Identifying competing aerobic nitrobenzene biodegradation pathways by compound-specific isotope analysis

Nitroaromatic compounds that contaminate soil and groundwater can be biodegraded by different, sometimes competing reaction pathways. We evaluated the combined use of compound-specific stable C and N isotope analysis to distinguish between enzymatic nitrobenzene oxidation by Comamonas sp. strain JS765 and partial reduction by Pseudomonas pseudoalcaligenes strain JS45 under aerobic conditions. Bulk C-13 and N-15 enrichment factors for nitrobenzene dioxygenation with JS765 were -3.9 parts per thousand +/- 0.09 parts per thousand (+/- 1 sigma) and -0.75 parts per thousand +/- 0.09 parts per thousand, respectively. The corresponding primary apparent kinetic isotope effects (AKIE) of 1.0241 +/- 0.0005 for C-13 and a secondary N-15 AKIE of 1.0008 +/- 0.0001 are in very good agreement with the proposed enzymatic addition of dioxygen to the aromatic ring to form a cis-dihydrodiol in the rate-limiting step of nitrobenzene degradation. For the partial reduction pathway with JS45, epsilon(C) and epsilon(N) values were -0.57 parts per thousand +/- 0.06 parts per thousand and -26.6 parts per thousand +/- 0.7 parts per thousand. The C-13 and N-15 AKIEs amount to 1.0034 +/- 0.0003 and 1.0273 +/- 0.0008, respectively, and are consistent with the two-electron reduction and dehydration of the aromatic NO2 group to nitrosobenzene. The combined evaluation of delta C-13 and delta N-15 changes in nitrobenzene, based on the isotope enrichment behavior found in this laboratory study, provide an excellent starting point for assessing of the extent of nitrobenzene biodegradation via competing pathways in contaminated environments.