Research Projects

Logos GenomEnviron and Ampere
Laboratoire Ampere
Ecole Centrale de Lyon . France
(33) 472 18 65 14

     

 


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

 

Coordinator:
Françoise Fayolle-Guichard (IFPEN)
Funding Source:

ANR for France and NIH for Hungary

Period:

1 Jan 2011 to 31 Dec. 2013

Partners:

Abstracts:

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.

Objectives:

From a collection of microcosms with MTBE-or ETBE-degradation capacities as a point of departure, the global objective of the project is to obtain, through an integrated research program, a detailed bank of data on the structure of microbial populations under such a selective pressure. Intermediate objectives will help achieving this aim: -The phylogenic analysis of the microcosms with MTBE- or ETBE- degradation capacities will allow determining the relative occurrence of the different groups of microorganisms that are present in microcosms with MTBE or ETBE biodegradation capacities. More specifically, such a study will determine if there is a specific microbial pattern arising from the phylogenic analysis of microcosms isolated from different geographic area (by DGGE and isolation studies). Another important question concerns the possible involvement of non-cultivable microorganisms in MTBE- or ETBE-biodegradation and it will be addressed by a coupled SIP/NanoSIMS approach using 13C-labelled MTBE or ETBE. Two significant microorganisms with either MTBE or ETBE biodegradation capacities will be sequenced providing new insights in these specific biodegradation pathways. -The study of the presence and expression of specific genes in the microcosms and in the strains isolated will be carried out in two ways: (i) the presence and the mode of expression of genes that are already known will be systematically investigated in microcosms and in microorganisms isolated to determine which specific pattern of genes is selected as a consequence of MTBE or ETBE pollution. (ii) moreover, the genomic DNA of a MTBE-degrading and an ETBE-degrading microorganisms will be sequenced and we will study the transcriptome of the two sequenced strains. This will also provide numerous new data on the MTBE and ETBE biodegradation pathways by determining the pool of genes over-expressed during MTBE or ETBE biodegradation. -Regarding the capacities of the oxygenases involved in the initial attack on MTBE and ETBE, we will be able to determine their biodegradation capacities and specificities towards a wide range of substrate, including MTBE and ETBE. Thus, we will obtain data regarding the oxygenases and better understand their way of MTBE and ETBE attack. Due to the significant environmental role of oxygenases in the biodegradation of xenobiotics, it is important to go on gathering data on this class of enzymes. By testing these enzymes towards a wider range of substrates, we will be able to determine their specificity. Moreover, these oxygenase will be studied through heterologous expression for their capacities towards numerous other substrates with various chemical structures, similar or different than those of MTBE or ETBE. It will allow determining if some of these oxygenases have potential in the transformation of chemicals to biotechnologically interesting molecules. The design of new bacterial systems (engineered plasmids/microorganisms) to investigate the heterologous expression of genes is very important to develop for further ecological studies. By the end of the project, we will have a significant bank of data concerning the MTBE or ETBE degradation capacities at different biological levels (microorganisms, enzymes and genes) obtained from microcosms of different geographic origins. This database will greatly enrich the knowledge on the ecology of MTBE-and ETBE-biodegraders and will help describing the role of a selective pressure. An additional objective of the project is to provide useful molecular tools as DNA microarrays (16SrDNA specific sequences, genes sequences) for the diagnostic of MTBE- or ETBE-polluted sites

Description
of the work:

-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.

Deliverables:

Microorganisms isolated from MTBE & ETBE degrading consortia

Biodegradation capacity of MTBE and ETBE of the different consortia and pure strains isolated

Phylogenic composition of MTBE- and ETBE-degrading consortia

Evaluation the capacities of the MTBE- and ETBE-monooxygenases towards a wide range of substrates

Genes involved in the biodegradation pathways of ETBE & MTBE

 

Sequencing & annotation of 2 relevant degraders

Identification of the microbial consortia with NanoSIMSISH

RTqPCR expression of identified genes

Transcriptomic study of the two strains sequenced

Results of the heterologous expression of genes in engineered microbial systems.

 

Publications & Communications:

Project website: MiOxyFun

 

 
     

MiOxyFun