Evaluation of Toluene Degradation Pathways by Two-Dimensional Stable Isotope Fractionation

Toluene degradation by several pure and mixed microbial cultures was investigated by two-dimensional compound specific isotope analysis (2D-CSIA). For most of the cultures, the respective toluene degradation pathway and toluene attacking enzymatic step was known. The slope of the linear regression for hydrogen (Δδ2H) vs carbon (Δδ13C) discrimination (Λ = Δδ2H/Δδ13C ≈ εHbulk/εCbulk) was determined in order to characterize aerobic and anaerobic toluene degradation pathways. The highest Λ value was estimated for the monohydroxylation of the methyl group by Pseudomonas putida (Λ = 53 ± 5). The lowest value was observed for Rhodococcus opacus (Λ = 2 ± 2) due to its insignificant hydrogen fractionation, which indicates that a ring dioxygenase was responsible for the initial attack of toluene. The fungus Cladosprium sphaerospermum containing a cytochrome P450-dependent methyl monooxygenase grouped within these extreme values (Λ = 16 ± 6). Λ values for organisms attacking toluene under anoxic conditions by benzylsuccinate synthase were significantly different and ranged from Λ = 4 ± 3 (Blastochloris sulfoviridis) to 31 ± 11 (strain TRM1). Values were in the same range for organisms using nitrate (Λ = 11−14) or sulfate (Λ = 28−31) as electron acceptor, indicating that it might be possible to distinguish toluene degradation under different electron acceptor conditions by 2D-CSIA.