Impact of bioavailability restrictions on microbially induced stable isotope fractionation. 2. Experimental evidence.

Stable isotope fractionation analysis (SIFA) of contaminants is an emerging technique to characterize in situ microbial activity. The kinetic isotope effect in microbial degradation reactions, or enzyme catalysis, is caused by the preferential cleavage of bonds containing light rather than heavy isotopes. This leads to a relative enrichment of the heavier isotopes in the residual substrate pool. However, a number of nonisotopically sensitive steps preceding the isotopically sensitive bond cleavage may affect the reaction kinetics of a degradation process, thus reducing the observed (i.e., the macroscopically detectable) isotope fractionation. Low bioavailability of contaminants poses kinetic limitations on the biodegradation process and can significantly reduce the observed kinetic isotope fractionation. Here we present experimental evidence for the influence of bioavailability-limited pollutant biodegradation on observed stable isotope fractionation. Batch laboratory experiments were performed to quantify the toluene hydrogen isotope fractionation of Pseudomonas putida mt-2 (pWWO) subjected to different small concentrations of toluene with and without deuterium label, which corresponded to realistic environmental mass transfer scenarios. Detected isotope fractionations depended significantly on the toluene concentration, hence confirming the influence of substrate mass transfer limitation on observed isotope fractionation, hypothesized by Thullner et al. (Environ. Sci. Technol. 2008, 42,6544-6551). Our results indicate that the bioavailability of a substrate should be considered during quantitative analysis of microbial degradation based on SIFA.