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Appl Microbiol Biotechnol (2011) 89:879–891 DOI 10.1007/s00253-010-2979-3

MINI-REVIEW

Biotechnological production of mannitol and its applications

Badal C. Saha & F. Michael Racine

Received: 18 August 2010 /Revised: 20 October 2010 /Accepted: 20 October 2010 /Published online: 10 November 2010 # Springer-Verlag (outside the USA) 2010

Abstract Mannitol, a naturally occurring polyol (sugar fermentation and using enzyme technology, downstream ), is widely used in the food, pharmaceutical, medical, processing, and applications of mannitol. and chemical industries. The production of mannitol by fermentation has become attractive because of the problems Keywords Mannitol production . Homofermentative lactic associated with its production chemically. A number of acid bacterium . Heterofermentative bacterium . homo- and heterofermentative (LAB), Fermentation . , and filamentous fungi are known to produce mannitol. In particular, several heterofermentative LAB are excellent producers of mannitol from . These bacteria convert Introduction fructose to mannitol with 100% yields from a mixture of and fructose (1:2). Glucose is converted to lactic acid Mannitol is a naturally occurring found in many and , and fructose is converted to mannitol. The fruits and vegetables. It is found in pumpkins, mushrooms, enzyme responsible for conversion of fructose to mannitol is onions, and in marine algae, especially brown seaweed. Brown NADPH- or NADH-dependent mannitol dehydrogenase algae (e.g. Laminaria claustoni) contains about 10%–20% (MDH). Fructose can also be converted to mannitol by using mannitol depending on the harvesting time (Schwarz 1994). MDH in the presence of the cofactor NADPH or NADH. A Manna, obtained by heating the bark of tree Fraxinus ornus, two enzyme system can be used for cofactor regeneration can contain up to 50% mannitol and was the commercial with simultaneous conversion of two substrates into two source of mannitol for many years until the 1920s (Soetaert products. Mannitol at 180 gl−1 can be crystallized out from 1990). It is widely used in the food, pharmaceutical, medical, the fermentation broth by cooling crystallization. This paper and chemical industries (Soetaert et al. 1995). Mannitol (US reviews progress to date in the production of mannitol by $7.30/kg; global market, 13.6 million kgyear−1) is currently produced commercially by a chemical method. A wide variety of microorganisms can produce mannitol by Mention of trade names or commercial products in this article is solely fermentation. Research efforts have been directed toward for the purpose of providing specific information and does not production of mannitol by fermentation and enzymatic means imply recommendation or endorsement by the U.S. Department of (Vandamme and Soetaert 1995;SahaandRacine2008; Agriculture. Ghoreishi and Shahrestani 2009; Song and Vielle 2009). In B. C. Saha (*) this paper, the authors review the chemical, microbial, and Bioenergy Research Unit, National Center for Agricultural enzymatic production of mannitol and its applications. Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, IL 61604, USA e-mail: [email protected] Chemical process for production of mannitol

F. M. Racine zuChem, Inc., Mannitol is produced industrially by high pressure hydro- Peoria, IL 61606, USA genation of fructose/glucose mixtures in aqueous solution at 880 Appl Microbiol Biotechnol (2011) 89:879–891 high temperature (120–160°C) with Raney nickel as a amounts of mannitol from glucose (Chalfan et al. 1975; catalyst and hydrogen gas (Soetaert 1990). α-Fructose is Loesche and Kornman 1976). The pathway for glucose converted to mannitol and β-fructose is converted to . metabolism and mannitol production by homofermentative The glucose is hydrogenated exclusively to sorbitol. Due to LAB is shown in Fig. 1. Forain et al. (1996) reported that a poor selectivity of the nickel catalyst, the hydrogenation of a strain of L. plantarum deficient in both L- and D-lactate 50:50 fructose/glucose mixture results in an approximately dehydrogenase (LDH) produces mannitol as an end-product 25:75 mixture of mannitol and sorbitol. It is relatively of glucose catabolism. LAB use several strategies for difficult to separate sorbitol and mannitol (Johnson 1976). regeneration of NAD+ during metabolism of sugars. Hols The requirement for separation of mannitol and sorbitol et al. (1999) showed that disruption of the ldh gene in results in even higher production costs and decreased yields Lactococcus lactis strain NZ20076 leads to the conversion (Soetaert et al. 1995). According to Takemura et al. (1978), of acetate into as a rescue pathway for NAD+ the yield of crystalline mannitol in the chemical process is regeneration. Neves et al. (2000) reported that a LDH- only 17% (w/w) based on the initial sugar substrates. If deficient mutant of Lc. lactis transiently accumulates is used as starting material and the hydrogenation is intracellular mannitol, which was formed from fructose-6- performed at alkaline pH, mannitol yields up to 31% can be phosphate by the combined action of mannitol-1-phosphate obtained (Schwarz 1994). The hydrogenation of pure fructose (M-1-P) dehydrogenase and phosphatase. They showed that results in mannitol yields of 48%–50% (Devos 1995). the formation of M-1-P by the LDH-deficient strain during Makkee et al. (1985) developed a process involving both glucose catabolism is a consequence of impairment in bio- and chemocatalysts for the conversion of glucose/ NADH oxidation caused by a greatly reduced LDH activity, fructose mixture into mannitol. Good yields (62%–66%) the transient formation of M-1-P serving as a regeneration were obtained by using glucose isomerase (GI) immobilized pathway for NAD+ regeneration. Gaspar et al. (2004) on silica in combination with a copper-on-silica catalyst described the construction of Lc. lactis strains able to form (water, pH ~7.0, 70°C, 50 kgcm−2 of hydrogen, trace mannitol as an end-product of glucose metabolism, using a amounts of buffer, Mg(II), borate, and EDTA). In another food-grade LDH-deficient strain as genetic basis for knock- method, mannitol is produced from by hydro- ing out the gene mtlA or mtlF. Resting cells of the double genation with stoichiometric yield (100% conversion) mutant strains (ΔldhΔmtlA and ΔldhΔmtlF)produced (Devos 1995). Mannose can be obtained from glucose by mannitol from glucose, with approximately one-third of chemical epimirization with a yield of 30%–36% (w/w). the carbon being successfully channeled to the production Thus, the mannitol yield from glucose can be as high as of mannitol. 36%. If the nonepimirized glucose can be enzymatically isomerized to fructose by using GI, the mannitol yields Mannitol production by heterofermentative LAB could reach 50% (w/w) (Takemura et al. 1978). However, the total cost of using the multisteps process is not A number of heterofermentative LAB of the genera economical. Devos (1995) suggested a process in which Lactobacillus, Leuconostoc, and Oenococcus can produce fructose is first isomerized to mannose using mannose mannitol directly from fructose (Saha 2003). In addition to isomerase. However, mannose isomerase is not yet com- mannitol, these bacteria may produce lactic acid, acetic mercially available for large scale use. acid, , and ethanol. The process is based on the ability of the LAB to use fructose as an electron acceptor and reducing it to mannitol using the enzyme Microbial production of mannitol mannitol 2-dehydrogenase (MDH). Saha and Nakamura (2003) reported that nine strains of heterofermentative LAB Lactic acid bacteria (LAB), , and fungi are known to (L. brevis NRRL B-1836, L. buchneri NRRL B-1860, L. produce mannitol from fructose or glucose (Smiley et al. cellobiosus NRRL B-1840, L. fermentum NRRL B-1915, L. 1967; Song et al. 2002; Wisselink et al. 2002; Saha 2003). intermedius NRRL B-3693, Leu. Amelilibiosum NRRL B- Both homo- and heterofermentative LAB produce mannitol 742, Leu. Citrovorum NRRL B-1147, Leu. mesenteroides (Saha 2003; Saha and Racine 2008). Table 1 summarizes subsp. dextranicum NRRL B-1120, and Leu. Paramesen- the production of mannitol by fermentation using a few teroides NRRL B-3471) produce mannitol from fructose. LAB, yeast, and fungal strains. The strain L. intermedius NRRL B-3693 produced 198 g mannitol from 300 g fructose l−1 in pH-controlled (pH 5.0) Mannitol production by homofermentative LAB fermentation at 37°C. The time of maximum mannitol production varied greatly from 15 h at 150 g fructose to Some homofermentative LAB such as Streptococcus 136 h at 300 g fructose l−1. The bacterium converted mutants and Lactobacillus leichmanii produce small fructose to mannitol from the early growth stage. One-third Appl Microbiol Biotechnol (2011) 89:879–891 881

Table 1 Mannitol production by fermentation

a −1 −1 Organism Fermentation type Substrate (g/l) Mannitol (g/l) Qp (gl h ) Reference

Lactic acid bacterium Lactobacillus sp. B001 Batch Fb (100)+Gc (50) 65 2.71 Itoh et al. 1992 Lactobacillus sp. Y-107 Batch F (100) 73 0.61 Yun and Kim 1998 Batch F (100) 71 0.95 Yun et al.1996b Lactobacillus fermentum Batch F (100)+G (50) 90 7.60 von Weymarn et al. 2002a Lactobacillus intermedius B-3693 Batch F (300) 198 1.46 Saha and Nakamura 2003 Fed-batch F (300) 201 2.18 Saha and Nakamura 2003 Batch F (250) 161 4.00 Saha and Racine 2010 Batch Md (75)+F (75) 105 4.77 Saha 2006a Batch SSFf Ie (300) 207 2.88 Saha 2006c I (250)+F (150) 228 2.07 Saha 2006c Fed-batch F (184)+G (92) 176 5.90 Racine and Saha 2007 CCRFg F (100)+G (50) 95 28.40 Racine and Saha 2007 Leuconostoc mesenteroides Batch F (100)+G (50) 90 3.75 Soetaert et al. 1995 Leuconostoc mesenteroides MCRBh F (100)+G (50) 91 20.60 von Weymarn et al. 2003 Leuconostoc sp. Y-002 Batch F (50) 20 0.80 Yun and Kim 1998 Yeast Candida magnoliae Batch F (150) 67 0.40 Song et al. 2002 Fed-batch F (250) 209 1.03 Song et al. 2002 Fed-batch F (250)+G (50) 213 0.85 Lee et al. 2003a Candida zeylannoides Batch n-Paraffin (100) 52 0.52 Hattori and Suzuki 1974 Torulopsis mannitofaciens Batch (100) 31 0.18 Onishi and Suzuki 1970 Torulopsis versalitis Batch G (194) 54 0.23 Onishi and Suzuki 1968 Asergillus candidus Batch G (32) 22 0.08 Smiley et al. 1967 Penicillium scabrosum Batch Sucrose (150) 60 0.21 Hendriksen et al. 1988

− a Volumetric productivity of mannitol (g l 1 h−1 ) b fructose c glucose d Molasses e Inulin f Simultaneous saccharification and fermentation g Continuous cell recycle fermentation h Membrane cell recycle bioreactor of fructose can be replaced with other substrates such as acetic acid were 202, 53, and 39 gl−1, respectively. With −1 glucose, , plus glucoamylase (simultaneous glucose (150 gl ) alone, the bacterium produced D-andL- saccharification and fermentation, SSF), mannose, and lactic acids in equal ratios (total, 70 gl−1) and ethanol (38 gl−1) galactose. Two-thirds of fructose can also be replaced by but no acetic acid. sucrose. The bacterium co-utilized fructose and glucose A competitive production process for mannitol by (2:1) simultaneously and produced very similar quantities fermentation would require inexpensive raw materials. of mannitol, lactic acid, and acetic acid in comparison with Saha (2006a) studied the production of mannitol by L. fructose only. The glucose was converted to lactic acid and intermedius NRRL B-3693 using molasses as a carbon acetic acid, and fructose was converted into mannitol. source. The bacterium produced mannitol (104 gl−1) from a Application of fed-batch fermentation by feeding equal mixture of molasses and fructose syrup (1:1; total sugars, amounts of substrate and medium four times decreased the 150 gl−1; fructose/glucose, 4:1) in 16 h. Several kinds of maximum mannitol production time of fructose (300 gl−1) inexpensive organic and inorganic nitrogen sources and from 136 to 92 h. The yields of mannitol, lactic acid, and corn steep liquor (CSL) were evaluated for their potential to 882 Appl Microbiol Biotechnol (2011) 89:879–891

Fig. 1 Pathway for glucose Glucose metabolism of homofermenta- tive lactic acid bacteria Glucose-6-P NADH + H+ NAD+

Fructose-6-P Mannitol-1-P ATP

ADP Pi Fructose-1,6-di-P Mannitol

Glyceraldehyde-3-P Dihydroxy-acetone-P ADP NAD+

ATP NADH + H+ 3-P-Glycerate

Phosphoenolpyruvate ADP

NAD+ NADH + H+ ATP

Lactate Pyruvate α -Acetolactate O2 CoA NAD+ CoA Diacetyl C + Formate NADH + H+ 2 NADH + H CO + Acetyl-CoA 2 Acetyl-CoA Acetoin NAD + + NADH + H ADP NADH + H + + NAD ATP NAD Acetaldehyde Acetate 2,3-Butanediol NADH + H+

NAD+ Ethanol replace more expensive nitrogen sources derived from Bacto- high substrate concentrations. The fed-batch process peptone and Bacto-yeast extract. Soy peptone (5 gl−1) and resulted in the accumulation of 176 g mannitol from 184 g CSL (50 gl−1) were found to be suitable substitutes for fructose and 92 g glucose l−1 of final fermentation broth in Bacto-peptone (5 gl−1) and Bacto-yeast extract (5 gl−1), 30 h with a volumetric productivity of 5.9 gl−1h−1. Further respectively. The bacterium produced 105 g mannitol from increases in volumetric productivity of mannitol were 150 g molasses and fructose syrup (1:1) in 22 h using 5 g obtained in a continuous cell-recycle fermentation process soy peptone and 50 g CSL l−1. The effects of four salt that reached more than 40 gl−1h−1. nutrients (ammonium citrate, sodium phosphate, MgSO4, As an alternative to the use of pure fructose, Saha and MnSO4) on the production of mannitol by L. inter- (2006c) investigated the production of mannitol by L. medius NRRL B-3693 in a simplified medium containing intermedius NRRL B-3693 using a commercial inulin 300 g fructose, 5 g soy peptone, and 50 g CSL l−1 in pH- preparation as a substrate at pH 5.0 and 37°C. It (extracted controlled fermentation at 5.0 at 37°C were evaluated using from the root of chicory plant, Cicharium intybus) a fractional factorial design (Saha 2006b). Only MnSO4 was contained 92.6% inulin, 2.8% fructose, 0.5% glucose, and found to be essential for mannitol production and 33 mgl−1 4.1% disaccharides. Inulin is a polyfructan, consisting of was found to support maximum mannitol production. The linear β-2,1-linked polyfructose chains terminated at the bacterium produced 200 g mannitol, 62 g lactic acid, and reducing end of a glucose residue (Vandamme and Derycke 40 g acetic acid from 300 g fructose l−1 in 67 h. Racine and 1983). The bacterium produced 106 g mannitol from dilute Saha (2007) improved the fermentation process further for acid hydrolyzate (pH 2.0, 121°C, 15 min) of 150 g inulin l−1 the production of mannitol by L. intermedius NRRL B- in 34 h. It also produced mannitol from inulin by 3693. A fed-batch protocol overcame limitations caused by simultaneous saccharification and fermentation (SSF) at Appl Microbiol Biotechnol (2011) 89:879–891 883 pH 5.0 and 37°C adding inulinase exogenously at the dose Erten (1998) studied the utilization of fructose (5 mmoll−1) level of 8 U (g substrate) −1. The L. intermedius B-3693 as an electron acceptor in two Leu. mesenteroides strains strain produced 207 g mannitol from 300 g inulin l−1 in 72 h under anaerobic conditions. These strains produced 0.26 mol by SSF. The fermentation time decreased from 72 to 62 h mannitol, 0.65–0.67 mol lactic acid, 0.37–0.57 mol ethanol, using a mixture of fructose and inulin (1:1; total, 300 gl−1). and 0.26–0.27 mol acetic acid per mol fructose at 25°C. When the fructose/inulin mixture (3:5, total 400 gl−1) was Fermentation of a mixture of fructose and glucose (1:1) used as substrate, the bacterium produced 228 g mannitol l−1 resulted in the production of the same metabolic end- from both inulin and fructose with a yield of 0.57 g per g products. Korakli et al. (2000) reported that mannitol is substrate after 110 h of SSF. This is the highest concentra- produced by sourdough Lactobacilli from fructose with tion of mannitol produced by a heterofermentative LAB so concomitant formation of acetate. They obtained a 100% far reported in the literature. The fermentation medium was yield of mannitol from fructose by L. sanfranciscensis simplified further and it now contains only CSL (50 gl−1) (isolated from sourdough) grown in a fed-batch culture −1 and MnSO4 (33 mgl ) in addition to carbon source (Saha containing a fructose–glucose mixture with a volumetric and Racine 2010). productivity of 0.5 gl−1h−1 and a final mannitol concentra- Martinez et al. (1963) reported that L. brevis fermented tion of 60 gl−1. Optimum production was at a concentration 1 mol fructose to 0.67 mol mannitol, 0.33 mol lactate, and of glucose/fructose mixture from 130 to 140 gl−1 and higher 0.33 mol acetate. Soetaert et al. (1995) reported a fed-batch concentrations of substrate were inhibitory. After adaptation fermentation method with automatic feeding strategy for of the cells in sucrose, the bacterium produced mannitol to rapid production of mannitol and D-lactic acid from fructose only 65% yield in relation to the fructose content of sucrose. or fructose/glucose mixture (2:1) by using Leu. pseudome- It also synthesized complex polysaccharides when grown on senteroides. The maximum volumetric productivity of sucrose. L. pontis isolated from sourdough produced mannitol was 11.1 gl−1h−1 with a final concentration of mannitol, lactic acid, and ethanol from fructose (Hammes 150 gl−1 in 24 h and a conversion efficiency of 94%. By et al. 1996). An unidentified Lactobacillus sp., designated using a special mutant strain, quantitative conversion and a B001, produced mannitol from fructose with a volumetric further concentration increase up to 185 g mannitol l−1 productivity of 6.4 gl−1h−1 (Itoh et al. 1992). Salou et al. could be obtained. Grobben et al. (2001) reported the (1994)reportedthatO. oenos converted 83 mol% of fructose spontaneous formation of a mannitol-producing variant of to mannitol when grown in a medium containing fructose Leu. pseudomesenteroides grown in the presence of and glucose (1:1) with volumetric productivity of about fructose. The mannitol producing variant differed from the 0.2 gl−1h−1.Pimenteletal.(1994) studied growth and mannitol-negative original strain in two physiological metabolism of sugars and acids by Leu. oenos under different aspects: the presence of MDH activity and the simultaneous conditions of temperature and pH. The bacterium produced utilization of fructose and glucose. The presence of MDH is mannitol from fructose. The addition of acids, particularly clearly a prerequisite for mannitol production. citrate, significantly repressed mannitol formation. Yun et al. (1996a) reported about 4–5 g of mannitol von Weymarn et al. (2002a) studied mannitol production accumulation per kg kimchi, a Korean pickled vegetable, by 8 heterofermentative LAB strains (L. brevis ATCC- during its fermentation. Yun and Kim (1998) isolated two 8287, L. buchneri TKK-1051, L. fermentum NRRL B- different LAB, Lactobacillus sp. Y-107 and Leuconostoc 1932, L. sanfranciscensis E-93491, Lactobacillus sp. sp. Y-002, during the fermentation of kimchi. These two (B001) BP-3158, Leu. mesenteroides ATCC-9135, Leu. strains utilized fructose and sucrose as substrates for pseudomesenteroides ATCC-12291, and O. oeni E-9762). mannitol formation. Under optimal conditions, the maximum They found that the ability to produce mannitol from fructose mannitol produced by Lactobacillus sp. Y-107 (at 35°C, varied markedly among these heterofermentative LAB initial pH 8.0, anaerobic, 100 g fructose l−1, 120 h) and species. The effects of growth temperature, pH, and nitrogen Leuconostoc sp. Y-002 (at 35°C, initial pH 6.0, anaerobic, flushing on mannitol production by four selected strains were 50 g fructose l−1, 25 h) were 73 and 26 g from 100 g studied in batch bioreactor cultivation. Using L. fermentum fructose l−1 with yields of 86 and 65% based on fructose and with fructose (20 gl−1) and glucose (10 gl−1) as carbon consumed, respectively. The volumetric productivities of source, mannitol yields from fructose were 86, 89, and mannitol by both strains were less than 1.0 gl−1h−1. Neither 94 mol% at 25, 30, and 35°C, respectively. Mannitol yields isolate produced other polyols such as glycerol and sorbitol but not the volumetric mannitol productivities were improved as by-products. These two bacterial strains were not able to with constant nitrogen gas flushing of the growth medium. utilize high concentrations of sugars above 100 gl−1 due to Applying the most promising strain (L. fermentum), high low osmotolerance of the isolates. Yun et al. (1996b) average and maximum mannitol productivities (7.6 and obtained a mannitol yield of 70 g from 100 g fructose 16.0 gl−1h−1, respectively) were achieved. von Weymarn et within 80 h at 28°C using Lactobacillus sp. KY-107. al. (2002b) then compared the ability to produce mannitol 884 Appl Microbiol Biotechnol (2011) 89:879–891 from fructose by ten heterofermentative bacteria (eight from Busse et al. (1961) and Erten (1998) found a lower above, Leu. mesenteroides ATCC-8086, and ATCC 8293) in mannitol yield from fructose in Leu. mesenteroides. In these resting state. They achieved high mannitol productivity cases, the enzyme reaction proceeds by the following (26.2 gl−1h−1) and mannitol yield (97 mol%) in high cell equation: density membrane cell-recycle culture using the best strain, ¼ þ þ : Leu. mesenteriodes ATCC 9135. A stable high-level 3 Fructose 1 Mannitol 2 Lactic acid 0 5 Acetic acid production of mannitol was maintained for 14 successive þ 1:5 Ethanol þ CO2 bioconversion batches using the same initial cell biomass. von Weymarn et al. (2002b) also reported that increasing the The net gain is 1.25 mol ATP per mol fructose initial fructose concentration from 100 to 120 and 140 gl−1 fermented. A typical pathway for mannitol production by resulted in decreased mannitol productivities due to both a heterofermentative LAB from glucose and fructose substrate and end-product inhibition of the key enzyme mixture (1:2) is shown in Fig. 2. MDH in Leu. mesenteriodes ATCC 9135. Von Weymarn et al. (2003) scaled up the mannitol production by Leu. Mannitol production by yeasts mesenteroides ATCC-9135. On a 2-l laboratory scale, high mannitol yields from fructose (93%–97%) and volumetric Some yeasts are known to produce a variety of sugar mannitol productivities (>20 gl−1h−1) were achieved. In the (Onishi and Suzuki 1968). Onishi and Suzuki pilot plant scale (100 l), the production levels of mannitol were similar to those in the laboratory. Also, high purity Glucose 2 Fructose mannitol crystals were obtained at similar yield levels. Ojamo ATP et al. (2003) achieved a volumetric mannitol productivity of about 20 gl−1h−1 using high cell density fermentation of Leu. ADP pseudomesenteroides ATCC 12291. Recently, Fontes et al. Glucose-6-P + (2009) reported production of 18 g mannitol from NAD juice containing 50 g reducing sugars (28 g fructose) with 67% fructose conversion into mannitol and productivity of NADH + H+ 1.8 gl−1h−1 by Leu. mesenteroides B-512F. 6-P-Gluconate + Mannitol produced by heterofermentative LAB is de- NAD rived from metabolism via the hexose phosphate pathway CO2 (Soetaert et al. 1999; Wisselink et al. 2002). The process NADH + H+ makes use of the capability of the bacterium to utilize Ribulose-5-P fructose as an alternative electron acceptor, thereby reduc- 2 Mannitol ing it to mannitol with the enzyme MDH. In this process, Xylulose-5-P the reducing equivalents are generated by oxidation of one- Pi third fructose to lactic acid and acetic acid. The enzyme reaction proceeds according to the following (theoretical) Glyceraldehyde 3-P Acetyl-P equation: ADP NAD+ ADP

3 Fructose ¼ 2 Mannitol þ Lactic acid þ Acetic acid þ CO2 ATP NADH + H+ ATP 3-P-Glycerate Acetate The net ATP gain is 2 mol ATP per mol fructose fermented. For fructose and glucose (2:1) cofermentation, the equation becomes Phosphoenolpyruvate ADP 2 Fructose þ Glucose ¼ 2 Mannitol þ Lactic acid ATP þ Acetic acid þ CO2 Pyruvate For sucrose and fructose (1:1) cofermentation, the NAD+ equation is NADH + H+ Sucrose þ Fructose ¼ 2 Fructose þ Glucose Lactate ¼ 2 Mannitol þ Lactic acid Fig. 2 Pathway for glucose and fructose (1:2) metabolism of þ Acetic acid þ CO2 heterofermentative lactic acid bacteria Appl Microbiol Biotechnol (2011) 89:879–891 885

(1970) studied the production of mannitol from glycerol 1994). Stress solutes such as NaCl and KCl in the range by Torulopsis yeasts. T. mannitofaciens produced 31% from 0.25 to 1 M did not promote polyol production. mannitol from glycerol under optimal conditions. T. versatilis is also a good producer of mannitol from glycerol. Mannitol production by filamentous fungi Hattori and Suzuki (1974) studied the large-scale produc- tion of and its conversion of mannitol by Candida Several filamentous fungi produce mannitol from glucose. zeylanoides grown on alkane. The strain produced about Yamada et al. (1961) showed that glucose is first converted 180 g meso-erythritol l−1 and a small amount of mannitol. to F-6-P, which is then reduced to M-1-P in the presence of Erythritol was almost entirely converted to mannitol by NADH, and M-1-P is hydrolyzed to mannitol by a specific keeping the KH2PO4 concentration in the medium at 40– phosphatase in Pircularia oryzae. Smiley et al. (1967) 200 mgl−1, and the mannitol concentration was 63 gl−1 after studied the biosynthesis of mannitol from glucose by 100 h incubation in a 5-l fermenter, which corresponded to Aspergillus candidus. The fungal strain converted glucose 52% of the alkane consumed. Zygosaccharomyces rouxii to mannitol with 50% yield based on glucose consumed in ATCC 12572 produced ethanol, glycerol, , and 10–16 days by feeding glucose daily with a volumetric mannitol from glucose (Groleau et al. 1995). productivity of 0.15 gl−1h−1 and a yield of 31.0 mol%. The Stankovic et al. (1989) studied mannitol production from presence of glucose in the medium was essential to prevent pentose sugars by Rhodotorula minuta (CCA 10-11-1). metabolism of mannitol. Nelson et al. (1971) reported the This was the only strain among 28 species of Candida, production of mannitol from glucose and other sugars by Bretanomyces, Decceromyces, Kluyveromyces, Saccharo- conidia of A. candidus. Low pH (~3.0) favored the myces, Schwanniomyces, and Trichosporon that produced percentage yield but decreased the fermentation rate. A. mannitol from D-pentose sugars. The yeast produced 16%, candidus produced mannitol from 2% glucose in 75% 4%, 5%, and 5% mannitol from ribose, xylose, arabinose, yield (based on sugar consumed) in 7 days at 28 °C. The and lyxose, respectively, when grown on these sugars fungus converts glucose to mannitol via F-6-P and M-1-P (10%) at 28°C and pH 4–7 for 14 days. In addition, the (Strandberg 1969). Lee (1967) determined the carbon strain produced 3%, 11%, 5%, and 6% D-arabinitol from balance for fermentation of glucose to mannitol by Aspergillus these sugars, respectively. Song et al. (2002) isolated more sp. The products found were: cells (17% of carbon input), than 1000 strains from various sources such as soil, CO2 (26%), mannitol (35%), glycerol (10%), erythritol seawater, honey, pollen, and fermentation sludge and tested (2.5%), glycogen (1%), and unidentified compounds (8%). them for their ability to produce mannitol. They identified a Cell-free enzyme studies indicated that mannitol was novel strain of Candida magnoliae (isolated from fermen- produced via the reduction of fructose-6-phosphate. tation sludge) which produced 67 g mannitol in 168 h from Hendriksen et al. (1988) screened 11 different Penicillium 150 g fructose l−1 in batch flask culture (30°C, 220 rpm, species for production of mannitol. All strains produced 10 g yeast extract l−1). A fructose concentration higher than mannitol and glycerol from sucrose. The highest amount of 200 gl−1 reduced the mannitol conversion yield and mannitol (43 gl−1) was produced by P. scabrosum IBT production rate. In fed-batch culture with 3−12% fructose, JTER4 and the highest combined yield of mannitol and mannitol production reached a maximum of 209 gl−1 after glycerol (65 gl−1) was obtained with P. aethiopicum IBT 200 h, corresponding to 83% yield and a volumetric MILA 4 when grown on sucrose (150 gl−1) and yeast productivity of 1.03 gl−1h−1. The strain produced only extract (20 gl−1) at pH 6.2 and 25 °C for 12 days. However, small quantities of by-products, such as glycerol, erythritol, the volumetric productivity of mannitol from sucrose by the and organic acids. Under optimum conditions, a final high mannitol producer P. scabrosum was only 0.14 gl−1 mannitol production of 213 g was obtained from 250 g h−1. Penicillium sp. uses the same metabolic route for fructose l−1 after 110 h (Lee et al. 2003a). Supplementation conversion of glucose to mannitol as A. candidus (Boosaeng with Ca2+ and Cu2+ increased mannitol production by C. et al. 1976). El-Kady et al. (1995) screened 500 filamentous magnoliae (Lee et al. 2007). Recently, Khan et al. (2009) fungal isolates belonging to ten genera and 74 species and reported that the resting cells of C. magnoliae produced identified Aspergillus, Eurotium, and Fennellia species as mannitol from glycerol with a yield of ~45%. De Zeeuw high (>1.82 gl−1) producers of mannitol cultivated on and Tynan (1973) reported that C. lipolytica produced liquid glucose–Czapek’s medium fortified with 15% NaCl mannitol as main sugar alcohol. A strain of Aureobasidium and incubated at 28 °C as static cultures for 15 days. pullulans, a yeast-like fungus, produced polyols of which Domelsmith et al. (1988) demonstrated that four fungal mannitol was the main polyol associated with minute cultures—Alternaria alternata, Cladosporium herbarum, quantities of glycerol with a yield of about 23% (based on Epicoccum purpurascens,andFusarium pallidoroseum substrate utilized) from 20% (w/v) sucrose in batch flask isolated from cotton leaf dust produced mannitol and are culture at 30 °C and pH 6.0 in about 240 h (Yun and Song a probable source of mannitol found in cotton dust. 886 Appl Microbiol Biotechnol (2011) 89:879–891

Mannitol production by recombinant microorganisms when grown in a medium containing both glucose and fructose. The yield of mannitol from fructose was improved Although a few homofermentative LAB produce mannitol from 74 to 86 mol%. A fructokinase-negative mutant could from glucose, its production level is very low. Several enable higher pH to be used in the mannitol production heterofermentative LAB produce excellent quantities of process without lowering the yield. mannitol (~66%) from fructose. They also produce lactic acid Wisselink et al. (2004) cloned and overexpressed mtlD and acetic acid as coproducts. This is why a number of from L. plantarum in Lc. lactis in different genetic back- recombinant microorganisms have been developed to either grounds (a wild-type strain, an LDH-deficient strain, and a overproduce mannitol or limit or eliminate the production of strain with reduced phosphofructokinase activity). Small coproducts (lactic acid and acetic acid). In this section, we amounts (<1%) of mannitol were formed by growing cells review the literature dealing with the construction of of mtlD-overexpressing LDH-deficient and phosphofructo- recombinant organisms for mannitol production. Kaup et al. kinase reduced strains. The resting cells of the LDH- (2004) constructed an efficient strain for deficient transformant converted 25% of glucose (3.6 gl−1) mannitol production from fructose in a whole cell biotrans- to mannitol. They concluded that the mtlD overexpressing formation. The strain expressed NAD+-dependent MDH from LDH-deficient Lc. lactis strain seemed to be the most Leu. pseudomesenteroides ATCC 12291 (Hahn et al. 2003) promising strain for mannitol production. Liu et al. (2005) for the reduction of fructose to mannitol, NAD+-dependent have cloned and expressed the mtlK gene encoding MDH formate dehydrogenase (FDH) from Mycobacterium vaccae from L. brevis in E. coli. The genetically engineered E. coli N10 (Galkin et al. 1995) for NADH regeneration, and the strain was able to catalyze the reduction of fructose to glucose facilitator from Zymomonas mobilis (Weisser et al. mannitol. 1995; Parker et al. 1995) for the uptake of fructose without Costenoble et al. (2003) demonstrated that mannitol is concomitant phosphorylation. The strain produced about produced under anaerobic conditions by a glycerol- 66 g mannitol from 90 g fructose l−1 within 8 h with a yield defective mutant of Saccharomyces cerevisiae expressing of 73% and a specific mannitol productivity of >4 g per g the mtlD gene from E. coli coding for NADH-dependent cell dry weight (cdw) h−1.Kaupetal.(2005)reportedthat M-1-P dehydrogenase. Improving efflux of the formed supplementation of this recombinant strain with extracellular mannitol seems to be necessary for obtaining anaerobic GI resulted in the formation of 145.6 g mannitol from 180 g growth and a sustained mannitol production. Baumchen glucose l−1. They have co-expressed the xylA gene of E. coli and Bringer-Meyer (2007) overexpressed MDH gene (mdh) in this recombinant E. coli strain which formed 83.7 g from Leu. pseudomesenteroides and coexpressed FDH gene mannitol from 180 g glucose l−1. Sasaki et al. (2005)cloned (fdh)inCorynebacterium glutamicum ATCC 13032. The a gene encoding MDH from L. reuteri and expressed in E. recombinant C. glutamicum cells produced mannitol at a coli. The purified recombinant enzyme works optimally at constant production rate of 0.22 g (g cdw)−1h−1. Expression 37°C and pH 5.4 for conversion of fructose to mannitol. of the glucose/fructose facilitator gene glf from Z. mobilis Aarnikunnas et al. (2003) used metabolic engineering of in C. glutamicum led to a 5-fold increased productivity of −1 −1 L. fermentum for production of mannitol and pure L-lactic 1.25 g (g cdw) h , yielding 87 g mannitol from 93.7 g acid or pyruvate. The authors first developed genetic tools fructose. In repetitive fed-batch biotransformation, 285 g to modify L. fermentum and then proceeded to inactivate mannitol was formed over a period of 96 h with an average first ldhD gene and then ldhL gene in order to create a productivity of 1.0 g (g cdw)−1h−1. bacterium that could produce mannitol and either pure L-lactic acid or pyruvic acid in a single process. In bioreactor cultivations, the single mutant strain constructed by inactiva- Enzymatic production of mannitol tion of the ldhD gene produced mannitol and L-lactic acid. The double mutant strain created by inactivating the ldhL Mannitol can be enzymatically produced from fructose in a gene produced mannitol and pyruvate. In addition, the mutant one pot synthesis by using NADH-dependent MDH or produced 2,3-butanediol and the volumetric productivity of NADPH-dependent MDH. Saha (2004) purified MDH from mannitol was decreased. Helanto et al. (2005) described the L. intermedius NRRL B-3693 and showed that the purified construction and characterization of a random mutant of Leu. enzyme can convert fructose to mannitol completely in the pseudomesenteroides that is unable to grow on fructose and presence of NADPH. The cofactor dependency of the the positive effects of the mutation on mannitol production. enzyme is a major limitation. A number of strategies such as They have performed the inactivation of its fructokinase enzymatic, electrochemical, chemical and photochemical, activity with random mutagenesis and screening the mutants and biological methods are available for cofactor regenera- unable to grow on fructose. The fructose uptake of the mutant tion (Chenault and Whitesides 1987;O’Neill and Woodward was unaltered and the mutant converted fructose to mannitol 2000). A two-enzyme system can be used for cofactor Appl Microbiol Biotechnol (2011) 89:879–891 887 regeneration with simultaneous conversion of two sub- Both NADH- and NADPH-dependent MDHs have been strates into two products of interest (Wichmann et al. 1981). purified from a number of microorganisms. As for example, One example is the simultaneous conversion of fructose NADH-dependent MDH has been purified from Lb. brevis, and formate using the enzymes MDH and FDH (Parmentier Leu. mesenteriodes, P. fluorescens, Rhodobacter spaeroides, et al. 2005). FDH converts formate to CO2 and reduces Saccharomyces cerevisiae,andT. delbruckii (Martinez et al. NAD to NADH. MDH uses NADH to convert fructose to 1963; Sakai and Yamanaka 1968; Brunker et al. 1997; mannitol and regenerates NAD. Schneider and Giffhom 1989; Schneider et al. 1993; Quain Synthesizing reaction: and Boulton 1987; Nidetzky et al. 1996). NADPH- dependent MDH has been purified from A. parasitius, C. Fructose þ NADH þ Hþ ¼ Mannitol þ NADþ magnoliae, Z. mobilis, and Gluconobacter suboxydans Regenerating reaction: (Niehaus and Dilts 1982; Lee et al. 2003b; Viikari and Korhola 1986; Adachi et al. 1999). Schneider and Giffhom þ þ ¼ þ Formic acid NAD CO2 NADH (1989) reported an increase of 8.3-fold of MDH activity by Slatner et al. (1998) achieved a volumetric mannitol constructing a strain (pAK82) from R. sphaeroides Si4 and productivity of 2.25 g 1−1 h−1 using a recombinant MDH by producing high cell concentrations via fed-batch cultiva- from P. fluorescens overexpressed in E. coli and FDH from tion in a bioreactor in comparison to batch cultivation of the C. boidinnii with a final product concentration of 72 gl−1 wild-type strain. Mannose can be reduced to mannitol and a fructose conversion of 80% in the system. The other enzymatically. However, the reversible reaction favors man- nitol oxidation and is thus not suitable (Stoop et al. 1998). product CO2 is easily separated from mannitol. Mannitol was crystallized from the ultrafiltered product solution in Baumchen et al. (2007) developed an in vivo system for 97% purity and 85% recovery, thus allowing reuse of the biotransformation of fructose to mannitol by the enzymes for repeated batch production of mannitol. Another expression of mdh gene encoding MDH from Leu. example of the two enzyme system for regeneration of pseudomesenteroides in B. megaterium. The NADH reduc- cofactor is MDH and glucose dehydrogenase (GDH) system tion necessary for MDH activity was regenerated via the using glucose/fructose mixture (1:1) and simultaneous oxidation of formate to CO2 by coexpression of fdh gene synthesis of mannitol and gluconic acid (Howaldt et al. encoding Mycobacterium vaccae N10 FDH. Recombinant 1988). NAD requiring GDH converts glucose to gluconic B. megaterium produced up to 10.6 g mannitol per l at the acid and generates NADH. MDH uses NADH to convert shaking flask scale. Whole cell biotransformation in a fed- batch bioreactor increased the mannitol production to fructose to mannitol and regenerates NAD. −1 −1 −1 Synthesizing reaction: 22 gl with a specific productivity of 0.32 g (g cdw) h and a mannitol yield of 0.91 molmol−1.However,the þ þ Fructose þ NADH þ H ¼ Mannitol þ NAD substrate uptake was the limiting factor of the overall biotransformation. Song et al. (2008) also demonstrated Regenerating reaction: that mannitol can be produced from glucose in a two step þ enzymatic process using a xylose Glucose þ NAD þ H2O ¼ Gluconic acid þ NADH isomerase (GI) and T. martina MDH. However, in the − Gluconic acid (market size, 35,000 tons year 1), a absence of a cofactor regeneration system, the final multifunctional carbonic acid, is a bulk chemical used in mannitol concentration only reached 19 mM. the chemical, pharmaceutical, food, beverage, textile and other industries (Kulbe et al. 1987). It can be used for cleaning purposes and in the construction industry, where it Downstream processing of mannitol can be used as a cement additive to increase cement resistance and stability under extreme climatic conditions, Mannitol, at 180 gl−1, can be recovered from the fermen- e.g., frost and floods (Hustede et al. 1989). tation broth by cooling crystallization. Saha and Nakamura The MDH used was from P. fluorescens, Torulaspora (2003) reported that small white needle-like crystals of delbruckii, and Schizophyllum commune, and the FDH was mannitol appeared upon refrigeration of the cell-free from Bacillus megaterium (Haltrich et al. 1996; Nidetzky et fermentation broth at 4°C. von Weymarn et al. (2002b) al. 1996). Downstream processing consists of the separation crystallized mannitol with a yield of 72 mass% from of enzymes from the product solution with the aid of reactor- permeates obtained from three successive batches of integrated membranes and of the isolation of the two fermentation using Leu. mesenteroides ATCC-12291. After products—mannitol and gluconic acid—by electrodialysis, recrystallization, the purity of mannitol was more than 99%. ion-exchange chromatography, and fractional crystallization Deusing et al. (1996) described a method for desalination by (Kulbe et al. 1987). electrodialysis and crystallization of mannitol produced by 888 Appl Microbiol Biotechnol (2011) 89:879–891 large-scale fermentation by Leu. mesenteroides with a purity Mannitol is used in medicine as a powerful osmotic of >99.5%. Soetaert et al. (1999) reported that the use of (to increase the formation of urine in order to prevent electrodialysis followed by crystallization resulted in cost- and treat acute renal failure and also in the removal of toxic effective recovery of highly pure crystalline mannitol and D- substances from the body) and in many types of surgery for lactic acid under optimized downstream processing of the the prevention of failure (to alter the osmolarity of the fermentation broth of Leu. mesenteroides. Slatner et al. glomerular filtrate) and to reduce dye and brain oedema (1998) crystallized mannitol from an enzymatic conversion (increased brain water content). Hypertonic mannitol can at 4°C (1 h) from the ultrafiltrate after evaporation of the enhance the transport of drugs across the –brain barrier product solution in vacuo to approximately half the original for the treatment of life-threatening brain diseases (Rapoport volume and subsequent addition of 1 volume equivalent of 2001; Miller 2002). Inhaled mannitol improves the hydra- isopropanol. After centrifugation, the residual alcohol in the tion and surface properties of sputum in patients with cystic solid product was evaporated at room temperature, leaving fibrosis (Daviskas et al. 2010). is a mannitol with a purity of at least 97%. Ojamo et al. (2003) well-known vasodilator, used in the treatment of hyperten- suggested that the acidic side stream, obtained from a LAB sion (Johnson 1976). Mannitol is also a scavenger of fermentation process, could be used as feed preservative. hydroxyl radicals (Shen et al. 1997). Itoh et al. (1992) used filtration to separate the cells from the fermentation broth. The cell-free broth was then concentrated by evaporation. Mannitol was first crystallized, and the Concluding remarks and future prospects crystals were separated by centrifugation. The mother liquor was further fractionated by a chromatographic method into Heterofermentative LAB have the capability to utilize two fractions—acetate fraction and a lactate/mannitol frac- high concentrations of fructose such that the mannitol tion. Mannitol was separated from lactate by crystallization, concentration in the fermentation broth could reach more −1 and the lactate was isolated by precipitation with Ca(OH)2. than 180 gl , which is well enough to be separated as such from the cell-free fermentation broth by cooling crystalli- zation. The lactic acid and acetic acid can be recovered by Applications of mannitol electrodialysis (Datta and Tsai 1997; Soetaert et al. 1995). The MDH responsible for catalyzing the conversion of Mannitol is used as a sweet-tasting bodying and texturing fructose to mannitol requires NADPH (NADH) as cofactor. agent. It reduces the crystallization tendency of sugars and is It is thus possible to develop a one-pot enzymatic process used as such to increase the shelf life of foodstuffs. Crystalline for production of mannitol from fructose if a cost-effective mannitol exhibits a very low hygroscopicity, making it useful cofactor regeneration system can be developed (Saha in products that are stable at high humidity. It is only about half 2004). The heterofermentative LAB cells can be immobi- as sweet as sucrose. Mannitol exhibits reduced physiological lized in a suitable support, and the immobilized cells calorie value (1.6 kcalg−1) compared to sucrose (4 kcalg−1). It can be used in a bioreactor to continuously produce has a low solubility in water of only 18% (w/v) at 25 °C and mannitol from fructose. The production of acetic acid 13% (w/v) at 14°C (Perry et al. 1997). In comparison, the and D-lacticacidbysomeLABcanbeblocked(inacti- solubility limit of sorbitol in water is about 70% (w/v) at vation of acetate kinase and D-LDH) by mutagenesis 25°C. Mannitol is sparingly soluble in organic solvents (Aarnikunnas et al. 2003). In that case, the fermentation such as ethanol and practically insoluble in ether, ketones, broth should contain only mannitol and L-lactic acid. and hydrocarbons (Schwarz 1994). It forms orthorhombic Both are value-added products and thus the fermentative crystals and the crystals have a melting point at 165–168 °C production of mannitol will become more attractive. Even (Schwarz 1994). Mannitol is extensively used in chewing though mannitol is currently not produced industrially by gum. It is chemically inert and is commonly used in the fermentation, the prospect of producing mannitol by pharmaceutical formulation of chewable tablets and granu- fermentation using a food grade LAB in near future looks lated powders. Mannitol prevents moisture absorption from very promising. the air, exhibits excellent mechanical compressing properties, does not interact with the active components, and has a sweet cool taste owing to its high negative heat of solution References (~121 kJkg−1) that masks the unpleasant taste of many drugs (Debord et al. 1987). The complex of with mannitol is used in the production of dry electrolytic Aarnikunnas I, von Weymarn N, Ronnholm K, Leisola M, Palva A (2003) Metabolic engineering of Lactobacillus fermentum for capacitors. It is an extensively used polyol for production production of mannitol and pure L-lactic acid or pyruvate. of resins and surfactants (Soetaert et al. 1995). 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