Identification of toluene degraders in a methanogenic enrichment culture

Abstract

Methanogenic biodegradation involves the cooperative metabolism of syntrophic bacteria that catalyse the initial attack and subsequent degradation of hydrocarbons, and methanogens that convert intermediates such as hydrogen and carbon dioxide, formate, and/or acetate to methane. The identity of syntrophic microbes and the nature of their interactions with other syntrophs and methanogens are not well understood. Furthermore, it is difficult to isolate the organisms responsible for the initial activation and subsequent degradation of hydrocarbon substrates under methanogenic conditions due to the thermodynamic relationships that exist among microbes in methanogenic communities. We used time-resolved RNA stable isotope probing and RT-qPCR to identify the organisms involved in the initial attack on toluene and subsequent degradation reactions in a highly enriched toluene-degrading methanogenic culture. Our results reveal the importance of a Desulfosporosinus sp. in anaerobic toluene activation in the culture. Other organisms that appear to play roles in toluene degradation include Syntrophaceae, Desulfovibrionales and Chloroflexi. The high bacterial diversity observed in this culture and the extensive labelling of different phylogenetic groups over the course of the stable isotope probing experiment highlight the complexity of the relationships that exist in methanogenic ecosystems.

Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

“…The high abundance of Firmicutes in TOLDC and NAPDC is consistent with recent reports of anaerobic monoaromatic hydrocarbon-degrading cultures that implicate Firmicutes (for example, Peptococcaceae) as primary hydrocarbon degraders (Abu Laban et al, 2009;Winderl et al, 2010;Sun and Cupples, 2012;Fowler et al, 2014) or playing key roles alongside Deltaproteobacteria (Ficker et al, 1999;Ulrich and Edwards, 2003;Sakai et al, 2009). In contrast, members of the Deltaproteobacteria, particularly Syntrophus/Smithella sp., have been implicated in methanogenic alkane degradation, particularly of longer-chain alkanes (Zengler et al, 1999;Gray et al, 2011;Cheng et al, 2013).…”

Functional Gene Markers for Fumarate-Adding and Dearomatizing Key Enzymes in Anaerobic Aromatic Hydrocarbon Degradation in Terrestrial Environments

“…Additionally, several Peptococcaceae have been identified as primary degraders of BTEX compounds in syntrophic consortia under different electron acceptor conditions, for example for benzene degradation coupled to nitrate reduction [Luo et al, 2014;van der Zaan et al, 2012], sulphate reduction [Herrmann et al, 2010;Taubert et al, 2012] or iron reduction [Kunapuli et al, 2007], or for toluene degradation under methanogenic conditions [Abu Laban et al, 2015;Fowler et al, 2014;Sun et al, 2014b].…”

“…Later, Fowler et al [2012] substantiated the placement of this bssA lineage upon catabolic analysis of a methanogenic, toluene-degrading enrichment culture from the Fort Lupton aquifer. Via RT-qPCR, Fowler et al [2014] could even show the active expression of clostridial bssA mRNA in the same microcosms. Similar clostridial bssA sequences affiliated to Desulfosporosinus spp.…”

Stable Isotope Probing Approaches to Study Anaerobic Hydrocarbon Degradation and Degraders

“…Phylotypes affiliated to Desulfosporosinus assimilated toluene under sulfate-reducing conditions in laboratory microcosms containing tar oil-contaminated sediment [Pilloni et al, 2011;Winderl et al, 2010] or agricultural soil [Sun and Cupples, 2012]. Notably, similar phylotypes assimilated 13 C from labeled toluene also under methanogenic conditions [Abu Laban et al, 2015;Fowler et al, 2014;, indicating the functional versatility of these taxa, being capable of toluene degradation coupled to both sulfate reduction or fermentation. Such functional versatility has been also assumed for anaerobic benzenedegrading Peptococcaceae , as discussed above .…”

“…A highly enriched methanogenic toluenedegrading culture derived from a gas condensate-contaminated aquifer was examined by RNA-SIP [Fowler et al, 2014], where also a phylotype related to Desulfosporosinus was identified as the key toluene-assimilating organism. Besides the dominant Desulfosporosinus , phylotypes affiliated to several other taxa (Acidobacteria , Actinobacteria , Syntrophaceae , Desulfovibrionales , Chloroflexi) were also shown to assimilate 13 C from labeled toluene to a lesser extent [Fowler et al, 2014;Sun et al, 2014]. This supports the concept of complex syntrophic relationships occurring in methanogenic BTEX degradation.…”