Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: Distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site

Hendrickx, Barbara; Junca, Howard; Vosáhlová, Jolana; Lindner, Antje; Rüegg, Irene; Bucheli‐Witschel, Margarete; Faber, Folkert; Egli, Thomas; Mau, Margit; Schlömann, Michael; Brennerova, Maria V.; Brenner, V.; Pieper, Dietmar H.; Top, Eva M.; Dejonghe, Winnie; Bastiaens, Leen; Springael, Dirk

doi:10.1016/j.mimet.2005.04.018

Cited by 135 publications

(89 citation statements)

References 49 publications

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“…Results from previous investigations demonstrated that nutrient additions do not necessarily lead to drastic alterations of community functions (43,44). Moreover, the sequences recovered from the SIP experiments represented Gammaproteobacteria and Betaproteobacteria, which is a finding consistent with those of field investigations of benzene-degrading communities (12).…”

Section: Discussionsupporting

confidence: 83%

“…Cultivation approaches that supply BTEX compounds in vapor phase were used in previous investigations (12,22). Hendrickx et al (12) reported the cultivation of Acidovorax and Variovorax spp.…”

Section: Discussionmentioning

confidence: 99%

“…Numerous studies have applied assays with molecular biomarker approaches to contaminated sites, with the objective of exploring the diversities of biodegradation genes and of the naturally occurring populations involved in metabolizing hydrocarbons (10)(11)(12)(13)(14)(15)(16). In addition, cultivation efforts have successfully isolated hydrocarbon-degrading organisms from habitats, including from soil, groundwater, marine waters, and deep sea sediments (12,(17)(18)(19)(20)(21)(22). Despite the many studies successfully reporting new information on the genetic basis (e.g., dioxygenase) of hydrocarbon metabolism, much remains to be learned regarding both the identities of populations responsible for biodegradation and the diversity of biodegradation genes (4,10,14,16).…”

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confidence: 99%

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Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

Posman

DeRito

Madsen

2017

Appl Environ Microbiol

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Investigations of environmental microbial communities are crucial for the discovery of populations capable of degrading hazardous compounds and may lead to improved bioremediation strategies. The goal of this study was to identify microorganisms responsible for aerobic benzene degradation in coal tar-contaminated groundwater. Benzene degradation was monitored in laboratory incubations of well waters using gas chromatography mass spectrometry (GC-MS). Stable isotope probing (SIP) experiments using [13C]benzene enabled us to obtain 13C-labled community DNA. From this, 16S rRNA clone libraries identified Gammaproteobacteria and Betaproteobacteria as the active benzene-metabolizing microbial populations. Subsequent cultivation experiments yielded nine bacterial isolates that grew in the presence of benzene; five were confirmed in laboratory cultures to grow on benzene. The isolated benzene-degrading organisms were genotypically similar (>97% 16S rRNA gene nucleotide identities) to the organisms identified in SIP experiments. One isolate, Variovorax MAK3, was further investigated for the expression of a putative aromatic ring-hydroxylating dioxygenase (RHD) hypothesized to be involved in benzene degradation. Microcosm experiments using Variovorax MAK3 revealed a 10-fold increase in RHD (Vapar_5383) expression, establishing a link between this gene and benzene degradation. Furthermore, the addition of Variovorax MAK3 to microcosms prepared from site waters accelerated community benzene degradation and correspondingly increased RHD gene expression. In microcosms using uninoculated groundwater, quantitative (q)PCR assays (with 16S rRNA and RDH genes) showed that Variovorax was present and responsive to added benzene. These data demonstrate how the convergence of cultivation-dependent and -independent techniques can boost understandings of active populations and functional genes in complex benzene-degrading microbial communities. IMPORTANCE Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

Posman

DeRito

Madsen

2017

Appl Environ Microbiol

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“…The catabolic genes checked were C12O and C23O (Sei et al, 1999), pdo (Krivobok et al, 2003), p3-24 (Dore et al, 2003), ndo (Teramoto et al, 2009), nidA (Brenza et al, 2003, phn (Jennifer et al, 2007) and all benzene,toluene, ethyl benzene and xylene (BTEX) catabolic genes as mentioned in Hendrickx et al (2006). The reaction mixture (50 µL) contained 1X taq buffer, 2.5 mM MgCl2, 200 mM concentration of each deoxynucleoside triphosphate, 50 pmol of each primer, 2.5 U of Taq DNA polymerase (Sigma Aldrich, Mumbai, India) and 1 µL of DNA template.…”

Section: Detection Of Catabolic Genes By Pcrmentioning

confidence: 99%

Isolation of hydrocarbonoclastic bacteria from bilge oil contaminated water

Sivaraman

Ganguly

Nikolausz

et al. 2011

Int. J. Environ. Sci. Technol.

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Two bacterial strains, i.e. Pseudomonas mendocina and Ochrobactrum sp. were isolated from bilge oil contaminated water of Mormugao harbour, Goa, India and grown in a culture medium with hexadecane as the sole carbon source. Pseudomonas mendocina was used in further studies as it was the dominant strain. This strain effectively degraded tetradecane, hexadecane and octadecane leaving a residual concentration of about 73 %, 54 % and 40 % respectively in 120 h. Sequence analysis of the dominant bands from the denaturing gradient gel electrophoresis profiles revealed the differences between the genera of bilge oil contaminated sea water and its enrichment culture on hexadecane indicating a shift in community structure based on the type of substrate available. Pseudomonas mendocina amplified for the following catabolic genes namely C23O, nid and ndo. Based on the catabolic gene study the potential of the bacterial strain isolated, i.e. Pseudomonas mendocina seems to be interesting as it will be able to degrade polyaromatic hydrocarbons as well. Physicochemical properties of Pseudomonas mendocina indicates production of exopolysaccharides based on the value of its isoelectric point.

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“…In practice, often two or more independent approaches are combined (Smets and Pritchard, 2003;Watanabe and Hamamuray, 2003), among these are evidence of concentration decrease of contaminants over time and distance (Wiedemeier et al, 1999), enrichment of heavy stable isotopes in the remaining fraction of organic contaminants (Hunkeler et al, 2002;Meckenstock et al, 2002;Sherwood Lollar et al, 1999), radiotracer studies (Bianchin et al, 2006;Conrad et al, 1997), succession of redox zones in the field (Kuhn and Suflita, 1989;Vroblesky and Chapelle, 1994); accumulation of signature metabolites (Beller, 2002;Elshahed et al, 2001), investigation of the intrinsic microbial biodegradation potential in microcosm studies e in parts with 14 C-labeled substrates e (Aelion and Bradley, 1991;Ambrosoli et al, 2005;Lovley, 2001); characterization of the bacterial community by molecular techniques (Amann et al, 1995;Bakermans et al, 2002), tracing 13 C in fatty acid profiles of bacteria (Geyer et al, 2005), and detection of bacterial enzymes (Hanson et al, 1999;Heinaru et al, 2005;Hendrickx et al, 2006;Löffler et al, 2000). Each of these methods has its advantages and limitations, and therefore it is advisable to base investigations on several approaches, even more when dealing with complex field sites.…”

Section: Introductionmentioning

confidence: 99%

Evidence for in situ degradation of mono-and polyaromatic hydrocarbons in alluvial sediments based on microcosm experiments with 13C-labeled contaminants

Morasch

Höhener

Hunkeler

2007

Environmental Pollution

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A method based on 13 C-labeled substrates was developed to determine the intrinsic biodegradation potential of aromatic pollutants under oxic and under anoxic conditions. AbstractA microcosm study was conducted to investigate the degradation of mono-and polyaromatic hydrocarbons under in situ-like conditions using alluvial sediments from the site of a former cokery. Benzene, naphthalene, or acenaphthene were added to the sediments as 13 C-labeled substrates. Based on the evolution of 13 C-CO 2 determined by gas chromatography isotope-ratio mass spectrometry (GC-IRMS) it was possible to prove mineralization of the compound of interest in the presence of other unknown organic substances of the sediment material. This new approach was suitable to give evidence for the intrinsic biodegradation of benzene, naphthalene, and acenaphthene under oxic and also under anoxic conditions, due to the high sensitivity and reproducibility of 13 C/ 12 C stable isotope analysis. This semi-quantitative method can be used to screen for biodegradation of any slowly degrading, strongly sorbing compound in long-term experiments.

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Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: Distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site

Cited by 135 publications

References 49 publications

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

Isolation of hydrocarbonoclastic bacteria from bilge oil contaminated water

Evidence for in situ degradation of mono-and polyaromatic hydrocarbons in alluvial sediments based on microcosm experiments with 13C-labeled contaminants

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