Production of high optical purity D-lactic acid

D-lactic acid is continuously produced by conducting fermentation in a D-lactic acid-producing medium by using as a seed culture a part of a broth obtained in a previous fermentation step of D-lactic acid and elevating a concentration of said growth-promoter to such an effect that a optical purity of the D-lactic acid may be prevented from decreasing. Instead, air may be used.

The invention relates to a process for producing D-lactic acid with a high 
optical purity by fermentation. In particular, the process is continuously 
carried out by the use of a fermentation broth of D-lactic acid as a seed 
culture in a subsequent fermentation. In addition, the invention provides 
an improvement also in the batchwise fermentation production of D-lactic 
acid. 
STATEMENT OF PRIOR ARTS 
It is known in Japanese patent publications A (unexamined) Nos. 58-16688 
and 58-36394 that D-lactic acid is produced by lactic acid bacteria. U.S. 
Pat. No. 3,262.,862 teaches the use of lactic acid bacteria for 
fermentation of D-lactic acid. 
It is noted that all conventional processes are conducted batchwise so that 
a prepropagation step accompanied by troublesome procedures such that a 
preserved lactic acid bacterium is allowed to grow while successively 
increasing the amount of the culturing broth should be carried out at each 
batch. In order to efficiently produce D-lactic acid, a continuous 
culturing of a fermentation medium has been attempted. However this 
process has not so far been applied at an industrial scale, since it is 
not possible to prevent the growth of infectious microbes for a prolonged 
period at present. Thus the production of D-lactic acid has been 
exclusively carried out batchwise. 
Since no process for preventing infectious microbes for a prolonged period 
in continuous fermentation of D-lactic acid has been established as yet, 
semicontinuous fermentation was attempted by repeating batch fermentation. 
As a result, it was found that repetition of batch fermentation caused 
reduction in the optical purity of the product, i.e. D-lactic acid. 
As a result of our studies to overcome these problems, we have found a 
process by which high-optical purity D-lactic acid can be produced by 
repeated batch fermentation, thus completing the present invention. 
SUMMARY OF THE INVENTION 
The invention provides a process for producing D-lactic acid, which 
comprises the step of conducting the fermention of D-lactic acid in a 
D-lactic acid-producing medium containing a carbohydrate, an inorganic 
salt, a growth-promoter and a neutralizing agent, by using as a seed 
culture a part of a broth obtained in a previous fermentation step of 
D-lactic acid in which a D-lactic acid-producing bacterium already grown 
in a pre-propagation step is used and elevating the concentration of said 
growth-promoter to such an amount that the optical purity of the D-lactic 
acid may be prevented from decreasing. It is preferred that the 
neutralizing agent is sodium hydroxide, sodium carbonate, sodium 
bicarbonate or ammonia. In a preferred embodiment of the invention, 
process air is introduced into the medium without the use of said broth 
and the elevation of the concentration of the growth-promoter. 
According to the invention, D-lactic acid is produced by 
(1) first growing a D-lactic acid-producing bacterium by a pre-preparation 
step, 
(2) cultivating D-lactic acid with said bacterium in a culture medium which 
comprises a carbohydrate, an inorganic salt, a growth-promoter and a 
neutralizing agent and 
(3) conducting further fermentation of D-lactic acid in a culture medium 
which comprises a carbohydrate, an inorganic salt, a growth-promoter and a 
neutralizing agent, using as a seed culture a part of the broth obtained 
in the previous cultivation step and elevating the concentration of, said 
growth-promoter to such an amount that the optical purity of the D-lactic 
acid may be prevented from decreasing. 
In other words, the invention provides a process for producing D-lactic 
acid wherein a D-lactic acid-producing bacterium previously grown by a 
prepropagation step is cultured in a medium comprising carbohydrates, 
inorganic salts, growth promoter(s) and neutralizing agent(s) and part of 
the broth thus obtained is employed as a seed culture in the subsequent 
D-lactic acid fermentation, wherein the concentration of the growth 
promoter(s) is elevated to an extent capable of inhibiting a decrease in 
the optical purity of D-lactic acid. 
The invention provides an improvement in the process in which a part of the 
broth obtained in the previous fermentation of D-lactic acid is used as a 
seed culture. That is, the process is improved by introducing air into the 
cultivation medium or using a specified neutralizing agent selected from 
sodium hydroxide, sodium carbonate, sodium bicarbonate and ammonia. This 
improvement is effective in the batch method of the process as well. This 
improved batch method can be conducted effectively without the elevation 
of a concentration of the growth-promoter. 
In the practical point of view, the batchwise process according to the 
invention is preferably carried out as illustrated below. 
(1) D-lactic acid is produced by culturing a D-lactic acid-producing 
bacterium belonging to the genus Sporolactobacillus in a D-lactic 
acid-producing medium comprising a carbohydrate, an inorganic acid, a 
growth-promoter and a neutralizing agent and introducing air into the 
medium. 
(2) D-lactic acid is produced by culturing a D-lactic acid-producing 
bacterium belonging to the genus Sporolactobacillus in a D-lactic 
acid-producing medium comprising a carbohydrate, an inorganic salt, a 
growth-promoter and a neutralizing agent selected from sodium hydroxide, 
sodium carbonate, sodium hydrogen-carbonate and ammonia. 
The above shown process enables production of D-lactic acid with a high 
optical purity when it is repeated batchwise. 
MICROORGANISM TO BE USED 
Any D-lactic acid-producing bacterium may be used in the present invention. 
For example, Sporolactobacillus inulinus ATCC 15538, Lactobacillus 
delbrueckii L. leichmannii JCM 1016, L. leichmannii JCM 1557 or L. lactis 
DSM-20073 may be used. 
CULTURE METHOD 
A seed culture of a D-lactic acid-producing bacterium is prepared in a 
conventional manner of batch fermentation. That is, the D-lactic 
acid-producing bacterium is cultured in, for example, a GYP medium as 
shown in Table 1. when the bacterium is sufficiently grown, the culturing 
broth is successively increased at a rate of 10 to 1000 times to thereby 
prepare the seed culture. Then the seed culture thus obtained is allowed 
to produce D-lactic acid in a D-lactic acid-producing medium. A 
composition of the medium should depend on a lactic-acid-producing 
bacterium used in the process. It is prepared from a carbohydrate, an 
inorganic salt, a growth promoter and a neutralizing agent. The 
carbohydrate preferably includes a sugar or saccharide such as glucose, 
fructose, sucrose, inulin, maltose, mannose, raffinose, trehalose and a 
substance containing therein any saccharide, for instance a starch 
hydrolyzate and molasses. The inorganic salt preferably includes magnesium 
sulfate, ammonium sulfate, potassium phosphate and ferrous sulfate. The 
growth promoter preferably includes yeast extract, peptone, meat extract 
and soybean powder. Each ingredient may be used with another in 
combination. The growth promoter is used since a lactic acid-producing 
bacterium generally exhibits high auxotrophy. It is further necessary to 
add neutralizing agent(s) thereto to thereby adjust the medium in a pH 
range of 4.5 to 7.0, since lactic acid-producing bacteria are acid 
sensitive. The neutralizing agent preferably includes CaCO.sub.3, 
Ca(OH).sub.2, NaOH, Na.sub.2 CO.sub.3, NaHCO.sub.3, KOH and ammonia. 
The fermentation process of the invention is normally conducted under 
anaerobic condition, for instance by passing an inert gas such as nitrogen 
gas into the system. It is added, however, that air may be used in the 
above shown embodiment without any inert gas. 
The fermentation temperature depends on the lactic acid-producing bacterium 
to be used. For example, it is preferable to carry out the fermentation at 
37.degree. C. when Sporolactobacillus inulinus ATCC 15538 is employed, 
while L. delbrueckii IFO 3534 is preferably cultured at 45.degree. to 
50.degree. C. Upon completion of the first D-lactic acid fermentation in 
this manner, part of the obtained broth is used as a seed culture in the 
subsequent D-lactic acid fermentation. The D-lactic acid obtained from the 
second fermentation in a medium of the same composition as that used in 
the first fermentation shows a lowered optical purity. However, this 
problem can be solved by elevating the concentration of growth 
promoter(s). The concentration of growth promoters in a medium used in a 
process of repeated fermentation is higher by generally 20 % or more 
preferably 50% or more, than the initial concentration. Alternately other 
growth promoter(s) may be added thereto. The amount of the growth 
promoter(s) to be added depends on the bacterium to be used. Generally 
0.1% or more, preferbly 0.5% or more, of the growth promoter(s) should be 
added. 
TABLE 1 
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GYP medium 
______________________________________ 
glucose 20 g/l 
yeast extract 10 g/l 
peptone 10 g/l 
sodium acetate 10 g/l 
magnesium sulfate 0.2 g/l 
FeSO.sub.4.7H.sub.2 O 10 mg/l 
MnSO.sub.4.4.5H.sub.2 O 
10 mg/l 
NaCl 10 mg/l 
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However it is also possible to prevent the D-lactic acid purity from 
falling by supplying air to the fermentation medium even in the case where 
CaCO.sub.3 is used as a neutralizing agent without elevating the 
concentration of the growth promoter(s). 
It is preferable to supply air to the medium at a rate of less than 10% by 
volume per min (hereinafter referred to as 0.1 V.V.M.) based on the amount 
of the liquid medium. It is undesirable that the rate exceeds 0.1 V.V.M., 
since the growth of the lactic acid-producing bacterium is completly 
inhibited thereby. 
The term "air" as used herein refers to one containing 21% by volume of 
oxygen. As a matter of course, the maximum amount of the air supply is 
restricted depending on the concentration of oxygen in the air. 
The use of this process makes it possible to produce high-optical purity 
D-lactic acid by the use of any neutralizing agent(s) without using a 
large amount of expensive growth promoter(s). 
However it is also possible to prevent the optical purity of D-lactic acid 
from falling by employing neutralizing agent(s) selected from among sodium 
hydroxide, sodium carbonate, sodium hydrogencarbonate and ammonia even 
when the fermentation is repeated in a medium of the same composition as 
that used in the first fermentation. 
This process makes it possible to effectively produce high-optical purity 
D-lactic acid without using a large amount of expensive growth 
promoter(s). 
The neutralizing agent(s) as used herein may be in the form of an aqueous 
solution, a powder or a gas. It may be selected from the viewpoint of 
convenience in operation. 
D-lactic acid is important as a starting material in synthesizing various 
optically active substances so that the demand therefor has been recently 
increasing. Under these circumstances, the present invention makes it 
possible to efficiently produce D-lactic acid on an industrial scale.

PREFERRED EMBODIMENTS OF THE INVENTION 
To further illustrate the present invention, the following Examples will be 
given. In each Example, the optical purity of D-lactic acid was determined 
by subjecting the total lactic acid to HPLC with the use of an ion 
exchange resin (SAX 801) wherein the content of L-lactic acid was 
enzymatically determined with the use of an L-lactic acid dehydrogenase 
and the optical purity of D-lactic acid was determined according to the 
following equation: 
##EQU1## 
EXAMPLES 1 AND 2 AND COMATIVE EXAMPLE 1 
Sporolactobacillus inulinus ATCC 15538 was inoculated into a GYP medium and 
static cultured therein at 37.degree. C. for three days. 1 ml of the 
culturing broth was inoculated into 25 ml of a GYP medium containing 1% of 
CaCO.sub.3 and static cultured therein at 37.degree. C. for one day, thus 
preparing a seed culture. 150 ml of the obtained seed culture was 
inoculated into 3 l of a fermentation medium having the following 
composition and fermentation was performed at 37.degree. C. under 
agitating at.200 rpm. 
______________________________________ 
Fermentation medium 
______________________________________ 
glucose 100 g/l 
yeast extract 5 g/l 
MgSO.sub.4.7H.sub.2 O 
0.2 g/l 
FeSO.sub.4.7H.sub.2 O 
10 mg/l 
MnSO.sub.4.4.5H.sub.2 O 
10 mg/l 
NaCl 10 mg/l 
CaCO.sub.3 60 g/l 
______________________________________ 
37 hours after the initiation of the fermentation, the glucose was 
completely consumed and 98 g/l of lactic acid containing 99.2% of D-lactic 
acid was accumulated in the medium. After the completion of the first 
D-lactic acid fermentation, 150 ml portions of the resulting broth were 
added to 2850-ml portions of (A) the fermentation medium as shown above 
(Comparative Example 1), (B) the fermentation medium as shown above except 
that the concentration of the yeast extract was elevated to 7.5 g/l 
(Example 1), (C) the fermentation medium as shown above except that 5 g/l 
of peptone was further added thereto (Example 2) respectively, and allowed 
to ferment therein. The optical purities of D-lactic acid thus produced 
were (A) 97.3% (Comparative Example), (B) 99.7% (Example 1) and (C) 99.4% 
(Example 2). 
EXAMPLES 3 AND 4 AND COMATIVE EXAMPLE 2 
The first D-lactic acid fermentation was carried out in the same manner as 
described in Example 1. 39 hours after the initiation of the fermentation, 
the glucose was completely consumed and 97 g/l of lactic acid containing 
99.3% of D-lactic acid accumulated in the medium. The D-lactic acid 
fermentation broth was subjected to the subsequent fermentation wherein 
yeast extract was employed in a concentration as shown in Table 2. Table 2 
shows the result. 
TABLE 2 
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D-lactic acid 
D-lactic acid 
optical purity 
optical purity 
Yeast extract 
in 2nd in 3rd 
concentration(% 
fermentation 
fermentation 
______________________________________ 
Comp. Ex. 2 
0.5 97.8% -- 
Ex. 3 0.6 99.0% 99.2% 
Ex. 4 0.75 99.3%* 99.2% 
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EXAMPLE 5 
Sporolactobacillus inulinus ATCC 15538 was inoculated into a GYP medium and 
static cultured therein a 37.degree. C. for three days. 2 ml of the 
culturing broth was inoculated into 50 ml of a GYP medium containing 1% of 
CaCO.sub.3 and static cultured therein at 37.degree. C. for one day, thus 
preparing a seed culture. 50 ml of the obtained seed culture was 
inoculated into 950 ml of a fermentation medium having the following 
composition and fermentation was performed at 37.degree. C. 
______________________________________ 
Fermentation medium 
______________________________________ 
glucose 100 g/l 
yeast extract 5 g/l 
MgSO.sub.4.7H.sub.2 O 
0.2 g/l 
FeSO.sub.4.7H.sub.2 O 
10 mg/l 
MnSO.sub.4.4.5H.sub.2 O 
10 mg/l 
NaCl 10 mg/l 
CaCO.sub.3 60 g/l 
______________________________________ 
39 hours after the initiation of the fermentation, the glucose was 
completely consumed and 95 g/l of lactic acid containing 99.1% of D-lactic 
acid was accumulated in the medium. After the completion of the first 
D-lactic acid fermentation, 50 ml of the resulting broth was added to 950 
ml of the fermentation medium as defined above and the fermentation was 
repeated while supplying air at a rate of 0.03 V.V.M. (V.V.M. is volume 
per volume per minute). Thus D-lactic acid of a optical purity 99.0% was 
obtained. 
EXAMPLE 6 
The procedures of Example 5 were followed except that air was supplied at a 
rate of 0.075 V.V.M. Table 3 shows test results. 
TABLE 3 
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1st fermentation 
2nd fermentation 
D-lactic acid air supply rate 
D-lactic acid 
optical purity (%) 
(V.V.M.) purity (%) 
______________________________________ 
Ex. 5 
99.1 0.030 99.0 
Ex. 6 
98.9 0.075 99.0 
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EXAMPLE 7 
Sporolactobacillus inulinus ATCC 15538 was inoculated into a GYP medium and 
static cultured therein at 37.degree. C. for three days. 2 ml of the 
culturing broth was inoculated into 50 ml of a GYP medium containing 1% of 
CaCO.sub.3 and static cultured therein at 37.degree. C. for one day, thus 
preparing a seed culture. 50 ml of the obtained seed culture was 
inoculated into 950 ml of a fermentation medium having the following 
composition and fermentation was performed at 37.degree. C. while 
controlling the pH value of the culture at 5.8 to 6.2 with an 8.5% aqueous 
solution of ammonia. 
______________________________________ 
Fermentation medium 
______________________________________ 
glucose 100 g/l 
yeast extract 5 g/l 
MgSO.sub.4.7H.sub.2 O 
0.2 g/l 
FeSO.sub.4.7H.sub.2 O 
10 mg/l 
MnSO.sub.4.4.5H.sub.2 O 
10 mg/l 
NaCl 10 mg/l 
______________________________________ 
41 hours after the initiation of the fermentation, the glucose was 
completely consumed and 82 g/l of lactic acid containing 99.5% of D-lactic 
acid was accumulated in the medium. After the completion of the first 
D-lactic acid fermentation, 50 ml of the resulting broth was added to 950 
ml of the fermentation medium as defined above and the fermentation was 
repeated. Thus D-lactic acid of a optical purity of 99.2% was obtained. 
The third fermentation was carried out in the same manner with the use of 
a broth obtained in the second fermentation as a seed culture. Thus, 
D-lactic acid of a optical purity of 99.3% was obtained. 
EXAMPLE 8 
A seed culture prepared in the same manner as described in Example 7 was 
cultured in a fermentation medium having the following composition while 
controlling the pH value of the culture at 5.8 to 6.6 with a 20% aqueous 
solution of sodium hydroxide at 37.degree. C. 
______________________________________ 
glucose 200 g/l 
yeast extract 10 g/l 
MgSO.sub.4.7H.sub.2 O 
0.2 g/l 
FeSO.sub.4.7H.sub.2 O 
10 mg/l 
MnSO.sub.4.4.5H.sub.2 O 
10 mg/l 
NaCl 10 mg/l 
______________________________________ 
50 hours after the initiation of the fermentation, the glucose was 
completely consumed and 128 g/l lactic acid containing 99.2% of D-lactic 
acid was accumulated in the medium. The fermentation was repeated five 
times in the same manner as described in Example 7. The purities of 
D-lactic acid obtained in the third and fifth fermentations were 98.9% and 
99.1% respectively.