Lactate Acetate

2 Fructose

NAM* NADPH+IT

NADI* NADPH+IT

2Mannitol

Fzgwre 2. Pathwayfor production ofmannitolfrom fructose by heterofermentative lactic acid bacteria.

(Reproduced from reference 25. Copyright 2003.)

3 Fructose 1 Mannitol + 2 Lactic acid + 0.5 Acetic acid +1.5 Ethanol + C02 The net gain is 1.25 mol of ATP per mol of fructose fermented..

Some homofermentative LAB such as Streptococcus mutants and Lb. leichmanii produce small amounts of mannitol (28,29). Forainetal. (30) reported that a strain of Lb. plantarum deficient in both L- and D-lactate dehydrogenase (LDH) produces mannitol as an end-product of glucose catabolism. LAB uses several strategies for regeneration of NAD+ during metabolism of carbohydrates. Veiga da Cumba et al. (31) reported that O. oeni produces erythritol to consume the reduced coenzymes formed in the glycolytic pathway. Hols et al. (32) showed that disruption of the Idh gene in Lb. lactis strain NZ20076 leads to the conversion of acetate into ethanol as a rescue pathway for NAD+ regeneration. Neves et al. (33) reported that a LDH-deficient (LDHd ) mutant of Lactococcus lactis transiently accumulates intracellular mannitol, which was formed from fructose-6-phosphate (F-6-P) by the combined action of mannitol-1-phosphate dehydrogenase (MPDH) and phosphatase. They showed that the formation of mannitol-1 -phosphate (M-1 -P) by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient formation of M-l-P serving as a regeneration pathway for NAD+ regeneration. Grobben et al. (34) reported the spontaneous formation of a mannitol-producing variant of Leu. pseudomesenteroides grown in the presence of fructose. The mannitol producing variant differed from the mannitol-negative original strain in two physiological aspects: the presence of MDH activity and the simultaneous utilization of fructose and glucose. The presence of MDH is clearly a prerequisite for mannitol production. Kets et al. (35) found that the accumulation of mannitol is a typical feature of salt-stressed Pseudomonas putida strains grown in glucose mineral medium.

Yeasts

Some yeasts are known to produce a variety of sugar alcohols (36). Onishi and Suzuki (31,38) studied the production of mannitol from glycerol by Torulopsis yeasts. T. mannitofaciens produced 31% mannitol from glycerol under optimal conditions. T. versatilis is also a good producer of mannitol from glycerol. Wako et al. (39) isolated a yeast strain (T-18) identified as Torulopsis sp. produced 23.7% mannitol and 42.4% glycerol in a medium (pH 9.0) containing 20% glucose. Hattori and Suzuki (40) studied the large-scale production of erythritol and its conversion of mannitol by Candida zeylanoides grown on alkane. The strain produced about 180 g/L meso-erythritol and a small amount of mannitol. Erythritol was almost entirely converted to mannitol by keeping the KH2P04 concentration in the medium at 40-200 mg/L, and the yield was 63 g/L after 100 h incubation in a 5-L fermentor, which corresponded to 52% of the alkane consumed. Zygosaccharomyces rouxii ATCC 12572 produces ethanol, glycerol, arabitol, and mannitol from glucose (41).

Stankovic et al. (42) studied mannitol production from pentose sugars by Rhodotorula minuta (CCA 10-11-1). This was the only strain among 28 species of Candida, Bretanomyces, Decceromyces, Kluyveromyces, Saccharomyces, Schwanniomyces and Trichosporon that produced mannitol from D-pentose sugars. The yeast produced 16,4,5, and 5% mannitol from ribose, xylose, arabinose, and lyxose, respectively, when grown on these sugars (10%) at 28 °C and pH 4-7 for 14 days. In addition, the strain produced 3,11,5 and 6% D-arabinitol from these sugars, respectively. Song et al. (43) isolated over 1000 strains from various sources such as soil, seawater, honey, pollen and fermentation sludge and tested for their ability to produce mannitol. They identified a novel strain of Candida magnoliae (isolated from fermentation sludge) which produced 67 g/L mannitol in 168 h from 150 g/L fructose in batch flask culture (30 °C, 220 rpm, 10 g yeast extract/L). A fructose concentration higher than 200 g/L reduced the mannitol conversion yield and production rate. In fed-batch culture with 30-120 g fructose/L, mannitol production reached a maximum of 209 g/L after 200 h corresponding to 83% yield and a volumetric productivity of 1.03 g/L/h. The strain produced only small quantities of by-products, such as glycerol, erythritol and organic acids. De Zeeuw and Tynan (44) reported that C. lipolytica produces mannitol as main sugar alcohol. Yun and Song (45) reported that a strain of A ureobasidium pullulans, a yeast- like fungus, produced polyols of which mannitol was the main polyol associated with minute quantities of glycerol with a yield of about 23% (based on substrate utilized) from 20% (w/v) sucrose in batch flask culture at 30 °C and pH 6.0 in about 240 h. Stress solutes such as NaCl and KC1 in the range from 0.25 to 1 M did not promote polyol production.

Filamentous Fungi

Several filamentous fungi produce mannitol from glucose. Yamada et al showed that glucose is first converted to F-6-P, which is then reduced to M1"^ m the presence of NADH, and M-l-P is hydrolyzed to mannitol by f specific phosphatase in Pircularia oryzae. Smiley et al. (47) studied the biosynthesis of mannitol from glucose by Aspergillus candidus. The fungal strain converted glucose to mannitol with 50% yield based on glucose consumed in 10-16 days by feeding glucose daily with a volumetric productivity of 0.15 g/L.h and a yield of 31.0 mol%. The presence of glucose in the medium was essential to prevent metabolism of mannitol. Nelson et al. (48) reported the production of mannitol from glucose and other sugars by conidia of A. candidus. Low pH (~ 3.0) favored the percentage yield but decreased the fermentation time. A. Candidus produced mannitol from 2% glucose in 75% yield (based on sugar consumed) in 7 days at 28 °C. The fungus converts glucose to mannitol via F-6-P and M-l-P (49). Lee (50) determined the carbon balance for fermentation of glucose to mannitol by Aspergillus sp. The products found were: cells (17% of carbon input), C02 (26%), mannitol (35%), glycerol (10%), eiythritol (2.5%), glycogen (1 %), and unidentified compounds (8%). Cell-free enzyme studies indicated that mannitol was produced via the reduction of F-6-P.

Hendriksenetal. (51) screened 11 different Pewcz7/w/w species for production of mannitol. All strains produced mannitol and glycerol from sucrose. The highest amount of mannitol (43/L) was produced by P. scabrosum IBT JTER4 and the highest combined yield of mannitol and glycerol (65 g/L) was obtained with P. aethiopicum IBT MILA 4 when grown on sucrose (150 g/L) and yeast extract (20 g/L) at pH 6.2 and 25 °C for 12 days. However, the volumetric productivity of mannitol from sucrose by the high mannitol producer P. scabrosum was only 0.14/g/L.h. Foda et al. (52) investigated the conditions suitable for the maximum conversion of glucose to mannitol by P. chrysogenum Q 176 in submerged culture. The fungus produced 48.6% mannitol from glucose (50 g/L) after 6 days at 25-30°C and pH 7.0. Penicillium sp. uses the same metabolic route for conversion of glucose to mannitol as A. candidus (55). El-Kady et al. (54) screened 500 filamentous fungal isolates belonging to 10 genera and 74 species and identified Aspergillus, Eurotium, and Fennellia species as high (> 10 mmol/L) producers of mannitol cultivated on liquid glucose-Czapek's medium fortified with 15% NaCl and incubated at 28 °C as static cultures for 15 days. Domelsmith et al. (55) demonstrated that four fungal cultures - Alternaria alternata, Cladosporium herbarum, Epicoccum purpurascens, and Fusarium pallidoroseum isolated from cotton leaf dust produce mannitol and are a probable source of mannitol found in cotton dust. A. niger was reported to produce mannitol using sodium acetate as carbon source (56).

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