Assessing in situ rates of anaerobic hydrocarbon bioremediation

Abstract

Identifying metabolites associated with anaerobic hydrocarbon biodegradation is a reliable way to garner evidence for the intrinsic bioremediation of problem contaminants. While such metabolites have been detected at numerous sites, the in situ rates of anaerobic hydrocarbon decay remain largely unknown. Yet, realistic rate information is critical for predicting how long individual contaminants will persist and remain environmental threats. Here, single-well push-pull tests were conducted at two fuel-contaminated aquifers to determine the in situ biotransformation rates of a suite of hydrocarbons added as deuterated surrogates, including toluene-d(8), o-xylene-d(10), m-xylene-d(10), ethylbenzene-d(5) (or -d(10)), 1, 2, 4-trimethylbenzene-d(12), 1, 3, 5-trimethylbenzene-d(12), methylcyclohexane-d(14) and n-hexane-d(14). The formation of deuterated fumarate addition and downstream metabolites was quantified and found to be somewhat variable among wells in each aquifer, but generally within an order of magnitude. Deuterated metabolites formed in one aquifer at rates that ranged from 3 to 50 µg l(-1) day(-1), while the comparable rates at another aquifer were slower and ranged from 0.03 to 15 µg l(-1) day(-1). An important observation was that the deuterated hydrocarbon surrogates were metabolized in situ within hours or days at both sites, in contrast to many laboratory findings suggesting that long lag periods of weeks to months before the onset of anaerobic biodegradation are typical. It seems clear that highly reduced conditions are not detrimental to the intrinsic bioremediation of fuel-contaminated aquifers.

Figure 1

Deuterated hydrocarbons used in this study and their corresponding fumarate addition and downstream metabolites assayed in push–pull tests by LC‐MS‐MS and/or GC‐MS to elucidate rates of in situ hydrocarbon biotransformation. ¹Analogous pathway for the o‐xylene‐d₈ isomer. ²Ethylbenzene‐d₁₀ was used in the Ft. Lupton tests, whereas ethylbenzene‐d₅ was used in the Hickam tests. ³Analogous pathway for the 1, 2, 4‐TMB‐d₁₂ isomer.

Figure 2

Mass spectral profiles of (A) authentic benzylsuccinic acid, (B) deuterated (d₈) fumarate addition metabolite produced during the anaerobic decomposition of toluene‐d₈ in laboratory incubations, (C) authentic m‐methylbenzylsuccinic acid and (D) deuterated (d₁₀) fumarate addition metabolite produced during the anaerobic degradation of m‐xylene‐d₁₀ in laboratory enrichments. Mass spectra and structures shown are of the trimethylsilylated derivatives analysed by GC‐MS.

Figure 3

Mass spectral profiles of (A) synthesized standard of 3, 5‐dimethylBSA, (B) fumarate addition metabolite produced during the anaerobic degradation of 1, 3, 5‐TMB‐d₁₂ in laboratory incubations, (C) 3, 5‐dimethylbenzoate‐d₉ produced during the anaerobic degradation of 1, 3, 5‐TMB‐d₁₂ in laboratory enrichments and (D) metabolite detected during push–pull studies with deuterated hydrocarbons conducted at the Hickam site. Mass spectra and structures shown are of the trimethylsilylated derivatives analysed by GC‐MS.

Figure 4

Formation of deuterated alkylbenzylsuccinic acids (A, C) and additional downstream metabolites (B, D) over time in push–pull tests carried out at the Ft. Lupton (A, B) and Hickam (C, D) sites as measured by LC‐MS‐MS (normalized to bromide concentrations). Regression analysis to determine rates of formation were carried out using initial time points only (e.g. dotted lines in A).