Degradation of o -xylene and m -xylene by a novel sulfate-reducer belonging to the genus Desulfotomaculum

Abstract

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO 2 . Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbonoxidizing bacterium in this genus. Keywords Sulfate reduction · BTEX · Benzylsuccinate synthase · Desulfotomaculum IntroductionPetroleum hydrocarbons are among the most abundant groundwater contaminants and can be found in gasworks plants, landfill leachates, and accidental fuel spills (US-EPA 1999). The mono-aromatic hydrocarbons benzene, toluene, ethylbenzene, and xylene isomers (BTEX) are putatively mutagenic or carcinogenic substances and make-up more than 50% by weight of the water-soluble gasoline fraction (Coleman et al. 1984). Due to their relatively high solubility, they are mobile with the groundwater flow and form contaminant plumes in aquifers. The degradative potential of anaerobic bacteria towards aromatic hydrocarbons in situ was investigated in several studies and a decrease of BTEX compounds could be demonstrated under denitrifying, Fe(III)-reducing, sulfate-reducing, and methanogenic conditions (Dolfing et al. 1990;Kazumi et al. 1997;Reinhard et al. 1997;Gieg et al. 1999;Phelps and Young 2001). Numerous anaerobic bacterial cultures have been enriched in the past, and pure strains have been isolated. Of all BTEX compounds, toluene has been studied most extensively with respect to its anaerobic degradation. Pure cultures of toluene-degrading bacteria that use NO 3 -, Fe(III), or SO 4 2-as electron acceptors have been isolated (Lovley and Lonergan 1990;Rabus et al. 1993;Seyfried et al. 1994;Zhou et al. 1995;Beller et al. 1996). Under anoxic conditions, degradation of toluene proceeds via addition of fumarate to the methyl group (Biegert et al. 1996;Beller and Spormann 1997b;Leuthner et al. 1998;Rabus and Heider 1998;Kane et al. 2002).Consumption of o-xylene was discovered simultaneously with toluene degradation in methanogenic consortia (Edwards and Grbic-Galic 1994). Later, transformation of o-xylene to o-methyl homologs of benzylsuccinate by toluene-degrading strains was reported; however, no further oxidation steps could be observed (Beller and Spormann...

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment

“…From our results it is impossible, however, to define whether two closely related, but functionally identical sub-populations were represented by the two T-RFs, or whether it was one population carrying two minimally differing (B98% sequence similarity) rrn operons (Pei et al, 2009). Members of the Peptococcaceae have been previously described as important constituents of microbial communities in contaminated aquifers (Robertson et al, 2001;Franzmann et al, 2002), and two respective isolates have been described to oxidize toluene (Liu et al, 2004;Morasch et al, 2004). More recently, related microbes have also been identified to be involved in the anaerobic degradation of benzene (Kunapuli et al, 2007;Abu Laban et al, 2009;Herrmann et al, 2010).…”

“…However, this model would require a secondary sulfate reducer consuming the produced acetate to become equally or even more strongly labeled than the primary degrader over time, which we did not observe. Moreover, the two isolated hydrocarbon-degrading Desulfosporosinus and Desulfotomaculum relatives described to date were both found to completely oxidize toluene to CO 2 (Liu et al, 2004;Morasch et al, 2004).…”

“…Unfortunately, we did not quantify acetate (or other organic carbon) during our SIP incubation. Although the two described related toluene-oxidizing Clostridia do not grow on acetate (Liu et al, 2004;Morasch et al, 2004), the utilization of acetate for assimilatory purposes cannot be excluded. However, if abundant acetate or other organic carbon had been available during the incubation, sulfate reduction would have been expected independent of toluene degradation, which was clearly not observed (Figure 1).…”

“…One of the few available Gram-positive BTEX-degrading sulfate reducers, Desulfotomaculum sp. strain Ox39 has been isolated from this site (Morasch et al, 2004). However, it remains to be verified whether an importance of such non-proteobacterial populations for on-site aromatics degradation can be substantiated using cultivation-independent tools.…”

Methylotrophic methanogenesis governs the biogenic coal bed methane formation in Eastern Ordos Basin, China

“…Both SXLL-B16 and SXLL-B21 showed the closest match to the 16S rRNA gene from the cultured organism Desulfotomaculum sp. (90% identity) which is a sulfate reducer and able to degrade o-xylene, m-xylene, and toluene (Morasch et al 2004) and from Pelotomaculum terephthalicicum (90% identity) which was isolated from methanogenic sludges as syntrophic phthalate isomer-degrading microbes (Qiu et al 2004). Both SXLL-B02 and SXLL-B18 were closely related to the 16S rRNA gene from the cultured organism Saccharofermentans acetigenes (91% Bernard et al (1978) identity), which was isolated from an upflow anaerobic sludge blanket reactor and could ferment polysaccharides (Chen et al 2010).…”