Process development for mannitol production by lactic acid bacteria
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Doctoral thesis (monograph)
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Technical biochemistry report, Teknillisen biokemian tiedote, 1/2002
AbstractD-Mannitol (here: mannitol) is a naturally occurring sugar alcohol with six carbon atoms. It is only half as sweet as sucrose. However, mannitol and other sugar alcohols exhibit reduced caloric values compared to the respective value of most sugars, which make them applicable as sweeteners in so-called "light" foods. Moreover, sugar alcohols are metabolized independently of insulin and are thus also applicable in diabetic food products. Besides applications in the food industry, mannitol is also used in the pharmaceutical industry. In medicine, mannitol is used to decrease cellular edema (excessive accumulation of fluid) and increases the urinary output. In this doctoral thesis, the development of a new bioprocess for the production of mannitol is described. For this purpose, aspects such as strain selection, choice of process method, optimization of process parameters, scale-up, and metabolic engineering were studied. At present, mannitol is produced commercially by catalytic hydrogenation of fructose-containing syrups. The existing chemical production methods are, however, characterized by several drawbacks. The uppermost being that when fructose is catalytically hydrogenated only about 50 % of it is converted into mannitol, whereas the rest is converted into another sugar alcohol, sorbitol. In addition, ultra-pure (expensive) raw materials (fructose and hydrogen gas) are required for efficient conversion. When more cost-effective raw materials, such as glucose-fructose syrups are used as starting material for catalytic hydrogenation, the main product is sorbitol and mannitol is formed as a by-product. Hence, mannitol production becomes very dependent on the market demand of sorbitol. Furthermore, mannitol is relatively difficult to purify from sorbitol. In addition, ion exchange is required for removal of the metal catalyst from the production solution. This results in even higher production costs and decreased yields. The microbial mannitol production process described in this thesis is based on high cell density cultures of slowly growing heterofermentative lactic acid bacteria. The bioconversion of fructose to mannitol was performed in a slowly agitated membrane cell-recycle bioreactor equipped with pH and temperature control. Neither aeration nor nitrogen flushing of the bioconversion medium was required, which drastically lowers the investment costs of such a plant. An important detail in the new bioprocess was the re-use of cell biomass in successive bioconversions. In a semi-continuous production experiment, the initial cell biomass provided stable mannitol productivities and yields for at least 14 successive batches. Moreover, using a simple purification protocol comprising cooling crystallization of a supersaturated solution and crystal recovery by means of drum centrifugation, high yields of high-purity (>98 %) mannitol crystals were obtained. Moreover, in scale-up trials the microbial mannitol production process was successfully run at a small pilot-scale (100 L). The yield of crystalline mannitol from the initial sugar consumed in the bioprocess was about 52 % (w/w). This compares favorably to a commercial chemical process with a yield of about 39 %. Hence, under optimized conditions the best production strain (Leuconostoc mesenteroides) converted up to 95 % (mol/mol) of fructose consumed into mannitol. Unfortunately, this is only achieved when a significant amount of glucose is co-metabolized by the cells. The catabolism of glucose enables cofactor regeneration in the cells and is thus, essential for the bioconversion of fructose to mannitol. Moreover, some mannitol is also lost in the purification steps. Another significant improvement brought about by the new bioprocess was a reduced by-product burden. In the commercial chemical process, a total of 1.58 kg by-products are formed for each kilogram of mannitol crystals produced. In the bioprocess, only 0.67 kg by-products are produced per kilogram crystalline mannitol. Using tools of genetic engineering, two key enzymes involved in the primary metabolism of another efficient mannitol-producer, Lactobacillus fermentum, were inactivated. A mutant deficient in D-lactate dehydrogenase and grown in a fructose-glucose medium produced high levels of both mannitol and pure L-lactate. Inactivation of both lactate dehydrogenases resulted in major rerouting of glucose catabolism, which led to the accumulation of pyruvate and production of 2,3-butanediol. Moreover, mutants with lowered fructokinase activity and deficient in acetate kinase were constructed and studied.
D-mannitol, mannitol dehydrogenase, Lactobacillus, Leuconostoc, membrane cell-recycle bioreactor, resting cells, metabolic engineering, lactate dehydrogenase, acetate kinase, fructokinase, L-lactate, pyruvate