By-product formation in ethanol fermentation by Zymomonas mobilis: Dissertation

The bacterium Zymomonas mobilis is a promising alternative to the traditional yeast in ethanol production processes when glucose is used as carbon source.In this study the reasons for decreased ethanol yields on sucrose and fructose based media were investigated.In addition to the previously recognized high-molecular weight fructopo Iymer, levan, two types of new by-products of sucrose fermentations by Z. mobilis were detected: sorbitol and oligosaccharides.When, fructose was used as carbon source, for mation of mannitol, dihydroxyacetone and glycerol decreased the ethanol yield. In the utilization of sucrose, transfructosylation reactions lead to the formation of glucose and fructose, oligosaccharides or levan.The amounts of oligomers and levan increased with the number of fructosyl transfers in the cell, i.e. with increasing sucrose concentration or dilution rate in continuous fermentation.Levan formation was inhibited by glucose and a number of other sugars, whereas no inhibition by ethanol was observed. Oligosaccharides consisted of trimers, tetramers and pentamers, containing respectively two, three or four fructose molecules per molecule of glucose.Two trimers and two tetramers were isolated, representing structurally different series in which the fructosyl unit is transferred to either the 1-or the 6-position of the fructose moiety of sucrose or oligomers.Reduction of fructose to form sorbitol is coupled to the oxidation of glucose to glu conate by a dehydrogenase having a non-dissociable coenzyme.Thus, sorbitol is formed in high quantities when both fructose and glucose are present in the medium. However, small amounts of sorbitol were also formed from glucose or fructose alone, as a consequence of the non-specificity of the enzyme system.The production of sorbi tol provides the cell with an alternative route for the formation of 6-phosphogluconate and thus a means of overcoming the rate limiting step in the metabolic pathway.Mannitol is formed from fructose by an NADPH-specific mannitol dehydrogenase, which is apparently coupled to the oxidation of glucose-6- phosphate.This may also serve as a way of increasing the rate of 6-phosphogluconate production in the cell.Accumulation of dihydroxyacetone decreased the ATP yield of the carbohydrate meta bolism, thus together with its inhibitory action decreasing the cell yield on fructose containing media.Acetaldehyde, which was formed particularly in aerated cultures of Zymomonas, was also shown to be toxic for growth.Different proportions of by-products were formed from sucrose in batch and con tinuous fermentations, mainly due to the different glucose concentrations in the media under different fermentation conditions.High ethanol yields were obtained only at very low dilution rates.Different Z. mobilis strains were compared with respect to their by-product patterns, but in spite of some variations in the by product composition, the ethanol yields were always within the same rather narrow limits.Thus, the develop ment of new strains is necessary in order to obtain improved ethanol yields on sucrose based media.