MXPA00003606A - Low ph lactic acid fermentation - Google Patents

Abstract

A process for producing lactic acid which includes incubating acid-tolerant lactate producing microorganisms, such as acid-tolerant homolactic bacteria, in nutrient medium to produce a fermentation broth with high levels of free lactic acid is provided. Isolated acid-tolerant homolactic bacteria capable of producing high levels of free lactic acid are also provided.

Description

FERMENTATION OF LACTIC ACID AT LOW pH BACKGROUND OF THE INVENTION Lactic acid and its salts have been used for a long time in a wide variety of applications in the chemical, cosmetic, food and pharmaceutical industries. More recently, new bioengineered materials based on lactate, such as biodegradable lactide polymers, have inflamed 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. Patents: 5, 142, 023; 5,247, 058; 5,258,488; 5,357,035; 5, 338, 822; 5.445, 123; 5, 539, 081; 5,525,706; 5,475, 080; 5, 359, 026; 5,484, 881; 5,585, 1 91; 5, 536,807; 5,247,059; 5,274, 073; 5.51 0.526; and 5, 594, 095. While chemical processes can be used to produce lactic acid, the cost increase of petrochemical raw material and the need to solve the mixture of racemic lactate produced by conventional chemical methods, makes the methods of fermentation an attractive alternative for the manufacture of lactate enriched in one of its optical isomers. The processes used to produce biodegradable lactide polymers usually require the free acid form of either L- or D-lactate as a starting material. Unfortunately, as with most organic acid fermentations, the inhibition of the final product by organic acid (lactic acid in this case) can be a major obstacle to efficient fermentation. Bacterial strains normally used in lactate fermentations can be inhibited by low pH in addition to the lactate concentration. To overcome this problem, industrial lactate fermentation processes are run at a higher pH, for example, at least about 5.0 and frequently at or above 6.0. This results in the production of a lactate product, which is essentially present all in the form of a salt. Normally, one or several additional process steps are required to remove the cationic 'counterion' and isolate the desired free lactic acid. Moreover, because high concentrations of certain salts, for example, sodium cations, can have an inhibitory effect on fermentation, the type and / or amount of salt present can also influence the efficiency of the fermentation. The production of racemic lactate of corn starch thinned with enzyme has been reported using lactobacillus amylovorus. Cheng et al. , J. Ind. Microbiol. , 7: 27-34 (1 991). Although relatively high production levels have been reported at pH as low as 4.2, this fermentation does not provide lactate enriched in any optical isomer. Several approaches have been reported to improve the efficiency of lactate fermentations. Several of these involve the removal of free lactic acid from the fermentation broth on a continuous basis. For example, electrodialysis has been used to reduce inhibition of the final product through the removal of lactate from the fermentation broth. The high cost of dialysis membranes coupled with a low lactate gradient has generally lowered the attractive qualities of this approach. Ion exchange and the use of polyvinylpyridine to remove lactate from the fermentation medium have also been reported. Yet another method that was recently described involves a multistage extraction procedure. This process involves extracting lactate from the broth with a tertiary amine in an attempt to keep the pH of the broth from falling to a value that inhibits the production of additional lactate. However, the levels of lactate production reportedly achieved via this method are still quite low. The use of this method may also require that the extracted fermentation broth be subjected to a second extraction to reduce at least the residual concentration of tertiary amine extractor before recycling the extracted broth again in the fermentation reaction. U.S. Patent No. 4, 769, 329 (Cooper et al.) Describes a process for the preparation of optically pure D- or L-lactic acid by fermentation using Lactobacillus at a pH between 4 and 6. However, Cooper et al. they do not describe high performance at a low pH. J P 87 44 188 describes the production of optically active lactic acid at a pH between 4.5 and 7.0 and a concentration of 1 01 g / l. J P 92 271 787 describes a method for making D-lactic acid by cultivating Pseudomonas I-producers at a pH of 8.0 in a medium containing 1,2-propanediazole. After the fermentation is complete, the culture is acidified with sulfuric acid to a pH of 2.0 to obtain 21 g / l of D-lactic acid. EP 0 308 064 describes an improved tomato beverage prepared by fermenting the tomato drink with Lactobacillus. Nothing in EP 0 308 064 discusses the yield or optical purity of the lactic acid obtained in such fermentation. GB 2 251 864 describes an acid tolerant strain of Lactobacillus, stable at low temperature, obtained by cultivating a mixed population of Lactobacillus in a medium comprising milk at a pH of 3.4 to 4.2.

Nothing in GB 2 251 864 describes the yield or optical purity of lactic acid obtained from stable Lactobacillus at low temperature. DE 27 00 644 (same as GB 1 547063) describes the fermentation of plant and / or animal material using lactic acid forming acid-tolerant bacteria Streptococcus faecalis or Leuconostoc bacteria in a medium having a pH between 4 and 4.7 to form food for farm and domestic animals and poultry. The performance of lactic acid at the specified pH is not described. All these approaches for producing lactic acid in its free acid form, based on the fermentation of lactobacillus, suffer from one or more disadvantages. Alternate approaches based on the fermentations of other microorganisms more tolerant to acid have also been reported. Yeasts, such as, Saccharomyces cerevisiae, are able to grow at a much lower pH than lactobacilli. Recombinant yeast strains have been produced by introducing the lactate dehydrogenase gene from a bacterial (lactobacillus) or mammalian (bovine) source in Saccharomyces cerevisiae. Strains of recombinant yeast are able, it is said, to produce lactate at or below the pKa of lactic acid (approximately 3.8). However, ethanol is the main fermentation product generated by these recombinant yeast strains. This both decreases the efficiency of lactate production and introduces additional potential sequelae with respect to the separation and purification of free lactic acid. The production of lactic acid by a pelleted form of the fungus, Rhizopus orgyzae, has also been reported. This fungal fermentation also normally produces glycerol and / or ethanol as main byproducts. The yield of free lactic acid was optimized in this case by continuous removal of the fermentation broth using a polyvinylpyridine column ("PVP"). Concentrations greater than about 25 g / l generated at low fermentation pH were not reported using the Rhizopus / PVP method.

Brief description of the invention The present invention relates to the production of lactate via fermentation. Particularly concerns fermentation with acid-tolerant lactate-producing microorganisms, such as 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 downstream processing required to isolate the lactate in its free acid form from the broth. In general, it has been determined that the processes conducted to make the fermentation broths (or other mixtures of lactic acid / lactate salt) at pHs in the order of about 4.8 or less (preferably 4.5 or less, most preferably 4.3 or less, typically 3.5 to 4.2), an overall efficient process can be developed, in which the lactic acid generated in the polymer production and, if desired, the lactate salt can be used. recovered can be recycled in the fermentation system as a buffering agent, or put differently for a pH control. The process provided herein for producing lactic acid includes incubating acid-tolerant lactate-producing microorganisms, such as acid-tolerant, homolytic lactobacillus, in nutrient medium at a pH which provides a substantial portion of the lactate product in the form of free acid. In the present, when the term "acid tolerant" is used in reference to bacteria, it is intended to refer to bacteria that are capable of producing lactate at a pH sufficient to provide a substantial portion of the lactate product in the free acid form. Acid-tolerant bacteria are usually capable of producing at least about 25 g / l of free lactic acid. Such bacteria can generally produce at least about 50 g / L of lactate (i.e., 50 g / L of total lactate) in nutrient medium at an "average incubation pH" of no more than about 4.2. If the fermentation is not performed 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 in ten (10) or more equal time intervals about the course of fermentation. The present fermentation process can be run in a continuous manner. Under such conditions, steady-state conditions (in terms of pH, lactate concentration and nutrient concentrations) are generally reached and maintained after an initial start-up phase has been completed. When the fermentation is conducted in this manner, the average incubation pH is the average pH of the broth after the initial start phase has been completed, ie the pH during the start phase is ignored to determine the incubation pH average. If the fermentation is carried out to a point where the pH and / or concentration of lactic acid inhibits the production of additional lactate, the "average incubation pH" is determined based on an average of the pH values measured in ten (10 ) or more equal time intervals over the period necessary to produce 90% of the limiting lactate concentration. As used herein, the "limiting lactate concentration" is the concentration of lactate under a given set of incubation conditions (nutrient medium, temperature, degree of aeration) at which the pH and / or concentration of lactic acid Generated by fermentation inhibits the production of additional lactate. 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 concentration of lactic acid inhibits the production of additional lactate. Inhibition of lactate production is considered to have occurred when the amount of lactate produced does not increase by more than about 3% on additional incubation for a period of up to about twelve (12) hours under the same conditions. This definition presumes that sufficient nutrients are still available for lactate production in the fermentation broth.

In the present, the terms "nutrient medium" and "fermentation broth" are used interchangeably. These terms refer to both (i) means in the form originally provided to acid-tolerant bacteria as a source of nutrient, and (ii) means produced after some or all of the originally provided nutrients have been consumed and the products of Fermentation including lactate have been excreted in the media by bacteria. In the present process, the pH of the fermentation broth after incubation of the acid-tolerant bacteria to produce lactate, is usually not greater 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 at which growth and / or lactate production ceases by the acid-tolerant bacteria. The cessation of growth and / or lactate production may be the result of a change in the reaction temperature, the depletion of one or more necessary nutrients in the fermentation broth, a deliberate change in pH, or the separation of the fermentation broth of bacterial cells. In those cases where fermentation is stopped by the addition of sufficient acid or base for the broth to stop the production of lactate, the pH of final incubation is defined as the pH of the nutrient medium just before the addition. Alternatively, lactate growth and / or 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 reaction of fermentation has reached a self-limiting point for the given set of incubation conditions. As noted above, it is quite common for bacterial fermentations that produce an organic acid, such as lactic acid, to be subjected to inhibition of the final product. The term "lactate" as used in this application refers to 2-hydroxypropionate either in its free acid or salt form (ie, "total lactate"). The terms "lactic acid" and "free lactic acid" are used interchangeably herein to refer to the acid form, i.e., 2-hydroxypropionic acid. The lactate salt form is specifically referred to herein as a lactate salt, for example, as any sodium salt of lactic acid or sodium lactate. The present invention also provides homologous acid-tolerant bacteria. Homologous acid-tolerant bacteria are generally capable of producing at least about 25 g / L of free lactic acid at an incubation temperature of at least about 40 ° C. Another embodiment of the present acid-tolerant bacterium is capable of producing at least about 50 g / L of lactate at a temperature above about 40 ° C and an average incubation pH of no more than about 4.2. Normally, acid-tolerant bacteria are capable of satisfying both of these lactate productivity measurements.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic representation of a flow diagram of a fermentation process, which includes the coupled removal of free lactic acid.

Figure 2 is a graph showing the ribotype patterns for several lactate-producing bacterial strains isolated from maize infusion water. Figure 3 is a graph showing the fermentation profile of glucose, fructose and lactate for the incubation of strain # 41 in a nutrient medium containing 10% vol of corn infusion liquor, 100 g / l of glucose and 33.4 g / l of calcium carbonate. Figure 4 is a graph showing the lactate production of the incubation of strain # 41 in a nutrient medium containing 90 g / l of glucose, 33.4 g / l of calcium carbonate and either 12% vol of infusion liquor of corn or 35% vol of light infusion water. Figure 5 is a graph showing the fermentation profile of glucose, fructose and lactate for incubation of homoloctic strain # 41 in a nutrient medium containing 90 g / l of glucose, 36.6 g / l of calcium carbonate and amounts water variables of corn infusion. Figure 6 is a graph showing the percentage of undissociated lactic acid ("free lactic acid") as a function of pH.

DETAILED DESCRIPTION The generation of lactic acid solutions, via bacteriological systems, having pHs in the order of 5.0 or below, preferably 4.8 or below, and normally 3.5 to 4.5, leads to a higher percentage of production of the lactate material, in the form of lactic acid. The generation of relatively large amounts of product from the fermentation process in the form of lactic acid, instead of lactate salt, is advantageous because it can reduce the need for, or degree of, certain steps of monitoring processes. acidification and / or "salt separation". That is, if a greater amount of the material is generated as free lactic acid, a processing step to generate the lactic acid from lactate, and the expenses and consequences associated with it, are reduced or avoided. Even if some acidification is conducted, substantially less acid addition would be involved than would be the case with a high pH system. In general, it has been determined that with processes conducted to make fermentation broths at pHs in the order of about 4.8 or less (preferably 4.5 or less, most preferably 4.3 or less, typically 3.5 to 4.2), a global efficient process can be developed, in which the generated lactic acid can be used in the production of polymer and the recovered lactate salt can be recycled in the fermentation system as a buffering agent, or set differently for pH control. 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 homoloctic bacterium in a suitable nutrient medium. Acid-tolerant homoloctic bacteria can be isolated from maize infusion water from a commercial corn milling facility. While different bacteria of this type can produce either racemic lactate or lactate predominantly either in the D or L isomeric form, the present process preferably employs a homolactic bacterium that predominantly produces L- or D-lactate, and most preferably, it produces L-lactate in optically pure form. The present process allows the efficient production of high concentrations of free acid form of an optical isomer of lactic acid. This efficiency can be expressed in a variety of ways. The concentration of lactic acid in the fermentation broth serves as a measure of the overall productivity of the process. The present process usually generates a solution that includes at least about 25 g / l, preferably at least about 30 g / l, and more preferably at least about 40 g / l of free lactic acid. Most preferably, the process produces these levels either of 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, an optical isomer of lactate is produced in essentially pure form. As noted above, normally, the lactate produced by the present process is predominantly in the L-lactate form. For example, one embodiment of the process includes incubating an acid-tolerant homologous bacterium in a nutrient medium to produce lactate, which includes at least about 75% by weight of L-lactate (ie, L-lactate having an optical purity of less approximately 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% (eg, includes at least about 95% by weight of L-lactate). Most preferably, the present process produces L- or D-lactate in essentially optically pure form (ie the lactate produced contains 99% by weight or greater than a single optical isomer). If a fermentation broth has a pH value between 3.0 and 4.5, there will be a significant amount of lactic acid in the undissociated form (see Fig. 6). Actually at a pH of 3.0, the molar ratio of free (non-dissociated) lactic acid to lactate ion at 25 ° C is approximately 7.0; and at a pH of about 4.5, the ratio at 25 ° C, is about 0.23. The total 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 concentration of free lactic acid. 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 1 00 g / l of lactate at a relatively low pH. The lower the pH of the solution, the higher the percentage of lactate that is present in its free acid form. For example, where the pH of the medium is equal to the pKa of lactic acid (about 3.8), 50% of the lactate is present in the free acid form. At a pH of 4.2, approximately 31% of the lactate as a free acid and at pH 4.0 and 3.9, approximately 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, 1 8% at pH 4.5 and 6.6% at pH 5.0. The pH of the broth during the incubation step can be expressed in several different ways, for example, in terms of the average incubation pH or the final incubation pH. The present fermentation process is usually capable of producing high lactate levels 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 normally 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 modes of the present fermentation process are capable of producing at least about 80 g / L of 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. The present fermentation process can be run in a continuous manner, where a fraction of the fermentation broth is removed as the fermentation proceeds. This can be done either continuously or at periodic intervals. Normally enough nutrient medium is 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 start-up phase has been completed. When the fermentation is conducted in this manner, the average incubation pH (the pH during the start phase is ignored) and the final incubation pH of the broth are essentially the same. Under such conditions, the fermentation is normally performed 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. Although the present incubation process can be performed at relatively low temperatures, for example, about 30 ° C to about 38 ° C, the acid-tolerant homoloctic bacterium is usually incubated in a suitable nutrient medium at a temperature of at least about 43 ° C. , and more preferably at about 45 ° C to about 52 ° C. Most preferably, the fermentation is carried out at about 47 ° C to about 50 ° C. There are several advantages of operating fermentation at these temperatures. The possibilities of complications due to the growth of other competitive organisms is diminished in this temperature range. Also, at higher temperatures, the reaction generally proceeds at a higher speed, allowing the efficient utilization of the process equipment. If the fermentation is carried out at too high a temperature, usually at about 54 ° C or higher, the growth and / or production of lactate by homolactic bacteria may be negligible. However, it may be possible, using standard selection techniques, to identify mutagenic homologous bacterial strains, which are capable of growth and lactate production at temperatures of 55 ° C and above. As described herein, "nutrient medium" refers to a water-based composition that includes minerals and their salts necessary for the growth of the bacterium of the present invention. The nutrient medium normally 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, that is, minute fractions of 1 percent (1000 ppm or less). The bacterium of the present invention can usually use various carbon and energy sources for the growth and / or production of lactate, 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 fussy and may only be able to grow one or two sugars on the list. In other cases, a starch (such as corn starch) or a hydrolyzate thereof may be used as a primary source of carbohydrates. As used herein, "corn infusion water" refers to water obtained from corn infusion tanks as well as other solutions derived therefrom having substantially the same spectrum of nutrients. For example, corn infusion liquor (sometimes referred to as "heavy infusion water") is a concentrated form of maize infusion water obtained by removing water and other volatile components, usually under vacuum. The corn infusion liquor typically has a dry solids content of about 35% by weight to about 50% by weight. The corn infusion liquor used in the experiments described in the Examples herein had a dry solids content of 36% by weight and is referred to herein as "CSL". Corn infusion waters obtained directly from corn infusion tanks and / or associated lines just prior to concentration to produce corn infusion liquor, generally have dry solids contents in the range of about 10% by weight to about 15% by weight and are referred to herein as "light infusion water" ("LSW"). The light infusion water normally has an SO2 content of no more than about 500 ppm. The infusion water used to supplement the nutrient medium used in the present process, preferably has an SO2 content of no more than about 300 ppm and, more preferably, no more than about 200 ppm. The light infusion water used in the experiments described in the Examples herein had a dry solids content of 1.2% by weight. In situations where one or more homolactic strains isolated from maize infusion water are to be used to produce lactate, the nutrient medium typically includes maize infusion water corresponding to at least about 15 g / L dry infusion water solids. . Preferably, the nutrient medium includes maize infusion water corresponding to at least about 25 g / l, and more preferably, at least about 30 g / l dry solids of infusion water. An example of a suitable nutrient medium for use in the present fermentation process is the MRS medium (such as, the MRS medium commercially available from Becton Dickinson &; Co.) or similar. The M RS medium is usually supplemented with maize infusion water to provide a source of nitrogen and a general source of nutrients, as well as with additional carbohydrate (such as glucose or fructose) as a source of carbon and energy. Normal means 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). However, it may not be necessary to add specific amounts of such salts to the medium. Frequently, the nutrient medium also includes a nonionic surfactant, such as a fatty acid monoester of a polyoxyethylene derivative of sorbitan (eg, Tween® 80, which is polyoxyethylene sorbitan monooleate (20)). The medium can be prepared by using separate salts as sources of each of the various inorganic components. Alternatively, a simple salt that acts as a source of more than one component can be used to prepare the nutrient medium. For example, potassium hydrogen phosphate (K2HPO) can 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 exchange of cations and anions will occur between the various dissolved salts. For example, if magnesium sulfate and ammonium citrate are added to the water during the preparation of the medium, the resulting solution will also include some species of ammonium sulfate and magnesium citrate, in addition to the species of magnesium sulfate and ammonium citrate. A type of nutrient medium, which is particularly suitable for use in the present fermentation process, includes corn infusion water supplemented with glucose and / or fructose as a source of carbon and additional energy.

An example of a medium suitable for use in the present invention includes: maize infusion water corresponding to about 30 to about 45 g / l dry solids of infusion water; about 80 to about 120 g / L of glucose, fructose or a mixture thereof; about 0 to about 10 g / l of yeast extract; about 0 to about 1 g / L of a non-ionic surfactant, such as, Tween® 80; about 0 to about 2 g / l of potassium hydrogen phosphate (K2H PO4); about 0 to about 0.2 g / l of magnesium sulfate (MgSO4); about 0 to about 0.05 g / l of manganese sulfate (MnSO4); about 0 to about 2 g / l of ammonium citrate; and optionally, about 10 to about 50 g / i of calcium carbonate (CaCO3). For the reasons discussed above, the amounts refer to the amounts of various materials added to form the medium and not to the actual concentrations of these species in the nutrient medium. In making such a nutrient medium, all components, except the non-ionic surfactant and calcium carbonate, are generally dissolved in an appropriate amount of water and autoclaved. The nonionic surfactant is usually added to the autoclaved medium while still being at a temperature of about 1000 ° C. The resulting solution is then allowed to cool to about 60 ° C or less before the calcium carbonate is added. It has been found that suitable nutrient media for use in the present process preferably include at least about 50 g / L of carbohydrate. More preferably, the nutrient medium includes at least about 70 g / l and, most preferably, at least about 90 g / l of the carbohydrate. The carbohydrate is usually made of glucose, fructose, galactose, melibiose, sucrose, raffinose, stachyose or a mixture thereof. Glucose, fructose and sucrose are particularly suitable for use as a source of carbon and energy in the nutrient medium. It is generally not useful to incorporate more than about 150 g / L of carbohydrate into the medium.

It has been found that it may be advantageous to include a base, such as calcium carbonate (CaCO3), sodium hydroxide (NaOH), ammonium hydroxide (N H4OH) and / or sodium bicarbonate (NaHCO3). Normally, at least about 30 g / 1 of calcium carbonate (or an equivalent amount of another base) is added to the nutrient medium. In some embodiments of the process, for example, modalities that produce higher levels of lactate, it may be preferred to include up to about 40 g / L of calcium carbonate in the nutrient medium. Although higher levels of base may be employed, due to the limitations in the solubility of the calcium carbonate salts and the desire to maintain a relatively low broth pH, it is generally not useful to incorporate more than about 1000 g / l carbonate of calcium carbonate. calcium in the middle. Very often, the full 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 creep into the solution. The overall effect is to neutralize a portion of the lactic acid in formation and prevent the pH of the broth from falling below a desired level (eg, below about 3.8-3.9). It may not be necessary to add a base, such as calcium carbonate to achieve this effect. A solution containing a lactate salt (eg, calcium, sodium or ammonium lactate) can be added to help buffer the pH of the fermentation broth. An example of a process in which this could occur would involve the separation of a fraction of the fermentation broth from the incubating bacteria, and recycle the portion back to the fermentation after the removal of some or all of the free lactic acid in the fraction. Alternatively, the calcium lactate could be isolated from the fermentation broth (e.g., in solid form), and mixed together with the nutrient medium that is added to the fermentation. Generally, the addition of lactate salt as a buffer 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 the salt form. Nutrient media including at least about 70 g / L of glucose and / or fructose and at least about 20 g / L of calcium carbonate are particularly suitable for use in the present process. Depending on the bacterial strain employed in the process, the incorporation of maize infusion water (eg, in an amount equivalent to at least about 25 g / l dry solids of maize infusion water) may also be preferred in this medium. nutrient It is particularly useful to add corn infusion water containing only the same chiral form of lactate to be generated by the fermentation process. The strain of homolactic bacteria and the fermentation conditions are normally chosen so that the free lactic acid is produced at a global rate of at least about 0.5 g / l / h, preferably at least about 1.0 g / l / h, more preferably at least about 2.0 g / l / h, and most preferably at least about 4.0 g / l / h. As used herein, the overall production rate 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 occurs, the overall production rate of free lactic acid (lactate) is calculated over the time required to produce 90% of the limiting concentration of free lactic acid (lactate). The productivity of the present process can also be expressed in terms of the overall production rate of lactate. The present fermentation process is generally carried out under conditions that produce iactate at a global rate of at least about 1.0 g / l / h, preferably at least about 2.0 g / l / h and more preferably, at least about 3.0 g / l / h. 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. Suitable examples of homolactic bacteria for use in the present fermentation method can be easily isolated from examples of maize infusion water, such as are found in commercial corn milling facilities. In addition, certain different homolactic bacteria isolated from different sources may also have the necessary capabilities to allow efficient production at low pH of high levels of free lactic acid.

Because the homolactic bacteria found in maize infusion water usually require a nutrient medium that includes maize infusion water for growth, the initial step in a process to identify and isolate such bacteria usually involves plating samples in a medium containing infusion water, such as 10% vol CSL-M RS agar, and then incubate the inoculated medium anaerobically at about 45-50 ° C. Bacterial isolates can be easily probed by heterolactic production by passing the isolate in a biphasic medium, which only contains infusion water in the minor phase. The growing strains are then monitored by the generation of gas at the bottom of the biphasic tubes. The isolated strains can be conveniently stored at low temperature (e.g., 4 ° C or lower) or maintained as a stock storage in a growth medium of infusion water / tomato juice / MRS agar. When desired, one or more acid tolerant strains, isolated in this manner from maize infusion water, can be used as an inoculum in a lactic acid fermentation. Using this type of methodology, samples of infusion water obtained from five different corn grinding facilities in the United States were examined, as well as three corn milling facilities located in Turkey, England and the Netherlands, by lactate-producing microorganisms. . The isolated microorganisms were characterized initially as heteroláctícos (ie capable of producing other fermentation products besides lactate) or homolactic producers. The homolactic strains were further characterized, inter alia, based on the production of global lactate, optical activity of the lactate produced and, in many cases, pH of final incubation in the absence of base (CaCO3) added to the fermentation medium. A total of 1 55 bacterial strains were isolated. Of the 1 09 strains that were characterized, 98 strains (90%) produced lactate as the only fermentation product ("homolactic" strains). As used herein, the term "homolactic" refers to a bacterial strain that substantially produces only lactic acid as the fermentation product. The remaining 1 1 strains (11%) produced other fermentation products in addition to lactate ("heterocyclectic" strains). Of the 98 homolactic strains, 22 were producing L-lactate, 1 8 were producing D-lactate and 58 produced racemic lactate. The present homolactic bacteria are generally capable of producing at least about 25 g / l of free lactic acid. Most preferably, the bacteria are homolactic bacteria capable of producing at least about 30 g / L of free L-lactic acid. In another embodiment of the invention, the homolactic bacterium is capable of generating a solution containing at least about 40 g / l, preferably at least about 75 g / l of lactate and preferably at least about 90 g / l of lactate at a pH of average incubation 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. The present acid-tolerant, homolactic bacterium is usually capable of growth and lactic acid production at temperatures between about 35 ° C and about 53 ° C. The optimum temperature for growth generally ranges from about 43 ° C to about 52 ° C and, preferably, about 47 ° C to about 50 ° C, although it has been shown that homolactic bacteria can grow at temperatures at or near room temperature. Normally negligible lactate production occurs by the bacteria when the temperature is above about 53 ° C or below about 30 ° C. The fermentation process is preferably conducted at about 47 ° C to about 52 ° C, because yeasts and laterobacilli are less thermotolerant and generally will not grow well, if at all, at these temperatures. In this way, in addition to intensifying lactate production, the fermentation of acid-tolerant homoloctic bacteria at high temperature can reduce the possibility of problems associated with contamination by other organisms. The present homolactic bacterium is normally capable of growth and lactate production at least within a pH range of about 3.7 to about 6.5 and preferably at least through a pH range of about 3.8 to about 5.0. Although the bacterium may be capable of producing lactate at a pH close to neutral (eg, 6.0-6.5), the bacteria employed in the present process are preferably capable of high levels of lactate at a pH where a substantial portion of the lactate exists. in its free acid form. Preferred forms of acid-tolerant homoloctic bacteria are capable of significant lactate production (eg, at least about 50 g / L) at a pH of 4.2 or less. A variety of reactor configurations can be employed including packed bed reactors, continuous stirred tank reactors, biological contact rotating reactors, sequencing batch reactors and fluidized bed reactors in the present process. The complete reaction can be carried out in a simple container having suitable means for controlling the temperature of the fermentation broth or, alternatively, the fermentation can be carried out in a first container, the broth can be maintained at the desired temperature by passing through a heat exchanger, for example, a plate heat exchanger and recycled to the fermentation reaction. The latter arrangement can provide a more rapid cooling of the reaction mixture and in some cases can be performed at the same time that the broth is passed through a membrane separation module to remove a portion of the broth (for example, where the Heat exchanger and membrane module are connected in series). A commonly used configuration includes a membrane recycling bioreactor. Reactors of this type typically include two modules, a fermentation vessel 10 and a membrane module 15 (see, for example, Figure 1). These two modules can be connected by a pipe or be parts of a simple apparatus. In one embodiment of the invention, homologous tolerant bacteria can be incubated in a first portion of nutrient medium in the fermentation vessel to generate a first product solution, which includes at least about 25 g / l of free L-lactic acid. The resulting fermentation broth can be separated to provide a first fraction, which includes free lactic acid and is substantially free of bacterial cells. This can be done by pumping a portion of the fermentation through a cell separator (e.g., a hollow fiber cell separator). The cell-containing fraction is normally recycled back to the fermentation vessel (see, for example, Figure 1), while the fraction containing lactic acid is separated for further processing. Normally additional nutrient medium is added to maintain the volume of the liquid 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 start-up phase has been completed. When run in this manner, the present fermentation is usually conducted so 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.

The fraction containing lactic acid, which is separated, can be processed using a variety of known methods to separate free lactic acid from other components of the solution. For example, lactic acid can be extracted from the solution using an extractor containing tertiary amine. An example of a suitable extractor is a solution of Alamina 336 in octyl alcohol. Other methods that can be used to isolate lactic acid include contacting the solution with a solid adsorbent, such as an ion exchange resin (e.g., a polyvinylpyridine column), distilling a lactic acid-containing fraction, or removing via membrane separation. Any of these types of separation methods can be used to process the lactic acid-containing fraction to generate a suppressed fraction of lactic acid and an isolated lactate fraction. The deleted fraction of lactic acid may contain some lactate in the form of a lactate salt, such as calcium lactate. The isolated lactate fraction can be further processed using any of a variety of known methods to produce a purer form of free lactic acid. The fraction containing lactic acid can also be processed to separate lactate salt (for example, calcium lactate) in solid form or solution, leaving a solution enriched in free lactic acid. The lactate salt can be separated using a suitable technique, such as extraction, crystallization, membrane separation and adsorption in a solid material (eg, anion exchange resin). The lactate salt can 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 falling below a desired level. For example, by recycling a sufficient amount of calcium lactate as a buffering agent, the pH of the fermentation broth can be maintained at a value close to the pK of lactic acid. Based on the theory, the lactate salt will buffer the 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 the production of 0.7 equivalent amount of new lactic acid production. A variety of methods are available to process lactate / lactic acid solutions, involving the generation of large amounts of lactic acid; for example, in solution at pHs no greater than about 4.8 (preferably not greater than about 4.2 or 4.3) of the fermentation broth; and with a concomitant isolation (and if you wish to recycle) of lactate salt (usually calcium lactate, potassium lactate, sodium lactate and / or ammonium lactate). Such processes are described, for example, in the co-filed, commonly assigned US patent application (for Cargill, I nc of Minnetonka, Minnesota) entitled LACTIC ACI D PROCESSI NG; METHODS; ARRANGEM ENTS; AN D, PRODUCTS (Processing of lactic acid, methods, fixes, and products), identifying John N. Starr, Aharon M. Eyal, Riki Canari, Betty Hazan and Rod Fisher as inventors (hereinafter referred to as Starr et al.'s application). Starr et al's application was filed on the same date as the present application (October 14, 1997). Advantageous global processes will depend, in part, on the selection, among the approximations, of the one that most easily provides an efficient and cost-effective overall processing scheme in large-scale implementation. The main concerns to select global processes refer to the design of the system to meet the two objectives of: 1. Isolation of lactic acid products for consecutive processing, for example, to generate polymer; and 2. Isolation of lactate salt, preferably in a desirable form for recycling to the fermentation broth. Three general approaches interest: 1. Separation of lactic acid from the solution leaving the lactate salt behind; and if it is desa, the direction of the residual solution having the lactate salt therein, after separation, towards a fermentor; 2. Isolation of the lactate salt from the solution; direction of the lactate salt, if desired, to a fermentor; and a consecutive isolation of the lactic acid product from the residual solution after separation of the lactate salt; and, 3. Simultaneous separation of lactic acid in one stream and lactate salt in another, leaving residual mixture. The techniques described in Starr et al. to achieve one or both of these objectives can be practiced in a variety of solutions of lactate material (ie, solutions of lactic acid and dissolved lactate salt). These solutions may comprise fermentation broth or broth that has been removed from a fermentor and has been modified in some way, for example, by filtration or pH adjustment. In reality, techniques can be applied to solutions that have also been done in other ways. However, the techniques and proposals described herein are developed particularly with a focus on the efficient processing of fermentation broth solutions, especially with regard to the acids, in which pH modification is not required by addition of acid and preferably it has not happened. The normal 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, the normal compositions in 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 lactate salt dissolved at 25 ° C, is within a range of about 1,000: 1 to 0.007: 1. The most preferred processing will involve solutions with a pH in the order of about 1.98-5.00 (ratio of H LA: LA within the range of about 75: 1 to 0.070: 1); and, more preferred processing will involve solutions having a pH within the range of about 3.0-4.5 (ratio of H LA: LA within the range of about 7.0: 1 to 0.23: 1). 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 may be used, for example, with pH adjustment by the addition of acid to the given more preferred pH range. In the present, there will sometimes be reference to "preferential separation" of: lactic acid of a composition containing lactic acid and lactate salt; or, lactate salt of a composition containing lactic acid and lactate salt. The term "preferential separation" and variants thereof, in this context, refers to a separation technique that preferentially removes one of the two components (lactic acid or lactate salt) from the other. In a normal preferred processing according to the present invention, a mixture of lactic acid and lactate salt is divided into two "product streams". In a product stream, (ie, the stream rich in free lactic acid), preferably the molar ratio of free lactic acid to lactate salt obtained is at least 271 and preferably at least 3/1. With certain of the techniques described herein, proportions of at least 5/1 and in reality in proportions of 1 0/1 or more are readily obtainable.

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 not greater than about 0.5. With normal preferred processing as described herein, proportions of no more than 0.3, preferably no more than 0.2 and most preferably 0.1 or less are easily obtained. Here, the term "stream" as used in the context indicated by the previous two paragraphs, refers to an isolated phase or product segment, without considering whether that phase or segment of product is a solution, solid or a mixture of materials. In this way, a "lactic acid rich stream" is simply a phase or mixture rich in lactic acid (versus lactate salt) compared to the original processed mixture; and, a "lactate salt rich stream" is a stream rich in lactate salt (versus lactic acid) compared to the original processed mixture. When the product stream enriched in free lactic acid is obtained as a result of the separation of free lactic acid from the mixture, for example, from a fermentation broth, the aqueous mixture remaining after the lactic acid removal will sometimes be referred to as "suppressed" with respect to free lactic acid. Similarly, when the lactate salt enriched stream results from the separation of the lactate salt from a mixture containing the lactic acid and the lactate salt, the remaining mixture will sometimes be referred to as "suppressed" with respect to salt of lactate. Preferably, when the processed solution is a fermentation broth, the product stream enriched in lactate salt is provided and formed such that the weight ratio of fermentor impurities, to lactate salt therein, is less than found in the fermentation broth, preferably by a factor of at least 5. This can be handled by techniques described herein which concern the control over the particular approach selected for the isolation of the lactate salt, as well as through use as various purification techniques, such as, retro washing or recrystallization.

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 in a fermentation system, in order to maintain water balance. If the concentration of an aqueous solution is used in order to facilitate water balance in the broth, preferably, relatively low cost concentration techniques are used, such as reverse osmosis and vapor recompression.

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 in the presene. The variation within the concepts of the invention will be evident.

Example 1 - Standard fermentation conditions Unless indicated otherwise, the fermentation reactions described in the following examples were run using a variety of growth media according to the following standard protocol. Cells (250 ul) were transferred from a stock storage of the particular strain in 40% tomato juice / 40% phase bottom of LSW-MRS agar / biphasic upper phase of M RS (biphasic TJ-SW-MRS) in fresh TJ-SW-MRS biphasic medium and incubated under static conditions for 1 8-24 hours at 47 ° C.

Medium MRS (pH = 6.2) 1 0 g / l of gelatin pancreatic digestion 8 g / l of beef extract 4 g / l of yeast extract 20 g / l of glucose 2 g / l of K2HP04 1 g / l of Tween® 80 5 g / l of sodium acetate 5 g / l of ammonium citrate 0.2 g / l of MgSO4 0.05 g / l of MnSO4 One-drop incubation in the fresh TJ-SW-MRS biphasic medium was used to inoculate 80 ml of Medium B supplemented with 10% CSL, glucose (60 g / l of total concentration) and calcium carbonate (20 g / l ) in a sealed serum bottle and incubated with shaking for 18 hours at 47 ° C in an environmental stirrer.

Medium B (PH = 4.7) 8-1 2% vol of corn infusion liquor 5 g / l of yeast extract 50-1 00 g / l of glucose 2 g / l of K2HPO4 1 g / l TweenR 80 2 g / l of ammonium citrate 0.2 g / l of MgSO4 0.05 g / l of MnSO4 20-40 g / l of CaCOs Termendores containing Medium B were inoculated with the desired levels of glucose and calcium carbonate (for example, 90 g / l of glucose and 33.4 g / 1 of calcium carbonate) with 10% (v / v) of the culture of 18 hours. The fermentation was run at 47-49 ° C with stirring at 50 rpm and the fermentation jars filled at 70-80%. Running the fermentation jars at this level of fluid volume ensured that the medium did not become highly aerobic.

Example 2 - Isolation of homolactic, acid-tolerant strains, without pH control Homolactic bacterial strains were isolated from maize infusion water samples obtained from eight different industrial corn milling facilities. The facilities were located in Blair, Nebraska; Edyville, Iowa; Cedar Rapids, Iowa; Dayton, Ohio; Mem Phis, Tennessee; Istanbul, Turkey; Tillbury, England; and Bergen Op Zoon, The Netherlands. Strains were isolated by obtaining samples of infusion water from commercial corn grinding facilities. Samples were plated on 1 0% CSL-MRS agar plates (pH 5.0) and incubated anaerobically at 47 ° C. The colonies were scratched again for isolation on plates of 10% CSL-MRS agar. The isolates were then transferred to a biphasic medium of 40% LSW-40% tomato juice-bottom phase MRS / MRS phase (pH 6.0) for maintenance purposes.

The isolated strains were classified by heterolactic production by monitoring the formation of gas (CO2) at the bottom of the tube. The homolactic isolates were then classified in Medium M RS supplemented with 10% > CSL and 30 g / l of glucose per lactate yield and the optical purity of the lactate produced. The results are shown in Table 1 below. The isolated bacterial strains were identified either as producers of homolactate ("homolactic") or producers of heterolactate ("heterolactic"). Based on the fermentation in MRS medium supplemented with 1.0% vol of maize infusion liquor ("CSL"), the isolated homoloctic bacterial strains were characterized in terms of global lactate production, final fermentation pH and% L - lactate produced (see Table 1 below). Since approximately 50% of the lactate in the added maize infusion liquor ("CSL") is normally D-lactate, strains that produced at least about 70% L-lactate were considered as producing L-lactate strains. This assumption was confirmed by subsequent experiments under conditions where the levels of D-lactate contamination in the product arising from infusion water present in the nutrient medium were lower (e.g., higher lactate production levels or by using infusion water from corn has more than 80% L-lactate (as a fraction of total lactate)). The fermentations were carried out at 48 ° C under the standard conditions described in Example 1. The results are shown in Table 1 below.

Example 3 - Isolation of homolactic strains. acid tolerant, using added base An additional set of homolactic strains was isolated from maize infusion water samples obtained from corn grinding 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 the fermentations carried out in Medium B supplemented with 10% vol of CSL, 90 g / l of glucose and 33 g / l. l of CaCO3. The global lactate production and / or percentage of L-lactate produced were measured for this set of strains. The results are shown in Table 2 below.

Table 2 Ce homolactic isolates Cepa no. g / l of lactate% L-lactate 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 68.72 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 - Added base effect on lactate production A variety of the strains described in Example 2 that had been identified as producing L-lactate was classified to examine the effect of added base (CaCO3) on lactate production . The fermentations were carried out at 48 ° C in MRS medium supplemented with 1 0% CSL and 30 g / l glucose. For the determinations made in the presence of added base, M RS medium supplemented with 10% CSL, 30 g / l glucose and 20 g / l CaCO3 were used.

Table 3 Effect of CaCO3 on lactate production Cepa Lactate production (g / l) Without base 20 g / l of CaCO3 6 21 42 1 0 20 32 14 23 37 1 9 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 - Production of L-lactate The level of production of L-lactate was characterized by a variety of strains described in Example 2. The fermentations were carried out at 48 ° C in MRS medium supplemented with 10% CSL, 30 g / l glucose and 20 g / l CaCO3.

Table 4 Production of L-lactate Strain #% L-lactate Lactate produced (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 strains of lactobacilli deposited in ATCC The lactate productivity of a number of known strains of lactobacilli isolated from sources other than maize infusion water was examined. Samples were obtained from eleven different strains of American Tissue Culture Collection (Rockville, Maryland) and were classified by total lactate production and pH of final incubation based on fermentation at 37 ° C in MRS medium supplemented with 75 g / l of glucose and 30 g / l calcium carbonate. The results are shown below in Table 5. All strains exhibited poor growth at 47 ° C and were inhibited by the presence of corn infusion water in the nutrient medium. While the nutrient requirements of the ATCC deposited strains are different from strains isolated from maiz infusion water, several strains deposited in ATCC appear to be capable of producing relatively high concentrations of free lactic acid. In particular, Lactobacillus helviticus (ATCC # 1 5009, 66 g / l of lactate at a final incubation pH of 4.03), Lactobacillus paracasei tolerans (ATCC # 25599, 66 g / l of lactate at a final incubation pH of 4.04) ), and Lactobacillus salivarius salivarius (ATCC # 1 1 741; 64 g / l of lactate at a final incubation pH of 4.1 2) appear to offer potential as producers of high-yield free lactic acid. The optical purity of the lactate produced by a variety of strains was determined. None of the strains capable of producing a relatively high concentration of free lactic acid was an L-lactate producing strain.

Table 5 Lactate production by strains of lactobacillus ATCC Example 7 - Tolerance to SO? of homoloctic strain # 41 The effect of varying levels of sulfur dioxide (SO2) on the lactate productivity of homoloctic 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 of CSL, 30 g / l of glucose and 20 g / l of CaCO3, via the standard fermentation protocol described in Example 1. The results shown in Table 6 below demonstrate that strain # 41 is capable of producing lactate in the presence of SO2 concentrations of up to at least about 600 ppm.

In a similar fermentation performed in the presence of 800 ppm, strain # 41 started to produce lactate after a latent phase of 1 44 hours.

Table 6 Tolerance to SO2 of homoloctic strain # 41 Conc. SO? Production of lactate (g / l) 24 h 48 h 72 h 200 ppm 11 48 66 400 ppm 9 27 55 600 ppm 9 11 43 Example 8 - Effect of temperature on lactate production The lactate productivity of homoloctic strain # 41 was determined over a temperature range between 41 ° C and 54 ° C. The fermentations were carried out in Medium B supplemented with 10% vol of CS L, 60 g / l of glucose and 20 g / l of calcium carbonate. The results shown in Table 7 below establish that the optimum temperature range for lactate production by strain # 41 is from 44 ° C to 54 ° C.

Table 7 Temperature dependence of lactate production Lactate production (g / l) Temp ('> C ± 24 h 48 h 72 h 41 14 51 68 44 25 55 68 47 26 50 63 50 31 52 57 54 9 19 2. 3 Example 9 - Effect of infusion water concentration on lactate production Fermentations were conducted employing a variety of the L-lactate-producing strains described in Example 2, to examine the effect of varying amounts of corn infusion liquor in the growth medium on lactate production. The fermentations were conducted at 48 ° C in Medium A (see below) supplemented with 50 g / l of glucose, 20 g / l of CaCO3, and either 1%, 5% or 10% of CS L.

Medium A (pH = 5.0) 1 0 g / l of yeast extract 0.2% of K2HPO4 1 g / l of TweenR 80 0.2% of ammonium citrate 0.005% of MnSO4 4H2O 0.02% MgSO4 7H2O source of carbon / energy added source of added nitrogen CaCO3 added to modulate the pH Table 8 Effect of infusion water on lactate production Strain # Lactate production (g / l) 1% CSL 5% CSL 10% CSL 1 0 1 9 31 23 1 0 32 24 6 22 41 9 33 45 5 35 Example 10 - Characterization of homogotactic strains based on ribotyping A variety of the homoloctic bacterial strains producing L-lactate isolated from maize infusion water were categorized based on the analysis of riboimprinting pattern (see, for example, Jaquet et al. al., Zbl. Bakt. 276, 356-365 (1992)). This technique is based on the digestion of DNA from a simple colony of the strain in question using a restriction enzyme EciRI 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 the genetic relationships between organisms and has been used to provide identification between four genera of bacteria (Salmonella, Listeria, Staphylococcus and E. coli), as well as for the taxonomic identification of Gram positive and Gram negative closely related. The ribotyping results of seven of the lactate-producing strains isolated from maize infusion water are shown in Figure 2. It is possible to identify strains with the same RiboGroup designation given at the same taxon level as identical. The ribotypes exhibited by the seven strains shown in Figure 2 did not match the patterns of any of the 30 different bacterial strains of lactic acid in a commercial laboratory compost database. Among the strains in the database that did not provide an equalization were Lactobacillus acidophilus, Lactobacillus animalis, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus amylovorus and Lactobacillus salivarius. The ribotypes of the strains listed in Figure 2 also did not provide an equalization with the patterns of 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 figure 2 also did not provide an equalization 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.

The ribotype patterns of the seven strains shown in Figure 2 fall into three RiboGroups. Two strains (# 1 14 and # 1 19) have identical ribotypes. One of these strains is a heterolactic strain (# 1 1 9), while the other is a homolactic strain, which produces racemic lactate (# 1 14). The D-lactate producing strain (# 79) exhibited a ribotype pattern, which was different from the other six. The remaining four strains (# 90, 127, 1 32 and 140) were classified in the same RiboGroup and it was considered that they were likely to be identified at the same taxon level, despite the fact that their standard patterns were not identical. Of the four strains with an MI L 4-1 1 32 pattern, three were L-lactate-producing strains (# 90, 132 and 140), while the fourth (# 127) produced racemic lactate.

Example 1 1 - Effect of added base on lactate production The effect of adding varying amounts of CaCO3 on the lactate productivity of homoloctic strain # 41 was examined. The experiments were performed at 47 ° C in Medium A supplemented with 8 vol% CSL, 200 g / l glucose and varialbs quantities of calcium carbonate added (30-90 g / l). The results are shown in Table 9 below.

Table 9 Effect of CaCO3 on lactate production Lactate production (g / l) Conc. CaCO3 0 h 24 h 51 h 1 20 h Final pH g / l 3. 1 7 48. 1 75.5 75.7 3.98 40 g / l 6. 1 2 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 1 2% CSL, 90 g / l of lucy g and 33.4 g / l of CaCO3 Figure 3 shows the pH profile 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 Figure 3 is based on the results obtained from incubation at 47 ° C of strain # 41 in Medium B supplemented with 10% vol of CS L, 1 00 g / l of glucose and 33.4 g / l of calcium carbonate.

Eiem plo 1 3 - Fermentation profile of strain # 41 with 90 g / l of glucose. 33.4 g / l of CaCO3 and 1 2% of CSL / 36% of LSW Figure 4 shows a 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 Figure 4 is based on the results obtained from the incubation of strain # 41 at 47 ° C in Medium C supplemented with 90 g / l of glucose, 33.4 g / l of calcium carbonate and either 1 2 % vol of CSL (36% by weight of dry solids) or 36% or vol of LSW (12% by weight of dry solids). The results summarized in Table 10 below show final free lactic acid levels of approximately 40 g / l free with any source of maize infusion water. Because lactate was produced with an L-lactate producing strain (# 41), at least about 35 g / i of free L-lactic acid was present at the conclusion of these fermentations (the rest is free D-lactate present in the added infusion water).

Table 10 Production of lactate with strain # 41 Source of water of corn infusion 12% CSL 36% LSW Lactate (g / l) 0 h 10.3 8.4 16 h 44.0 52.4 24 h 80.5 92.2 44 h 91 .5 96.8 Final pH 3.92 3.98 Final free lactate (g / l) 42 41 Example 14 - Production of lactate from strain # 41 with 8-12% CSL, 90 g / l glucose and 36.6 g / l CaCO3 Figure 5 shows the production of lactate as a function of time during the course of representative fermentation experiments with strain # 41. The fermentations were performed using a modified version of the process described in Example 1. The cells of strain # 41 were grown before in 800 ml of medium and then separated from the medium. The pre-grown cells were then suspended in 800 μl of fresh medium. The profile shown in Figure 5 is based on the results obtained from incubation of the pre-grown cells at 47 ° C in Medium B supplemented with 90 g / l of glucose, 36.6 g / l of calcium carbonate and either % vol of CSL (36% by weight of dry solids), 12% vol of CSL, 24% vol of LSW (12% by weight dry solids) or 36% voi of LSW.

Table 1 1 Lactate production with strain # 41 Source of final pH infusate Free lactate lactic acid corn 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 The effects of varying the amounts of an added carbohydrate source (glucose) on the production of lactate for the strain were examined homoláctica # 41. Fermentations were run by incubating strain # 41 at 48 ° C in Medium A supplemented with 10% vol of CSL, 20 g / L of CaCO3 and the indicated level of glucose using the standard fermentation procedure described in Example 1 . The medium also contained 1 -1 5 g / l of additional fermentable sugar (mainly glucose and fructose) from the corn infusion liquor. The results are shown in Table 1 2 below. The results of this experiment suggest that at least for the added base level (20 g / L of CaCO3), the lactate productivity can be enhanced by the addition of at least about 50 g / L of a carbohydrate source, such as , glucose.

Table X1 Effect of glucose on lactate production Glucose Production of lactate (g / l) added 24 h 48 h 72 h 30 g / l 14 39 42 50 g / l 1 1 51 55 80 g / l 1 1 50 67 1 00 g / l 9 47 65 The invention has been described with reference to several specific and preferred embodiments and techniques. However, the invention should not be construed as limited to the specific embodiments described in the specification. It should be understood that many variations and modifications can be made, as long as they remain within the spirit and scope of the invention.

TABLE 1 Isolated homolactic strains Table 1 (cont.) Isolated homolactic strains

Claims (9)

1. REVIVALATION IS 1 . A process for producing lactic acid comprising: providing a nutrient medium; inoculate the nutrient medium with lactate producing, acid-tolerant microorganisms; and incubating the acid-tolerant lactate-producing microorganisms in the nutrient medium to generate a solution including at least 50 g / L of lactate at a final incubation pH of no more than 4.3, wherein the lactate has an optical purity of at least 50%.
2. 2. The process of claim 1 comprising incubating acid-tolerant homolactic bacteria in the nutrient medium, to generate a solution at a final incubation pH of no more than 4.3, including at least 50 g / L of L-lactate or at least 50 g / l of D-lactate.
3. 3. The process of claim 2, wherein the nutrient medium includes at least 1.5 g / l dry solids of corn infusion water.
4. The process of claim 2, wherein the nutrient medium comprises at least 50 g / L carbohydrate, which includes glucose, fructose, galactose, melibiose, sucrose, raffinose, stachyose or a mixture thereof.
5. 5. The process of claim 2, which comprises incubating the bacteria at 35 ° C to 53 ° C.
6. The process of claim 2, comprising incubating the bacterium in nutrient medium at an average incubation pH of no more than 4.3 to generate a solution that includes at least 75 g / L of L-lactate.
7. 7. The process of claim 1, wherein the nutrient medium comprises base. The process of claim 7, wherein the base comprises calcium carbonate, sodium hydroxide, ammonium hydroxide, sodium bicarbonate or a mixture thereof. The process of claim 1, wherein the nutrient medium comprises lactate salt. The process of claim 9, wherein the lactate salt comprises calcium lactate, sodium lactate, ammonium lactate or a mixture thereof. eleven . The process of claim 1, which comprises incubating the bacterium in the nutrient medium to produce L-lactate having an optical purity of at least 80%. The process of claim 1, which comprises incubating the bacterium in the nutrient medium to produce lactate at a global rate of at least 2.0 g / l / h. The process of claim 1, which comprises incubating the acid-tolerant homolactic bacterium in the nutrient medium to generate a solution at a final incubation pH of no more than 4.0, including at least 50 g / l of L -lactate or at least 50 g / l of D-lactate. The process of claim 1, which comprises incubating the bacterium in nutrient medium having an average incubation pH of no more than 4.2. 1 5. Acid-tolerant homoloctic bacteria isolated from maize infusion water capable of generating a solution including at least 50 g / l of lactate at a final incubation pH of no more than 4.3, wherein the lactate has a purity optics of at least 50%. The homolactic bacteria of claim 1, wherein said bacteria are capable of being incubated in nutrient medium having an average incubation pH of no more than 4.3, to generate a solution that includes at least 50 g / l of lactate having an optical purity of at least 50%. 7. The homolactic bacteria of claim 15, wherein the bacteria include gram-positive, gram-positive bacteria, indifferent to air. The homolactic bacteria of claim 1, wherein said bacteria are capable of producing lactate having an optical purity of at least 80%. 9. The homolactic bacteria of claim 1, wherein said bacteria are capable of producing at least 80 g / L of L-lactate at an incubation temperature of at least 47 ° C and an average incubation pH of no more than of 4.2. The homolactic bacteria of claim 1, wherein said bacteria are capable of producing at least 50 g / l of the lactate when incubated in nutrient medium, which includes at least 1 5 g / l of dry solids of water of infusion of corn. twenty-one . The process of claim 1, further comprising: incubating acid-tolerant lactate-producing microorganisms in a first portion of nutrient medium to produce a first product solution that includes at least 50 g / L of lactate at an incubation pH end of no more than 4.3, where the lactate in the first product solution has an optical purity of at least 50%; separating the first product solution to produce '(i) a first fraction, which includes lactate and is substantially free of microorganisms, and (i) a second fraction which includes the microorganisms; and processing the first fraction to produce a third suppressed fraction of lactic acid and a fourth fraction enriched with lactic acid. 22. The process of claim 21, further comprising forming a mixture that includes (i) at least a portion of the third fraction; (ii) a second portion of the nutrient medium; and (iii) the microorganisms; and incubating the mixture to produce a second product solution including at least 50 g / L of lactate at a final incubation pH of no more than 4.3, wherein the lactate in the second product solution has an optical purity of at least fifty% . 23. The process of claim 21, wherein the third suppressed fraction of lactic acid includes lactate salt. The process of claim 1, further comprising: incubating acid-tolerant lactate-producing microorganisms in a first portion of nutrient medium to produce a product solution that includes at least 50 g / L of lactate at a pH of final incubation of no more than 4.3, wherein the lactate in the product solution has an optical purity of at least 50%; and processing the product solution to produce a suppressed fraction of lactic acid and an enriched fraction of lactic acid.