Anaerober Abbau von Alkanen und Alkenen durch Sulfatreduzierer
Alkane und Alkene kommen als Bestandteile von Erdöl häufig in der Natur vor. Aufgrund der globalen Verschmutzung durch Erdöl bzw. daraus resultierenden Kraftstoffen, spielt der Abbau dieser Verbindungen eine große Rolle. Diese Kohlenwasserstoffe können aerob mit Hilfe bakterieller Mono- oder Dioxyge...
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Alkanes and alkenes are components of crude oil. Consequently, they are unfortunately part of the environment. The degradation of these components is very important, because of the increasing global pollution caused by crude oil and other fuels. These hydrocarbons are degraded anaerobically by mono- and dioxygenases. The most known anaerobic bacteria degrade alkanes via fumarate addition. However, there are some other anaerobic microorganisms that also degrade alkanes in a different way, but the exact mechanisms of action are still unknown. Former findings of alkene degradation show that a hydration of the double bound of the alkene could be the initial reaction. Desulfococcus oleovorans strain Hxd3 and Desulfatibacillum alkenivorans strain AK-01 are well-known sulfate-reducing bacteria, which degrade alkanes and alkenes anaerobically. D. alkenivorans degrades alkanes via fumarate addition. In contrast, D. oleovorans uses a different, not fully characterized metabolic pathway. Nevertheless, it is still known that the strain contains genes, which encode for an enzyme, which has a high sequence similarity to the ethylbenzenedehydrogenase (EBDH) of Aromatoleum aromaticum. The main aim of this work was to characterize the alkane and alkene degradation by sulfate-reducing bacteria under anaerobic conditions and to analyse the possible involvement of the EBDH-like enzyme of D. oleovorans on the anaerobic alkane and alkene metabolism. We detected induced proteins in cells of D. oleovorans, which were grown on hexadecane. Using MALDI-TOF analyses, these proteins were identified as subunits of the EBDH-like enzyme. The enzyme was called alkanehydroxylase in this thesis, because of its supposed ability to catalyse the hydroxylation of the alkane to the corresponding iso-alcohol. We could also detect small amounts of the corresponding intermediate 2-hexadecanol in an extract of a hexadecane-degrading culture of D. oleovorans by GC/MS analyses. The theory that a hydration reaction is the initial step of the alkene degradation to the corresponding 1-alcohol by sulfate-reducing bacteria forms the basis for our analyses. The evaluation of the fatty acid pattern of different substrates showed that D. oleovorans prefers alkenes and iso-alcohols instead of alkanes. One explanation for this observation could be that the bacteria need more energy to activate alkanes. The genes of the three subunits of the active alkane hydroxylase lie together with other genes on an apparent operon. All these proteins together may form a membrane-bound complex. In this complex electrons of the hydroxylation reaction, which takes place on the periplasm side, were may transferred to menaquinone, which is located in the membrane. The endergonic electron transfer is probably linked to energy consumption. D. alkenivorans discriminates between alkanes and alkenes but not as much as D. oleovorans. In hexadecane-degrading cultures of D. alkenivorans only the alkylsuccinate synthase was detected as induced protein. This enzyme catalysis the addition of fumarate to the methyl group of the alkane. Using the fatty acid pattern, we were able to show that D. oleovorans has a different degradation pathway than D. alkenivorans, which degrades alkanes by fumarate addition. Products of the degradation of an odd-chain iso-alcohol were mostly even-chain fatty acids in D. alkenivorans. In contrast, odd-chain alkanes were degraded to odd-chain fatty acids. Nevertheless, D. oleovorans mainly produced even-chain fatty acids independent of the substrate (odd-chain iso-alcohols or odd-chain alkanes). The results could indicate that the 2-iso-alcohol is a metabolite of the anaerobic alkane metabolism of D. oleovorans. We could not detect any specific induced proteins in both strains, when alkenes were used as substrates. However, we found that the initial enzyme of the alkene degradation in both strains is not dependent on tungsten such as the acetylene hydratase of Pelobacter acetylenicus, which catalyses the addition of water to the triple bound of acetylene. Genes of D. oleovorans which could encode for an acetylenehydratase-like enzyme, which is necessary for the alkene degradation, were transferred into an isolated strain of Pseudomonas stutzeri, which is able to grow on 1-hexadecanol but not on hexadecene. It was possible to detect growth of hexadecene-growing cultures under aerobic conditions with two of the potential alkene degrading enzymes of D. oleovorans.