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Badal C. Saha,1 Lawrence K. Nakamura2
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois 61604 2 Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois 61604; telephone: +1 (309) 681-6276; fax: +1 (309) 681-6427; e-mail: email@example.com
Received 14 May 2002; accepted 25 November 2002 DOI: 10.1002/bit.10638
Abstract: Lactobacillus intermedius B-3693 was selected as a good producer of mannitol from fructose after screening 72 bacterial strains. The bacterium produced mannitol, lactic acid, and acetic acid from fructose in pHcontrolled batch fermentation. Typical yields of mannitol, lactic acid, and acetic acid from 250 g/L fructose were 0.70, 0.16, and 0.12 g, respectively per g of fructose. The fermentation time was greatly dependent on fructose concentration but the product yields were not dependent on fructose level. Fed-batch fermentation decreased the time of maximum mannitol production from fructose (300 g/L) from 136 to 92 h. One-third of fructose could be replaced with glucose, maltose, galactose, mannose, raffinose, or starch with glucoamylase (simultaneous saccharification and fermentation), and two-thirds of fructose could be replaced with sucrose. L. intermedius B-3693 did not co-utilize lactose, cellobiose, glycerol, or xylose with fructose. It produced lactic acid and ethanol but no acetic acid from glucose. The bacterium produced 21.3 ± 0.6 g lactic acid, 10.5 ± 0.3 g acetic acid, and 4.7 ± 0.0 g ethanol per L of fermentation broth from dilute acid (15% solids, 0.5% H2SO4, 121°C, 1 h) pretreated enzyme (cellulase, -glucosidase) saccharified corn fiber hydrolyzate. © 2003 Wiley Periodicals, Inc. *Biotechnol Bioeng 82:
Keywords: Lactobacillus intermedius; mannitol production; lactic acid production; ethanol production; mixed acid production; fructose fermentation; batch fermentation; fed-batch fermentation; pH-controlled fermentation
INTRODUCTION Mannitol, a naturally occurring polyol, is widely used in the food, pharmaceutical, medical, and chemical industries (Soetaert et al., 1995). It is used as a sweet-tasting bodying
Correspondence to: B. C. Saha † Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.
and texturing agent. Mannitol reduces the crystallization tendency of sugars and is used as such to increase the shelflife of foodstuffs. Crystalline mannitol exhibits a very low hygroscopicity, making it useful in products that are stable at high humidity. It is extensively used in chewing gum. Because of its desirable properties, mannitol is commonly used in the pharmaceutical formulation of chewable tablets and granulated powders. It prevents moisture absorption from the air, exhibits excellent mechanical compressing properties, does not interact with the active components, and has a sweet cool taste that masks the unpleasant taste of many drugs (Debord et al., 1987). The complex of boric acid with mannitol is used in the production of dry electrolytic capacitors. It is an extensively used polyol for production of resins and surfactants. Mannitol is used in medicine as a powerful osmotic diuretic and in many types of surgery for the prevention of kidney failure and to reduce dye and brain edema. Mannitol hexanitrate is a well-known vasodilator, used in the treatment of hypertension. Mannitol is currently produced industrially by highpressure hydrogenation of fructose/glucose mixtures in aqueous solution at high temperature (120–160°C) with Raney nickel as catalyst (Makkee et al., 1985). Typically, the hydrogenation of a 50/50 fructose/glucose mixture results in an approximately 25/75 mixture of mannitol and sorbitol. This means that about half of the fructose is converted to mannitol and half of it to sorbitol. The glucose is hydrogenated exclusively to sorbitol. As a consequence, the commercial production of mannitol is always accompanied by the production of sorbitol thus resulting in a less efficient process (Soetaert et al., 1995). In recent years, research efforts have been directed towards production of polyols by fermentation and enzymatic means (Vandamme and Soetaert, 1995). We recently screened 72 bacterial cultures from ARS Culture Collection for production of mannitol from glucose or fructose. While
© 2003 Wiley Periodicals, Inc. * This article is a US Government Work and, as such, is in the public domain in the United States of America.
5-inch stir bars. B. Inc. casei B-1922. 0. L. Samples were withdrawn at a specific time to determine cell growth. We selected Lactobacillus intermedius B-3693 as a good producer of mannitol. apiarius NRS-1438. The pH was maintained at 5. L. IL) These strains (with NRRL numbers) were: L. Micrococcus luteus B-287..05 g manganese sulfate. thiaminolyticus B-14605. gasseri B-14168. L. hirae B-14926. pentosus B-473. an Aminex HPX-87H column was used. M. B. or 8 N NaOH. chondroitinus B-14420. Lactococcus lactis B-1821. 4. choshinensis B-23247. amyloliquefaciens B-14394. San Jose. we describe the production of mannitol and lactic acid by fermentation with L. L. mesenteroides B-1209. P. cellobiosus B-1840. amylophilus B-4436. The column was maintained at 65°C.6 mm) column was obtained from Advanced Separation Technologies. NJ). illinoisensis NRS-1356. L. paracasei B-4564. L. lautus NRS-666. Millipore Corp. Paenibacillus polymyxa B-367. 1. w/v) was inoculated with a loopful of cells taken from a stock slant and incubated at 37°C on a rotary shaker (130 rpm) for 24 h. cereus B-3711. 10 g beef extract. L. arabinosus B-787. nine cultures produced mannitol from fructose. The medium and the substrate (glucose or fructose 5%. Cultures were stirred magnetically using 1. E. kristinae B-14845. mesenteroides subsp. and the sugars were eluted with deionized water (Milli-Q water. reuszeri NRS-1206. B.and L-lactic acids were purchased from Sigma Chemical Company (St. macerans B-172. citrovorum B-1147. amylovorus B-4545. and production yield. B. The column was maintained at 85°C. fructivorans B-4000.5). (1960) and contained 10 g peptone. plantarum B-4496. E. 0. Both D. Peoria. MO). mali B-4565. L. B. coryniformis B-4391. CA). B. an Aminex HPX-87P column was used. L. P. amylolyticus B-377. L. 5 g sodium acetate. animalis B14177. glucanolyticus B-14680. IL). L. L. P. intermedius B-3693. P. 1. MATERIALS AND METHODS Materials Aminex HPX-87P (300 × 7. B. w/v) were sterilized separately at 121°C for 15 min. leichmanii B-4525.0 mL Tween 80.0 by adding 2. faecium B-1295. mycoides NRS-273. Screening Medium and Culture Conditions The screening medium designated as MRS was based on that of de Man et al. L. alginolyticus NRS-1347. L. acidophilus B-4495. and product yield. reuteri B-14172.1 g magnesium sulfate. and 2 g disodium phosphate per liter (final pH 6. L. paramesenteroides B-3471. Louis. P. B. lactis B-3468. An Astec CLC-D (150 × 4. 1999). delbrueckii B-763. L. CA). P. L. at 130 rev/min. brevis B-1836. oenos B-3474. gramminis B-14857. residual substrate. P.6 mL/min. and the organic acids were eluted with 5 mM H2SO4 or 10 mM HNO3 at a flow rate of SAHA AND NAKAMURA: PRODUCTION OF MANNITOL AND LACTIC ACID 865 . 5 g yeast extract. sugar utilization. buchneri B-1860. pentosaceus B-14009. Samples were withdrawn periodically to determine cell growth. smithii NRS-173. P. coagulans NRS-609. agri B-1158. pumilus B-14292. L. B. megaterium B-14308. L. Bedford. a 250-mL Erlenmeyer flask containing 100 mL of the medium with fructose (2%. Sugar utilization and product analysis were performed by highpressure liquid chromatography (HPLC) (Spectra-Physics.none of the cultures produced mannitol from glucose. licheniformis NRS-1264. L. L. salivarius B-1949. rhamnosus B-442. NC). Enterococcus faecalis B-537. E. Corn fiber from a corn wet milling facility was supplied by Williams Bioenergy (Pekin. Bacillus subtilis NRS-744. P. jensenii B-4550. dextranicum B-1120. L. To our knowledge. L. For organic acid and ethanol analyses. fermentum B-1915. In this paper. pabuli B-14213. L. For quantification of sugar and mannitol. Brevibacillus brevis NRS604. Preparation of Corn Fiber Hydrolyzate Corn fiber was pretreated and saccharified with enzymes by the procedure described previously (Saha and Bothast.8 mm) and Aminex HPX-87H (300 × 7. Streptococcus dysgalactiae B-688. (Wippany. intermedius B-3693. L. L. validus NRS-1000. Celluclast (cellulase) and Novozyme 188 ( glucosidase) were from Novozymes (Franklinton. the production of mannitol by this organism has not been reported. Bacterial Strains Seventy-two bacterial strains were obtained from the ARS Culture Collection (National Center for Agricultural Utilization Research. (1994). helveticus B-1935. P. Analytical Methods Cell growth was monitored by measuring optical density of the appropriately diluted culture broth at 660 nm.8 mm) columns were purchased from Bio-Rad Laboratories (Hercules. P. L. The experimental set-up is shown in Fig. Batch culture experiments were performed in pH-controlled 500-mL fleakers with an initial medium volume of 300 mL at either 30 or 37°C essentially as described by Bothast et al. L. MA) at a flow rate of 0. P. P. peoriae B-14750. Leuconostoc amelibiosum B-742. A 125-mL Erlenmeyer flask containing 50 mL MRS medium with substrate was inoculated with a loopful of cells taken from a stock slant and incubated at 30°C on a rotary shaker (130 rpm). L. L. casseliflavus B-3502. Fermentation Experiments The fermentation experiments were carried out in simplified MRS medium (without beef extract and Tween 80). L. mesenteroides subsp. 2 g ammonium citrate. curdlanolyticus B-23243. and B. For seed culture. B. L. P. bulgaricus B-548. P. Pediococcus acidilactici B-1153. pulvifaciens B-14166.
00 0. fermentum B-1915. 200. Mannitol production from fructose by L. RESULTS Screening for Mannitol Production The strains that produced mannitol from fructose were L. Moreover.00 0.00 0.12 ± 0.16 ± 0. and the mobile phase was 5 mM CuSO4 at a flow rate of 1. 2). cellobiosus B-1840. Also.0 g/L at 150. lactic acid. L.00 0. 130 rpm.00 0. citrovorum B-1147. Small white needlelike crystals of mannitol appeared upon refrigeration of the cell-free fermentation broth of 300 g/L fructose at 4°C (Fig. and 300 g/L substrate concentration are shown in Fig. 3A– D.9. and it did not consume mannitol even when all supplied fructose had been utilized.0. 175.0 mL/min. L.6 mL/min. initial pH 6. 250.01 Acetic acid (g/g)b 0. the product (mannitol. brevis B-1836. However. L. 2003 . and L. 82. all these strains produced lactic acid and acetic acid. intermedius B-3693 was selected for further study on mannitol production based on its ability to produce mannitol more rapidly than the other strains (data not shown).a Fructose (g/L) 150 200 250 300 a Time (h) 15 40 64 136 Mannitol (g/g)b 0.66 ± 0.9.03 0.69 ± 0. intermedius B-3693 is presented in Table I.72 ± 0.02 0.3 ± 11.and L-(+)-lactic acid were measured quantitatively by HPLC.6 ± 6.17 ± 0. intermedius B-3693 Effect of Fructose Concentration Strain B-3693 produced mannitol. intermedius B-3693.03 Lactic acid (g/g)b 0. L. pH controlled at 5. Typical time courses of fructose utilization and mannitol. lactic acid. dextranicum B-1120.15 ± 0. Among these nine strains. the strain L. JUNE 30. respectively. The mannitol yields were 107. b In g product per g fructose provided. Leuconostoc amelibiosum B-742. In addition. Peaks were detected by refractive index and identified and quantified by comparison to retention times of authentic standards. lactic acid. 200. pH-controlled fleaker fermentation system set-up.17 ± 0. 138. Batch Production of Mannitol by L. 500-mL fleaker with 300 mL of medium. 0.6 ± 5. NO. intermedius NRRL B-3693 in pH-controlled batch fermentation.11 ± 0. and 300 g/L fructose. 250. VOL. mesenteroides subsp. buchneri B-1860. L.00 0. L.5. paramesenteroides B-3471. Both D-(−). there was a long lag period of about 72 h in growth and fructose utilization at 300 g/L fructose concentration in comparison to the lag period of about 16 h at 250 g/L fructose.12 ± 0. and 198.6 ± 0. Peaks were detected by UV detector at 240 nm and identified and quantified by comparison to retention times of authentic standards.13 ± 0. An Astec CLC-D column was used at room temperature. the product patterns and yields were not greatly influenced by fructose concentration. The bacterium transformed fructose to mannitol from the early growth stage.00 0. 7.Figure 1.70 ± 0. and acetic acid when grown on fructose in pH-controlled fermentation. and acetic acid) concentration continued to increase slightly upon further continuation of Table I. and acetic acid production at 150.5. The effect of fructose concentration ranging from 150 to 300 g/L on mannitol production by L. 866 BIOTECHNOLOGY AND BIOENGINEERING. The time of maximum mannitol yield varied greatly from 15 h at 150 g/L fructose to 136 h at 300 g/L fructose concentration.00 At 37°C.
which were partially neutralized during fermentation by adding NaOH to control the pH at 5.4 ± 2.0 ± 0. Use of the fed-batch approach to decrease the fermentation time or increase the glucose concentration did not improve these fermentation parameters. glycerol.5 ± 0.1 ± 0. the fed-batch culture technique was used. HPLC analysis of the fermentation broth with an Astec CLC-D column indicates that the bacterium produced both D. no formation of acetic acid was observed. respectively. The conversion efficiency of fructose to mannitol was 96%.3. Mannitol crystals formed in cell-free fermentation broth at 4°C. mannose. Figure 2.5 ± 4.6 g.4 ± 9.1 g lactic acid and 38.1) was obtained at 24 h. The bacterium co-utilized fructose and glucose simultaneously and produced very similar quantities of mannitol. Fructose used: 300 g/L. Two-thirds of the fructose were also replaced by sucrose. Two-thirds of the fructose can also be replaced by sucrose.9 g ethanol in 64 h.0. the bacterium utilized all glucose provided (150 g/L) and produced lactic acid (70. maltose. lactic acid. and acetic acid in comparison with fructose only. lactic acid.7 g/L. the L-form eluted first and was well separated from the D-form (Fig.7 ± 0.8 in 136 h at fructose concentrations of 200. A660 of 6. 5.5 in 15 h) was obtained at fructose concentration of 150 g/L. lactic acid.3 ± 0. 250. The maximum cell growth (cell density. It is clear that one-third of the fructose can be replaced with glucose. and galactose. starch with glucoamylase.9 ± 0. The glucose was converted to lactic acid and acetic acid. and 33.4 ± 0. 44.2) occurred in 64 h. The yields of mannitol.5 and 30.7 ± 0.96. 7). During the entire fermentation. In this column.1 ± 0.3 ± 0.0 in 64 h. it did not contribute to net mannitol SAHA AND NAKAMURA: PRODUCTION OF MANNITOL AND LACTIC ACID 867 . 5.and D-lactic acids were 22. intermedius B-3693 In order to decrease the fermentation time required to complete 300 g/L fructose utilization.5 ± 0.and L-lactic acid in equal proportions (1:1 ratio) throughout the time course studied. A time course of fructose (100 g/L) and glucose (50 g/L) co-fermentation is shown in Fig. under the specified conditions as mentioned in the section Materials and Methods. starch plus glucoamylase (simultaneous saccharification and fermentation. the bacterium utilized 80% substrate and produced 70. maltose. As shown in Fig. The average maximum cell densities (A660) were 4.2 ± 1. and acetic acid were 202.4 ± 0.3) was observed at 48 h. arabinose.0.6 ± 0. The maximum cell growth (A660 of 3. respectively in 160 h. 52. The retention times of L.3) was observed at 88 h. Substitution of Fructose With Other Energy Sources In order to investigate the role of other sugars in mannitol production. respectively. the fermentation in most cases.3. The maximum cell growth (A660 of 8. At 200 g/L glucose. 6. However. The maximum cell growth (A660 of 7. The bacterium was not able to co-utilize lactose. galactose. and 6. the production of lactic acid and acetic acid increased considerably. The fermentation time decreased considerably from 136 to 92 h by feeding equal amounts of substrate and medium four times. intermedius B-3693 The bacterium utilized glucose and produced lactic acid and ethanol.0 g/L) in 40 h. There was a lag of growth and glucose utilization for about 24 h and maximum cell growth (A660 of 8. xylose. The results of mannitol production by the twosubstrate system are presented in Table II. and 38. and glycerol. SSF). and 300 g/L. 4. and xylose with fructose.3 ± 1. Even though arabinose was co-utilized with fructose very well by the bacterium.2 min. Fed-Batch Production of Mannitol by L. and acetic acid from cofermentation of fructose and glucose (2:1) at 300 g/L total substrate concentration were 179. The yields of mannitol. respectively. The results of fed batch culture with 300 g/L fructose are shown in Fig.production. one-third of the fructose was replaced with other substrates such as glucose. cellobiose.6 g/L) and ethanol (36. cellobiose.4 in 24 h. Fermentation of Glucose by L. mannose.
1 ± 1.3 ± 1.2 ± 0.6 16.0. intermedius NRRL B-3693 in pH-controlled batch fermentation. 82.3 ± 0.7 ± 0.8 ± 0.5 with H2SO4.6 ± 1. ( ) lactic acid. The liquid portion of corn fiber hydro- Table II. ( ) acetic acid.5. overlimed with Ca(OH)2 to pH 10.2 15.6 ± 1. ( ) mannitol.6 ± 0. NO.1 14.a Time (h) 20 64 24 15 89 15 40 64 Mannitol (g/L) 97.7 24.4 13.9 Lactic acid (g/L) 23.3 41.2 18.1 ± 1.5% H2SO4 at 121°C for 1 h. Time courses of fructose (150–300 g/L) utilization and mannitol production by L.8 ± 0. neutralized back to pH 6.9 13.3 13. VOL. treated with Celluclast and Novozyme 188 (1 mL each for 100 g corn fiber) at pH 5.2 95.7 ± 0.8 ± 0.5 23.9 ± 0.5.3 ± 0.2 ± 0. intermedius B-3693 in pH-controlled batch fermentation at 37°C: (A) 150 g/L.5 ± 0.7 86. 130 rpm. pH maintained at 5.2 ± 0.4 ± 2. 868 BIOTECHNOLOGY AND BIOENGINEERING. Symbols: ( ) fructose. and centrifuged (8. Mannitol production using two substrate system (fructose and another sugar) by L.4 14. 500-mL fleaker with 300 mL of medium.8 89.1 ± 0.9 ± 0.Figure 3.6 84. (B) 200 g/L.0 and 45°C for 72 h.6 ± 0. JUNE 30.1 27. 20 min) to remove the solids.6 25. initial pH 6. (C) 250 g/L. intermedius B-3693 Corn fiber (15% solids) was pretreated with 0. 7.9 82.1 Acetic acid (g/L) 15.7 94.3 ± 2. Fermentation of Corn Fiber Hydrolyzate by L.5 20. (D) 300 g/L. 2003 .2 Substrate (g/L) Fructose Fructose Fructose Fructose Fructose Fructose Fructose Fructose a (100) plus glucose (50) (50) plus sucrose (100) (100) plus starch (50) and glucoamylase (100) plus maltose (50) (100) plus mannose (50) (100) plus galactose (50) (100) plus affinose (50) (100) plus arabinose (50) At 30°C.000 rpm.7 61.6 ± 1.
intermedius NRRL B-3693 in pH-controlled batch fermentation at 35°C.3 g acetic acid. Symbols: ( ) fructose. lactic acid. Symbols: ( ) glucose. Figure 7. ( ) mannitol. The bacterium produced lactic acid and ethanol but no acetic acid from glucose. Figure 6. and acetic acid with fructose as growth substrate.7 ± 0. glucose. intermedius NRRL B-3693 in pH-controlled fed-batch fermentation at 37°C. ( ) lactic acid. The mobile phase was 5 mM CuSO4 at a flow rate of 1. and lactic acid and ethanol production by L.10.0 g ethanol per L of fermentation broth. ( ) mannitol. L-(+)-lactic acid. Symbols: ( ) fructose. There was practically no difference in product profiles as well as in product ratios in both cases of fructose and glucose metabolism under aerobic and anaerobic conditions. ( ) acetic acid. SAHA AND NAKAMURA: PRODUCTION OF MANNITOL AND LACTIC ACID 869 . and 4. An Astec CLC-D column was used at room temperature. ( ).5 ± 0. D-(−)-lactic acid.3 ± 0. Fructose used: 300 g/L (final concentration). D. The product patterns were analyzed by HPLC. The bacterium was able to grow well in this overlimed corn fiber hydrolyzate and produced 21. ( ) lactic acid. HPLC pattern of fermentation broth analysis of L. ( ) lactic acid. intermedius NRRL B-3693 grown on glucose (150 g/L). ( ) ethanol. 50.6 ± 0. Time courses of fructose utilization and mannitol production by L. ( ) acetic acid. intermedius NRRL B-3693 in pH-controlled batch fermentation at 37°C.1 g) was used as substrate. It utilized both fructose and glucose well and produced mannitol. Fructose and Glucose Metabolism by L. L. Time course of glucose (150 g/L) utilization.6 g lactic acid. Figure 5. lyzate (total sugar content. intermedius B-3693 Under Anaerobic Conditions The bacterium was grown in MRS medium under anaerobic condition using 2% fructose or 2% glucose as carbon source.Figure 4.0 mL/min. Time courses of fructose (100 g/L) and glucose (50 g/L) coutilization and mannitol production by L. Peaks were detected by UV detector.
Leuconostoc sp. There was no formation of sorbitol. xylitol.and acetic acid. This is also the first detailed production formation study for a mannitol producing Lactobacillus sp.. Thus a desired mannitol concentration in the fermentation broth can be achieved by using 300 g/L substrate concentration by both batch and fed-batch fermentation. intermedius B-3693 with results of earlier workers. 1998 Song et al. intermedius NRRL B-3693. JUNE 30. the reducing equivalents are generated by conversion of about one-third of the fructose to lactic acid Table III. 1998). maltose. For fructose and glucose (2:1) co-fermentation. mannose. 8. At fructose concentration at 300 g/L. 1988 Bacteria Lactobacillus intermedius B-3693 Lactobacillus sp. It appears that mannitol produced by L. intermedius NRRL B-3693 is presented in Fig. 1974 Onishi and Suzuku. xylose. or arabitol in the cases of glucose. B001 Lactobacillus sp. 1998 Korakli et al.. and raffinose (Table II). intermedius B-3693. 1967 Hendriksen et al. 7.. Y-107. The process makes use of the capability of L.. respectively. galactose. intermedius to utilize fructose as an alternative electron acceptor. Comparison of mannitol production by L. and Leucononostoc mesenteroides (Soetaert et al. 1995. 2002 Hattori and Suzuki. 2002). The fermentation time decreased considerably from 136 to 92 h by using the fed-batch approach. Microorganism Substrate (g/L) Fructose Fructose Fructose Fructose Fructose Fructose Fructose Fructose Fructose Fructose Fructose Fructose (150) (200) (250) (300) (300) (100) + glucose (50) (100) + glucose (50) (100) (?) (100) + glucose (50) (08) (50) Timea (h) 15 40 64 136 92 20 24 120 120 35 — 25 168 100 240 168 288 288 Yieldb (%) 72 69 70 66 67 65 65 73 60 g/L 60 30–40 40 45 52 28 31 69 40 Reference This work This work This work This work This work (fed-batch) This work Itoh et al. DISCUSSION This is the first study of mannitol production by L. 870 BIOTECHNOLOGY AND BIOENGINEERING. the equation becomes: 2 Fructose + Glucose → 2 Mannitol + Lactic Acid + Acetic Acid + CO2. In this process. Mannitol yields were calculated on the basis of initial sugars employed. 82. and arabinose co-fermentation with fructose. thereby reducing it to mannitol with the enzyme mannitol dehydrogenase. It is thought that enzyme reaction proceeds according to the following (theoretical) equation: 3 Fructose → 2 Mannitol + Lactic Acid + Acetic Acid + CO2. such as Lactobacillus sp. 1998 Yun and Kim. using fructose as an electron acceptor (Grobben et al. NO. 1970 Smiley et al... An overview of the proposed pathway of fructose utilization by L. 1992 Yun and Kim. the final mannitol concentration in the fermentation broth exceeded the solubility limit of 180 g/L at 25°C. VOL. Y-002 Yeast Candida magnoliae Candida zeylannoides Torulopsis versalitis Torulopsis mannitofaciens Fungi Aspergillus candidus Penicillium scabrosum a Fructose (150) n-Paraffin (100) Glucose (194) Glycerol (100) Glucose (32) Sucrose (150) b Time required to reach maximum mannitol yield. 1995 Erten. 2000 Soetart et al. intermedius was derived from hexose phosphate pathway like other mannitol-producing bacteria. This offers an extra advantage for downstream processing of fermentation broth because mannitol can be crystallized out by simple cooling of the fermentation broth Figure 8. Y-107 Lactobacillus sanfranciscensis Leuconostoc mesenteroides Leuconostoc sp. Y-002. 2001. Several heterofermentative lactic acid bacteria produce mannitol in large amounts. Wisselink et al. 2003 . 1968 Onishi and Suzuki... Glucose can be replaced with starch plus glucoamylase (simultaneous saccharification to glucose and fermentation to lactic acid and acetic acid). 1996. Overview of the pathway of fructose utilization by L. Yun and Kim..
Frisvad JC. and galactose) in corn fiber acid hydrolyzate and produced lactic acid. 2001. Inaba H. Biotechnol Bioeng 9: 365–374. Hendriksen HV. Soetaert W. Carter. Microbial production of D-mannitol and Dfructose from glycerol. Fuels and chemicals from biomass. The heterofermentative lactic acid bacterium produced lactic acid (0. Lefebvre C. A medium for the cultivation of Lactobacilli. Soetaert W. Guyot JC. Korakli M. Schwarz E. Wolf G.(Fig. acetic acid. and Jarvis W. Flosenzier AV. Eggink G. Wisselink HW. Debord B. 2002. European Patent 486024. 1970. Production methods of D-mannitol. Onishi H.) thanks Gregory J. References Bothast RJ. Process Biochem 33:735–739. and ethanol. Weusthuis RA.C. Production of mannitol by Penicillium strains. Biotechnical modification of carbohydrates. DC: American Chemical Society. Yun JW. Grobben GJ. Saha BC. Yun JW. FEMS Microbiol Rev 16:163–186. Hyun H-H. Starch/Staerke 37:136–141. de Man JC. Woodward J. 1995). Tsai S-P. To the authors’ knowledge. 1988. Fermentation of L-arabinose. 1997). Production of mannitol by Lactobacillus sanfranciscensis. editors. Bouche R. J Appl Bacteriol 23:130–135. A comparative study of mannitol production by two lactic acid bacteria. Campagna. Liepins P. Suzuki T. A comparative study of mannitol production by L. Rogosa M. Adv Food Sci 22:1–4. 2000. Washington. The reported fermentation time for L. Agro Food Ind Hi-Tech 6:41–44. 1960. Drug Dev Ind Pharm 13:1533–1546. 1974. Hammes WP. Cadmus MC. Production of D-mannitol and D-lactic acid by fermentation with Leuconostoc mesenterroides. Biosynthesis of D-mannitol from D-glucose by Aspergillus candidus. The data presented in this table with other bacteria are with enriched MRS medium. Itoh Y. Buchholz K. 1996. p 224–236. Agr Biol Chem 38:1203–1208. Hattori K. arabinose. Wooldrige. it produced both D. intermedius B-3693 with those of the earlier workers is presented in Table III. Appl Microbiol 16:1847–1852. Saha BC. Tanaka A. Mannitol production by lactic acid bacteria: a review. Lactobacillus sp. Wisselink HW. Eggink G. Appl Environ Microbiol 67:2867–2870. Araya H. Alder-Nissen J. Baek H. Mathiasen TE.. Van Bekkum H. acetic acid and lactic acid. Study of different forms of mannitol: comparative behavior under compression. Hugenholtz J. This finding is quite different from the reported D-lactic acid production from Leuconostoc mesenteroides (Soetaert et al.47 g/g) and no acetic acid from glucose (150 g/L). Int Dairy J 12:151–161. In: Saha BC. Spontaneous formation of a mannitolproducing variant of Leuconostoc pseudomesenteroides grown in the presence of fructose. Suzuki T. Ogasawara K. It is not clear why acetic acid was not produced. Weusthuis RA. vandamme EJ. Metabolism of fructose as an electron acceptor by Leuconostoc mesenteroides. 1968. Guyot-Hermann AM. Vandamme EJ. However. Hubert J. intermedius B-30560 might be shortened by using the enriched MRS medium. Sakamoto Y. 1994. Hugenholtz J. Bothast RJ. Emborg C. Sharpe ME. Smiley KL. Tonia R. KY-107. It has the potential to be used for making lactic acid. 2002. Lee J-K. B001 and method of producing mannitol. SAHA AND NAKAMURA: PRODUCTION OF MANNITOL AND LACTIC ACID 871 . Song K-H. Large scale production of erythritol and its conversion to D-mannitol production by n-alkane-grown Candida zeylanoides. Further. 1995. Biotechnol Bioeng 12:913–920. Biotechnol Lett 18:35–40. Sarah E.24 g/g glucose). Ingram LO. Hoefnagel HN. acetic acid. 1995. 1987. and ethanol from waste and underutilized agricultural residues such as corn fiber hydrolyzate. Biotechnol Lett 16:401–406. 1999. Pretreatment and enzymatic saccharification of corn fiber.S. 1985. it also produced ethanol in good yield (0.and L-lactic acid in equal amounts. Appl Biochem Biotechnol 76:65–77. 1998. Suzuki T. J Chem Technol Biotechnol 43:223–228. xylose. Kim DH. D-xylose and D-glucose by ethanologenic recombinant Klebsiella oxytoca strain P2. these authors did not present any experimental evidence for the D-lactic acid production. Production of D-mannitol and glycerol by yeasts. Koga J. J Ferment Bioeng 85:203–208. Kim S-Y. Production of mannitol by a novel strain of Candida magnoliae. 1997. Microbial transformation of fructose to mannitol by Lactobacillus sp. Hong S-G. 1967. Makkee M. Song J-Y. Grobben GJ. 1992. Song SK. 2). Peters SWPG. Kang SC. The bacterium was able to utilize sugars (glucose. Datta R. (Summer Intern) for technical assistance. Lactic acid production and potential uses: a technology and economic assessment. 1998. Kennedy. Biotechnol Lett 24:9–12. Lactic acid and acetic acid can also be recovered by electrodialysis (Datta and Tsai. Kieboom APG. Jr. Onishi H. Erten H. One of the authors (B. A simplified MRS medium without beef extract and Tween was used throughout this study.