Method for producing 2-keto-L-gulonic acid

2-keto-L-gulonic acid is in high yield produced by contacting a microorganism of the genus Pseudogluconobacter, either as it is or after processing, with L-sorbose.

The present invention relates to a method for producing 2-keto-L-gulonic 
acid which is of value as an intermediate for the synthesis of L-ascorbic 
acid and the strains of the genus Pseudogluconobacter to be used in the 
production method. 
2-Keto-L-gulonic acid which is a valuable intermediate for the synthesis of 
L-ascorbic acid has heretofore been produced by the commercially 
established method of Reichstein [Helvetica Chimica Acta 17, 311 (1934)]. 
However, this method involves many steps, requires large quantities of 
solvents, and is therefore not satisfactory as a modern technology. As 
alternatives of this Reichstein's method, several methods employing 
microorganisms, in the main, have been proposed. For example, one may 
refer to the method which comprises oxidizing glucose to 5-keto-D-gluconic 
acid with the aid of a microorganism, reducing it either chemically or 
microbiologically to L-idonic acid, and oxidizing the same further 
microbiologically to 2-keto-L-gulonic acid [U.S. Pat. No. 2,421,611]. 
Another method that is known comprises oxidizing glucose to 
2,5-diketo-D-gluconic acid with the aid of a microorganism and converting 
the same to 2-keto-L-gulonic acid microbiologically or chemically 
[Japanese Patent Publication No. 39-14493, No. 53-25033, No. 56-15877 and 
No. 59-35920]. 
However, the chemical reduction steps in these known methods, namely the 
reduction of 5-keto-D-gluconic acid to L-idonic acid in the former method 
and the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gluonic acid 
in the latter method, have disadvantages in respect to stereo-specificity, 
so that the by-production of D-gluconic acid in the former and 
2-keto-D-gluconic acid in the latter results in decreased yields of the 
desired compound. Furthermore, even when the above reduction is carried 
out with the aid of a microorganism, the overall product yield drops 
because the microorganism must be supplied with an excess of glucose (the 
starting material) as a reduction energy source. In this respect, the 
production of 2-keto-L-gulonic acid using L-sorbose as a starting material 
involves only an oxidation process. In fact, attempts using the bacterium 
belonging to the genus Gluconobacter, the genus Pseuckmonas, the genus 
Serratia, the genus Achromobacter and the genus Alcaligenes, have already 
been made in this direction. Thus, one may refer to the literatures 
including Biotechnology and Bioengineering 14, 799 (1972), Acta 
Microbiologica Sinica, 20, 246 (1980), and 21, 185 (1981 ), Japanese 
Patent Publication No. 41-159 and No. 41-160, U.S. Pat. No. 3,043,749 and 
Japanese Patent Publication No. 49-39838. 
However, the results achieved with the strains so far named in the 
literature are not satisfactory, indeed, and the yields are too low to 
warrant a commercial exploitation of them. 
Under the circumstances the present inventors sought earnestly for a 
commercially profitable method for producing 2-keto-L-gulonic acid and 
discovered that a bacterial strain K591s which was isolated from a soil 
sample collected in Wakayama Prefecture and bacterial strains 12-5, 12-15, 
12-4 and 22-3 which were isolated from soil samples collected in Shiga 
Prefecture are able to convert L-sorbose to 2-keto-L-gulonic acid in 
yields by far exceeding the earlier results. Moreover, as the result of a 
taxonomical investigation, the present inventors found that these are new 
bacteria which have not been described in the literature. The present 
invention has been developed on the basis of the above findings. 
Thus, the present invention is concerned with (1) a method for producing 
2-keto-L-gulonic acid which comprises contacting a microorganism of the 
genus Pseudogluconobacter which is able to oxidize L-sorbose to 
2-keto-L-gulonic acid, either as it is or after processing, with L-sorbose 
to produce and accumulate 2-keto-L-gulonic acid and harvesting the same; 
(2) a method for producing 2-keto-L-gulonic acid which comprises 
contacting a microorganism of the genus Pseudocluconobacter which is able 
to oxidize L-sorbose to 2-keto-L-gulconic acid with L-sorbose in the 
presence of at least one of the microorganisms belonging to the genus 
Bacillus, the genus Pseudomonas, the genus Proteus, the genus Citrobacter, 
the genus Enterobacter, the genus Erwinia, the genus Xanthomonas, the 
genus Flavobacterium, the genus Micrococcus, or the genus Escherichia; and 
(3) the Pseudogluconobacter saccharoketogenes which is aerobic and grows 
in the presence of coenzyme A. 
Of the above-mentioned 5 bacterial strains, the strains K591s and 12-5 have 
the following taxonomical characteristics. 
(a) Morphology 
(1) Rods, which measure 0.3 to 0.5.times.0.7 to 1.4 .mu.m. 
(2) No cellular polymorphism 
(3) Motile with 2 to 4 polar flagella 
(4) Non-sporulating 
(5) Gram-negative 
(6) Non-acid-fast 
(b) Cultural characteristics 
(1) Nutrient agar plate: Substantially no growth. Yeast extract nutrient 
agar: Round, entire margin, smooth surface, opalescent. 
(2) Yeast extract nutrient agar slant: Growth moderate and filiform, 
smooth, opalescent. 
(3) Yeast extract nutrient liquid culture: Moderate growth, uniform 
turbidity throughout medium. 
(4) Nutrient gelatin stab: Sparse surface growth; gelatin not liquefied. 
(5) Litmus milk: Acidified and coagulated. 
(c) Physiological characteristics 
(1) Nitrate reduction: weak but positive 
(2 ) Denitrification: negative 
(3) Methyl red (MR) test: positive 
(4) Voges-Proskauer (VP) test: negative 
(5) Indole: not produced 
(6) Hydrogen sulfide: not produced 
(7) Starch: not hydrolized 
(8) Citric acid: not utilized 
(9) Ammonium salts: utilized 
(10) Pigments: not produced 
(11) Urease: produced 
(12) Oxidase: positive 
(13) Catalase: positive 
(14) The temperature range for growth: 16.degree.-36.degree. C.; the 
optimum temperature range for growth: 24.degree.-34.degree. C. The pH 
range for growth: 5.5-8.7; the optimum pH range: 6.0-7.5. 
(15) Aerobic 
(16) Hugh-Leifson's OF test: oxidative 
(17) Acid is produced but gas is not produced from L-arabinose, D-xylose, 
D-glucose, D-fructose, D-galactose, D-mannose, maltose, sucrose, lactose, 
trehalose, D-mannitol, and glycerol. Neither acid nor gas is produced from 
D-sorbitol, inositol or starch 
(d) Other characteristics 
(1) Weak production of acetic acid from ethanol 
(2) Biotin, thiamine, riboflavine and coenzyme A (CoA) are required for 
growth. 
(3) Production of dihydroxyacetone from glycerol 
(4) The guanine+cytosine content of DNA: 67.+-.1 mole % 
(5) The presence of a ubiquinone containing 10 isoprene units (CoQ.sub.10) 
(6) Marked production of 2-keto-L-gulonic acid from L-sorbose 
(7) Streptomycin-resistant 
The taxonomical characteristics of the 12-15 strain are described below. 
(a) Morphology 
(1) Rod-shaped; cells measuring 0.3 to 0.5.times.0.7 to 1.4 .mu.m 
(2) No cellular polymorphism 
(3) Motile with 2 to 4 polar flagella 
(4) Non-sporulating 
(5) Gram-negative 
(6) Non-acid-fast 
(b) Cultural characteristics 
(1) Nutrient agar plate: Substantially no growth. Yeast extract nutrient 
agar plate: Round, entire margin, smooth and opalescent. 
(2) Yeast extract nutrient agar slant: Growth moderate and filiform, smooth 
and opalescent. 
(3) Yeast extract nutrient liquid culture: Moderate growth, uniform 
turbidity throughout medium. 
(4) Nutrient gelatin stab: Sparse growth at top only. Gelatin not 
liquefied. 
(5) Litmus milk: Acidified but not coagulated. 
(c) Physiological characteristics 
(1) Nitrate reduction: negative 
(2) Denitrification: negative 
(3) Methyl red (MR) test: positive 
(4) Voges-Proskauer (VP) test: negative 
(5) Indole: not produced 
(6) Hydrogen sulfide: not produced 
(7) Starch: not hydrolyzed 
(8) Citrate: not utilized 
(9) Ammonium salts: utilized 
(10) No pigment production 
(11) Urease: produced 
(12) Oxidase: positive 
(13) Catalase: positive 
(14) Growth occurs at 23.degree.-32.degree. C., optimally at 
28.degree.-32.degree. C. The pH range for growth: pH 6.0-7.5; the optimum 
pH range: 6.5-7.1. 
(15) Aerobic 
(16) Hugh-Leifson's of test: oxidative 
(17) Acid is produced but gas is not produced from L-arabinose, D-xylose, 
D-glucose, D-fructose, D-mannose, maltose, sucrose, lactose, trehalose, 
and glycerol. Neither acid nor gas is produced from D-mannitol, 
D-sorbitol, inositol and starch. 
(d) Other characteristics 
(1) Weak production of acetic acid from ethanol 
(2) Biotin, thiamine, riboflavine and CoA are required for growth 
(3) Production of dihydroxyacetone from glycerol 
(4) The guanine+cytosine content of DNA: 67.+-.1 mole % 
(5) The presence of a ubiquinone containing 10 isoprene units (CoQ.sub.10) 
(6) Marked production of 2-keto-L-gluonic acid from L-sorbose 
(7) Streptomycin-resistant 
The taxonomical characteristics of the 12-4 strain are described below. 
(a) Morphology 
(1) Rods, each cell measuring 0.3 to 0.5.times.0.7 to 1.4 .mu.m 
(2) No cellular polymorphism 
(3) Motile with 2 to 4 polar flagella 
(4) Non-sporulating 
(5) Gram-negative 
(6) Non-acid-fast 
(b) Cultural characteristics 
(1) Nutrient agar plate: Minute colonies do not permit detailed 
observation. Yeast extract nutrient agar: Round, entire margin, smooth, 
opalescent. 
(2) Yeast extract nutrient agar slant: Growth moderate and filiform, 
smooth, opalescent. 
(3) Yeast extract nutrient liquid culture: Moderate growth; uniform 
turbidity throughout medium. 
(4) Nutrient gelatin stab: Weak growth at top only. Gelatin not liquefied. 
(5) Litmus milk: Acidified but not coagulated. 
(c) Physiological characteristics 
(1) Nitrate reduction: negative 
(2) Denitrification: negative 
(3) Methyl red (MR) test: positive 
(4) Voges-Proskauer (VP) test: negative 
(5) Indole: not produced 
(6) Hydrogen sulfide: produced 
(7) Starch: not hydrolyzed 
(8) Citrate: not utilized 
(9) Ammonium salts: utilized 
(10) No pigment production 
(11) Urease: produced 
(12) Oxidase: positive 
(13) Catalase: positive 
(14) Growth occurs at 16.degree.-36.degree. C., optimally at 
24.degree.-34.degree. C. The pH range for growth: 5.5-8.2; the optimum pH 
range: 6.0-7.5. 
(15) Aerobic 
(16) Hugh-Leifson's of test: oxidative 
(17) Acid is produced but gas is not produced from L-arabinose, D-xylose, 
D-glucose, D-fructose, D-galactose, D-mannose, maltose, sucrose, lactose, 
trehalose, and glycerol. Neither acid nor gas is produced from D-mannitol, 
D-sorbitol, inositol and starch. 
(d) Other characteristics 
(1) Weak production of acetic acid from ethanol 
(2) Biotin, thiamine, riboflavine and either CoA or pantothenic acid are 
required for growth. 
(3) Production of dihydroxyacetone from glycerol 
(4) The guanine+cytosine content of DNA: 67.+-.1 mole % 
(5) The presence of a ubiquinone containing 10 isoprene units (CoQ.sub.10) 
(6) Marked production of 2-keto-L-gulonic acid from L-sorbose 
(7) Streptomycin-resistant 
The taxonomical characteristics of the 22-3 strain are described below. 
(a) Morphology 
(1) Rods, each cell measuring 0.3 to 0.5.times.0.7 to 1.4 .mu.m. 
(2) No cellular polymorphism 
(3) Motile with 2 to 4 polar flagella 
(4) Non-sporulating 
(5) Gram-negative 
(6) Non-acid-fast 
(b) Cultural characteristics 
(1) Nutrient agar plate: Minute colonies do not permit detailed 
observation. Yeast extract nutrient agar: Round, entire margin, smooth, 
opalescent. 
(2) Yeast extract nutrient agar slant: Growth moderate and filiform, 
smooth, opalescent. 
(3) Yeast extract nutrient liquid culture: Moderate growth; uniform 
turbidity throughout medium. 
(4) Nutrient gelatin stab: Weak growth at top only. Gelatin not liquefied. 
(5) Litmus milk: Acidified but not coagulated. 
(c) Physiological characteristics 
(1) Nitrate reduction: positive (weak) 
(2) Denitrification: negative 
(3) Methyl red (MR) test: positive 
(4) Voges-Proskauer (VP) test: negative 
(5) Indole: not produced 
(6) Hydrogen sulfide: not produced 
(7) Starch: not hydrolyzed 
(8) Citrate: not utilized 
(9) Ammonium salts: utilized 
(10) No pigment production 
(11) Urease: produced 
(12) Oxidase: positive 
(13) Catalase: positive 
(14) Growth occurs at 16.degree.-38.degree. C. optimally at 
24.degree.-34.degree. C. The pH range for growth: 5.5-8.7; the optimum pH 
range: 6.0-7.8. 
(15) Aerobic 
(16) Hugh-Leifson's OF test: oxidative 
(17) Acid is produced but gas is not produced from L-arabinose, D-xylose, 
D-glucose, D-fructose, D-galactose, D-mannose, maltose, sucrose, lactose, 
trehalose, and glycerol. Neither acid nor gas is produced from D-mannitol, 
D-sorbitol, inositol and starch. 
(d) Other characteristics 
(1) Weak production of acetic acid from ethanol 
(2) Biotin, thiamine, riboflavine and either CoA or pantothenic acid are 
required for growth. 
(3) Production of dihydroxyacetone from glycerol 
(4) The guanine+cytosine content of DNA: 67.+-.1 mole % 
(5) The presence of a ubiquinone containing 10 isoprene units (CoQ.sub.10) 
(6) Marked production of 2-keto-L-gulonic acid from L-sorbose 
(7) Streptomycin-resistant 
The above taxonomical characteristics of the 5 strains of soil origin were 
reviewed by reference to Bergey's Manual of Determinative Bacteriology 8th 
ed. (1974) and Bergey's Manual of Systematic Bacteriology Vol 1 (1984). 
The above review showed that the K591s, 12-5, 12-15, 12-4 and 22-3 strains 
were tentatively classified into the genus Pseudomonas in view of the 
finding that they are gram-negative, motile, and rod bacteria having polar 
flagella. And in the light of the finding that they require certain growth 
factors, that the combined guanine and cytosine content of DNA is 67.+-.1 
mole % and that their quinone system is a ubiquinone having 10 isoprene 
units, they are similar to Pseudomonas diminuta and Pseudomonas 
vesicularis which belong to RNA Group IV of Section IV of this genus. 
However, the weak production of acetic acid from ethanol and the 
production of dihydroxyacetone from glycerol are the characteristics which 
differentiate the strains from the bacteria of the genus Pseudomonas. 
The above characteristics are those of species of the genus Gluconobacter. 
However, in light of the fact that these 5 strains give positive responses 
to the oxidase test, are not able to grow at pH 4.5 and show good growth 
in either yeast extract nutrient medium or peptone yeast extract medium 
without carbohydrates, and have a combined DNA guanine and cytosine 
content of 67.+-.1 mole %, they are different from the species of the 
genus Gluconobacter. 
Thus, these 5 strains of K591s, 12-5, 12-15, 12-4 and 22-3 could not be 
relegated to any of the known genera and had to be considered to be 
bacteria of a novel species of a novel genus. Accordingly, the strains 
K591s, 12-5, 12-15, 12-4 and 22-3 were collectively designated as 
Pseudogluconobacter saccharoketogenes. 
Referring to the nutritional requirements of these 5 strains, K591s, 12-5 
and 12-15 have the unique property to require CoA for the growth. The CoA 
requirement of these 3 strains can not be substituted by pantothenic acid. 
On the other hand, 12-4 and 22-3 can grow in the presence of pantothenic 
acid as well as in the presence of CoA. 
In the following description, these Pseudogluconobacter saccharoketogenes 
strains are sometimes referred to as oxidative strains. 
The strains which can be used in accordance with the present invention 
include not only the above-described 5 strains but also other strains 
inclusive of the mutants derived from the 5 strains by irradiation with 
ultraviolet light or X-rays or treatment with chemical mutagens such as 
N-methyl-N'-nitro-N-nitrosoguanidine (nitrosoguanidine), 
methylmethanesulfonate, nitrogen mustard and so on. As an example of such 
mutants, there may be mentioned the strain TH 14-86 which was derived from 
Pseudogluconobacter saccharoketogenes K591s by treatment with 
nitrosoguanidine. This mutant strain TH 14-86 exhibits the same 
taxonomical characteristics as the parent strain except that it shows an 
increased ability to produce 2-keto-L-gulonic acid from L-sorbose. 
The above-mentioned Pseudogluconobacter saccharoketogenes K591s, 12-5 and 
TH 14-86 were deposited at the Institute for Fermentation, Osaka, (IFO), 
17-85 Jusohonmachi 2-chome, Yodogawa-ku, Osaka, Japan on Sep. 19, 1985 and 
Pseudogluconobacter saccharoketogenes 12-15, 12-4 and 22-3 on Dec. 16, 
1985. Furthermore, Pseudogluconobacter saccharoketogenes K591s, 12-5 and 
TH 14-86 were deposited at Fermentation Research Institute (FRI) of the 
Agency of Industrial Science and Technology, the Ministry of International 
Trade and Industry, 1-3 Higashi 1-chome, Yatabe-machi tsukaba-gun, 
Ibaraki-ken 305, Japan on Oct. 7, 1985 and Pseudogluconobacter 
saccharoketogenes 12-15, 12-4 and 22-3 on Dec. 20, 1985. These deposits 
were converted to deposits under the Budapest Treaty and these 
microorganisms have been stored at FRI since Aug. 9, 1986. 
The deposit numbers at IFO and at FRI are as follows: 
______________________________________ 
Microorganism IFO FRI 
______________________________________ 
Pseudogluconobacter 
14464 P-8481 BP-1130 
saccharoketogenes K591s 
Pseudogluconobacter 
14465 P-8480 BP-1129 
saccharoketogenes 12-5 
Pseudogluconobacter 
14466 P-8479 BP-1128 
saccharoketogenes TH14-86 
Pseudogluconobacter 
14482 P-8577 BP-1132 
saccharoketogenes 12-15 
Pseudogluconobacter 
14483 P-8576 BP-1131 
saccharoketogenes 12-4 
Pseudogluconobacter 
14484 P-8578 BP-1133 
saccharoketogenes 22-3 
______________________________________ 
In the practice of the present invention, the above-mentioned strains can 
be grown in L-sorbose-containing media or, alternatively, L-sorbose may be 
contacted with a preparation derived from cells of said strains. 
The term "preparation derived from cells" or "cell preparation" is used 
herein to mean any and all of washed cells from culture broths of said 
bacteria, acetone dried cells, immobilized cells on supports such as 
polyacrylamide gel, K-carrageenin and the like, and other equivalent 
preparations. 
The starting material L-sorbose may be added all at once at initiation of 
cultivation, in several installments in the course of cultivation or 
continuously to the culture medium. 
Referring to the reaction by contact between L-sorbose and said 
microorganism, the concentration of L-sorbose in the reaction system is 3 
to 30 percent (w/v), preferably 5 to 25% (w/v), based on the medium. 
As an example of procedure for contacting L-sorbose with said bacterial 
cell preparation, there may be mentioned a method which comprises adding 
L-sorbose, 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.5, 
0.5M) and CaCO.sub.3 to the cell preparation, diluting with water, and 
shaking the mixture in a conical flask. 
The concentration of L-sorbose in such a reaction system for effecting 
contact between L-sorbose and said cell preparation is 0.1to 10% (w/v), 
preferably 0.3 to 3% (w/v). The amount of the cell preparation is 1 to 30 
mg/ml on a pre-reaction dry cell basis. The pH of the reaction system is 
controlled in the range of pH about 5.5 to 7.5, the reaction temperature 
is about 20.degree. to 40.degree. C., and the reaction time is about 1 to 
100 hours. 
In working the present invention into practice by incubating a 
Pseudogluconobacter strain in an L-sorbose-containing liquid medium to 
produce and accumulate 2-keto-L-gluonic acid in the broth, it has been 
found that the accumulation yield of 2-keto-L-gulonic acid is remarkably 
higher when other bacteria are allowed to be present in combination with 
the Pseudogluconobacter oxidative strain than it is the case when the 
oxidative strain alone is cultivated. 
The bacteria that are allowed to be present concomitantly may for example 
be bacteria of the following genera: Bacillus, Pseudomonas, Proteus, 
Citrobacter, Enterobacter, Erwinia, Xanthomonas and Flavobacterium. As the 
specific species, the following may be mentioned. 
Bacillus cereus IFO 3131 
Bacillus licheniformis IFO 12201 
Bacillus megaterium IFO 12108 
Bacillus pumilus IFO 12090 
Bacillus amyloliquefaciens IFO 3022 
Bacillus subtilis IFO 13719 
Bacillus circulans IFO 3967 
Pseudomonas trifolii IFO 12056 
Pseudomonas maltophilia IFO 12692 
Proteus inconstans IFO 12930 
Citorobacter freundii IFO 13544 
Enterobacter cloacae IFO 3320 
Erwinia herbicola IFO 12686 
Xanthomonas pisi IFO 13556 
Xanthomonas citri IFO 3835 
Flavobacterium menigosepticum IFO 12535 
Micrococcus varians IFO 3765 
Escherichia coli IFO 3366 
Any of these stains may be incubated in an appropriate medium at 20.degree. 
to 40.degree. C. for 1 to 4 days and the resulting culture is used as an 
inoculum for cultivation in the presence of said concomitant bacteria. The 
inoculum size is generally desirably 1/10 to 1/1000 of that of the 
oxidative strain. When the concomitant strain in this amount is incubated 
with the oxidative strain, the growth of the oxidative strain is promoted 
so that compared with a pure culture of the oxidative strain, the mixed 
culture is able to oxidize L-sorbose in higher concentrations to 
2-keto-L-gluonic acid in a shorter time period. The bacteria used as said 
concomitant bacteria are preferably these which cannot assimilate or only 
sparingly assimilate L-sorbose and 2-keto-L-gulonic acid. Otherwise, the 
same cultivation conditions as those of the pure culture of the oxidative 
strain can be employed. The medium used for cultivation of the 
above-mentioned microorganisms may be a liquid or solid medium containing 
nutrients which can be utilized by the said strain. However, for mass 
production, a liquid medium is preferred. The medium contains the carbon 
sources, nitrogen sources, inorganic salts, organic acid salts and trace 
nutrients which are generally used in the cultivation of microorganisms. 
While the starting material L-sorbose serves as the carbon source, other 
auxiliary carbon sources such as glucose, glycerin, sucrose, lactose, 
maltose, molasses, etc. can also be employed. The nitrogen sources are 
exemplified by various inorganic and organic nitrogen-containing compounds 
or nitrogenous materials such as ammonium salts (e.g. ammonium sulfate, 
ammonium nitrate, ammonium chloride, ammonium phosphate, etc.), corn steep 
liquor (CSL), peptone, meat extract, yeast extract, dried yeast, soybean 
flour, cottonseed meal, urea, and so on. As the inorganic salts, there may 
be employed salts of potassium, sodium, calcium, magnesium, iron, 
manganese, cobalt, zinc, copper and/or phosohoric acid. 
As the trace nutrients, in addition to CoA, pantothenic acid, biotin, 
thiamine and riboflavine which are essential growth factors for said 
microorganisms, there can be added those substances which promote the 
growth of the microorganisms and the production of 2-keto-L-gulonic acid 
thereby, such as flavine mononucleotide (FMN), flavine adenine 
dinucleotide (FAD), other vitamins, L-cysteine, L-glutamic acid, sodium 
thiosulfate, etc., either in the form of pure chemical compounds or in the 
form of natural materials containing them, in suitable amounts. 
As regards the cultural method, any of stationary culture, shaking culture, 
submerged culture, and so on can be employed. For mass production, the 
so-called submerged culture is preferred. 
Of course, cultural conditions depend on the bacterial strain, medium 
composition, and other factors, and can be chosen in each case so that the 
object compound may be obtained with the highest efficiency. Thus, for 
example, the incubation temperature may advantageously be in the range of 
25.degree. to 35.degree. C. and the medium pH may be about 5 to 9. 
As the cultivation is conducted under the above conditions for 10 to 120 
hours, 2-keto-L-gulonic acid is accumulated in the highest concentration. 
As the pH value of the medium generally lowers with the formation of the 
object compound, it may be advantageous to add a suitable basic substance 
such as sodium hydroxide, potassium hydroxide or ammonia from time to time 
so as to maintain the medium at an optimal pH level for the elaboration of 
2-keto-L-gulonic acid by the bacterial strain or have a suitable buffer 
agent contained in the medium to thereby keep the medium pH constant. 
Aside from the above, the sterilized culture broths of bacteria other than 
the oxidative strains can be used advantageously as medium components. The 
bacteria that can be utilized in this manner include those of the genus 
Bacillus, the genus Pseudomonas, the genus Citrobacter, the genus 
Escherichia, and the genus Erwinia, for instance. Specifically, the 
following bacteria may be mentioned. 
______________________________________ 
Bacillus cereus IFO 3131 
Bacillus subtilis IFO 3023 
Bacillus pumilus IFO 12089 
Bacillus megaterium IFO 12108 
Bacillus amyloliquefaciens 
IFO 3022 
Pseudomonas trifolii 
IFO 12056 
Citrobacter freundii 
IFO 12681 
Escherichia coli IFO 3546 
Erwinia herbicola IFO 12686 
______________________________________ 
Thus, these bacteria are incubated in media which permit their growth at 
20.degree. to 40.degree. C. for 2 to 4 days and the resulting culture 
broths are sterilized and added to the medium for the oxidative strain in 
a proportion of 0.5 to 5.0 percent (V/V). In this manner, growth of the 
oxidative strain can be encouraged. 
The 2-keto-L-gulonic acid thus elaborated and accumulated in the culture 
broth or the reaction mixture can be harvested and purified by the per se 
known method utilizing its properties. 2-Keto-L-gulonic acid may be 
harvested in the form of free acid or separated in the form of salt with, 
for example, sodium, potassium, calcium, ammonium or the like. 
Any harvesting method compatible with the object of the invention can be 
employed. For example, the culture broth is freed of cells, as required, 
by filtration, centrifugation or treatment with activated carbon and the 
solution is concentrated. The precipitated crystals are collected by 
filtration and recrystallized to recover the object compound. Further, 
solvent extraction, chromatography, precipitation or salting-out, and 
other procedures may be applied in a suitable combination and/or in 
repetition. 
When 2-keto-L-gulonic acid is obtained in its free form, it can be 
converted to a salt with, for example, sodium, potassium, calcium, 
ammonium or the like by the conventional method. When the object compound 
is recovered in the form of a salt, it can be converted to the free acid 
or a different salt by the known method. 
The identity of the product compound obtained by the method of the present 
invention with 2-keto-L-gulonic acid has been established by the 
determination of physicochemical constants such as elemental analysis, 
melting point, optical rotation, infrared absorption spectrum, etc. 
The quantitative determination of 2-keto-L-gulonic acid in the reaction 
mixture or the culture broth was performed by high performance liquid 
chromatography (mobile phase: dilute sulfuric acid pH 2.2; flow rate: 0.5 
ml/min.; detector: differential refractometer) using a sulfonated 
polystyrene gel column (Shimadzu Seisakusho, Ltd. , Japan, SCR-101H 
column, 7.9 mn.times.30 cm). As the standard, crystals of sodium 
2-keto-L-gulonate monohydrate were used. The detection of 2-keto-L-gulonic 
acid was done by thin layer chromatography. Thus, as a cellulose plate 
(Merck, U.S.A.) was spotted with a sample and after development with a 
solvent system of phenol-water-formic acid (75:25:5) at room temperature 
for 3 hours, dried and treated with a color reagent, 2-keto-L-gulonic acid 
gave a spot at Rf about 0.30, the spot being black-brown with silver 
nitrate, yellow with o-phenylenediamine, or pink with anilinephthalic 
acid. 
2-Keto-L-gulonic acid can be produced in good yield by the method of the 
present invention using a microorganism belonging to the genus 
Pseudogluconobacter which is able to oxidize L-sorbose to 2-keto-L-gulonic 
acid.

The following examples are intended to illustrate the present invention in 
further detail. The % figures mentioned in connection with media represent 
weight/volume percents. 
Example 1 
A 200 ml conical flask was charged with 20 ml of a seed culture medium 
containing 2.0% of glucose, 1.0% of peptone, 1.0% of dried yeast and 2.0% 
of CaCO.sub.3 and sterilized by autoclaving at 120.degree. C. for 20 
minutes. The flask was inoculated with 1 loopful of Pseudogluconobacter 
saccharoketogenes K591s (IFO 14464; FERM BP-1130) grown on a slant medium 
in Table 1 at 28.degree. C. for 4 days, and incubated at 30.degree. C. 
with shaking (200 rpm) for 2 days. Two ml of the resulting broth was 
transplanted into a flask containing the same seed culture medium as above 
and incubated under the same conditions to give a seed culture. 
A 200 ml conical flask was charged with 25 ml of a fermentation medium 
containing 2.0% of CSL, 0.5% of dried yeast, 0.5% of ammonium sulfate, 
0.05% of Na.sub.2 S.sub.2 O.sub.3.5H.sub.2 O, 0.2% of ferrous sulfate, 
4.0% of CaCO.sub.3, and 10.0% of L-sorbose (separately sterilized) and 
sterilized by autoclaving at 120.degree. C. for 20 minutes. This conical 
flask containing the above fermentation medium was inoculated with 1.25 ml 
of the above-prepared seed culture and incubated with shaking at 
30.degree. C. for 3 days. As assayed by high performance liquid 
chromatography, the resulting fermentation broth contained 60.5 mg/ml of 
2-keto-L-gluconic acid (conversion ratio: 56.1%). This fermentation broth 
(1000 ml) was centrifuged to remove the cellular and other sediments. The 
supernatant (980 ml) obtained was passed through an Amberlite IR 120 (Rohm 
& Haas Co., U.S.A., H-form, 500ml) column, which was then washed with 
about 300 ml of deionized water. The effluent and washings were combined 
and passed through an activated carbon (500 ml) column, followed by 
washing with about 300 ml of deionized water to remove the cations and 
color. The effluent and washings were combined (1600 ml), adjusted to pH 
6.5 with sodium hydroxide, and concentrated under reduced pressure at 
50.degree. C. to about 70 ml. This concentrate rate was allowed to stand 
at 5.degree. C. for 24 hours, whereupon colorless prisms were obtained. 
The prisms were collected by filtration, washed with a small quantity of 
cold methanol, and dried over phosphorus pentoxide at room temperature 
under reduced pressure to give 37.5 g of monosodium 2-keto-L-gulonate 
monohydrate. 
Melting point 147.degree.-155.degree. C. (decomp.). Elemental analysis 
(C.sub.6 H.sub.9 O.sub.7 Na.H.sub.2 O) Calcd.: C, 30.78%; H, 4.74% Found: 
C, 30.94%; H, 4.85% 
Optical rotation: [.alpha.].sub.D.sup.24 -23.3.degree. (C=1.0, water). In 
HPLC retention time, TLC Rf value, and color, the above product was in 
agreement with the authentic sample. 
Example 2 
A test tube (16 mm.times.160 mm) containing 5 ml of a complete medium in 
Table 2 was inoculated with a loopful of Pseudogluconobacter 
saccharoketogenes K591s grown on a slant medium in Table 1 and incubated 
at 30.degree. C. with shaking for 2 days. This culture (1 ml) was 
transferred to a test tube containing 5 ml of the same medium, which was 
then incubated with shaking for 4 hours. The resulting broth (5 ml) was 
aseptically centrifuged (12,000 rpm) at 5.degree. C. for 15 minutes to 
harvest the cells. The cells were suspended in 10 ml of tris-maleic acid 
buffer (pH 6.5; 0.05M) and recentrifuged. The above procedure was repeated 
twice and the washed cells were suspended in 5 ml of the above-mentioned 
buffer containing 1 mg/ml of nitrosoguanidine and shaken at 30.degree. C. 
for 2 hours for mutagenic treatment. The suspension was centrifuged 
(12,000 rpm) at 5.degree. C. for 15 minutes to collect the cells which 
were then washed twice with 10 ml portions of trismaleic acid buffer to 
recover a fraction containing nitrosoguanidine-treated cells. This was 
diluted with 0.85% saline to a suitable concentration and spread over a 
plate (diameter: 9 cm) containing 15 ml of the complete medium (solid). 
The inoculated plate medium was incubated at 28.degree. C. for 5 days to 
grow colonies. The colonies were counted and compared with the untreated 
control. The mortality of the microorganisms due to the nitrosoguanidine 
treatment was 90.4%. The colonies on the complete medium plate were 
replicated onto the minimum essential medium plates in Table 3 and after 
incubation at 28.degree. C. for 3 days, the frequency of auxotrophs 
(nutritional mutants) was investigated. The frequency was about 6.6%. 
The colonies treated with the mutagen on the complete medium plate were 
streaked onto a fresh complete medium plate over a length of about 2 cm at 
the rate of 12 strains per plate. After incubation at 28.degree. C. for 2 
days, one loopful of the grown cells were transferred to a test tube 
containing 3 ml of a medium (pH 6.5) composed of 7.0% of L-sorbose 
(separately sterilized), 1.0% of dried yeast 10% of peptone, 0 1% of 
ferrous chloride and 3.0% of CaCO.sub.3 and incubated with shaking at 
30.degree. C. for 4 days. Among the tested mutant strains, the strain 
TH14-86 was found to produce 2-keto-L-gulonic acid twice as much as the 
parental strain K591s under the above conditions. This strain TH14-86 (IFO 
14466; FERM BP-1128) was chosen as an oxidative strain with an augmented 
ability to oxidize L-sorbose. 
TABLE 1 
______________________________________ 
Slant medium (g/l) 
______________________________________ 
D-sorbitol 
25 
Peptone 10 
Yeast extract 
10 
CACO.sub.3 
2 
Agar 20 
pH 7.0 
______________________________________ 
TABLE 2 
______________________________________ 
Complete medium (g/l) 
______________________________________ 
D-sorbitol 25 
Peptone 10 
Yeast extract 10 
pH 6.5 (In the case of a solid medium, 20 g of 
agar was added) 
______________________________________ 
TABLE 3 
______________________________________ 
Minimum essential medium (g/l) 
______________________________________ 
Sucrose 5 
K.sub.2 HPO.sub.4 3 
KH.sub.2 PO.sub.4 1 
(NH.sub.4).sub.2 SO.sub.4 1 
NaCl 1 
MgSO.sub.4.7H.sub.2 O 0.1 
MnCl.sub.2.nH.sub.2 O 0.002 
Sodium L-glutamate 0.1 
L-cysteine 0.1 
CoA 0.002 
FMN 0.002 
Thiamine 0.002 
Biotin 0.001 
pH 7.0 (In the case of a solid medium, 20 g of 
agar was added) 
______________________________________ 
Example 3 
The mutant strain TH14-86 derived from Pseudogluconobacter 
saccharoketogenes K591s in Example 2 was grown on a slant medium at 
28.degree. C. for 4 days. A loopful of the cells were taken from the slant 
culture and inoculated into a 200 ml conical flask containing 20 ml of the 
seed culture medium described in Example 1 and incubated at 30.degree. C. 
with shaking for 2 days. 
A conical flask of 1 liter capacity was charged with 200 ml of a medium 
composed of 3.0% of glucose, 1.0% of peptone, 1.0% of dried yeast and 2.0% 
of CaCO.sub.3 and sterilized by autoclaving at 120.degree. C. for 20 
minutes. This conical flask was inoculated with 20 ml of the above culture 
and incubated at 28.degree. C. with shaking for 2 days to give a seed 
culture. Separately, a loopful of Bacillus megaterium IFO 12108 grown on a 
slant medium at 28.degree. C. for 2 days was inoculated into a 200 ml 
conical flask containing 20 ml of a medium composed of 4.0% of sucrose, 
4.0% of cottonseed meal, 0.65% of K.sub.2 HPO.sub.4, 0.55% of KH.sub.2 
PO.sub.4, 0.05% of ammonium sulfate, 0.05% of NaCl, 0.05% of magnesium 
sulfate and 0.05% of calcium pantothenate (pH 7.0) (sterilized by 
autoclaving at 120.degree. C. for 20 minutes) and incubated at 30.degree. 
C. for 3 days. The resulting culture broth was sterilized by autoclaving 
at 120.degree. C. for 20 minutes, stored in the cold, and used as a 
component of the under-mentioned fermentation medium. Thus, a 5 liter jar 
fermentor was charged with 3 liters of a yeast medium composed of 12.5% of 
L-sorbose (separately sterilized at 120.degree. C. for 15 minutes), 0.5% 
of ammonium sulfate, 0.03% of KH.sub.2 PO.sub.4, 0.05% of Na.sub.2 S.sub.2 
O.sub.3.5H.sub.2 O, 0.05% of magnesium sulfate, 0.1% of 
FeSO.sub.4.7H.sub.2 O, 5 .mu.g/ml of MnSO.sub.4.4H.sub.2 O, 5 .mu.g/ml of 
thiamine, 0.1 .mu.g/ml of biotin, 0.1 .mu.g/ml of FMN, 5.0% of CaCO.sub.3, 
and 4.0% (V/V) of the above sterilized broth of Bacillus megaterium and 
sterilized by autoclaving at 120.degree. C. for 30 minutes. This 
fermentation medium was inoculated with 300 ml of the above seed culture 
and cultivated at 32.degree. C. with aeration at 2.4N-l/min. and stirring 
at 800 r.p.m. for 3 days. The resultant fermentation broth contained 102.0 
mg/ml of 2-keto-L-gulonic acid (conversion ratio: 75.7%). This broth (1 l) 
was purified in the same tanner as Example 1 to give 73.2 g of monosodium 
2-keto-L-gulonate monohydrate crystals. 
Example 4 
Pseudogluconobacter saccharoketogenes 12-5 (IFO 14465; FERM BP-1129) was 
incubated in the same manner as Example 1 to give a seed culture. A 200 ml 
conical flask was charged with 20 ml of a fermentation medium (9.0% of 
L-sorbose) described in Example 3 and sterilized by autoclaving at 
120.degree. C. for 20 minutes. The flask was inoculated with 1.5 ml of the 
above seed culture and incubated at 32.degree. C. for 2 days. The 
resulting fermentation broth contained 73.2 mg/ml of 2-keto-L-gulonic acid 
(conversion ratio: 75.4%). 
Example 5 
Pseudogluconobacter saccharoketogenes 12-4 (FERM BP-1131; IFO 14483), 12-15 
(FERM BP-1132; IFO 14482) and 22-3 (FERM BP-1133; IFO 14484) were 
respectively incubated with shaking in the same manner as Example 4 for 3 
days. The yields of 2-keto-L-gulonic acid in the broth were 52.1 mg/ml for 
the strain 12-4(conversion ratio: 53.7%); 48.7 mg/ml for the strain 
12-15(conversion ratio:50.2%); and 69.3 mg/ml for the strain 
22-3(conversion ratio:71.4%) 
Example 6 
A 200 ml conical flask was charged with 25 ml of a medium (pH 7.0) composed 
of 1.0% of L-sorbose (separately sterilized), 0.5% of peptone, and 0.5% of 
yeast extract and sterilized by autoclaving at 120.degree. C. for 15 
minutes. The flask was inoculated with a loopful of Pseudogluconobacter 
saccharoketogenes TH14-86 grown on a slant medium in Table 1 at 28.degree. 
C. for 4 days and incubated at 30.degree. C. with shaking for 2 days to 
give a seed culture. 
A 200 ml conical flask was charged with 25 ml of a medium (pH 7.0) composed 
of 5.0% of L-sorbose (separately sterilized), 1.0% of peptone, 0.5% of 
yeast extract and 2.0% of CaCO.sub.3 and sterilized by autoclaving at 
120.degree. C. for 15 minutes. This flask was inoculated with 1.0 m of the 
above seed culture and incubated at 30.degree. C. for 2 days. 
The resulting culture (500 ml) was allowed to stand at room temperature for 
20 minutes and the sediment was removed by decantation. The remaining 
fluid was centrifuged at a slow speed of 1,000 rpm at room temperature to 
remove the sediment composed predominantly of CaCO.sub.3. The cell 
suspension thus obtained was further centrifuged (6,000 rpm) at 5.degree. 
C. for 10 minutes and the cells collected were washed twice with about 
100 ml portions of cold saline (0.85%) and re-centrifuged (6,000 rpm) at 
5.degree. C. to give washed cells. The cells were further suspended in 35 
ml of cold saline (0.85%) to give a washed cell suspension. To 4 ml of 
this washed cell suspension were added 300 mg of L-sorbose, 0.5 ml of 2- 
(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.5; 0.5M) and 180 mg 
of CaCO.sub.3, followed by dilution with water to make 10 ml. The mixture 
was reacted in a 100 ml conical flask at 30.degree. C. with shaking for 24 
hours. The reaction mixture obtained in this manner was found to contain 
24.6 mg/ml of 2-keto-L-gulonic acid(conversion ratio: 76.0%). 
Example 7 
Pseudogluconobacter saccharoketogenes K591s, 12-5 and TH14-86 were 
respectively grown on a slant medium at 28.degree. C. for 4 days. 
Separately, the concomitant bacteria in Table 4 were grown on the same 
slant medium at 28.degree. C. for 2 days. One loopful of each strain was 
inoculated into a 200 ml conical flask containing 20 ml of a seed culture 
medium in Example 1 and incubated with shaking (200 rpm) at 30.degree. C. 
for 2 days. In this manner, various culture broths were obtained. 
A 200 ml conical flask was charged with 25 ml of a fermentation medium 
composed of 2.0% of CSL, 0.3% of dried yeast, 0.5% of ammonium sulfate, 
0.05% of Na.sub.2 S.sub.2 O.sub.3.5H.sub.2 O 0.2% of ferrous sulfate, 5.0% 
of CaCO.sub.3, and 15.0% of L-sorbose (separately sterilized) and 
sterilized by autoclaving at 120.degree. C. for 20 minutes. The conical 
flask containing the above medium was inoculated with the above seed 
culture (1.5 ml) of one of said Pseudogluconobacter saccharoketogenes 
(oxidative) strains and incubated with shaking at 30.degree. C. for 5 days 
to give a pure culture. 
In the case of mixed culture, 0.1 ml of a seed culture of said concomitant 
bacteria was inoculated simultaneously at the inoculation with the 
oxidative strain and the inoculated medium was incubated at 30.degree. C. 
with shaking for 5 days. 
The amount of 2-keto-L-gulonic acid produced in each broth was assayed by 
high performance liquid chromatography. The results are set forth in Table 
4. The presence of concomitant bacteria resulted in increased yields of 
2-keto-L-gulonic acid. 
TABLE 4 
______________________________________ 
The production of 2-keto-L-gulonic acid by 
cultivation of the strain Pseudogluconobacter 
saccharoketogenes with and without the con- 
comitant bacteria 
Pseudogluconobacter 
saccharoketogenes 
K591s 12-5 TH14-86 
Concomitant Bacteria 
(mg/ml) (mg/ml) (mg/ml) 
______________________________________ 
No additive 55.3 74.1 87.6 
(34.2%) (45.8%) (54.1%) 
Bacillus cereus 87.3 101.5 125.9 
IFO 3131 (54.0%) (62.7%) (77.8%) 
Bacillus licheniformis 
-- -- 125.0 
IFO 12201 (77.3%) 
Bacillus megaterium 
69.3 90.2 135.4 
IFO 12108 (42.8%) (55.8%) (83.7%) 
Bacillus pumilus 93.1 129.0 134.7 
IFO 12090 (57.5%) (79.8%) (83.3%) 
Bacillus amyloliquefaciens 
-- -- 126.9 
IFO 3022 (78.5%) 
Bacillus subtilis 81.7 94.4 135.3 
IFO 13719 (50.5%) (58.4%) (83.7%) 
Pseudomonas trifolii 
67.2 98.8 122.6 
IFO 12056 (41.5%) (61.1%) (75.8%) 
Pseudomonas maltophilia 
71.9 79.8 135.3 
IFO 12692 (44.4%) (49.3%) (83.7%) 
Proteus inconstans 
-- -- 124.5 
IFO 12930 (77.0%) 
Citrobacter freundii 
-- -- 132.3 
IFO 13544 (81.8%) 
Enterobacter cloacae 
-- -- 132.0 
IFO 3320 (81.6%) 
Erwinia herbicola 71.8 111.6 129.1 
IFO 12686 (44.4%) (69.0%) (79.8%) 
Xanthomonas pisi -- -- 121.5 
IFO 13556 (75.1%) 
Flavobacterium meningosepticum 
-- -- 122.8 
IFO 12535 (75.9%) 
______________________________________ 
The figure in the parenthesis shows a conversion ratio. 
Example 8 
A 2 l Sakaguchi flask was charged with 500 ml of a preculture medium 
composed of 2.0% of glucose, 1.0% of peptone, 1.0% of dried yeast, 2.0% of 
CaCO.sub.3, and 0.01% of Actcol (defoaming agent, Takeda Chemical 
Industries, Ltd.) and sterilized by autoclaving at 120.degree. C. for 20 
minutes. The sells of Pseudogluconobacter saccharoketogenes TH14-86 grown 
on a slant medium in Table 1 were suspended in 10 ml of sterile water and 
the whole amount was inoculated into the Sakaguchi flask and incubated on 
a reciprocating shaker (85 spm) at 28.degree. C. for 3 days to give a 
preculture. A 200-liter fermentor was charged with 120 l (pH 6.5) of a 
seed culture composed of 3.0% of glucose, 1.0% of CSL, 0.5% of dried 
yeast, 0.05% of sodium thiosulfate, 0.1% of ferrous sulfate, 2.0% of 
calcium carbonate and 0.03% of Actcol, and sterilized at 125.degree. C. 
for 30 minutes. To this fermentor was transferred 1.8 l of the 
above-mentioned preculture, followed by cultivation at 120 rpm 
(agitation), 100N-l/min. (aeration), 1.0 Kg/cm.sup.2 G(pressure) and 
30.degree. C. for 3 days to give a seed culture. 
On the other hand, one loopful of the concomitant strain Bacillus 
megaterium IFO 12108 grown on a slant medium in Table 1 at 28.degree. C. 
for 2 days was inoculated into a 2 liter Sakaguchi flask containing 500 ml 
of the above-mentioned preculture medium and incubated on a reciprocating 
shaker (85 spm) at 28.degree. C. for 2 days to give a preculture. A 50 
liter fermentor was charged with 30 l of the same medium as the above 
preculture medium and sterilized at 120.degree. C. for 20 minutes. This 
fermentor was inoculated with 500 ml of the preculture of the concomitant 
strain and cultivated at 120 rpm (agitation), 30N-l/min. (aeration), 1.0 
Kg/cm.sup.2 G (pressure), and 30.degree. C. for 2 days to give a seed 
culture of the concomitant strain. 
A 2 m.sup.3 fermentor was charged with 1000 l of a fermentation medium 
composed of 15.0% of L-sorbose (separately sterilized), 5.0% of calcium 
carbonate, 2.0% of CSL, 0.2% of dried yeast, 0.3% of ammonium sulfate, 
0.05% of sodium thiosulfate, 0.1% of ferrous sulfate, and 0.03% of Actcol 
and sterilized at 125.degree. C. for 30 minutes. To this fermentor were 
transferred 110 l of the above seed culture of the strain 
Pseudogluconobacter saccharoketogenes TH14-86 and 10 l of the seed culture 
of the concomitant strain Bacillus megaterium IFO 12108, and the 
cultivation was carried out at 110 rpm (agitation), 900N-l/min. 
(aeration), 0.5 Kg/cm.sup.2 G (pressure), and 30.degree. C. The culture 
broth after 4 days of incubation contained 123.1 mg/ml of 2-keto-L-gulonic 
acid (conversion ratio: 76.1%). 
Example 9 
A 200 ml conical flask was charged with 20 ml of the preculture medium of 
Example 8 and sterilized by autoclaving at 120.degree. C. for 30 minutes. 
A loopful of Pseudogluconobacter saccharoketogenes TH14-86 grown on a 
slant medium in Table 1 at 28.degree. C. for 4 days was inoculated into 
the above flask and incubated at 30.degree. C. with shaking for 2 days. 
The resulting culture (20 ml) was transferred to a 1 liter conical flask 
containing 200 ml of the same medium and incubated at 30.degree. C. with 
shaking for 2 days to give a seed culture of TH 14-86. 
One loopful of Bacillus megaterium IFO 12108 grown on a slant medium at 
28.degree. C. for 2 days was inoculated into a 200 ml conical flask 
containing 20 ml of the preculture medium and incubated with shaking at 
28.degree. C. for 2 days to give a seed culture of the concomitant 
bacteria. A fermentation medium (3 l) composed of 3.0% of L-sorbose 
(separately sterilized), 2.0% of CSL, 0.2% of dried yeast, 0.3% of 
ammonium sulfate, 0.05% of sodium thiosulfate, 0.1% of ferrous sulfate, 
0.02% of Actcol and 9.0% of calcium carbonate was adjusted to 2.1 l and 
sterilized by autoclaving at 120.degree. C. for 30 minutes. The 
sterilized medium was charged into a 5 liter jar fermenter. 
This jar fermentor was inoculated with 300 ml of the above seed culture of 
the strain TH14-86 and 4 ml of the seed culture of the concomitant strain, 
and the cultivation was carried out at 30.degree. C., 2.4N-l/min. 
(aeration) and 800 rpm (agitation). 
Separately, 510 g of L-sorbose was dissolved in water to prepare 800 ml of 
a sorbose solution and sterilized by autoclaving at 120.degree. C. for 20 
minutes. This sterilized solution was continuously added to the jar 
fermentor from the 6th to the 42th hour of the cultivation. Following the 
addition of L-sorbose, the cultivation was continued under the same 
conditions as above for additional 28 hours (totally 70 hours). The 
resulting broth contained 163.5 mg/ml of 2-keto-L-gulonic acid (conversion 
ratio: 75.8%).