title: Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations.
abstract: Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with [methyl-13C]toluene. These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population. After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1omega7c, 16:1omega7t, 16:0, cy17:0, and 18:1omega7c. Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp. (beta-Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon 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 using 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 pristine 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 promising approach to identify and to quantify denitrifying toluene degraders within microbial communities.
authors.affiliation: Fachbereich Bodenbiologie, Institut für Terrestrische Okologie, Eidgenössische Technische Hochschule (ETH), Schlieren, Switzerland.
authors.forename: O
authors.initials: O
authors.lastname: Pelz
authors.forename: A
authors.initials: A
authors.lastname: Chatzinotas
authors.forename: N
authors.initials: N
authors.lastname: Andersen
authors.forename: S M
authors.initials: SM
authors.lastname: Bernasconi
authors.forename: C
authors.initials: C
authors.lastname: Hesse
authors.forename: W R
authors.initials: WR
authors.lastname: Abraham
authors.forename: J
authors.initials: J
authors.lastname: Zeyer
mesh_terms: D001419:Bacteria / Q000378:metabolism*; D002247:Carbon Isotopes; D019015:Geologic Sediments; D014050:Toluene / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D014050:Toluene