Candidatus Alkanophaga archaea from Guaymas Basin hydrothermal vent sediment oxidize petroleum alkanes.

Methanogenic and methanotrophic archaea produce and consume the greenhouse gas methane, respectively, using the reversible enzyme methyl-coenzyme M reductase (Mcr). Recently, Mcr variants that can activate multicarbon alkanes have been recovered from archaeal enrichment cultures. These enzymes, called alkyl-coenzyme M reductase (Acrs), are widespread in the environment but remain poorly understood. Here we produced anoxic cultures degrading mid-chain petroleum n-alkanes between pentane (C5) and tetradecane (C14) at 70 °C using oil-rich Guaymas Basin sediments. In these cultures, archaea of the genus Candidatus Alkanophaga activate the alkanes with Acrs and completely oxidize the alkyl groups to CO2. Ca. Alkanophaga form a deep-branching sister clade to the methanotrophs ANME-1 and are closely related to the short-chain alkane oxidizers Ca. Syntrophoarchaeum. Incapable of sulfate reduction, Ca. Alkanophaga shuttle electrons released from alkane oxidation to the sulfate-reducing Ca. Thermodesulfobacterium syntrophicum. These syntrophic consortia are potential key players in petroleum degradation in heated oil reservoirs.

Establishing anaerobic hydrocarbon-degrading enrichment cultures of microorganisms under strictly anoxic conditions.

Traditionally, the description of microorganisms starts with their isolation from an environmental sample. Many environmentally relevant anaerobic microorganisms grow very slowly, and often they rely on syntrophic interactions with other microorganisms. This impedes their isolation and characterization by classic microbiological techniques. We developed and applied an approach for the successive enrichment of syntrophic hydrocarbon-degrading microorganisms from environmental samples. We collected samples from microbial mat-covered hydrothermally heated hydrocarbon-rich sediments of the Guaymas Basin and mixed them with synthetic mineral medium to obtain sediment slurries. Supplementation with defined substrates (i.e., methane or butane), incubation at specific temperatures, and a regular maintenance procedure that included the measurement of metabolic products and stepwise dilutions enabled us to establish highly active, virtually sediment-free enrichment cultures of actively hydrocarbon-degrading communities in a 6-months to several-years' effort. Using methane as sole electron donor shifted the originally highly diverse microbial communities toward defined mixed cultures dominated by syntrophic consortia consisting of anaerobic methane-oxidizing archaea (ANME) and different sulfate-reducing bacteria. Cultivation with butane at 50 °C yielded consortia of archaea belonging to Candidatus Syntrophoarchaeum and Candidatus Desulfofervidus auxilii partner bacteria. This protocol also describes sampling for further molecular characterization of enrichment cultures by fluorescence in situ hybridization (FISH), and transcriptomics and metabolite analyses, which can provide insights into the functioning of hydrocarbon metabolism in archaea and resolve important mechanisms that enable electron transfer to their sulfate-reducing partner bacteria.