10152
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
alcohol), is widely used in the food, pharmaceutical, medical,
and chemical industries. The production of mannitol by
fermentation has become attractive because of the problems
associated with its production chemically. A number of
homo- and heterofermentative lactic acid bacteria (LAB),
yeasts, and filamentous fungi are known to produce mannitol.
In particular, several heterofermentative LAB are excellent
producers of mannitol from fructose. These bacteria convert
fructose to mannitol with 100% yields from a mixture of
glucose and fructose (1:2). Glucose is converted to lactic acid
and acetic acid, and fructose is converted to mannitol. The
enzyme responsible for conversion of fructose to mannitol is
NADPH- or NADH-dependent mannitol dehydrogenase
(MDH). Fructose can also be converted to mannitol by using
MDH in the presence of the cofactor NADPH or NADH. A
two enzyme system can be used for cofactor regeneration
with simultaneous conversion of two substrates into two
products. Mannitol at 180 gl−1 can be crystallized out from
the fermentation broth by cooling crystallization. This paper
reviews progress to date in the production of mannitol by
Mention of trade names or commercial products in this article is solely
for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of
Agriculture.
B. C. Saha (*)
Bioenergy Research Unit, National Center for Agricultural
Utilization Research, Agricultural Research Service,
United States Department of Agriculture,
Peoria, IL 61604, USA
e-mail: Badal.Saha@ars.usda.gov
F. M. Racine
zuChem, Inc.,
Peoria, IL 61606, USA
fermentation and using enzyme technology, downstream
processing, and applications of mannitol.
Keywords Mannitol production . Homofermentative lactic
acid bacterium . Heterofermentative lactic acid bacterium .
Fermentation . Mannitol dehydrogenase
Introduction
Mannitol is a naturally occurring sugar alcohol found in many
fruits and vegetables. It is found in pumpkins, mushrooms,
onions, and in marine algae, especially brown seaweed. Brown
algae (e.g. Laminaria claustoni) contains about 10%–20%
mannitol depending on the harvesting time (Schwarz 1994).
Manna, obtained by heating the bark of tree Fraxinus ornus,
can contain up to 50% mannitol and was the commercial
source of mannitol for many years until the 1920s (Soetaert
1990). It is widely used in the food, pharmaceutical, medical,
and chemical industries (Soetaert et al. 1995). Mannitol (US
$7.30/kg; global market, 13.6 million kgyear−1) is currently
produced commercially by a chemical hydrogenation method.
A wide variety of microorganisms can produce mannitol by
fermentation. Research efforts have been directed toward
production of mannitol by fermentation and enzymatic means
(Vandamme and Soetaert 1995; Saha and Racine 2008;
Ghoreishi and Shahrestani 2009; Song and Vielle 2009). In
this paper, the authors review the chemical, microbial, and
enzymatic production of mannitol and its applications.
Chemical process for production of mannitol
Mannitol is produced industrially by high pressure hydro-
genation of fructose/glucose mixtures in aqueous solution at
880
high temperature (120–160°C) with Raney nickel as a
catalyst and hydrogen gas (Soetaert 1990). α-Fructose is
converted to mannitol and β-fructose is converted to sorbitol.
The glucose is hydrogenated exclusively to sorbitol. Due to
poor selectivity of the nickel catalyst, the hydrogenation of a
50:50 fructose/glucose mixture results in an approximately
25:75 mixture of mannitol and sorbitol. It is relatively
difficult to separate sorbitol and mannitol (Johnson 1976).
The requirement for separation of mannitol and sorbitol
results in even higher production costs and decreased yields
(Soetaert et al. 1995). According to Takemura et al. (1978),
the yield of crystalline mannitol in the chemical process is
only 17% (w/w) based on the initial sugar substrates. If
sucrose is used as starting material and the hydrogenation is
performed at alkaline pH, mannitol yields up to 31% can be
obtained (Schwarz 1994). The hydrogenation of pure fructose
results in mannitol yields of 48%–50% (Devos 1995).
Makkee et al. (1985) developed a process involving both
bio- and chemocatalysts for the conversion of glucose/
fructose mixture into mannitol. Good yields (62%–66%)
were obtained by using glucose isomerase (GI) immobilized
on silica in combination with a copper-on-silica catalyst
(water, pH ~7.0, 70°C, 50 kgcm−2 of hydrogen, trace
amounts of buffer, Mg(II), borate, and EDTA). In another
method, mannitol is produced from mannose by hydro-
genation with stoichiometric yield (100% conversion)
(Devos 1995). Mannose can be obtained from glucose by
chemical epimirization with a yield of 30%–36% (w/w).
Thus, the mannitol yield from glucose can be as high as
36%. If the nonepimirized glucose can be enzymatically
isomerized to fructose by using GI, the mannitol yields
could reach 50% (w/w) (Takemura et al. 1978). However,
the total cost of using the multisteps process is not
economical. Devos (1995) suggested a process in which
fructose is first isomerized to mannose using mannose
isomerase. However, mannose isomerase is not yet com-
mercially available for large scale use.
Microbial production of mannitol
Lactic acid bacteria (LAB), yeast, and fungi are known to
produce mannitol from fructose or glucose (Smiley et al.
1967; Song et al. 2002; Wisselink et al. 2002; Saha 2003).
Both homo- and heterofermentative LAB produce mannitol
(Saha 2003; Saha and Racine 2008). Table 1 summarizes
the production of mannitol by fermentation using a few
LAB, yeast, and fungal strains.
Mannitol production by homofermentative LAB
Some homofermentative LAB such as Streptococcus
mutants and Lactobacillus leichmanii produce small
Appl Microbiol Biotechnol (2011) 89:879–891
amounts of mannitol from glucose (Chalfan et al. 1975;
Loesche and Kornman 1976). The pathway for glucose
metabolism and mannitol production by homofermentative
LAB is shown in Fig. 1. Forain et al. (1996) reported that a
strain of L. plantarum deficient in both L- and D-lactate
dehydrogenase (LDH) produces mannitol as an end-product
of glucose catabolism. LAB use several strategies for
regeneration of NAD+ during metabolism of sugars. Hols
et al. (1999) showed that disruption of the ldh gene in
Lactococcus lactis strain NZ20076 leads to the conversion
of acetate into ethanol as a rescue pathway for NAD+
regeneration. Neves et al. (2000) reported that a LDH-
deficient mutant of Lc. lactis transiently accumulates
intracellular mannitol, which was formed from fructose-6-
phosphate by the combined action of mannitol-1-phosphate
(M-1-P) dehydrogenase and phosphatase. They showed that
the formation of M-1-P by the LDH-deficient strain during
glucose catabolism is a consequence of impairment in
NADH oxidation caused by a greatly reduced LDH activity,
the transient formation of M-1-P serving as a regeneration
pathway for NAD+ regeneration. Gaspar et al. (2004)
described the construction of Lc. lactis strains able to form
mannitol as an end-product of glucose metabolism, using a
food-grade LDH-deficient strain as genetic basis for knock-
ing out the gene mtlA or mtlF. Resting cells of the double
mutant strains (ΔldhΔmtlA and ΔldhΔmtlF) produced
mannitol from glucose, with approximately one-third of
the carbon being successfully channeled to the production
of mannitol.
Mannitol production by heterofermentative LAB
A number of heterofermentative LAB of the genera
Lactobacillus, Leuconostoc, and Oenococcus can produce
mannitol directly from fructose (Saha 2003). In addition to
mannitol, these bacteria may produce lactic acid, acetic
acid, carbon dioxide, 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
mannitol 2-dehydrogenase (MDH). Saha and Nakamura
(2003) reported that nine strains of heterofermentative LAB
(L. brevis NRRL B-1836, L. buchneri NRRL B-1860, L.
cellobiosus NRRL B-1840, L. fermentum NRRL B-1915, L.
intermedius NRRL B-3693, Leu. Amelilibiosum NRRL B-
742, Leu. Citrovorum NRRL B-1147, Leu. mesenteroides
subsp. dextranicum NRRL B-1120, and Leu. Paramesen-
teroides NRRL B-3471) produce mannitol from fructose.
The strain L. intermedius NRRL B-3693 produced 198 g
mannitol from 300 g fructose l−1 in pH-controlled (pH 5.0)
fermentation at 37°C. The time of maximum mannitol
production varied greatly from 15 h at 150 g fructose to
136 h at 300 g fructose l−1. The bacterium converted
fructose to mannitol from the early growth stage. One-third
Appl Microbiol Biotechnol (2011) 89:879–891 881

Table 1 Mannitol production by fermentation

Organism Fermentation type Substrate (g/l) Mannitol (g/l) Qpa (gl−1 h−1) 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 Glycerol (100) 31 0.18 Onishi and Suzuki 1970
Torulopsis versalitis Batch G (194) 54 0.23 Onishi and Suzuki 1968
Fungus
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
glucose, maltose, starch plus glucoamylase (simultaneous
saccharification and fermentation, SSF), mannose, and
galactose. Two-thirds of fructose can also be replaced by
sucrose. The bacterium co-utilized fructose and glucose
(2:1) simultaneously and produced very similar quantities
of mannitol, lactic acid, and acetic acid in comparison with
fructose only. The glucose was converted to lactic acid and
acetic acid, and fructose was converted into mannitol.
Application of fed-batch fermentation by feeding equal
amounts of substrate and medium four times decreased the
maximum mannitol production time of fructose (300 gl−1)
from 136 to 92 h. The yields of mannitol, lactic acid, and
acetic acid were 202, 53, and 39 gl−1, respectively. With
glucose (150 gl−1) alone, the bacterium produced D- and L-
lactic acids in equal ratios (total, 70 gl−1) and ethanol (38 gl−1)
but no acetic acid.
A competitive production process for mannitol by
fermentation would require inexpensive raw materials.
Saha (2006a) studied the production of mannitol by L.
intermedius NRRL B-3693 using molasses as a carbon
source. The bacterium produced mannitol (104 gl−1) from a
mixture of molasses and fructose syrup (1:1; total sugars,
150 gl−1; fructose/glucose, 4:1) in 16 h. Several kinds of
inexpensive organic and inorganic nitrogen sources 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 O

2
CoA NAD+ Diacetyl
CoA
C 2 NADH + H+
Formate NADH + H+
Acetyl-CoA CO2 NAD+
Acetyl-CoA Acetoin
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-
peptone and Bacto-yeast extract. Soy peptone (5 gl−1) and
CSL (50 gl−1) were found to be suitable substitutes for
Bacto-peptone (5 gl−1) and Bacto-yeast extract (5 gl−1),
respectively. The bacterium produced 105 g mannitol from
150 g molasses and fructose syrup (1:1) in 22 h using 5 g
soy peptone and 50 g CSL l−1. The effects of four salt
nutrients (ammonium citrate, sodium phosphate, MgSO4,
and MnSO4) on the production of mannitol by L. inter-
medius NRRL B-3693 in a simplified medium containing
300 g fructose, 5 g soy peptone, and 50 g CSL l−1 in pH-
controlled fermentation at 5.0 at 37°C were evaluated using
a fractional factorial design (Saha 2006b). Only MnSO4 was
found to be essential for mannitol production and 33 mgl−1
was found to support maximum mannitol production. The
bacterium produced 200 g mannitol, 62 g lactic acid, and
40 g acetic acid from 300 g fructose l−1 in 67 h. Racine and
Saha (2007) improved the fermentation process further for
the production of mannitol by L. intermedius NRRL B-
3693. A fed-batch protocol overcame limitations caused by
high substrate concentrations. The fed-batch process
resulted in the accumulation of 176 g mannitol from 184 g
fructose and 92 g glucose l−1 of final fermentation broth in
30 h with a volumetric productivity of 5.9 gl−1 h−1. Further
increases in volumetric productivity of mannitol were
obtained in a continuous cell-recycle fermentation process
that reached more than 40 gl−1 h−1.
As an alternative to the use of pure fructose, Saha
(2006c) investigated the production of mannitol by L.
intermedius NRRL B-3693 using a commercial inulin
preparation as a substrate at pH 5.0 and 37°C. It (extracted
from the root of chicory plant, Cicharium intybus)
contained 92.6% inulin, 2.8% fructose, 0.5% glucose, and
4.1% disaccharides. Inulin is a polyfructan, consisting of
linear β-2,1-linked polyfructose chains terminated at the
reducing end of a glucose residue (Vandamme and Derycke
1983). The bacterium produced 106 g mannitol from dilute
acid hydrolyzate (pH 2.0, 121°C, 15 min) of 150 g inulin l−1
in 34 h. It also produced mannitol from inulin by
simultaneous saccharification and fermentation (SSF) at
Appl Microbiol Biotechnol (2011) 89:879–891
pH 5.0 and 37°C adding inulinase exogenously at the dose
level of 8 U (g substrate) −1. The L. intermedius B-3693
strain produced 207 g mannitol from 300 g inulin l−1 in 72 h
by SSF. The fermentation time decreased from 72 to 62 h
using a mixture of fructose and inulin (1:1; total, 300 gl−1).
When the fructose/inulin mixture (3:5, total 400 gl−1) was
used as substrate, the bacterium produced 228 g mannitol l−1
from both inulin and fructose with a yield of 0.57 g per g
substrate after 110 h of SSF. This is the highest concentra-
tion of mannitol produced by a heterofermentative LAB so
far reported in the literature. The fermentation medium was
simplified further and it now contains only CSL (50 gl−1)
and MnSO4 (33 mgl−1) in addition to carbon source (Saha
and Racine 2010).
Martinez et al. (1963) reported that L. brevis fermented
1 mol fructose to 0.67 mol mannitol, 0.33 mol lactate, and
0.33 mol acetate. Soetaert et al. (1995) reported a fed-batch
fermentation method with automatic feeding strategy for
rapid production of mannitol and D-lactic acid from fructose
or fructose/glucose mixture (2:1) by using Leu. pseudome-
senteroides. The maximum volumetric productivity of
mannitol was 11.1 gl−1 h−1 with a final concentration of
150 gl−1 in 24 h and a conversion efficiency of 94%. By
using a special mutant strain, quantitative conversion and a
further concentration increase up to 185 g mannitol l−1
could be obtained. Grobben et al. (2001) 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.
Yun et al. (1996a) reported about 4–5 g of mannitol
accumulation per kg kimchi, a Korean pickled vegetable,
during its fermentation. Yun and Kim (1998) isolated two
different LAB, Lactobacillus sp. Y-107 and Leuconostoc
sp. Y-002, during the fermentation of kimchi. These two
strains utilized fructose and sucrose as substrates for
mannitol formation. Under optimal conditions, the maximum
mannitol produced by Lactobacillus sp. Y-107 (at 35°C,
initial pH 8.0, anaerobic, 100 g fructose l−1, 120 h) and
Leuconostoc sp. Y-002 (at 35°C, initial pH 6.0, anaerobic,
50 g fructose l−1, 25 h) were 73 and 26 g from 100 g
fructose l−1 with yields of 86 and 65% based on fructose
consumed, respectively. The volumetric productivities of
mannitol by both strains were less than 1.0 gl−1 h−1. Neither
isolate produced other polyols such as glycerol and sorbitol
as by-products. These two bacterial strains were not able to
utilize high concentrations of sugars above 100 gl−1 due to
low osmotolerance of the isolates. Yun et al. (1996b)
obtained a mannitol yield of 70 g from 100 g fructose
within 80 h at 28°C using Lactobacillus sp. KY-107.
883
Erten (1998) studied the utilization of fructose (5 mmoll−1)
as an electron acceptor in two Leu. mesenteroides strains
under anaerobic conditions. These strains produced 0.26 mol
mannitol, 0.65–0.67 mol lactic acid, 0.37–0.57 mol ethanol,
and 0.26–0.27 mol acetic acid per mol fructose at 25°C.
Fermentation of a mixture of fructose and glucose (1:1)
resulted in the production of the same metabolic end-
products. Korakli et al. (2000) reported that mannitol is
produced by sourdough Lactobacilli from fructose with
concomitant formation of acetate. They obtained a 100%
yield of mannitol from fructose by L. sanfranciscensis
(isolated from sourdough) grown in a fed-batch culture
containing a fructose–glucose mixture with a volumetric
productivity of 0.5 gl−1 h−1 and a final mannitol concentra-
tion of 60 gl−1. Optimum production was at a concentration
of glucose/fructose mixture from 130 to 140 gl−1 and higher
concentrations of substrate were inhibitory. After adaptation
of the cells in sucrose, the bacterium produced mannitol to
only 65% yield in relation to the fructose content of sucrose.
It also synthesized complex polysaccharides when grown on
sucrose. L. pontis isolated from sourdough produced
mannitol, lactic acid, and ethanol from fructose (Hammes
et al. 1996). An unidentified Lactobacillus sp., designated
B001, produced mannitol from fructose with a volumetric
productivity of 6.4 gl−1 h−1 (Itoh et al. 1992). Salou et al.
(1994) reported that O. oenos converted 83 mol% of fructose
to mannitol when grown in a medium containing fructose
and glucose (1:1) with volumetric productivity of about
0.2 gl−1 h−1. Pimentel et al. (1994) studied growth and
metabolism of sugars and acids by Leu. oenos under different
conditions of temperature and pH. The bacterium produced
mannitol from fructose. The addition of acids, particularly
citrate, significantly repressed mannitol formation.
von Weymarn et al. (2002a) studied mannitol production
by 8 heterofermentative LAB strains (L. brevis ATCC-
8287, L. buchneri TKK-1051, L. fermentum NRRL B-
1932, L. sanfranciscensis E-93491, Lactobacillus sp.
(B001) BP-3158, Leu. mesenteroides ATCC-9135, Leu.
pseudomesenteroides ATCC-12291, and O. oeni E-9762).
They found that the ability to produce mannitol from fructose
varied markedly among these heterofermentative LAB
species. The effects of growth temperature, pH, and nitrogen
flushing on mannitol production by four selected strains were
studied in batch bioreactor cultivation. Using L. fermentum
and with fructose (20 gl−1) and glucose (10 gl−1) as carbon
source, mannitol yields from fructose were 86, 89, and
94 mol% at 25, 30, and 35°C, respectively. Mannitol yields
but not the volumetric mannitol productivities were improved
with constant nitrogen gas flushing of the growth medium.
Applying the most promising strain (L. fermentum), high
average and maximum mannitol productivities (7.6 and
16.0 gl−1 h−1, respectively) were achieved. von Weymarn et
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−1 h−1) and mannitol yield (97 mol%) in high cell equation:
density membrane cell-recycle culture using the best strain,
Leu. mesenteriodes ATCC 9135. A stable high-level 3 Fructose ¼ 1 Mannitol þ 2 Lactic acid þ 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−1 h−1) were achieved. In the alcohols (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
ATP
mannitol crystals were obtained at similar yield levels. Ojamo
et al. (2003) achieved a volumetric mannitol productivity of
about 20 gl−1 h−1 using high cell density fermentation of Leu. ADP
pseudomesenteroides ATCC 12291. Recently, Fontes et al. Glucose-6-P
NAD+
(2009) reported production of 18 g mannitol from cashew
juice containing 50 g reducing sugars (28 g fructose) with
67% fructose conversion into mannitol and productivity of NADH + H+
1.8 gl−1 h−1 by Leu. mesenteroides B-512F. 6-P-Gluconate
NAD+
Mannitol produced by heterofermentative LAB is de-
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
(1970) 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.
Hattori and Suzuki (1974) studied the large-scale produc-
tion of erythritol and its conversion of mannitol by Candida
zeylanoides grown on alkane. The strain produced about
180 g meso-erythritol l−1 and a small amount of mannitol.
Erythritol was almost entirely converted to mannitol by
keeping the KH2PO4 concentration in the medium at 40–
200 mgl−1, and the mannitol concentration was 63 gl−1 after
100 h incubation in a 5-l fermenter, which corresponded to
52% of the alkane consumed. Zygosaccharomyces rouxii
ATCC 12572 produced ethanol, glycerol, arabitol, and
mannitol from glucose (Groleau et al. 1995).
Stankovic et al. (1989) 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, Saccharo-
myces, 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. (2002) isolated more
than 1000 strains from various sources such as soil,
seawater, honey, pollen, and fermentation sludge and tested
them for their ability to produce mannitol. They identified a
novel strain of Candida magnoliae (isolated from fermen-
tation sludge) which produced 67 g mannitol in 168 h from
150 g fructose l−1 in batch flask culture (30°C, 220 rpm,
10 g yeast extract l−1). A fructose concentration higher than
200 gl−1 reduced the mannitol conversion yield and
production rate. In fed-batch culture with 3−12% fructose,
mannitol production reached a maximum of 209 gl−1 after
200 h, corresponding to 83% yield and a volumetric
productivity of 1.03 gl−1 h−1. The strain produced only
small quantities of by-products, such as glycerol, erythritol,
and organic acids. Under optimum conditions, a final
mannitol production of 213 g was obtained from 250 g
fructose l−1 after 110 h (Lee et al. 2003a). Supplementation
with Ca2+ and Cu2+ increased mannitol production by C.
magnoliae (Lee et al. 2007). Recently, Khan et al. (2009)
reported that the resting cells of C. magnoliae produced
mannitol from glycerol with a yield of ~45%. De Zeeuw
and Tynan (1973) reported that C. lipolytica produced
mannitol as main sugar alcohol. A strain of Aureobasidium
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 (Yun and Song
885
1994). Stress solutes such as NaCl and KCl in the range
from 0.25 to 1 M did not promote polyol production.
Mannitol production by filamentous fungi
Several filamentous fungi produce mannitol from glucose.
Yamada et al. (1961) showed that glucose is first converted
to F-6-P, which is then reduced to M-1-P in the presence of
NADH, and M-1-P is hydrolyzed to mannitol by a specific
phosphatase in Pircularia oryzae. Smiley et al. (1967)
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 gl−1 h−1 and a yield of 31.0 mol%. The
presence of glucose in the medium was essential to prevent
metabolism of mannitol. Nelson et al. (1971) 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 rate. 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-1-P
(Strandberg 1969). Lee (1967) determined the carbon
balance for fermentation of glucose to mannitol by Aspergillus
sp. The products found were: cells (17% of carbon input),
CO2 (26%), mannitol (35%), glycerol (10%), erythritol
(2.5%), glycogen (1%), and unidentified compounds (8%).
Cell-free enzyme studies indicated that mannitol was
produced via the reduction of fructose-6-phosphate.
Hendriksen et al. (1988) screened 11 different Penicillium
species for production of mannitol. All strains produced
mannitol and glycerol from sucrose. The highest amount of
mannitol (43 gl−1) was produced by P. scabrosum IBT
JTER4 and the highest combined yield of mannitol and
glycerol (65 gl−1) was obtained with P. aethiopicum IBT
MILA 4 when grown on sucrose (150 gl−1) and yeast
extract (20 gl−1) 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 gl−1
h−1. Penicillium sp. uses the same metabolic route for
conversion of glucose to mannitol as A. candidus (Boosaeng
et al. 1976). El-Kady et al. (1995) screened 500 filamentous
fungal isolates belonging to ten genera and 74 species and
identified Aspergillus, Eurotium, and Fennellia species as
high (>1.82 gl−1) 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. (1988) demonstrated that four fungal
cultures—Alternaria alternata, Cladosporium herbarum,
Epicoccum purpurascens, and Fusarium pallidoroseum
isolated from cotton leaf dust produced mannitol and are
a probable source of mannitol found in cotton dust.
886
Mannitol production by recombinant microorganisms
Although a few homofermentative LAB produce mannitol
from glucose, its production level is very low. Several
heterofermentative LAB produce excellent quantities of
mannitol (~66%) from fructose. They also produce lactic acid
and acetic acid as coproducts. This is why a number of
recombinant microorganisms have been developed to either
overproduce mannitol or limit or eliminate the production of
coproducts (lactic acid and acetic acid). In this section, we
review the literature dealing with the construction of
recombinant organisms for mannitol production. Kaup et al.
(2004) constructed an efficient Escherichia coli strain for
mannitol production from fructose in a whole cell biotrans-
formation. The strain expressed NAD+-dependent MDH from
Leu. pseudomesenteroides ATCC 12291 (Hahn et al. 2003)
for the reduction of fructose to mannitol, NAD+-dependent
formate dehydrogenase (FDH) from Mycobacterium vaccae
N10 (Galkin et al. 1995) for NADH regeneration, and the
glucose facilitator from Zymomonas mobilis (Weisser et al.
1995; Parker et al. 1995) for the uptake of fructose without
concomitant phosphorylation. The strain produced about
66 g mannitol from 90 g fructose l−1 within 8 h with a yield
of 73% and a specific mannitol productivity of >4 g per g
cell dry weight (cdw) h−1. Kaup et al. (2005) reported that
supplementation of this recombinant strain with extracellular
GI resulted in the formation of 145.6 g mannitol from 180 g
glucose l−1. They have co-expressed the xylA gene of E. coli
in this recombinant E. coli strain which formed 83.7 g
mannitol from 180 g glucose l−1. Sasaki et al. (2005) cloned
a gene encoding MDH from L. reuteri and expressed in E.
coli. The purified recombinant enzyme works optimally at
37°C and pH 5.4 for conversion of fructose to mannitol.
Aarnikunnas et al. (2003) used metabolic engineering of
L. fermentum for production of mannitol and pure L-lactic
acid or pyruvate. The authors first developed genetic tools
to modify L. fermentum and then proceeded to inactivate
first ldhD gene and then ldhL gene in order to create a
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-
tion of the ldhD gene produced mannitol and L-lactic acid.
The double mutant strain created by inactivating the ldhL
gene produced mannitol and pyruvate. In addition, the mutant
produced 2,3-butanediol and the volumetric productivity of
mannitol was decreased. Helanto et al. (2005) described the
construction and characterization of a random mutant of Leu.
pseudomesenteroides that is unable to grow on fructose and
the positive effects of the mutation on mannitol production.
They have performed the inactivation of its fructokinase
activity with random mutagenesis and screening the mutants
unable to grow on fructose. The fructose uptake of the mutant
was unaltered and the mutant converted fructose to mannitol
Appl Microbiol Biotechnol (2011) 89:879–891
when grown in a medium containing both glucose and
fructose. The yield of mannitol from fructose was improved
from 74 to 86 mol%. A fructokinase-negative mutant could
enable higher pH to be used in the mannitol production
process without lowering the yield.
Wisselink et al. (2004) cloned and overexpressed mtlD
from L. plantarum in Lc. lactis in different genetic back-
grounds (a wild-type strain, an LDH-deficient strain, and a
strain with reduced phosphofructokinase activity). Small
amounts (<1%) of mannitol were formed by growing cells
of mtlD-overexpressing LDH-deficient and phosphofructo-
kinase reduced strains. The resting cells of the LDH-
deficient transformant converted 25% of glucose (3.6 gl−1)
to mannitol. They concluded that the mtlD overexpressing
LDH-deficient Lc. lactis strain seemed to be the most
promising strain for mannitol production. Liu et al. (2005)
have cloned and expressed the mtlK gene encoding MDH
from L. brevis in E. coli. The genetically engineered E. coli
strain was able to catalyze the reduction of fructose to
mannitol.
Costenoble et al. (2003) demonstrated that mannitol is
produced under anaerobic conditions by a glycerol-
defective mutant of Saccharomyces cerevisiae expressing
the mtlD gene from E. coli coding for NADH-dependent
M-1-P dehydrogenase. Improving efflux of the formed
mannitol seems to be necessary for obtaining anaerobic
growth and a sustained mannitol production. Baumchen
and Bringer-Meyer (2007) overexpressed MDH gene (mdh)
from Leu. pseudomesenteroides and coexpressed FDH gene
(fdh) in Corynebacterium glutamicum ATCC 13032. The
recombinant C. glutamicum cells produced mannitol at a
constant production rate of 0.22 g (g cdw)−1 h−1. Expression
of the glucose/fructose facilitator gene glf from Z. mobilis
in C. glutamicum led to a 5-fold increased productivity of
1.25 g (g cdw)−1 h−1, yielding 87 g mannitol from 93.7 g
fructose. In repetitive fed-batch biotransformation, 285 g
mannitol was formed over a period of 96 h with an average
productivity of 1.0 g (g cdw)−1 h−1.
Enzymatic production of mannitol
Mannitol can be enzymatically produced from fructose in a
one pot synthesis by using NADH-dependent MDH or
NADPH-dependent MDH. Saha (2004) purified MDH from
L. intermedius NRRL B-3693 and showed that the purified
enzyme can convert fructose to mannitol completely in the
presence of NADPH. The cofactor dependency of the
enzyme is a major limitation. A number of strategies such as
enzymatic, electrochemical, chemical and photochemical,
and biological methods are available for cofactor regenera-
tion (Chenault and Whitesides 1987; O’Neill and Woodward
2000). A two-enzyme system can be used for cofactor
Appl Microbiol Biotechnol (2011) 89:879–891
regeneration with simultaneous conversion of two sub-
strates into two products of interest (Wichmann et al. 1981).
One example is the simultaneous conversion of fructose
and formate using the enzymes MDH and FDH (Parmentier
et al. 2005). FDH converts formate to CO2 and reduces
NAD to NADH. MDH uses NADH to convert fructose to
mannitol and regenerates NAD.
Synthesizing reaction:
Fructose þ NADH þ Hþ ¼ Mannitol þ NADþ
Regenerating reaction:
Formic acid þ NADþ ¼ CO2 þ NADH
Slatner et al. (1998) achieved a volumetric mannitol
productivity of 2.25 g 1−1 h−1 using a recombinant MDH
from P. fluorescens overexpressed in E. coli and FDH from
C. boidinnii with a final product concentration of 72 gl−1
and a fructose conversion of 80% in the system. The other
product CO2 is easily separated from mannitol. Mannitol
was crystallized from the ultrafiltered product solution in
97% purity and 85% recovery, thus allowing reuse of
enzymes for repeated batch production of mannitol. Another
example of the two enzyme system for regeneration of
cofactor is MDH and glucose dehydrogenase (GDH) system
using glucose/fructose mixture (1:1) and simultaneous
synthesis of mannitol and gluconic acid (Howaldt et al.
1988). NAD requiring GDH converts glucose to gluconic
acid and generates NADH. MDH uses NADH to convert
fructose to mannitol and regenerates NAD.
Synthesizing reaction:
Fructose þ NADH þ Hþ ¼ Mannitol þ NADþ
Regenerating reaction:
Glucose þ NADþ þ H2 O ¼ Gluconic acid þ NADH
Gluconic acid (market size, 35,000 tons year−1), a
multifunctional carbonic acid, is a bulk chemical used in
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
can be used as a cement additive to increase cement
resistance and stability under extreme climatic conditions,
e.g., frost and floods (Hustede et al. 1989).
The MDH used was from P. fluorescens, Torulaspora
delbruckii, and Schizophyllum commune, and the FDH was
from Bacillus megaterium (Haltrich et al. 1996; Nidetzky et
al. 1996). Downstream processing consists of the separation
of enzymes from the product solution with the aid of reactor-
integrated membranes and of the isolation of the two
products—mannitol and gluconic acid—by electrodialysis,
ion-exchange chromatography, and fractional crystallization
(Kulbe et al. 1987).
887
Both NADH- and NADPH-dependent MDHs have been
purified from a number of microorganisms. As for example,
NADH-dependent MDH has been purified from Lb. brevis,
Leu. mesenteriodes, P. fluorescens, Rhodobacter spaeroides,
Saccharomyces cerevisiae, and T. delbruckii (Martinez et al.
1963; Sakai and Yamanaka 1968; Brunker et al. 1997;
Schneider and Giffhom 1989; Schneider et al. 1993; Quain
and Boulton 1987; Nidetzky et al. 1996). NADPH-
dependent MDH has been purified from A. parasitius, C.
magnoliae, Z. mobilis, and Gluconobacter suboxydans
(Niehaus and Dilts 1982; Lee et al. 2003b; Viikari and
Korhola 1986; Adachi et al. 1999). Schneider and Giffhom
(1989) reported an increase of 8.3-fold of MDH activity by
constructing a strain (pAK82) from R. sphaeroides Si4 and
by producing high cell concentrations via fed-batch cultiva-
tion in a bioreactor in comparison to batch cultivation of the
wild-type strain. Mannose can be reduced to mannitol
enzymatically. However, the reversible reaction favors man-
nitol oxidation and is thus not suitable (Stoop et al. 1998).
Baumchen et al. (2007) developed an in vivo system for
the biotransformation of fructose to mannitol by the
expression of mdh gene encoding MDH from Leu.
pseudomesenteroides in B. megaterium. The NADH reduc-
tion necessary for MDH activity was regenerated via the
oxidation of formate to CO2 by coexpression of fdh gene
encoding Mycobacterium vaccae N10 FDH. Recombinant
B. megaterium produced up to 10.6 g mannitol per l at the
shaking flask scale. Whole cell biotransformation in a fed-
batch bioreactor increased the mannitol production to
22 gl−1 with a specific productivity of 0.32 g (g cdw)−1 h−1
and a mannitol yield of 0.91 molmol−1. However, the
substrate uptake was the limiting factor of the overall
biotransformation. Song et al. (2008) also demonstrated
that mannitol can be produced from glucose in a two step
enzymatic process using a Thermotoga neapolitana xylose
isomerase (GI) and T. martina MDH. However, in the
absence of a cofactor regeneration system, the final
mannitol concentration only reached 19 mM.
Downstream processing of mannitol
Mannitol, at 180 gl−1, can be recovered from the fermen-
tation broth by cooling crystallization. Saha and Nakamura
(2003) reported that small white needle-like crystals of
mannitol appeared upon refrigeration of the cell-free
fermentation broth at 4°C. von Weymarn et al. (2002b)
crystallized mannitol with a yield of 72 mass% from
permeates obtained from three successive batches of
fermentation using Leu. mesenteroides ATCC-12291. After
recrystallization, the purity of mannitol was more than 99%.
Deusing et al. (1996) described a method for desalination by
electrodialysis and crystallization of mannitol produced by
888
large-scale fermentation by Leu. mesenteroides with a purity
of >99.5%. Soetaert et al. (1999) reported that the use of
electrodialysis followed by crystallization resulted in cost-
effective recovery of highly pure crystalline mannitol and D-
lactic acid under optimized downstream processing of the
fermentation broth of Leu. mesenteroides. Slatner et al.
(1998) crystallized mannitol from an enzymatic conversion
at 4°C (1 h) from the ultrafiltrate after evaporation of the
product solution in vacuo to approximately half the original
volume and subsequent addition of 1 volume equivalent of
isopropanol. After centrifugation, the residual alcohol in the
solid product was evaporated at room temperature, leaving
mannitol with a purity of at least 97%. Ojamo et al. (2003)
suggested that the acidic side stream, obtained from a LAB
fermentation process, could be used as feed preservative.
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
crystals were separated by centrifugation. The mother liquor
was further fractionated by a chromatographic method into
two fractions—acetate fraction and a lactate/mannitol frac-
tion. Mannitol was separated from lactate by crystallization,
and the lactate was isolated by precipitation with Ca(OH)2.
Applications of mannitol
Mannitol is used as a sweet-tasting bodying and texturing
agent. It reduces the crystallization tendency of sugars and is
used as such to increase the shelf life of foodstuffs. Crystalline
mannitol exhibits a very low hygroscopicity, making it useful
in products that are stable at high humidity. It is only about half
as sweet as sucrose. Mannitol exhibits reduced physiological
calorie value (1.6 kcalg−1) compared to sucrose (4 kcalg−1). It
has a low solubility in water of only 18% (w/v) at 25 °C and
13% (w/v) at 14°C (Perry et al. 1997). In comparison, the
solubility limit of sorbitol in water is about 70% (w/v) at
25°C. Mannitol is sparingly soluble in organic solvents
such as ethanol and practically insoluble in ether, ketones,
and hydrocarbons (Schwarz 1994). It forms orthorhombic
crystals and the crystals have a melting point at 165–168 °C
(Schwarz 1994). Mannitol is extensively used in chewing
gum. It is chemically inert and is commonly used in the
pharmaceutical formulation of chewable tablets and granu-
lated powders. Mannitol prevents moisture absorption from
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
(~121 kJkg−1) 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 (Soetaert et al. 1995).
Appl Microbiol Biotechnol (2011) 89:879–891
Mannitol is used in medicine as a powerful osmotic
diuretic (to increase the formation of urine in order to prevent
and treat acute renal failure and also in the removal of toxic
substances from the body) and in many types of surgery for
the prevention of kidney failure (to alter the osmolarity of the
glomerular filtrate) and to reduce dye and brain oedema
(increased brain water content). Hypertonic mannitol can
enhance the transport of drugs across the blood–brain barrier
for the treatment of life-threatening brain diseases (Rapoport
2001; Miller 2002). Inhaled mannitol improves the hydra-
tion and surface properties of sputum in patients with cystic
fibrosis (Daviskas et al. 2010). Mannitol hexanitrate is a
well-known vasodilator, used in the treatment of hyperten-
sion (Johnson 1976). Mannitol is also a scavenger of
hydroxyl radicals (Shen et al. 1997).
Concluding remarks and future prospects
Heterofermentative LAB have the capability to utilize
high concentrations of fructose such that the mannitol
concentration in the fermentation broth could reach more
than 180 gl−1, 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
electrodialysis (Datta and Tsai 1997; Soetaert et al. 1995).
The MDH responsible for catalyzing the conversion of
fructose to mannitol requires NADPH (NADH) as cofactor.
It is thus possible to develop a one-pot enzymatic process
for production of mannitol from fructose if a cost-effective
cofactor regeneration system can be developed (Saha
2004). The heterofermentative LAB cells can be immobi-
lized in a suitable support, and the immobilized cells
can be used in a bioreactor to continuously produce
mannitol from fructose. The production of acetic acid
and D-lactic acid by some LAB can be blocked (inacti-
vation of acetate kinase and D-LDH) by mutagenesis
(Aarnikunnas et al. 2003). In that case, the fermentation
broth should contain only mannitol and L-lactic acid.
Both are value-added products and thus the fermentative
production of mannitol will become more attractive. Even
though mannitol is currently not produced industrially by
fermentation, the prospect of producing mannitol by
fermentation using a food grade LAB in near future looks
very promising.
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