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Abstract

Anaerobic alkane degradation has mainly been described for bacteria belonging to the Deltaproteobacteria class which activate the alkanes by adding fumarate to the skeleton of the molecule. So far, knowledge of anaerobic alkane degradation has been restricted to the degradation of long-chain alkanes and the butane and propane degrading strain BuS5. In this master thesis, I studied the culture Butane-50, which was obtained from the Guaymas Basin hydrothermal vent area in the Gulf of California. The Butane-50 culture grows on butane and is dominated by consortia of archaea and bacteria. The bacteria belong to the HotSeep-1 clade, which has been described as sulphate reducers in thermophilic AOM. At the start of my thesis the consortial archaea in Butane-50 were not identified, since two different archaeal clades were found in the culture with similarly high 16S rRNA sequence abundance: GoM- Arch87 (affiliated with Methanosarcinales) and 19c-33 (affiliated with Thermoplasmata). The objectives of this master thesis were to identify the archaeal partner of the Butane-50 consortia and investigate the functioning of the butane oxidation in the culture. To identify the archaeal partner, CARD-FISH analyses with specific probes for the GoM-Arch87 group (GOM-407) were performed. All consortial archaea had positive signal for the GOM-407 probe, confirming that organisms targeted with this probe have a major role in the consortia. The general structure of the consortia is highly similar to those of AOM consortia, suggesting a similar syntrophic interaction within Butane-50 consortia. To study the functioning of the butane oxidation, metagenomic and metatranscriptomic analyses were carried out on the Butane-50 enrichment. Two draft genomes of representatives of the GoM-Arch87 group, (BOX-1 and BOX-2) were obtained. BOX-1 had the highest coverage and therefore it was selected for a more detailed analysis. BOX-1 included an almost complete set-up of the methanogenesis pathway, including the modifications known from ANME-1. Furthermore, it includes a pathway for butyrate fermentation and carbon fixation genes via the reductive acetyl-CoA pathway, as well as genes for a NiFe-hydrogenase. Transcriptomic analyses showed high expression levels for all these pathways. Therefore, I propose a model for the butane oxidation in BOX-1 in which butane is degraded using the reverse methanogenesis pathway (at least partially) to produce butyrate. Butyrate is further oxidized in the butyrate fermentation pathway to produce acetyl-CoA. BOX-1 does not encode for sulphate reduction. Hence, the reducing equivalents are likely transferred to HotSeep-1, maybe as hydrogen since the genes encoding for the NiFe-hydrogenase subunits had high expression levels. This is the first time that the (reverse) methanogenesis pathway has been reported to be involved in the oxidation of non-methane (short chain) hydrocarbon. Moreover, it could imply the existence of a hydrocarbon sink in the deep ocean that so far has been missed.