Assessment of MTBE biodegradation pathways by two-dimensional isotope analysis in mixed bacterial consortia under different redox conditions.

The fuel oxygenate, methyl tert-butyl ether (MTBE), although now widely banned or substituted, remains a persistent groundwater contaminant. Multidimensional compound-specific isotope analysis (CSIA) of carbon and hydrogen is being developed for determining the extent of MTBE loss due to biodegradation and can also potentially distinguish between different biodegradation pathways. Carbon and hydrogen isotopic fractionation factors were determined for MTBE degradation in aerobic and anaerobic laboratory cultures. The carbon isotopic enrichment factor (epsilonC) for aerobic MTBE degradation by a bacterial consortium containing the aerobic MTBE-degrading bacterium, Variovorax paradoxus, was -1.1 +/- 0.2 per thousand and the hydrogen isotope enrichment factor (epsilonH) was -15 +/- 2 per thousand. This corresponds to an approximated lambda value (Lambda = epsilonH/epsilonC) of 14. Carbon isotope enrichment factors for anaerobic MTBE-degrading enrichment cultures were -7.0 +/- 0.2 per thousand and did not vary based on the original inoculum source, redox condition of the enrichment, or supplementation with syringic acid as a co-substrate. The hydrogen enrichment factors of cultures without syringic acid were insignificant, however a strong hydrogen enrichment factor of -41 +/- 3 per thousand was observed for cultures which were fed syringic acid during MTBE degradation. The Lambda = 6 obtained for NYsyr cultures might be diagnostic for the stimulation of anaerobic MTBE degradation by methoxylated compounds by an as yet unknown pathway and mechanism. The stable-isotope enrichment factors determined in this study will enhance the use of CSIA for monitoring anaerobic and aerobic MTBE biodegradation in situ.

Community characterization of anaerobic methyl tert-butyl ether (MTBE)-degrading enrichment cultures.

Use of the fuel oxygenate methyl tert-butyl ether (MTBE) has led to widespread environmental contamination. Anaerobic biodegradation of MTBE observed under different redox conditions is a potential means for remediation of contaminated aquifers; however, no responsible microorganisms have been identified as yet. We analyzed the bacterial communities in anaerobic-enriched cultures originating from three different contaminated sediments that have retained MTBE-degrading activity for over a decade. MTBE was transformed to tert-butyl alcohol and the methyl group used as a carbon and energy source. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes showed that the MTBE-utilizing microcosms established from different sediment sources had substantially different community profiles, suggesting that multiple species are capable of MTBE biodegradation. The 16S rRNA genes from one enrichment culture were cloned and sequenced. Phylogenetic analysis showed a diverse community, with phylotypes belonging to the Proteobacteria, Bacteroidetes, Firmicutes, Chloroflexi and Thermotogae. Continued enrichment on MTBE further reduced the community to three predominant phylotypes, as evidenced by T-RFLP analysis, which were most closely related to the Deltaproteobacteria, Firmicutes and Chloroflexi. These three common operational taxonomic units were detectable in the enrichments from Atlantic and Pacific coastal samples. Identification of the microorganisms important in mediating anaerobic MTBE transformation will provide the foundation for developing tools for site assessment and bioremediation monitoring.

Effects of co-substrates and inhibitors on the anaerobic O-demethylation of methyl tert-butyl ether (MTBE).

Methyl tert-butyl ether (MTBE) contamination is widespread in aquifers near urban areas around the world. Since this synthetic fuel oxygenate is resistant to most physical methods of treating fuel-contaminated water, biodegradation may be a useful means of remediation. Currently, information on anaerobic MTBE degradation is scarce. Depletion has been observed in soil and sediment microcosms from a variety of locations and under several redox conditions, but the responsible organisms are unknown. We are studying anaerobic consortia, enriched from contaminated sediments for MTBE-utilizing microorganisms for over a decade. MTBE degradation occurred in the presence of other fuel components and was not affected by toluene, benzene, ethanol, methanol, or gasoline. Many aryl O-methyl ethers, such as syringic acid, that are O-demethylated by acetogenic bacteria, were also O-demethylated by the MTBE-utilizing enrichment cultures. The addition of these compounds as co-substrates increased the rate of MTBE degradation, offering a potentially useful method of stimulating the MTBE degradation rate in situ. Propyl iodide caused light-reversible inhibition of MTBE degradation, suggesting that the MTBE degradation process is corrinoid dependent. The anaerobic MTBE degradation process was not directly coupled to methanogenesis or sulfidogenesis and was inhibited by the bactericidal antibiotic, rifampicin. These results suggest that MTBE degradation is mediated by acetogenic bacteria.

Swapping the gene-specific and regional silencing specificities of the Hst1 and Sir2 histone deacetylases.

Sir2 and Hst1 are NAD(+)-dependent histone deacetylases of budding yeast that are related by strong sequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repression. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2 interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomere chromosomal regions. To identify the determinants of gene-specific versus regional repression, we created a series of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform both regional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereas gene-specific repression required Rfm1 and Sum1. Our findings demonstrate that the nonconserved N-terminal region and two amino acids within the enzymatic core domain account for cofactor specificity and proper targeting of these proteins. These results suggest that the differences in the silencing and repression functions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather may be the result of distinct cofactor specificities.