Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations

Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubate d under anoxic denitrifying conditions with [methyl-C-13]toluene. These mic rocosms served as a laboratory model system to evaluate the combination of isotope (C-13-labeling of polar-lipid-derived fatty acids) and molecular te chniques (16S rRNA-targeting gene probes) to identify the toluene-metaboliz ing population. After total depletion of toluene, the following bacterial p hospholipid fatty acids (PLFA) were C-13-enriched: 16:1 omega 7c, 16:1 omeg a 7t, 16:0. cy17:0, and 18. 1 omega 7c. Pure culture experiments demonstrat ed that these compounds were also found in PLFA profiles of PHC-degrading A zoarcus spp. (beta -Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carb on isotope ratios. Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment. The microbial community in the microcosm incubations was characterized by u sing DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes. Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pris tine sediment were PHC-degrading Azoarcus spp. In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp. cells remained at less than 1% of the DAPI-stained cells. The results show that isotope analysis in combination with whole-cell hybridization is a pr omising approach to identify: and to quantify denitrifying toluene degrader s within microbial communities.