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.

Keywords: BTEX; Desulfotomaculum; deep aquifer; natural attenuation; sulfate-reduction.

FIGURE 1

(A) Degradation of benzene, toluene, and ethylbenzene (BTE) during the second subculture (07/2002); Filled squares : ethylbenzene, filled diamonds : toluene, filled triangles : benzene, cross : o-xylene as internal standard. (B) Degradation of BTE along increase of observable biomass during the eighth subculture (04/2008); Filled squares : ethylbenzene, filled diamonds : toluene, filled triangles : benzene, cross : o-xylene as internal standard and filled circles: cells.mL^-1. (C) Effects of inhibitors addition on BTE biodegradation during the eighth subculture (04/2008); Arrow indicates the addition at day 139 of sodium molybdate (MoO₄, 10 mM) or bromoethanesulfonate (BES, 2 mM); Filled squares : ethylbenzene + BES, filled diamonds : toluene + BES, filled triangles : benzene +BES, cross : o-xylene as internal standard + BES or + MoO4, open squares : ethylbenzene + MoO₄, open diamonds : toluene + MoO₄, open triangles : benzene + MoO₄. Start levels of BTEX were 100 ppm.

FIGURE 2

(A) Degradation of benzene, toluene, and ethylbenzene (BTE) during the original microcosm (10/2000) incubation; (B) Degradation of BTE during the first subcultures (02/2002); (C) Degradation of BTE during the second subcultures (07/2002); (D) Degradation of BTE during the fifth subcultures (01/2005). (E) Degradation of ethylbenzene, or toluene, or benzene during the sixth subcultures (04/2008). Filled squares: ethylbenzene, filled diamonds: toluene, filled triangles: benzene, cross: o-xylene as internal standard. Start levels of BTEX were 100 ppm.

FIGURE 3

δ¹³C and δ²H of residual benzene fraction versus benzene degradation rate under sulfate-reduction. Filled circle: δ¹³C; clear square: δ²H. Benzene degradation refers to B [%], see Experimental procedures section. Start level of benzene was 12 ppm.

FIGURE 4

(A,B) Double logarithmic plot according to the Rayleigh equation expressing changes in isotopic composition and compounds concentration along time for carbon and hydrogen during anaerobic degradation of benzene; (C) Dual isotope plots of Δδ²H versus Δδ¹³C for anaerobic benzene biodegradation giving the Λ values as the slope of the regression. Dashed lines in all graphes represent the corresponding 95% confidence intervals from duplicate analysis.

FIGURE 5

Biplot of AKIE_C vs. AKIE_H values from data retrieved in this study and in literature dedicaced to benzene biodegradation. Redox condition are deduced from the culture condition where the biodegradation was observed. 1: methanogenic consortium (Mancini et al., 2008); 2: methanogenic consortium (Mancini et al., 2008); 3: sulfate-reducing consortium (Fischer et al., 2008); 4: sulfate-reducing consortium, this study; 5: sulfate-reducing consortium (Bergmann et al., 2011); 6: iron-reducing consortium (Bergmann et al., 2011); 7: nitrate-reducing consortium (Mancini et al., 2003); 8: Nitrate-reducing consortium (Mancini et al., 2008); 9: nitrate-reducing consortium (Mancini et al., 2008);10: nitrate-reducing consortium (Mancini et al., 2003); 11: Ralstonia Pickettii (Fischer et al., 2008); 12: Azoarcus denitriificans (Fischer et al., 2008); 13: Burkholderia sp. (Hunkeler et al., 2001); 14: Cupriavidus necator ATCC 17697 (Fischer et al., 2008); 15: Acinetobacter sp. (Hunkeler et al., 2001).

FIGURE 6

Maximum-likelihood tree based on 16s rRNA gene (1115 bases) showing the phylogenetic relationship between the both sequences detected in microcosms (clone Bc105: minor phylotype and clone Bc107: dominant phylotype) and closest relatives. Reliability values (aLRT values) greater than 50% are given at nodes.