U.S. patent application number 14/027754 was filed with the patent office on 2014-01-16 for method of producing lactic acid by continuous fermentation.
This patent application is currently assigned to TORAY INDUSTRIES, INC.. The applicant listed for this patent is Takashi Mimitsuka, Ken Morita, Nanami Sasaki, Hideki Sawai, Katsushige Yamada. Invention is credited to Takashi Mimitsuka, Ken Morita, Nanami Sasaki, Hideki Sawai, Katsushige Yamada.
Application Number | 20140017746 14/027754 |
Document ID | / |
Family ID | 40952119 |
Filed Date | 2014-01-16 |
United States Patent
Application |
20140017746 |
Kind Code |
A1 |
Sasaki; Nanami ; et
al. |
January 16, 2014 |
METHOD OF PRODUCING LACTIC ACID BY CONTINUOUS FERMENTATION
Abstract
A method of producing lactic acid by continuous fermentation
includes filtering a culture of polyploidy prototrophic yeast
having a capacity to produce lactic acid by introduction of a
lactate dehydrogenase gene through a porous membrane having an
average pore size of not less than 0.01 .mu.m and less than 1
.mu.m; and recovering the lactic acid from filtrate while the
unfiltered liquid is retained in or returned to the culture and a
fermentation feedstock is added to the culture.
Inventors: |
Sasaki; Nanami; (Kanagawa,
JP) ; Morita; Ken; (Kanagawa, JP) ; Mimitsuka;
Takashi; (Kanagawa, JP) ; Sawai; Hideki;
(Kanagawa, JP) ; Yamada; Katsushige; (Kanagawa,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sasaki; Nanami
Morita; Ken
Mimitsuka; Takashi
Sawai; Hideki
Yamada; Katsushige |
Kanagawa
Kanagawa
Kanagawa
Kanagawa
Kanagawa |
|
JP
JP
JP
JP
JP |
|
|
Assignee: |
TORAY INDUSTRIES, INC.
Tokyo
JP
|
Family ID: |
40952119 |
Appl. No.: |
14/027754 |
Filed: |
September 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12865934 |
Aug 6, 2010 |
8551745 |
|
|
PCT/JP2009/051750 |
Feb 3, 2009 |
|
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14027754 |
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Current U.S.
Class: |
435/139 |
Current CPC
Class: |
C12P 7/56 20130101 |
Class at
Publication: |
435/139 |
International
Class: |
C12P 7/56 20060101
C12P007/56 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 4, 2008 |
JP |
2008-023917 |
Feb 15, 2008 |
JP |
2008-034462 |
Claims
1. A method of producing lactic acid by continuous fermentation
comprising: filtering a culture of polyploidy prototrophic yeast
having a capacity to produce lactic acid by introduction of a
lactate dehydrogenase gene through a porous membrane having an
average pore size of not less than 0.01 .mu.m and less than 1
.mu.m; and recovering the lactic acid from filtrate while the
unfiltered liquid is retained in or returned to the culture and a
fermentation feedstock is added to the culture.
2. The method according to claim 1, wherein said filtration is
carried out with a transmembrane pressure difference of said porour
membrane within a range of 0.1 kPa to less than 20 kPa.
3. The method according to claim 1, wherein said polyplid yeast is
diploid.
4. The method according to claim 1, wherein the yield of lactic
acid to saccharides in said continuous fermentation is not less
than 70%.
5. The method according to claim 1, wherein the concentration of
accumulated lactic acid in the culture subjected to said continuous
fermentation is not less than 40 g/L.
6. The method according to claim 1, wherein the rate of production
of lactic acid during said continuous fermentation is not less than
7.5 g/L/h.
7. The method according to claim 5, wherein said continuous
fermentation is continued for not less than 400 hours.
8. The method according to claim 1, wherein said polyplid
prototrophic yeast belongs to Saccharomyces.
9. The method according to claim 1, wherein said polyplid
prototrophic yeast is Saccharomyces cerevisiae.
Description
TECHNICAL FIELD
[0001] This disclosure relates to a method for producing lactic
acid by continuous fermentation, wherein polyploid yeast having a
capacity to produce lactic acid is employed to stabilize culturing
of and fermentation by the yeast, thereby enabling long-term
efficient production of lactic acid.
BACKGROUND
[0002] Methods of fermentation can be largely classified into Batch
fermentation and Fed-Batch fermentation, and continuous
fermentation. Batch fermentation and Fed-Batch fermentation can be
carried out with a simple equipment and finished in a short time,
and is therefore less prone to be suffered from contamination,
which is advantageous. However, there is a problem in that, as the
product concentration in the culture increases with time, the
productivity and the yield decrease due to the influence(s) of the
osmotic pressure, product inhibition and/or the like. Therefore, it
is difficult to maintain the stability, a high yield and a high
productivity for a long time. Continuous fermentation has an
advantage in that accumulation of a desired substance in the
fermenter at a high concentration can be avoided and thereby a high
yield and a high productivity can be maintained for a long time,
but it is very difficult to stably continue culture by continuous
fermentation for a long time, so that researches have been carried
out therefor.
[0003] As a proposal for continuous fermentation, there is a method
wherein microorganisms or cultured cells are filtered through a
separation membrane and the product is recovered from the filtrate,
while the microorganisms or cultured cells which were subjected to
the filtration are retained in or returned to the culture to
maintain a high concentration of the microorganisms or cultured
cells in the culture.
[0004] For example, technologies wherein continuous fermentation is
carried out in a continuous fermentation device having a ceramic
membrane are disclosed (JP 5-95778 A, JP 62-138184 A, JP 10-174594
A and JP 2005-333886 A). However, the disclosed technologies have
problems of decreases in the filtration flow rate and the
filtration efficiency due to clogging of the ceramic membrane, so
that reverse washing or the like is carried out to prevent the
clogging.
[0005] A method for producing succinic acid by continuous
fermentation (JP 2007-252367 A) is also disclosed. In this
technology, a high filtration pressure (about 200 kPa) is employed
for membrane separation. Since a high filtration pressure is
disadvantageous in terms of not only the cost but also physical
damage to microorganisms or cells by the pressure during
filtration, it is inappropriate for continuous fermentation wherein
microorganisms or cells are continuously returned to the culture.
In JP 2007-252367 A, as proposals for maintaining continuous
fermentation for a long time, technologies for the separation
membrane, the filtration pressure and the like are disclosed, but
the length of time of the continuous fermentation is about 300
hours, and therefore a method to maintain continuous culture for a
longer period of time is demanded.
[0006] On the other hand, studies on microorganisms to be used for
producing organic acids have been intensively carried out employing
yeasts having high tolerances for acids (Biotechnology Progress,
11, 294-298 (1995) and Journal of Fermentation and Bioengineering,
86, 284-289 (1998)). In yeasts, there are haploid yeasts having
only single sets of chromosomes and polyploid yeasts having
pluralities of sets of chromosomes. Polyploid yeasts are used
mainly as baker's yeasts and brewer's yeasts (JP 2000-139326 A, JP
2002-027974 A and JP 2002-253212 A). These are used for production
of foods and beverages for the purposes of enhancement of flavor
and improvement of the production process, and there is no
description of their usage for continuous culture.
[0007] Further, methods for producing lactic acid using polyploids
are disclosed (JP 2006-006271 A, JP 2006-020602 A, JP 2007-089466 A
and JP 2001-204464 A). These employ polyploids to increase the
number of the lactic acid synthetic gene, but the culture is
carried out by Fed-Batch fermentation and the culturing time is as
short as not more than 100 hours, and there is no description of
continuous fermentation using a membrane.
[0008] In addition, production of lactic acid using prototrophic
yeast which does not show auxotrophy was reported (US 2005/0112737
A), but also in this case, the culturing time is less than 100
hours, and Fed-Batch fermentation is the only method disclosed as a
fermentation method.
[0009] Thus, continuous fermentation to enhance the productivity,
and the microorganism to be used for fermentation have been
separately studied. In cases where microorganisms were cultured by
continuous fermentation which is advantageous as a fermentation
method, the filtration pressure increased during the culture and
continuation of the culture for a long time became impossible or
the productivity of lactic acid decreased as the culture was
continued longer, so that it has so far been difficult to allow
exertion of the both advantages sufficiently. Thus, development of
a technology for continuous fermentation which solves these
problems and allows stable production of lactic acid for a long
time has been demanded.
[0010] It could therefore be helpful to provide a method for
production of lactic acid by continuous fermentation, by which a
high productivity of lactic acid can be stably maintained for a
long time.
SUMMARY
[0011] We discovered that, to allow lactic acid-producing yeast to
maintain a high productivity of lactic acid while stably repeating
their proliferation for a long time during continuous fermentation,
maintenance of the high productivity of lactic acid for a long time
can be achieved by employing polyploid yeast having lactic
acid-producing capacity.
[0012] We thus provide: [0013] (1) A method for producing lactic
acid by continuous fermentation, wherein culture of polyploid yeast
having a capacity to produce lactic acid is filtered through a
porous membrane having an average pore size of not less than
0.01.mu..mu..mu. and less than 1 .mu.m and the product is recovered
from the filtrate, while the unfiltered liquid is retained in or
returned to the culture and a fermentation feedstock is added to
the culture. [0014] (2) The method for producing lactic acid
according to (1), wherein said filtration is carried out with a
transmembrane pressure difference of the porous membrane within the
range of 0.1 kPa to less than 20 kPa. [0015] (3) The method for
producing lactic acid according to (1) or (2), wherein the
polyploid yeast is diploid. [0016] (4) The method for producing
lactic acid according to any one of (1) to (3), wherein the
polyploid yeast is prototrophic. [0017] (5) The method for
producing lactic acid according to any one of (1) to (4), wherein
the yield of lactic acid to saccharides in the continuous
fermentation is not less than 70%. [0018] (6) The method for
producing lactic acid according to any one of (1) to (5), wherein
the concentration of accumulated lactic acid in the culture
subjected to the continuous fermentation is not less than 40 g/L.
[0019] (7) The method for producing lactic acid according to any
one of (1) to (6), wherein the rate of production of lactic acid
during the continuous fermentation is not less than 7.5 g/L/h.
[0020] (8) The method for producing lactic acid according to any
one of (5) to (7), wherein the continuous fermentation is continued
for not less than 400 hours. [0021] (9) The method for producing
lactic acid according to any one of (1) to (8), wherein the
polyploid yeast belongs to Saccharomyces. [0022] (10) The method
for producing lactic acid according to any one of (1) to (9),
wherein the polyploid yeast is Saccharomyces cerevisiae.
[0023] By using polyploid yeast, continuous fermentation is
possible which allows stable maintenance of a high productivity of
lactic acid as the desired fermentation product for a long time,
thereby enabling stable production of lactic acid at a low
cost.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a schematic side view for explanation of a
continuous fermentation device of the membrane separation type.
[0025] FIG. 2 is a schematic side view for explanation of another
continuous fermentation device of the membrane separation type.
[0026] FIG. 3 is a schematic perspective view for explanation of a
separation membrane element.
[0027] FIG. 4 is a schematic perspective view for explanation of
another example of the separation membrane element.
[0028] FIG. 5 is a vector for expression of a lactate dehydrogenase
gene.
[0029] FIG. 6 shows changes in the concentrations of accumulated
lactic acid in Examples 13 and 14 and Comparative Examples 16 and
17.
[0030] FIG. 7 shows changes in the yields of lactic acid to glucose
in Examples 13 and 14 and Comparative Examples 16 and 17.
[0031] FIG. 8 shows changes in the rates of production of lactic
acid in Examples 13 and 14 and Comparative Examples 16 and 17.
[0032] FIG. 9 shows changes in the concentrations of accumulated
lactic acid in Examples 15 and 16 and Comparative Examples 18 and
19.
[0033] FIG. 10 shows changes in the yields of lactic acid to
glucose in Examples 15 and 16 and Comparative Examples 18 and
19.
[0034] FIG. 11 shows changes in the rates of production of lactic
acid in Examples 15 and 16 and Comparative Examples 18 and 19.
DESCRIPTION OF SYMBOLS
[0035] 1. Fermentation reaction vessel [0036] 2. Separation
membrane element [0037] 3. Hydraulic head difference controlling
apparatus [0038] 4. Gas supplying apparatus [0039] 5. Stirrer
[0040] 6. Level sensor [0041] 7. Culture medium supplying pump
[0042] 8. pH adjustment solution supplying pump [0043] 9. pH
sensor/controlling apparatus [0044] 10. Temperature controller
[0045] 11. Fermentation liquid circulating pump [0046] 12. Membrane
separation vessel [0047] 13. Supporting plate [0048] 14. Channel
material [0049] 15. Separation membrane [0050] 16. Recess [0051]
17. Liquid collection pipe [0052] 18. Separation membrane bundle
[0053] 19. Upper resin sealing layer [0054] 20. Lower resin sealing
layer [0055] 21. Support frame [0056] 22. Liquid collection
pipe
DETAILED DESCRIPTION
[0057] We provide a method for producing lactic acid by continuous
fermentation while, in fermentation by culturing yeast having a
capacity to produce lactic acid, maintaining a high productivity of
lactic acid for a long time by using polyploid yeast, wherein,
during the continuous fermentation, culture of the polyploid yeast
having a capacity to produce lactic acid is filtered through a
separation membrane and the product is recovered from the filtrate,
while allowing the unfiltered liquid to be retained in or returned
to the culture and adding a fermentation feedstock to the culture,
which separation membrane has an average pore size of not less than
0.01 .mu.m and less than 1 .mu.m.
[0058] First, the porous membrane as a separation membrane will be
described. The porous membrane preferably has a separation
performance and a permeability depending on the properties and the
use of the liquid to be processed. The porous membrane preferably
has a porous resin layer in view of the blocking performance,
permeability and separation performance, for example, resistance to
dirt.
[0059] The porous membrane comprising a porous resin layer
preferably has a porous resin layer on the surface of a porous base
material, which layer acts as a separation function layer. The
material of the porous base material comprises an organic material,
inorganic material and/or the like, and preferably an organic
material, more preferably an organic fiber is used. Examples of the
porous base material preferred among these include woven fabrics
and non-woven fabrics using organic fibers such as cellulose
fibers, cellulose triacetate fibers, polyester fibers,
polypropylene fibers and polyethylene fibers, and a non-woven
fabric whose density can be relatively easily controlled, whose
production is easy and which is inexpensive is more preferably
used. The thickness of the porous base material is preferably not
less than 50 .mu.m and not more than 3,000 .mu.m in view of
supporting the porous resin layer and strengthening the separation
membrane. The porous base material may be either impregnated with
the porous resin layer or not impregnated with the porous resin
layer, which is selected depending on the use.
[0060] As the porous resin layer, an organic polymer membrane may
be suitably used. Examples of the material of the organic polymer
membrane include polyethylene resins, polypropylene resins,
polyvinyl chloride resins, polyvinylidene fluoride resins,
polysulfone resins, polyethersulfone resins, polyacrylonitrile
resins, polyolefin resins, cellulose resins and cellulose
triacetate resins, and the material may be a mixture of resins
containing these resins as the major component. The major component
means that the component is contained in an amount of not less than
50% by weight, preferably not less than 60% by weight. Preferred
examples of the material of the organic polymer membrane especially
include those which can be easily formed by a solution and are
excellent in physical durability and chemical resistance, such as
polyvinyl chloride resins, polyvinylidene fluoride resins,
polysulfone resins, polyethersulfone resins, polyacrylonitrile
resins and polyolefin resins, and mixtures of resins containing
these resins as the major component. A polyvinylidene fluoride
resin or a mixture of resins containing it as the major component
is more preferably used.
[0061] As the polyvinylidene fluoride resin, a homopolymer of
vinylidene fluoride or a copolymer with vinyl monomers capable of
copolymerizing with vinylidene fluoride is preferably used.
Examples of the vinyl monomers capable of copolymerizing with
vinylidene fluoride include tetrafluoroethylene,
hexafluoropropylene and chlorotrifluoroethylene.
[0062] Examples of the polyolefin resins include polyethylene,
polypropylene, chlorinated polyethylene and chlorinated
polypropylene, and chlorinated polyethylene is preferably used.
[0063] It is important for the porous membrane to have an average
surface pore size of not less than 0.01 .mu.m. With an average
surface pore size of the porous membrane of not less than 0.01
.mu.m, the membrane is less prone to clogging by the yeast cells
used for the fermentation, and has a property to stably maintain
the filtration performance for a long time. Further, with an
average pore size of the porous membrane of not less than 0.01
.mu.m, both a high blocking performance which does not allow
leakage of the polyploid yeasts and a high permeability can be
achieved, and the permeability can be maintained with high accuracy
and reproducibility for a long time. In cases where the average
surface pore size of the porous membrane is less than 0.01 .mu.m,
the permeability of the porous membrane decreases, and in some
cases, efficient operation is impossible even if the membrane is
not dirty. In cases where the average pore size of the porous
membrane is not less than 0.01 .mu.m, preferably not less than 0.02
.mu.m, more preferably not less than 0.04 .mu.m, efficient
operation is possible.
[0064] It is important for the porous membrane to have an average
pore size of less than 1 .mu.m, preferably less than 0.4 .mu.m,
more preferably less than 0.2 .mu.m to prevent leakage of the
polyploid yeast, that is, occurrence of a trouble decreasing the
elimination rate, and to prevent direct clogging of the pores by
the polyploid yeast. Further, in some cases, the polyploid yeast
produces substances other than lactic acid which is the substance
of interest, such as proteins and saccharides that are prone to
aggregation, and there are also cases where cell debris is produced
by death of a part of the polyploid yeast in the culture.
Therefore, to avoid clogging of the porous membrane by such
substances, the average pore size of not more than 0.1 .mu.m is
more preferred. Thus, the average surface pore size of the porous
membrane is preferably not more than 0.4 .mu.m, more preferably not
more than 0.2 .mu.m, especially preferably not more than 0.1
.mu.m.
[0065] The average surface pore size can be determined by observing
the surface of the porous membrane with a scanning electron
microscope at a magnification of 10,000.times. to measure the
diameters of all the pores observable within an area of 9.2
.mu.m.times.10.4 .mu.m and calculating the average of the
diameters. Alternatively, the average pore size can be determined
by a method wherein a picture of the surface of the membrane is
taken using a scanning electron microscope at a magnification of
10,000.times., and not less than 10, preferably not less than 20
pores are randomly selected, followed by measuring the diameters of
these pores and calculating the number average. In cases where a
pore is not circular, the average pore size can be determined by a
method wherein a circle having the area equal to that of the pore
(equivalent circle) is determined using an image processing device
or the like, and the diameter of the equivalent circle is regarded
as the diameter of the pore.
[0066] In terms of the average surface pore size of the porous
membrane, in cases where the standard deviation of the pore sizes
is small, that is, in cases where the pore sizes are uniform, a
uniform permeated liquid is more likely to be obtained, and the
standard deviation .sigma. is preferably not more than 0.1 .mu.m.
Further, in view of ease of management of fermentation operation,
the standard deviation of the average pore size is preferably as
small as possible. The standard deviation .sigma. of the average
pore size is calculated according to (Equation 1) below wherein N
represents the number of pores observable within an area of 9.2
.mu.m.times.10.4 .mu.m under the above-mentioned observation of the
surface of the porous membrane with a scanning electron microscope
at a magnification of 10,000.times., Xk represents the respective
diameters observed, and X(ave) represents the average of the pore
sizes:
.sigma. = k = 1 N ( X k - X ( ave ) ) 2 N . ( Equation 1 )
##EQU00001##
[0067] In the porous membrane, permeability to the culture is one
of its important properties. As an index for permeability of the
porous membrane, the pure water permeability coefficient of the
porous membrane before use can be used. The pure water permeability
coefficient of the porous membrane is preferably not less than
2.times.10.sup.-9 m.sup.3/m.sup.2/s/pa when the amount of
permeation is measured using, as the raw water, drinking water
filtered through a dialysis membrane (Filtryzer B2-1.5H
manufactured by Toray Industries, Inc.) at 25.degree. C. with a
head height of 1 m, and in cases where the pure water permeability
coefficient is not less than 2.times.10.sup.-9 m.sup.3/m.sup.2/s/pa
and not more than 6.times.10.sup.-7 m.sup.3/m.sup.2/s/pa, an amount
of permeation of water which is practically sufficient can be
obtained. More preferably, the pure water permeability coefficient
is not less than 2.times.10.sup.-9 m.sup.3/m.sup.2/s/pa and not
more than 1.times.10.sup.-7 m.sup.3/m.sup.2/s/pa.
[0068] The membrane surface roughness in the porous membrane is the
average value of the heights from the surface in the vertical
direction. The membrane surface roughness is one of the factors
which enable easy detachment of polyploid yeast adhered to the
surface of the separation membrane by the membrane surface washing
effect of the liquid current produced by stirring or a circulating
pump. The surface roughness of the porous membrane is preferably
not more than 0.1 .mu.m. In cases where the membrane surface
roughness is not more than 0.1 .mu.m, polyploid yeast adhered to
the membrane can be easily detached and the shear force produced on
the surface of the membrane can be reduced during filtration of the
polyploid yeast. Therefore, destruction of the yeast is suppressed,
and clogging of the porous membrane is also suppressed, so that
stable filtration can be carried out for a long time, and, since
the continuous fermentation can be carried out with a lower
transmembrane pressure difference, even in cases where the membrane
was clogged, a better washing recovery performance can be obtained
compared to in cases where the operation was carried out with a
higher transmembrane pressure difference. Since stable continuous
fermentation is possible by suppressing clogging, the surface
roughness of the porous membrane is preferably as small as
possible.
[0069] The membrane surface roughness is measured using the
following atomic force microscope (AFM) under the following
conditions:
Device: Atomic force microscope (Nanoscope IIIa manufactured by
Digital Instruments)
[0070] Conditions [0071] Probe: SiN cantilever (manufactured by
Digital Instruments) [0072] Scanning mode Contact mode (measurement
in air) [0073] Underwater tapping mode (measurement in water)
[0074] Scanning area: 10 .mu.m.times.10 .mu.m, 25 .mu.m.times.25
.mu.m (measurement in air) [0075] 5 .mu.m.times.5 .mu.m, 10
.mu.m.times.10 .mu.m (measurement in water) [0076] Scanning
resolution: 512.times.512 [0077] Sample preparation: When the
measurement was carried out, the membrane sample was soaked in
ethanol at room temperature for 15 minutes and then soaked in RO
water for 24 hours, followed by washing and drying it in the
air.
[0078] The membrane surface roughness (drough) is calculated
according to the following (Equation 2) using the above atomic
force microscope (AFM), based on the heights of respective points
in the direction of the z-axis:
d rough = n = 1 N Z n - Z _ N ( Equation 2 ) ##EQU00002## [0079]
d.sub.rough: Surface roughness (.mu.m) [0080] Z.sub.n: Height in
the direction of the z-axis (.mu.m) [0081] Z: Average height in the
scanned area (.mu.m) [0082] N: Number of samples measured.
[0083] The porous membrane may be either a flat membrane or a
hollow fiber membrane. In cases where the porous membrane is a flat
membrane, its average thickness is selected depending on the use
thereof, and it is preferably not less than 20 .mu.m and not more
than 5,000 .mu.m, more preferably not less than 50 .mu.m and not
more than 2,000 .mu.m. In cases where the porous membrane is a
hollow fiber membrane, the inner diameter of the hollow fiber is
preferably not less than 200 .mu.m and not more than 5,000 .mu.m,
and the membrane thickness is preferably not less than 20 .mu.m and
not more than 2000 .mu.m. A fabric or a knit produced by forming an
organic fiber or an inorganic fiber into a cylindrical shape may be
contained in the hollow fiber.
[0084] The method for producing the porous membrane will be now
described by way of examples.
[0085] First, a method for preparing a flat membrane which is one
preferred embodiment of the porous membrane will now be described.
On the surface of a porous base material, a coating of a starting
solution containing a resin and a solvent is formed, while
impregnating the starting solution into the porous base material.
Thereafter, only the coated surface of the porous base material
having the coating is brought into contact with a coagulation bath
containing a nonsolvent to coagulate the resin, while forming a
porous resin layer on the surface of the porous base material.
[0086] The starting solution is prepared by dissolving a resin into
a solvent. The temperature of the starting solution is usually
preferably selected within the range of 5 to 120.degree. C. in view
of the film-forming property. The solvent dissolves the resin and
acts on the resin to promote formation of a porous resin layer by
the resin. Examples of the solvent which may be used include
N-methylpyrrolidinone (NMP), N,N-dimethylacetamide (DMAc),
N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO),
N-methyl-2-pyrrolidone, tetrahydrofuran, tetramethylurea, trimethyl
phosphate, cyclohexanone, isophorone, .gamma.-butyrolactone, methyl
isoamyl ketone, dimethyl phthalate, propylene glycol methyl ether,
propylene carbonate, diacetone alcohol, glycerol triacetate,
acetone and methyl ethyl ketone. Among these, N-methylpyrrolidinone
(NMP), N,N-dimethylacetamide (DMAc), N,N-dimethylformamide (DMF)
and dimethyl-sulfoxide (DMSO), in which resins show high
solubilities, can be preferably used. These may be used either
individually or as a mixture of 2 or more thereof.
[0087] Further, a component(s) other than a solvent, such as
polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and/or
glycerin may be added to the solvent. A nonsolvent may also be
added to the solvent. A nonsolvent is a liquid which does not
dissolve a resin. The non-solvent has an action of regulating the
rate of coagulation of a resin, thereby regulating the sizes of the
pores. Examples of the nonsolvent which may be used include water
and alcohols such as methanol and ethanol. Among these, water and
methanol are preferred as the nonsolvent in view of the cost
thereof. The compound other than a solvent, and the nonsolvent may
be a mixture.
[0088] To the starting solution, a pore-forming agent may be added.
The pore-forming agent is extracted upon immersion in the
coagulation bath, thereby making the resin layer porous. Addition
of a pore-forming agent allows the regulation of average pore size.
The pore-forming agent preferably has a high solubility in the
coagulation bath. Examples of the pore-forming agent include
inorganic salts such as calcium chloride and calcium carbonate.
Further examples of the pore-forming agent include polyoxyalkylenes
such as polyethylene glycol and polypropylene glycol; water-soluble
macromolecular compounds such as polyvinyl alcohol, polyvinyl
butyral and polyacrylic acid; and glycerin.
[0089] The method of preparation of a hollow fiber membrane which
is one preferred example of the porous membrane will now be
described. A hollow fiber membrane can be prepared by extruding a
starting solution comprising a resin and a solvent from the outer
pipe of a double pipe mouthpiece, while extruding a fluid for
formation of the hollow portion from the inner pipe, followed by
cooling and solidifying the resultant in a cooling bath.
[0090] The starting solution can be prepared by dissolving the
resin mentioned in the above-mentioned preparation method for a
flat membrane in the solvent mentioned in the above-mentioned
preparation method for a flat membrane at a concentration of not
less than 20% by weight and not more than 60% by weight. As the
fluid for formation of the hollow portion, a gas or a liquid can be
usually used. Further, on the outer surface of the obtained hollow
fiber membrane, another porous resin layer may be coated
(laminated). The lamination may be carried out for changing
properties of the hollow fiber membrane such as the
hydrophilicity/hydrophobicity and the pore sizes to desired
properties. The other porous resin layer to be laminated can be
prepared by dissolving a resin in a solvent followed by bringing
the resulting starting solution into contact with a coagulation
bath containing a nonsolvent to coagulate the resin. Examples of
the material of the resin which can be preferably used include ones
similar to those of the material of the above-mentioned organic
polymer membrane. The method of the lamination is not restricted,
and examples thereof include immersion of the hollow fiber membrane
in the starting solution and application of the starting solution
to the surface of the hollow fiber membrane, and after the
lamination, a part of the starting solution adhered may be scraped
off or blown off with an air knife to adjust the amount of
lamination.
[0091] The porous membrane can be made into a separation membrane
element by bonding and sealing the hollow portion of the hollow
fiber membrane using a member such as a resin and placing the
resultant on a support.
[0092] The porous membrane can be made into a separation membrane
element by combining it with a support. A separation membrane
element wherein a supporting plate is used as the support and the
porous membrane is placed on at least one side of the supporting
plate is one preferred form of the separation membrane element
having the porous membrane. Placement of the porous membranes on
the both sides of the supporting plate to increase the permeability
is also a preferred example of the separation membrane element.
[0093] In the method for producing lactic acid, the filtration is
preferably carried out with a transmembrane pressure difference of
the porous membrane of not less than 0.1 kPa and less than 20 kPa.
In cases where the filtration is carried out with a transmembrane
pressure difference of not less than 20 kPa to filtrate the
fermentation culture medium, power is necessary for applying the
pressure and therefore the economic effect upon the production of
lactic acid decreases. Further, by application of a higher
transmembrane pressure difference of not less than 20 kPa, the
polyploid yeast used in the continuous fermentation culture may be
disrupted, leading to decrease in the capacity to produce lactic
acid. With a transmembrane pressure difference of less than 0.1
kPa, the filtration takes time, leading to decrease in the
production rate. Since in cases where the transmembrane pressure
difference which is the filtration pressure is within the range of
not less than 0.1 to less than 20 kPa, the transmembrane pressure
difference can be obtained by the hydraulic head difference, so
that the inside of the fermenter is not especially required to be
kept pressured and the capacity to produce lactic acid does not
decrease. Further, since the inside of the fermenter is not
especially required to be kept pressurized, an embodiment wherein
the porous membrane is placed inside the fermenter is possible, and
hence the fermentation device can be downsized, which is
advantageous.
[0094] The term "transmembrane pressure difference" herein means
the pressure difference in the porous membrane between the side of
the liquid to be processed and the side of the permeated liquid.
When an operation is carried out at a constant processing flow rate
per unit area of the porous membrane and the liquid to be processed
is filtered through the porous membrane for a long time, pollutants
existing in the liquid to be processed adhere to the pores on the
porous membrane and accumulate on the surface, leading to pollution
of the membrane. This causes clogging of the membrane and increases
in the transmembrane pressure difference, leading to significant
decrease in the processing flow rate and difficulty in continuation
of a stable operation, so that the filtration is preferably carried
out while measuring the transmembrane pressure difference. The
transmembrane pressure difference can be measured by placing
manometers in the sides of the liquid to be processed and the
permeated liquid in the porous membrane and measuring the
pressures, followed by calculating the pressure difference.
[0095] The polyploid yeast having a capacity to produce lactic acid
will now be described. In the method for producing lactic acid, use
of a polyploid yeast having a capacity to produce lactic acid
enables, under simple operating conditions, continuous fermentation
wherein a high productivity of lactic acid which is the desired
fermentation product can be stably maintained for a long time, and
therefore lactic acid can be stably produced at a low cost.
[0096] Polyploid yeast is yeast having 2 or more sets of
chromosomes in the cell. Polyploid yeast is larger than haploid
yeast generally used in genetic analyses, and therefore clogging of
a porous membrane is not likely to occur, so that it is suitable
for long-term culture. By using polyploid yeast, the shear force
produced on the surface of the membrane can be reduced, destruction
of the yeast is suppressed, and clogging of the porous membrane is
also suppressed, so that stable filtration can be carried out for a
long time, and moreover, since reuse of the membrane is possible,
the cost can be reduced, which are advantageous. Further, even in
cases where the membrane was clogged, a better washing recovery
performance can be obtained compared to the cases of haploid yeast.
The number of sets of chromosomes in the polyploid yeast is not
restricted, and diploid yeast having 2 sets of chromosomes are
preferred.
[0097] Examples of the polyploid yeast include yeasts such as
baker's yeasts, sake yeasts, wine yeasts and beer yeasts frequently
used in the fermentation industry. The polyploid yeast to be used
may be isolated from a natural source or may be one whose
properties were partially modified by mutation or genetic
recombination.
[0098] Polyploid yeast having a capacity to produce lactic acid
means polyploid yeast to which a lactate dehydrogenase gene
(hereinafter also referred to as LDH) was introduced. The method of
preparation of polyploid yeast to which a lactate dehydrogenase
gene was introduced is not restricted, and polyploidization of
haploid yeast to which a lactate dehydrogenase gene was introduced
enables simple multiplication of the copy number of the lactate
dehydrogenase gene without changing the genotypes per chromosome,
eventually leading to enhancement of the lactic acid producing
capacity.
[0099] Further, if the polyploid yeast having a capacity to produce
lactic acid is prototrophic, a culture medium containing less
nutrients than conventional media, that is, a low-cost culture
medium can be employed, and continuous fermentation which allows
stable maintenance of a high productivity of lactic acid for a long
time is possible under simple operating conditions, enabling
low-cost and stable production of lactic acid, so that prototrophic
yeast is preferably used.
[0100] Auxotrophy of yeast means that a mutation occurred to a
nutrient-synthesis gene in the wild type yeast, resulting in
deficiency of the synthesis capacity of a nutrition. Reversion from
auxotrophy means reversion of the auxotrophic mutation to the wild
type or a state very similar thereto. Since auxotrophy is used as a
marker for mainly genetic manipulation and the like, auxotrophic
yeast is preferably used for genetic manipulation.
[0101] Known examples of nutrients necessary for yeast showing
auxotrophy include methionine, tyrosine, isoleucine, phenylalanine,
glutamic acid, threonine, aspartic acid, valine, serine, arginine,
uracil, adenine, lysine, tryptophan, leucine and histidine.
Examples of genotypes in yeast showing auxotrophy include the
followings: [0102] methionine auxotrophy: met1, met2, met3, met4,
met5, met6, met7, met8, met10, met13, met14, met20; [0103] tyrosine
auxotrophy: tyr1; [0104] isoleucine-valine auxotrophy: ilv1, ilv2,
ilv3, ilv5; [0105] phenylalanine auxotrophy: pha2; [0106] glutamic
acid auxotrophy: GLU3; [0107] threonine auxotrophy: thr1, thr4;
[0108] aspartic acid auxotrophy: asp1, asp5; [0109] serine
auxotrophy: ser1, ser2; [0110] arginine auxotrophy: arg1, arg3,
arg4, arg5, arg8, arg9, arg80, arg81, arg82, arg84; [0111] uracil
auxotrophy: ura1, ura2, ura3, ura4, ura6; [0112] adenine
auxotrophy: ade1, ade2, ade3, ade4, ade5, ade6, ade8, ade9, ade12,
ADE 15; [0113] lysine auxotrophy: lys1, lys2, lys4, lys5, lys7,
lys9, lys11, lys13, lys14; [0114] tryptophan auxotrophy: trp1,
trp2, trp3, trp4, trp5; [0115] leucine auxotrophy: leu1, leu2,
leu3, leu4, leu5; and [0116] histidine auxotrophy: his1, his2,
his3, his4, his5, his6, his7, his8.
[0117] The prototrophic yeast preferably is yeast having no
genotype showing the above-described auxotrophies or yeast wherein
such a genotype is complemented. Whether or not yeast is
prototrophic can be judged based on whether or not the yeast can be
grown in SD medium (Table 1) which is a minimal medium for
yeast.
TABLE-US-00001 TABLE 1 YEAST NITROGEN W/O AA 1.7 g (manufactured by
Difco) Glucose 30 g Agar 20 g up to 1 L
[0118] Examples of the method for preparing a prototrophic yeast by
reversion from the auxotrophy in an auxotrophic yeast include a
method wherein a nutrient-synthesis gene is introduced by a gene
recombination technique to allow reversion from the auxotrophy and
a method wherein the process in which yeasts having different
auxotrophies are mated with each other and ascus formation is
allowed to occur, thereby allowing reversion from an auxotrophy of
interest is repeated until completion of reversion from all the
auxotrophies, to obtain a prototrophic yeast.
[0119] The LDH introduced in the polyploid yeast having a capacity
to produce lactic acid is not restricted as long as it encodes a
protein having an activity of converting reduced nicotinamide
adenine dinucleotide (NADH) and pyruvic acid into oxidized
nicotinamide adenine dinucleotide (NAD+) and lactic acid, and
examples of the available LDH include LDHs derived from lactic acid
bacteria having high yields of lactic acid to saccharides and LDHs
derived from mammals and LDHs derived from amphibians. Among these,
LDHs derived from Homo sapiens and frogs are preferably used. Among
the frogs, LDHs derived from frogs belonging to Pipidae are
preferably used, and among the frogs belonging to Pipidae, LDHs
derived from Xenopus laevis can be preferably used.
[0120] The LDHs include genes having genetic polymorphisms and
mutant-type genes produced by mutagenesis or the like. "Genetic
polymorphism" means that a part of the base sequence of a gene is
changed due to a natural mutation(s) on the gene, and "mutagenesis"
means that a mutation(s) is/are artificially introduced to a gene.
Examples of the method of mutagenesis include a method using a
site-directed mutagenesis kit (Mutan-K (TAKARA BIO INC.)) and a
method using a random mutagenesis kit (BD Diversify PCR Random
Mutagenesis (CLONTECH)). The LDH may have a deletion(s) and/or an
insertion(s) in a part of its base sequence as long as it encodes a
protein having an activity of converting NADH and pyruvic acid into
NAD+ and lactic acid.
[0121] In the polyploid yeast having a capacity to produce lactic
acid, the LDH may be retained in a plasmid or YAC maintained
outside the yeast chromosomes, but as mentioned above, it is
preferably incorporated into a yeast chromosome and retained
therein. The method of incorporation of a LDH into a yeast
chromosome is not restricted and, for example, it may be introduced
by a method disclosed in JP 2008-29329 A. In the polyploid yeast
having a capacity to produce lactic acid, at least one, preferably
not less than two, more preferably not less than three, still more
preferably not less than 4 LDH(s) is/are retained.
[0122] The fermentation feedstock may be any fermentation feedstock
as long as it promotes the growth of the yeast to be cultured and
allows satisfactory production of lactic acid as the desired
fermentation product, and examples thereof which are preferably
used include liquid media containing carbon sources, nitrogen
sources, inorganic salts and, if needed, organic micronutrients
such as amino acids and vitamins as appropriate. Examples of the
carbon sources which are preferably used include saccharides such
as glucose, sucrose, fructose, galactose, lactose and maltose;
saccharified starch solutions containing these saccharides; sweet
potato molasses; sugar beet molasses; high test molasses; cane
juices; extracts and concentrated liquids of cane juices; raw
sugars purified or crystallized from cane juices; refined sugars
purified or crystallized from cane juices; and further, organic
acids such as acetic acid and fumaric acid; alcohols such as
ethanol; and glycerin. The term "saccharide" means the first
oxidation product of a polyol, which is a carbon hydrate having an
aldehyde group or a ketone group, and one having an aldehyde group
is classified as aldose and one having a ketone group is classified
as ketose. It is preferably glucose, sucrose, fructose, galactose,
lactose or maltose. The carbon source may be added at one time when
the culture is begun or may be added intermittently or continuously
during the culture.
[0123] Examples of the nitrogen source used include ammonia gas,
aqueous ammonia, ammonium salts, urea and nitric acid salts; and
other organic nitrogen sources used supplementarily such as oil
cake, soybean-hydrolyzed liquids, casein digests, other amino
acids, vitamins, corn steep liquors, yeasts or yeast extracts, meat
extracts, peptides such as peptones, and various fermentation
microorganisms and hydrolysates thereof.
[0124] Examples of the inorganic salt which may be added as
appropriate include phosphoric acid salt, magnesium salt, calcium
salt, iron salt and manganese salt.
[0125] In cases where the polyploid yeast having a capacity to
produce lactic acid is auxotrophic, the nutrient may be added as a
preparation or a natural product containing it. An anti-forming
agent may also be added as required.
[0126] The conditions of the fermentation culture of the polyploid
yeast having a capacity to produce lactic acid are not restricted
as long as the yeast can be cultured, and it is preferably carried
out at a pH of 4 to 8 and a temperature of 20 to 40.degree. C. The
pH of the fermentation culture medium is adjusted to a value
predetermined within the above-described range with an inorganic
acid or organic acid; alkaline substance; urea; calcium carbonate;
or ammonia gas.
[0127] During the fermentation culture of the polyploid yeast
having a capacity to produce lactic acid, if the feed rate of
oxygen is required to be increased, a method can be employed in
which the oxygen concentration is maintained at not less than 21%
by adding oxygen into the air; the pressure of the culture medium
is increased; the stirring rate is increased; or the ventilation
volume is increased. On the other hand, if the feed rate of oxygen
is required to be decreased, a gas which does not contain oxygen,
such as carbon dioxide gas, nitrogen or argon may be mixed with the
air and supplied.
[0128] Batch fermentation or Fed-Batch fermentation may be carried
out at the initial phase of the culture to increase the yeast cell
concentration, followed by starting the continuous culture
(withdrawal), or the yeast cells may be seeded at a high
concentration and subjected to the continuous culture at the
beginning of the culture. It is possible to start supplying the
fermentation feedstock liquid and withdrawing the culture at an
appropriate timing. The timing of the start of supplying of the
fermentation feedstock liquid and the timing of the start of
withdrawing of the culture are not necessarily the same. The
supplying of the fermentation feedstock liquid and the withdrawing
of the culture may be carried out either continuously or
intermittently. Nutrients as described above necessary for the
growth of the yeast cells may be added to the fermentation
feedstock liquid to allow continuous growth of the yeast cells.
[0129] The concentration of the yeast cells in the fermentation
culture medium is preferably maintained high within the range which
does not cause death of the yeast cells at a high rate due to an
environment of the fermentation culture medium which is
inappropriate for the growth of the yeast cells, in view of
achieving efficient production. For example, by maintaining the
concentration of the yeast cells at not less than 5 g/L in terms of
dry weight, a good production efficiency can be obtained. As long
as an operational trouble of a continuous fermentation device or
decrease in the production efficiency is not induced, the upper
limit of the concentration of the yeast cells is not
restricted.
[0130] The operation of continuous culture by allowing the growth
of fresh yeast cells having a fermentative production capacity is
usually preferably carried out in a single fermentation reaction
vessel in view of control of the culture. However, the number of
the fermentation reaction vessel(s) is not restricted as long as
the continuous culture is carried out to produce the product while
allowing the growth of yeast cells. A plurality of fermentation
reaction vessels may be used, for example, because of a small
volume of each fermentation reaction vessel. In this case, a high
productivity of the fermentation product can be obtained even by
continuous culture using a plurality of fermentation reaction
vessels connected in parallel or in series through pipes.
[0131] "Continuation of continuous fermentation" means a state
wherein supply of the fermentation feedstock liquid and withdrawal
of the culture are carried out continuously or intermittently.
Lactic acid is preferably efficiently produced during the period
when the continuous fermentation is continued. "Efficient
production of lactic acid" means a state wherein the concentration
of accumulated lactic acid, the rate of production of lactic acid,
and the yield of lactic acid to saccharides are evaluated, and one,
preferably two, more preferably all of these is/are high.
[0132] "The concentration of accumulated lactic acid" means the
concentration of lactic acid contained in the culture, and "a state
wherein the concentration of accumulated lactic acid is high" means
a state wherein the concentration of accumulated lactic acid is not
less than 40 g/L, and the concentration of accumulated lactic acid
is preferably 42 g/L, more preferably 44 g/L.
[0133] "The rate of production of lactic acid" means the amount of
lactic acid produced per unit time, and "a state wherein the rate
of production of lactic acid is high" means a state wherein the
rate of production of lactic acid represented by Equation 3 is not
less than 7.5 g/L/h, preferably not less than 8, more preferably
not less than 9 g/L/h:
[0134] P.sub.B: Rate of production of lactic acid (g/l/h)
P B = V P t = P ( F M + F N ) V ( Equation 3 ) ##EQU00003##
[0135] F.sub.M: Feed rate of culture medium (1/h)
[0136] E.sub.N: Feed rate of neutralizing agent (1/h)
[0137] P: Concentration of product (g/l)
[0138] P.sub.B: Production rate (g/l/h)
[0139] S: Substrate concentration (g/l)
[0140] S.sub.R: Substrate concentration in culture medium to be
supplied (g/l)
[0141] t: Time (h)
[0142] V: Amount of culture (1)
[0143] Y.sub.P/S: Yield of product (g/g).
[0144] "The yield of lactic acid to saccharides" means the ratio of
lactic acid produced from a carbon source consumed per unit time,
and "a state wherein the yield of lactic acid to saccharides is
high" is a state wherein the yield of lactic acid to saccharides
represented by Equation 4 is not less than 70%, preferably not less
than 75%, more preferably not less than 80%:
[0145] Y.sub.P/S: Yield of product (g/g)
Y p / S = V P S R - S = P ( F M + F N ) F .times. S R - S ( F M + F
N ) . ( Equation 4 ) ##EQU00004##
The weights of all the saccharides in the culture medium are
converted to the mass of glucose. Example: 1 mol of sucrose is
converted to 2 mol of glucose.
[0146] Separation and purification of lactic acid contained in the
filtered/separated fermentation liquid produced can be carried out
by a combination of conventionally-known methods for concentration,
distillation, crystallization and the like, and examples of the
methods include a method wherein pH of the filtered/separated
fermentation liquid is adjusted to not more than 1 followed by
extraction with diethyl ether, ethyl acetate or the like, a method
wherein lactic acid is allowed to adsorb to an ion-exchange resin
followed by washing and elution, a method wherein lactic acid is
allowed to react with an alcohol in the presence of an acid
catalyst to produce an ester, which is then subjected to
distillation, a method wherein lactic acid is crystallized as the
calcium salt or the lithium salt, and a separation/purification
method wherein the nanofilter disclosed in WO2009/004922 is
combined with distillation.
[0147] Preferred examples of the continuous fermentation device
used in the method for producing lactic acid will now be described
referring to the drawings.
[0148] FIG. 1 is a schematic side view for explanation of a
preferred example of the continuous fermentation device of the
membrane separation type used in the method for producing lactic
acid by continuous fermentation. FIG. 1 is a representative example
of the device in which the separation membrane element is placed
outside the fermentation reaction vessel.
[0149] In FIG. 1, the continuous fermentation device of the
membrane separation type is basically constituted by a fermentation
reaction vessel 1, a membrane separation vessel 12 and a pressure
difference controlling apparatus 3. In the separation membrane
element 2, a porous separation membrane is incorporated. Preferred
examples of the porous separation membrane employed include the
separation membrane and the separation membrane element disclosed
in WO 2002/064240. The membrane separation vessel 12 is connected
to a fermentation reaction vessel 1 via a fermentation culture
medium circulating pump 11.
[0150] In FIG. 1, a culture medium can be fed to the fermentation
reaction vessel 1 by a culture medium supplying pump 7, and, as
required, the fermentation culture medium in the fermentation
reaction vessel 1 can be stirred by a stirrer 5, and further, as
required, a necessary gas can be supplied by a gas supplying
apparatus 4. The supplied gas can be recovered and recycled,
followed by supplying it again with the gas supplying apparatus 4.
Further, as required, pH of the fermentation liquid can be adjusted
by a pH sensor/controlling apparatus 9 and a pH adjustment solution
supplying pump 8, and further, as required, the temperature of the
fermentation culture medium can be adjusted by a temperature
controller 10, to carry out highly productive fermentative
production. The fermentation liquid in the device is circulated
between the fermentation reaction vessel 1 and the membrane
separation vessel 12 by the fermentation liquid circulating pump
11. The fermentation culture medium containing a fermentation
product is filtered and separated by the separation membrane
element 2 into the microorganism and the fermentation product,
which can be recovered from the device system.
[0151] The concentration of the microorganisms in the device system
can be maintained high by allowing the microorganisms, which were
filtered and separated, to stay in the device system, thereby
enabling a highly productive fermentative production. The
filtration and separation by the separation membrane element 2 can
be carried out by the hydraulic head pressure difference from the
fluid level in the membrane separation vessel 12 without using
particular power, but, as required, the filtration/separation rate
of the separation membrane element 2 and the amount of the
fermentation culture medium in the device system can be
appropriately controlled by a level sensor 6 and the pressure
difference controlling apparatus 3. As required, a necessary gas
can be supplied by the gas supplying apparatus 4 into the membrane
separation vessel 12. The supplied gas can be recovered and
recycled, followed by supplying it again with the gas supplying
apparatus 4. The filtration and separation by the separation
membrane element 2 can also be carried out, as required, by suction
filtration with a pump or the like or by pressurizing the inside of
the device system. The polyploid yeast may be cultured in a culture
vessel by continuous fermentation and supplied to the fermentation
vessel as required. By culturing the polyploid yeast in a culture
vessel and supplying it into the fermentation vessel as required,
continuous fermentation can be carried out constantly with fresh
polyploid yeast highly capable of producing lactic acid, and
continuous fermentation can be continued for long time with a high
productive performance.
[0152] FIG. 2 is a schematic side view for explanation of another
example of the continuous fermentation device of the membrane
separation type. Among the continuous fermentation devices used in
the method for producing lactic acid, a representative example in
which the separation membrane element is placed inside the
fermentation reaction vessel is shown in the schematic view in FIG.
2.
[0153] In FIG. 2, the continuous fermentation device of the
membrane separation type is basically constituted by a fermentation
reaction vessel 1 and a pressure difference controlling apparatus
3. In the separation membrane element 2 in the fermentation
reaction vessel 1, a porous membrane is incorporated. Examples of
this porous separation membrane which can be employed include the
separation membrane and the separation membrane element disclosed
in WO 2002/064240. Details of this separation membrane element will
be described later.
[0154] The example of the continuous fermentation by the continuous
fermentation device of the membrane separation type in FIG. 2 will
now be explained. By a culture medium supplying pump 7, a culture
medium is continuously or intermittently fed to the fermentation
reaction vessel 1. The culture medium may be subjected to heat
disinfection or heat sterilization, or sterilization treatment
using a filter before the feeding, as required. During the
fermentative production, as required, the fermentation culture
medium in the fermentation reaction vessel 1 is stirred by a
stirrer 5 in the fermentation reaction vessel 1. During the
fermentative production, as required, a necessary gas may be
supplied into the fermentation reaction vessel 1 by a gas supplying
apparatus 4. During the fermentative production, as required, pH of
the fermentation culture medium in the fermentation reaction vessel
1 may be controlled by a pH sensor/controlling apparatus 9 and a pH
adjustment solution supplying pump 8, and as required, the
temperature of the fermentation culture medium in the fermentation
reaction vessel 1 may be controlled by a temperature controller 10,
to carry out highly productive fermentative production. pH and
temperature were shown as examples of physicochemical conditions of
the fermentation culture medium to be controlled by
instrumentation/control apparatuses, but, as required, dissolved
oxygen and/or ORP may be controlled, and by an analysis apparatus
such as an on-line chemical sensor, the concentration of lactic
acid in the fermentation culture medium may be measured followed by
controlling physicochemical conditions using as an index the
concentration of lactic acid in the fermentation culture medium.
When the continuous or intermittent feeding of the culture medium
is carried out, the amount of the culture medium to be fed and the
rate of feeding thereof are appropriately controlled using as an
index the measured value of the physicochemical environment of the
fermentation liquid by the instrumentation apparatus.
[0155] In FIG. 2, in terms of the fermentation culture medium, the
yeast cells and the fermentation product are filtered and separated
by a separation membrane element 2 placed in the fermentation
reaction vessel 1, and the fermentation product is recovered from
the device system. The concentration of the yeast in the device
system can be maintained high by allowing the yeast, which was
filtered and separated, to stay in the device system, thereby
enabling a highly productive fermentative production. The
filtration and separation by the separation membrane element 2 can
be carried out by the hydraulic head pressure difference from the
fluid level in the fermentation reaction vessel 1 without using
particular power, but, as required, the filtration/separation rate
of the separation membrane element 2 and the amount of the
fermentation culture medium in the fermentation reaction vessel 1
can be appropriately controlled by a level sensor 6 and the
pressure difference controlling apparatus 3. The
filtration/separation by the above-described separation membrane
element can also be carried out, as required, by suction filtration
with a pump or the like or by pressurizing the inside of the device
system. The yeast cells may be cultured in a culture vessel
prepared separately and supplied to the fermentation vessel as
required. By culturing the yeast cells in a culture vessel and
supplying them into the fermentation vessel as required, continuous
fermentation can be carried out constantly with fresh yeast cells,
and continuous fermentation can be continued for long time with a
high productive performance.
[0156] The separation membrane element preferably used in the
continuous fermentation device used in the method for producing
lactic acid will now be described referring to the drawings. In the
continuous fermentation device used in the method for producing
lactic acid, the separation membrane and the separation membrane
element disclosed in WO 2002/064240 may be preferably used.
[0157] In terms of the example of the separation membrane element,
the separation membrane and the separation membrane element
disclosed in WO 2002/064240 as preferred examples will now be
summarized referring to the drawing. FIG. 3 is a schematic
perspective view for explanation of one example of the separation
membrane element.
[0158] The separation membrane element is constituted, as shown in
FIG. 3, of a channel material 14 and the separation membrane 15
arranged in this order on each of the both sides of a supporting
plate 13 having rigidity. The supporting plate 13 has a recess 16
on each of the both sides. The fermentation culture medium is
filtered through the separation membrane 15. The channel material
14 is used for allowing efficient flow of the permeated liquid,
which was filtered through the separation membrane 15, to the
supporting plate 13. The permeated liquid allowed to flow to the
supporting plate 13 then moves through the recesses 16 on the
supporting plate 13, and is recovered to the outside of the
continuous fermentation device through a liquid collection pipe 17
which is a means of discharge.
[0159] Another example of the separation membrane element shown in
FIG. 4 will now be described. FIG. 4 is a cross-sectional view for
explaining the other example of the separation membrane
element.
[0160] In terms of the separation membrane element, as shown in
FIG. 4, a separation membrane bundle 18 constituted by hollow fiber
membranes is adhered and immobilized in a bundled manner by an
upper resin sealing layer 19 and a lower resin sealing layer 20. By
the adherence and immobilization by the lower resin sealing layer
20, the hollow portion of the hollow fiber membrane is sealed to
provide a structure which prevents leakage of the fermentation
culture medium. On the other hand, the upper resin sealing layer 19
does not seal the inner pore of the hollow fiber membrane, to
provide a structure which allows the permeated liquid to flow
through a liquid collection pipe 22. This separation membrane
element may be placed in the continuous fermentation device via a
support frame 21. The permeated liquid filtered by the separation
membrane bundle 18 flows through the hollow portion of the hollow
fiber membrane and is recovered to the outside of the fermentation
culture vessel via the liquid collection pipe 22. As the power to
recover the permeated liquid, suction filtration by the hydraulic
head pressure difference or a pump, liquid, gas or the like, or a
method to pressurize the inside of the device system or the like
may be used.
[0161] Members constituting the separation membrane element of the
continuous fermentation device used in the method for producing
lactic acid by continuous fermentation are preferably resistant to
autoclaving. If the inside of the fermentation device can be
sterilized, the risk of contamination with unfavorable fungus
bodies can be avoided, and more stable continuous fermentation is
possible. The members constituting the separation membrane element
are preferably resistant to treatment at a temperature of
121.degree. C. for 15 minutes, which are the conditions of
autoclaving. Examples of the members of the separation membrane
element which may be preferably selected include metals such as
stainless steel and aluminum; and resins such as polyamide resins,
fluorocarbon resins, polycarbonate resins, polyacetal resins,
polybutylene terephthalate resins, PVDF, modified polyphenylene
ether resins and polysulfone resins.
[0162] In the continuous fermentation device used in the method for
producing lactic acid by continuous fermentation, the separation
membrane element may be placed either outside the fermentation
vessel or inside the fermentation reaction vessel. In cases where
the separation membrane element is placed outside the fermentation
reaction vessel, a membrane separation vessel may be separately
provided to place the separation membrane element therein, and the
fermentation culture medium may be continuously filtered by the
separation membrane element while allowing the fermentation culture
medium to circulate between the fermentation reaction vessel and
the membrane separation vessel.
[0163] In the continuous fermentation device used in the method for
producing lactic acid by continuous fermentation, the membrane
separation vessel is preferably autoclavable. By making the
membrane separation vessel autoclavable, contamination can be
easily avoided.
EXAMPLES
[0164] Our methods will now be described by way of Examples.
Reference Example 1
Preparation of Polyploid Yeast Having Capacity to Produce Lactic
Acid
[0165] A polyploid yeast having a capacity to produce lactic acid
was prepared by polyploidization of yeast having a capacity to
produce lactic acid obtained by introduction of the L-lactate
dehydrogenase gene derived from Xenopus laevis (hereinafter also
referred to as "XLDH") to the Saccharomyces cerevisiae NBRC10505
strain. Further, as will be described in detail below, reversion
from auxotrophy and/or a temperature-sensitive mutation was/were
given to the resulting polyploid yeast. The yeast strains used for
preparation of polyploid yeast and the yeast strains prepared by
the method described below are shown in FIG. 2. Procedures for the
methods of the insertion of the LDH, the giving of the
temperature-sensitive mutation and the reversion from auxotrophy
will be described in detail below.
(Part 1)
[0166] Construction of Vector for Expression of L-LDH (XLDH)
Derived from Xenopus laevis
[0167] Cloning of XLDH (SEQ ID NO:1) was carried out by PCR. For
the PCR, a phagemid DNA prepared from a cDNA library (manufactured
by STRATAGENE) derived from kidney of Xenopus laevis according to
the manufacturer's protocol was used as the template.
[0168] For the PCR amplification reaction, KOD-Plus polymerase
(manufactured by Toyobo Co., Ltd.) was used, and the reaction
buffer, the dNTP mix and the like included therein were used. The
phagemid DNA prepared according to the manufacturer's protocol as
described above was used to prepare 50 .mu.l of a reaction system
containing 50 ng/sample of the phagemid DNA, 50 pmol/sample of
primers and 1 U/sample of KOD-Plus polymerase. The reaction
solution was subjected to denaturation by a PCR amplification
device iCycler (manufactured by BIO-RAD) at a temperature of
94.degree. C. for 5 minutes, and this was followed by 30 cycles of:
94.degree. C. (heat denaturation) for 30 seconds, 55.degree. C.
(annealing of the primers) for 30 seconds and 68.degree. C.
(extension of the complementary strand) for 1 minute. Thereafter,
the reaction solution was cooled to a temperature of 4.degree. C.
The primers (SEQ ID NOs:2 and 3) for amplification of the gene were
prepared such that the SalI-recognition sequence and the NotI
recognition sequence were added to the 5'-end and the 3'-end,
respectively.
[0169] The PCR amplification fragment was purified and
phosphorylated by T4 polynucleotide Kinase (manufactured by TAKARA
BIO INC.) at its ends, and ligated to the pUC118 vector (which had
been preliminarily digested with a restriction enzyme HindIII and
the digested ends had been subjected to dephosphorylation
treatment). The ligation was carried out using DNA Ligation Kit
Ver.2 (manufactured by TAKARA BIO INC.). Competent cells of E. coli
DH5.alpha. (manufactured by TAKARA BIO INC.) were transformed with
the ligation solution and plated on an LB plate containing 50
.mu.g/mL of an antibiotic ampicillin, followed by culturing
overnight. Plasmid DNAs were collected from grown colonies by
miniprep and digested with restriction enzymes SalI and NotI,
followed by selection of a plasmid to which the LDH gene derived
from Xenopus laevis was inserted. This series of operation was
entirely carried out according to the manufacturer's protocol.
[0170] The pUC118 vector to which XLDH was inserted was digested
with restriction enzymes SalI and NotI, and the DNA fragments were
separated by 1% agarose gel electrophoresis, followed by
purification of the fragment containing XLDH according to a
conventional method. The obtained fragment was ligated to the
XhoI/NotI-digestion site in the expression vector pTRS11 shown in
FIG. 5, and plasmid DNA was collected by the same method as
described above, followed by digestion thereof with restriction
enzymes XhoI and NotI and selection of pTRS102 which is an
expression vector to which XLDH was inserted.
[0171] The method for inserting LDH to the PDC1, SEDT and TDH3 loci
using the thus obtained pTRS102 as the template for XLDH will now
be described.
(Part 2)
Insertion of LDH to Yeast Chromosome PDC1 Locus
[0172] Using pTRS102 as the template for amplification and
oligonucleotides (SEQ ID NOs:4 and 5) as the primer set, PCR was
carried out to amplify a PCR fragment of 1.3 kb comprising XLDH and
the TDH3 terminator sequence. SEQ ID NO:4 was designed such that
the sequence corresponding to the region of 60 bp in the immediate
upstream of the initiation codon of the PDC 1 gene was added.
[0173] Subsequently, using the plasmid pTRS424 as the template for
amplification and oligonucleotides (SEQ ID NOs:6 and 7) as the
primer set, PCR was carried out to amplify a PCR fragment of 1.2 kb
comprising the TRP 1 gene which is the selection marker for the
yeast. SEQ ID NO:7 was designed such that the sequence
corresponding to the region of 60 bp in the immediate downstream of
the termination codon of the PDC1 gene was added.
[0174] Each DNA fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method.
Using a mixture of the obtained 1.3 kb fragment and 1.2 kb fragment
as the template for amplification and oligonucleotides (SEQ ID
NOs:4 and 7) as the primer set, PCR was carried out to amplify a
PCR fragment of about 2.5 kb in which XLDH, the TDH3 terminator and
the TRP 1 gene were linked and the sequences corresponding to the
region of 60 bp in the immediate upstream and the region of 60 bp
in the immediate downstream of the PDC1 gene were added to the
5'-end and the 3'-end, respectively.
[0175] The PCR fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method,
followed by transformation and culturing on a culture medium to
which tryptophan was not supplemented. By this, a transformant
having XLDH introduced to the downstream of the PDC 1 gene promoter
on the chromosome was selected.
[0176] To confirm that the thus obtained transformant was yeast
having the Xenopus laevis-derived L-LDH introduced to the
downstream of the PDC1 gene promoter on the chromosome, the
following operations were carried out. First, the genomic DNA of
the transformant was prepared with a genomic DNA extraction kit Dr.
GenTLE (manufactured by TAKARA BIO INC.), and using the resulting
genomic DNA as the template for amplification and oligonucleotides
(SEQ ID NOs:7 and 8) as the primer set, PCR was carried out to
confirm that a DNA fragment of about 2.8 kb was amplified. An
untransformed strain yields a DNA fragment of about 2.1 kb by
amplification by the above PCR.
(Part 3)
Insertion of LDH to Yeast Chromosome SED1 Locus
[0177] In terms of the introduction into the SED 1 locus, using
pTRS102 prepared in Reference Example 1 as the template for
amplification and oligonucleotides (SEQ ID NOs:5 and 9) as the
primer set, PCR was carried out to amplify a PCR fragment of 1.3 kb
comprising the LDH gene derived from Xenopus laevis and the TDH3
terminator sequence. SEQ ID NO:9 was designed such that the
sequence corresponding to the region of 60 bp in the immediate
upstream of the initiation codon of the SED1 gene was added.
[0178] Subsequently, using the plasmid pRS423 as the template for
amplification and oligonucleotides (SEQ ID NOs:6 and 10) as the
primer set, PCR was carried out to amplify a PCR fragment of about
1.3 kb comprising the HIS3 gene which is the selection marker for
the yeast. SEQ ID NO:10 was designed such that the sequence
corresponding to the region of 60 bp in the immediate downstream of
the termination codon of the SED1 gene was added.
[0179] Each DNA fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method.
Using a mixture of the obtained two kinds of fragments having
lengths of about 1.3 kb as the template for amplification and
oligonucleotides (SEQ ID NOs:9 and 10) as the primer set, PCR was
carried out to amplify a PCR fragment of about 2.6 kb in which the
LDH derived from Xenopus laevis, the TDH3 terminator and the HIS3
gene were linked and the sequences corresponding to the regions of
60 bp in the immediate upstream and downstream of the SED1 gene
were added to the 5'-end and the 3'-end, respectively.
[0180] The PCR fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method,
followed by transformation and culturing on a culture medium to
which histidine was not supplemented. By this, a transformant
having XLDH introduced to the downstream of the SED1 gene promoter
on the chromosome was selected.
[0181] To confirm that the thus obtained transformant was a yeast
having XLDH introduced to the downstream of the SED1 gene promoter
on the chromosome, the following operations were carried out.
First, the genomic DNA of the transformant was prepared with a
genomic DNA extraction kit Dr. GenTLE (manufactured by TAKARA BIO
INC.), and using the resulting genomic DNA as the template for
amplification and oligonucleotides (SEQ ID NOs:11 and 12) as the
primer set, PCR was carried out to confirm that a DNA fragment of
about 2.9 kb was amplified. An untransformed strain yields a DNA
fragment of about 1.4 kb by amplification by the above PCR.
(Part 4)
Insertion of LDH to Yeast Chromosome TDH3 Locus
[0182] In terms of the introduction into the TDH3 locus, a plasmid
in which the TDH3 terminator of pTRS102 was replaced with the ADH1
terminator was prepared.
[0183] First, the genomic DNA was prepared from the NBRC10505
strain with a genomic DNA extraction kit "Dr. GenTLE" (manufactured
by TAKARA BIO INC.), and using the extracted genomic DNA as the
template and oligonucleotides (SEQ ID NOs:13 and 14) as the primer
set, PCR was carried out to amplify a PCR fragment containing the
ADH1 promoter. SEQ ID NO:13 was designed such that the NotI
recognition sequence was added to its 5'-end and SEQ ID NO:14 was
designed such that the HindIII recognition sequence was added to
its 3'-end.
[0184] The PCR amplification fragment was purified and
phosphorylated by T4 polynucleotide Kinase (manufactured by TAKARA
BIO INC.) at its ends, and ligated to the pUC118 vector (which had
been preliminarily digested with a restriction enzyme HincII and
the digested ends had been subjected to dephosphorylation
treatment). Competent cells of E. coli DH5.alpha. (manufactured by
TAKARA BIO INC.) were transformed with the ligation solution and
plated on an LB plate containing 50 .mu.g/mL of an antibiotic
ampicillin, followed by culturing overnight. From grown colonies,
plasmid DNAs were collected by miniprep and digested with
restriction enzymes NotI and HindIII, followed by selection of a
plasmid to which the ADH1 terminator was inserted. The prepared
plasmid will be referred to as pUC118-ADH1t.
[0185] Subsequently, pUC118-ADH1t was digested with restriction
enzymes NotI and HindIII, and the DNA fragments were separated by
1% agarose gel electrophoresis, followed by purification of the
fragment containing the ADH1 terminator according to a conventional
method. The obtained fragment containing the ADH1 terminator was
ligated to the NotI/HindIII-digestion site in pTRS102 and plasmid
DNAs were collected by the same method as described above, followed
by digestion thereof with restriction enzymes NotI and HindIII and
selection of a plasmid in which the TDH3 terminator was replaced
with the ADH1 terminator. The thus prepared plasmid will be
hereinafter referred to as pTRS150.
[0186] Using this pTRS150 as the template and oligonucleotides (SEQ
ID NOs:15 and 16) as the primer set, PCR was carried out to amplify
a PCR fragment of 1.3 kb comprising the frog-derived L-LDH gene and
the ADH1 terminator sequence. The primer having the sequence of SEQ
ID NO:16 was designed such that the sequence corresponding to the
region of 60 bp in the immediate upstream of the initiation codon
of the TDH3 gene was added.
[0187] Subsequently, using the plasmid pRS426 as the template for
amplification and oligonucleotides (SEQ ID NOs:17 and 18) as the
primer set, PCR was carried out to amplify a PCR fragment of 1.2 kb
comprising the URA3 gene which is the selection marker for the
yeast. The primer having the sequence of SEQ ID NO:18 was designed
such that the sequence corresponding to the region of 60 bp in the
immediate downstream of the termination codon of the TDH3 gene was
added.
[0188] Each DNA fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method.
Using a mixture of the obtained 1.3 kb fragment and 1.2 kb fragment
as the template for amplification and oligonucleotides (SEQ ID
NOs:16 and 18) as the primer set, PCR was carried out to amplify a
PCR fragment of about 2.5 kb in which XLDH, the ADH1 terminator and
the URA3 gene were linked.
[0189] The PCR fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method,
followed by transformation and culturing on a culture medium to
which uracil was not supplemented. By this, a transformant having
XLDH introduced to the downstream of the TDH3 gene promoter on the
chromosome was selected.
[0190] To confirm that the thus obtained transformant was yeast
having XLDH introduced to the downstream of the TDH3 gene promoter
on the chromosome, the following operations were carried out.
First, the genomic DNA of the transformant was prepared with a
genomic DNA extraction kit "Dr. GenTLE" (manufactured by TAKARA BIO
INC.), and using the resulting genomic DNA as the template for
amplification and oligonucleotides (SEQ ID NOs:19 and 20) as the
primer set, PCR was carried out to confirm that a DNA fragment of
about 2.8 kb was amplified. An untransformed strain yields a DNA
fragment of about 2.1 kb by amplification by the above PCR.
(Part 5)
Obtaining Yeast Having PDC5 Gene Having Temperature-sensitive
Mutation
[0191] A temperature-sensitive mutation was given by mating the
yeast SW015 strain having a temperature-sensitive mutation in the
pdc5 gene described in JP 2008-048726 A with the yeast to which a
temperature-sensitive mutation was to be given. In this diploid
yeast, ascus formation was allowed to occur in an ascus formation
medium, and the resulting asci were anatomized with a
micromanipulator to obtain respective haploid cells. Auxotrophy was
analyzed for each of the haploid cells to select a strain having
XLDH inserted into one of the PDC1 gene, SED1 gene and TDH1 locus
and further having a temperature-sensitive mutation in the PDC5
gene (incapable of growing at 34.degree. C.).
[0192] The method of reversion from auxotrophy will now be
described.
(Part 6)
[0193] Preparation of Strain Showing Reversion from Lysine
Auxotrophy
[0194] In cases of reversion from lysine auxotrophy, the following
method was used. Using the genomic DNA of BY4741 manufactured by
Funakoshi as the template and oligonucleotides (SEQ ID NOs:21 and
22) as the primer set, PCR was carried out to amplify a PCR
fragment of about 2 kb corresponding to the former half of the LYS2
gene. The PCR fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method,
followed by transformation to cancel the amber mutation of the LYS2
gene. By culturing the yeast on a culture medium to which lysine
was not supplemented, a transformant showing reversion of lysine
synthesis capacity was selected.
[0195] To confirm that the thus obtained transformant was yeast
whose amber mutation of the LYS2 gene was canceled, the following
operations were carried out. First, diploid cells were obtained by
mating the obtained transformant with the 20GY77 strain having the
wild type LYS2 gene. In the diploid cells, ascus formation was
allowed to occur in an ascus formation medium. The resulting asci
were anatomized with a micromanipulator to obtain respective
haploid cells, and auxotrophy was analyzed for each of the haploid
cells. All the obtained haploid cells were confirmed to have the
lysine synthesis capacity. In cases where reversion of the lysine
synthesis capacity occurred without canceling the mutation of the
LYS2 gene, cells having no lysine synthesis capacity are obtained
among the haploid cells obtained as above.
(Part 7)
[0196] Obtaining Strain Showing Reversion from Leucine
Auxotrophy
[0197] In the case of reversion from leucine auxotrophy, the
following method was used. Using the plasmid PRS425 as the template
and oligonucleotides (SEQ ID NOs:23 and 24) as the primer set, PCR
was carried out to amplify a PCR fragment of about 2 kb of the LEU2
gene. The PCR fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method,
followed by transformation to cancel the mutation of the LEU2 gene.
By culturing the yeast on a culture medium to which leucine was not
supplemented, a transformant showing reversion of the leucine
synthesis capacity was selected.
[0198] To confirm that the thus obtained transformant was yeast
whose mutation of the LEU2 gene was canceled, the following
operations were carried out. First, diploid cells were obtained by
mating the obtained transformant with the Saccharomyces cerevisiae
20GY7 strain having the wild type LEU2 gene. In the diploid cells,
ascus formation was allowed to occur in an ascus formation medium.
The resulting asci were anatomized with a micromanipulator to
obtain respective haploid cells, and auxotrophy was analyzed for
each of the haploid cells. All the obtained haploid cells were
confirmed to have the leucine synthesis capacity. In cases where
reversion of the leucine synthesis capacity occurred without
canceling the mutation of the LEU2 gene, cells having no leucine
synthesis capacity are obtained among the haploid cells obtained as
above.
(Part 8)
[0199] Obtaining Strain Showing Reversion from Adenine
Auxotrophy
[0200] In the case of reversion from adenine auxotrophy, the
following method was used. Using the plasmid PRS422 as the template
and oligonucleotides (SEQ ID NOs:25 and 26) as the primer set, PCR
was carried out to amplify a PCR fragment of about 2 kb of the ADE2
gene. The PCR fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method,
followed by transformation to cancel the mutation of the ADE2 gene.
By culturing the yeast on a culture medium to which adenine was not
supplemented, a transformant showing reversion of the adenine
synthesis capacity was selected.
[0201] To confirm that the thus obtained transformant was yeast
whose mutation of the AED2 gene was canceled, the following
operations were carried out. First, diploid cells were obtained by
mating the obtained transformant with the Saccharomyces cerevisiae
20GY7 strain having the wild type ADE2 gene. In the diploid cells,
ascus formation was allowed to occur in an ascus formation medium.
The resulting asci were anatomized with a micromanipulator to
obtain respective haploid cells, and auxotrophy was analyzed for
each of the haploid cells. All the obtained haploid cells were
confirmed to have the adenine synthesis capacity. In cases where
reversion of the adenine synthesis capacity occurred without
canceling the mutation of the ADE2 gene, cells having no adenine
synthesis capacity are obtained among the haploid cells obtained as
above.
(Part 9)
[0202] Obtaining Strain Showing Reversion from Uracil
Auxotrophy
[0203] In the case of reversion from uracil auxotrophy, the
following method was used. Using the plasmid pRS426 as the template
and oligonucleotides (SEQ ID NOs:27 and 28) as the primer set, PCR
was carried out to amplify a PCR fragment of about 2 kb of the URA3
gene. The PCR fragment was separated by 1% agarose gel
electrophoresis and purified according to a conventional method,
followed by transformation to cancel the mutation of the URA3 gene.
By culturing the yeast on a culture medium to which uracil was not
supplemented, a transformant showing reversion of the uracil
synthesis capacity was selected.
[0204] To confirm that the thus obtained transformant was yeast
whose mutation of the URA3 gene was canceled, the following
operations were carried out. First, diploid cells were obtained by
mating the obtained transformant with the Saccharomyces cerevisiae
20GY7 strain having the wild type URA3 gene. In the diploid cells,
ascus formation was allowed to occur in an ascus formation medium.
The resulting asci were anatomized with a micromanipulator to
obtain respective haploid cells, and auxotrophy was analyzed for
each of the haploid cells. All the obtained haploid cells were
confirmed to have the uracil synthesis capacity. In cases where
reversion of the uracil synthesis capacity occurred without
canceling the mutation of the URA3 gene, cells having no uracil
synthesis capacity are obtained among the haploid cells obtained as
above.
(Part 10)
Obtaining Polyploid Strain Having Capacity to Produce Lactic
Acid
[0205] By mating the transformants obtained by the combination of
the methods of Parts 1 to 9, a diploid prototrophic strain having
no auxotrophy was obtained. In the diploid prototrophic strain,
ascus formation was allowed to occur in an ascus formation medium,
and the resulting asci were anatomized with a micromanipulator to
obtain respective haploid cells. Auxotrophy was analyzed for each
of the haploid cells, and strains grown in SD medium (Table 1) were
judged as having reversion from auxotrophy and regarded as haploid
prototrophic strains.
TABLE-US-00002 TABLE 2 Strain Mating name type Genotype H1003
MATa/.alpha. .DELTA.pdc1::XLDH-TRP1 .DELTA.sed1::XLDH-HIS3
.DELTA.tdh3::XLDH-URA3 pdc5.sup.ts-9 Polyploid yeast (prototrophic)
SU014 MATa/.alpha. .DELTA.pdc1::XLDH-TRP1 .DELTA.sed1::XLDH-URA3
.DELTA.tdh3::XLDH-HIS3 pdc5.sup.ts-9 lys2-801 leu2-.DELTA.
Polyploid yeast (auxotrophic) SE001 MATa/.alpha.
.DELTA.pdc1::XLDH-TRP1 .DELTA.tdh3::XLDH-URA3 pdc5.sup.ts-9
Polyploid yeast (prototrophic) H1003-1B MATa .DELTA.pdc1::XLDH-TRP1
.DELTA.sed1::XLDH-HIS3 .DELTA.tdh3::XLDH-URA3 pdc5.sup.ts-9 Haploid
yeast (prototrophic) SU014-3B MATa .DELTA.pdc1::XLDH-TRP1
.DELTA.sed1::XLDH-URA3 .DELTA.tdh3::XLDH-HIS3 pdc5.sup.ts-9
lys2-801 leu2-.DELTA.1 Haploid yeast (auxotrophic) SE001-1A MATa
.DELTA.pdc1::XLDH-TRP1 .DELTA.tdh3::XLDH-HIS3 pdc5.sup.ts-9
lys2-801 leu2-.DELTA.1 Haploid yeast (prototrophic) SW015
MAT.alpha. ura3-52 trp1-.DELTA.63 his3-.DELTA.200 pdc5ts-9 lys2-801
leu2-.DELTA.1 Haploid yeast (auxotrophic) pdc5.sup.ts-9:
Temperature-sensitive mutation
Reference Example 2
Method for Quantifying Lactic Acid
[0206] Lactic acid was confirmed by measuring the amount of lactic
acid in the centrifugation supernatant of the culture by HPLC under
the following conditions; [0207] Column: Shim-Pack SPR--H
(manufactured by Shimadzu Corporation) [0208] Mobile phase: 5 mM
p-toluenesulfonic acid (flow rate: 0.8 mL/min.) [0209] Reaction
solution: 5 mM p-toluenesulfonic acid, 20 mM Bis-Tris, 0.1 mM
EDTA2Na (flow rate: 0.8 mL/min.) [0210] Detection method: electric
conductivity [0211] Temperature: 45.degree. C.
[0212] Measurement of the optical purity of L-lactic acid was
carried out by HPLC under the following conditions: [0213] Column:
TSK-gel Enantio L1 (manufactured by produced by Tosoh Corporation)
[0214] Mobile phase: 1 mM aqueous copper sulfate solution [0215]
Flow rate: 1.0 ml/min. [0216] Detection method: UV 254 nm [0217]
Temperature: 30.degree. C.
[0218] The optical purity of L-lactic acid was calculated by the
following equation: [0219] Optical purity (%)=100.times.(L-D)/(L+D)
[0220] (wherein L represents the concentration of L-lactic acid,
and D represents the concentration of D-lactic acid).
Reference Example 3
Preparation of Porous Membrane (Part 1)
[0221] A polyvinylidene fluoride (PVDF) resin and
N,N-dimethylacetamide (DMAc) were used as a resin and a solvent,
respectively, and these were sufficiently stirred at a temperature
of 90.degree. C. to obtain a starting solution having the following
composition:
[Starting Solution]
[0222] PVDF: 13.0% by weight [0223] DMAc: 87.0% by weight.
[0224] Subsequently, the above starting solution was cooled to a
temperature of 25.degree. C. and applied to a polyester fiber
non-woven fabric (porous base material) having a density of 0.48
g/cm.sup.3 and a thickness of 220 .mu.m which had been
preliminarily attached on a glass plate, and the resultant was
immediately immersed in a coagulation bath at a temperature of
25.degree. C. having the following composition for 5 minutes to
obtain a flat membrane having a porous resin layer formed on the
porous base material:
[Coagulation Bath]
[0225] Water: 30.0% by weight [0226] DMAc: 70.0% by weight.
[0227] This porous membrane was peeled from the glass plate and
immersed three times in hot water at a temperature of 80.degree. C.
to wash out DMAc, to obtain a separation membrane. By observation
of an area of 9.2 .mu.m.times.10.4 .mu.m on the surface of the
porous resin layer with a scanning electron microscope at a
magnification of 10,000.times., it was revealed that the average
diameter of all the observable pores was 0.1 .mu.m. Subsequently,
the above separation membrane was evaluated for its pure water
permeability coefficient, and it was turned out to be
50.times.10.sup.-9 m.sup.3/m.sup.2/s/Pa. The standard deviation of
the average pore size was 0.035 .mu.m, and the membrane surface
roughness was 0.06 .mu.m.
Reference Example 4
Preparation of Porous Membrane (Part 2)
[0228] A vinylidene fluoride homopolymer having a weight average
molecular weight of 417,000 and .gamma.-butyrolactone were
dissolved at a temperature of 170.degree. C. at ratios of 38% by
weight and 62% by weight, respectively, to prepare a starting
solution. This starting solution was extruded from a mouthpiece
while being accompanied by .gamma.-butyrolactone as a fluid for
formation of the hollow portion, followed by cooling and
solidifying the resultant in a cooling bath at 20.degree. C.
comprising aqueous 80% by weight .gamma.-butyrolactone solution, to
prepare a hollow fiber membrane.
[0229] Subsequently, 14% by weight of a vinylidene fluoride
homopolymer having a weight average molecular weight of 284,000, 1%
by weight of cellulose acetate propionate (manufactured by Eastman
Chemical Company, CAP482-0.5), 77% by weight of
N-methyl-2-pyrrolidone, 5% by weight of polyoxyethylene coconut oil
fatty acid sorbitan (manufactured by Sanyo Chemical Industries,
Ltd.; trade name "IONET T-20C" (registered trademark)) and 3% by
weight of water were mixed and dissolved at a temperature of
95.degree. C., to adjust a starting solution. This starting
solution was evenly applied to the surface of the hollow fiber
membrane and immediately allowed to coagulate in a water bath, to
prepare the hollow fiber membrane (porous membrane). The average
pore size of the surface in the side of the liquid to be processed
of the obtained hollow fiber membrane (separation membrane) was
0.05 .mu.m. The pure water permeability coefficient of the hollow
fiber membrane which is the above separation membrane was evaluated
to be 5.5.times.10.sup.-9 m.sup.3/m.sup.2sPa. The standard
deviation of the average pore size was 0.006 .mu.m.
Example 1
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 1)
[0230] Using the yeast HI003 strain prepared in Reference Example
1, lactic acid was produced with the continuous fermentation device
of FIG. 1 and the SC4 medium having the composition shown in Table
3. The culture medium was autoclaved at 121.degree. C. for 15
minutes before use. As the members for the separation membrane
element, stainless steel and polysulfone resin molded products were
used. As the separation membrane, the porous membrane prepared in
the above-described Reference Example 3 was used.
TABLE-US-00003 TABLE 3 YEAST NITROGEN W/O AA & AS 1.7 g
(manufactured by Difco) Yeast Synthetic Drop-out Medium 3.84 g
Supplements without tryptophan (manufactured by SIGMA) Ammonium
sulfate 1.5 g Glucose 100 g up to 1 L
[0231] The operation conditions in Example 1 were as follows:
[0232] Fermentation reaction vessel capacity: 2 (L) [0233] Membrane
separation vessel capacity: 0.5 (L) [0234] Separation membrane
used: polyvinylidene fluoride filter (Reference Example 3) [0235]
Effective filtration area of the membrane separation element: 60
cm.sup.2 [0236] Temperature adjustment: 32 (.degree. C.) [0237]
Ventilation volume in the fermentation reaction vessel: 0.05
(L/min.) [0238] Ventilation volume in the membrane separation
vessel: 0.3 (L/min.) [0239] Stirring rate in the fermentation
reaction vessel: 100 (rpm) [0240] pH adjustment: adjusted to pH 5
with 8 N Ca(OH).sub.2 [0241] Feed rate of lactic acid fermentation
medium: variably controlled within the range of 50 to 300 ml/hr.
[0242] Amount of the liquid circulated by the fermentation liquid
circulating device: 0.1 (L/min.) [0243] Control of the amount of
permeation of the liquid through the membrane: control of the flow
rate by the transmembrane pressure difference (controlled at a
transmembrane pressure difference of 0.1 kPa to less than 20
kPa).
[0244] The culture vessel comprising the separation membrane
element was autoclaved at 121.degree. C. for 20 minutes.
[0245] For evaluation of the concentration of lactic acid which is
the product, the above-described HPLC shown in Reference Example 2
was used, and for measurement of the glucose concentration,
"Glucose Test Wako C" (registered trademark) (manufactured by Wako
Pure Chemical Industries, Ltd.) was used.
[0246] First, the HI003 strain was cultured in 5 ml of SC4 medium
in a test tube overnight with shaking to obtain a culture
(pre-pre-preculture). The obtained culture was inoculated in 100 ml
of fresh SC4 medium and subjected to culture in a 500 ml Sakaguchi
flask for 24 hours at a temperature of 30.degree. C. with shaking
(pre-preculture). The pre-preculture was inoculated in 1.5 L of SC4
medium in the continuous fermentation device shown in FIG. 1, and
the fermentation reaction vessel 1 was stirred with the stirrer 5
attached thereto. The ventilation volume, temperature and pH in the
fermentation reaction vessel 1 were adjusted and culture was
carried out for 24 hours without operating the fermentation culture
medium circulating pump 11 (preculture). Immediately after the
completion of the preculture, the fermentation culture medium
circulating pump 11 was operated, and production of lactic acid by
continuous fermentation was carried out by continuous culture under
the conditions of, in addition to the operating conditions under
which the preculture was carried out, aeration of the membrane
separation vessel 12, continuous supply of SC4 medium and control
of the amount of permeation of the liquid through the membrane such
that the amount of the fermentation culture medium in the
continuous fermentation device of the membrane separation type
became 2 L. The amount of permeation of the liquid through the
membrane during the continuous fermentation test was appropriately
controlled by the pressure difference controlling apparatus 3 such
that transmembrane pressure difference became not less than 0.1 kPa
and less than 20 kPa. The concentration of the produced lactic acid
and the concentration of the remaining glucose in the fermentation
culture medium permeated through the membrane were measured as
appropriate.
[0247] As a result of 800 hours of the continuous fermentation
test, stable production of lactic acid by continuous fermentation
was possible as shown in Table 6.
Example 2
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 2)
[0248] Using the yeast HI003 strain prepared in Reference Example
1, lactic acid was produced with the continuous fermentation device
shown in FIG. 2 and the SC4 medium having the composition shown in
Table 3. The culture medium was autoclaved at 121.degree. C. for 15
minutes before use. As the members for the separation membrane
element, stainless steel and poly-sulfone resin molded products
were used. As the separation membrane, the porous separation
membrane prepared in the above-described Reference Example 3 was
used.
[0249] The operation conditions in Example 2 were as follows:
[0250] Fermentation reaction vessel capacity: 2 (L) [0251]
Separation membrane used: polyvinylidene fluoride filter (Reference
Example 3) [0252] Effective filtration area of the membrane
separation element: 120 cm.sup.2 [0253] Temperature adjustment: 32
(.degree. C.) [0254] Ventilation volume in the fermentation
reaction vessel: 0.05 (L/min.) [0255] Feed rate of the lactic acid
fermentation medium: variably controlled within the range of 50 to
300 ml/hr. [0256] Stirring rate in the fermentation reaction
vessel: 800 (rpm) [0257] pH adjustment: adjusted to pH 5 with 8 N
Ca(OH).sub.2 [0258] Control of the amount of permeation of the
liquid through the membrane: control of the flow rate by the
transmembrane pressure difference (controlled at a transmembrane
pressure difference of 0.1 kPa to less than 20 kPa).
[0259] The culture vessel comprising the separation membrane
element was autoclaved at 121.degree. C. for 20 minutes.
[0260] For evaluation of the concentration of lactic acid which is
the product, the above-described HPLC shown in Reference Example 2
was used, and for measurement of the glucose concentration,
"Glucose Test Wako C" (registered trademark) (manufactured by Wako
Pure Chemical Industries, Ltd.) was used.
[0261] First, the HI003 strain was cultured in 5 ml of SC4 medium
in a test tube overnight with shaking to obtain a culture
(pre-pre-preculture). The obtained culture was inoculated in 100 ml
of fresh SC4 medium and subjected to culture in a 500 ml Sakaguchi
flask for 24 hours at a temperature of 30.degree. C. with shaking
(pre-preculture). The pre-preculture was inoculated to 1.5 L of SC4
medium in the continuous fermentation device of the membrane
separation type shown in FIG. 2, and the fermentation reaction
vessel 1 was stirred with the stirrer 5 attached thereto at 400
rpm. The ventilation volume, temperature and pH in the fermentation
reaction vessel 1 were adjusted and culture was carried out for 24
hours (preculture). Immediately after the completion of the
preculture, production of lactic acid by continuous fermentation
was carried out by continuous supply of SC4 medium and continuous
culture while controlling the amount of permeation of the liquid
through the membrane such that the amount of the fermentation
culture medium in the continuous fermentation device of the
membrane separation type became 1.5 L. The amount of permeation of
the liquid through the membrane during the continuous fermentation
test was appropriately controlled by the pressure difference
controlling apparatus 3 such that the transmembrane pressure
difference became not less than 0.1 kPa and less than 20 kPa. The
concentration of the produced lactic acid and the concentration of
the remaining glucose in the fermentation culture medium permeated
through the membrane were measured as appropriate. Further, the
yield of lactic acid to saccharides and the rate of production of
lactic acid, which were calculated from the fed glucose calculated
from the concentrations of lactic acid and glucose, were
measured.
[0262] The result of 800 hours of the continuous fermentation test
is shown in Table 6. Stable production of lactic acid by continuous
fermentation was possible by the method for production of lactic
acid using the continuous fermentation device shown in FIG. 2.
Example 3
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 3)
[0263] The operations and evaluations were carried out under the
same conditions as in Example 1 except that the porous membrane
prepared in the above-described Reference Example 4 was used as the
separation membrane. The result of 750 hours of the continuous
fermentation test is shown in Table 6. Stable production of lactic
acid by continuous fermentation was possible by our method for
production of lactic acid.
Example 4
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 4)
[0264] The operations and evaluations were carried out under the
same conditions as in Example 2 except that the porous separation
membrane prepared in the above-described Reference Example 4 was
used as the separation membrane. The result of 770 hours of a
fermentation test is shown in Table 6. Stable production of lactic
acid by continuous fermentation was possible by our method for
production of lactic acid.
Comparative Example 1
Production of Lactic Acid by Batch Fermentation Using Polyploid
(Prototrophic) Yeast Having Capacity to Produce Lactic Acid
[0265] Batch fermentation, which is the most typical mode of
fermentation using yeast cells, was carried out to evaluate its
L-lactic acid productivity. Using the SC4 medium shown in Table 3,
batch fermentation test was carried out using only the fermentation
reaction vessel 1 in the continuous fermentation device of the
membrane separation type in FIG. 1. The culture medium was
autoclaved at 121.degree. C. for 15 minutes before use. Also in
this Comparative Example 1, the yeast HI003 strain prepared in the
above-described Reference Example 1 was used as the yeast, and the
concentration of lactic acid, which is the product, and saccharides
in the culture were quantified using the HPLC shown in the
above-described Reference Example 2.
[0266] The operation conditions in Comparative Example 1 were as
follows: [0267] Fermentation reaction vessel capacity: 2 (L) [0268]
Temperature adjustment: 30 (.degree. C.) [0269] Ventilation volume
in the fermentation reaction vessel: 0.05 (L/min.) [0270] Stirring
rate in the fermentation reaction vessel: 100 (rpm) [0271] pH
adjustment: adjusted to pH 5 with 8 N Ca(OH).sub.2.
[0272] The culture vessel was autoclaved at 121.degree. C. for 20
minutes.
[0273] First, the HI003 strain was cultured in 5 ml of a lactic
acid fermentation medium in a test tube overnight with shaking
(pre-preculture). The pre-preculture was inoculated in 100 ml of a
fresh lactic acid fermentation medium and subjected to culture in a
500 ml Sakaguchi flask for 24 hours with shaking (preculture). The
preculture was inoculated in 1 L of the lactic acid fermentation
medium in the continuous fermentation device of the membrane
separation type, and the fermentation reaction vessel 1 was stirred
with the stirrer 5 attached thereto at 100 rpm for aeration of the
fermentation reaction vessel 1. The temperature and pH were
adjusted and batch fermentation culture was carried out without
operating the fermentation culture medium circulating pump 11. The
amount of growth of the yeast at this time was 14 in terms of
absorbance at 600 nm. The result of the batch fermentation is shown
in Table 6. The fermentation time was short, and the yield of
lactic acid to saccharides and the rate of production of lactic
acid were inferior to those in Examples.
Comparative Example 2
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Prototrophic) (Part 1)
[0274] The evaluations were carried out under the same conditions
as in Example 1 except that the yeast HI003-1B strain prepared in
Reference Example 1 was used. As a result, the transmembrane
pressure difference became not less than 20 kPa 600 hours later and
the culture was terminated. The result of the continuous
fermentation test is shown in Table 6. As a result, it was revealed
that lactic acid can be produced longer and more stably when
polyploid yeast is used.
Comparative Example 3
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Prototrophic) (Part 2)
[0275] The operations and evaluations were carried out under the
same conditions as in Example 2 except that the yeast HI003-1B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
600 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 6. As a result, it
was revealed that lactic acid can be produced longer and more
stably when polyploid yeast is used.
Comparative Example 4
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Prototrophic) (Part 3)
[0276] The operations and evaluations were carried out under the
same conditions as in Example 3 except that the yeast HI003-1B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
500 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 6. As a result, it
was revealed that lactic acid can be produced longer and more
stably when polyploid yeast is used.
Comparative Example 5
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Prototrophic) (Part 4)
[0277] The operations and evaluations were carried out under the
same conditions as in Example 4 except that the yeast HI003-1B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
500 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 6. As a result, it
was revealed that lactic acid can be produced longer and more
stably when polyploid yeast is used.
Comparative Example 6
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) (Part 1)
[0278] The operations and evaluations were carried out under the
same conditions as in Example 1 except that the yeast SU014-3B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
300 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 6.
[0279] As a result, it was revealed that lactic acid can be
produced longer and more stably when prototrophic yeast or
polyploid yeast is used.
Comparative Example 7
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) (Part 2)
[0280] The operations and evaluations were carried out under the
same conditions as in Example 2 except that the yeast SU014-3B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
280 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 6. As a result, it
was revealed that lactic acid can be produced longer and more
stably when prototrophic yeast or polyploid yeast is used.
Comparative Example 8
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) (Part 3)
[0281] The operations and evaluations were carried out under the
same conditions as in Example 3 except that the yeast SU014-3B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
350 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 6. As a result, it
was revealed that lactic acid can be produced longer and more
stably when prototrophic yeast or polyploid yeast is used.
Comparative Example 9
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) (Part 4)
[0282] The operations and evaluations were carried out under the
same conditions as in Example 4 except that the yeast SU014-3B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
270 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 6. As a result, it
was revealed that lactic acid can be produced longer and more
stably when prototrophic yeast or polyploid yeast is used.
Example 5
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 5)
[0283] The operations and evaluations were carried out under the
same conditions as in Example 1 except that the lactic acid
fermentation medium described in Table 4 was used. The result of
800 hours of a fermentation test is shown in Table 7. Even with the
lactic acid fermentation medium having less nutrients than SC4
medium, stable production of lactic acid by continuous fermentation
was possible by our method for producing lactic acid.
TABLE-US-00004 TABLE 4 Raw sugar (manufactured by Muso Co., Ltd.)
50 g Ammonium sulfate 1.5 g up to 1 L
Example 6
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 6)
[0284] The operations and evaluations were carried out under the
same conditions as in Example 2 except that the lactic acid
fermentation medium described in Table 4 was used. The result of
800 hours of a fermentation test is shown in Table 7. Even with the
lactic acid fermentation medium having less nutrients than SC4
medium, stable production of lactic acid by continuous fermentation
was possible by our method for producing lactic acid.
Example 7
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 7)
[0285] The operations and evaluations were carried out under the
same conditions as in Example 3 except that the lactic acid
fermentation medium described in Table 4 was used. The result of
800 hours of a fermentation test is shown in Table 7. Even with the
lactic acid fermentation medium having less nutrients than SC4
medium, stable production of lactic acid by continuous fermentation
was possible by our method for producing lactic acid.
Example 8
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 8)
[0286] The operations and evaluations were carried out under the
same conditions as in Example 4 except that the lactic acid
fermentation medium described in Table 4 was used. The result of
800 hours of a fermentation test is shown in Table 7. Even with the
lactic acid fermentation medium having less nutrients than SC4
medium, stable production of lactic acid by continuous fermentation
was possible by our method for producing lactic acid.
Example 9
Production of Lactic Acid by Continuous Fermentation Using
Polyploid Yeast (Auxotrophic) Having Capacity to Produce Lactic
Acid (Part 1)
[0287] The operations and evaluations were carried out under the
same conditions as in Example 5 except that the yeast SU014 strain
prepared in Reference Example 1 was used. The result of 700 hours
of a continuous fermentation test is shown in Table 7. Although the
yield of lactic acid to saccharides and the rate of production of
lactic acid were rather lower than in the cases of the prototrophic
polyploid yeast, stable production of lactic acid by continuous
fermentation was possible for a long time.
Example 10
Production of Lactic Acid by Continuous Fermentation Using
Polyploid Yeast (Auxotrophic) Having Capacity to Produce Lactic
Acid (Part 2)
[0288] The operations and evaluations were carried out under the
same conditions as in Example 6 except that the yeast SU014 strain
prepared in Reference Example 1 was used. The result of 700 hours
of a fermentation test is shown in Table 7. Although the yield of
lactic acid to saccharides and the rate of production of lactic
acid were rather lower than in the cases of the prototrophic
polyploid yeast, stable production of lactic acid by continuous
fermentation was possible for a long time.
Example 11
Production of L-Lactic Acid by Continuous Fermentation Using
Polyploid Yeast (Auxotrophic) Having Capacity to Produce Lactic
Acid (Part 3)
[0289] The operations and evaluations were carried out under the
same conditions as in Example 7 except that the yeast SU014 strain
prepared in Reference Example 1 was used. The result of 650 hours
of a continuous fermentation test is shown in Table 7. Although the
yield of lactic acid to saccharides and the rate of production of
lactic acid were rather lower than in the cases of the prototrophic
polyploid yeast, stable production of lactic acid by continuous
fermentation was possible for a long time.
Example 12
Production of Lactic Acid by Continuous Fermentation Using
Polyploid Yeast (Auxotrophic) Having Capacity to Produce Lactic
Acid (Part 4)
[0290] The operations and evaluations were carried out under the
same conditions as in Example 8 except that the yeast SU014 strain
prepared in Reference Example 1 was used. The result of 670 hours
of a fermentation test is shown in Table 7. Although the yield of
lactic acid to saccharides and the rate of production of lactic
acid were rather lower than in the cases of the prototrophic
polyploid yeast, stable production of lactic acid by continuous
fermentation was possible for a long time.
Comparative Example 10
Production of Lactic Acid by Batch Fermentation Using Polyploid
(Prototrophic) Yeast Having Capacity to Produce Lactic Acid
[0291] The operations and evaluations were carried out under the
same conditions as in Comparative Example 1 except that the lactic
acid fermentation medium described in Table 4 was used. The result
of batch fermentation is shown in Table 7. The fermentation time
was short, and the yield of lactic acid to saccharides and the rate
of production of lactic acid were inferior to those in the
Examples.
Comparative Example 11
Production of Lactic Acid by Batch Fermentation Using Polyploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid
[0292] The operations and evaluations were carried out under the
same conditions as in Comparative Example 2 except that the yeast
SU014 strain prepared in Reference Example 1 was used. The result
of batch fermentation is shown in Table 7. The fermentation time
was short, and the yield of lactic acid to saccharides and the rate
of production of lactic acid were inferior to those in the
Examples.
Comparative Example 12
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid (Part
1)
[0293] The operations and evaluations were carried out under the
same conditions as in Example 5 except that the yeast SU014-3B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
300 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 7. As a result, it
was revealed that lactic acid can be produced longer and more
stably when polyploid yeast is used.
Comparative Example 13
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid (Part
2)
[0294] The operations and evaluations were carried out in the same
manner as in Example 6 except that the yeast SU014-3B strain
prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
280 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 7. As a result, it
was revealed that lactic acid can be produced longer and more
stably when polyploid yeast is used.
Comparative Example 14
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid (Part
3)
[0295] The operations and evaluations were carried out in the same
manner as in Example 7 except that the yeast SU014-3B strain
prepared in Reference Example 1 was used. As a result, in the case
of the haploid yeast, the transmembrane pressure difference became
not less than 20 kPa at 350 hours and the culture was terminated.
The result of the continuous fermentation test is shown in Table 7.
As a result, it was revealed that lactic acid can be produced
longer and more stably when polyploid yeast is used.
Comparative Example 15
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid (Part
4)
[0296] The operations and evaluations were carried out in the same
manner as in Example 8 except that the yeast SU014-3B strain
prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
270 hours and the culture was terminated. The result of the
continuous fermentation test is shown in Table 7. As a result, it
was revealed that lactic acid can be produced longer and more
stably when polyploid yeast is used.
Example 13
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 9)
[0297] The operations and evaluations were carried out under the
same conditions as in Example 1 except that the effective
filtration area of the membrane separation element was increased to
240 cm.sup.2 and the lactic acid fermentation medium 2 described in
Table 5 was used. The results of 1400 hours of a fermentation test
are shown in FIGS. 6 to 8. Stable production of lactic acid by
continuous fermentation was possible for a long time by our method
for producing lactic acid.
TABLE-US-00005 TABLE 5 Raw sugar (manufactured by Muso Co., Ltd.)
100 g Ammonium sulfate 1.5 g up to 1 L
Example 14
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 10)
[0298] The operations and evaluations were carried out under the
same conditions as in Example 3 except that the effective
filtration area of the membrane separation element was increased to
240 cm.sup.2 and the lactic acid fermentation medium 2 described in
Table 5 was used. The results of 1350 hours of a fermentation test
are shown in FIGS. 6 to 8. Stable production of lactic acid by
continuous fermentation was possible for a long time by our method
for producing lactic acid.
Example 15
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 11)
[0299] The operations and evaluations were carried out under the
same conditions as in Example 13 except that the yeast SE001 strain
prepared in Reference Example 1 was used. The results of 1350 hours
of a fermentation test are shown in FIGS. 9 to 11. Stable
production of lactic acid by continuous fermentation was possible
for a long time by our method for producing lactic acid.
Example 16
Production of Lactic Acid by Continuous Fermentation Using
Polyploid (Prototrophic) Yeast Having Capacity to Produce Lactic
Acid (Part 12)
[0300] The operations and evaluations were carried out under the
same conditions as in Example 14 except that the yeast SE001 strain
prepared in Reference Example 1 was used. The results of 1350 hours
of a fermentation test are shown in FIGS. 9 to 11. Stable
production of lactic acid by continuous fermentation was possible
for a long time by our method for producing lactic acid.
Comparative Example 16
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid (Part
5)
[0301] The operations and evaluations were carried out under the
same conditions as in Example 13 except that the yeast SU014-3B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
300 hours and the culture was terminated. The results of the
continuous fermentation test are shown in FIGS. 9 to 11. As a
result, it was revealed that lactic acid can be produced longer and
more stably when polyploid yeast is used.
Comparative Example 17
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid (Part
6)
[0302] The operations and evaluations were carried out under the
same conditions as in Example 14 except that the yeast SU014-3B
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
280 hours and the culture was terminated. The results of the
continuous fermentation test are shown in FIGS. 9 to 11. As a
result, it was revealed that lactic acid can be produced longer and
more stably when polyploid yeast is used.
Comparative Example 18
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid (Part
7)
[0303] The operations and evaluations were carried out under the
same conditions as in Example 15 except that the yeast SE001-1A
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
300 hours and the culture was terminated. The results of the
continuous fermentation test are shown in FIGS. 9 to 11. As a
result, it was revealed that lactic acid can be produced longer and
more stably when polyploid yeast is used.
Comparative Example 19
Production of Lactic Acid by Continuous Fermentation Using Haploid
Yeast (Auxotrophic) Having Capacity to Produce Lactic Acid (Part
8)
[0304] The operations and evaluations were carried out under the
same conditions as in Example 16 except that the yeast SE001-1A
strain prepared in Reference Example 1 was used. As a result, the
transmembrane pressure difference became not less than 20 kPa at
270 hours and the culture was terminated. The results of the
continuous fermentation test are shown in FIGS. 9 to 11. As a
result, it was revealed that lactic acid can be produced longer and
more stably when polyploid yeast is used.
[0305] The culture conditions in Examples 1 to 16 and Comparative
Examples 1 to 19 are shown in Table 8.
TABLE-US-00006 TABLE 6 Com- Com- Com- Com- Com- Com- Com- Com- Com-
parative parative parative parative parative Exam- Exam- Exam-
Exam- parative parative parative parative Exam- Exam- Exam- Exam-
Exam- ple 1 ple 2 ple 3 ple 4 Example 1 Example 2 Example 3 Example
4 ple 5 ple 6 ple 7 ple 8 ple 9 Fermentation 800 800 750 770 72 600
600 500 500 300 280 350 270 time (hr.) Total glucose 8424 7896 6653
7500 100 6312 5922 4158 4687 3240 2820 3000 2420 fed Total L-lactic
5882 5764 4790 5600 26 4366 4323 2993 3857 1920 1700 1770 1488 acid
produced (g) Unused 20 10 20 10 0 20 10 20 10 40 70 40 20 glucose
Yield of L- 0.7 0.73 0.72 0.75 0.22 0.65 0.67 0.66 0.68 0.6 0.62
0.59 0.62 lactic acid to glucose Rate of 3.7 4.8 3.2 4.8 0.36 3.5
4.6 3 4.5 3.2 4 2.5 3.8 production of L-lactic acid (g/L/hr.)
TABLE-US-00007 TABLE 7 Com- Com- Com- Com- Com- Com- parative
parative parative parative parative parative Exam- Exam- Exam-
Exam- Exam- Exam- Exam- Exam- Exam- Exam- Exam- Exam- Exam- Exam-
ple 5 ple 6 ple 7 ple 8 ple 9 ple 10 ple 11 ple 12 ple 10 ple 11
ple 12 ple 13 ple 14 ple 15 Fermentation 800 800 750 770 700 700
650 670 72 70 300 280 350 270 time (hr.) Total 6362 10530 5020 9876
5610 8057 4170 8283 100 100 2354 3586 2302 3295 glucose fed Total
L- 4440 7680 3600 7392 3360 4900 2535 4958 26 22 1350 2128 1312
1998 lactic acid produced (g) Unused 20 10 20 20 10 25 15 20 0 0 27
40 40 20 glucose Yield of L- 0.7 0.73 0.72 0.75 0.6 0.61 0.61 0.6
0.26 0.22 0.58 0.6 0.58 0.61 lactic acid to glucose Rate of 3.7 4.8
3.2 4.8 3.2 3.5 2.6 3.7 0.36 0.32 3 3.8 2.5 3.7 production of
L-lactic acid (g/L/hr.)
TABLE-US-00008 TABLE 8 Area of Yeast Culture Separation Culture
separation Sample apparatus membrane medium Result membrane Example
1 HI003 FIG. 1 Reference Table 3 Table 6 60 cm.sup.2 Example 3
Example 2 HI003 FIG. 2 Reference Table 3 Table 6 120 cm.sup.2
Example 3 Example 3 HI003 FIG. 1 Reference Table 3 Table 6 60
cm.sup.2 Example 4 Example 4 HI003 FIG. 2 Reference Table 3 Table 6
120 cm.sup.2 Example 4 Example 5 HI003 FIG. 1 Reference Table 4
Table 7 60 cm.sup.2 Example 3 Example 6 HI003 FIG. 2 Reference
Table 4 Table 7 120 cm.sup.2 Example 3 Example 7 HI003 FIG. 1
Reference Table 4 Table 7 60 cm.sup.2 Example 4 Example 8 HI003
FIG. 2 Reference Table 4 Table 7 120 cm.sup.2 Example 4 Example 9
SU014 FIG. 1 Reference Table 4 Table 7 60 cm.sup.2 Example 3
Example 10 SU014 FIG. 2 Reference Table 4 Table 7 120 cm.sup.2
Example 3 Example 11 SU014 FIG. 1 Reference Table 4 Table 7 60
cm.sup.2 Example 4 Example 12 SU014 FIG. 2 Reference Table 4 Table
7 120 cm.sup.2 Example 4 Example 13 HI003 FIG. 1 Reference Table 5
FIG. 6, FIG. 240 cm.sup.2 Example 3 7, FIG. 8 Example 14 HI003 FIG.
1 Reference Table 5 FIG. 6, FIG. 240 cm.sup.2 Example 4 7, FIG. 8
Example 15 SE001 FIG. 1 Reference Table 5 FIG. 9, FIG. 240 cm.sup.2
Example 5 10, FIG. 11 Example 16 SE001 FIG. 1 Reference Table 5
FIG. 9, FIG. 240 cm.sup.2 Example 6 10, FIG. 11 Comparative HI003
FIG. 1 (Batch Table 3 Table 6 Example 1 Culture) Comparative
HI003-1B FIG. 1 Reference Table 3 Table 6 60 cm.sup.2 Example 2
Example 3 Comparative HI003-1B FIG. 2 Reference Table 3 Table 6 120
cm.sup.2 Example 3 Example 3 Comparative HI003-1B FIG. 1 Reference
Table 3 Table 6 60 cm.sup.2 Example 4 Example 4 Comparative
HI003-1B FIG. 2 Reference Table 3 Table 6 120 cm.sup.2 Example 5
Example 4 Comparative SU014-3B FIG. 1 Reference Table 3 Table 6 60
cm.sup.2 Example 6 Example 3 Comparative SU014-3B FIG. 2 Reference
Table 3 Table 6 120 cm.sup.2 Example 7 Example 3 Comparative
SU014-3B FIG. 1 Reference Table 3 Table 6 60 cm.sup.2 Example 8
Example 4 Comparative SU014-3B FIG. 2 Reference Table 3 Table 6 120
cm.sup.2 Example 9 Example 4 Comparative HI003 FIG. 1 (Batch Table
4 Table 7 Example 10 Culture) Comparative SU014 FIG. 1 (Batch Table
4 Table 7 Example 11 Culture) Comparative SU014-3B FIG. 1 Reference
Table 4 Table 7 60 cm.sup.2 Example 12 Example 3 Comparative
SU014-3B FIG. 2 Reference Table 4 Table 7 120 cm.sup.2 Example 13
Example 3 Comparative SU014-3B FIG. 1 Reference Table 4 Table 7 60
cm.sup.2 Example 14 Example 4 Comparative SU014-3B FIG. 2 Reference
Table 4 Table 7 120 cm.sup.2 Example 15 Example 4 Comparative
SU014-3B FIG. 1 Reference Table 5 FIG. 6, FIG. 240 cm.sup.2 Example
16 Example 3 7, FIG. 8 Comparative SU014-3B FIG. 1 Reference Table
5 FIG. 6, FIG. 240 cm.sup.2 Example 17 Example 4 7, FIG. 8
Comparative SE001-1A FIG. 1 Reference Table 5 FIG. 9, FIG. 240
cm.sup.2 Example 18 Example 5 10, FIG. 11 Comparative SE001-1A FIG.
1 Reference Table 5 FIG. 9, FIG. 240 cm.sup.2 Example 19 Example 6
10, FIG. 11
INDUSTRIAL APPLICABILITY
[0306] By our methods for producing lactic acid by continuous
fermentation using polyploid yeast as the yeast sample, continuous
fermentation wherein a high productivity of lactic acid which is
the desired fermentation product can be stably maintained for a
long time is possible under simple operating conditions. Therefore,
lactic acid as a fermentation product can be stably produced at a
low cost widely in the fermentation industry.
Sequence CWU 1
1
281999DNAXenopus laevis 1atggcaactg tgaaggataa actcatccac
aatgtggtca aggaggagtc gctcccccag 60aacaaggtca ccattgtggg tgtgggggcc
gtgggcatgg cctgtgccat cagtgtcctg 120cagaaggatt tggcagatga
gcttgcactt gttgatgtga tagaagacaa actgaagggg 180gaaatgatgg
atctccagca tggcagtctg ttccttcgta cccccaagat tgtctcaggg
240aaagattaca gcgtcactgc aaactccaag ctggtagttg tgacggccgg
ggcccgtcag 300caggagggag agagtcgcct gaatctggtt cagcgcaatg
tcaacatctt caaattcatc 360attcccaaca ttgtcaagta cagccccaac
tgcaccctgc tcatcgtctc caacccagtg 420gacattctga catatgtggc
ctggaagatc agtggattcc ccaaaaaccg tgtcattggc 480agcggctgca
atttggactc tgcccgtttc cgttacctca tggggcagaa gtttgggatc
540cacacccaga gctgccacgg ttgggtcatt ggggaacacg gagactcgag
tgtgccagtg 600tggagtgggg tgaatgtggc tggcgtgtcc ctgaaaaccc
tgcaccccga tattgggagt 660gacgcagaca aggagaactg gaaggaggtg
cacaagcagg ttgtggacag cgcctatgaa 720gtgatcaagc tgaagggcta
cacctcctgg gctattggcc tgtccgtagc tgacctgtct 780gagagtatcc
tgaagaacct ccgccgagtc catcccattt ccacaatggt caagggcatg
840tacggcgtga ataatgatgt tttcctcagt gtcccctgtg tgttgggcaa
cttgggcatc 900acagacgtgg ttaacatgac gctgaaggca gatgaagagg
atcgcttacg caagagcgca 960gacaccctgt gggccatcca gaaggagctg cagttctag
999226DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 2gtcgacatgg caactgtgaa ggataa 26326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3gcggccgcct agaactgcag ctcctt 26485DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
4tctcaattat tattttctac tcataacctc acgcaaaata acacagtcaa atcaatcaaa
60atggcaactg tgaaggataa actca 85520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5aggcgtatca cgaggccctt 20660DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6gaattaattc ttgaagacga
aagggcctcg tgatacgcct agattgtact gagagtgcac 60780DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
7tatttttcgt tacataaaaa tgcttataaa actttaacta ataattagag attaaatcgc
60ctgtgcggta tttcacaccg 80825DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 8caaatatcgt ttgaatattt ttccg
25985DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 9tattgattta tagtcgtaac tacaaagaca agcaaaataa
aatacgttcg ctctattaag 60atggcaactg tgaaggataa actca
851080DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10aaaaaataac ataatactga aagaaagcat taagaaggcg
gatgtgtcaa acaccaccgt 60ctgtgcggta tttcacaccg 801120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11tagattggcc gtaggggctg 201220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 12cacgcaacgc gtaagaaaca
201326DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13gcggccgcga atttcttatg atttat 261426DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14aagcttaagc ttgcatgccg gtagag 261520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15aggcgtatca cgaggccctt 201685DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 16tttttttagt tttaaaacac
caagaactta gtttcgaata aacacacata aacaaacaaa 60atggcaactg tgaaggataa
actca 851760DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 17gaattaattc ttgaagacga aagggcctcg
tgatacgcct agattgtact gagagtgcac 601880DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18ctaagtcata aagctataaa aagaaaattt atttaaatgc aagatttaaa gtaaattcac
60ctgtgcggta tttcacaccg 801925DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 19atttcttaaa cttcttaaat tctac
252025DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 20atgaatcgaa aatgtcatta aaata 252124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
21gacaattctg gttaggtcca agag 242224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
22ttaagctgct gcggagcttc cacg 242322DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23atgtctgccc ctaagaagat cg 222424DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 24ttaagcaagg attttcttaa
cttc 242524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25atggattcta gaavagttgg tata 242624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26ttacttgttt tctagataag cttc 242724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27atgtcgaaag ctacatataa gcaa 242823DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
28ttattttgct ggccgcatct tct 23
* * * * *