Glycerol Production by Microbial Fermentation
Microbial production of glycerol has been known for 150 years, and glycerol was produced commercially during World War I. Glycerol production by microbial synthesis subsequently declined since it was unable to compete with chemical synthesis from petrochemical feedstocks due to the low glycerol yields and the difficulty with extraction and purification of glycerol from broth. As the cost of propylene has increased and its availability has decreased especially in developing countries and as glycerol has become an attractive feedstock for production of various chemicals, glycerol production by fermentation has become more attractive as an alternative route. Substantial overproduction of glycerol by yeast from monosaccharides can be obtained by: (1) forming a complex between acetaldehyde and bisulfite ions thereby retarding ethanol production and restoring the redox balance through glycerol synthesis; (2) growing yeast cultures at pH values near 7 or above; or (3) using osmotolerant yeasts. In recent years, significant improvements have been made in the glycerol production using osmotolerant yeasts on a commercial scale in China. The most outstanding achievements include: (1) isolation of novel osmotolerant yeast strains producing up to 130 g/L glycerol with yields up to 63% and the productivities up to 32 g/(L day); (2) glycerol yields, productivities and concentrations in broth up to 58%, 30 g/(L day) and 110–120 g/L, respectively, in an optimized aerobic fermentation process have been attained on a commercial scale; and (3) a carrier distillation technique with a glycerol distillation efficiency greater than 90% has been developed. As glycerol metabolism has become better understood in yeasts, opportunities will arise to construct novel glycerol overproducing microorganisms by metabolic engineering.
Glycerol, a 1,2,3-propanetriol, is a simple alcohol with many uses in the cosmetic, paint, automotive, food, tobacco, pharmaceutical, pulp and paper, leather and textile industries (Table 1) or as a feedstock for the production of various chemicals. Glycerol is also known as glycerin or glycerine. Glycerol has also been considered as a feedstock for new industrial fermentations in the future. For example, glycerol can be fermented to 1,3 propanediol (Biebl et al., 1998, 1999), which is used for the chemical synthesis of poly(trimethylene terephthalate), a new polyester with novel fiber and textile applications that combines excellent properties (good resilience, inherent stain resistance, low static generation) with an environmentally benign manufacturing process (Biebl et al., 1998, 1999). The transformation of glycerol to dihydroxyacetone by the bacterium Acetobacter suboxidans is another example of a potential process (Charney, 1978). In a submerged fermentation, the bacteria produce dihydroxyacetone in yields of 75–90% from a 5–15% solution of glycerol. The dihydroxyacetone can be transformed further by a dihydroxyacetone kinase to dihydroxyacetone phosphate, which serves as an essential substrate for some aldolases to produce various optically active sugar derivatives (Itoh et al., 1999).
Glycerol can be produced either by microbial fermentation or by chemical synthesis from petrochemical feedstocks or can be recovered as a by-product of soap manufacture from fats. Traditionally, glycerol is produced as a by-product of the hydrolysis of fats in soap and other related materials and contributes significantly to the present glycerol production volume of about 600 000 tons annually. This process is now of lesser importance in industrial nations and many developing countries, because of the replacement of soap with detergents (Rehm, 1988; Agarwal, 1990). Currently, approximately 25% of world glycerol production occurs by the oxidation or chlorination of propylene to glycerol, but this route has declined in relative importance since the early 1970s (Hester, 2000) partially because of environmental concerns. Furthermore, as the cost of propylene has increased and its availability has decreased especially in developing countries, glycerol production by fermentation has become more attractive as an alternative route (Agarwal, 1990; Wilke, 1999). A significant amount of glycerol is also synthesized from allyl alcohol. Currently, the price of glycerol is between US$1.10/kg and US$1.25/kg and is expected to increase in line with inflation over the next 10 years (Hester, 2000). Glycerol production costs by microbial fermentation are difficult to estimate. Recently, High Plains Corporation (Wichita, KS) reported that glycerol production costs between US$0.40/kg and US$0.53/kg would result in a profitable operation.
Glycerol was produced via the fermentation route for the first time on a large scale using the sulfite-steered yeast process during World War I when demand for glycerol in explosive manufacture exceeded the supply from the soap industry (Prescott and Dunn, 1959). However, wartime process technology could never adapt to the peacetime competition from the chemically synthesized process developed after World War II as yields of glycerol from sugar by fermentation were low and recovery by distillation was inefficient.
Glycerol production by yeast fermentation has been known since the investigations of Pasteur (1858). In Saccharomyces cerevisiae, glycerol is a by-product of the fermentation of sugar to ethanol in a redox-neutral process. The role of NADH-consuming glycerol formation is to maintain the cytosolic redox balance especially under anaerobic conditions, compensating for cellular reactions that produce NADH (van Dijken and Scheffers, 1986). Substantial overproduction of glycerol from monosaccharides can be obtained by yeast: (1) forming a complex of acetaldehyde with the bisulfite ion that limits ethanol production and that promotes reoxidation of glycolytically formed NADH by glycerol synthesis; (2) growing at pH values around 7 or above; and (3) by using osmotolerant yeasts (Rehm, 1988; Agarwal, 1990).