The effect of fungal hyphae on the mobilization of soil-dwelling bacteria and their access to hydrophobic phenanthrene in soil was tested in columns containing air-filled agricultural soil. The experimental design included a spatial separation between zones of bacterial inoculation and contamination. Motile Pseudomonas putida PpG7 (NAH7) and fast-growing, hydrophilic Pythium ultimum were used as the model phenanthrene-degrading and vector organisms, respectively. Efficient translocation of strain PpG7 in the range of centimetres in presence of P. ultimum indicated that the fungal mycelia bridged air-filled pores and thereby provided a continuous network of water-paths that enabled bacteria to spread in the soil. Biodegradation of the soil-associated phenanthrene was found only in the presence of the fungal mycelia, hence proving that the fungal network facilitated the access of the bacteria to the contaminant. Our data suggest that the specific stimulation of indigenous fungi is a promising method to mobilize pollutant degrading bacteria and thereby improve soil bioremediation in-situ.

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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...

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.

The microbial degradation of tert‐butyl alcohol (TBA), an important environmental pollutant and an intermediate in the degradation of methyl tert‐butyl ether (MTBE), was proposed to involve a monooxygenase for the initial oxidation of TBA, but up to now a specific enzyme with that activity has not been described except the well‐known AlkB for the Gram‐positive strain Mycobacterium austroafricanum IFP2012 (Lopez Ferreira et al., Appl. Microbiol. Biotechnol. 2007, 75, 909–919). In the course of our studies of the MTBE pathway, the proteome patterns in one‐ and two‐dimensional gels of Aquincola tertiaricarbonis L108 which was grown on lactate, on hydroxyisobutyrate (2‐HIBA) and TBA, were compared. A protein of about 55 kDa was detected after growth on TBA and 2‐HIBA, which, after mass spectrometric analysis of the tryptic digested peptides, was assigned with a high score to phthalate dioxygenase. Sequence analysis of PCR products obtained with primers derived from the amino acid sequences in the above peptides supported the assignment to the hydroxylase subunit of phthalate dioxygenase‐like proteins by covering 96.7 % of a corresponding gene from Methylibium petroleiphilum PM1. The conserved amino acid motifs ‐R‐x12‐CxHRxxxLxxG‐x8‐CxYHR‐x6‐G‐ for the Rieske [2Fe‐2S] binding domain and (‐D/E)xxxDxxHxxxxH‐ for the mononuclear iron binding domain were found. A second protein of about 38 kDa was detected after growth on TBA with a lower score and attributed to a putative iron‐sulfur oxidoreductase subunit. Primers derived from the peptides resulted in an amplicon, which covered 75.7 % of a corresponding gene from M. petroleiphilum PM1. Conserved motifs ‐RxYSL‐x20‐22‐RGGS‐ for FMN binding and ‐GGIGxTPxxxM‐ for NAD binding were detected, which suggests that this protein is the small subunit of a two‐component phthalate dioxygenase‐like enzyme typically containing FMN. Dioxygenase‐related enzymes are known to catalyze also monooxygenase reactions (see e.g. Zhou et al. J. Bacteriol. 2002, 184, 1547–1555), which makes it likely that the two proteins induced in the presence of TBA are involved in TBA oxidation.