Biodegradability of fuel oxygenates (ETBE and MTBE): Microorganisms-Monooxygenases-Functionality – MiOxyFun

Ethers, methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE), are added to gasoline to enhance the octane index. MTBE and ETBE are highly soluble in water (40 and 10 g.L-1, respectively). Their use at a huge scale required to get information about their fate in the environment and in impacted aquifers. MTBE was found in several aquifers as a contaminant after releases of MTBE-supplemented gasoline due to its poor biodegradability. To our knowledge, the groundwater contamination by ETBE was not documented in the countries that use it, like France and Hungary. Studies are required to understand the environmental impact of MTBE and ETBE and to characterize the microorganisms, enzymes and genes involved in their biodegradation.

The project is based on a unique common collection of MTBE- or ETBE- biodegrading microcosms from different geographic areas that was obtained in France and Hungary. This collection will allow to study:

-The oxygenases responsible for the first enzymatic attack on MTBE or ETBE. We will characterize the biodegradation capacities of the oxygenases active towards ETBE and MTBE by determining (i) the degradation kinetics, (ii) the production of tert-butyl alcohol or TBA, an intermediate of MTBE and ETBE biodegradation, (iii) the interactions with other components of gasoline (mono-aromatics compounds or BTEXs and n-alkanes).
We will also characterize the biodegradation capacities towards a wide range of chemicals with different structures to understand the specificity of the oxygenase and its capacity to attack compounds with a high steric hindrance (tertio-butyl group).
The activity of these oxygenases will also been studied by expressing the genes encoding these oxygenases through heterologous expression in engineered microbial systems which is essential for proving the role and function of enzymes.

-The phylogenic composition of the microcosms (i) by using "Denaturating Gradient Gel Electrophoresis" (DGGE) and (ii) by isolating microorganisms and determining their MTBE or ETBE biodegradation capacities. Then, (iii) the structure of the functional microbial communities will be studied by coupling a SIP (Stable Isotope Probing) approach to NanoSIMS observation, which principle relies on the labelling of the microbial cells with 13C-MTBE or 13C-ETBE; this study will determine which microrganisms metabolize the 13C-labelled substrate in situ and if the microorganisms isolated correspond to those active in situ in the consortia.
Two significant MTBE- or ETBE-degrading microorganisms will be chosen for further genomic DNA sequencing and annotation.

-The genes induced on MTBE or ETBE.
*Different genes involved in the MTBE or ETBE biodegradation are known: (i) ethB encoding a cytochrome P450 oxidizing ETBE, (ii) mdpA, encoding a hydroxylase related to the Alkane Hydroxylases (AHs), (iii) mpdB and mpdC encoding dehydrogenases responsible for the production of 2-hydroxyisobutyric acid (HIBA) in the TBA pathway and (iv) icmA encoding a mutase responsible for HIBA assimilation. We will search for the presence of this panel of genes in the microcosms or strains isolated. Then, we will study their differential expression during growth on MTBE or ETBE by comparison to a classical substrate (RTqPCR).
*A transcriptomic study will be carried out using high throughput RTqPCR (OpenArray™ nanocapillary RTq-PCR system) on the two strains sequenced. This will determine the pool of genes over-expressed in the presence of MTBE or ETBE.

To our knowledge, such an integrated study (microorganisms, oxygenases and genes) was never carried out previously on a collection of MTBE and ETBE-degrading microcosms from different origins. It will allow to get an important databank on the diversity of MTBE-and ETBE-biodegraders, the mode of action of the oxygenases and the genes involved in the MTBE- and ETBE- biodegradtion and will bring new insights on the environmental impact of the fuel oxygenates.