Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CH4BioVal (Biological Valorization of Methane Emissions)

Teaser

CH4BioVal has the aim of contributing to the development of a sustainable society by exploring the possibility of using methane as a substrate for the production of high added-value chemicals and commodities.Methane is a green-house gas, which has a global warming potential 80...

Summary

CH4BioVal has the aim of contributing to the development of a sustainable society by exploring the possibility of using methane as a substrate for the production of high added-value chemicals and commodities.

Methane is a green-house gas, which has a global warming potential 80 times that of CO2. More than 70 million tonnes of methane are being released annually into the atmosphere from anthropogenic sources, such as waste water treatment plants, landfills, mines etc. These emissions, on one hand, have a very negative impact on climate by contributing to global warming, secondly, they suppose a waste of energy and carbon, which could be reused for the fabrication of high added value chemicals. Two of these chemicals, which are the focus of this project, are PHB (a biodegradable plastic) and ectoine (an osmoprotector with uses in the food and cosmetic industry).

The way to profit from the enormous economic potential of the methane that is being released into the atmosphere worldwide, is to use so-called methanotrophic organisms. Those are bacteria that are able to use methane as a carbon and energy source. Some of these organisms produce naturally valuable chemicals. For example bacteria from the genus Methylocystis produce PHB, and the halo-tolerant bacterium Methylomicrobium alcaliphilum produces ectoine.

In order to use methanotrophs as economically sustainable platforms for industrial biotechnology, the perfomance of these organisms has to be improved by increasing the yields of the valuable compounds that they produce naturally, and specially by developing the genetic and bioinformatic tools necessary to expand the currently narrow scope of products that can be produced form methane. A key tool for the achievement of the mentioned goals is the developed of Genome
Scale Metabolic Models (GSMMs), which are comprehensive compilations of all the metabolic reactions that take place in a microorganism. These models allow to predict the outcome of genetic manipulations (gene insertions and knockouts), thus allowing the metabolic engineering of the modelled organisms.

During the course of this project, GSMMs of 6 of the most relevant species of methanotrophs have been constructed and validated, also work on isolating new species of methanotrophs able to produce PHB and ectoine has been carried out, leading to 5 scientific articles already published and two more currently in the process of being submitted.

Work performed

The following lines of work have been pursued during the execution of the project:

1. Isolation of new methantrophs from environmental samples showing methane consumption activity.

2. Reconstruction of Genome Scale Metabolic Models (GSMMs) of methanotrophs of the genus Methylocystis, Methylomicrobium and Methylocella.

3. Validation experiments for the developed GSMMs
4. Development and optimization of genetic engineering techniques for the manipulation of bacteria of the genus Methylocystis and Methylomicrobium.

5. Preparation of a FET project (Future Emerging Technologies), submitted to the 2019 call, and expected to be resubmitted in the 2020 call.

Two secondments have been carried out:

The first one in September of 2018 at the Spanish BioTech company Biopolis S.L., located in Valencia. This colaboration led to the preparation of the mentioned FET project, which was unfortunatelly not funded in the 2019 call, and the publication of the GSMM of the strain Methylocystis parvus, strain that was provided by Biopolis (see list of publications below)

The second secondment, between June and August of 2019 was carried out at the laboratory of Prof. Colin Murrell at the East Anglia University. This led to the reconstruction of the first GSMM of the bacterium Methylocella silvestris. A very versatile methanotroph wich is able not only to use methane as a carbon and energy source, but also other alkanes such as ethane or propane. A paper reporting the results of this colaboration is under preparation.

The project has resulted in the follwing 5 (already published) articles.

1. Bordel S, Rodríguez Y, Hakobyan A, Rodríguez E, Lebrero R, Muñoz R. Genome scale metabolic modeling reveals the metabolic potential of three Type II methanotrophs of the genus Methylocystis. 2019. Metab Eng. 54, 191-199 (IF 7.674)
2. Bordel S, Rojas A, Muñoz R. Reconstruction of a Genome Scale Metabolic Model of the polyhydroxybutyrate producing methanotroph Methylocystis parvus OBBP. 2019. Microb Cell Fact. 18, 104 (IF 3.831)
3. Bordel S, Rodríquez E, Muñoz R. Genome sequence of Methylocystis hirsuta CSC1, a polyhydroxyalkanoate producing methanotroph. 2019. MicrobiologyOpen. 8, e00771 (IF 2.682)
4. Pérez R, Cantera S, Bordel S, García Encina PA, Muñoz R. The effect of temperature dureing culture enrichment on methanotrophic polyhydroxyalkanoate production. 2019. Int. Biodeter. Biodegr. 140, 144-151 (IF 3.562)
5. Cantera S, Bordel S, Lebrero R, Gancedo J, García Encina PA, Muñoz R. Bio-converison of methane to high profit margin compounds: an innovative, environmentally friendly and cost-effective platform for metane abatement. 2019. World J Microbiol Biotechnol. 35, 16 (IF 2.1)

Two more articles are under preparation:
The first of them will describe the mechanisms of adaptation to high salinity of the methanotroph Methylomicrobium alcaliphilum using RNA sequencing.
The second article will present the first GSMM of Methylocella silvestris and is the result of the previously mentioned collaboration with Prof. Colin Murrell at East Anglia University.

Final results

The main results of the project going beyond the state of the art are:

The sequencing and publication of the genome of the methanotroph Methylocystis hirsuta (publication number 3)

The reconstruction and validation of the first GSMMs of Type II methanotrophs (only one model of a Type I methanotroph existed before the starting date of the project). These models have allowed to elucidate the role of the stored PHB in these organisms and to identify the reactions playing anaplerotic roles of the serine cycle during growth on methane and other single carbon compounds. This will facilitate future metabolic engineering strategies and/or selection strategies to improve PHB accumulation (Publications 1 and 2).

In the frame of the collaboration with Prof. Colin Murrell, the first GSMM of methylocella silvestris was reconstructed. This allowed to identify the importance of the glyoxylate shunt played in this organism (in contrast for example to the genus methylocella), which makes it able to grow on substrates of two carbons, such as ethanol or acetate, but also is essential for growth on methane. It was also revealed that Methylocella silvestris degrades propane using two parallel metabolic pathways which could be exploited in the future for the obtenction of chemicals such as lactic acid or high value derivatives of methylmalonyl.

The remaining articles to be published are also expected to clarify the role of specific sodium pumps that allow the adaptation of Methylomicrobium alcaliphilum to high salinity environments as well as to permit the optimization of this strain to produce ectoine and other high value compounds.

Overall we can conclude that key tools for the industrial use of methanotrophs have been developed, provided that this research is continued at a more ambitious scale such as the FET project that was prepared during the fellowship in collaboration with the company Biopolis S.L.

Website & more info

More info: http://ch4bioval.uva.es.