Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR PRODUCING L-SORBOSE
The present invention relates to an improved process for
preparing L-sorbose, which process facilitates especially the
industrial production of this substance, and to specific
nonrecombinant microorganisms which can be used particularly
advantageously in the context of this preparation process.
L-Sorbose is a starting product for preparing 2-keto-L-gulonic
acid, from which L-ascorbic acid is synthesized on an industrial
scale. Preparing ascorbic acid or precursors thereof, for example
L-sorbose, in a microbiological method by fermentation offers the
advantage of preparing the desired product in an enantiomerically
pure form.
However, the fermentative processes used to date for preparing
L-sorbose are associated with a plurality of disadvantages. One
of the most serious defects of processes known to date is that
the starting product, D-sorbitol, was only able to be used at a
relatively low concentration, that is in the range of about 1-15$
by weight. The L-sorbose concentration after completion of the
fermentation was correspondingly low, even in the case of
complete reaction. A second serious disadvantage was the
requirement that the reaction had to be carried out batchwise.
This means that for each production batch a new inoculum must
firstly be prepared in a plurality of stages. This is
particularly labor-intensive and time-consuming and is a serious
disadvantage for the industrial production of L-sorbose. The main
reason for these disadvantages is that the microorganisms
required for the fermentation, especially bacteria of the species
Gluconobacter oxydans, have only an extremely restricted lifetime
under the conditions of a fermentation on an industrial scale,
such as high D-sorbitol concentration, inexpensive non-complex
nutrient medium, pH fluctuations in the range from about 3 to 7,
fluctuations in the oxygen supply in the range from 0.1 to 1.5 1
of 02/1 of medium/h. In addition, deviations of the reaction
conditions from the ideal parameters are responsible for
shortening the lifetime of a bacterial culture, especially in
large fermenters. Frequently a majority of the bacteria used are
no longer viable or capable of.-multiplication at the end of the
fermentation. In order to create the ideal start conditions for
the next reaction batch, i.e. to be able to prepare an inoculum
of high cell density, therefore, the prior art requires culture
of a fresh inoculum.
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EP-A-0 758 679 describes an L-sorbose-producing G. oxydans strain
6624 (deposition number FERM-BP 4415) which can be cultured in
20% strength sorbitol medium. After culturing for 4 days, this
strain has converted D-sorbitol into L-sorbose in high yield.
This strain is furthermore transformed with an expression
construct which bears the DNA encoding the enzymes L-sorbose
dehydrogenase and L-sorbosone dehydrogenase. The resultant
transformant is used for producing 2-keto-L-gulonic acid from
D-sorbitol. However, the culture medium used for this
fermentation has a D-sorbitol concentration of only 5%. Culture
for 5 days is necessary to complete the reaction. Overall it must
thus be stated that the bacteria disclosed by EP-A-0 758 679 have
satisfactory properties neither with respect to D-sorbitol
tolerance nor with respect to L-sorbose synthesis performance.
The usability of the microorganisms described there for the
semicontinuous or continuous conversion of D-sorbitol is likewise
not proposed.
It is an object of the present invention therefore to create the
preconditions for a more efficient fermentative preparation of
L-sorbose.
We have found that this object is achieved especially by
providing a semicontinuous fermentative process for preparing
L-sorbose from D-sorbitol using microorganisms of the species
Gluconobacter oxydans. Furthermore, this object is achieved by
providing optimized bacteria of the species Gluconobacter
oxydans.
The present invention thus firstly relates to highly tolerant -
with respect to D-sorbitol - bacteria of the species
Gluconobacter oxydans which are obtainable by conditioning a
low-tolerance G. oxydans strain by adapting it stepwise to an
increased D-sorbitol concentration in the culture medium by
culturing it at a D-sorbitol concentration which increases from
stage to stage.
A "high-tolerance" G. oxydans strain of the invention, i.e. one
adapted to high D-sorbitol concentrations, is present if this can
be cultured in media which have an initial D-sorbitol
concentration in the range from about 20 to 40% by weight, for
example from about 25 to 38% by weight or from 28 to 35% by
weight. Growth at D-sorbitol concentrations of less than 20% by
weight is obviously equally possible. Preferably, a strain of
this type should also, during growth, ferment the D-sorbitol
~
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present in the medium to L-sorbose at high yield, for example
from 70 to 100%.
For example, the high-tolerance strains of the invention are
obtainable by increasing the D-sorbitol concentration stepwise in
the course of conditioning from initially about 5 to 10% by
weight to a final concentration in the range from about 20 to 40%
by weight, for example from about 25 to 38% by weight or from 28
to 35% by weight. The conditioning can be carried out for example
as follows:
A sterile liquid medium which is suitable for the growth of
G. oxydans and is known per se is prepared, the initial
D-sorbitol content of which is set so that after the inoculation
it has a D-sorbitol content of for example from about 5 to 15% by
weight, for example about 10% by weight. Suitable formulae for
culture media are described for example in "Industrielle
Mikrobiologie" [Industrial Microbiology], Hans-Jurgen Rehm,
Springer Verlag Berlin, Heidelberg, New York, 2nd Edition,
p. 500. For example, use could be made of a 10% strength sorbitol
medium (pH 5) to which 0.5% by weight of yeast extract with or
without 0.1% by weight of glucose have been added. This medium is
inoculated with a freshly prepared inoculum of a non-conditioned
G: oxydans strain. The inoculated medium is cultured at about
Z5 from 30 to 37~C under aerobic conditions and for example with
gentle shaking or stirring. The fermentation is continued until
no further significant L-sorbose formation can be observed. An
aliquot is taken from the fermentation broth and used to
inoculate a further sterile culture medium, which is prepared in
the interim, of the second culture stage, the D-sorbitol content
of which (after the reinoculation) is higher than the content in
the medium of the preceding culture stage. The ratio of inoculum
to culture to be inoculated should be roughly in the range from
1:5 to 1:20, for example from 1:5 to 1:10. The above procedure is
repeated, increasing the sorbitol concentration stepwise until a
bacterial culture is obtained which grows in the medium having
the desired high initial D-sorbitol concentration. The stepwise
increase of the D-sorbitol concentration can be selected as a
function of the response of the starting strain to the
concentration increase. In addition, the increase can be made in
steps of the same or different-~ izes. For example, the D-sorbitol
concentration can be increased stepwise in identical steps from
0.5% to 3%, for example 1% or 2% steps.
According to a further embodiment, bacteria of the species
G. oxydans are prepared which, in addition to an increased
D-sorbitol tolerance, also have an essentially constant, i.e.
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stable, high L-sorbose synthesis performance. Bacteria having a
profile of properties of this type are obtainable by culturing a
high sorbitol tolerance bacterium, as described above, repeatedly
at elevated sorbitol concentration, for example in a roughly from
25 to 35% strength by weight D-sorbitol medium, until L-sorbose
formation is complete.
A satisfactorily "high" L-sorbose synthesis performance of the
invention is achieved when from about 90 to 100 mol%, for example
about from 93 to 98 mol%, of the sorbitol used is reacted after
no more than about 48 hours, in particular after no more than
about 36 hours, for example after from about 15 to 25 hours or
from 18 to 22 hours. Since the synthesis performance can be
influenced by the culture conditions, the above criteria apply in
particular under the following standard conditions: initial
D-sorbitol content: 28-30% by weight; pH of the medium: 4.5-5.0;
aeration 0.5-1.5 1 of air/1 of medium/h; volumetric ratio of
inoculum to charged culture medium about from I:5 to 1:6;
standard medium: comprising per kg of medium
Corn steep liquor, 50% dry matter 4.354 g
Ammonium sulfate 0.495 g
Diammonium phosphate 0.124 g
Magnesium sulfate heptahydrate 0.082 g
Calcium carbonate 0.472 g
Antifoamer 0.150 g
A satisfactorily "constant" or "stable" L-sorbose synthesis
performance of the invention is given if culturing can be
repeated by serial inoculation of an aliquot of the culture broth
into freshly prepared culture medium, preferably at a volumetric
ratio of inoculum to charged culture medium of about from 1:5 to
1:6, more precisely without significant decrease of the L-sorbose
synthesis performance of the production strain used. The
synthesis performance may fluctuate within the abovedefined
ranges for conversion rate and reaction period. A constant
synthesis performance should still be observable after
reinoculation for from 1 to 5 times or more frequently, for
example from 10 to 50 times or more frequently.
The conditioning methods of the invention can in principle be
carried out with all non-conditioned G. oxydans strains. However,
preferably conditioning is performed using Gluconobacter oxydans
NRRL-B72. This strain is freely available from the deposition
site (Northern Regional Research Laboratory, USA).
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The present invention relates especially to G. oxydans bacteria
conditioned in the above manner which are capable of converting
D-sorbitol to L-sorbose under aerobic conditions and where the
bacterium furthermore occurs in the form of an individual, pairs,
5 or short chains of non-motile, Gram-negative rods, exhibits good
growth in semisolid sorbitol-glucose-yeast extract-agar medium
and in liquid sorbitol-corn steep liquor medium at a pH of about
3.5-6, a temperature of about 25-40~C and a D-sorbitol
concentration of up to about 40% by weight, for example at from
over 20 to 40% by weight, in particular at from about 25 to 35%
by weight.
A particularly preferred G. oxydans strain obtained in accordance
with the abovedescribed conditioning process starting from
I5 NRRL-B72 and recorded under the internal name "2B3" has the
following properties:
1. Morphological properties of 2B3:
- Gram stain: negative
- cell shape: rods
- cell size: 1-2 ~tcn
- occurrence of the cells: individually, in pairs or in short
chains
- motility: negative
- spore formation: negative
- colonies:
a) on meat extract-peptone agar (pH 7.0): after incubation
for 2 days thin poorly visible indistinct colonies
occasionally appear;
b) after incubation for 2 days on sorbitol-glucose-yeast
extract agar (pH 5), domed, round colonies of the shape
and size of a pinhead appear. The colonies are whitish
yellow and glossy.
2. Physiological properties:
- growth temperature: T = 25-40~C, optimum at 32~C
- pH range: growth at pH 3.5-6, optimum at pH 4.5-5.0
- aerobic
- catalase: positive
- oxidase: negative
- nitrate reduction: negative
- gelatin liquefaction: negative
- HZS formation: negative
- indole production: negative
- oxidation of ethanol to acetic acid: positive
- starch: not hydrolyzed
- glycerol, D-mannitol, sorbitol: utilized
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- lactate oxidation: negative
- oxidation of acetate to C02 and HZO: negative
The strain produced thus matches the essential characteristics of
G. oxydans according to the information in Bergey's Manual of
Systematic Bacteriology (9th Edition), p. 275 ff. Reference may
be made to this standard work of identification with respect to
other non=limiting characteristics.
In addition to the above specifically described bacteria of the
species G. oxydans, the invention also relates to mutants and
variants thereof which have essentially the same properties and
are suitable for carrying out the inventive process for preparing
L-sorbose, which process is described in more detail in the
sections hereinafter.
Examples of suitable methods for preparing mutants and variants
of the specifically described inventive microorganisms include,
without being restricted thereto: mutagenesis by irradiation with
ultraviolet light or X-rays; treatment with a chemical mutagen
such as nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine),
methyl methanesulfonate, nitrogen mustard and the like; gene
integration methods, and transduction using bacteriophages. These
methods are known from the prior art and are described for
example in: J.H. Miller, Experiments in Molecular Genetics, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1972); J.H. Miller, A Short Course in Bacterial Genetics, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1991); M. Singer and P. Berg, Genes & Genomes, University
Science Books, Mill Valley, California (1991); J. Sambrook, E.F.
Fritsch and T. Maniatis, Molecular Cloning; A Laboratory Manual,
2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York (1989); P.B. Kaufman et al., Handbook of
Molecular and Cellular Methods in Biology and Medicine, CRC
Press, Boca Raton, Florida (1995); Methods in Plant Molecular
Biology and Biotechnology, B.R. Glick and J.E. Thompson, Eds.,
CRC Press, Boca Raton, Florida (1993); and P.F. Smith-Keary,
Molecular Genetics of Escherichia coli, The Guilford Press, New
York, NY (1989).
The present invention also relates to a process for the
semicontinuous fermentative preparation of L-sorbose, which
comprises
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a) inoculating with a bacterium of the species Gluconobacter
oxydans a first culture medium which contains D-sorbitol and
culturing it until the D-sorbitol conversion is essentially
complete;
b) after completion of the conversion, isolating the L-sorbose
formed from the majority of the resultant culture broth;
c) with the remaining amount of the culture broth, inoculating a
second D-sorbitol-containing culture medium in a weight ratio
of from 1:4 to 1:8 and culturing it until the D-sorbitol
conversion is essentially complete; and
d) after completion of the conversion, isolating from the
culture broth the L-sorbose formed;
the initial D-sorbitol concentration after inoculation of the
culture media being in each case up to about 40% by weight. The
inoculum has for example a cell density corresponding to from 1
to 10 g of biomass (dry matter) per liter of inoculum.
According to a preferred embodiment of the inventive process, the
fermentation cycle including the steps a) to c) is repeated as
often as desired until a sufficient amount of L-sorbose has been
formed. For the purposes of the invention a "desired" repetition
of fermentation cycles is for example from 1 to 5 times or more
frequently such as for example from 10 to 50 times or from 50 to
200 times repetition of the fermentation cycle.
It can be seen herefrom that the inventive process offers the
particular advantage in comparison with the prior art of not
requiring new inoculum to inoculate the production culture
medium. Rather, the inoculum required can be branched off
directly from the culture broth of the preceding fermentation
cycle, preferably after completion of the D-sorbitol conversion.
Surprisingly, according to the invention it was also found that
numerous fermentation cycles can be passed through although the
initial D-sorbitol concentration is unusually high, for example
in the range 25-35% by weight. In addition it is surprising that
each of the fermentation cycles gives L-sorbose in a virtually
quantitative yield, i.e. in the range of about 95-98 mol% within
a reaction time per cycle of about 15-25 hours, in particular
about 20 hours. The ratio of inoculum to culture medium chosen
according to the invention ensures that the production batch
virtually does not pass through a lag phase, but rather contains
a bacterial culture of high vitality.
The fermentation temperature is preferably about 25-40~C, in
particular about 32-36~C. Particular preference is given to a
fermentation temperature of about 35~C which is between the growth
~
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optimum of the bacterium (32°Cy and the temperature optimum of the
enzymatically catalyzed partial oxidation of D-sorbitol to
L-sorbose (36°C). At temperatures below 25°C and above
40°C, no
significant bacterial multiplication is observable. Whereas if
the temperature falls below 25°C, on subsequent heating, growth
and productivity increase back to normal values, increasing the
temperature to over 40°C leads to permanent damage of bacterial
growth and productivity.
In addition, according to the invention preference is given to
carrying out the fermentation with supply of about 0.5-1.5 liters
of atmospheric air per liter of medium and per minute. The
bacterial growth and the fermentative conversion of D-sorbitol
can be further improved in a manner known per se by enriching the
air with oxygen, for example by up to about 20% by volume. The
oxygen transfer is also controllable in a manner known per se by
varying the pressure. In stirred fermenters, the oxygen transfer
is influenced in particular by the stirring power of the agitator
used. Stirring powers of about from 0.5 to 4 kW/m3 of medium are
preferred. An example of a stirrer type customarily used is the
paddle stirrer.
As mentioned above, the bacteria preferred according to the
invention have a growth optimum at pH 4.5-5Ø At a pH from above
6 to about 7.5, growth and productivity are reversibly inhibited,
while the pH in the course of the fermentation can also briefly
assume values of 3.5 or below, for example to about 3.0, without
adversely affecting sorbitol oxidation in the running batch or
growth and productivity in the next fermentation cycle.
In addition, preferably, care must be taken to ensure that the
sorbitol substrate does not have an excessive content of
D-glucose impurities, since in the course of the fermentation
this causes an unusual fall in pH owing to the oxidation of
D-glucose to gluconic acid.
The generation time of the microorganisms preferably used
according to the invention for the fermentation increases with
increasing sorbitol concentration in the medium. Thus the
generation time is for example 3 times longer in a 27% strength
medium than in a 10% strength medium under otherwise identical
fermentation parameters. Above a concentration range of about
30-32o by weight of sorbitol, the generation time increases
sharply. The growth stops at a concentration of about 40% by
weight. In a 27% strength sorbitol medium the bacteria reach
optimum bacterial density within about from 12 to 18 hours after
inoculation at a ratio of 1:5.6 (inoculum to culture medium). The
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oxidation of D-sorbitol to L-sorbose does not start until after
the culture has reached a certain cell density, for example about
from 30 to 50% of the final density.
Preferably, the D-sorbitol substrate used should have no
D-mannitol impurity if L-sorbose is to be isolated in crystalline
form. This is because D-mannitol is converted enzymatically to
D-fructose. The latter in small amounts inhibits the
crystallization process.
A further advantage of the invention is that the inventive
preparation process can be carried out using a relatively simple
inexpensive nutrient medium or culture medium. Preferably, this
comprises per kg of liquid medium:
a) about 100-300 g of D-sorbitol
b) about 4-5 g of corn steep liquor (50% dry matter)
c) about 0.4-0.6 g of (NH4)ZS04
d) about 0.1-0.15 g of (NH4)2HP04
e) about 0.06-0.1 g of MgS04.7H20
f) about 0.5-0.9 g of CaC03 with or without
g) an active amount of antifoamer.
Before the inoculation the culture medium is sterilized in a
manner known per se. For this purpose, before the sterilization
the pH of the medium is adjusted to 5.5, for example using about
70-80% strength acetic acid. During the fermentation the pH is
controlled only by the added calcium carbonate. Additional
control, for example by addition of sodium hydroxide solution to
the running fermentation process, is not necessary. During the
initial growth phase, the pH of the culture is about 5.5-6 and
during the log phase (logarithmic multiplication of the bacteria)
of growth the pH decreases to a range of about 4.0-4.5.
In supplementation to the abovementioned essential components
which are completely sufficient in themselves for a fermentation,
if necessary other components can be added to the nutrient medium
used according to the invention.
Thus, for example, one or more other hydrocarbon sources may be
present, for example starch hydrolysates, cellulose hydrolysates
or molasses; and alcohols, such as glycerol.
Furthermore, one or more nitrogen sources may additionally be
present, for example ammonia, ammonium salts of inorganic or
organic acids, such as ammonium chloride, ammonium nitrate,
ammonium phosphate, ammonium sulfate and ammonium acetate; urea;
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nitrate and nitrite salts and other nitrogen-containing materials
such as amino acids, meat extract, peptone, fish meal, fish
hydrolysates, casein hydrolysates, soybean hydrolysates, yeast
extract, dried yeast, ethanol yeast distillate, soybean flour,
5 cottonseed meal and the like.
Furthermore, inorganic salts may be additionally be present, for
example salts of potassium, calcium, sodium, magnesium,
manganese, iron, cobalt, zinc, copper and other trace elements
10 and also phosphoric acid.
Suitable trace elements and growth factors may likewise be added
individually or in combination, for example coenzyme A,
pantothenic acid, biotin, thiamine, riboflavin, flavin
mononucleotide, flavin adenine dinucleotide and other vitamins,
amino acids, such as cysteine, sodium thiosulfate, p-aminobenzoic
acid, niacinamide and the like. These may be used in pure form or
in the form of natural materials which contain these substances.
The preferred fermentation technique used in each case primarily
depends on the size of the batch. Whereas for smaller batches an
aerobic shaken culture is suitable in principle, to carry out the
process of the invention on an industrial scale fermentation
under submerged aerobic conditions is preferred.
The conversion of D-sorbitol is followed in a conventional
manner, for example by HPLC analysis of a withdrawn sample. To
determine the L-sorbose content a suitable method is, for
example, an HPLC column type OA KC 7.8 x 300 mm, from Merck,
mobile phase 0.05 N sulfuric acid, 0.4 ml/min flow rate.
The L-sorbose formed is isolated continuously or batchwise using
conventional methods. The resultant culture broth, if appropriate
after removing the cell mass, for example by filtration or
centrifugation, is first concentrated in a conventional manner.
This is for example by evaporation at elevated temperature, for
example from 50 to 80~C, and reduced pressure, for example from
0.01 to 0.1 bar. The crystals precipitating out are filtered and
if necessary recrystallized. Other purification steps, for
example solvent extraction, chromatography, precipitation or
salting out, can if required be-employed individually or in
combination.
A particularly preferred embodiment of the process of the
invention is described in the following experimental part.
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Example 1: Preparation of a variety of nutrient media:
a) Corn steep liquor-salt mixture
corn steep liquor, 50% dry matter 240 g (about 200 ml)
ammonium sulfate 22.2 g
diammonium phosphate 6.6 g
magnesium sulfate heptahydrate 3.6 g
calcium carbonate 48.6 g
To prepare the medium all ingredients apart from calcium
carbonate are mixed together and the pH is adjusted to about
6 using 25% strength sodium hydroxide solution. Calcium
carbonate is then added. The pH is measured again and if
required adjusted to 6.5.
b) Sorbitol-corn steep liquor-salt agar
The laboratory cultures of Gluconobacter oxydans are
maintained on this solid medium in agar slope tubes. The
inoculum cultures for the first inoculation of the fermenter
are produced on this solid medium in Roux flasks immediately
prior to inoculation.
sorbitol syrup, 70% by weight dry matter ?5.Og (about 5%
sorbitol)
corn steep liquor-salt mixture 7.5g
agar powder 25.Og
demineralized water 1000 ml
Sorbitol syrup, corn steep liquor-salt mixture and water are
mixed together, heated to 60~C and ffiltered. The agar powder
is then slowly added to the boiling filtrate. The hot medium
is then distributed between the culture vessels (9 ml per
30 ml agar slope tube and 100 ml per 650 ml Roux flask). The
vessels are then sealed and autoclaved at 121~C for
20 minutes.
c) Sorbitol-glucose-yeast extract agar (pH 5)
This medium is used to study the purity of the culture during
a fermentation. The medium is suitable for growth of
G. oxydans and is unsuitable for most of the contaminating
microorganisms which usually occur. G. oxydans forms
characteristic domed round whitish-yellow glossy cultures on
the medium after incubation at 32~C for 1 to 2 days.
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sorbitol syrup, 70% by weight dry matter 75.0 g (about 5%
sorbitol)
glucose 1.0 g
yeast extract 5.0 g
agar powder 15.0 g
demineralized water 1000 ml
Sorbitol syrup, yeast extract and glucose are mixed together
25
35
and then water is added. The p8 is adjusted to 5.3-5.4 with
10 12% strength by weight acetic acid. The agar powder is added
slowly to the boiling mixture. The medium is distributed to
screw-top glass bottles while it is still hot and autoclaved
at 121~C for 20 minutes. After cooling to about 50~C the
medium is poured onto sterile plastic Petri dishes.
d) Meat extract-peptone agar (pH 7)
This medium is used for studying the purity of the culture
during a fermentation. It is a nutrient medium for most of
20 the contaminating microorganisms which usually occur. It is
unsuitable for the growth of G. oxydans. After incubation at
32~C for 2 days G. oxydans at most forms poorly visible
indistinct colonies on this medium. Usually no growth is
observed.
meat extract 3.0 g
peptone 5.0 g
agar powder 15.0 g
demineralized water 1000 ml
The medium is prepared similarly to the above medium c),
except that the pH is adjusted to 7.
e) Production medium
Formula for 1000 kg of a 28% strength sorbitol medium
(density 1097 kg/m3):
corn steep liquor, 50% dry matter 4.354 kg
ammonium sulfate 0.495 kg
diammonium phosphate 0.124 kg
magnesium sulfate heptahydrate 0.082 kg
calcium carbonate 0.472 kg
antifoamer 0.150 kg
Sorbitol syrup (70% dry matter; equivalent to 400 of
sorbitol) is diluted with tap water and the corn steep liquor
is added. The mineral salts, predissolved or suspended in tap
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water, are then added. The pH is then adjusted to 5.5 using
70-80% strength acetic acid. Finally the antifoamer is added.
The medium is sterilized in a manner known per se at 141°C
for 2 minutes.
Example 2: Production of an inoculum culture of G. oxydans
The precultures of G. oxydans are maintained at 5°C in the form of
agar cultures (medium b). Using a sterile inoculation loop, from
one agar culture 5 new agar cultures are produced by incubation
at 32°C for 2 days. The bacteria of 8 such agar cultures are used
to inoculate the same medium in Roux flasks. For this purpose
bacteria of an agar culture are suspended in 2 x 5 ml of sterile
0.9% strength sodium chloride solution. The suspension is
transferred to a Roux flask. In total B inoculated flasks are
incubated at 32°C for 2 days and if necessary kept at 5°C. The
bacteria which have grown in two Roux flasks are suspended in 2 x
50 ml of sterile 0.9% strength sodium chloride solution and
transferred to a sterile glass vessel. This culture serves as
ZO inoculum for the sorbitol fermentation in a 50 m3 fermenter. The
cell density of the inoculum is about 109 bacteria per ml.
Example 3: Fermentation of D-sorbitol
a} First fermentation step
The 50 m3 fermenter is filled with 15,000 kg of production
medium (cf. Example 1 medium e)), in which, however, the
sorbitol concentration is only 15% by weight and the content
of corn steep liquor and mineral salts has been doubled. The
medium is set to 35°C. A 200 ml inoculum culture produced in
accordance with Example 2 is transferred to the fermenter
under sterile conditions. The fermenter is aerated with
1600-1800 m3 of sterile air per hour. The pressure in the
fermenter is about 1.3 atm. The initial pH is about 5.5-6Ø
The fermentation period in this first step is about from 30
to 40 hours. At the end of the fermentation the pH has
dropped to about 4.0-4.5. The resulting culture broth is
either worked up to isolate the Z,-sorbose formed or used as
inoculum for the second fermentation stage.
b) Second fermentation step
A second 50 m3 fermenter is filled with 33,000 kg of sterile
production medium (28% by weight of sorbitol) and inoculated
with 6400 liters of inoculum (corresponding to 7000 kg of
culture from the first fermentation step). The ratio of
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14
inoculum to production medium is 1:4.7. Fermentation is then
carried out over a period of about from 18 to 22 hours until
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the sorbitol concentration has decreased to below 0.1% by
weight. The L-sorbose yield is about 8.3 kg/m3 of fermenter
5 volume per hour. A further inoculum of 6400 liters is
branched off for the next fermentation cycle from the
resulting fermentation broth. The remaining amount of
fermentation broth is worked up for the isolation of
L-sorbose.
Example 4: Isolation of L-sorbose
A fermentation broth present after a process according to Example
3 comprises about from 25 to 26% by weight of L-sorbose, less
15 than about 0..1% by weight of D-sorbitol, residues of the growth
medium (dissolved or suspended) and from 1 to 2 g of bacterial
mass per liter. The fermentation broth is held at about 50°C to
prevent further bacterial growth and loss of sorbose.
20 The fermentation broth is first concentrated in a conventional
falling-film evaporator in a first concentration step to a
sorbose content of about 42% by weight at from 60 to 80~C and a
pressure of 0.02 bar. In a second step, it is further
concentrated in a conventional long-tube evaporator at 57~C and a
25 pressure of 0.02 bar. By means of this second evaporation step
supersaturation of the concentrated L-sorbose solution is
achieved. The crystal content is from about 30 to 50% by volume.
The crude crystal suspension obtained in this manner is then
transferred into a conventional sedimentation vessel. In the
30 bottom region thereof, after some time a very dense crystal
suspension forms which is essentially free from bacterial mass.
This crystal suspension is separated off from the supernatant,
centrifuged, washed with water and dried at about 90~C to a
residual moisture content below 0.1% by weight.
The resultant mother liquor, the wash liquid and the supernatant
from the sedimentation vessel can then, as described above, be
worked up further, if appropriate after combination with the
fermentation broth from another batch.
The overall yield based on the--D-sorbitol used is usually in the
range from about 89 to 92 mol% after complete workup. The product
prepared in this manner serves as starting product for preparing
L-ascorbic acid.
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Example 5: Conditioning of Gluconobacter oxydans
a) In a similar manner to the above medium c) (cf. Example 1), a
sterile agar-free liquid medium (850 ml total volume in a 2 1
5 culture vessel) which has a D-sorbitol content of about 10%
by weight is prepared. This medium is inoculated with 150 ml
of a freshly prepared inoculum culture of a non-conditioned
G. oxydans strain.
10 The inoculated medium (pH 5) is incubated at 32~C under
aerobic conditions (atmospheric air) with vigorous shaking
(80 rpm). The fermentation is continued until significant
L-sorbose formation can no longer be observed. A volume of
150 ml is taken off from the fermentation broth and used to
15 inoculate a further sterile culture medium which has been
prepared in the interim and has a D-sorbitol content of 11%
by weight. The above procedure is repeated with stepwise (1%
steps) increase in sorbitol concentration until a bacterial
culture is obtained which grows in about 30% strength by
weight D-sorbitol medium.
b) The G. oxydans culture thus obtained which is adapted to high
sorbitol concentrations can then, for further optimization of
the L-sorbose synthesis performance, be serially inoculated
into 30% strength by weight D-sorbitol medium (150 ml of
inoculum per 850 ml of medium). Optimum synthesis performance
is achieved when about from 90 to 98 mol% of the sorbitol
used is converted after culturing for about 24 hours.
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