Identification of tertiary butyl alcohol (TBA)-utilizing organisms in BioGAC reactors using 13C-DNA stable isotope probing

Aslett, Denise; Haas, Joseph E.; Hyman, Michael R.

doi:10.1007/s10532-011-9455-3

Cited by 19 publications

(15 citation statements)

References 38 publications

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“…With PCR products from the enrichment cultures, about 95 and 60% of the complete mdpJ and mdpK genes, respectively, were sequenced, showing Ͼ99% identity to the mdpJK genes from strain L108. This high degree of sequence identity is not surprising, as it has already been found that not only mdpJ but also other genes related to MTBE degradation are obviously well conserved among MTBE-and TBA-metabolizing bacteria (3,39,45).…”

Section: Detection Of Alkenes In Cultures Grown On Fuel Oxygenatesmentioning

confidence: 60%

Formation of Alkenes via Degradation of tert -Alkyl Ethers and Alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp

Schäfer

Muzica

Schuster

et al. 2011

Appl Environ Microbiol

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Bacterial degradation pathways of fuel oxygenates such as methyl tert-butyl and tert-amyl methyl ether (MTBE and TAME, respectively) have already been studied in some detail. However, many of the involved enzymes are still unknown, and possible side reactions have not yet been considered. In Aquincola tertiaricarbonis L108, Methylibium petroleiphilum PM1, and Methylibium sp. strain R8, we have now detected volatile hydrocarbons as by-products of the degradation of the tert-alkyl ether metabolites tert-butyl and tert-amyl alcohol (TBA and TAA, respectively). The alkene isobutene was formed only during TBA catabolism, while the beta and gamma isomers of isoamylene were produced only during TAA conversion. Both tert-alkyl alcohol degradation and alkene production were strictly oxygen dependent. However, the relative contribution of the dehydration reaction to total alcohol conversion increased with decreasing oxygen concentrations. In restingcell experiments where the headspace oxygen content was adjusted to less than 2%, more than 50% of the TAA was converted to isoamylene. Isobutene formation from TBA was about 20-fold lower, reaching up to 4% alcohol turnover at low oxygen concentrations. It is likely that the putative tert-alkyl alcohol monooxygenase MdpJ, belonging to the Rieske nonheme mononuclear iron enzymes and found in all three strains tested, or an associated enzymatic step catalyzed the unusual elimination reaction. This was also supported by the detection of mdpJK genes in MTBE-degrading and isobutene-emitting enrichment cultures obtained from two treatment ponds operating at Leuna, Germany. The possible use of alkene formation as an easy-to-measure indicator of aerobic fuel oxygenate biodegradation in contaminated aquifers is discussed.The extensive use of methyl tert-butyl ether (MTBE) and related compounds as fuel oxygenates has resulted in contamination of numerous groundwater sites in the United States and Europe (13,26,33,51). Although MTBE is now banned in some countries and may be phased out in others (52), it will persist at polluted sites for a long time due to its poor biodegradability. Despite this enormous environmental prevalence, only a few strains capable of growth on MTBE, ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) have been found, and fuel oxygenate metabolism has not been elucidated in full detail (20,30,31,46). Since the pioneering works of Salanitro et al. and Steffan et al. (42,43,48), it is generally agreed that aerobic MTBE degradation proceeds via a monooxygenase-catalyzed ether cleavage resulting in formation of tert-butyl alcohol (TBA). The latter is hydroxylated to the corresponding diol, 2-methyl-1,2-propanediol (MPD), which is further oxidized to 2-hydroxyisobutyric acid (2-HIBA). This branched carboxylic acid is then introduced into common metabolic routes after isomerization to 3-hydroxybutyric acid (39). Since ETBE shares the tert-butyl structure with MTBE, it should have a similar degradation path via TBA (8, 30). The biochemistry of TAME catabol...

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Section: Detection Of Alkenes In Cultures Grown On Fuel Oxygenatesmentioning

confidence: 60%

Formation of Alkenes via Degradation of tert -Alkyl Ethers and Alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp

Schäfer

Muzica

Schuster

et al. 2011

Appl Environ Microbiol

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“…Thus far, all mdpJ genes and the corresponding proteins detected in pure cultures and enrichments associated with MTBE or TBA degradation were highly similar (3,38). The amino acid sequences of MdpJ found for strains PM1 and L108, for example, showed 97% identity.…”

Section: Discussionmentioning

confidence: 85%

“…In strain PM1, the oxygenase and reductase are encoded by the mdpJ and mdpK genes, respectively, which show about 97% identity to the corresponding sequences found in strain L108. Recently, the importance of MdpJ in TBA metabolism has also been demonstrated by 13 C metabolomic and proteomic stable isotope probing (SIP) approaches investigating oxygenate degradation in mixed cultures (3,4). It is likely that MdpJ is also involved in TAA degradation, as strains L108 and PM1 could metabolize TAME and TAA (31,38).…”

mentioning

confidence: 99%

Bacterial Degradation of tert-Amyl Alcohol Proceeds via Hemiterpene 2-Methyl-3-Buten-2-ol by Employing the Tertiary Alcohol Desaturase Function of the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

Schuster

Schäfer

Hübler

et al. 2011

Journal of Bacteriology

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Tertiary alcohols, such as tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA) and higher homologues, are only slowly degraded microbially. The conversion of TBA seems to proceed via hydroxylation to 2-methylpropan-1,2-diol, which is further oxidized to 2-hydroxyisobutyric acid. By analogy, a branched pathway is expected for the degradation of TAA, as this molecule possesses several potential hydroxylation sites. In Aquincola tertiaricarbonis L108 and Methylibium petroleiphilum PM1, a likely candidate catalyst for hydroxylations is the putative tertiary alcohol monooxygenase MdpJ. However, by comparing metabolite accumulations in wild-type strains of L108 and PM1 and in two mdpJ knockout mutants of strain L108, we could clearly show that MdpJ is not hydroxylating TAA to diols but functions as a desaturase, resulting in the formation of the hemiterpene 2-methyl-3-buten-2-ol. The latter is further processed via the hemiterpenes prenol, prenal, and 3-methylcrotonic acid. Likewise, 3-methyl-3-pentanol is degraded via 3-methyl-1-penten-3-ol. Wild-type strain L108 and mdpJ knockout mutants formed isoamylene and isoprene from TAA and 2-methyl-3-buten-2-ol, respectively. It is likely that this dehydratase activity is catalyzed by a not-yet-characterized enzyme postulated for the isomerization of 2-methyl-3-buten-2-ol and prenol. The vitamin requirements of strain L108 growing on TAA and the occurrence of 3-methylcrotonic acid as a metabolite indicate that TAA and hemiterpene degradation are linked with the catabolic route of the amino acid leucine, including an involvement of the biotin-dependent 3-methylcrotonyl coenzyme A (3-methylcrotonyl-CoA) carboxylase LiuBD. Evolutionary aspects of favored desaturase versus hydroxylation pathways for TAA conversion and the possible role of MdpJ in the degradation of higher tertiary alcohols are discussed.

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“…Any TRFLP fragment illustrating an increase in relative abundance in the heavy fraction of the samples (exposed to labeled substrate) compared to the controls (exposed to unlabeled substrate) is identified (using 16S rRNA gene clone libraries) as the putative degrader. The method has been used to identify the microorganisms involved in the degradation of numerous contaminants (4,15,21,34,40,51,58,64,65). In the current study, a range of sources (contaminated site sediment, agricultural soils, and wastewater treatment samples) were examined for anaerobic MTBE degradation potential.…”

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confidence: 99%

Anaerobic Methyl tert -Butyl Ether-Degrading Microorganisms Identified in Wastewater Treatment Plant Samples by Stable Isotope Probing

Sun

Cupples

2012

Appl Environ Microbiol

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ABSTRACTAnaerobic methyltert-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phylaFirmicutes(familyRuminococcaceae) andAlphaproteobacteria(genusSphingopyxis) were the dominant MTBE degraders. Previous studies have suggested a role forFirmicutesin anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (generaMethanosarcinaandMethanocorpusculum) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.

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Identification of tertiary butyl alcohol (TBA)-utilizing organisms in BioGAC reactors using 13C-DNA stable isotope probing

Cited by 19 publications

References 38 publications

Formation of Alkenes via Degradation of tert -Alkyl Ethers and Alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp

Formation of Alkenes via Degradation of tert -Alkyl Ethers and Alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp

Bacterial Degradation of tert-Amyl Alcohol Proceeds via Hemiterpene 2-Methyl-3-Buten-2-ol by Employing the Tertiary Alcohol Desaturase Function of the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

Anaerobic Methyl tert -Butyl Ether-Degrading Microorganisms Identified in Wastewater Treatment Plant Samples by Stable Isotope Probing

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