U.S. patent application number 15/738888 was filed with the patent office on 2020-06-25 for process for producing lactic acid or its salts from fermentation using thermotolerance bacillus bacteria.
This patent application is currently assigned to PTT Global Chemical Public Company Limited. The applicant listed for this patent is PTT Global Chemical Public Company Limited Chulalongkorn University. Invention is credited to Phatthanon Prasitchoke, Nuttha Thongchul, Vasana Tolieng.
Application Number | 20200199633 15/738888 |
Document ID | / |
Family ID | 57607324 |
Filed Date | 2020-06-25 |
United States Patent
Application |
20200199633 |
Kind Code |
A2 |
Thongchul; Nuttha ; et
al. |
June 25, 2020 |
PROCESS FOR PRODUCING LACTIC ACID OR ITS SALTS FROM FERMENTATION
USING THERMOTOLERANCE BACILLUS BACTERIA
Abstract
This invention relates to a process for producing lactic acid or
its salts that can be performed easily, reduce complicated steps,
and provide high lactic acid yield and high productivity, wherein
said process comprising the following steps: (a) cultivating
thermotolerance Bacillus genus bacteria to obtain a seed culture;
(b) increasing cell number of bacteria by inoculating the seed
culture obtained from step (a) into a fermenter containing an
initial carbon source under an aerobic condition; (c) fermenting
the seed culture obtained from step (b) in the fermenter under a
microaerobic condition to obtain lactic acid or its salts; wherein
the step (b) comprising at least one addition of the carbon source
under any one of the following conditions, which are independent to
each other, to increase a concentration of the carbon source:
--when the concentration of carbon source in the fermenter reduces
to 50% or less comparing to the initial concentration.
Inventors: |
Thongchul; Nuttha; (Bangkok,
TH) ; Tolieng; Vasana; (Bangkok, TH) ;
Prasitchoke; Phatthanon; (Bangkok, TH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PTT Global Chemical Public Company Limited
Chulalongkorn University |
Chatuchak Bangkok
Bangkok |
|
TH
TH |
|
|
Assignee: |
PTT Global Chemical Public Company
Limited
Bangkok
TH
Chulalongkorn University
Bangkok
TH
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20190100779 A1 |
April 4, 2019 |
|
|
Family ID: |
57607324 |
Appl. No.: |
15/738888 |
Filed: |
June 28, 2016 |
PCT Filed: |
June 28, 2016 |
PCT NO: |
PCT/TH2016/000058 PCKC 00 |
371 Date: |
December 21, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 1/22 20130101; C12P
7/56 20130101 |
International
Class: |
C12P 7/56 20060101
C12P007/56; C12N 1/22 20060101 C12N001/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2015 |
TH |
1501003766 |
Claims
1. A process for producing lactic acid or its salts, comprising the
steps of: (a) cultivating thermotolerance Bacillus genus bacteria
to obtain a seed culture; (b) increasing the cell number of the
thermotolerance Bacillus genus bacteria by inoculating the seed
culture obtained from step (a) into a fermenter containing an
initial carbon source under an aerobic condition; (c) fermenting
the seed culture obtained from step (b) in the fermenter under a
microaerobic condition to obtain lactic acid or its salts; wherein
step (b) further comprises at least one addition of the carbon
source under any one of the following conditions, which are
independent of each other, to increase the concentration of the
carbon source: when the concentration of the carbon source in the
fermenter reduces to 50% or less compared to the initial
concentration; when step (b) is carried out for a time of at least
one third; when an optical density (OD) of bacteria cells in the
fermenter increases by at least 10 times.
2. The process for producing lactic acid or its salts according to
claim 1, wherein the addition of the carbon source in step (b)
increases the concentration of the carbon source in the fermenter
to 75% or more compared to the initial concentration.
3. The process for producing lactic acid or its salts according to
claim 1, wherein the addition of the carbon source in step (b) is
performed under the condition that the concentration of the carbon
source in the fermenter reduces to 25% or less compared to the
initial concentration.
4. The process for producing lactic acid or its salts according to
claim 1, wherein the addition of the carbon source is performed
under the condition that step (b) is carried out for a time of at
least one half.
5. The process for producing lactic acid or its salts according to
claim 1, wherein the addition of the carbon source is performed
under the condition that the optical density (OD) of bacteria cells
in the fermenter increases by 10 to 50 times.
6. The process for producing lactic acid or its salts according to
claim 1, wherein the concentration of the initial carbon source in
step (b) is in a range of from 10 to 20 g/L.
7. The process for producing lactic acid or its salts according to
claim 1, wherein the concentration of the initial carbon source in
step (b) is 15 g/L.
8. The process for producing lactic acid or its salts according to
claim 1, wherein said process is carried out at a temperature in a
range of from 45 to 60.degree. C.
9. The process for producing lactic acid or its salts according to
claim 8, wherein said process is carried out at the temperature in
the range of from 48 to 52.degree. C.
10. The process for producing lactic acid or its salts according to
claim 1, wherein the thermotolerance Bacillus genus bacteria in
step (a) is selected from Bacillus acidiproducens, Bacillus
coagulans, or a mixture of these bacteria.
11. The process for producing lactic acid or its salts according to
claim 1, wherein step (a) is carried out for 2 to 5 hours.
12. The process for producing lactic acid or its salts according to
claim 11, wherein step (a) is carried out for 3 to 4 hours.
13. The process for producing lactic acid or its salts according to
claim 1, wherein step (b) is carried out for 2 to 5 hours.
14. The process for producing lactic acid or its salts according to
claim 13, wherein step (b) is carried out for 3 to 4 hours.
15. The process for producing lactic acid or its salts according to
claim 14, wherein step (b) is carried out for 4 hours.
16. The process for producing lactic acid or its salts according to
claim 1, wherein step (c) further comprises at least one addition
of the carbon source.
17. The process for producing lactic acid or its salts according to
claim 1, wherein the concentration of the carbon source in step (c)
is in a range of from 100 to 200 g/L.
18. The process for producing lactic acid or its salts according to
claim 17, wherein the concentration of the carbon source in step
(c) is 100 g/L.
19. The process for producing lactic acid or its salts according to
claim 1, wherein step (c) is controlled so as to have a pH of 6 to
7 with the use of basic solution.
20. The process for producing lactic acid or its salts according to
claim 19, wherein step (c) is controlled so as to have a pH of 6.5
with the use of basic solution.
21. The process for producing lactic acid or its salts according to
claim 19, wherein the basic solution is selected from sodium
hydroxide, ammonium hydroxide, potassium hydroxide, or a mixture
thereof.
22. The process for producing lactic acid or its salts according to
claim 21, wherein the basic solution is sodium hydroxide or
potassium hydroxide.
23. The process for producing lactic acid or its salts according to
claim 22, wherein the basic solution is sodium hydroxide.
24. The process for producing lactic acid or its salts according to
claim 1, wherein the carbon source is a fermentable sugar.
25. The process for producing lactic acid or its salts according to
claim 24, wherein the fermentable sugar is selected from
monosaccharide, disaccharide, trisaccharide, or a mixture
thereof.
26. The process for producing lactic acid or its salts according to
claim 24 wherein the monosaccharide is selected from glucose,
fructose, galactose, or a mixture thereof.
27. The process for producing lactic acid or its salts according to
claim 24, wherein the disaccharide is selected from sucrose,
lactose, maltose, cellobiose, or a mixture thereof.
28. The process for producing lactic acid or its salts according to
claim 24, wherein the trisaccharide is selected from raffinose,
isomaltotriose, maltotriose, nigerotriose, kestose, or a mixture
thereof.
29. The process for producing lactic acid or its salts according to
claim 24, wherein the fermentable sugar is glucose.
30. The process for producing lactic acid or its salts according to
claim 1, wherein the fermenter further comprises a mixer.
31. The process for producing lactic acid or its salts according to
claim 30, wherein the mixer has a speed in a range of from 150 to
450 rpm.
32. The process for producing lactic acid or its salts according to
claim 31, wherein the mixer has a speed of 300 rpm.
33. The process for producing lactic acid or its salts according to
claim 2, wherein the addition of the carbon source in step (b) is
performed under the condition that the concentration of the carbon
source in the fermenter reduces to 25% or less compared to the
initial concentration.
34. The process for producing lactic acid or its salts according to
claim 2, wherein the addition of the carbon source is performed
under the condition that step (b) is carried out for a time of at
least one half.
35. The process for producing lactic acid or its salts according to
claim 2, wherein the addition of the carbon source is performed
under the condition that the optical density (OD) of bacteria cells
in the fermenter increases by 10 to 50 times.
36. The process for producing lactic acid or its salts according to
claim 6, wherein the concentration of the initial carbon source in
step (b) is 15 g/L.
37. The process for producing lactic acid or its salts according to
claim 10, wherein step (a) is carried out for 2 to 5 hours.
38. The process for producing lactic acid or its salts according to
claim 16, wherein the concentration of the carbon source in step
(c) is in a range of from 100 to 200 g/L.
Description
SUMMARY OF THE INVENTION
[0001] This invention relates to a process for producing lactic
acid or its salts from fermentation using thermotolerance Bacillus
bacteria, said process comprising the following steps:
[0002] (a) cultivating thermotolerance Bacillus bacteria to obtain
seed culture;
[0003] (b) increasing cell number of bacteria by inoculating the
seed culture obtained from step (a) into a fermenter containing an
initial carbon source under an aerobic condition;
[0004] (c) fermenting the seed culture obtained from step (b) in
the fermenter under a microaerobic condition to obtain lactic acid
or its salts;
[0005] wherein the step (b) comprising at least one addition of the
carbon source under any one of the following conditions, which are
independent to each other, to increase a concentration of the
carbon source: [0006] when the concentration of the carbon source
in the fermenter reduces to 50% or less comparing to the initial
concentration; [0007] when the step (b) is carried out for a time
of at least one third; [0008] when an optical density (OD) of
bacterial cell in the fermenter increases at least 10 times.
FIELD OF THE INVENTION
[0009] This invention is in the field of biotechnology relating to
the fermentation process and bacteria that can produce lactic
acid.
BACKGROUND OF THE INVENTION
[0010] It is well known that lactic acid is widely used in plastic
industry, food and drug industry, and cosmetic industry. For
plastic industry, lactic acid is widely used, especially for the
production of polyester such as polylactic acid or
poly(lactic-co-glycolic acid). Polymer produced from lactic acid
has an advantage that it is biodegradable and biocompatible. Said
polymer can be used in many applications such as fiber in textile,
film, catgut packaging, and scaffold in medical.
[0011] At present, there are several production processes of lactic
acid such as chemical synthesis and biotechnology. The
biotechnology has several advantages, including utilization of
renewable resources for microbial fermentation such as cassava,
corn, wheat, or cane. Moreover, the microbial fermentation can
produce lactic acid with high optical purity.
[0012] Most of lactic acid productions are the fermentation of
sugar such as glucose, sucrose, maltose, or other carbohydrates
such as starch or cellulose, wherein microorganisms that can
produce lactic acid can be both bacteria and fungi.
[0013] Bacteria in genus Lactobacillus, Leuconostoc, and
Streptococcus are well known for the production of lactic acid from
sugar under an anaerobic condition, leading to energy saving and
providing higher yield than the one produced by fungi. However,
said bacteria genus are fastidious bacteria, which need vitamins
and essential amino acids for their growth. Moreover, said bacteria
genus cannot produce enzyme to convert starch into sugar, which
needs the pretreatment step of converting starch to sugar prior to
fermentation. This increases the production cost.
[0014] One problem of polymer production from lactic acid is high
production cost. It is necessary to develop the lactic acid
production process with lower production cost, high yield and high
productivity. In addition, there are several attempts to develop
microorganisms and culturing steps in order to produce robust
microorganisms that have ability to grow and proliferate with high
rate.
[0015] One attempt to reduce the lactic acid production cost is the
reduction of production step such as the use of thermotolerance
Bacillus bacteria in lactic acid production. This is because
normally the raw material of lactic acid production requires a
pretreatment process prior to fermentation. Several pretreatment
can be used, including mechanical treatment, heat treatment,
chemical treatment, or enzyme treatment, depending on the physical,
chemical, and nutritional property of such carbon sources. Said
pretreatment processes are generally performed at high temperature
in a range from about 50 to about 100.degree. C. General bacteria
cannot grow at said temperature. Therefore, an additional step is
needed to reduce the temperature to a room temperature prior to
using such carbon sources in lactic acid production. This results
in complication of production process and higher production
cost.
[0016] Therefore, there had been developments in production process
of lactic acid using thermotolerance Bacillus bacteria such as
Bacillus coagulans, Bacillus acidiproducens, Bacillus
stearothermophilus, and Bacillus licheniformis as disclosed in
Ouyang et al, (Appl. Biochem. Biotechnol., 2012, 168, 2387-2397),
Wang et al, (Bioresour. Technol., 2011, 102, 8152-8158), and U.S.
Pat. No. 8,119,376. However, it was found that the production of
lactic acid as disclosed in said documents resulted in low
productivity and yield, which was the limitation for the production
of lactic acid in the industrial scale.
[0017] One attempt to reduce lactic acid production cost is to
eliminate the purifying step of lactic acid obtained from
fermentation. The neutralizing agent is one of important factors.
Generally, calcium carbonate (CaCO.sub.3) that is used in the
production of lactic acid generates calcium lactate as by product.
Therefore, the acidification step is further needed to convert
calcium lactate into lactic acid by using acid solution such as
sulfuric acid (H.sub.2SO.sub.4). This contributes to the
complication of production process and high production cost.
Moreover, said process causes calcium sulfate (CaSO.sub.4) which is
the undesired by product, leading to a problem to eliminate said
substance, especially in the industrial scale production.
[0018] U.S. Pat. No. 5,002,881 and WO2006124633 disclosed the use
of ammonium hydroxide (NH.sub.4OH) as neutralizing agent during
fermentation in lactic acid production. However, ammonium hydroxide
is a volatile compound which could be harmful to operator's health.
Besides, ammonium hydroxide is a weak basic; thus, it is needed to
be used in high amount to control pH that results in a difficulty
in separation and purification steps.
[0019] Ye et al, (Bioresour. Technol., 2013, 132, 38-44), Qin et
al, (Bioresour. Technol., 2010, 101, 7570-7576) disclosed the use
of strong basic such as sodium hydroxide (NaOH) and potassium
hydroxide (KOH) during the fermentation of lactic acid production.
However, it was found that the use of said basic caused the
negative effect on the bacteria cell, resulting in low productivity
and yield.
[0020] From the reasons mentioned above, this invention aims to
improve the process for producing lactic acid or its salt from
fermentation to provide high productivity and yield, wherein said
process can be performed easily and reduce complicated steps.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0021] Technical terms or scientific terms used herein have
definitions as understood by those having an ordinary skill in the
art, unless stated otherwise.
[0022] Any tools, equipment, methods, or chemicals mentioned here
mean tools, equipment, methods, or chemicals commonly operated or
used by those skilled in the art, unless explicated stated
otherwise that they are tools, equipment, methods, or chemicals
specifically used in this invention.
[0023] Use of singular noun or singular pronoun with "comprising"
in the claims or the specification refers to "one" and also "one or
more", "at least one", and "one or more than one".
[0024] Throughout this application, the term "about" is used to
indicate that any value presented or showed herein may potentially
vary or deviate. Such variation or deviation may result from errors
of equipment, method, or from individual operator implementing
equipment or method. These variations or deviations caused by the
changes of physical properties.
[0025] "Microaerobic condition" means condition that air has been
controlled to be limited without further adding of any gas,
including air or inert gas to evacuate the existing air.
[0026] "Concentration of carbon source" unless stated specifically,
means the concentration of carbon source in such system such as in
a fermenter at a specific time.
[0027] Hereafter, invention embodiments are shown without any
purpose to limit the scope of the invention.
[0028] This invention relates to the process for producing lactic
acid or its salts from fermentation using thermotolerance Bacillus
bacteria. Said process comprising the following steps:
[0029] (a) cultivating thermotolerance Bacillus bacteria to obtain
a seed culture;
[0030] (b) increasing cell number of bacteria by inoculating the
seed culture obtained from step (a) into a fermenter containing an
initial carbon source under an aerobic condition;
[0031] (c) fermenting the seed culture obtained from step (b) in
the fermenter under a microaerobic condition to obtain lactic acid
or its salts;
[0032] wherein the step (b) comprising at least one addition of the
carbon source under any one of the following conditions, which are
independent to each other, to increase a concentration of carbon
source: [0033] when the concentration of the carbon source in the
fermenter reduces to 50% or less comparing to the initial
concentration; [0034] when the step (b) is carried out for a time
of at least one third; [0035] when an optical density (OD) of
bacteria cell in the fermenter increases at least 10 times.
[0036] In one embodiment, the addition of carbon source in step (b)
in any above conditions increases the concentration of the carbon
source in the fermenter to 75% or more comparing to the initial
concentration. The addition of said carbon source includes the
addition that results in the concentration larger than the initial
concentration.
[0037] Preferably, the addition of carbon source is performed under
the condition that the concentration of the carbon source in the
fermenter reduces to 25% or less comparing to the initial
concentration.
[0038] Preferably, the addition of the carbon source is performed
under the step (b) is carried out for the time of at least one
half.
[0039] Preferably, the addition of the carbon source is performed
under the condition that the optical density (OD) of bacteria cell
in the fermenter increases at least 10 to 50 times.
[0040] In one embodiment, the concentration of the initial carbon
source in step (b) may be in a range of 10 to 20 g/L, preferably
about 15 g/L.
[0041] In one embodiment, said process for producing lactic acid or
its salts may be carried out at the temperature in a range of about
45 to about 60.degree. C., preferably at the temperature about 48
to 52.degree. C.
[0042] In one embodiment, the thermotolerance Bacillus genus in the
step (a) may be selected from Bacillus acidiproducens, Bacillus
coagulans, or a mixture of these bacteria.
[0043] In one embodiment, the initial optical density of bacteria
cell in the step (a) is about 0.20 to 0.70, preferably 0.20 to
0.40.
[0044] In one embodiment, the step (a) may be carried out for about
2 to 5 hours, preferably about 3 to 4 hours.
[0045] In one embodiment, a bacteria cultivation media may contain
nitrogen source that may be selected from yeast extract, peptone
extract, beef extract and legume extract.
[0046] In one embodiment, the bacteria cultivation media and
bacteria cell increasing media may contain inorganic nitrogen
source that may be selected from ammonium chloride, ammonium
nitrate, and ammonium dihydrogen phosphate. The use of said
inorganic compounds for nitrogen source has benefit in the
production of lactic acid in industrial scale because they are
cheap, affordable, and can be quality controlled.
[0047] In one embodiment, the step (b) may be carried out for about
2 to 5 hours, preferably about 3 to 4 hours, and most preferably
about 4 hours.
[0048] In one embodiment, the step (c) further comprises at least
one addition of the carbon source.
[0049] In one embodiment, the concentration of carbon source in
step (c) may be in a range of about 100 to 200 g/L, preferably
about 100 g/L.
[0050] In one embodiment, the step (c) is controlled to have pH of
6 to 7 with a use of basic solution, preferably have pH of 6.5 with
a use of basic solution.
[0051] In one embodiment, the basic solution may be selected from
sodium hydroxide (NaOH), ammonium hydroxide (NH.sub.4OH), potassium
hydroxide (KOH), or a mixture thereof, preferably sodium hydroxide
or potassium hydroxide, and most preferably sodium hydroxide.
[0052] In one embodiment, the carbon source used in the production
of lactic acid or its salt is a fermentable sugar.
[0053] The fermentable sugar is any sugar that can be found in
nature or any sugar derived from a substance comprising sugar. Said
sugar may be modified or unmodified.
[0054] The fermentable sugar may be selected from, but not limited
to monosaccharide, disaccharide, trisaccharide, or a mixture
thereof.
[0055] In one embodiment, the monosaccharide may be selected from
glucose, fructose, galactose, or a mixture thereof.
[0056] In one embodiment, the disaccharide may be selected from
sucrose, lactose, maltose, cellobiose, or a mixture thereof.
[0057] In one embodiment, the trisaccharide may be selected from
raffinose, isomaltotriose, maltotriose, nigerotriose, kestose, or a
mixture thereof.
[0058] Preferably, the fermentable sugar is glucose.
[0059] In one embodiment, the fermenter in step (b) and/or step (c)
further comprises a mixer.
[0060] In one embodiment, the mixer has a speed in a range of about
150 to 450 rpm, preferably about 300 rpm.
[0061] In one embodiment, the change of fermenter condition from
aerobic condition in step (b) to microaerobic condition in step (c)
may be performed by stopping aeration in the fermenter in order
that the remaining air from step (b) has been consumed, or adding
nitrogen to replace air during fermentation in step (c).
[0062] In one embodiment, the fermentation in step (c) may be
performed in batch, semi-batch, or continuous.
[0063] In another embodiment, said production of lactic acid or its
salt may further comprises the step of separating and purifying the
mixture obtained from step (c).
[0064] The separation and purification may be selected from, but
not limited to centrifugation, filtration, membrane filtration,
flocculation, extraction, distillation, crystallization,
filtration, ion exchange resin, or electrodialysis.
[0065] The following is the property testing according to the
invention, wherein the methods and equipment used in the test are
commonly used methods and are not intended to limit the scope of
the invention.
[0066] Glucose and lactic acid are analyzed by high performance
liquid chromatography using a Shimadzu equipped with Biorad, Aminex
HPX-87H ion exclusion organic acid (300 mm.times.7.8 mm) at a
temperature around 45.degree. C., and reflective index detector
Shimadzu-RID-10A for detecting a signal comparing to a standard
signal.
[0067] Optical density (OD) of bacteria during cultivation and
fermentation is analyzed by spectrophotometry at a wavelength 600
nm.
[0068] Yield is calculated from a ratio of an amount of produced
lactic acid to an amount of carbon source used during
fermentation.
[0069] Yield of produced lactic acid per bacteria cell (Y.sub.p/x)
represents the effectiveness of bacteria cell in the production of
lactic acid, which is calculated from a ratio of an amount of
produced lactic acid and a difference between the bacteria optical
density after fermentation step and after increasing of bacteria
cell step.
[0070] The following examples are presented to illustrate the
present invention without limiting the scope of the invention.
[0071] In the following examples, unless stated specifically, media
for culturing bacteria and increasing bacteria cell has composition
(per liter) as following: 10 g glucose, 15 g yeast extract, 4 g
ammonium chloride (NH.sub.4Cl), 5 g calcium hydroxide
(Ca(OH).sub.2), and 20 mL saline solution.
Multiple Additions of the Carbon Source in the Step of Increasing
Bacteria Cell
[0072] To study the effect of multiple additions of carbon source
in the step of increasing bacteria cell on lactic acid or its salt
production, a sample without adding carbon source in the step of
increasing bacteria cell is used to compare with lactic acid
production according to the invention.
Comparative Example (without Adding the Carbon Source in the Step
of Increasing Bacteria Cell)
[0073] The cultivation of bacteria is carried out by adding
thermotolerance Bacillus genus in a flask containing bacteria
culturing media, wherein the initial concentration of bacteria is
about 1% by volume and the initial optical density is about
0.30-0.40. The sample is centrifuged at about 250 rpm for 3 hours
at a temperature of about 50.degree. C. to obtain a seed culture.
Then, bacteria cell is increased by adding the seed culture in a 5
L fermenter containing about 2.5 L culture media with initial
glucose concentration of about 15 g/L and calcium hydroxide
(Ca(OH).sub.2) to control pH to be about 6.5. The step of
increasing bacteria cell is performed at the temperature about
50.degree. C. for about 3 hours under 1 vvm aeration and 300 rpm
mixing. Then, the aeration is stopped and about 1 L of 350 g/L
glucose solution is added into the fermenter in order to achieve
the initial glucose concentration of 100 g/L at the beginning of
the fermentation step. The fermentation is operated under a
microaerobic condition at the temperature of about 50.degree. C.
and the mixing speed of about 300 rpm by using various basic
solutions in Table 1 to control pH to be about 6.5. The
fermentation is performed until glucose is not detected. Then, the
products are centrifuged at about 10,000 rpm for about 5 minutes.
The obtained products are analyzed for produced lactic acid and the
optical density of bacteria during fermentation.
Examples According to the Invention
[0074] The thermotolerance Bacillus genus is added into a flask
containing bacteria culturing media, wherein the initial
concentration of bacteria is about 1% by volume and the initial
optical density is about 0.30-0.40. The sample is centrifuged at
about 250 rpm for 3 hours at a temperature of about 50.degree. C.
to obtain a seed culture. Then, bacterial cell is increased by
adding the seed culture in a 5 L fermenter containing about 2.5 L
culture media with initial glucose concentration of about 15 g/L
and calcium hydroxide (Ca(OH).sub.2) to control pH to be about 6.5.
The step of increasing bacteria cell is performed at the
temperature about 50.degree. C. for about 2 hours under 1 vvm
aeration and 300 rpm mixing. Then, about 0.5 L of 90 g/L glucose
solution was added. The step of increasing bacteria cell is
operated for another 2 hours. Then, the fermentation is performed
under a microaerobic condition at the temperature of about
50.degree. C. and the mixing speed of about 300 rpm by using
various basic solutions in Table 1 to control pH to be about 6.5.
The fermentation is performed until glucose is not detected. Then,
the products are centrifuged at about 10,000 rpm for about 5
minutes. The obtained products are analyzed for produced lactic
acid and the optical density of bacteria during fermentation.
[0075] From Table 1, when comparing examples A1, A2, A3 to
comparative examples 1a, 2a, and 3a, respectively which are the
lactic acid production by using Bacillus acidiproducens strain, it
can be found that the step of increasing bacteria cell comprising
at least one addition of the carbon source results in the higher
Y.sub.p/x, productivity, and yield. Moreover, it can be found that
the lactic acid production according to the invention can use
sodium hydroxide and potassium hydroxide solutions which are strong
acids to control pH during fermentation and gives rise to high
lactic acid yield.
[0076] Furthermore, when comparing examples B1, B2, B3 to
comparative examples 1b, 2b, and 3b, respectively which are the
lactic acid production by using Bacillus coagulans strain, it can
be observed that at least one addition of the carbon source in the
step of increasing bacteria cell results in greatly higher
Y.sub.p/x. This indicates an increase of efficiency in utilizing
carbon source.
[0077] Therefore, regarding above results, it can be summarized
that the lactic acid production according to the invention can
enhance the efficiency of lactic acid production from
thermotolerance Bacillus genus, which can be performed easily and
can reduce complicated steps as indicated in the objective of this
invention.
TABLE-US-00001 TABLE 1 The lactic acid bacteria production from
thermotolerance Bacillus genus in various conditions Optical
density Optical density Basic (OD) before (OD) after solution in
Fermentation the step of the step of Optical density Lactic acid
fermentation period increasing increasing (OD) after concentration
Yield Productivity Example step (hr) bacteria cell bacteria cell
fermentation (g/L) (g/g) (g/L/hr) Y.sub.p/x Bacillus acidiproducens
strain Comparative 7M 17 0.32 7.7 20.7 87.3 0.87 5.1 6.7 Example 1a
NH.sub.4OH Comparative 10M 19 0.32 7.0 22.0 99.9 0.94 5.6 6.6
Example 2a NaOH Comparative 10M 18 0.30 11.4 25.1 102.9 0.95 5.7
7.5 Example 3a KOH Example A1 7M 17 0.28 10.0 21.1 99.1 0.99 5.9
8.2 NH.sub.4OH Example A2 10M 18 0.35 11.3 22.6 108.9 1.04 6.1 8.8
NaOH Example A3 10M 19 0.31 12.5 23.7 102.0 0.92 5.5 9.1 KOH
Bacillus coagulans strain Comparative 7M 18 0.31 9.3 21.9 93.5 0.90
5.2 7.4 Example 1b NH.sub.4OH Comparative 10M 12 0.32 10.4 20.4
94.4 0.94 7.9 9.4 Example 2b NaOH Comparative 10M 18 0.32 9.0 21.6
102.6 1.00 5.7 8.1 Example 3b KOH Example B1 7M 20 0.31 13.6 28.3
102.8 1.00 5.1 6.9 NH.sub.4OH Example B2 10M 20 0.38 10.9 17.2
108.0 1.00 5.4 17.5 NaOH Example B3 10M 20 0.26 9.5 16.5 102.9 0.97
5.2 14.7 KOH
BEST MODE OF THE INVENTION
[0078] Best mode of the invention is as disclosed in the detailed
description.
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