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Assessment of in situ biodegradation of fuel additives (mtbe, etbe) using innovative concepts based on stable isotope labelled substrates

Final Activity Report Summary - FUEL OXYGENATES SIP (Assessment of in situ biodegradation of fuel additives (MTBE, ETBE) using innovative concepts based on stable isotope labelled substrates)

Numerous high polluted gasoline-impacted sites have been identified in Europe in the last years, particularly in Germany, rendering obvious how important knowledge of gasoline additives natural attenuation processes is with regard to cost-effective management of contaminated sites. In that sense, the main scientific aim of the project was the assessment of in situ biodegradation of fuel oxygenates in contaminated groundwater tables.

This purpose was accomplished by the application of innovative methodologies, such as compound-specific stable isotope analysis (CSIA) and protein - stable isotope probing (SIP) on microcosm experiments with pure and mixed bacterial cultures which were able to grow on or co-metabolically degrade the three different fuel oxygenates, namely methyl tertiary butyl ether (MTBE), ethyl tertiary butyl ether (ETBE) and tertiary amyl methyl ether (TAME) under different oxygen levels, i.e. for both oxic and hypoxic conditions.

The results of this study suggested a link between stable isotope fractionation values and different enzymatic mechanisms for aerobic biodegradation of fuel ethers, as well as a correlation of those ones with the identification of metabolically active microbial key players in microcosms from gasoline contaminated sites.

Moreover, new lines of research were started, focussing on the possible impact of the reduced oxygen levels that were typically found in polluted aquifers on MTBE degradation rates and isotopic enrichment factors. Oxygen concentration was rapidly depleted in fuel contaminated aquifers and MTBE degrading aerobic strains had to cope with oxygen limiting conditions. Therefore, knowledge on the variability of isotope fractionation under hypoxic conditions was needed for a proper quantification of fuel oxygenate biodegradation employing CSIA. Although individual enrichment factors of the two model bacterial strains were not significantly affected by these conditions, the experiments showed indirect effects, caused by competition interactions, when they were together in a mixture. The higher oxygen affinities that were observed for the low fractionating model strain due to related enzyme made these bacteria more competitive to degrade MTBE versus the ones incredibly reducing the total observed isotope enrichment factor in a mixture.

These first studies supported the assumption that truly oxic environments were not an essential requirement to degrade fuel oxygenates and explain discrepancies or low fractionation values found in some field studies. In addition to the protein-based stable isotope probing (protein-SIP) experiments, this part of the study suggested a tendency of the fuel ethers degraders’ community to be dominated by one or few species which became more competitive under specific conditions.