Katrin Knittel

Marine seeps are locally restricted areas, yet highly productive oases of life in the deep sea. They are shaped by the emission of gas and reduced fluids from subsurface reservoirs to the seafloor.

Most seeps are dominated by methane seepage and only minor concentrations of other hydrocarbon gases are present as trace gases. In addition to hydrocarbon gases, some marine seeps also release higher hydrocarbons and oil. Prominent examples are natural oil seeps in the Northern and Southern Gulf of Mexico or in the Guaymas Basin. A unique type of oil seeps are the asphalt volcanoes of the deep Gulf of Mexico. Here, massive lava-like flow fields of solidified asphalt and asphalt-laden heavy oils were reported.

Anaerobic oxidation of methane

Methane and non-methane hydrocarbons are controlling factors for sulphur cycling at marine seeps. The sulfate-dependent anaerobic oxidation of methane (AOM) is a key biogeochemical process at methane seeps. The metabolic process of AOM is assumed to be a reversal of methanogenesis coupled to the reduction of sulphate to sulphide involving anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (for a review see Knittel and Boetius, 2009).

Three ANME groups, i.e. ANME-1, ANME-2, and ANME-3, have been identified so far to be involved in sulfate-dependent AOM (Hinrichs et al., 1999; Boetius et al., 2000; Knittel et al., 2005). They have been shown to live in a consortium with sulfate-reducing bacteria of clade Desulfosarcina/Desulfococcus (DSS), clade SEEP-SRB2, Desulfobulbus relatives or of clade HotSeep1.

Candidatus "Methanoperedens nitroreducens" (ANME-2d) has been reported to be involved in nitrate-dependent AOM (Haaron et al., 2013). 

Since many years the Department of Molecular Ecology is involved in projects on different aspects of AOM in close collaboration with the HGF MPG Joint Research Group for Deep-Sea Ecology and the Department of Microbiology. This collaboration includes the molecular characterization of new habitats or enrichments, diversity and phylogenetic analysis but also cloning of specific genes and microscopy (e.g. Ruff et al., 2015; Wegener et al., 2016; Krukenberg et al., 2016).

ANME phylogeny
Phylogeny of anaerobic methanotrophs (ANME). Scale bar, 10% estimated sequence divergence. Modified after Knittel and Boetius, 2009.
AOM aggregates
Diversity of aggregates mediating the anaerobic oxidation of methane in marine sediments. ANME: green; sulfate-reducing bacteria: green. Scale bar (applicable to all images if not otherwise indicated), 10 µm.
Images from Knittel & Boetius, 2010 or © Knittel, MPI-MM.

Non-methane hydrocarbon degrading sulfate reducers

Some marine seeps contain a broad range of alkanes, alkenes and aromatic compounds. Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria has an important role in carbon and sulfur cycling. In collaboration with the in close collaboration with the HGF MPG Joint Research Group for Deep-Sea Ecology, the Department of Microbiology, the HGF-Centers in Leipzig and Munich as well as with GEOMAR in Kiel several ecophysiological aspects of hydrocarbon-degrading communities are studied.  

Identification of key non-methane hydrocarbon degraders

Stable isotope probing (SIP) on incubations with butane or dodecane allowed the identification of four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short-chain and long-chain alkanes (Kleindienst et al., 2015). All clades were affiliated with the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent -oxidation, and the reverse Wood-Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analogue to those discovered for isolates, are of great relevance in marine benthic habitats.

The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage as well as to massive hydrocarbon input, e.g. as encountered during anthropogenic oil spills.

In collaboration with GEOMAR (T. Treude) we studied the response of the microbial community to a simulated petroleum seepage using a sediment oil-flow through set-up. Bacteria degrading petroleum compounds were identified using NGS tag sequencing and CARD-FISH (Mishra et al., submitted; Stagars et al., submitted).


Phylogeny of hydrocarbon degraders
Phylogeny of isolated or enriched anaerobic hydrocarbon degraders based on 16S rRNA genes. Nitrate-reducing bacteria are printed in blue, sulfate-reducing bacteria in red, iron-reducing bacteria in purple, phototrophic bacteria in green, fermentative bacteria in orange and syntrophic bacteria degrading hydrocarbons in a consortium under methanogenic conditions in light blue. Georgfuchsia toluolica, printed in light green, has been shown to use Fe(III), Mn(IV), and nitrate as terminal electron acceptor for growth on aromatic compounds. Substrate usage is given within parenthesis. The bar represents 10% estimated sequence divergence.
© K. Knittel, MPI-MM

Diversity of alkane-degrading communities based on MasD

Marine cold seep
Hydrocarbons emission at cold seeps.
© D. Meier, D. Probandt, MPI-MM

A common way of anaerobic non-methane alkane activation is its addition across the double bond of fumarate to form alkyl-substituted succinates, a step catalyzed by a glycyl radical enzyme, 1-methyl alkyl succinate synthase (Mas, Grundmann et al., 2008; Rabus et al., 2016; also known as alkylsuccinate synthase (Ass, Callaghan et al., 2008). Other known mechanisms for non-methane alkane activation include anaerobic hydroxylation followed by carboxylation and the oxygen-independent hydroxylation (reviewed in Callaghan, 2013). In a project with  the HGF MPG Joint Research Group for Deep-Sea Ecology and the Department of Microbiology we showed that the thermophilic archaeon "Candidatus Syntrophoarchaeum" can activate butane via alkyl-coenzyme M formation (Laso-Pérez et al., 2016).

Most cultivated anaerobic alkane degraders activate alkanes via fumarate addition. Thus, the gene encoding the catalytic subunit of the responsible enzyme Mas (masD) serves as relevant genetic marker and its study allows a cultivation-independent survey of the diversity and distribution of alkane-degrading communities in any anoxic hydrocarbon-impacted environment.

We studied the diversity of MasD in several seep sediments to identify cosmopolitan species as well as to identify factors structuring the alkane-degrading community. Using next generation sequencing we obtained a total of 420 MasD species-level operational taxonomic units (OTU0.96) at 96% amino acid identity (Stagars et al., 2016). The MasD community structure is clearly driven by the hydrocarbon source available at the various seeps. Two of the detected OTU0.96 were cosmopolitan and abundant while 75% were locally restricted, suggesting the presence of few abundant and globally distributed alkane degraders as well as specialized variants that have developed under specific conditions at the diverse seep environments. 

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