Microbial Production of Glyceric Acid,an Organic Acid That Can Be Mass Produced from Glycerol

Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% d-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of d-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.A shift from petroleum to bio-based feedstocks will be necessary for a sustainable industrial society and effective management of greenhouse gas emissions (2, 20). Biodiesel fuel (BDF) is produced from vegetable oils and animal fats and can replace the diesel in diesel engine motors. Although the European Union currently produces 82% of the BDF produced in the world (7), the use of BDF will probably continue to grow worldwide, because petroleum is a limited resource. Massive amounts of glycerol can be obtained as a by-product of BDF production (approximately 10% by weight) through transesterification with alcoholysis generally catalyzed by NaOH or KOH. As the use of this glycerol is an important component of the economics of the BDF industry, there is a worldwide demand for the efficient use of glycerol (24).Among the recent developments in the conversion of glycerol into valuable chemicals, epichlorohydrin (ECH) and 1,2-propanediol (propylene glycol) are now commercially synthesized from glycerol by chemical processes, and 1,3-propanediol (1,3-PDO) and dihydroxyacetone (DHA) are produced from glycerol by biotechnological processes (4, 5, 17, 18, 24, 25). ECH, propylene glycol, and 1,3-PDO are used mainly as intermediates for resins and polymers. However, an increase in the price of glycerol, such as that which occurred due to the collapse of the BDF market (its price increased nearly threefold in Germany by the end of 2007 [24]), can have a large negative effect on the production of such low-price commodity chemicals. Hence, research on the production of more value-added and functional chemicals from (raw) glycerol is important.Recently, we have focused on the production of a glycerol derivative, glyceric acid (GA), using a bioprocess (Fig. ​(Fig.1)1) (9, 10). GA from an extract of Cynara scolymus leaves has liver stimulant and cholesterolytic activity in dogs (11), and d-GA calcium salt accelerates ethanol and acetaldehyde oxidation in rats (8). GA-based esters also exhibit antitrypsin activity (12), and novel oligoesters based on GA derivatives may be useful for pharmaceutical purposes, such as drug delivery systems (23). These reports suggest that GA is a promising chemical, but it is very expensive as a reagent for investigational use.Open in a separate windowFIG. 1.Proposed pathway for the conversion of glycerol to GA (glyceric acid) by acetic acid bacteria. The bioconversion of glycerol to DHA (dihydroxyacetone) is also represented.Before we began our research, little was known about GA as a biotechnological product, except for one Japanese patent from 25 December 1987 (Daicel Chemical Industries, Japanese patent application 51069) and a report on its by-production during DHA production by Gluconobacter oxydans (3, 21). According to the patent, resting cells of Gluconobacter cerinus IFO3262 (later Gluconobacter frateurii NBRC3262) converted 100 g/liter of glycerol to 57 g/liter of d-GA in a fermentor over a 2-day incubation. Recently, we revealed that Acetobacter tropicalis NBRC16470 produced 22.7 g/liter of optically pure d-GA from 200 g/liter of glycerol in a fermentor over a 4-day incubation (9). However, because this method of GA production is far from practical, we are attempting to develop a GA manufacturing bioprocess based on strain, fermentation, and process development.In this study, we searched for a GA producer among 162 acetic acid bacterial strains and investigated the GA productivity and enantiomeric composition of 88 selected strains. We also investigated oxidative fermentation conditions in a 5-liter jar fermentor and applied electrodialysis (ED) to recover glycerate from culture broth. Furthermore, we clarified the gene and enzyme involved in GA production from glycerol for the first time.