Carbon and hydrogen isotopic fractionation during anaerobic biodegradation of benzene

Abstract

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors (epsilon ) for carbon (range of -1.9 to -3.6 per thousand ) and hydrogen (range of -29 to -79 per thousand ) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.

FIG. 1.

Biodegradation of benzene in enriched cultures under nitrate-reducing (closed squares, replicates from experiment 1; open squares, replicates from experiment 2), sulfate-reducing (closed triangles, replicates from one experiment), and methanogenic (closed circles, replicates from experiment 1; open circles, replicates from experiment 2) conditions. (A) Concentration of benzene versus time during biodegradation of benzene in enriched cultures. Error bars represent ±5% error on individual benzene concentrations. Note that the scale on the x axis is different for the methanogenic experiment. (B) δ¹³C of remaining benzene versus fraction of benzene remaining during biodegradation of benzene. The error on the x axis is ±7%, based on the propagation of ±5% error through the equation for the fraction of benzene remaining (concentration at time t/initial concentration). All error bars on δ¹³C values represent ±0.5‰ (incorporating both accuracy and reproducibility [see text]). The dashed lines represent ±0.5‰ error on the mean δ¹³C values for all controls. (C) δ²H of remaining benzene versus fraction of benzene remaining during biodegradation of benzene. The error on the x axis is ±7%, based on the propagation of ±5% error through the equation for fraction of benzene remaining (concentration at time t/initial concentration). All error bars on δ²H values represent ±5‰ (incorporating both accuracy and reproducibility [see text]). The dashed lines represent ±5‰ error on the mean δ²H values for all controls.

FIG. 2.

(A) Two proposed reaction sequences for aerobic biodegradation of benzene to form catechol via cis-1,2-dihydroxycyclohexadiene (modified from reference 17) and a benzene epoxide intermediate (modified from reference 35). The reaction illustrates oxygen addition to benzene ring in the initial transformation steps. (B) Proposed pathway for anaerobic biodegradation of benzene to form the first intermediate phenol in the initial transformation step (reprinted from reference 22).