Production of D-Mannitol and Glycerol by Yeasts

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APPLIED MICROBIOLOGY, Dec. 1968, p. 1847-1952 Vol. 16, No. 12 Copyright t 1968 American Society for Microbiology Printed in U.S.A. Production of D-Mannitol and Glycerol by Yeasts HIROSHI ONISHI ANT)

1 APPLIED MICROBIOLOGY, Dec. 1968, p Vol. 16, No. 12 Copyright t 1968 American Society for Microbiology Printed in U.S.A. Production of D-Mannitol and Glycerol by Yeasts HIROSHI ONISHI ANT) TOSHIYUKI SUZUKI Noda Institute for Scientific Research, Noda-shi, Chiba-ken, Japan Received for publication 30 September 1968 D-Mannitol has not so far been known as a major product of sugar metabolism by yeasts. Three yeast strains, a newly isolated yeast from soy-sauce mash, Torulopsis versatilis, and T. anomala, were found to be good mannitol producers. Under optimal conditions, the isolate produced mannitol at good yield of 30% of the sugar consumed. Glucose, fructose, mannose, galactose, maltose, glycerol, and xylitol were suitable substrates for mannitol formation. High concentrations of yeast extract, Casamino Acids, NaCl, and KCI in media affected significantly the mannitol yield, whereas high levels of inorganic phosphate did not show any detrimental effect. In studies on production of D-mannitol (mannitol) by yeasts, Osaki (16) isolated pure mannitol from the fermented saccharose broth of Saccharomyces sake, and Roxburgh et al. (22) and Spencer et al. (25) reported that osmophilic yeasts produced some mannitol from glucose, although major products were D-arabitol and glycerol. In these cases, mannitol was merely a minor product giving a very low yield of 0.1 to 0.5% of sugar used. On the other hand, the production of mannitol from glucose by Aspergillus has been extensively studied, showing rather good yield of 20 to 50% of sugar consumed (1-3, 17, 18). Recently, Smiley et al. (23) showed an improved mannitol production process by examining various factors affecting a submerged A. candidus fermentation, and Lee (4) observed carbon balance of mannitol fermentation and the biosynthetic pathway by an Aspergillus species. We found that a newly isolated yeast produced a large amount of mannitol by aerobic dissimilation of sugars. The present report describes isolation of a new yeast, some of the factors influencing mannitol production, and identification of a product. MATERIALS AND METHODS Isolation ofa new yeast. An appropriate dilution of a 12-month-old soy-sauce mash was plated on 15% NaCl maltose-agar medium (5% maltose, 0.1% KH2PO4, 0.05% MgSO4.7H20, 0.01% CaCl2.2H20, 0.01% NaCl, 0.4% Vitamin Free Casamino Acids (Difco), 0.1% Yeast Extract (Difco), and 2% agar). Subculture purification of colonies was effected by subculturing on koji-agar, followed by selection of single colonies. The stock culture was transferred on koji-agar every 2 to 3 weeks. Yeast strains exanined. A total of 45 yeast strains were screened for their ability to produce mannitol They consisted of six newly isolated yeasts, Torulopsis versatilis (CBS 1752) and T. anomala (CBS 1731), which were supplied through the kindness of W. Slooff, and the following 37 strains of the collection of our institute: Candida tropicalis, C. guilliermondii, C. borea, C. utilis, C. polymorpha, C. albicans, Cryptococcus neoformans, C. Iaurentii, Debaryomyces sake, D. hansenii, D. miso var. 1, D. membranaefaciens, Hansenula anomala, H. subpelliculosa, H. miso (, Monilia sitophila, M. vini, Pichia miso, P. farinosa, P. membranaefaciens, P. mandschurica, P. quercibus, P. polymorpha, Saccharomyces sake, S. fragilis, S. monacensis, S. paradoxus, S. rouxii, S. rouxii var. halomembranis, S. acidifaciens, Torulopsis famata, T. sake, T. nodaensis, T. halophila, Trichosporon behrendii, T. cutanewn, and Trigonopsis variabilis. Media. The composition of the standard medium routinely used for fermentation was as follows: 10% glucose, 0.1% KH2PO4, 0.05% MgSO4.7H20, 0.01% CaCl2-2H20, 0.01% NaCl, 0.4% Vitamin Free Casamino Acids, and 0.1% Yeast Extract, ph 5.0. Some changes in the medium were also made and departures from the medium were noted in text or tables. Fermentation conditions. For the screening test, one loop of yeast from a 5-day koji-agar slant culture was inoculated into large test tubes, each containing 8 ml of the sterilized standard medium. The tubes were shaken on a reciprocal shaker operating at 300 rev/ min with a stroke of 2 cm at 30 C for 5 days. For examination of factors affecting mannitol production and for isolation of mannitol, shake-flask cultures were run. A 0.1-ml amount of a 5-day yeast culture in koji-extract was inoculated into 500-ml shake flasks each containing 50 ml of medium. The flasks were shaken on a reciprocal shaker operating at 140 rev/min with a stroke of 7.5 cm. Fermentation temperature was 30 C unless otherwise stated. Fermentation time is given in the tables. Analytical methods. After removal of yeast cells by filtration or centrifugation, the cleared fermented broth was analyzed according to the modified Somogyi-Nelson method for reducing sugar (7, 24),

2 1848 ONISHI AND SUZUKI APPL. MICROBIOL. the anthrone method for nonreducing sugar (5), and the method of Neish for polyalcohol (6). Paper chromatography was performed by the ascending method on Whatman no. 1 filter paper by using a solvent system composed of nine parts (by volume) of methylethylketone, one part of glacial acetic acid, and one part of water saturated with boric acid (21). Reducing sugar was detected by spraying with aniline hydrogen phthalate reagent (19) and polyalcohol by K104-tetra base reagent (27). Glycerol and mannitol were fractionated on a celite column and each fraction was assayed by periodate oxidation (6). Isolation ofmannitol. After removing yeast cells by ifitration, the cleared broth was concentrated in vacuum at 50 C to syrup. The syrup was extracted with hot 99% ethyl alcohol. The ethyl alcohol extract gave a crystalline product, and pure mannitol was obtained by recrystallization from 95% ethyl alcohol. Mannitol hexaacetate was prepared by the same procedure described in a previous paper (12). RESULTS Survey ofcultures and identification ofa product A total of 45 yeast strains was screened for their mannitol-producing ability. Mannitol production in test tube cultures was qualitatively examined by paper chromatography. Only five strains, isolate T-6, T. versatilis, T. anomala, T. nodaensis, and C. neoformans gave an RF of 0.09, which corresponded to that of authentic mannitol. Polyhydric products and yield obtained in shakeflask cultures of the five strains are shown in Table 1. T. nodaensis and C. neoformans produced very small amounts of mannitol, whereas the isolate T-6, T. versatilis, and T. anomala produced mannitol at rather good yields of 27, 15, and 14% of the glucose consumed, respectively. (A taxonomic study of isolate T-6 which gave the best yield will be published elsewhere.) Mannitol was then isolated from fermented broth of strain T-6. A 1.5 g amount of a pure crystalline product was obtained from the broth, which contained 3.6 g of mannitol. The product was identified as mannitol. The melting point was 165 C and was not depressed when mixed with an Strain TABLE 1. Survey of mannitol-producing yeasts" time authentic sample of mannitol. Elementary analysis gave the following results: C6H1406; calculated: C, 39.56, H, 7.76; found: C, 39.81, H, Optical rotation, as [x] 25,was (c = 7.81 in saturated borax solution), and the infrared spectrum was indistinguishable from that of authentic mannitol. The hexaacetyl derivative of the crystalline product agreed in properties with authentic mannitol, showing a melting point of 124 C. Analysis: C18H26012; calculated: C, 49.77; H, 6.03; found: C, 49.84, H, These findings provide the first demonstration of mannitol as a major product in aerobic dissimilation of glucose by yeasts. Substrates of mannitol production. Substrates used by the isolate T-6 for the production of mannitol in aerobic fermentation were studied. Sugars and polyalcohols examined were D-xylOse, L-arabinose, D-arabinose, D-ribose, L-rhamnose, D-glucose, D-fructose, D-mannose, D-galactose, maltose, saccharose, lactose, glycerol, xylitol, and D-arabitol. The results of these fermentations are shown in Table 2. D-Xylose, D-arabinose, D-ribose, L-rhamnose, and D-arabitol permitted little or no growth. L-Arabinose, saccharose, and lactose supported good growth of the yeast but little mannitol production. D-Fructose, D-mannose, D-galactose, maltose, glycerol, and xylitol all showed good growth and mannitol production. Effect of glucose concentration. As shown in Table 3, 20% glucose concentration gave the best mannitol production with 28% yield based upon glucose used. Glucose concentration of 30% increased glycerol yield and gave the highest yield of total polyalcohols. Effect of nitrogen sources. Several nitrogen sources were tested for their suitability for mannitol production. When an inorganic ammonium salt was used as a nitrogen source, the rapid decrease in ph of the medium during fermentation might reduce polyalcohol yield (9). Therefore, potassium citrate-citric acid buffer was added to all media shown in Table 4. Both Glucose glucose usedb Initial Final Mannitol Glycerol days g/100 ml g/100 ml % % Isolate T T. anomala T. versatilis T. nodaensis C. neoformans a Medium, the standard medium. b Percentage yields of D-arabitol and erythritol were zero

3 VOL. 16, 1968 D-MANNITOL AND GLYCEROL PRODUCTION BY YEASTS TABLE 2. Substrates used by the isolate T-6a 1849 Substrate concn substrate used Substrate time Growth_l Initial Final Mannitol Glycerol days g/10o ml g/100 ml 70 D-Xylose 10 No growth D-Arabinose 10 Little L-Arabinose 7 Good b b D-Ribose 10 Little L-Rhamnose 10 No growth D-Glucose 6 Very good D-Fructose 7 Very good D-Mannose 7 Very good D-Galactose 7 Very good Maltose 7 Very good Saccharose 7 Good Trace Trace Lactose 7 Good Trace Trace Glycerol 7 Good Xylitol 7 Good c 20.Oc 12.6c D-Arabitol 10 No growth a Media: each substrate was added to standard medium without glucose. bpaper chromatogram showed that most of the product from L-arabinose was a pentitol (38% yield) with a very small amount of glycerol and mannitol. c Glycerol, xylitol, and mannitol were separated by paper chromatography on Whatman 3MM paper with a solvent system described in Materials and Methods. The corresponding areas were cut out from paper and the polyalcohols were eluted with water. Each fraction was assayed by periodate oxidation (6). TABLE 3. Effect of glucose conicenttration Initial glucose Fermentation time Final glucose glucose used Mannitol Glycerol g/100 ml days g/100 ml % % ammonium nitrogen and amino nitrogen were found to be available for mannitol production. Effect of concentration of yeast extract and Casamino Acids. Because the C:N ratio of medium significantly affected polyalcohol production by P. miso (15), the effect of yeast extract and Casamino Acids concentration on mannitol formation was examined (Table 5). At the highest level of Casamino Acids (4%), sugar utilization was fast but polyalcohol yield decreased, showing only glycerol formation. At the lowest level (Casamino Acids-free and yeast extract, 0.1%), fermentation was slow and mannitol yield was low. At the highest level of yeast extract (4%), growth was again excessive, with the result that mannitol was not produced. Media containing the highest concentration of either nitrogen source gave significant amounts of ethyl alcohol (11.8 to 23.6% yield based on glucose used), though little ethyl alcohol was produced in the standard medium. A medium containing 0.4% Casamino Acids and 0.10% yeast extract appeared to be best for mannitol production. Effect of phosphate concentration. It has been shown that an excess of inorganic phosphate has a detrimental effect upon the yield of polyalcohol in some yeasts (20, 26). The effect of phosphate concentration on mannitol yield was studied with modified standard medium in which KH2PO4 content was varied from 0 to 2% (Table 6). Even at the highest level used, mannitol yield did not drop. Effect of aeration. In aerobic fermentation, aeration was necessary for high yields of mannitol. The effect of volume of medium in shake flasks was studied by using flasks containing 30, 50, 100, and 200 ml of the standard medium (Table 7). The highest aeration condition with 30 ml of the medium gave the best yield of mannitol. Effect of temperature. The effect of temperature on the mannitol fermentation is shown in Table 8. A temperature of 35 C is too high, since yeast growth was small and sugar utilization was very slow. The fermentations at 22.5 and 26 C were similar in mannitol yield. Sugar utilization was faster and the yield of mannitol was highest at 30C.

1850 ONISHI AND SUZUKI APPL. MICROBIOL. Nitrogen source TABLE 4. Effect of nitrogen source' Initial Glucose Final Polyalcohol yield based on glucose usedb g/100 ml g/100 ml Casamino Acids, 0.4% 10.

4 1850 ONISHI AND SUZUKI APPL. MICROBIOL. Nitrogen source TABLE 4. Effect of nitrogen source' Initial Glucose Final Polyalcohol yield based on glucose usedb g/100 ml g/100 ml Casamino Acids, 0.4% (Mannitolc++, glycerolc+++) Ammonium sulfate, 0.2% (Mannitolc++, glycerolc+++) Ammonium lactate, 0.4% (Mannitolc++, glycerolc+++) Urea, 0.05% (Mannitole++, glycerolc+++) Sodium L-glutamate, 0.4% (Mannitolc++, glycerolc+++) a Medium: 10% glucose, 0.1% KH2PO4, 0.05% MgSO4-7H20, 0.01% CaCl2*2H20, 0.01% NaCl, and vitamins (biotin, 0.4 jug; calcium pantothenate, 80 ;&g; inositol, 400 jug; niacin, 80,Ag; p-aminobenzoic acid, 40,g; pyridoxine hydrochloride, 80,g; thiamine hydrochloride, 80,g; and riboflavin, 40 Ag per 100 ml of medium). Two and one-half ml of a buffer solution consisting of 2.5% citric acid and 10% potassium citrate was added to 100 ml of medium. Fermentation time: 7 days. b Total polyalcohol yield was expressed as glycerol. - Relative amount of mannitol and glycerol produced was checked according to paper chromatogram. TABLE 5. Effect ofconcentration ofyeast extract and Casamino Acids upon yield ofmannitol Expt no. Medium ~~~~~~ ~~~~tation Fermen- ti mne Glucose glucose used Initial Final Mannitol Glycerol Ethyl alcohol days g/100 ml g/100 ml % S % 1 Standard medium Casamino Acids-free stand ard medium 3 (2)a + Casamino Acids, 0.2% (2) + Casamino Acids, 1.5% (2) + Casamino Acids, 4.0% (2) + yeast extract, 0.4% (2) + yeast extract, 1.5% (2) + yeast extract, 4.0% a Medium used in experiment 2. TABLE 6. Effect ofphosphate concentrations KH12PO4 added to the basal medium KH2 added Glucose Guoeglucose used Initial Final Mannitol Glycerol g/100 ml g/joo ml % % No addition % % %7o astandard medium without KH2PO4 was employed as the basal medium. Fermentation time: 7 days. Effect ofph ofmedium. The effect of initial ph of the medium on polyalcohol yield was examined by using five media of ph 3.2, 5.2, 6.7, 7.6, and 8.3, respectively. The ph was adjusted aseptically with 1 N HCl or 1 N NaOH solution after sterilization of the standard medium. The yeast could not grow in medium above ph 7.6. Initial ph of 5.2 gave the best yield. Effect of high concentration of NaCl and KC1. As previously reported, high concentrations of NaCl and KCl greatly enhanced glycerol production in S. rouxii (9) and changed patterns of polyalcohol productivity in P. miso and other yeasts (11). T-6 was isolated from soy-sauce mashes containing 18% NaCl and showed a high tolerance for salt. A study was made of the effect of high concentrations of NaCl and KCl on mannitol production. As shown in Table 9, only glycerol was produced in media containing high levels of NaCl or KCI. The phenomenon was analogous to that in D-arabitol and erythritol biosynthesis by P. miso (11). These phenomena give interesting examples of how high concentrations of NaCl or KCl affect pathways of glucose metabolism in salt-tolerant yeasts.

5 VOL. 16, 1968 D-MANNITOL AND GLYCEROL PRODUCTION BY YEASTS 1851 TABLE 7. Effect of aerationa per flask Final glucose Mannitol produced Glycerol produced glucose used Mannitol Glycerol ml g/l00 ml gil00 ml g/l00 ml N % (Stationary) a Medium, standard medium; initial glucose (g/100 ml), 10.08; fermentation time, 6 days. TABLE 8. Effect of temperaturea Temp Temp tation Fermen- time Final, FinalJ glucose glucose used Mannitol Glycerol C days g/l00 ml % % Trace amedium, standard (g/100 ml), TABLE 9. medium; initial glucose Effect of high concentration of NaCI and KCIa NaCI or KCI Fermen- Final glucose consumed added to standard tation time glucose medium tto Mannitol Glycerol days g/l00 ml % % No addition % NaCl % NaCl % KCI % KCl a Initial glucose (g/100 ml), DISCUSSION Extensive studies on polyalcohol production from glucose by various genera and species of yeasts have revealed six characteristic types based upon production of glycerol, erythritol, or D-arabitol, or all three (8, 10). We demonstrated another pattern of polyalcohol production in a newly isolated yeast, T-6, which produces mannitol and glycerol in high yield. Important factors affecting mannitol production were found to be concentrations of nitrogen sources and of NaCl or KCl in culture media. At high levels of these components, the yeast produced only glycerol. Inorganic phosphate did not adversely affect mannitol production. Our previous and present reports showed that yeasts could produce a great variety of polyalcohols of C3, C4, C5, C6, and C7 from pentoses and hexoses: glycerol, erythritol, D-arabitol (10), and mannitol from glucose; glycerol, D-arabitol, and dulcitol from galactose (14); xylitol, mesoglycero-ido-heptitol and D-glycero-D-ido-heptitol from xylose (12); L-arabitol from L-arabinose; and ribitol from D-ribose (13). Thus, almost all of the polyalcohols commonly found in nature can be produced by yeast metabolism. ACKNOWLEDGMENTS We wish to express our sincere thanks to K. Arima and Y. Ikeda of the University of Tokyo for their kind guidance. We also thank M. Mogi and N. Iguchi of our institute for their encouragement. The technical assistance of K. Kouchi is gratefully acknowledged. LITERATURE CITED 1. Birkinshaw, J. H., J. H. V. Charles, A. H. Hetherington, and H. Raistrick Biochemistry of microorganisms. IX. Production of mannitol from dextrose by species of Aspergillus. Trans. Roy. Soc. (London) B220: Coyne, F. P., and H. Raistrick Studies in the biochemistry of microorganisms. XX. On the production of mannitol from hexoses and pentoses by a white species of Aspergillus. Biochem. J. 25: Kinoshita, K Formation of itaconic acid and mannitol by a new fungus. J. Chem. Soc. Japan 50: Lee, W. H Carbon balance of a mannitol fermentation and the biosynthetic pathway. Appl. Microbiol. 15: Morris, D. L Quantitative determination of carbohydrates with Dreywood's anthrone reagent. Science 107: Neish, A. C Analytical methods for bacterial fermentations, 2nd NRC (Natl. Res. Council Can.) Bull

1852 ONISHI AND SUZUKI Appi MICROBIOL. 7. Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375-380. 8. Nickerson, W. J., and R. G.

6 1852 ONISHI AND SUZUKI Appi MICROBIOL. 7. Nelson, N A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: Nickerson, W. J., and R. G. Brown Uses and products of yeasts and yeast-like fungi. Adv. Appl. Microbiol. 7: Onishi, H Studies on osmophilic yeasts. VI. Glycerol production by the salt-tolerant yeasts in the medium with high concentrations of sodium chloride. Bull. Agr. Chem. Soc. Japan 23: Onishi, H Studies on osmophilic yeasts. VIII. Polyalcohol production by various genera and species of yeasts. Bull. Agr. Chem. Soc. Japan 24: Onishi, H Studies on osmophilic yeasts. XV, The effects of high concentrations of sodium chloride on polyalcohol production. Agr. Biol. Chem. (Tokyo) 27: Onishi, H., and M. B. Perry The production of meso-glycero-ido-heptitol and D-glycero-Dido-heptitol by Pichia miso. Can. J. Microbiol. 11: Onishi, H., and T. Suzuki The production of xylitol, L-arabinitol and ribitol by yeasts. Agr. Biol. Chem. (Tokyo) 30: Onishi, H., and T. Suzuki Formation of dulcitol in aerobic dissimilation of D-galactose by yeasts. J. Bacteriol. 95: Onishi, H., N. Saito, and I. Koshiyama Studies on osmophilic yeasts. XI. Various factors affecting polyalcohol production by Pichia miso. Agr. Biol. Chem. (Tokyo) 25: Osaki, M Production of mannitol by a sake-yeast. J. Ferment. Technol. 16: Otani, Y Studies on the production of mannitol by fungi. I. On the production of mannitol by a new fungus, Aspergillus mannitosus. J. Agr. Chem. Soc. Japan 31: Otani, Y Studies on the production of mannitol by fungi. IL. On the production of mannitol from glucose and sucrose by Aspergillus mannitosus. J. Agr. Chem. Soc. Japan 31: Partridge, S. M Aniline hydrogen phthalate as a spraying reagent for chromatography of sugars. Nature 164: Peterson, W. H., W. F. Hendershot, and G. J. Hajny Factors affecting production of glycerol and D-arabitol by representative yeasts of the genus Zygosaccharomyces. Appl. Microbiol. 6: Rees, W. R., and T. Reynolds A solvent for the paper chromatographic separation of glucose and sorbitol. Nature 181: Roxburgh, J. M., J. F. T. Spencer, and H. R. Sallans Recovery of polyhydric alcohols from yeast fermentations. Can. J. Technol. 34: Smiley, K. L., M. C. Cadmus, and P. Liepins Biosynthesis of D-mannitol from D-glucose by Aspergillus candidus. Biotechnol. Bioeng. 9: Somogyi, M Notes on sugar determination. J. Biol. Chem. 195: Spencer, J. F. T., and H. R. Sallans Production of polyhydric alcohols by osmophilic yeasts. Can. J. Microbiol. 2: Spencer, J. F. T., and P. Shu Polyhydric alcohol production by osmophilic yeasts: effect of oxygen tension and inorganic phosphate concentration. Can. J. Microbiol. 3: Yoda, A A new method for detection of non-reducing sugars and polyalcohols on paper chromatogram. J. Chem. Soc. Japan 73:18-19.

Erythritol Production by a Yeastlike Fungus

APPLIED MICROBIOLOGY Vol. 12 No. 3 p. May, 1964 Copyright 1964 American Society for Microbiology Printed in U.S.A. Erythritol Production by a Yeastlike Fungus G. J. HAJNY, J. H. SMITH 1, AND J. C. GARVER