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Scientific
Publications - Work Done by Microbiology Reader Bioscreen C
Process Biochemistry, Volume
37, Issue 11 , June 2002, Pages 1207-1213
Production of
 -mannitol
by heterofermentative lactic acid bacteria
Niklas von Weymarn, Mervi Hujanen and Matti Leisola
Laboratory of Bioprocess Engineering, Department of Chemical Engineering,
Helsinki University of Technology, P.O. Box 6100, FIN-02015 HUT Helsinki,
Finland
Received 18 September 2001; revised 2 November 2001;
accepted 8 November 2001. Available online 14 December 2001.
ABSTRACT
Eight heterofermentative lactic acid bacteria were compared as to their
ability to convert
-fructose
to -mannitol.
Four promising strains were identified and the effects of growth temperature,
pH, and nitrogen flushing on mannitol production by these strains were studied
in batch bioreactor cultivations. In contrast to earlier findings, a high growth
temperature was observed to improve the yield of mannitol from fructose with
Lactobacillus fermentum. When Lb. fermentum was grown at 25,
30, and 35 °C, yields of 86.4±0.8, 88.9±2.4, and 93.6±0.6 mol%, respectively,
were achieved. In general, constant nitrogen gas flushing of the growth media
was found to improve the mannitol yields, but not the volumetric mannitol
productivities. Applying the most promising strain in a batch mannitol
production experiment, high average and maximum volumetric mannitol
productivities (7.6 and 16.0 g/l/h, respectively) were achieved.
Author Keywords: Lactic acid bacteria; Heterofermentative;
Lactobacillus; Leuconostoc; Mannitol; Productivity
1. INTRODUCTION
-Mannitol
is a six-carbon sugar alcohol, which is about half as sweet as sucrose. It is
found in small quantities in most fruits and vegetables [1 and 2] and has
various applications, e.g. in foods, pharmaceuticals, medicine, and chemistry.
At present, mannitol is produced commercially by catalytic hydrogenation of
fructose syrups or invert sugar with the co-production of another sugar alcohol,
sorbitol. Typically, the hydrogenation of a 50/50–fructose/glucose mixture
results in a 30/70 mixture of mannitol and sorbitol [3]. Besides the fact that
mannitol is the by-product of the chemical process and thus can be liable to
supply problems, it is also relatively difficult to separate from sorbitol. In
contrast to most sugars and other sugar alcohols mannitol dissolves poorly in
water (13% (w/w) at 14 °C) [4]. Cooling crystallization is therefore commonly
used to separate mannitol from sorbitol and other components. However, according
to Takemura et al. the yield of crystalline mannitol, in the chemical process,
is still only approximately 17% (w/w) based on the initial sugar substrates [5].
In order to improve the total process yield of mannitol it would be
advantageous to develop a process with mannitol as the main product and with no
sorbitol formation. Some alternative processes based on the use of microbes have
been suggested in the literature. Yeast, fungi, and lactic acid bacteria (LAB)
especially, have proved to produce mannitol effectively without co-formation of
sorbitol [6]. Among LAB only heterofermentative species are known to convert
fructose into mannitol [3, 7 and 8]. Species belonging to the genera
Leuconostoc, Oenococcus, and Lactobacillus particularly, have
been reported to produce mannitol effectively. In addition to mannitol these
microbes co-produce mainly lactic and acetic acid, carbon dioxide, and ethanol.
These by-products are, however, easily separated from mannitol.
Several groups have studied the bioconversion of fructose into mannitol with
growing cells, but only two groups report volumetric mannitol productivities
over 5 g/l/h. Using a fed-batch cultivation protocol Soetaert et al. reached a
volumetric productivity of about 6.3 g/l/h with L. pseudomesenteroides [9 and
10]. A similar productivity level (6.4 g/l/h) was earlier also achieved with an
unidentified Lactobacillus species, named B001 [6]. Moreover, these groups
report mannitol yields (mannitol produced from fructose consumed) of 94 and 96
mol%, respectively. Recently, Korakli et al. reported a 100% yield with a strain
of Lb. sanfranciscensis [11]. However, this strain grew very slowly and the
volumetric productivity in a fed-batch cultivation was only 0.5 g/l/h. Other
species of heterofermentative LAB reported to be producers of mannitol include
L. mesenteroides, O. oeni, Lb. brevis, Lb. buchneri, and Lb. fermentum [3, 12,
13 and 14].
Although several papers are available reporting mannitol production with LAB,
only one focuses on the comparison of two different species. Yun and Kim
cultivated two food-isolated LAB strains in shake flasks, and under optimal
growth conditions they concluded that the more effective strain (Lactobacillus
sp.) converted 86 mol% of fructose consumed into mannitol, whereas the other
strain (Leuconostoc sp.) had a yield of only 65 mol% [15]. They also
reported that from the variety of carbohydrate substrates tested, notable
mannitol formation was detected only when either fructose or sucrose were used
as the substrate.
In this paper, the production of mannitol by eight heterofermentative LAB was
studied. A simplified production (SP) medium was developed and in batch
cultivations the effects of temperature, pH, and nitrogen gas flushing on growth
and mannitol production were examined. Results reported here enable a
preliminary comparison of the behaviour of the studied microbes in bioreactor
environments. Moreover, this study identifies two LAB strains with clearly
better volumetric mannitol productivities in batch mode compared to prior
studies and the strains used in them.
2. MATERIALS AND METHODS
2.1. Microorganisms and medium
The microbial strains used in this study, summarized in Table 1, were
initially grown in a modified MRS growth medium containing 20 g/l fructose and
10 g/l glucose (pH 6.2). O. oeni grew poorly at pH 6.2 and was therefore
cultured at an initial pH of 5.0. A SP medium was prepared from individual stock
solutions. The following concentrations were used in the complete SP medium:
tryptone (Pronadisa, Hispanlab S.A., Spain), 10 g/l; yeast extract (Difco,
Becton Dickinson and Company, USA), 5 g/l; K2HPO4, 2 g/l;
fructose, 20 g/l; glucose, 10 g/l; MgSO4, 0.2 g/l; FeCl3,
10 mg/l; CaCl2, 20 mg/l; MnSO4, 10 mg/l, and NaCl, 10
mg/l. The amounts of the variable components (Mg, Fe, Ca, Mn, and Na) were
adapted from Dols et al. [16]. The final SP medium, used in 600-ml bioreactor
cultivations is presented in Table 2. In the 2-l Lb. fermentum
bioreactor cultivation, SP medium was used with modifications. The initial
fructose, glucose, and yeast extract concentrations were increased to 100, 50,
and 10 g/l, respectively.
Table 1. Microbial strains used in this study
Table 2. The simplified production (SP) medium used in the bioreactor
cultivations (amounts in brackets were used in Lb. fermentum
cultivations)
_1207.gif)
2.2. Growth conditions
The initial comparison of strains and the development of the SP medium were
conducted in a Bioscreen C analyzer (Labsystems Oy, Finland). A working volume
of 400  l was
applied. The temperature was controlled at 30 °C and the optical density (600
nm) of the cell suspensions was measured automatically at 1-h interval. Samples
for analysis of mannitol concentration were taken at an early stationary growth
phase. Bioscreen experiments were carried out in quadruplicate, but due to the
large number of possible samples a cell-free culture broth from four parallel
cultures were combined and analysed by high performance liquid chromatography
(HPLC).
Bioreactor cultivations were carried out in either a Biostat Q system (B.
Braun Biotech International, Germany) with four 600-ml (working volume) culture
vessels or a Biostat MD system with a 2-l (working volume) culture vessel. Both
systems were equipped with automatic probes for the measurement of temperature,
pH, and dissolved oxygen tension (DOT). In the Biostat Q system, magnetic bars
and a magnetic drive unit were used for mixing. The agitation speed was 400 rpm.
In the Biostat MD system, two disc impellers were used for mixing (200 rpm).
During anaerobic experiments, the growth media were constantly flushed with
nitrogen gas, whereas during semi-anaerobic experiments no gases were added to
the bioreactors. The DOT probes were calibrated with nitrogen gas and air. The
pH was controlled automatically by addition of 5 M NaOH or 2 M H2SO4.
The 2-l Lb. fermentum production study was performed semi-anaerobically
at 40 °C and pH 5.0.
All pre-cultures were grown in standard MRS (pH 6.2) (Pronadisa, Hispanlab,
S.A., Spain). A 5% (v/v) inoculum of a 10-h culture (L. mesenteroides,
L. pseudomesenteroides, Lb. brevis, and Lb. fermentum), a 20-h
culture (Lb. sp. B001, Lb. sanfranciscensis, Lb. buchneri)
or a 3-day-old culture (O. oeni) was used. All conditions were examined
in two successive cultivations. These results are thus presented as mean values
and standard deviations. The 2-l cultivation was inoculated 10% (v/v) with a
10-h culture.
2.3. Analytical methods
Cell growth was monitored by measuring the optical density at 600 nm (OD600)
against water. The cell-free extracts were prepared as follows: cells at late
exponential growth phase were harvested, washed twice in 50 mM phosphate buffer
(pH 6.5) and resuspended in 4 ml of the same buffer. The suspension was
sonicated in the presence of glass beads for 8×15 s with 30 s cooling on ice
between the treatments. Cell debris was separated by centrifugation and
supernatants were assayed immediately for NADH oxidase activity. NADH oxidase
activity was assayed at 30 °C in 50 mM sodium phosphate buffer (pH 6.5) by
following the oxidation of 0.13 mM NADH at 340 nm. Protein concentrations were
measured by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad
Laboratories, USA).
Glucose, fructose, ethanol, and mannitol concentrations were measured with
HPLC using an Aminex HPX-87C column (Bio-Rad Laboratories, USA) at 60 °C with
Milli-Q water as the eluent. The flow rate of the eluent was controlled at 0.6
ml/min. The HPLC apparatus consisted of a Perkin–Elmer Series 200 Autosampler
and LC Pump (Perkin–Elmer Corporation, USA), and a HP 1047A refractometer
(Hewlett–Packard Company, Japan). Also a Deashing Micro-Guard pre-column
(Bio-Rad Laboratories, USA) was used. Organic acids were analysed with an Aminex
HPX-87H column as described earlier [17].
The maximum specific growth rates (max)
were calculated with Microsoft Excel. A chart for the natural logarithm of
optical density values versus time was drawn. The maximum specific growth rate
is the steepest slope of a linear trendline (3–5 successive values) in the
exponential growth phase.
3. RESULTS AND DISCUSSION
The presentation of the results is divided into three parts. In the first
part, experiments conducted in
l-scale are
presented, whereas the second part covers the results from the 600-ml bioreactor
studies. In the third part, knowledge gathered in parts one and two was applied
to perform a 2-l mannitol production experiment.
3.1. Bioscreen
3.1.1. Comparison of different mannitol producing LAB species
The results of culturing the strains in the modified MRS medium are presented
in Table 3. L. mesenteroides (max=0.57/h),
L. pseudomesenteroides (0.46/h), Lb. brevis (0.45/h), and Lb.
fermentum (0.55/h) grew significantly faster than the other four species and
as expected, they were also superior in volumetric mannitol productivity. With
most strains tested a significant fraction of fructose consumed by the cells
escaped probably into the phosphoketolase pathway and thus, into formation of
excess lactic and acetic acid, ethanol, and carbon dioxide. However, in unison
with Korakli et al. [11] we found that Lb. sanfranciscensis converted
almost 100% fructose consumed into mannitol. Based on the productivities shown
in Table 3, L. mesenteroides, L. pseudomesenteroides, Lb.
brevis, and Lb. fermentum were chosen for the bioreactor studies.
Table 3. Mannitol production by heterofermentative LAB
_1207.gif)
Column headings: r, the volumetric mannitol productivity; q, the
specific mannitol productivity (here the volumetric productivity divided by the
optical density); Y, the yield of mannitol produced from fructose
consumed in the initial comparison experiments.
3.1.2. Development of a simplified production (SP) medium
In order to simplify the growth medium for production studies on a bioreactor
scale a Bioscreen analyzer and the SP medium were used. The variable metal
components (Mg, Fe, Ca, Mn, and Na) were omitted one at a time from a basic SP
medium (tryptone, yeast extract, K2HPO4, and sugars). The
results showed that only minor changes in maximum specific growth rates were
seen, when the variable components were missing from the basic medium (data not
shown). The volumetric mannitol productivities, however, were significantly
affected by the removal of Mn2+ from the growth media of all four
strains. In comparison to the respective values in the complete SP medium, the
volumetric productivities of Lb. brevis, Lb. fermentum, L.
mesenteroides and L. pseudomesenteroides in medium without Mn2+
decreased 6, 32, 9, and 17%, respectively. To a lesser degree also the removal
of Mg2+ was found to decrease the volumetric mannitol productivities
(2–4%). Furthermore, a significant decrease in volumetric mannitol productivity
was only seen with Lb. brevis, when using the simple SP medium instead of
the nutrient-rich MRS medium (32%). In an additional experiment the basic SP
medium was supplemented with variable concentrations of Mg2+ and Mn2+.
The results indicated that the volumetric mannitol productivity was improved
even further when double amounts of both Mg2+ and Mn2+
were used in cultures of Lb. fermentum. Table 2 summarizes the
composition of the SP medium used in the 600-ml bioreactor cultivations.
Manganese and magnesium ions are essential co-factors for enzymes in the
primary sugar metabolism of LAB. Magnesium functions as a co-factor for
fructokinase, phosphoketolase, and acetate kinase, whereas manganese functions
as a co-factor for some enzymes in the pathway from glyceraldehyde-3-P to
pyruvate and for lactate dehydrogenase. Clearly these metal ions play a central
role in the production of reducing power (NAD(P)H) and ATP, and hence, are
essential for many cellular functions and more importantly, for the transport
and reduction of fructose. Mannitol dehydrogenase, on the other hand, does not
require any metal ions.
3.2. Bioreactor cultivations
3.2.1. Effect of temperature and pH on growth and mannitol production
The maximum specific growth rates of all the four strains were clearly
improved when the growth temperature was increased from 25 to 35 °C (Fig. 1).
The growth temperature was also observed to have a strong influence on the
volumetric mannitol productivity of the cells. The effect of growth temperature
on productivity was particularly evident with Lb. fermentum, where a
change from 25 to 35 °C brought about an approximately twofold increase in the
volumetric mannitol productivity (1.00±0.02 to 2.03±0.04 g/l/h). Of the four
strains tested, L. mesenteroides was least affected by changes in the
growth temperature. In fact, increasing the growth temperature from 30 to 35 °C
with L. mesenteroides resulted in a small decrease in volumetric
productivity (1.97–1.94 g/l/h). The specific mannitol productivities (volumetric
productivity divided by the optical density) were also clearly higher at 35 °C
than at 25 °C. In cultivations with Lb. brevis, Lb. fermentum,
L. mesenteroides, and L. pseudomesenteroides the specific mannitol
productivities were improved from 0.18 to 0.22, 0.16 to 0.30, 0.37 to 0.50, and
0.34 to 0.45 g/l/h, respectively, when grown at 35 °C instead of 25 °C.
_1207.gif)
Fig. 1. Effect of temperature on maximum specific growth rate, volumetric
mannitol productivity, and mannitol yield of four different LAB species. The
standard deviations were found to be significantly smaller than the changes in
the actual results and are therefore not shown in the figure. Columns: grey,
maximum specific growth rates (1/h); white, volumetric mannitol
productivities (g/l/h); dark, yields of mannitol produced from fructose
consumed (mole/mole).
On the other hand, better mannitol yields were achieved with Lb. brevis,
L. mesenteroides, and L. pseudomesenteroides, when the growth
temperatures were lowered (Fig. 1). However, entirely opposite findings were
made with Lb. fermentum, where a high growth temperature resulted in the
best yield. When the temperature was controlled at 25 °C, the yield with Lb.
fermentum was measured to be 86.4±0.8 mol%. Respectively, at 35 °C Lb.
fermentum converted up to 93.6±0.6 mol% of the fructose consumed into
mannitol.
The highest maximum specific growth rates were achieved when the pH was
controlled at 5.5 (Fig. 2). Lower pH values (5.0 and 4.5) decreased the maximum
specific growth rates of all four strains. The maximum specific growth rate of
L. mesenteroides was especially affected by a low pH. The maximum
specific growth rate of this strain, at pH 4.5, was only approximately half of
its respective value at pH 5.5. A high pH value improved volumetric mannitol
productivities, whereas better mannitol yields were observed at low pH values.
The effect of pH on the specific mannitol productivities was found to be small
(data not shown).
_1207.gif)
Fig. 2. Effect of pH on maximum specific growth rate, volumetric mannitol
productivity, and mannitol yield of four different LAB species. The standard
deviations were found to be significantly smaller than the changes in the
actual results and are therefore not shown in the figure. Columns: grey,
maximum specific growth rates (1/h); white, volumetric mannitol
productivities (g/l/h); dark, yields of mannitol produced from fructose
consumed (mole/mole).
Earlier Soetaert [9] observed in studies done with L. pseudomesenteroides
that decreasing the growth temperature and the pH result in more efficient
conversion of fructose into mannitol, i.e. a better yield. On the other hand, he
also observed that a low temperature and a low pH lead to decreased volumetric
mannitol productivities. Similar observations are reported here. For example, in
cultivations with Lb. fermentum a change in growth temperature from 35 to
25 °C resulted in a 50% decrease in volumetric mannitol productivity. However,
the study also revealed that this behaviour cannot be generalized to all
heterofermentative LAB and all temperature ranges. With L. mesenteroides
a change from 35 to 30 °C resulted in a small improvement of volumetric mannitol
productivity. Even more divergent to earlier findings is the observation made
with Lb. fermentum, where high temperatures resulted, in addition to
increased productivities, also in better mannitol yields.
3.2.2. Effect of nitrogen gas flushing on growth and mannitol production
The growth of all four strains was considerably more rapid under
semi-anaerobic conditions (i.e. no gassing of the growth media) than under
strict anaerobic conditions (i.e. constant nitrogen flushing of the growth
media). When compared in maximum specific growth rates, Lb. brevis was
found to be least affected (up approximately 6%) by the change of anaerobic to
semi-anaerobic conditions, whereas the maximum specific growth rates of the
other three strains were increased in the range of 15–19%. The volumetric
mannitol productivity of Lb. fermentum increased from 1.33±0.02 to
1.65±0.06 g/l/h, when the cells were grown under semi-anaerobic conditions
rather than under anaerobic conditions. A more subtle increase was obtained with
the other strains, where the volumetric mannitol productivities of Lb. brevis,
L. mesenteroides, and L. pseudomesenteroides were improved with
0.9, 2.9, and 3.7%, respectively. The differences in specific mannitol
productivities observed under anaerobic and semi-anaerobic conditions were very
small (data not shown). On the other hand, the yield of mannitol from fructose
was higher under anaerobic conditions with three of the strains (Lb. brevis,
Lb. fermentum, and L. mesenteroides). The respective behaviour of
L. pseudomesenteroides did, however, deviate from the former pattern.
Under anaerobic conditions the mannitol yield of L. pseudomesenteroides
was 73.8±0.1 mol%, whereas under semi-anaerobic conditions it was up to 77.1±1.7
mol%.
Hence, nitrogen gas flushing of the growth media seems to be ineffective as a
way to assure high volumetric or specific mannitol productivities. In fact,
clear enhancement in volumetric productivities was seen with all four strains,
when grown under semi-anaerobic conditions rather than under strict anaerobic
conditions. The most significant change was again seen with Lb. fermentum,
where an almost 25% increase in volumetric mannitol productivity was obtained
under semi-anaerobic conditions. This observation is naturally at least partly a
direct correlation with the improved growth rate of this strain under
semi-anaerobic conditions compared to the respective rate under anaerobic
conditions. Although the yield of mannitol from fructose was higher in
cultivations with constant nitrogen gas flushing (exception: L.
pseudomesenteroides), it would be more cost-effective to build and run an
industrial-scale process without the need to invest in expensive bioreactor
gassing systems.
Surprisingly, during the semi-anaerobic experiments oxygen depletion in the
growth medium differed notably among the strains. At the beginning of these
experiments the DOT in the growth media was approximately 90±5%. During the
experiments L. mesenteroides was observed to run out of oxygen (DOT=0%)
after 2.0±0.1 h, whereas Lb. brevis, Lb. fermentum, and L.
pseudomesenteroides ran out of oxygen at 5.4±0.5, 6.7±1.0, and 7.0±0.4 h,
respectively. The NADH oxidase activities at t=7 h in the semi-anaerobic
experiments were as follows: Lb. brevis, 0.57 U/mg protein, Lb.
fermentum, 0.20 U/mg, and L. mesenteroides, 0.49 U/mg. No activity
was detected with L. pseudomesenteroides. Hence, the specific activity
measured for L. mesenteroides is similar to earlier reports, 0.6 and 0.44
U/mg [18 and 19]. The lack of NADH oxidase activity in L. pseudomesenteroides
is supported by two related observations. First, the yield of mannitol from
fructose with L. pseudomesenteroides was not improved, when the cells
were grown under anaerobic conditions compared with semi-anaerobic conditions.
If an NADH oxidase activity was present, it would most likely be competing with
mannitol dehydrogenase for the reducing equivalents (NAD(P)H) in the cells and
thus, negatively affect the fructose-to-mannitol yield. Second, the
disappearance of dissolved oxygen from the growth medium was notably slower with
L. pseudomesenteroides (no activity) than with L. mesenteroides
(detectable activity). In the case of L. pseudomesenteroides, the oxygen
dissolved in the growth medium was slowly replaced by carbon dioxide produced by
the cells. Hence, it is speculated that the rapid decrease in dissolved oxygen,
seen with L. mesenteroides, is due both to formation of carbon dioxide
and the presence of a significant NADH oxidase activity.
3.3. Production of mannitol in a 2-l batch cultivation
When L. fermentum was cultured at high initial fructose and glucose
concentrations, efficient bioconversion of fructose into mannitol was achieved
(Fig. 3). In 11 h, 193.6 g fructose was consumed by the cells resulting in
production of 175.3 g mannitol. Hence, the volumetric mannitol productivity and
mannitol yield were 7.6 g/l/h and 89.6 mol%, respectively (final VOLUME=2.11 l).
A maximal volumetric productivity of 16.0 g/l/h was achieved between t=8
h and t=9 h. No residual glucose was detected, when the initial fructose
was depleted, as seen in experiments with low initial sugar concentrations.
Although an increased growth temperature (40 °C) was used, the yield of mannitol
from fructose was not as high as expected based on earlier comparison studies
(see Fig. 1). It is speculated that the high initial sugar concentrations used
in this experiment most likely altered the metabolism of the cells in an
unfavourable direction. In earlier studies with growing LAB cells, the best
volumetric mannitol productivity achieved is 6.4 g/l/h [6]. Hence, we now report
an improvement to this level.
_1207.gif)
Fig. 3. Mannitol production with Lactobacillus fermentum NRRL-B-1932
in a 2-l batch bioreactor cultivation. Legends: open circles, fructose
(g/l); open triangles, glucose (g/l); closed circles, mannitol
(g/l); closed rectangles, optical density at 600 nm.
4. CONCLUSIONS
The ability to produce mannitol from fructose was found to vary notably among
the heterofermentative LAB species studied here. In general, good mannitol
productivity was favoured by high temperature and pH, whereas a good mannitol
yield was favoured by low temperature and pH. In batch cultivations, the
volumetric mannitol productivity is strongly influenced by the growth rate of
the cells. However, strains and species with similar growth rates are still
likely to differ in mannitol production capabilities. Mannitol dehydrogenase (EC
1.1.1.67) is the key enzyme responsible for converting fructose into mannitol.
Typically, among heterofermentative LAB a varying fraction of the fructose that
has been transported into the cell is phosphorylated by a fructokinase enzyme to
form fructose-6-P and thus, further channelled into the phosphoketolase pathway.
The ‘leaking’ carbon skeleton is then converted stepwise into end products such
as acetic and lactic acid, ethanol, and carbon dioxide. The leakage of fructose
to the phosphoketolase pathway is a serious consideration in mannitol
production, mostly because fructose is relatively expensive in comparison to the
final selling price of mannitol.
LAB are generally unable to produce many of the essential building blocks
needed for cellular growth (amino acids, vitamins etc.) and therefore grow
poorly in inexpensive mineral media. Hence, efficient growth is only achieved
when the growth media is supplemented with expensive complex nutrients, such as
protein hydrolysates. Taking the cost of biomass production into account,
volumetric productivities typically achieved in batch and fed-batch cultivations
are simply not sufficient for feasible production of mannitol. A natural way to
improve the competitiveness of the bioprocess alternative would be to study the
application of more advanced production methods, such as resting cells,
cell-recycling, continuous cultures, and cell immobilization. Topics of this
kind are currently under investigation in our laboratory. Recently, using high
cell densities of L. pseudomesenteroides ATCC-12291 immobilized to a
solid carrier, Ojamo et al. achieved a volumetric mannitol productivity of about
20 g/l/h
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