AIR2-CT93-0825 Production of 1,3-propandiol From Glycerol Surpluses. Yield Optimisation By Technological Development and By Genetic Strain Improvement

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AIR Cluster VI - Bioplastics : Biological Conversion : Biopolymers/Gums : Biotechnology : Paints/Coatings/Plastics : Vegetable Oil/Fat

	Contract No	AIR2-CT93-0825
	Total Cost	380 300
	EC Contribution	265 150
	Start Date	01/01/1994
	Duration	36 months

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OBJECTIVE:

Due to increased production of plant oils in developing countries and the larger use by the oleochemical industry, glycerol surpluses are on the world market, which led to a continuous decrease in glycerol prices. In future, the situation on the glycerol market will be even more tense if agricultural rape oil production comes into play as envisaged by several EC countries. In a cooperative research project we propose to develop a biotechnological process to produce 1,3-propanediol (PD) from glycerol by Clostridium butyricum, a harmless microorganism that is widely disseminated in nature. Until now, PD is produced only in negligible amounts due to its high price, however it is a useful component for all types of polycondensates : polyethers, polyesters polyurethanes and additionally can be used as a solvent and chemical intermediate for various compounds. The chemical synthesis of 1,3PD is difficult and costly on the other hand, the microbial conversion of glycerol to 1 3-PD is relatively simple and can lead to a decrease in prices. Natural plastics based on 1,3-PD are expected to be light-insensitive and more biodegradable in comparison to fully synthetic polymers. We propose to exploit enabling technology and expertise developed in our laboratories on the use of Clostridium butyricum to promote technological development and to generate strains with improved fermentation characteristics.

RESULTS FROM THE PROJECT :

The metabolism of Clostridium butyricum was studied in batch culture and in chemostat culture using either glucose or glycerol. Under glycerol limitation and at different growth rates, the production of 1,3 propanediol was not affected. The main observation for high dilution rates was the decrease in butyrate concentration and a concomitant increase in acetate concentration.The marked change in the acetate/butyrate ratio did not occur either in non limited glycerol chemostat or in batch culture. The evolution of this ratio was associated with the specific rate of glycerol utilization; at a high glycerol uptake rate, the butyrate pathway may become saturated and the cells initiate the acetic acid formation a corresponding shift in the activity of the acetate and the butyrate kinase occured, whereas, high levels of the 1,3 PD oxidoreductase were detected in all extracts. The dilution rate had almost no effect on the glycerol conversion into 1,3 PD showing that the carbon flux distribution through the dehydratase and the 1,3 PD dehydrogenase was balanced by the flux through the glycerol dehydrogenase and the dihydroxyacetone kinase. Due to the limitation of the butyrate pathway, C. butyricum growing on glycerol regenerated the reducing equivalents via the highly active 1,3 propanediol oxidoreductase. Our data indicated that the NADH produced by the glycerol dehydrogenase and the glyceraldehyde -3- phosphate dehydrogenase was not sufficient for the 1,3 PD formation and part of the ferredoxin produced by the pyruvate ferredoxin oxido reductase was reoxidized by the ferredoxin-NAD+ reductase activity to produce NADH. The phosphoroclastic activities were high when the m of the clostridium was high, whereas the hydrogenase activities increased during time fermentation when the m decreased. Ferredoxin-NAD+ reductase and NADH-ferredoxin reductase were always in the same ratio and exhibited the best activities at the end of the exponential phase. Hydrogenase and phosphoroclastic activities were significantly higher in cell-free extracts obtained from cells grown on glucose than on glycerol. The ferredoxin-NAD+ reductase associated to a low level of hydrogenase and high activity of the 1,3 PD oxidoreductase explained the high level of NADH recovery.

Contacts

Coordinator

• Henri PETITDEMANGE / Universite Henri Poincare - Nancy I Faculte des Sciences - Lab. de Chimie / FRANCE

EC Scientific Officer

• Ciarán MANGAN / European Commission - DG Research Quality of Life Programme - Unit B1.2 / BELGIUM

Participant

• Gesellschaft für Biotechnologische For schung / GERMANY