Page 91 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Rhodocyclaceae and play a role in PAH metabolism (Singleton, Hu and Aitken 2012; Chemerys et al. 2014). The Rhodocyclaceae family members Azoarcus and Thauera consist of anaerobic aromatic compound degrading denitrifiers (Anders et al. 1995; Liu et al. 2006). As the absolute cell counts did not change considerably between the two sampling points of 17 and 25 months (Fig. 1B), it is interesting to note that the microbial community shifted towards a higher abundance of Rhodocyclaceae. This might indicate that the microbial community changed towards degrading complex organic compounds from coal biosolubilization. The involvement of both betaproteobacterial species in the degradation of aromatic compounds was supported by the presence of genes encoding benzylsuccinate synthase (BssABC) and 4-hydroxybenzoyl- CoA reductase (HcrAB) that previously have been shown to be involved in benzoate and toluene degradation, respectively (Hermuth, Leuthner and Heider 2002; Barragán et al. 2004) (Table S2). For Azoarcus, the functional analyses revealed the presence of benzoate-coenzyme A ligase (BzdA) which supported its capacity for benzoate degradation (Barragán et al. 2004).
The identification of Thauera aromatica was possible to species level and was supported by both metagenomic 16S rRNA and functional gene analyses. Its capacity for complex organic compound degradation including relatively inert aromatic compounds may be crucial in coal breakdown processes (Hermuth, Leuthner and Heider 2002; Boll 2011; Kuntze et al. 2011). Both Azoarcus and Thauera have been observed before in sub-bituminous and bituminous methane-producing coal seams (Li, Hendry and Faiz 2008). Strikingly, we did not detect nitrate in the coal well at the start of the experiment (Table S1) so it is unclear what terminal electron acceptors could have been used by Thauera and Azoarcus.
Organic compounds could be further degraded by Geobacter metallireducens
Both the 17- and 25-month amplicon sequencing data indicated high relative abundance of Geobacteraceae (22% and 33% of bacterial amplicon reads, respectively, Fig. 2). The 25 months metagenomic data supported the high relative abundance (32% of 16S rRNA reads, Fig. 2) and allowed the identification of a single Geobacter species present, namely Geobacter metallireducens (100% sequence identity). G. metallireducens inhabits freshwater sediments and couples metal reduction of mainly iron and manganese to the complete anaerobic oxidation of a wide variety of organic electron donors including acetate, butyrate, ethanol, butanol,
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