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.