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ANR funded project

Bio-Matières et Energies (Bio-ME)
Edition 2012


Biomines


Mining of the microbial biodiversity for the identification of new enzymes and strains for CBP

Identification of novel biocatalysts for biomass degradation
The Biomines project plans to explore natural environments specialised in plant biomass degradation, by using both microbiological and molecular approaches. The project aims at identifying new enzymes and bacterial strains able to efficiently degrade lignocellulose, for a potential future use in second generation bioethanol production plant.

Improving the efficiency of lignocellulose hydrolysis by new enzymes and strains
The development of second generation biofuels, produced from lignocellulosic biomass, is a promising alternative to existing technologies, allowing both the reduction of greenhouse gas emissions and avoiding competition with food production. However, present bioethanol production schemes from these biomass sources are currently too expensive to constitute a viable solution. In particular, enzymatic hydrolysis of lignocellulose is an inefficient step with low yield. An interesting alternative is the so-called «consolidated bioprocessing« production scheme (CBP), which employs a single organism catalysing both biomass hydrolysis and fermentation of the liberated sugars. This process design thus represents a high potential for cost reduction. Today, no known microorganism is able to use all main plant cell wall sugars and to efficiently hydrolyse the lignocellulosic material. Therefore, in the present project, we plan to isolate microorganisms and (hemi)cellulolytic enzymes from specific and well adapted environments, possibly leading to the development of efficient CBP biocatalysts.
In order to identify interesting enzymes and microorganisms, specific enrichment cultures with microcoms from plant biomass degrading environments will be set up. By this way, the probability of isolating pertinent enzymes or strains will be greatly increased. High throughput sequencing will then allow covering an important part of the biodiversity present and identifying efficient and highly active enzymes.

Identification of biocatalysts by two complementary approaches
To maximize the chance of discovering relevant micro-organisms, specific enrichment cultures on lignocellulosic biomass will be set up, inoculated with microflorae from soil and compost. From these enrichment cultures, microorganisms will be isolated by using a classical microbiological approach. Carefully chosen substrates and culturing conditions will allow isolating the best adapted strains, and some of them could lead to a further development to efficient industrial biocatalysts. The capacity of strains to produce ethanol and other alcohols will also be tested.
As the majority of strains from natural systems cannot be cultivated, the enrichment cultures will also serve as a source for mining the uncultivable part of the (hemi)cellulose degrading microbial communities, using a metagenomics approach. The DNA of the whole microbial community will be extracted and anaysed by high throughput sequencing. By this way, an important data base, including coding sequences for enzymes catalysing various biochemical reactions, will be created. Bioinformatic analysis and enzymatic activity tests, which will be performed after production in adapted expression hosts, will allow the identification of the most interesting enzymes. The suitability of identified bacterial strains for use in an industrial production process will be evaluated.

Results

Aerobic and anaerobic enrichment cultures with either soil or compost as inoculum were realized. The amount of CO2 produced under aerobic conditions showed that enrichment cultures containing compost had a higher biomass degrading activity than those containing soil. In addition, analysis of the microcosms by 16S ribosomal RNA gene sequencing revealed that their compositions were quite distinct, as a function of the enrichment type and culture temperature. Several strains degrading lignocellulose at different temperatures were isolated from soil and compost enrichment cultures. Their cellulolytic and/or hemicellulolytic activity could be confirmed on model substrates and their identification by 16S gene sequencing, as well as a more detailed characterisation of the strains are underway.
The two best performing enrichment cultures were selected for metagenomic DNA extraction. 454 pyrosequencing resulted in more than one million reads per sample. An enrichment of Carbohydrate Active enZymes (CAZymes) and glycoside hydrolases was observed in the case of solid incubation of compost on lignocellulosic biomass, but not in suspension cultures. In parallel, functional screening of metagenomic libraries allowed us to isolate a about thirty clones harbouring glycoside hydrolases which are currently being subcloned and characterized.

Outlook

As for the microbiology part of the project, we will continue the characterisation of isolated strains. The capacity to degrade different cellulosic model substrates, such as CMC and Avicel, but also pretreated lignocellulose substrates will be determined and hydrolysis kinetics established. Their ability to produce alcohols will also be tested.
The characterisation of enzymes from metagenomic libraries and expressed as tagged recombinant proteins in E. coli will continue. Their specific activities on several substrates as well as optimal reaction conditions will be determined.

Scientific outputs and patents

Results will be presented on scientific meetings and published in peer reviewed scientific journals.

Partners

AFMB Architecture et Fonction des Macromolécules Biologiques UMR6098

CNRS - DR12 - LCB Centre National de la Recherche Scientifique - Délégation Provence et Corse - Laboratoire de Chimie Bactérienne

IFPEN IFP Energies nouvelles

ANR grant: 786 175 euros
Beginning and duration: juin 2012 - 48 mois

Submission abstract

There are worldwide efforts to enhance sustainable development, including the growing use of biofuels to replace fossil energy-based fuels. Bioethanol represents a major part of biofuels, but currently used resources, sugar and starch-based food crops, will soon be limiting for the increasing demand. The development of cost-effective second generation biofuels, produced from lignocellulosic biomass is a promising alternative which can lead to substantial reductions of greenhouse gas emissions and avoid competition of land use for food production. Present process configurations and especially the production of cellulolytic enzymes are, however, too expensive to constitute viable solutions. The Consolidated Bioprocessing scheme employing a single organism for both hydrolysis of lignocellulosic biomass and fermentation of liberated sugars, could allow important cost savings. Economic production requires, in addition, that all constituents of lignocellulosic biomass and especially pentose sugars are utilized. Today, no micro-organism able to use all sugar components and to efficiently catalyse the two transformation steps is available. In the present research project, we therefore propose to isolate micro-organisms and (hemi)cellulolytic enzymes from specific adapted environments to constitute the base for the development of a future CBP biocatalyst.
To maximize the chance of discovering relevant micro-organisms, specific enrichment cultures on lignocellulosic biomass will be set up, inoculated with microflorae from plant polymer degrading environments. Selected substrates and well defined conditions will be employed to isolate the best adapted strains likely to lead to well performing biocatalysts under industrial conditions. In addition, a culture collection including thermophiles and hyper-thermophiles will be screened for strains with (hemi)cellulolytic activity. The capacity of strains to produce ethanol and other alcohols will also be tested.
As the majority of strains from natural systems cannot be cultivated, the enrichment cultures will also serve as a source for mining the uncultivable part of the (hemi)cellulose degrading microbial communities, using a metagenomics approach. By this way, an important pool of enzymes of much higher diversity will be created. To ensure the highest success rates for the isolation of relevant genes, a characterization by TGGE analyses and 16S sequencing of the enrichment cultures will help choosing the best samples for extraction of nucleic acids. This metagenomic DNA will be mined by direct pyrosequencing and by functional screening for cellulolytic and hemicellulolytic enzymes. Bioinformatic analysis will identify Carbohydrate Active enZymes (CAZymes) and guide the selection of enzymes to be further characterized. Expression in the model strain Clostridium cellulolyticum will allow a first evaluation of the genes for their suitability to be integrated in a future CBP micro-organism.

 

ANR Programme: Bio-Matières et Energies (Bio-ME) 2012

Project ID: ANR-12-BIME-0006

Project coordinator:
Madame Senta BLANQUET (IFP Energies nouvelles)
senta.blanquet@nullifpen.fr

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.