Appl Microbiol Biotechnol (2011) 90:257–267 DOI 10.1007/s00253-010-3015-3 APPLIED MICROBIAL AND CELL PHYSIOLOGY Production of arabitol from glycerol: strain screening and study of factors affecting production yield Srujana Koganti & Tsung Min Kuo & Cletus P. Kurtzman & Nathan Smith & Lu-Kwang Ju Received: 26 August 2010 /Revised: 10 November 2010 /Accepted: 15 November 2010 /Published online: 3 December 2010 # Springer-Verlag 2010 Abstract Glycerol is a major by-product from biodiesel Keywords Arabitol . Xylitol . Biodiesel . Glycerol . production, and developing new uses for glycerol is Osmotolerant yeast . Debaryomyces hansenii imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 Introduction yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of Biodiesel motor fuel produced from renewable sources such xylitol as the dominant metabolite. Some produced polyol as vegetable oil and animal fat is an attractive alternative to mixtures that might present difficulties to downstream petroleum-derived fuel (Krawczyk 1996). In biodiesel separation and purification. Several Debaryomyces hansenii production using transesterification of triglycerides, glycerol strains produced arabitol as the predominant metabolite is the major by-product produced: About 1 kg of glycerol is with high yields, and D. hansenii strain SBP-1 (NRRL Y- formed for every 9 kg of biodiesel produced (Dasari et al. 7483) was chosen for further study on the effects of several 2005). Biodiesel consumption in the USA has increased growth conditions. The optimal temperature was found to dramatically from 75 million gallons in 2005 to 450 million be 30°C. Very low dissolved oxygen concentrations or gallons in 2007. Accompanying this increase was the anaerobic conditions inhibited polyol yields. Arabitol yield production of around 45 million gallons of glycerol in improved with increasing initial glycerol concentrations, 2007 (National Biodiesel Board 2007). Refined glycerol has reaching approximately 50% (w/w) with 150 g/L initial numerous applications in the food, drug, textile, and glycerol. However, the osmotic stress created by high salt cosmetic industries whereas crude glycerol produced from concentrations (≥50 g/L) negatively affected arabitol pro- the biodiesel industry is of low value because of impurities duction. Addition of glucose and xylose improved arabitol such as spent catalyst, salts after neutralization, residual production while addition of sorbitol reduced production. methanol, methyl esters, and free fatty acids (Liu et al. 2002; Results from this work show that arabitol is a promising Bournay et al. 2005). The economics of the biodiesel value-added product from glycerol using D. hansenii SBP- industry is strongly influenced by the value of its by- 1 as the producing strain. products, and developing new uses for biodiesel glycerol is imperative to the sustainability of the biodiesel industry : S. Koganti L.-K. Ju (*) (Demirbas 2003; Haas et al. 2006). Department of Chemical and Biomolecular Engineering, In this study, the biodiesel by-product glycerol was used as The University of Akron, the substrate for production of arabitol, a polyhydric alcohol. Akron, OH 44325-3906, USA e-mail: [email protected] A study by the Department of Energy identified arabitol, and : : its enantiomer xylitol, as one of the top 12 biomass-derivable T. M. Kuo C. P. Kurtzman N. Smith building block chemicals. Arabitol and xylitol can be trans- Foodborne Pathogens and Mycology Research Unit, National formed into several groups of chemicals like arabonic/ Center for Agricultural Utilization Research, ARS, USDA, 1815 N. University Street, arabinoic acid, xylaric/xylonic acid, propylene glycol, and Peoria, IL 61604-3999, USA ethylene glycol (Werpy and Petersen 2004). Arabitol and 258 Appl Microbiol Biotechnol (2011) 90:257–267 xylitol have melting points of 103°C and 93°C, respectively. Media Both are highly soluble in water and both form white crystals when purified (Le Tourneau 1966; Talja and Roos 2001). The yeasts were maintained on YM agar slants (5.0 g The catabolism of arabitol by Escherichia coli involves the peptone, 10.0 g glucose, 3.0 g yeast extract, 3.0 g of malt formation of arabitol phosphate which induces the synthesis extract, and 20.0 g of agar in 1 L deionized water). Cultures of compounds that inhibit bacterial metabolism (Scangos and were grown for inoculation in YM broth (without agar). Reiner 1979). This property makes it possible to use arabitol The medium used in the screening study and in later studies as a sweetener that reduces dental caries. Also, the caloric on cell growth and arabitol production by Debaryomyces value of arabitol is 0.2 kcal/g, whereas it is 2.4 kcal/g for hansenii SBP-1 (NRRL Y-7483) had the following compo- xylitol (McCormick and Touster 1961;Hucketal.2004; sition (per liter of solution): yeast extract, 3 g; (NH4)2SO4, Mitchell 2006;Crick1961). It is highly possible that arabitol 2g;K2HPO4, 2.4 g; KH2PO4, 1.6 g; MgSO4⋅7H2O, 1 g; can be used in many of the known applications of xylitol, as and glycerol, 100 g (unless specified otherwise). The a natural sweetener, a dental caries reducer, and a sugar glycerol used in this study was a crude glycerol from a substitute for diabetic patients (Gare 2003). If desirable, biodiesel plant without a glycerol refinery (provided by arabitol can also be converted to xylitol, for example, by Biodiesel Systems, Madison, WI, USA). The crude glycerol using Glucanobacter oxydans (Suzuki et al. 2002). This had 88% glycerol. To make 100 g/L glycerol in the culture bacterium was capable of oxidizing D-arabitol to D-xylulose medium, 113 g/L of the crude glycerol was added. The using the membrane-bound D-arabitol dehydrogenase and medium had an initial pH of 6.6–6.8. Glycerol (and other then converting D-xylulose to D-xylitol using the membrane- carbon sources used in some studies, i.e., glucose, xylose, bound D-xylitol dehydrogenase. Xylitol yield of around 25% and sorbitol) was autoclaved separately from other medium has been reported (Sugiyama et al. 2003). components. Arabitol is known to be produced by osmophilic yeast species such as Debaryomyces (Nobre and Costa 1985), Candida (Bernard et al. 1981), Pichia (Bisping et al. 1996), Wickerhamomyces (Hansenula; Van Eck et al. 1989), and Table 1 Genera and number of strains screened Saccharomycopsis (Endomycopsis;Hajny1964). When Genera # of strains exposed to osmotic stress, the yeast accumulates compat- ible solutes such as arabitol, glycerol, xylitol, erythritol, and Debaryomyces 67 mannitol to balance the osmotic pressure across the cell Geotrichum 41 membrane. An objective of this study was to select the Metschnikowia 37 yeast strains that produce large amounts of arabitol with Candida 24 high yields and minimal other polyols (for easier down- Dipodascus 14 stream separation) using glycerol as substrate. Experiments Pichia 5 characterized the most promising strains for growth and Trigonopsis 4 arabitol production under different culture conditions. Galactomyces 4 Zygosaccharomyces 2 Citeromyces 1 Materials and methods Saccharomycopsis 1 Hyphopichia 1 Yeast strain screening Wickerhamia 1 Lachancea 1 Extensive culture screening of 214 strains from 25 genera Torulaspora 1 was conducted for arabitol production from glycerol. The Naumovozyma 1 following five genera contained the largest numbers of Kodamaea 1 strains screened: Debaryomyces, Geotrichum, Metschniko- Sugiyamaella 1 wia, Candida, and Dipodascus. A complete list of the Hanseniaspora 1 genera and the numbers of screened strains from each genus Cephaloascus 1 is given in Table 1. All of these strains were obtained from Botryozyma 1 the Agricultural Research Service Culture Collection Trichomonascus 1 (NRRL) at National Center for Agricultural Utilization Sporopachydermia 1 Research, United States Department of Agriculture, Peoria, Endomyces 1 IL, USA. NRRL accession numbers for the species Schizoblastosporion 1 considered is shown in Table 2. Appl Microbiol Biotechnol (2011) 90:257–267 259 Table 2 Strains producing at least 5 g/L of total polyols, listed in the reaction time, the flasks were removed from the incubator/ alphabetical order shaker and processed for analysis of glycerol and polyol Species NRRL # SBP # Total polyol (g/L) product concentrations. Candida quercitrusa Y-5392 118 6 Culture conditions for D. hansenii SBP-1 (NRRL Y-7483) Debaryomyces hansenii Y-7483 1 10 D. hansenii Y-1015 2 11 Inoculum was prepared by transferring a loop of cells from D. hansenii Y-10452 3 9 an agar plate to 50 mL YM broth in a 250 mL flask covered D. hansenii Y-1448 5 5 with cheese cloth. The culture was grown at room D. hansenii Y-7426 7 5 temperature (22±1°C) for 24 h under vigorous magnetic D. hansenii Y-1454 8 5 stirring; 2.5 mL of the inoculum thus prepared was added to D. hansenii Y-10150 15 5 each flask in the subsequent study of culture conditions, D. coudertii Y-5984 33 5 which were made with a 50-mL medium volume in 250 mL Galactomyces reesei Y-17566 167 8 flasks shaken at 200 rpm. The temperature used in these Geotrichum candidum Y-552 12 14 studies was 30°C except in the study of temperature effects. G. candidum Y-714 181 8 Multiple samples were taken during cultivation to establish G. candidum Y-1282 182 5 the profiles of cell growth, substrate consumption, and G. candidum Y-17010 188 9 product formation. G. candidum Y-2071 189 15 G. cucujoidarum Y-27731 194 19 Effect of medium volume in shaker flasks G. cucujoidarum Y-27732 219 13 G. fermentans Y-17567 169 10 Shake flasks are not very suitable for studying the effects G. fragrans Y-17571 177 7 of dissolved oxygen concentrations (DO) on cell growth G. histeridarum Y-27729 195 10 and product formation.