RESUMEN
A bacterial consortium (Mix3) composed of microorganisms originating from different environments (soils and wastewater) was obtained after enrichment in the presence of a mixture of 16 hydrocarbons, gasoline, and diesel oil additives. After addition of the mixture, the development of the microbial composition of Mix3 was monitored at three different times (35, 113, and 222 days) using fingerprinting method and dominant bacterial species were identified. In parallel, 14 bacteria were isolated after 113 days and identified. Degradation capacities for Mix3 and the isolated bacterial strains were characterized and compared. At day 113, we induced the expression of catabolic genes in Mix3 by adding the substrate mixture to resting cells and the metatranscriptome was analyzed. After addition of the substrate mixture, the relative abundance of Actinobacteria increased at day 222 while a shift between Rhodococcus and Mycobacterium was observed after 113 days. Mix3 was able to degrade 13 compounds completely, with partial degradation of isooctane and 2-ethylhexyl nitrate, but tert-butyl alcohol was not degraded. Rhodococcus wratislaviensis strain IFP 2016 isolated from Mix3 showed almost the same degradation capacities as Mix3: these results were not observed with the other isolated strains. Transcriptomic results revealed that Actinobacteria and in particular, Rhodococcus species, were major contributors in terms of total and catabolic gene transcripts while other species were involved in cyclohexane degradation. Not all the microorganisms identified at day 113 were active except R. wratislaviensis IFP 2016 that appeared to be a major player in the degradation activity observed in Mix3.
Asunto(s)
Actinobacteria/metabolismo , Hidrocarburos/metabolismo , Consorcios Microbianos , Actinobacteria/clasificación , Actinobacteria/aislamiento & purificación , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Perfilación de la Expresión Génica , Metagenómica/métodos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Factores de TiempoRESUMEN
Two strains, identified as Rhodococcus wratislaviensis IFP 2016 and Rhodococcus aetherivorans IFP 2017, were isolated from a microbial consortium that degraded 15 petroleum compounds or additives when provided in a mixture containing 16 compounds (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, octane, hexadecane, 2,2,4-trimethylpentane [isooctane], cyclohexane, cyclohexanol, naphthalene, methyl tert-butyl ether [MTBE], ethyl tert-butyl ether [ETBE], tert-butyl alcohol [TBA], and 2-ethylhexyl nitrate [2-EHN]). The strains had broad degradation capacities toward the compounds, including the more recalcitrant ones, MTBE, ETBE, isooctane, cyclohexane, and 2-EHN. R. wratislaviensis IFP 2016 degraded and mineralized to different extents 11 of the compounds when provided individually, sometimes requiring 2,2,4,4,6,8,8-heptamethylnonane (HMN) as a cosolvent. R. aetherivorans IFP 2017 degraded a reduced spectrum of substrates. The coculture of the two strains degraded completely 13 compounds, isooctane and 2-EHN were partially degraded (30% and 73%, respectively), and only TBA was not degraded. Significant MTBE and ETBE degradation rates, 14.3 and 116.1 mumol of ether degraded h(-1) g(-1) (dry weight), respectively, were measured for R. aetherivorans IFP 2017. The presence of benzene, toluene, ethylbenzene, and xylenes (BTEXs) had a detrimental effect on ETBE and MTBE biodegradation, whereas octane had a positive effect on the MTBE biodegradation by R. wratislaviensis IFP 2016. BTEXs had either beneficial or detrimental effects on their own degradation by R. wratislaviensis IFP 2016. Potential genes involved in hydrocarbon degradation in the two strains were identified and partially sequenced.
Asunto(s)
Gasolina , Hidrocarburos , Petróleo/metabolismo , Rhodococcus/aislamiento & purificación , Rhodococcus/metabolismo , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , Biodegradación Ambiental , Francia , Hidrocarburos/química , Hidrocarburos/metabolismo , Datos de Secuencia Molecular , Nitratos , Rhodococcus/genética , Alcohol terc-Butílico/metabolismoRESUMEN
Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C2 to C16). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C1 to C16) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.
Asunto(s)
Alcanos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Mycobacterium/metabolismo , Alcohol terc-Butílico/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , ADN Bacteriano/genética , ADN Ribosómico/genética , Expresión Génica , Genoma Bacteriano , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mycobacterium/clasificación , Mycobacterium/genética , Mycobacterium/crecimiento & desarrollo , Oxidación-Reducción , Filogenia , ARN Ribosómico 16S/genética , Alineación de SecuenciaRESUMEN
A molecular characterization of pristine and petroleum hydrocarbon-contaminated Alpine soils sampled in Tyrol (Austria) was performed. To identify predominant bacteria, PCR-amplified 16S rRNA gene fragments from five pristine and nine contaminated soils were analysed using denaturing gradient gel electrophoresis (DGGE). Sequencing and phylogenetic analyses demonstrated that the majority of the DGGE bands represented bacteria in the Actinobacteria and Proteobacteria phyla: 18 and 73%, respectively, in pristine soils, compared with 20 and 76%, respectively, in contaminated soils. A different distribution pattern of bacterial classes in the Proteobacteria was observed between pristine and contaminated soils. The relative proportion of microorganisms belonging to the Alphaproteobacteria was larger in pristine (46%) than in contaminated (24%) soils, while Betaproteobacteria and Gammaproteobacteria were detected only in the hydrocarbon-contaminated soils. This result compared favourably with earlier work in which hydrocarbon-degradation genotypes, largely pseudomonads and Acinetobacter, belonging to the Gammaproteobacteria, were enriched following oil hydrocarbon contamination. In contrast, members of the Actinobacteria phylum, represented by Rhodococcus and Mycobacterium, were found in pristine soils where contamination events had not occurred. The results demonstrate a significant shift in the microbial community structure in Alpine soils following contamination. Furthermore, more potentially novel phylotypes were found in the pristine soils than in the contaminated soils.
Asunto(s)
Actinobacteria/clasificación , Hidrocarburos/metabolismo , Filogenia , Proteobacteria/clasificación , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , Actinobacteria/genética , Altitud , Austria , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Ecosistema , Electroforesis en Gel de Poliacrilamida/métodos , Datos de Secuencia Molecular , Petróleo/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Proteobacteria/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Methyl tert-butyl ether (MTBE) is a persistent pollutant of surface and groundwater, and the reasons for its low biodegradability are poorly documented. Using one of the rare bacterial strains able to grow in the presence of MTBE, Mycobacterium austroafricanum IFP 2012, the protein profiles of crude extracts after growth in the presence of MTBE and glucose were compared by SDS-PAGE. Ten proteins with molecular masses of 67, 64, 63, 55, 50, 27, 24, 17, 14 and 11 kDa were induced after growth in the presence of MTBE. Partial amino acid sequences of N-terminal and internal peptide fragments of the 64 kDa protein were used to design degenerate oligonucleotide primers to amplify total DNA by PCR, yielding a DNA fragment that was used as a probe for cloning. A two-step cloning procedure was performed to obtain a 10 327 bp genomic DNA fragment containing seven ORFs, including a putative regulator, mpdR, and four genes, mpdC, orf1, mpdB and orf2, in the same cluster. The MpdB protein (64 kDa) was related to a flavoprotein of the glucose-methanol-choline oxidoreductase family, and the MpdC protein (55 kDa) showed a high similarity with NAD(P) aldehyde dehydrogenases. Heterologous expression of these gene products was performed in Mycobacterium smegmatis mc2 155. The recombinant strain was able to degrade an intermediate of MTBE biodegradation, 2-methyl 1,2-propanediol, to hydroxyisobutyric acid. This is believed to be the first report of the cloning and characterization of a cluster of genes specifically involved in the MTBE biodegradation pathway of M. austroafricanum IFP 2012.