Tracing toluene-assimilating sulfate-reducing bacteria using 13C-incorporation in fatty acids and whole-cell hybridization

Pelz, Oliver; Chatzinotas, Antonis; Zarda-Hess, Annatina; Abraham, Wolf‐Rainer; Zeyer, Josef

doi:10.1111/j.1574-6941.2001.tb00890.x

Cited by 48 publications

(10 citation statements)

References 45 publications

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“…Syntrophobacteraceae were identified in a toluene-degrading sulfate-reducing bacterial consortium, but in that study, the Desulfobulbaceae were identified as key organisms of toluene degradation within the consortium (43). In another study, polar lipid-derived fatty acid SIP in combination with whole-cell hybridization linked toluene degradation to the Desulfobacter-like populations, while Syntrophobacteraceae were present in the microbiota but not responsible for toluene biodegradation (45). The Syntrophobacteraceae were also observed in a benzene-degrading in situ microcosm but were not linked to benzene biodegradation (23).…”

Section: Resultsmentioning

confidence: 90%

Diversity of Five Anaerobic Toluene-Degrading Microbial Communities Investigated Using Stable Isotope Probing

Sun

Cupples

2012

Appl Environ Microbiol

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Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [ 13 C]toluene under nitrate-or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genus Thauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the class Clostridia (genus Desulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the class Deltaproteobacteria, within the family Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.

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Section: Resultsmentioning

confidence: 90%

Diversity of Five Anaerobic Toluene-Degrading Microbial Communities Investigated Using Stable Isotope Probing

Sun

Cupples

2012

Appl Environ Microbiol

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“…Therefore, following the reasoning of Parkes et al [48], these carbon sources may not necessarily be present or used by the SRB in situ in this sediment. Instead, SRB may be directly involved in degradation of PHC constituents [29]. We are aware of the problem that the dominant types of SRB in microcosms may change with increasing incubation time, especially on substrates that are incompletely oxidized [48].…”

Section: Discussionmentioning

confidence: 99%

“…However, we must be aware that yet undiscovered SRB species and genera may inhabit anaerobic environments [30,31]. Only a few authors have characterized SRB communities in freshwater [32,33] and direct information on SRB in PHC-contaminated freshwater environments is even more limited [29].…”

Section: Introductionmentioning

confidence: 99%

Sulfate-reducing bacterial community response to carbon source amendments in contaminated aquifer microcosms

Kleikemper

2002

FEMS Microbiology Ecology

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Microbial sulfate reduction is an important metabolic activity in many reduced habitats. However, little is known about the sulfatereducing communities inhabiting petroleum hydrocarbon (PHC)-contaminated freshwater aquifer sediments. The purpose of this study was to identify the groups of sulfate-reducing bacteria (SRB) selectively stimulated when sediment from a PHC-contaminated freshwater aquifer was incubated in sulfate-reducing aquifer microcosms that were amended with specific carbon sources (acetate, butyrate, propionate, lactate, and citrate). After 2 months of incubation, the SRB community was characterized using phospholipid fatty acid (PLFA) analysis combined with multivariate statistics as well as fluorescence in situ hybridization (FISH). Molybdate was used to specifically inhibit SRB in separate microcosms to investigate the contribution of non-SRB to carbon source degradation. Results indicated that sulfate reduction in the original sediment was an important process but was limited by the availability of sulfate. Substantially lower amounts of acetate and butyrate were degraded in molybdate treatments as compared to treatments without molybdate, suggesting that SRB were the major bacterial group responsible for carbon source turnover in microcosms. All of the added carbon sources induced changes in the SRB community structure. Members of the genus Desulfobulbus were present but not active in the original sediment but an increase of the fatty acids 15:1g6c and 17:1g6c and FISH results showed an enrichment of these bacteria in microcosms amended with propionate or lactate. The appearance of cy17:0 revealed that bacteria affiliated with the Desulfobacteriaceae were responsible for acetate degradation. Desulfovibrio and Desulfotomaculum spp. were not important populations within the SRB community in microcosms because they did not proliferate on carbon sources usually favored by these organisms. Metabolic, PLFA, and FISH results provided information on the SRB community in a PHC-contaminated freshwater environment, which exhibited stimulation patterns similar to other (e.g. marine) environments. ß

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“…A 13 C-labeled acetate pulse was administered to sediments, with the resulting 13 C-enriched polar-lipid-derived fatty acid (PLFA) signature profiles subsequently being preferentially analyzed. Other studies utilizing the same approach have followed suit (9,24).…”

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confidence: 99%

RNA Stable Isotope Probing, a Novel Means of Linking Microbial Community Function to Phylogeny

Manefield¹,

Whiteley²,

Griffiths³

et al. 2002

Appl Environ Microbiol

518

419

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Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [ 13 C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13 C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus. Our results suggest that this organism is important for the function of this bioreactor.Our dependence upon laboratory-based studies of culturable bacterial species is acknowledged to be a primary limitation to progress in microbial ecology (2). Traditionally, it has been difficult to attribute a recognized microbially mediated process to the organism(s) responsible for that process in situ (12). Recently, however, culture-independent approaches to linking microbial community function with the genetic identity of key organisms have begun to emerge (7,18,23). Among the limited number of elegant methodologies capable of identifying microorganisms responsible for particular biogeochemical processes, stable-isotope probing (SIP) holds considerable promise.SIP involves the incorporation of stable-isotope-labeled substrates into cellular biomarkers that can be used to identify organisms assimilating the substrate (6). Stable isotopes were first used in this capacity to identify the microbial community component responsible for acetate oxidation in aquatic sediments (7). A 13 C-labeled acetate pulse was administered to sediments, with the resulting 13 C-enriched polar-lipid-derived fatty acid (PLFA) signature profiles subsequently being preferentially analyzed. Other studies utilizing the same approach have followed suit (9, 24).The utility of PLFA-based SIP may be limited, because resolving PLFA profiles composed of multiple species can be problematic (26). This limitation has been overcome with the demonstration that stable-isotope-labeled DNA can be recovered from total community nucleic acid extractions on the basis of its increased buoyant density and can be used as an alternative, unambiguous biomarker (21, 26, 32).Because DNA-based SIP relies on the isolation of labeled DNA by density centrifugation, the degree of isotopic enrichment is crucial. Of the many factors determining the success of an enrichment, including the duration of the pulse and the presence of unlabeled substrate inherent to the system, the rate of DNA synthesis in situ plays a pivotal role. We speculate that DNA synthesi...

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Tracing toluene-assimilating sulfate-reducing bacteria using 13C-incorporation in fatty acids and whole-cell hybridization

Cited by 48 publications

References 45 publications

Diversity of Five Anaerobic Toluene-Degrading Microbial Communities Investigated Using Stable Isotope Probing

Diversity of Five Anaerobic Toluene-Degrading Microbial Communities Investigated Using Stable Isotope Probing

Sulfate-reducing bacterial community response to carbon source amendments in contaminated aquifer microcosms

RNA Stable Isotope Probing, a Novel Means of Linking Microbial Community Function to Phylogeny

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