Physico-chemical and biological characterization of an aquifer polluted with ETBE

International audiencePetroleum compounds and among them, gasoline, is the most massively used chemicals worldwide and, as a consequence gasoline derives compounds are the most frequently found contaminants in groundwate

Catabolisme du méthyl tert-butyl éther (MTBE) (caractérisation des enzymes impliquées dans la dégradation du MTBE chez Mycobacterium austroafricanum IFP 2012)

PARIS-AgroParisTech Centre Paris (751052302) / SudocSudocFranceF

Cytochromes P450-mediated degradation of fuel oxygenates by environmental isolates

International audienceThe degradation of fuel oxygenates [methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME)] by Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005 and Gordonia sp. IFP 2009 (formerly Mycobacterium sp.) isolated from different environments was compared. Strains IFP 2001, IFP 2005 and IFP 2009 grew on ETBE due in part to the activity of a cytochrome P450, CYP249. All of these strains were able to degrade ETBE to tert-butyl alcohol and are harboring the CYP249 cytochrome P450. They were also able to degrade MTBE and TAME, but ETBE was degraded in all cases most efficiently, with degradation rates measured after growth on ETBE of 2.1, 3.5 and 1.6 mmol ETBE g-1 dry weight h-1 for strains IFP 2001, IFP 2005 and IFP 2009, respectively. The phylogenetic relationships between the different ethR (encoding the regulator) and ethB (encoding the cytochrome P450) genes were determined and showed high identity between different ethB genes (> 99%). Only ETBE was able to induce the expression of ethB in strains IFP 2001 and IFP 2005 as measured by reverse transcriptase quantitative PCR. Our results are a first indication of the possible role played by the ethB gene in the ecology of ETBE degradatio

DISTRIBUTION AND REGULATION OF CYTOCHROMES P450 INVOLVED IN FUEL OXYGENATES DEGRADATION

International audienceWe compared the degradation capacity towards fuel oxygenates (methyl tert-butyl ether or MTBE, ethyl tert-butyl ether or ETBE and tert-amyl methyl ether or TAME) of R. ruber IFP 2001, R. zopfii IFP 2005, Mycobacterium sp. IFP 2009 and R. erythropolis NI86/21. Strains IFP 2001, IFP 2005 and IFP 2009 are able to grow on ETBE due to the activity of a cytochrome P450, CYP249 and strain NI86/21 was able to grow on an herbicide, S-ethyl dipropylthiocarbamate or EPTC due to the activity of another cytochrome P450, CYP116. We demonstrated that the cytochrome involved in EPTC biodegradation was also able to degrade ETBE to tert-butyl alcohol (TBA). All the strains harbouring CYP249 were also able to degrade MTBE and TAME but the preferred substrate was ETBE in all cases with specific activity measured after growth on ETBE of 2.1, 3.5 and 1.6 mmoles ether g-1dry weight h-1 for strain IFP 2001, IFP 2005 and IFP 2009, respectively. We also investigated the induction of the eth genes in the R. ruber IFP2001 and determined that only ETBE was able to induce the system and that this induction was inhibited in presence of easier substrate, implying a catabolite repression system. Using alignments, we spotted several mutations in the regulators EthR and the cytochromes EthB itself which might explain the differences observed in the degradation rates. Our results show that the specificity of the eth cytochrome system is rather due to the regulator itself than to the specificity of the cytochrome

Traitement biologique in situ au sein d'un aquifère de polluants de type ETBE et MTBE

National audienceLe MtBE et l' EtBE sont des composés chimiques utilisés dans les essences reformulées sans plomb. Leurs propriétés physico-chimiques, forte solubilité dans l'eau et faible biodégradabilité naturelle, se traduisent par une accumulation importante dans les eaux souterraines, entraînant des risques de contamination et des problèmes d'utilisation de la ressource. Ces composés sont persistants dans l'environnement et leur toxicité est, par ailleurs, débattue. De ce fait la connaissance de leur devenir dans l'environnement dans les cas de pollutions accidentelles ou chroniques par des essences reformulées, nécessite la mise en place d'études spécifiques

Ethyl tert-butyl ether (ETBE) biodegradation by a syntrophic association of Rhodococcus sp. IFP 2042 and Bradyrhizobium sp. IFP 2049 isolated from a polluted aquifer

International audienceEthyl tert-butyl ether (ETBE) enrichment was obtained by adding contaminated groundwater to a mineral medium containing ETBE as the sole carbon and energy source. ETBE was completely degraded to biomass and CO2 with a transient production of tert-butanol (TBA) and a final biomass yield of 0.37 ± 0.08 mg biomass (dry weight).mg−1 ETBE. Two bacterial strains, IFP 2042 and IFP 2049, were isolated from the enrichment, and their 16S rRNA genes (rrs) were similar to Rhodococcus sp. (99 % similarity to Rhodococcus erythropolis) and Bradyrhizobium sp. (99 % similarity to Bradyrhizobium japonicum), respectively. Rhodococcus sp. IFP 2042 degraded ETBE to TBA, and Bradyrhizobium sp. IFP 2049 degraded TBA to biomass and CO2. A mixed culture of IFP 2042 and IFP 2049 degraded ETBE to CO2 with a biomass yield similar to the original ETBE enrichment (0.31 ± 0.02 mg biomass.mg−1 ETBE). Among the genes previously described to be involved in ETBE, MTBE, and TBA degradation, only alkB was detected in Rhodococcus sp. IFP 2042 by PCR, and none were detected in Bradyrhizobium sp. IFP 2049

Aquincola tertiaricarbonis gen. nov., sp nov., a tertiary butyl moiety-degrading bacterium

International audienceStrains L10T, L108 and CIP I-2052 were originally obtained from methyl tert-butyl ether (MTBE)-contaminated groundwater and from a wastewater treatment plant, respectively. All share the ability to grow on tert-butanol, an intermediate of MTBE degradation. Cells are strictly aerobic, motile by a polar flagellum and exhibit strong pili formation. Poly beta-hydroxybutyrate (PHB) granules are formed. The DNA G+C content is 69–70.5 mol% and the main ubiquinone is Q-8. The major cellular fatty acids are 16 : 1 cis-9 and 16 : 0 and the only hydroxy fatty acid is 10 : 0 3-OH. The major phospholipids are phosphatidylethanolamine (PE) 16 : 1/16 : 1 and phosphatidylglycerol 16 : 0/16 : 1. A significant amount of PE 17 : 0/16 : 1 is present. The 16S rRNA gene sequences of these strains are almost identical and form a separate line of descent in the Rubrivivax–Roseateles–Leptothrix–Ideonella–Aquabacterium branch of the Betaproteobacteria with 97 % similarity to 16S rRNA genes of the type strains of Rubrivivax gelatinosus, Leptothrix mobilis and Ideonella dechloratans. However, physiological properties, DNA–DNA relatedness values and the phospholipid and cellular fatty acid profiles distinguish the novel isolates from the three closely related genera. Therefore, it is concluded that strains L10T, L108 and CIP I-2052 represent a new genus and novel species for which the name Aquincola tertiaricarbonis gen. nov., sp. nov., is proposed. The type strain is strain L10T (=DSM 18512T=CIP 109243T)

Study of an aquifer contaminated by ethyl tert-butyl ether (ETBE): Site characterization and on-site bioremediation

International audienceEthyl tert-butyl ether (ETBE) was detected at high concentration (300 mg L−1) in the groundwater below a gas-station. No significant carbon neither hydrogen isotopic fractionation of ETBE was detected along the plume. ETBE and BTEX biodegradation capacities of the indigenous microflora Pz1-ETBE and of a culture (MC-IFP) composed of Rhodococcus wratislaviensis IFP 2016, Rhodococcus aetherivorans IFP 2017 and Aquincola tertiaricarbonis IFP 2003 showed that ETBE and BTEX degradation rates were in the same range (ETBE: 0.91 and 0.83 mg L−1 h−1 and BTEX: 0.64 and 0.82 mg L−1 h−1, respectively) but tert-butanol (TBA) accumulated transiently at a high level using Pz1-ETBE (74 mg L−1). An on-site pilot plant (2 m3) filled with polluted groundwater and inoculated by MC-IFP, successfully degraded four successive additions of ETBE and gasoline. However, an insignificant ETBE isotopic fractionation was also accompanying this decrease which suggested the involvement of low fractionating-strains using EthB enzymes, but required of additional proofs. The ethB gene encoding a cytochrome P450 involved in ETBE biodegradation (present in R. aetherivorans IFP 2017) was monitored by quantitative real-time polymerase chain reaction (q-PCR) on DNA extracted from water sampled in the pilot plant which yield up to 5 × 106 copies of ethB gene per L−1

Summary of raw and assembled data obtained in the six 454 pyrosequencing runs.

<p>Summary of raw and assembled data obtained in the six 454 pyrosequencing runs.</p