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.

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…The structure of the microbial communities and its dynamics in environmental samples enriched with aromatic compounds have been analyzed using whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes (156,275). A 16S rRNA biomarker was also developed to estimate the concentration of putative benzene degraders under strongly anaerobic (sulfate-reducing and methanogenic) conditions by real-time quantitative PCR.…”

“…13 C]toluene into the polar lipid-derived fatty acids of a sulfate-reducing bacterial community (275).…”

Solvent Stress Response of the Denitrifying Bacterium “ Aromatoleum aromaticum ” Strain EbN1

“…Recently, the complete genome sequence of strain EbN1 was determined, allowing detailed reconstruction of its metabolic network and in-depth proteomic analysis of substrate-dependent regulation of individual degradation pathways (28,35,54). Strain EbN1 represents a promising model organism for in situ processes, since toluene-amended microcosms from oil-contaminated and pristine sites displayed rapid consumption of nitrate (31) and contained high numbers of bacteria related to the phylogenetic cluster harboring strain EbN1 (20,31). Correspondingly, strain EbN1 removes toluene with high efficiency in laboratory twodimensional aquifer microcosms which mimic in situ concentration gradients (2).…”

Phospholipid fatty acids analysis-fatty acid methyl ester (PLFA-FAME) changes during bioremediation of crude oil contamination soil

“…We may emphasize that the existence of gram-positive bacteria increases in petroleum-contaminated environment. It was indicated that saturated fatty acid 16:0 is an indicator which can be found in all bacteria generally (Pelz et al, 2001;Keinanen et al, 2003), however, Kneif et al (2006) stated that PLFA 16:0 is only specific to methane-oxidizing bacteria (metanotroph) as a source of carbon and energy. Some researchers indicated that biomarkers of fatty acids n16:1w7c, n18:1w9c and n18:1w7c increase for gram-negative bacteria (Ringelberg et al, 2008).…”