxidation is not resolved yet. This work provides evidence that sulfate reduction with methane-derived reducing equivalents proceeds via the established sulfate reduction pathway in Black Sea microbial mats with high AOM activity. These mats represent a natural enrichment of consortia consisting of ANME-2 and deltaproteobacteria of the Desulfosarcina/Desulfococcus (DSS)-clade. They contained substantial amounts and activities of the key enzymes for sulfate reduction, ATP sulfurylase (Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). Sat as well as the small subunit of Apr and native Dsr were isolated. Their N-terminal amino acid sequences were characteristic of deltaproteobacterial enzymes. Moreover, the retrieval of the Apr-encoding genes revealed their relationship to those in SRB of the DSS clade, sustaining the hypothesis that sulfate reduction with methane-derived reducing equivalents takes place in the deltaproteobacterial cells. Different possibilities the assumed extracellular transfer of reducing equivalents from ANME to the SRB were studied. In accordance to published papers, hydrogen as well as the reduced carbon compounds formate, acetate and methanol could be excluded as transferred intermediates. Moreover, there is no transfer of reducing equivalents via methyl sulfide, which has recently been suggested as intermediate. It neither inhibited AOM nor served as electron donor for sulfate reduction in AOM cultures in the absence of methane. Additionally, the effect of diffusible redox-active electron carriers on AOM was studied. The thiols cysteine and homocysteine neither stimulated AOM nor sulfate reduction in the absence of methane. Micromolar concentrations of anthraquinones and flavins inhibited AOM, thus they are most likely not involved in the extracellular electron transfer in AOM. Finally, the possibility of an electron transfer from cell to cell via outer membrane proteins/'nanowires' was taken into account. Accordingly, an anoxic electrochemical cell was developed to study AOM. A methane-dependant current production by AOM-active Black Sea microbial mats was not observed therein, thus evidence of a direct electron transfer is still lacking.Dissertation353 105 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Bioelectrical corrosion of iron by lithotrophic sulfate-reducing bacteria(2012-06-14); ; ; Iron, a technological material of prime importance, deteriorates by interaction with its abiotic and biotic environment. While the corrosion of iron in oxic environments is, to our present knowledge, a largely chemical (abiotic) process, corrosion in anoxic environments is heavily affected by microbial activity. In technology, this is referred to as microbially influenced corrosion (MIC). Contrary to the more prominent rusting of iron with oxygen, MIC usually is a hidden process occurring in places such as closed cooling water systems or buried pipelines. The inferred economic costs are tremendous. Sulfate-reducing bacteria (SRB) are the suspected main culprits; MIC usually is most severe in sulfate-containing environments, and iron sulfides (FeS), the characteristic product of SRB-induced corrosion, are ubiquitously found at the affected sites. Metal destruction by SRB is conventionally attributed to three effects, (i) the chemical aggressiveness of their metabolic product sulfide, (ii) a facilitated cathodic reduction of protons to molecular hydrogen at deposited iron sulfides and (iii) the microbial consumption of cathodic hydrogen from the iron or iron sulfides. Recently, another mechanism, i.e. direct electron uptake from metallic iron, has been discovered in specialized lithotrophic SRB that were isolated from enrichment cultures with iron as the only source of electrons (Dinh et al., 2004). In the present study we investigated the suggested mechanisms of SRB-induced corrosion with particular emphasis on the latter, bioelectrical process. This was achieved by a combination of kinetic and electrochemical studies in axenic SRB microcosms, and the physicochemical analysis of the formed corrosion products. Little or no corrosion resulted from galvanically coupled iron sulfides and microbial consumption of hydrogen, respectively. However, the specialized lithotrophic SRB corroded metallic iron severely and formed large amounts of an electroconductive mineral crust. Their corrosiveness results from the formation of a galvanic element between the iron anode and the bacterial cathode. Electrons flow from the iron through the sulfidic crust to the crust-attached bacteria reducing sulfate. The biological cathodic reaction controls the rate and, with appropriately adjusted cultivation conditions, such bioelectrical corrosion progressed at technologically highly relevant rates in long-term incubations. The direct corrosion of iron through electron uptake is here referred to as electrical microbially influenced corrosion (EMIC). This mechanism is fundamentally different from the indirect corrosive effect of SRB owing to the excretion of the chemical hydrogen sulfide (chemical microbially influenced corrosion, CMIC). CMIC is also known to progress at high rates in laboratory cultures. Hence, we intended to unravel the relative contribution of EMIC and CMIC to total microbial corrosion in natural sulfate-rich environments. Careful chemical analysis of corrosion products combined with knowledge of the fundamental differences of the two corrosion mechanisms allowed such quantitative assessment. In a marine tidal mud flat, studied as an example of a sulfate-rich environment possibly favoring MIC, severe metal corrosion could indeed be observed and exclusively attributed to EMIC, i.e. bioelectrical corrosion by lithotrophic SRB. A better understanding of microbial corrosion mechanisms as well as their quantitative contribution to corrosion damage is expected to aid in the development of more effective MIC prevention and mitigation strategies. The striking physiological ability of the organisms to corrode iron by direct electron uptake apparently has polyphyletic origin; it was found in several phylogenetically unrelated bacterial isolates. However, the ecological role of such microorganisms in their natural (metal-free) habitat is currently unknown. Interestingly, we detected high numbers of directly corrosive SRB in marine anoxic sediment, despite obvious absence of man-made iron constructions. It is hypothesized that anaerobic biocorrosion is due to the promiscuous use of an ecophysiologically relevant catabolic trait for uptake of external electrons from natural abiotic and biotic sources.Dissertation381 170 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Extrazelluläre Chitindeacetylase mariner und terrestrischer Bakterien(2006-10-13); ; ; The aim of this work was to find a bacterial catabolic enzyme, suitable for the deacetylation of chitin to chitosan. This enzyme, called chitin deacetylase, is known to be present in fungi but not in bacteria. To find such an enzyme we first had to isolate bacteria which are able to grow on chitin.From marine sediments and also from compost, enriched with chitin, several bacteria strains were isolated on chitin plates or on acetate, the second product of a chitin deacetylase besides chitosan.Some of the strains were tested for chitin deacetylase activity with a colorimetric test or with a test for the formation of acetate. Regarding the advantages of an extracellular enzyme for a technical process (the purification of the enzyme is easier), the screening methods were designed to detect extracellular enzymatic activity in the culture media. A few strains had chitin deacetylating activity but the best results were those of strain 99.6. This strain had also a chitinase activity. The cda-encoding gene of this strain was cloned into E.coli at the Fraunhofer Institute in Hannover.First investigations with 99.6 were done to optimize the culture conditions for a technical process. We also tried to reduce chitinase activity by UV-induced mutations. But after this, also the deacetylating activity was much lower. Trying to concentrate the ezyme we recognized that the enzyme is very unstable. Most of its activity was lost during centrifugation or freezing.Dissertation287 384