Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L108

Abstract

Growth of Aquincola tertiaricarbonis L108 on the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), as well as on their main metabolites tert-butyl alcohol (TBA), tert-amyl alcohol (TAA) and 2-hydroxyisobutyrate (2-HIBA) was systematically investigated to characterize the range and rates of oxygenate degradation by this strain. The effective maximum growth rates for MTBE, ETBE and TAME at pH 7 and 30 6C were 0.045 h "1 , 0.06 h "1 and 0.055 h "1, respectively, whereas TAA, TBA and 2-HIBA permitted growth at rates up to 0.08 h , respectively. The experimental growth yields with all these substrates were high. Yields of 0.55 g dry mass (dm) (g MTBE) "1 , 0.53 g dm (g ETBE) "1 , 0.81 g dm (g TAME) , and 3.27 mmol 2-HIBA h "1 g "1. The relatively high rates with TBA, TAA and 2-HIBA indicate that the transformations of these metabolites did not limit the metabolism of MTBE and the related ether compounds. Despite the fact that these metabolites still carry a tertiary carbon atom that is commonly suspected to confer recalcitrance to the ether oxygenates, the transformation rates were in the same range as those with succinate and fructose. With MTBE, strain L108 grew at pHs between 5.5 and 8.0 at near-maximal rate, whereas no growth was found below pH 5.0 and above pH 9.0. The optimum growth temperature was 30 6C, but at 5 6C still about 15 % of the maximum rate remained, whereas no growth occurred at 42 6C. This indicates that MTBE metabolites are valuable substrates and that A. tertiaricarbonis L108 is a good candidate for bioremediation purposes. The possible origin of its exceptional metabolic capability is discussed in terms of the evolution of enzymic activities involved in the conversion of compounds carrying tertiary butyl groups. INTRODUCTIONMethyl tert-butyl ether (MTBE) and the related compounds ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) are widely used as oxygenating compounds in gasoline (Krayer von Krauss & Harremoës, 2001), leading to pollution by unnoticed leakages and accidental spills (Baehr et al., 1999;Klinger et al., 2002; Schmidt et al., 2002;Squillace et al., 1996). They threaten water resources by their unpleasant odour and taste and suspected carcinogenicity (McGregor, 2006). Consequently, much effort is put into studying the environmental fate of these compounds and developing measures against fuel oxygenate pollution. Microbial degradation has been considered in both respects (Deeb et al., 2000;Fayolle et al., 2001;Schmidt et al., 2004). However, MTBE and structurally related compounds were initially found to withstand microbial attack. This was thought to be mainly due to the ether bond in these compounds and the presence of a tertiary carbon atom.Generally, it has proved difficult to isolate strains from enrichment cultures using MTBE as sole carbon and energy source. Attempts for more than 15 years were of limited success. At present, there are only a few strains capable of growing solely on oxygenates. The...

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

“…S3 to S7 in the supplemental material). Diols and carboxylic acids were quantified by using high-performance liquid chromatography (HPLC) with refractive index (RI) detection as described elsewhere previously (30,31), applying an eluent of 0.01 N sulfuric acid at 0.6 ml per min and a Nucleogel Ion 300 OA column (300 by 7.7 mm; Macherey-Nagel). In addition, carboxylic acid metabolites were identified as methyl esters by GC mass spectrometry (see the supplemental material).…”

“…Liquid and gas samples were taken as previously described (38), by puncturing the butyl rubber stoppers of incubation bottles with syringes equipped with 0.6-by 30-mm Luer Lock needles. The biomass was monitored by measuring the optical density at 700 nm (OD 700 ), using a multiplication factor of 0.54 for calculating the dry biomass in g per liter (31). Volatile compounds (MTBE, TAME, TAEE, TBA, TAA, isoamylene, isoprene, 2-methyl-3-buten-2-ol, prenol, prenal, 3-methyl-3-pentanol, 3-methyl-1-penten-3-ol, and methylacetoin) were quantified by headspace gas chromatography (GC) using flame ionization detection (FID) (38).…”

“…Proposed pathways for the bacterial degradation of tert-butyl alkyl (MTBE and ETBE) and tert-amyl alkyl (TAME and TAEE) ethers (27,31). (A) Initially, the methyl/ethyl groups of the ethers are hydroxylated, resulting in the formation of instable hemiacetals (not shown).…”

“…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). However, the hydroxylation of TAA by MdpJ or other bacterial tert-alcohol monooxygenases has not been investigated so far.…”

Éter típusú üzemanyag-adalékok mikrobiális bontása

“…Kemotaxonómiai, fiziológiai és DNS-alapú vizsgálatok alapján a három törzs ugyanabba a fajba, az Aquincola tertiaricarbonis-ba tartozik [120]. Az ismert mikrobák közül az Aquincola tertiaricarbonis L108 rendelkezik a legnagyobb potenciállal az éter típusú üzemanyag-adalékok lebontásában, lévén az MTBE, az ETBE, a TAME, a TAEE és a DIPE, valamint legfontosabb metabolitjaik, a TBA, a TAA és a 2-HIBA nagy hatékonyságú degradációjára képes az abiotikus környezeti tényezők (hőmérséklet, pH) széles tartományában [121,122].…”