The use of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites: degradation of naphthalene, o-, and m-xylene by sulfate-reducing bacteria

Morasch, Barbara; Annweiler, Eva; Warthmann, R.; Meckenstock, Rainer U.

doi:10.1016/s0167-7012(00)00242-6

Cited by 51 publications

(45 citation statements)

References 41 publications

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“…Naphthalene binds to the XAD7 resin, which acts as a reservoir, and maintains the aqueous concentration of naphthalene below saturation (230 mM or 30 p.p.m.) while supplying enough naphthalene to support growth over the course of the experiment (Morasch et al, 2001). Naphthalene (6-14 mg) was added to 6 ml MSB containing 0.1 g XAD7 before the tubes were sealed with Teflon-lined stoppers and autoclaved.…”

Section: Methodsmentioning

confidence: 99%

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Naphthalene metabolism and growth inhibition by naphthalene in Polaromonas naphthalenivorans strain CJ2

Pumphrey

Madsen

2007

Microbiology

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This study was designed to characterize naphthalene metabolism in Polaromonas naphthalenivorans CJ2. Comparisons were completed using two archetypal naphthalenedegrading bacteria: Pseudomonas putida NCIB 9816-4 and Ralstonia sp. strain U2, representative of the catechol and gentisate pathways, respectively. Strain CJ2 carries naphthalene catabolic genes that are homologous to those in Ralstonia sp. strain U2. Here we show that strain CJ2 metabolizes naphthalene via gentisate using respirometry, metabolite detection by GC-MS and cell-free enzyme assays. Unlike P. putida NCIB 9816-4 or Ralstonia sp. strain U2, strain CJ2 did not grow in minimal medium saturated with naphthalene. Growth assays revealed that strain CJ2 is inhibited by naphthalene concentrations of 78 mM (10 p.p.m.) and higher, and the inhibition of growth is accompanied by the accumulation of orange-coloured, putative naphthalene metabolites in the culture medium. Loss of cell viability coincided with the appearance of the coloured metabolites, and analysis by HPLC suggested that the accumulated metabolites were 1,2-naphthoquinone and its unstable auto-oxidation products. The naphthoquinone breakdown products accumulated in inhibited, but not uninhibited, cultures of strain CJ2. Furthermore, naphthalene itself was shown to directly inhibit growth of a regulatory mutant of strain CJ2 that is unable to metabolize naphthalene. These results suggest that, despite being able to use naphthalene as a carbon and energy source, strain CJ2 must balance naphthalene utilization against two types of toxicity.

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Section: Methodsmentioning

confidence: 99%

“…This procedure has been shown to buffer the aqueous naphthalene concentration at levels proportional to the added mass (Morasch et al 2001).…”

Section: Naphthalene Concentration and Growth Inhibitionmentioning

confidence: 99%

Naphthalene metabolism and growth inhibition by naphthalene in Polaromonas naphthalenivorans strain CJ2

Pumphrey

Madsen

2007

Microbiology

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“…In addition, 1 mM Na 2 S was used as reducing agent, 10 mM Na 2 SO 4 was added as electron acceptor and 5 mM cAMP was added to stimulate activity (Bruns et al, 2002). To warrant constantly low in situ concentrations of toluene during SIP incubation, 0.3 g of Amberlite XAD7 adsorber resin (Sigma-Aldrich, Munich, Germany) was added to each bottle (Morasch et al, 2001). A 5 ml of either non-labeled ( 12 C) or fully ( 13 C 7 ) labeled toluene (Sigma-Aldrich) were injected through butyl rubber stoppers with a gastight syringe and allowed to adsorb to the carrier for 2 days.…”

Section: Incubation Of Sedimentsmentioning

confidence: 99%

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment

et al. 2010

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Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with 13 C 7 -toluene, the production of both sulfide and 13 CO 2 was clearly coupled to the 13 C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only B50%, pointing toward high ratios of heterotrophic CO 2 -fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped.

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“…The site was contaminated with monocyclic and polycyclic aromatic hydrocarbons, as well as heterocyclic compounds. Bacteria were cultivated at 30°C in bicarbonatebuffered freshwater mineral medium, pH 7.4, with sulfate (10 mM) as electron acceptor as described before (Morasch et al 2001). The medium was prepared under an atmosphere of N 2 /CO 2 (80:20 v/v) and reduced with Na 2 S (1 mM) (Widdel and Bak 1992).…”

Section: Purification and Culture Conditionsmentioning

confidence: 99%

“…Aromatic substrates were directly injected with micro-syringes through the rubber stoppers. In order to keep concentrations in the water phase low but to provide a substrate reservoir, 0.3 g adsorber resin Amberlite-XAD7 (Fluka, Buchs, Switzerland) was added per bottle, and the concentration of aromatic hydrocarbons stabilized at a level of 30-60 µM, as described before (Morasch et al 2001). Instead of Amberlite-XAD7, in some experiments, 1 ml 2,2,4,4,6,8,8,-heptamethylnonane (Aldrich, Milwaukee, Wis., USA) was used as a non-aqueous carrier liquid amended with 1.5% xylene.…”

Section: Purification and Culture Conditionsmentioning

confidence: 99%

Degradation of o -xylene and m -xylene by a novel sulfate-reducer belonging to the genus Desulfotomaculum

Morasch

Schink

Tebbe

et al. 2004

Archives of Microbiology

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117

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A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO 2 . Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbonoxidizing bacterium in this genus. Keywords Sulfate reduction · BTEX · Benzylsuccinate synthase · Desulfotomaculum IntroductionPetroleum hydrocarbons are among the most abundant groundwater contaminants and can be found in gasworks plants, landfill leachates, and accidental fuel spills (US-EPA 1999). The mono-aromatic hydrocarbons benzene, toluene, ethylbenzene, and xylene isomers (BTEX) are putatively mutagenic or carcinogenic substances and make-up more than 50% by weight of the water-soluble gasoline fraction (Coleman et al. 1984). Due to their relatively high solubility, they are mobile with the groundwater flow and form contaminant plumes in aquifers. The degradative potential of anaerobic bacteria towards aromatic hydrocarbons in situ was investigated in several studies and a decrease of BTEX compounds could be demonstrated under denitrifying, Fe(III)-reducing, sulfate-reducing, and methanogenic conditions (Dolfing et al. 1990;Kazumi et al. 1997;Reinhard et al. 1997;Gieg et al. 1999;Phelps and Young 2001). Numerous anaerobic bacterial cultures have been enriched in the past, and pure strains have been isolated. Of all BTEX compounds, toluene has been studied most extensively with respect to its anaerobic degradation. Pure cultures of toluene-degrading bacteria that use NO 3 -, Fe(III), or SO 4 2-as electron acceptors have been isolated (Lovley and Lonergan 1990;Rabus et al. 1993;Seyfried et al. 1994;Zhou et al. 1995;Beller et al. 1996). Under anoxic conditions, degradation of toluene proceeds via addition of fumarate to the methyl group (Biegert et al. 1996;Beller and Spormann 1997b;Leuthner et al. 1998;Rabus and Heider 1998;Kane et al. 2002).Consumption of o-xylene was discovered simultaneously with toluene degradation in methanogenic consortia (Edwards and Grbic-Galic 1994). Later, transformation of o-xylene to o-methyl homologs of benzylsuccinate by toluene-degrading strains was reported; however, no further oxidation steps could be observed (Beller and Spormann...

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The use of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites: degradation of naphthalene, o-, and m-xylene by sulfate-reducing bacteria

Cited by 51 publications

References 41 publications

Naphthalene metabolism and growth inhibition by naphthalene in Polaromonas naphthalenivorans strain CJ2

Naphthalene metabolism and growth inhibition by naphthalene in Polaromonas naphthalenivorans strain CJ2

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment

Degradation of o -xylene and m -xylene by a novel sulfate-reducer belonging to the genus Desulfotomaculum

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