FAIR-CT96-5047 Genetic modification of Clostridium acetobutylicum to improve ABE fermentation

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Biological Conversion : Bulk Chemicals : FAIR Area 2.2 - Bioprocessing : FAIR Marie Curie Research Training Grants : Liquid Biofuels and Biogas

Objectives:

ABE fermentation results in the anaerobic conversion of carbohydrates by strains of Clostridium into acetone, butanol and ethanol. This process was one of the first large-scale industrial fermentations to be developed, but use declined from the 1960s onwards due to its inability to compete economically with the chemical synthesis of these solvents. However, interest in the use of renewable resources as substrates for the production of chemicals and developments in the field of biotechnology have revived interest in this process. One factor that reduces the economic viability of the ABE fermentation is the high price of the carbohydrate substrates needed. Genetic research aimed at the development of strains able to use cheap cellulosic substrates (i.e. agricultural wastes) is seen as a way to make this process economically viable. The main enzymes involved in microbial cellulose degradation can be classified as exo- and endo-glucanases. Most of the soventogenic clostridial strains show production of endoglucanases, but cannot grow on cellulose as sole carbon source, indicating a possible lack of exoglucanase. This project studied the use of lignocellulosic substrates for ABE fermentation and the cloning and expression of exo-glucanase genes from cellulolytic organisms in selected clostridial strains.

Activities and Results:

The first task was to extend the substrate range of solventogenic clostridia by transforming the strains with cellulose genes from other organisms and study expression of these genes. Thereafter , attention was focused on the suppression of degenerative changes and autolysis in solventogenic clostridia. The growth and solvent production of clostridial strains on domestic organic waste (collected from houses in Wageningen, Netherlands) was investigated The collected material was treated by extrusion in order expand the (hemi)cellulose fibres present so they were more accessible to attack by cellulolytic enzymes. The extruded material was analysed for solvent- and hot water extractives, lignin, pectin, sugars and ash composition. A solution of 10% (w/v) DOW in demineralised water was used as medium for fermentation. All strains tested grew and produced solvents on this medium. The utilisation of the polymeric sugars present in the DOW by the bacteria was determined. In order to optimise a transformation method for strains different vectors have been tested. Best results were obtained with the pAMbeta1 derivative plasmid pIL253. Various strains have been transformed with the plasmids pGh9 and pGh9:ISS1, which contain a thermo-sensitive replicon derived from the one of plasmid pGK12. The cellulase gene celD from the fungus Neocallimastix patriciarum has been cloned in an expression secretion cassette containing the promoter and signal peptide sequence of the eglA gene from C. acetobutylicum NCP262. C. beijerinckii NCIMB 8052 has been transformed with this construct and the transformants obtained analysed. Recently, the genome of the strain ATCC 824 has been sequenced. Surprisingly, in the chromosome of this strain the whole set of genes coding for enzymes involved in cellulose degradation appears to be present. In particular, an operon containing two putative cellulose binding protein genes, four putative endo-glucanase genes and two putative exo-glucanase genes are present. It is not known if this operon is expressed or if the cellulose genes present in it code for active enzymes. Therefore, one of the putative endo-glucanase genes and one gene coding for a putative exo-glucanase have been cloned in E coli, and their activity on different cellulosic substrates has been tested.

Keywords: ABE fermentation, acetone, butanol, ethanol

Contacts

Scientific Supervisor

• Pieternel AM CLAASSEN / Agrotechnology & Food Innovations B.V. NETHERLANDS