New Bio-Indicators for Long Term Natural Attenuation of Monoaromatic Compounds in Deep Terrestrial Aquifers

Abstract

Deep subsurface aquifers despite difficult access, represent important water resources and, at the same time, are key locations for subsurface engineering activities for the oil and gas industries, geothermal energy, and CO2 or energy storage. Formation water originating from a 760 m-deep geological gas storage aquifer was sampled and microcosms were set up to test the biodegradation potential of BTEX by indigenous microorganisms. The microbial community diversity was studied using molecular approaches based on 16S rRNA genes. After a long incubation period, with several subcultures, a sulfate-reducing consortium composed of only two Desulfotomaculum populations was observed able to degrade benzene, toluene, and ethylbenzene, extending the number of hydrocarbonoclastic–related species among the Desulfotomaculum genus. Furthermore, we were able to couple specific carbon and hydrogen isotopic fractionation during benzene removal and the results obtained by dual compound specific isotope analysis (𝜀C = -2.4‰ ± 0.3‰; 𝜀H = -57‰ ± 0.98‰; AKIEC: 1.0146 ± 0.0009, and AKIEH: 1.5184 ± 0.0283) were close to those obtained previously in sulfate-reducing conditions: this finding could confirm the existence of a common enzymatic reaction involving sulfate-reducers to activate benzene anaerobically. Although we cannot assign the role of each population of Desulfotomaculum in the mono-aromatic hydrocarbon degradation, this study suggests an important role of the genus Desulfotomaculum as potential biodegrader among indigenous populations in subsurface habitats. This community represents the simplest model of benzene-degrading anaerobes originating from the deepest subterranean settings ever described. As Desulfotomaculum species are often encountered in subsurface environments, this study provides some interesting results for assessing the natural response of these specific hydrologic systems in response to BTEX contamination during remediation projects.

The sequence capture by hybridization: a new approach for revealing the potential of mono‐aromatic hydrocarbons bioattenuation in a deep oligotrophic aquifer

“…Periodically, TX degradation was monitored in the four microcosms using SPME/GC/FID as described in Aüllo et al . ().…”

“…In the context of this study, we sought to demonstrate the presence of bssA genes in a deep aquifer used to store natural gas, which is associated with trace amounts of other hydrocarbons, in order to reveal a potential for natural bioattenuation of TX. We hypothesized that the Clostridia , in particular members of the Peptococcaceae family, play a key role in the degradation of TX in deep aquifers (Basso et al ., ; Berlendis et al ., ; Aüllo et al ., ). It is known that some members of Peptococcaceae are capable of degrading mono‐aromatic hydrocarbons directly or via syntrophic relationships (Morasch et al ., ; Taubert et al ., ).…”

“…It is currently difficult, indeed impossible, to assess the degradation potential of FW sampled from a deep aquifer without having recourse to laboratory‐based degradation assays. In our previous studies, amplifications of the bssA gene using the primers cited previously have unfortunately often proved to be unsuccessful, as a result of the few targets available and problems of non‐specific amplification (Aüllo et al ., ). Next‐generation sequencing (NGS) techniques allow a deeper analysis of genetic diversity and thus offer the possibility of dispensing with culture‐based techniques.…”

“…The degradation of toluene and m , p and o ‐xylenes (TX) under anoxic conditions has been demonstrated in numerous marine and continental environments. It can be associated with the reduction of nitrate, sulphate, iron and CO 2 (Dolfing et al ., ; Beller and Spormann, ; Harms et al ., ; Kane et al ., ; Kube et al ., ; Morasch et al ., ; Morasch and Meckenstock, ; Washer and Edwards, ; Aüllo et al ., ). In all cases, the initial addition of fumarate to the hydrocarbon molecule is catalysed by either benzylsuccinate synthase (toluene) or benzylsuccinate synthase‐like enzymes (xylenes).…”

“…In oligotrophic and stable environments, like deep continental aquifers (−500 to −1200 m), the input of exogenous TX represents a potential source of carbon which is likely to modify the growth and survival strategies of microorganisms. Over the last several years, the potential for biodegradation of monoaromatic hydrocarbons in such environments has been demonstrated through culture‐based approaches (Morasch et al ., ; Berlendis et al ., ; Aüllo et al ., ). The low biomass present in these ecosystems makes the demonstration of biodegradation capacities difficult (i.e.…”