U.S. patent application number 10/202238 was filed with the patent office on 2003-07-10 for low ph lactic acid fermentation.
This patent application is currently assigned to Cargill, Inc.. Invention is credited to Carlson, Ting Liu, Peters, Eugene Max.
Application Number | 20030129715 10/202238 |
Document ID | / |
Family ID | 25489055 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129715 |
Kind Code |
A1 |
Carlson, Ting Liu ; et
al. |
July 10, 2003 |
Low pH lactic acid fermentation
Abstract
A process for producing lactic acid which includes incubating
acid-tolerant homolactic bacteria in nutrient medium to produce a
fermentation broth with high levels of free lactic acid is
provided. An isolated acid-tolerant homolactic bacteria capable of
producing high levels of free lactic acid is also provided.
Inventors: |
Carlson, Ting Liu; (Dayton,
OH) ; Peters, Eugene Max; (Dayton, OH) |
Correspondence
Address: |
Attention of Dennis R. Daley
MERCHANT & GOULD P.C.
P.O. Box 2903
Minneapolis
MN
55402-0903
US
|
Assignee: |
Cargill, Inc.
Wayzata
MN
|
Family ID: |
25489055 |
Appl. No.: |
10/202238 |
Filed: |
July 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10202238 |
Jul 23, 2002 |
|
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08949420 |
Oct 14, 1997 |
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Current U.S.
Class: |
435/139 |
Current CPC
Class: |
C12R 2001/00 20210501;
C12P 7/56 20130101; C12N 1/20 20130101; C12N 1/00 20130101 |
Class at
Publication: |
435/139 |
International
Class: |
C12P 007/56 |
Claims
What is claimed is:
1. A process for producing lactic acid comprising: incubating an
acid-tolerant homolactic bacteria in nutrient medium to generate a
solution including at least about 25 g/L free L-lactic acid or at
least 25 g/L free D-lactic acid.
2. The process of claim 1 comprising incubating the bacteria in the
nutrient medium to generate a solution including at least about 40
g/L L-lactate at a final incubation pH of no more than about
4.0.
3. The process of claim 1 comprising incubating the bacteria at a
temperature of at least about 47.degree. C.
4. The process of claim 1 wherein the nutrient medium comprises
corn steep water.
5. The process of claim 1 wherein the nutrient medium comprises at
least about 50 g/L carbohydrate which includes glucose, fructose,
galactose, melibiose, sucrose, raffinose, stachyose, or a mixture
thereof.
6. The process of claim 1 wherein the nutrient medium comprises
base.
7. The process of claim 1 wherein the nutrient medium comprises a
lactate salt.
8. The process of claim 1 comprising incubating the bacteria in the
nutrient medium to produce L-lactate having an optical purity of at
least about 50%.
9. The process of claim 1 comprising incubating the bacteria in the
nutrient medium to produce free lactic acid at a overall rate of at
least about 0.5 g/L/hr.
10. The process of claim 1 comprising incubating the bacteria at a
temperature of at least about 47.degree. C. to generate a solution
including at least about 30 g/L free L-lactic acid; wherein the
nutrient medium includes (i) at least about 25 g/L corn steep water
dry solids, (ii) at least about 50 g/L glucose, fructose, or a
mixture thereof, and (iii) at least about 20 g/L CaCO.sub.3.
11. The process of claim 10 wherein the nutrient medium further
comprises yeast extract.
12. The process of claim 10 wherein the nutrient medium further
comprises nonionic surfactant.
13. The process of claim 1 comprising incubating the bacteria in
the nutrient medium at an average incubation pH of no more than
about 4.2.
14. Acid-tolerant homolactic bacteria capable of generating a
solution including at least about 25 g/L free L-lactic acid or at
least about 25 g/L free D-lactic acid.
15. The homolactic bacteria of claim 14 wherein said bacteria are
rodshaped, gram positive, air indifferent bacteria.
16. The homolactic bacteria of claim 14 wherein said bacteria is
capable of producing L-lactate having an optical purity of at least
about 80%.
17. The homolactic bacteria of claim 14 wherein said bacteria is
capable of generating a solution including at least about 80 g/L
L-lactate at an incubation temperature of at least about 47.degree.
C. and an average incubation pH of no more than about 4.2.
Description
BACKGROUND OF THE INVENTION
[0001] Lactic acid and its salts have long been utilized in a wide
variety of applications in the chemical, cosmetic, food and
pharmaceutical industries. More recently, new bioengineering
materials based on lactate, such as biodegradable lactide polymers,
have kindled an increased demand for lactate and especially for the
free acid form of either L- or D-lactate. The use of lactic acid in
the production of various industrial polymers has been described,
for example, in U.S. Pat. Nos.: 5,142,023; 5,247,058; 5,258,488;
5,357,035; 5,338,822; 5,446,123; 5,539,081; 5,525,706; 5,475,080;
5,359,026; 5,484,881; 5,585,191; 5,536,807; 5,247,059; 5,274,073;
5,510,526; and 5,594,095. (The complete disclosures of these
seventeen patents, which are owned by the assignee of the present
application, Cargill, Inc. of Minneapolis, Minn., are incorporated
herein by reference.)
[0002] While chemical processes can be used to produce lactic acid,
the rising cost of petrochemical feedstocks and the need to resolve
the racemic lactate mixture produced by conventional chemical
methods, make fermentation methods an attractive alternative for
the manufacture of lactate enriched in one of its optical isomers.
The processes used to produce biodegradable lactide polymers
typically require the free acid form of either L- or D-lactate as a
starting material. Unfortunately, as with most organic acid
fermentations, the end-product inhibition by the organic acid
(lactic acid in this instance) can be a major obstacle to efficient
fermentation. Bacterial strains typically employed in lactate
fermentations may be inhibited by low pH in addition to lactate
concentration. To overcome this problem, industrial lactate
fermentation processes are typically run at a higher pH, e.g., at
least about 5.0 and often at or above 6.0. This results in the
production of a lactate product which is essentially all present in
the form of a salt. Additional process step(s) are typically
required to remove the cationic counterion and isolate the desired
free lactic acid. Moreover, since high concentrations of certain
salts, e.g., sodium cations, may have an inhibitory effect on
fermentation, the type and/or amount of salt present can also
influence the efficiency of the fermentation.
[0003] The production of racemic lactate from enzyme-thinned corn
starch using lactobacillus amylovorus has been reported. While
relatively high production levels at pH as low as 4.2 have been
reported, this fermentation does not provide lactate enriched in
either optical isomer.
[0004] A number of approaches for improving the efficiency of
lactate fermentations have been reported. Several of these involve
removal of free lactic acid from the fermentation broth on a
continuous basis. For example, electrodialysis has been used to
reduce the end product inhibition through removal of lactate from
the fermentation broth. The high cost of dialysis membranes coupled
with a low lactate gradient has generally lowered the
attractiveness of this approach. Ion exchange and the use of
polyvinylpyridine to remove lactate from the fermentation medium
have also been reported. Yet another method which was described
recently, involves a multistage extraction procedure. This process
involves an extraction of lactate from the broth with a tertiary
amine in an attempt to keep the broth pH from dropping to a value
which inhibits further lactate production. The lactate production
levels reportedly achieved via this method are still, however,
quite low. Utilization of this method may also require that the
extracted fermentation broth be subjected to a second extraction to
at least reduce the residual concentration of tertiary amine
extractant before recycling the extracted broth back into the
fermentation reaction.
[0005] All of these approaches to producing lactic acid in its free
acid form based on fermentation of lactobacillus suffer from one or
more disadvantages. Alternative approaches based on the
fermentations of other more acid tolerant microorganisms have also
been reported. Yeasts, such as Saccharomyces cerevisiae, are
capable of growth at much lower pH than lactobacillus. Recombinant
yeast strains have been produced by introducing the lactate
dehydrogenase gene from a bacterial (lactobactobacillus) or
mammalian (bovine) source into Saccharomyces cerevisiae. The
recombinant yeast strains are reportedly able to produce lactate at
or below the pK.sub.a of lactic acid (about 3.8). Ethanol is,
however, the major fermentation product generated by the these
recombinant yeast strains. This both lowers the efficiency of
lactate production and introduces additional potential issues with
regard to the separation and purification of free lactic acid.
Lactic acid production by a pellet form of the fungus, Rhizopus
orgyzae, has also been reported. This fungal fermentation also
typically produces glycerol and/or ethanol as major byproducts. The
yield of free lactic acid was optimized in this instance by
continuous removal from the fermentation broth using a
polyvinylpyridine ("PVP") column. No lactate concentrations higher
than about 25 g/L were reported to have been generated using the
Rhizopus/PVP method.
SUMMARY OF THE INVENTION
[0006] The present invention relates to the production of lactate
via fermentation. It particularly concerns fermentation with
acid-tolerant bacteria to produce a fermentation broth with high
levels of free lactic acid. The presence of the high level of free
lactic acid can facilitate the down stream processing required to
isolate lactate in its free acid form from the broth.
[0007] The process provided herein for producing lactic acid
includes incubating acid-tolerant bacteria, such as acid-tolerant
homolactic lactobacillus, in nutrient medium at a pH which
furnishes a substantial portion of the lactate product in the free
acid form. Herein, when the term "acid-tolerant" is employed in
reference to bacteria, the intent is to refer to bacteria which are
capable of producing lactate at a pH sufficient to furnish a
substantial portion of the lactate product in the free acid form.
The acid-tolerant bacteria are typically capable of producing at
least about 25 g/L free lactic acid. Such bacteria generally can
also produce at least about 50 g/L lactate in nutrient medium at an
"average incubation pH" of no more than about 4.2.
[0008] If fermentation is not carried out to a point where the
limiting lactate concentration is reached, the "average incubation
pH" is determined based on an average of the pH values measured at
ten(10) or more equal time intervals over the course of the
fermentation. The present fermentation process may be run in a
continuous fashion. Under such conditions, steady state conditions
(in terms of pH, lactate concentration and nutrient concentrations)
are generally achieved and maintained after an initial startup
phase has been concluded. When fermentation is conducted in this
manner, the average incubation pH is the average pH of the broth
after the initial startup phase has been completed, i.e., the pH
during the startup phase is ignored in determining the average
incubation pH.
[0009] If fermentation is carried out to a point where pH and/or
lactic acid concentration inhibits further lactate production, the
"average incubation pH" is determined based on an average of the pH
values measured at ten(10) or more equal time intervals over the
time period necessary to produce 90% of the limiting lactate
concentration. As used herein, the "limiting lactate concentration"
is the lactate concentration under a given set of incubation
conditions (nutrient medium, temperature, degree of aeration) at
which pH and/or lactic acid concentration generated by the
fermentation inhibits further lactate production. As used herein,
the term "limiting incubation pH" means the pH of the fermentation
broth for a given set of incubation conditions at which the pH
and/or lactic acid concentration inhibits further lactate
production. Inhibition of lactate production is considered to have
occurred when the amount of lactate produced does not increase by
more than about 3% upon further incubation for a period of up to
about twelve (12) hours under the same conditions. This definition
presumes that sufficient nutrients for lactate production are still
available in the fermentation broth.
[0010] Herein the terms "nutrient medium" and "fermentation broth"
are used interchangeably. These terms refer to both (i) media in
the form originally provided to the acid-tolerant bacteria as a
source of nutrient and (ii) media produced after some or all of the
originally provided nutrients have been consumed and fermentation
products including lactate have been excreted into the media by the
bacteria.
[0011] In the present process, the pH of the fermentation broth
after incubation of the acid-tolerant bacteria to produce lactate
is typically no more than about 4.2 ("final incubation pH"). As
referred to herein, the "final incubation pH" is the pH of the
fermentation broth at the point that growth and/or lactate
production by the acid-tolerant bacteria ceases. The cessation of
growth and/or lactate production may be the result of a change in
reaction temperature, the exhaustion of one or more necessary
nutrients in the fermentation broth, a deliberate change in pH, or
the separation of the fermentation broth from the bacterial cells.
In those instances where fermentation is arrested by the addition
of sufficient acid or base to the broth to stop lactate production,
the final incubation pH is defined to be the pH of the nutrient
medium just prior to the addition. Alternatively, growth and/or
lactate production may stop due to the accumulation of one or more
fermentation products and/or a change in broth pH resulting from
the accumulation of fermentation products, i.e., the fermentation
reaction has reached a self limiting point for the given set of
incubation conditions. As noted above, it is quite common for
bacterial fermentations which produce an organic acid such as
lactic acid to be subject to end-product inhibition.
[0012] The term "lactate" as used in this application refers to
2-hydroxypropionate in either its free acid or salt form. The terms
"lactic acid" and "free lactic acid" are employed interchangeably
herein to refer to the acid form, i.e., 2-hydroxypropionic acid.
The salt form of lactate is specifically referred to herein as a
lactate salt, e.g., as either the sodium salt of lactic acid or
sodium lactate.
[0013] The present invention also provides acid-tolerant homolactic
bacteria. The acid-tolerant homolactic bacteria are generally
capable of producing at least about 25 g/L free lactic acid at an
incubation temperature of at least about 40.degree. C. Another
embodiment of the present acid-tolerant bacteria is capable of
producing at least about 50 g/L lactate at a temperature above
about 40.degree. C. and an average incubation pH of no more than
about 4.2. Typically, the acid-tolerant bacteria is capable
satisfying both of these measures of lactate productivity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a schematic depiction of a flow diagram of an
fermentation process which includes the coupled removal of free
lactic acid.
[0015] FIG. 2 is a graph showing the ribotype patterns for a number
of lactate-producing bacterial strains isolated from corn steep
water.
[0016] FIG. 3 is a graph showing the fermentation profile of
glucose, fructose and lactate for incubation of strain #41 in a
nutrient medium containing 10 vol.% corn steep liquor, 100 g/L
glucose and 33.4 g/L calcium carbonate.
[0017] FIG. 4 is a graph showing lactate production from incubation
of strain #41 in a nutrient medium containing 90 g/L glucose, 33.4
g/L calcium carbonate and either 12 vol.% corn steep liquor or 36
vol.% light steep water.
[0018] FIG. 5 is a graph showing the fermentation profile of
glucose, fructose and lactate for incubation of homolactic strain
#41 in a nutrient medium containing 90 g/L glucose, 36.6 g/L
calcium carbonate and varying amounts of corn steep water.
[0019] FIG. 6 is a graph showing the percentage undissociated
lactic acid ("free lactic acid") as a function of pH.
DETAILED DESCRIPTION
[0020] The present process allows the efficient production of
lactate and, in particular, the efficient production of high
concentrations of free lactic acid via incubation of an
acid-tolerant homolactic bacteria in a suitable nutrient medium.
The acid-tolerant homolactic bacteria may be isolated from the corn
steep water of a commercial corn milling facility. While different
bacteria of this type may produce either racemic lactate, or
lactate predominantly in either the D- or L-isomeric form, the
present process preferably employs a homolactic bacteria which
produces predominantly L- or D-lactate, and most preferably
produces L-lactate in optically pure form.
[0021] The present process allows the efficient production of high
concentrations of free acid form of an optical isomer of lactic
acid. This efficiency may be expressed in a variety of manners. The
concentration of free lactic acid in the fermentation broth serves
as one measure of the overall productivity of the process. The
present process typically generates a solution including at least
about 25g/L, preferably at least about 30g/L, and more preferably
at least about 40g/L free lactic acid. Most preferably, the process
produces these levels of either free L-lactic acid or free D-lactic
acid. The optical purity of the lactate (and free lactic acid)
produced is preferably at least about 50%, more preferably at least
about 80% and, most preferably, one optical isomer of lactate is
produced in essentially pure form.
[0022] As noted above, typically, the lactate produced by the
present process is predominantly in the form of L-lactate. For
example, one embodiment of the process includes incubating an
acid-tolerant homolactic bacteria in nutrient medium to produce
lactate which includes at least about 75 wt.% L-lactate (i.e.,
L-lactate having an optical purity of at least about 50%).
Preferably, the optical purity of the lactate produced by the
present process is at least about 80%, and more preferably at least
about 90% (e.g., includes at least about 95 wt.% L-lactate). Most
preferably, the present process produces L- or D-lactate lactate in
essentially optically pure form (i.e., the lactate produced
contains 99 wt.% or higher of a single optical isomer).
[0023] The amount of free lactic acid present in a solution is a
function of both the pH of the solution and the overall
concentration of lactate in the mixture. Thus, specifying these two
parameters for a given solution, such as a fermentation broth,
effectively specifies the free lactic acid concentration. The
present process is capable of generating a solution which includes
at least about 50 g/L, preferably at least about 80 g/L, and more
preferably at least about 100 g/L lactate at a relatively low pH.
The lower the solution pH, the higher the percentage of the lactate
which is present in its free acid form. For example, where the
medium pH is equal to the pK.sub.a of lactic acid (about 3.8), 50%
of the lactate is present in the free acid form. At pH 4.2, about
31% of the lactate as a free acid and at pH 4.0 and 3.9, about 41%
and 47% respectively of the lactate is present in the free acid
form. The fraction of free lactic acid is even lower at higher pH,
18% at pH 4.5 and 6.6% at pH 5.0.
[0024] The pH of the broth during the incubation step can be
expressed in several different ways, e.g., in terms of the average
incubation pH or the final incubation pH. The present fermentation
process in typically capable of producing high levels of lactate at
an average incubation pH of no more than about 4.3, preferably no
more than about 4.2, and more preferably no more than about 4.0.
Alternatively, the pH of the broth during incubation can be
expressed in terms of the final incubation pH. The present process
typically allows the production of high lactate concentrations at a
final incubation pH of no more than about 4.2, preferably no more
than about 4.0, and more preferably no more than about 3.9.
Particularly effective embodiments of the present fermentation
process are capable of producing at least about 80 g/L lactate at
an average incubation pH of no more than about 4.0 and/or a final
incubation pH of no more than about 3.9.
[0025] The present fermentation process may be run in a continuous
fashion where a fraction of the fermentation broth is removed as
the fermentation proceeds. This may be done either continuously or
at periodic intervals. Sufficient nutrient medium is typically
added to the reactor to maintain a constant liquid volume. Under
such fermentation conditions, steady state conditions (in terms of
pH, lactate concentration and nutrient concentrations) are
generally achieved and maintained after an initial startup phase
has been concluded. When fermentation is conducted in this manner,
the average incubation pH (the pH during the startup phase is
ignored) and the final incubation pH of the broth are essentially
the same. Under such conditions, fermentation is typically carried
out at a pH of no more than about 4.2, preferably no more than
about 4.0, and more preferably no more than about 3.9.
[0026] Although the present incubation process may be carried out
at relatively low temperatures, e.g., about 30.degree. C. to about
38.degree. C., the acid-tolerant homolactic bacteria is typically
incubated in a suitable nutrient medium at a temperature of at
least about 43.degree. C., and more preferably at about 45.degree.
C. to about 52.degree. C. Most preferably, the fermentation is
carried out at about 47.degree. C. to about 50.degree. C. There are
a number of advantages of operating the fermentation at these
temperatures. The chances of complications due to growth of other
competing organisms is lessened in this temperature range. In
addition, at higher temperatures, the reaction generally proceeds
at a faster rate allowing efficient utilization of process
equipment. If fermentation is carried out at too high a
temperature, typically at about 54.degree. C. or above, growth
and/or lactate production by the homolactic bacteria may be
negligible. It may be possible, however, using standard selection
techniques to identify mutant homolactic bacterial strains which
are capable of growth and lactate production at temperatures of
55.degree. C. and above.
[0027] As described herein "nutrient medium" refers to a water
based composition including minerals and their salts necessary for
growth of the bacterium of the present invention. The nutrient
medium typically contains effective amounts of a carbon source, a
nitrogen source, a phosphate source, a sulfate source, calcium and
trace elements. The term "trace elements" refers to elements
essential for growth in trace concentrations i.e., minute fractions
of 1 percent (1000 ppm or less).
[0028] The bacteria of the present invention typically can utilize
a number of carbon and energy sources for growth and/or lactate
production, such as glucose, fructose, galactose, melibiose,
sucrose, raffinose, and/or stachyose. Some of the bacteria may be
able to use all or most of these sugars as a source of carbon and
energy while other strains are more fastidious and may only be able
to grow on one or two sugars from the list. In other instances, a
starch (such corn starch) or a hydrolysate thereof may be used as
primary carbohydrate source.
[0029] As used herein, "corn steep water" refers to water obtained
from corn steeping tanks as well as other solutions derived
therefrom having substantially the same spectrum of nutrients. For
example, corn steep liquor (also sometimes referred to as "heavy
steep water") is a concentrated form of corn steep water obtained
by removal of water and other volatile components, typically under
vacuum. Corn steep liquor typically has a dry solids content of
about 35 wt.% to about 50 wt.%. The corn steep liquor used in the
experiments described in the Examples herein had a dry solids
content of 36 wt.% and is referred to herein as "CSL." Corn steep
waters obtained directly from corn steeping tanks and/or associated
lines just before concentration to produce corn steep liquor
generally have dry solids contents in the range of about 10 wt.% to
about 15 wt.% and are referred to herein as "light steep
water"("LSW"). Light steep water typically has an SO.sub.2 content
of no more than about 500 ppm. The steep water used to supplement
the nutrient medium used in the present process preferably has an
SO.sub.2 content of no more than about 300 ppm and, more
preferably, no more than about 200 ppm. The light steep water used
in the experiments described in the Examples herein had a dry
solids content of 12 wt%.
[0030] In situations where one or more homolactic strains isolated
from corn steep water are to be used to produce lactate, the
nutrient medium typically includes corn steep water corresponding
to at least about 15 g/L steep water dry solids. Preferably, the
nutrient medium includes corn steep water corresponding to at least
about 25 g/L and, more preferably, at least about 30 g/L steep
water dry solids.
[0031] One example of a suitable nutrient medium for use the
present fermentation process is MRS medium (such as the MRS medium
commercially available from Becton Dickinson & Co.) or the
like. The MRS medium is generally supplemented with corn steep
water to provide a nitrogen source and general source of nutrients
as well as with additional carbohydrate (such as glucose or
fructose) as a carbon and energy source. Typical media suitable for
use in the present process also include magnesium salt(s),
manganese salt(s), phosphate salt(s), potassium salt(s) and/or
citrate salt(s). It may, however, not be necessary to add specific
amounts of such salts to the medium. Often, the nutrient medium
also includes a nonionic surfactant, such as fatty acid monoester
of a polyoxyethylene derivative of sorbitan (e.g., Tween.sup.R 80
which is polyoxyethylene (20) sorbitan monooleate).
[0032] The medium may be prepared by using separate salts as
sources of each of the various inorganic components. Alternatively,
a single salt which acts as a source of more than one component may
be used to prepare the nutrient medium. For example, potassium
hydrogen phosphate (K.sub.2HPO.sub.4) may be added as a source of
both potassium cations and phosphate anions. It will be recognized
that after the various components have been dissolved in water
during the preparation of the nutrient medium, an interchange of
cations and anions among the various dissolved salts present will
occur. For example, if magnesium sulfate and ammonium citrate are
added to water during the preparation of the medium, the resulting
solution will also include some ammonium sulfate and magnesium
citrate species in addition to magnesium sulfate and ammonium
citrate species. One type of nutrient medium which is particularly
suitable for use in the present fermentation process includes corn
steep water supplemented with glucose and/or fructose as an
additional carbon and energy source.
[0033] One example of a suitable medium for use in the present
invention includes:
[0034] corn steep water corresponding to about 30 to about 45 g/L
steep water dry solids;
[0035] about 80 to about 120 g/L glucose, fructose or a mixture
thereof;
[0036] about 0 to about 10 g/L yeast extract;
[0037] about 0 to about 1 g/L of a nonionic surfactant such as
Tween.sup.R 80;
[0038] about 0 to about 2 g/L potassium hydrogen phosphate
(K.sub.2HPO.sub.4);
[0039] about 0 to about 0.2 g/L magnesium sulfate (MgSO.sub.4);
[0040] about 0 to about 0.05 g/L manganese sulfate
(MnSO.sub.4);
[0041] about 0 to about 2 g/L ammonium citrate; and
[0042] optionally, about 10 to about 50 g/L calcium carbonate
(CaCO.sub.3).
[0043] For the reasons discussed above, the amounts refer to the
quantities of the various materials added to form the medium and
not to the actual concentrations of these species in the nutrient
medium. In making up such a nutrient medium, all of the components
except the nonionic surfactant and the calcium carbonate are
generally dissolved in an appropriate amount of water and autoclave
sterilized. The nonionic surfactant is typically added to the
autoclaved medium while it is still at a temperature of close to
about 100.degree. C. The resulting solution is then typically
allowed to cool to about 60.degree. C. or lower before the calcium
carbonate is added.
[0044] It has been found that suitable nutrient mediums for use in
the present process preferably include at least about 50 g/L of
carbohydrate. More preferably, the nutrient medium include at least
about 70 g/L and, most preferably, at least about 90 g/L of the
carbohydrate. The carbohydrate typically is made up of glucose,
fructose, galactose, melibiose, sucrose, raffinose, stachyose, or a
mixture thereof. Glucose, fructose, and sucrose are particularly
suitable for use as a carbon and energy source in the nutrient
medium. It is generally not useful to incorporate more than about
150 g/L carbohydrate in the medium.
[0045] It has been found that it may be advantageous to include a
base such as calcium carbonate (CaCO.sub.3), sodium hydroxide
(NaOH), ammonium hydroxide (NH.sub.4OH) and/or sodium bicarbonate
(NaHCO.sub.3). Typically at least about 30 g/L calcium carbonate
(or an equivalent amount of another base) is added to the nutrient
medium. In some embodiments of the process, e.g., embodiments which
produce higher levels of lactate, it may be preferred to include up
to about 40 g/L calcium carbonate in the nutrient medium. While
higher levels of base may be employed, due to limitations on the
solubility of calcium carbonate salts and the desire to maintain a
relatively low broth pH, it is generally not useful to incorporate
more than about 100 g/L calcium carbonate in the medium. Very
often, the entire amount of calcium carbonate present will not
initially dissolve in the nutrient medium. As the fermentation
proceeds, some of the calcium carbonate may react with the lactic
acid being formed to generate calcium lactate. As this occurs,
additional portions of the undissolved calcium carbonate may be
drawn into solution. The overall effect is to neutralize a portion
of the forming lactic acid and prevent the pH of the broth from
dropping below a desired level (e.g., below about 3.8-3.9).
[0046] It may not be necessary to add a base such as calcium
carbonate to achieve this effect. A solution containing a lactate
salt (e.g., calcium, sodium or ammonium lactate) may be added to
aid in buffering the pH of the fermentation broth. One example of a
process in which this might occur would involve the separation of a
fraction of the fermentation broth from the incubating bacteria,
and recycling the portion back into the fermentation after removal
of some or all of the free lactic acid in the fraction.
Alternatively, calcium lactate might be isolated from the
fermentation broth (e.g., in solid form), and mixed together with
nutrient medium being added to the fermentation. Generally,
addition of lactate salt as a buffering salt can be advantageous
because it minimizes the amount of neutralizing base added to the
fermentation broth thereby minimizing the amount of lactate
produced that is converted to salt form.
[0047] Nutrient media including at least about 70 g/L glucose
and/or fructose and at least about 20 g/L calcium carbonate are
particularly suitable for use in the present process. Depending on
the bacterial strain employed in the process, incorporation of corn
steep water (e.g., in an amount equivalent to at least about 25 g/L
corn steep water dry solids) in this nutrient medium may also be
preferred. It is particularly useful to add corn steep water
containing only the same chiral form of lactate to be generated by
the fermentation process.
[0048] The strain of homolactic bacteria and the fermentation
conditions are typically chosen such that free lactic acid is
produced at a overall rate of at least about 0.5 g/L/hr, preferably
at least about 1.0 g/L/hr, more preferably at least about 2.0
g/L/hr, and most preferably at least about 4.0 g/L/hr. As used
herein, overall rate of production of either lactate or free lactic
acid (or lactate) is calculated by dividing the total amount of
free lactic acid (lactate) produced by the incubation time. For
fermentations where a limiting lactate concentration is produced,
the overall production rate of free lactic acid (lactate) is
calculated over the time required to produce 90% of the limiting of
free lactic acid (lactate) concentration.
[0049] The productivity of the present process may also be
expressed in terms of the overall production rate for lactate. The
present fermentation process is generally carried out under
conditions which produce lactate at a overall rate of at least
about 1.0 g/L/hr, preferably at least about 2.0 g/L/hr and, more
preferably, at least about 3.0 g/L/hr. As indicated herein, lactate
is preferably produced at these rates in a broth at an average
incubation pH of no more than about 4.1, and more preferably, no
more than about 4.0.
[0050] Suitable examples of homolactic bacteria for use in the
present fermentation method may be readily isolated from samples of
corn steep water, such as are found in commercial corn milling
facilities. In addition, certain other homolactic bacteria isolated
from different sources may also have the necessary capabilities to
permit efficient low pH production of high levels of free lactic
acid.
[0051] Since the homolactic bacteria found in corn steep water
typically require a nutrient medium which includes corn steep water
for growth, the initial step in a process for identifying and
isolating such bacteria typically involves plating samples in a
steep water-containing medium, such as 10 vol.% CSL-MRS agar, and
then incubating the inoculated medium anaerobically at about
45-50.degree. C. Bacterial isolates can easily be probed for
heterolactic production by passing the isolate into a biphasic
medium which only contains steep water in the lower phase. The
growing strains are then monitored for the generation of gas at the
bottom of the biphasic tubes. The isolated strains may be
conveniently stored at low temperature (e.g., 4.degree. C. or
below) or maintained as a bench stock in a steep water/tomato
juice/MRS agar growth medium. When desired, one or more
acid-tolerant strains isolated in this fashion from corn steep
water may be used as an inoculant in a lactic acid
fermentation.
[0052] Using this type of methodology, steep water samples obtained
from five different corn milling facilities in the United States as
well as three corn milling facilities located in Turkey, England
and the Netherlands were examined for lactate producing
microorganisms. The isolated microorganisms were initially
characterized as heterolactic (i.e., able to produce other
fermentation products in addition to lactate) or homolactic
producers. The homolactic strains were further characterized, inter
alia, based on overall lactate production, optical activity of
lactate produced and, in many instances, final incubation pH in the
absence of base (CaCO.sub.3) added to the fermentation medium. A
total of 155 bacterial strains were isolated. Of the 109 strains
which were characterized, 98 strains (90% ) produced lactate as the
sole fermentation product ("homolactic" strains). The remaining 11
strains (11%) produced other fermentation products in addition to
lactate ("heterolactic" strains). Of the 98 homolactic strains, 22
were L-lactate producers, 18 were D-lactate producers, and 58
produced racemic lactate.
[0053] The present homolactic bacteria are generally capable of
producing at least about 25 g/L free lactic acid. Most preferably,
the bacteria are homolactic bacteria capable of producing at least
about 30 g/L free L-lactic acid. In another embodiment of the
invention, the homolactic bacteria is capable of generating a
solution containing at least about 40 g/L, preferably at least
about 75 g/L lactate, and preferably at least about 90 g/L lactate
at an average incubation pH of no more than about 4.3. As discussed
elsewhere herein, particularly effective strains of the present
homolactic bacteria are capable of producing these levels of
L-lactate (or D-lactate) at an average incubation pH of no more
than about 4.0 and/or a final incubation pH of no more than about
3.9.
[0054] The present acid-tolerant homolactic bacteria is typically
capable of growth and lactic acid production at temperatures
between about 35.degree. C. and about 53.degree. C. Optimum
temperature for growth generally ranges from about 43.degree. C. to
about 52.degree. C. and, preferably, about 47.degree. C. to about
50.degree. C., although it has been demonstrated that the
homolactic bacteria can grow at temperatures at or close to room
temperature. Negligible lactate production by the bacteria
typically occurs when the temperature is above about 53.degree. C.
or below about 30.degree. C. The fermentation process is preferably
conducted at about 47.degree. C. to about 52.degree. C., since
yeasts and heterolactic lactobacilli are less thermotolerant and
generally will not grow well, if at all, at these temperatures.
Thus, in addition to enhancing lactate production, fermentation of
the acid-tolerant homolactic bacteria at high temperature can
decrease the possibility of problems associated with contamination
by other organisms.
[0055] The present homolactic bacteria is typically capable of
growth and lactate production at least within a pH range of about
3.7 to about 6.5 and preferably at least across a pH range from
about 3.8 to about 5.0. Even though the bacteria may be able to
produce lactate at a pH close to neutral (e.g., 6.0-6.5), bacteria
employed in the present process preferably are capable of high
levels of lactate at a pH where a substantial portion of the
lactate exists is its free acid form. Preferred forms of the
acid-tolerant homolactic bacteria are capable of significant
lactate production (e.g., at least about 50 g/L) at a pH of 4.2 or
below.
[0056] A variety of reactor configurations including packed bed
reactors, continous stirred tank reactors, rotating biological
contact reactors, sequencing batch reactors and fluidized bed
reactors may be employed in the present process. The entire
reaction may be performed in a single vessel having appropriate
means to control the temperature of the fermentation broth or,
alternatively fermentation may be carried out in a first vessel,
the broth may be maintained at the desired temperature by passage
through a heat exchanger, for example, a plate heat exchanger and
recycled to the fermentation reaction. The latter arrangement can
provide more rapid cooling of the reaction mixture and can in some
instances be carried out at the same time that broth is passed
through a membrane separation module to remove a portion of the
broth (e.g., where the heat exchanger and membrane module are
connected in series).
[0057] One commonly used configuration includes a membrane recycle
bioreactor. Reactors of this type typically includes two modules, a
fermentation vessel 10 and a membrane module 15 (see e.g., FIG. 1).
These two modules may be connected by a pipe or be parts of a
single apparatus.
[0058] In one embodiment of the invention, acid-tolerant homolactic
bacteria may be incubated in a first portion of nutrient medium in
the fermentation vessel to generate a first product solution
including at least about 25 g/L free L-lactic acid. The resulting
fermentation broth may be separated to provide a first fraction
which includes free lactic acid and is substantially free of
bacterial cells. This may be carried out by pumping a portion of
the fermentation through a cell separator (e.g., a hollow fiber
cell separator). The cell-containing fraction is typically recyled
back into the fermentation vessel (see e.g., FIG. 1), while the
lactic acid-containing fraction is split off for further
processing. Additional nutrient medium is typically added to
maintain the liquid volume in the fermentation vessel at a constant
level. When fermentation is conducted in this manner, steady state
conditions (in terms of pH, lactate concentration and nutrient
concentrations) are generally achieved and maintained after an
initial startup phase has been concluded. When run in such a mode,
the present fermentation is typically conducted such that the pH of
the broth is maintained at about 4.2 or below and, preferably, in
the range between about 3.7 and 4.0.
[0059] The lactic acid-containing fraction which is split off may
be processed using a number of known methods to separate free
lactic acid from the other components of the solution. For example,
the lactic acid may be extracted from the solution using a tertiary
amine-containing extractant. One example of a suitable extractant
is a solution of Alamine 336 in octyl alcohol. Other methods which
may be used to isolate the lactic acid include contacting the
solution with a solid adsorbent, such as an ion exchange resin
(e.g., a polyvinylpyridine column), distilling off a lactic acid
containing fraction, or removal via membrane separation. Any of
these type of separation methods may be used to process the lactic
acid-containing fraction to generate a lactic acid-depleted
fraction and a lactate isolate fraction. The lactic acid-depleted
fraction may contain some lactate in the form of a lactate salt,
such as calcium lactate. The lactate isolate fraction may be
further processed using any of a variety of known methods to
produce a purer form of free lactic acid.
[0060] The lactic acid-containing fraction may also be processed to
separate out lactate salt (e.g., calcium lactate) in solid or
solution form, leaving a solution enriched in free lactic acid. The
lactate salt may be separated using a suitable technique such as
extraction, crystallization, membrane separation and adsorption on
a solid material (e.g., anion exchange resin). The lactate salt may
be returned to the fermentation vessel where it can serve to buffer
the pH of the solution and prevent the pH of the broth from
dropping below a desired level. For example, by recycling a
sufficient amount of calcium lactate as a buffering agent, the pH
of the fermentation broth may be maintained at a value close to the
pK.sub.a of lactic acid. Based on theory, the lactate salt will
buffer production of an equivalent amount of new lactic acid
production at a pH of 3.85. At pH 4.0, each equivalent of lactate
salt will buffer production of 0.7 equivalent amount of new lactic
acid production.
[0061] A variety of methods are available for processing
lactate/lactic acid solutions involving generation of large amounts
of lactic acid; for example, in solution at pHs no greater than
about 4.8 (preferably no greater than about 4.2 or 4.3) from the
fermentation broth; and, with a concomitant isolation (and if
desired recycling) of lactate salt (typically calcium lactate,
potassium lactate, sodium lactate and/or ammonium lactate). Such
processes are described, for example, in commonly assigned (to
Cargill, Inc. of Minnetonka, Minn.), co-filed, U.S. patent
application entitled LACTIC ACID PROCESSING; METHODS; ARRANGEMENTS;
AND, PRODUCTS, identifying Aharon M. Eyal, John N. Starr, Riki
Canari, Betty Hazan, Rod Fisher, Jeffrey J. Kolstad, David R.
Witzke, and Patrick R. Gruber as inventors (hereinafter referred to
as the Starr et al application). The Starr et al application was
filed on the same date of the present application (Oct. 14, 1997)
and is incorporated herein by reference. Advantageous overall
processes will depend, in part, upon selection, among the
approaches, of the one which most readily facilitates an overall
cost-effective and efficient processing scheme in large scale
implementation.
[0062] The principle concerns in selecting overall processes relate
to design of the system to accommodate the two objectives of:
[0063] 1. Isolation of lactic acid products for follow-up
processing, for example to generate polymer; and
[0064] 2. Isolation of lactate salt, preferably in a form desirable
for recycling to the fermentation broth. Three general approaches
concern:
[0065] 1. Separation of the lactic acid from the solution leaving
the lactate salt behind; and, if desired, direction of the residual
solution having the lactate salt therein, after the separation,
into a fermentor;
[0066] 2. Isolation of the lactate salt from the solution;
direction of the lactate salt, if desired, into a fermentor; and, a
follow-up isolation of the lactic acid product from the residual
solution after lactate salt separation; and,
[0067] 3. Simultaneous separation of lactic acid into one stream
and lactate salt into another, leaving residual mixture.
[0068] The techniques described in Starr et al. to achieve one or
both of these objectives can be practiced on a variety of solutions
of lactate material (i.e. solutions of lactic acid and dissolved
lactate salt). These solutions may comprise fermentation broth or
broth which has been removed from a fermentor and modified in some
manner, for example by filtration or pH adjustment. Indeed the
techniques can be applied to the solutions which are made in other
manners as well. The techniques and proposals described therein,
however, are particularly developed with a focus on efficient
processing of fermentation broth solutions, especially relatively
acidic ones, in which pH modification by addition of acid is not
required and preferably has not occurred. Typical compositions in
which these techniques can be applied, with respect to pH, would be
at least 0.86 and less than 6.0. That is, typical compositions on
which the techniques will be practiced, will have a pH within this
range. For such compositions the molar ratio of free lactic acid to
dissociated acid or dissolved lactate salt at 25.degree. C., is
within a range of about 1,000:1 to 0.007:1. More preferred
processing will involve solutions with a pH in the order of about
1.98-5.00 (HLA:LA ratio within the range of about 75:1 to 0.070:1);
and, most preferred processing will involve solutions having a pH
within the range of about 3.0-4.5 (HLA:LA ratio within the range of
about 7.0:1 to 0.23:1).
[0069] As indicated above, solutions within the most preferred pH
range described above are readily obtained via the present
fermentation process with substantial concentrations of the lactate
material therein. Alternatively, other fermentation broths can be
used, for example with pH adjustment by addition of acid typically
to the most preferred pH range given.
[0070] Herein, there will sometimes be reference to "preferential
separating" of: lactic acid from a composition containing lactic
acid and lactate salt; or, lactate salt from composition containing
lactic acid and lactate salt. The term "preferential separating"
and variants thereof, in this context, is meant to refer to
separation technique which preferentially removes one of the two
components (lactic acid or lactate salt) with respect to the other.
In typical preferred processing according to the present invention
a mixture of lactic acid and lactate salt is divided into two
"product streams". In one product stream, (i.e., the free lactic
acid rich stream), preferably the molar ratio of free lactic acid
to lactate salt obtained is at least 2/1 and preferably at least
3/1. With certain of the techniques described herein, ratios of at
least 5/1 and indeed in ratios of 10/1 or more are readily
obtainable.
[0071] The other product stream is the lactate salt rich stream. In
this stream, preferably the molar ratio of free lactic acid to
lactate salt is no greater than 0.5. With typical preferred
processing as described herein ratios of no greater than 0.3,
preferably no greater than 0.2 and most preferably 0.1 or lower are
readily obtained.
[0072] Herein the term "stream" when used in the context indicated
by the previous two paragraphs, is meant to refer to an isolated
phase or product segment, without regard to whether that phase or
product segment is a solution, solid or a mixture of materials.
Thus, a "lactate acid rich stream" is merely a phase or mixture
rich in lactic acid (versus lactate salt) by comparison to the
original mixture processed; and, a "lactate salt rich stream" is a
stream rich in lactate salt (versus lactic acid) by comparison to
the original mixture processed.
[0073] When the product stream enriched in free lactic acid is
obtained as a result of separating the free lactic acid from the
mixture, for example from a fermentation broth, the remaining
aqueous mixture after the free lactic acid removal will sometimes
be referred to as "depleted" with respect to free lactic acid.
Similarly, when the lactate salt enriched stream results from
separation of the lactate salt from a mixture containing the free
lactic acid and the lactate salt, the remaining mixture will
sometimes be referred to as "depleted" with respect to the lactate
salt.
[0074] Preferably, when the solution processed is a fermentation
broth, the product stream enriched in lactate salt is provided and
formed such that the weight ratio of impurities from the fermentor,
to lactate salt therein, is lower than found in the fermentation
broth, preferably by a factor of at least 5. This can be managed by
techniques described herein concerning control over the particular
approach selected for isolation of the lactate salt, as well as
through use as various purification techniques, such as back
washing or recrystallization.
[0075] Preferably, the lactate product stream is eventually
isolated as an aqueous solution or mixture of an aqueous phase and
a solid phase, for convenient recycling into a fermentation system,
in order to maintain water balance. If concentration of an aqueous
solution is used in order to facilitate the water balance in the
broth, preferably relatively low-cost concentration techniques such
as reverse osmosis and vapor recompression are used.
[0076] The invention will be further described by reference to the
following examples. These examples illustrate but do not limit the
scope of the invention that has been set forth herein. Variation
within the concepts of the invention will be apparent.
EXAMPLE 1
Standard Fermentation Conditions
[0077] Unless otherwise indicated, the fermentation reactions
described in the following examples were run using a variety of
growth media according to the following standard protocol.
[0078] Cells (250 ul) were passed from a bench stock of the
particular strain in 40% tomato juice/40% LSW-MRS agar bottom
phase/MRS top phase biphasic (TJ-SW-MRS biphasic) into fresh
TJ-SW-MRS biphasic medium and incubated under static conditions for
18-24 hours at 47.degree. C.
1 MRS Medium (pH = 6.2) 10 g/L pancreatic digest of gelatin 8 g/L
beef extract 4 g/L yeast extract 20 g/L glucose 2 g/L
K.sub.2HPO.sub.4 1 g/L Tween .RTM. 80 5 g/L sodium acetate 5 g/L
ammonium citrate 0.2 g/L MgSO.sub.4 0.05 g/L MnSO.sub.4
[0079] A 1.0 aliquot of the incubate in the fresh TJ-SW-MRS
biphasic medium was used to inculate 80 ml of Medium B supplemented
with 10% CSL, glucose (60 g/L total concentration) and calcium
carbonate (20 g/L) in a sealed serum bottle and incubated with
agitation 18 hours at 47.degree. C. in an environmental shaker.
2 Medium B (pH = 4.7) 8-12 vol. % corn steep liquor 5 g/L yeast
extract 50-100 g/L glucose 2 g/L K.sub.2HPO.sub.4 1 g/L Tween .RTM.
80 2 g/L ammonium citrate 0.2 g/L MgSO.sub.4 0.05 g/L MnSO.sub.4
20-40 g/L CaCO.sub.3
[0080] Fermenters containing Medium B with the desired levels of
glucose and calcium carbonate (e.g., 90g/L glucose and 33.4 g/L
calcium carbonate) were inoculated with 10% (v/v) of the 18 hours
old culture. Fermentation was run at 47-49.degree. C. with stirring
at 150 rpm and fermentation jars 70-80% full. Running the
fermentation jars at this liquid volume level ensured that the
medium did not become highly aerobic.
EXAMPLE 2
Isolation of Acid-Tolerant Homolactic Strains Without pH
Control
[0081] Homolactatic bacterial strains were isolated from samples of
corn steep water obtained from eight different industrial corn
milling facilities. The facilities were located in Blair, Nebr.;
Edyville, Iowa; Cedar Rapids, Iowa; Dayton, Ohio; Memphis, Tenn.;
Istanbul, Turkey; Tillbury, England; and Bergen Op Zoon, the
Netherlands.
[0082] The strains were isolated by obtaining samples of steep
water from commercial corn milling facilities. The samples were
plated on 10% CSL-MRS agar plates (pH 5.0) and incubated
anaerobically at 47.degree. C. Colonies were restreaked for
isolation on 10% CSL-MRS agar plates. Isolates were then passed
into a 40% LSW-40% tomato juice-MRS bottom phase/MRS top phase
biphasic medium (pH 6.0) for maintenance purposes. The isolated
strains were screened for heterolactic production by monitoring for
the formation of gas (CO.sub.2) in the bottom of the tube. The
homolactic isolates were then screened in MRS Medium supplemented
with 10 vol.% CSL and 30 g/L glucose for lactate yield and the
optical purity of the lactate produced. The results are shown in
Table 1 below.
[0083] The isolated bacterial strains were identified as either
homolactate producers ("homolactate") or heterolactate producers
("heterolactic"). Based on fermentation in MRS medium supplemented
with 10 vol.% corn steep liquor ("CSL"), the isolated homolactic
bacterial strains were characterized in terms of overall lactate
production, final fermentation pH and % L-lactate produced (see
Table 1 below). Since about 50% of the lactate in the added corn
steep liquor ("CSL") was typically D-lactate, strains which
produced at least about 70% L-lactate were considered to be
L-lactate producing strains. This assumption was confirmed by
subsequent experiments under conditions where D-lactate
contamination levels in the product arising from steep water
present in the nutrient medium were lower (e.g., higher lactate
production levels or using corn steep water having greater than 80%
L-lactate (as a fraction of the total lactate)).
[0084] The fermentations were carried out at 48.degree. C. under
the standard conditions described in Example 1. The results are
shown in Table 1 below.
EXAMPLE 3
Isolation of Acid-Tolerant Homolactic Strains Using Added Base
[0085] An additional set of homolactic strains were isolated from
corn steep water samples obtained from the corn milling facilities
in Edyville (Iowa), Cedar Rapids (Iowa), and Blair (Nebraska). The
isolation procedure employed was the same as described in Example
2. The isolated homolactic strains were characterized based on
fermentations carried out in Medium B supplemented with 10 vol.%
CSL, 90 g/L glucose and 33 g/L CaCO.sub.3. The overall lactate
production and/or percentage L-lactate produced were measured for
this set of strains. The results are shown in Table 2 below.
3TABLE 2 Isolated Homolactic Strains Strain No. g/L Lactate %
L-Lac. 90 62 81 92 67.9 59 95 62.47 44 99 63.17 78 103 58.53 75 104
65.18 75 109 66.26 83 114 58.6 46 117 47.99 62 127 49.54 44 129
68.75 77 132 59.12 95 133 60.37 95 134 28.87 63 136 54.1 41 139
66.08 47 140 57.18 94
EXAMPLE 4
Effect of Added Base on Lactate Production
[0086] A number of the strains described in Example 2 which had
been identified as L-lactate producers were screened to examine the
effect added base (CaCO.sub.3) on lactate production. The
fermentations were carried out at 48.degree. C. in MRS medium
supplemented with 10% CSL and 30 g/L glucose. For the
determinations made in the presence of added base, MRS medium
supplemented with 10% CSL, 30 g/L glucose and 20 g/L CaCO.sub.3
were used.
4TABLE 3 Effect of CaCO.sub.3 on Lactate Production Lactate
Production (g/L) Strain # No Base 20 g/L CaCO.sub.3 6 21 42 10 20
32 14 23 37 19 17 33 21 26 49 22 19 34 23 28 47 24 18 46 41 24 48
42 27 49 43 23 42 44 24 39 45 21 37 46 21 47 47 21 37 51 24 37
EXAMPLE 5
L-Lactate Production
[0087] The level of L-lactate production was characterized for a
number of the strains described in Example 2. The fermentations
were carried out at 48.degree. C. in MRS medium supplemented with
10% CSL, 30 g/L glucose and 20 g/L CaCO.sub.3.
5TABLE 4 L-Lactate Production Lactate Produced Strain # % L-Lactate
(g/L) 10 87% 39.12 14 79% 21.11 21 85% 38.56 23 85% 35.69 24 84%
31.78 41 86% 38.10 42 83% 30.62 43 80% 25.17 44 84% 31.75 46 86%
36.12
EXAMPLE 6
Lactate Production by ATCC Deposited Lactobacillus Strains
[0088] The lactate productivity of a number of known lactobacillus
strains isolated from sources other than corn steep water was
examined. Samples of eleven different strains were obtained from
the American Tissue Culture Collection (Rockville, Md.) and
screened for total lactate production and final incubation pH based
on fermentation at 37.degree. C. in MRS medium supplemented with 75
g/L glucose and 30 g/L calcium carbonate. The results are shown
below in Table 5. All of the strains exhibited poor growth at
47.degree. C. and were inhibited by the presence of corn steep
water in the nutrient medium. While the nutrient requirements of
the ATCC deposited strains are different from the strains isolated
from corn steep water, several of the ATCC deposited strains appear
to be capable of producing relatively high concentrations of free
lactic acid. In particular Lactobacillus helviticus (ATCC # 15009;
66 g/L lactate at a final incubation pH of 4.03), Lactobacillus
paracasei tolerans(ATCC # 25599; 66 g/L lactate at incubation pH of
4.04), and Lactobacillus salivarius salivarius(ATCC # 11741; 64 g/L
lactate at a final incubation pH appear to offer potential as high
productivity free lactic acid producers. The optical purity of the
lactate produced by a number of the strains was determined. None of
the strains capable of producing a relatively high concentration of
free lactic acid was an L-lactate producing strain.
6TABLE 5 Lactate Production by ATCC Lactobacillus Strains ATCC #
Lactobacillus Lac % L-Lac pH 12315 L. delbrueckii lactic 47 42 4.93
11741 L. salivarius 64 52 4.12 salivarius 25302 L. paracasei
paracasei 52 69 4.76 25258 L. jensenii 3 -- 6.25 15009 L.
helveticus 66 53 4.03 33409 L. delbrueckii 18 54 5.45 bulgaricus
25599 L. paracasei tolerans 66 53 4.04 39392 L. casei casei 50 12
4.71 33323 L. grasseri 18 -- 5.62 4536 L. acidophilus 40 -- 5.43
35046 L. animalis 51 -- 4.78
EXAMPLE 7
SO .sub.2 Tolerence of Homolactic Strain #41
[0089] The effect of varying levels of sulfur dioxide (SO.sub.2) on
the lactate productivity of the homolactic strain #41 was examined.
The effects of varying sulfur dioxide concentration on lactate
production were examined using strain #41. The fermentations were
carried out in MRS Medium supplemented with 10 vol.% CSL, 30 g/L
glucose and 20 g/L CaCO.sub.3 via the standard fermentation
protocol described in Example 1. The results shown in Table 6 below
demonstrate that the strain #41 is capable of producing lactate in
the presence of SO.sub.2 concentrations of up to at least about 600
ppm. In similar fermentation carried out in the presence of 800
ppm, strain #41 started producing lactate after a dormant phase of
144 hours.
7TABLE 6 SO.sub.2 Tolerance of Homolactic Strain #41 Lactate
Production (g/L) SO.sub.2 Conc. 24 hr. 48 hr. 72 hr. 200 ppm 11 48
66 400 ppm 9 27 55 600 ppm 9 11 43
EXAMPLE 8
Effect of Temperature on Lactate Production
[0090] The lactate productivity of the homolactic strain #41 was
determined over a range of temperatures between 41.degree. C. and
54.degree. C. The fermentations were carried out in Medium B
supplemented with 10 vol.% CSL, 60 g/L glucose and 20 g/L calcium
carbonate. The results shown in Table 7 below establish that the
optimum temperature range for lactate production by the strain #41
is from 44.degree. C. to 540.degree. C.
8TABLE 7 Temperature Dependence of Lactate Production Lactate
Production (g/L) Temp. (.degree. C.) 24 hr. 48 hr. 72 hr.
41.degree. 14 51 68 44.degree. 25 55 68 47.degree. 26 50 63
50.degree. 31 52 57 54.degree. 9 19 23
EXAMPLE 9
Effect of Steep Water Concentration on Lactate Production
[0091] Fermentations employing a number of the L-lactate producing
strains described in Example 2 were conducted to examine the effect
of varying amounts of corn steep liquor in the growth medium on
lactate production. The fermentations were conducted at 48.degree.
C. in Medium A (see below) supplemented with 50 g/L glucose, 20 g/L
CaCO.sub.3, and either 1%, 5% or 10% CSL.
9 Medium A (pH = 5.0) 10 g/L yeast extract 0.2% K.sub.2HPO.sub.4 1
g/L Tween .RTM. 80 0.2% ammonium citrate 0.005% MnSO.sub.4
4H.sub.2O 0.02% MgSO.sub.4 7H.sub.2O Added carbon/energy source
Added nitrogen source CaCO.sub.3 added to modulate pH
[0092]
10TABLE 8 Effect of Steep Water on Lactate Production Lactate
Production (g/L) Strain # 1% CSL 5% CSL 10% CSL 10 1 19 31 23 1 10
32 24 1 6 22 41 1 9 33 45 1 8 35
EXAMPLE 10
Characterization of Homolactic Strains Based on Ribotype
[0093] A number of the L-lactate producing homolactic bacterial
strains isolated from corn steep water were categorized based on
riboprint pattern analysis (see, e.g., Jaquet et al., Zbl. Bakt.,
276, 356-365(1992)). This technique is based on digestion of DNA
from a single colony of the strain in question using an EciRI
restriction enzyme and hybridization after size separation on an
agarose gel with a chemically labeled rRNA operon from E. coli..
The resulting pattern is a direct indicator of genetic
relationships between organisms and has been used to provide
identification between four genera of bacteria (Samonella,
Listeria, Staphylococcus and E. coli) as well as for the
taxonomical identification of closely related gram positive and
gram negative strains.
[0094] The results of ribotyping of seven of the lactate producing
strains isolated from corn steep water are shown in FIG. 2. Strains
given the same RiboGroup designation are likely to be identified to
the same taxon level as identical. The ribotypes exhibited by the
seven strains shown in FIG. 2 did not match the patterns of any of
30 different lactic acid bacterial strains in a commercial
laboratory's computer database. Among the strains in the database
which did not provide a match were Lactobacillus acidophilus,
Lactobacillus animalis, Lactobacillus delbrueckii, Lactobacillus
helveticus, Lactobacillus amiylovorus and Lactobacillus salivarius.
The ribotypes of the strains listed in FIG. 2 also did not provide
a match with the patterns from Lactobacillus agilis, Lactobacillus
brevis, Lactobacillus buchneri, Lactobacillus confusus,
Lactobacillus coryniformis, Lactobacillus curvatus, Lactobacillus
farciminis, Lactobacillus kefir, Lactobacillus murinus,
Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus
sake, and Lactobacillus suebicus. The ribotype patterns shown in
FIG. 2 also did not provide a match with Lactococcus garviae,
Lactococcus lactis, and Lactococcus raffinolactis or with
Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc
mesenteroides, Leuconostoc paramesenteroides, Pediococcus
acidilactici, Pediococcus dextrinicus and Pediococcus
pentoxaceus.
[0095] The ribotype patterns of the seven strains shown in FIG. 2
fall into three RiboGroups. Two strains (#114 and #119) have
identical ribotypes. One of these strains is a heterolactic strain
(#119) while the other is a homolactic strain which produces
racemic lactate (#114). The one D-lactate producing strain (#79)
exhibited a ribotype pattern which was different from the other
six. The remaining four strains (#90, 127, 132 and 140) were
classified in the same RiboGroup and were considered to be likely
to be identified to the same taxon level, despite the fact that
their ribotype patterns were not identical. of the four strains
with a MIL 4-1132 pattern, three were L-lactate producing strains
(#90, 132 and 140) while the (#127) produced racemic lactate.
EXAMPLE 11
Effect of Added Base on Lactate Production
[0096] The effect of the additon of varying amounts of CaCO.sub.3
on the lactate productivity of homolactic strain #41 was examined.
The experiments were carried out at 47.degree. C. in Medium A
supplemented with 8 vol.% CSL, 200 g/L glucose, and varying amounts
of added calcium carbonate (30-90 g/L). The results are shown in
Table 9 below.
11TABLE 9 Effect of CaCO.sub.3 on Lactate Production CaCO.sub.3
Lactate Production (g/L) Conc. 0 hr. 24 hr. 51 hr. 120 hr. Final pH
30 g/L 3.17 48.1 75.5 75.7 3.98 40 g/L 6.12 53.4 81.3 87.0 4.48 50
g/L 5.84 49.4 83.4 88.1 4.73 60 g/L 3.21 50.2 75.4 77.2 4.75 70 g/L
4.85 48.9 75.3 73.8 4.8 80 g/L 3.45 54.4 61.1 83.6 4.77 90 g/L 5.39
49.6 57.8 83.6 4.74
EXAMPLE 12
Fermentation Profile of Strain #41 with 12% CSL,90 g/L Glucose and
33.4 g/L CaCO.sub.3
[0097] FIG. 3 shows the profile of pH and the organic components in
the fermentation broth as a function of time during the course of a
representative fermentation experiment. The profile shown in FIG. 3
is based on results obtained from incubation at 47.degree. C. of
strain #41 in Medium B supplemented with 10 vol.% CSL, 100 g/L
glucose and 33.4 g/L calcium carbonate.
EXAMPLE 13
Fermentation Profile of Strain #41 with 90 g/L Glucose, 33.4 g/L
CaCO.sub.3 and 12% CSL/36% LSW
[0098] FIG. 4 shows lactate production as a function of time during
the course of representative fermentation experiments with strain
#41. The fermentations were carried out using the procedure
described in Example 1. The profile shown in FIG. 4 is based on
results obtained from incubation of strain #41 at 47.degree. C. in
Medium C supplemented with 90 g/L glucose, 33.4 g/L calcium
carbonate and either 12 vol.% CSL (36 wt. dry solids) or 36 vol.%
LSW (12 wt.% dry solids). The results summarized in Table 10 below
show final free lactic acid levels of about 40 g/L free with either
source of corn step water. Since the lactate was produced with an
L-lactate producing strain (#41), at least about 35 g/L free
L-lactic acid was present at the conclusion of these fermentations
(the remainder is free D-lactate present in the added steep
water).
12TABLE 10 Lactate Production with Strain #41 Corn Steep Water
Source 12% CSL 36% LSW Lactate (g/L) 0 hrs. 10.3 8.4 16 hrs. 44.0
52.4 24 hrs. 80.5 92.2 44 hrs. 91.5 96.8 Final pH 3.92 3.98 Final
Free 42 41 Lactate (g/L)
EXAMPLE 14
Lactate Production of Strain #41 with 8-12% CSL. 90 g/L Glucose and
36.6 g/L CaCO.sub.3
[0099] FIG. 5 shows lactate production as a function of time during
the course of representative fermentation experiments with strain
#41. The fermentations were carried out using a modified version of
the procedure described in Example 1. Cells of strain #41 were
pregrown in 800ml of medium and then separated from the medium. The
pregrown cells were then resuspended in 800 ml of fresh medium. The
profile shown in FIG. 5 is based on results obtained from
incubation of the pregrown cells at 47.degree. C. in Medium B
supplemented with 90 g/L glucose, 36.6 g/L calcium carbonate and
either 8 vol.% CSL (36 wt.% dry solids), 12 vol.% CSL, 24 vol.% LSW
(12 wt.% dry solids), or 36 vol.% LSW.
13TABLE 11 Lactate Production with Strain #41 Corn Steep Water
Source Final pH Lactate Free Lactic 8% CSL 3.83 93 g/L 47 g/L 24%
LSW 3.90 94 g/L 44 g/L 12% CSL 3.80 97 g/L 52 g/L 36% LSW 3.81 99.5
g/L 53 g/L
EXAMPLE 15
Effect of Added Glucose on Lactate Production
[0100] The effects varying the amounts of an added carbohydrate
source (glucose) on lactate production was examined for the
homolactic strain #41. The fermentations were run by incubating the
#41 strain at 48.degree. C. in Medium A supplemented with 10 vol.%
CSL, 20 g/L CaCO.sub.3 and the indicated level of glucose using the
standard fermentation procedure described in Example 1. The medium
also contained an additional 1-15 g/L fermentable sugar (mainly
glucose and fructose) from the corn steep liquor. The results are
shown in Table 12 below. The results of this experiment suggest
that at least for the level of base added (20 g/L CaCO.sub.3),
lactate productivity may be enhanced by the addition of at least
about 50 g/L of a carbohydrate source such as glucose.
14TABLE Xl Effect of Glucose on Lactate Production Lactate
Production (g/L) Glucose Added 24 hr. 48 hr. 72 hr. 30 g/L 14 39 42
50 g/l 11 51 55 80 g/L 11 50 67 100 g/l 9 47 65
[0101] The invention has been described with reference to various
specific and preferred embodiments and techniques. The invention is
not to be construed, however, as limited to the specific
embodiments disclosed in the specification. It should be understood
that many variations and modifications may be made while remaining
within the spirit and scope of the invention.
15TABLE 1 Isolated Homolactic Strains Strain g/L % L- No. Lactate
pH Lactate 1 16.7 4.04 34 2 19.4 3.97 36 3 4.51 5 8.1 5.22 18 6
18.4 4.02 69 7 17.5 4.03 38 8 23.8 4.51 43 9 25.1 4.29 34 10 23.6
4.33 73 11 26.2 4.3 37 12 24.6 4.32 36 13 21.6 4.22 54 14 24.3 4.15
77 15 24.2 4.13 51 16 21.3 4.25 64 17 18.1 4.34 39 18 25.2 4.28 74
19 10.4 5.06 35 20 25.3 4.14 69 21 23.1 4.17 76 22 22.4 4.21 75 23
28.6 4.12 78 24 22.8 4.19 41 25 22.6 4.19 44 26 8.1 17 27 23.7 4.19
48 28 22 4.21 44 29 21.1 4.18 51 30 23.6 4.15 47 32 20.4 4.15 46 34
19.5 41 35 40 36 35 37 37 38 42 39 62 40 36 41 24.5 4.17 76 42 25.9
4.25 75 43 25 4.26 74 44 26.2 4.28 74 45 25.9 4.27 74 46 27.4 4.25
76 47 26 4.27 73 48 13.3 4.54 47 49 28.4 4.19 47 50 29.2 4.21 47 51
26.1 4.22 76 52 30.6 48 55 2 56 31 57 32 58 0 59 0 60 0 61 45 62 88
63 5 64 92 65 41 66 4 67 5 68 5 69 49 70 48 71 44 72 5 73 5 74 5 75
3 76 53.27 2 77 4 78 3 79 3 80 3 81 15.8 82 16.7 83 39.9 55 84 14
85 14.2 86 8.1 87 8.4 88 46.1 55
* * * * *