Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid

Definitions

• the present invention relates generally to lactic acid production. More particularly, it relates to a novel fermentation process in which lactic acid is effectively produced in its undissociated ("free") form.
• lactic acid has long been used in the food industry in the production of confectionary products, soft drinks, beers, wines, dairy products, baby foods, jams, salad dressings, etc. It is also used in the preparation of pharmaceuticals, cosmetics, agrichemicals and many other products. Recently, there has also been substantial academic and commercial interest in lactic acid as a potential raw material for producing biodegradable plastics. See, for instance, Lipinsky, E.S., and Sinclair, R.G., Chem. En ⁇ . Prog., August, 2j6, (1986).
• lactic acid is produced via both synthetic and fermentation processes.
• the synthetic process converts lactonitrile to lactic acid, with the lactonitrile starting material being available as a byproduct in acrylonitrile production. Van Ness, J.H. , "Hydroxy Carboxylic Acids," in Encyclopedia of Chemical Technology. 3rd Ed., Wiley, Volume 13, pp. 80-103 (1981).
• bacteria or other microorganisms produce lactic acid as they metabolize carbon-containing (e.g. carbohydrate) raw materials.
• carbon-containing e.g. carbohydrate
• the lactic acid is immediately neutralized by an alkali such as NaOH, NH.OH or more commonly CaCO Intel thereby forming lactate salt and preventing pH drop and lactic acid buildup.
• an alkali such as NaOH, NH.OH or more commonly CaCO Intel thereby forming lactate salt and preventing pH drop and lactic acid buildup.
• the broth is acidified, to convert the lactate salt to free lactic acid which is then separated from the broth.
• this separation and purification is cumbersome and inefficient. Atkinson, B. and Mavituna, F., Biochemical Engineering and Biotechnology Handbook, the Nature Press, N.Y. (1983).
• lactate itself inhibits lactic acid producing organisms, albeit to a lesser extent than lactic acid and low pH.
• one preferred embodiment of the invention relates to an improvement in a fermentation process for producing lactic acid.
• the improved process includes the step of forming a fermentation broth containing free lactic acid.
• This lactic acid-rich broth is contacted with an effective amount of solid-phase polymer containing tertiary a ine groups to adsorb and increase rate of production of the free acid.
• lactic acid has been produced at surprisingly high rates, typically expressed in grams per liter of working fermentation volume per hour ("g/L/hr").
• lactic acid is produced in undissociated or "free” form, as contrasted to other processes in which lactate salt (e.g. sodium or calcium lactate) is produced.
• lactate salt e.g. sodium or calcium lactate
• Figure 1 is a diagrammatic representation of a fermentation apparatus used for preferred extractive fermentations of the invention.
• Figure 2 is a graph of fermentation and effluent broth pH's versus time (hrs) for a preferred extractive fermentation.
• Figure 3 is a graph of cell density, glucose concentration and total lactic acid concentration (free lactic acid + lactate) versus time for a preferred extractive fermentation.
• Figure 4 is a graph of cell density, glucose concentration, free lactic acid concentration, total lactic acid concentration and fermentation pH x 10 versus time for a comparative non-extractive fermentation.
• FIG. 5 is a diagram of the Rotating Biological Contactor (RBC) Apparatus used in Examples 7.
• one preferred embodiment of the invention relates to a fermentation process for producing lactic acid.
• a carbon source is fermented to produce lactic acid.
• fermentations are conducted using bacteria, fungi or other microorganisms capable of forming lactic acid upon metabolizing a carbon source such as a carbohydrate.
• bacteria of the Family Lactobacillaceae are employed.
• Fungi those of the Family Rhizopus can be employed, for example. It is well within the purview of one ordinarily skilled in this art to select and use a suitable lactic acid-producing bacterium or other organism from among the many known and available for this purpose.
• Lactobacillus delbrueckii NRRL-B-445, and Rhizopus oryzae NRRL 395 obtained from the United States Department of Agriculture, Peoria, Illinois, have been used.
• the fermentation is conducted at a temperature suitable for the particular organism being used, typically between about 30° and 60°C for bacterial fermentations.
• the applicants' preferred fermentations using L. delbrueckii have been conducted at about 42°C.
• the fermentation temperatures may vary widely, but are often within the range of about 25°C to about 50°C.
• the carbon source for the fermentation can be conventional. These include, for instance, carbohydrate-containing raw materials such as molasses, etc. Suitable solutions containing sugars such as glucose and sucrose can also be prepared and used without departing from the scope of the invention herein.
• raw materials such as barley, cassava, corn, oats and rice may be used as a carbon source.
• K. Buchta "Lactic Acid", Biotechnology. H. Dellweg (Ed.) (1985), pp. 410-17.
• lactic acid which is formed is quickly neutralized to lactate salt to remove highly inhibitory lactic acid and maintain the fermentation pH at a level optimal for growth of the lactic acid-producing organism.
• This has been accomplished using basic substances, for instance sodium or ammonium hydroxide or calcium carbonate.
• the applicants' preferred processes are conducted so as to form a fermentation broth that is rich in free lactic acid. This free lactic acid is then directly recovered as product as will be more particularly described below.
• the pH of the fermentation is initially controlled by the addition of a base such as sodium hydroxide.
• a base such as sodium hydroxide.
• the pH is preferably kept at or near the optimum growth pH for the fermentive microorganism employed.
• the pH was initially kept at about 5.5 to 6.0 in order to provide optimum growth conditions for L. delbrueckii and achieve a high cell density.
• an amount of lactate salt was formed which acted beneficially as a buffer during later stages of the fermentation. Once a desired cell density had been achieved and buffering lactate salts were formed, the pH control of the fermentation was released.
• the fermentation pH then began to fall, and after it reached a level at which lactic acid production substantially decreased, the broth was contacted with the solid-phase polymer having tertiary amine groups to adsorb free lactic acid.
• the fermentive production of free lactic acid was coupled to its removal i_n situ by selective adsorption onto the the pyridine polymer.
• This adsorption of free lactic acid decreases feedback inhibition in the system by the free acid and by pH's which would attend its buildup.
• an increased rate of lactic acid production accompanied the decrease in inhibition.
• This increased productivity can be observed for example by comparing the relative productivities (e.g. in g/L/hr) of analogous extractive and non-extractive fermentations.
• fermentive production of lactic acid employing Rhizopus is preferably conducted under non-growth conditions. That is, conditions of the fermentation are controlled so as to inhibit the growth of the fungus. This may be achieved, for instance, by using a fermentation medium that inhibits fungal growth. Such inhibition can be accomplished, for example, with a fermentation medium lacking a nitrogen source necessary for fungal growth, although other similar means may also be used.
• the fungus When non-growth conditions are to be used during the major lactic acid production, the fungus is initially grown in the desired amount for the fermentation. During this growth period, of course, a medium fully supportive of growth is employed. Once the desired fungal growth has been achieved, the growth medium is removed and replaced with the growth-inhibiting fermentive medium. The fermentation is then conducted over a period of time, and, optionally, the growth-inhibiting medium can be temporarily replaced from time to time with a growth-supporting medium to rejuvinate the fungus.
• the tertiary amine functions of the adsorbent polymer can be provided by N-heterocyclic or by N-aliphatic groups, preferably in their free base form.
• AMBERLYST® A-21 resin available from Rohm and Haas, Philadelphia, Pennsylvania, can be used in the invention. This A-21 resin contains aliphatic tertiary amine functions.
• A-21 resin contains aliphatic tertiary amine functions.
• the tertiary amine functions are pyridine functions, for example as occur in polyvinylpyridine polymers.
• These polyvinylpyridine polymers have provided particular advantage in work to date, especially such polymers crosslinked with a suitable agent therefor, e.g. with divinylbenzene, and being either gel or macroreticular form resins.
• crosslinking of at least about 2% has been preferred from work to date, although it can be greater for instance up to about 50% or more. A more preferred range, however, is about 2% to about 25% crosslinking.
• REILLEXTM 402 polymer has shown to be preferred, being a 2% cross-linked copolymer of 4-vinylpyridine and a commercially available divinylbenzene.
• REILLEXTM 402 polymer exhibits a convenient granular form having good thermal stability.
• Other preferred polymers have to date included, for example, a second cross-linked poly(4-vinylpyridine) copolymer commercially available under the REILLEXTM 425 trademark.
• This latter material is 25% crosslinked with divinylbenzene, and exhibits a convenient, highly porous macroreticular bead form also having good thermal stability.
• REILLEXTM polymers reference can be made to relevant literature available either through the industry or from the manufacturer itself. One such reference is a brochure published by Reilly Industries, Inc. entitled REILLEXTM: A New Family of Cross-linked Polyvinylpyridines from Reilly (REILLEXTM Report 2. 1986). It will be understood that in addition to these several REILLEXTM polymers, other solid polymers which contain pyridine groups to selectively adsorb the acid are also suitable for use in the applicants' invention.
• crosslinking is a desirable range and, as illustrated by Examples 4 and 5 below, an about 8% crosslinked gel-form poly 4-vinylpyridine resin has provided very high adsorptive capacities, and is thus particularly preferred thus far from this standpoint.
• the broth is separated from the fermenting medium and is filtered to remove cells (e.g. any fermentive microorganisms present) prior to contact with the polymer.
• the cells can then be returned to the fermentor, and the filtered broth passed through a column containing the polymer. It is preferred that such a column be kept at a relatively low temperature, e.g. between about 0°C and about 30°C, more preferably about 15°C to about 25°C, in order to keep the adsorptive capacity of the polymer relatively high.
• the free lactic acid is selectively removed from the broth as it passes through the column (i.e.
• the polymer substantially adsorbs free lactic acid as compared to lactate salt) , and the eluent broth is returned to the fermentor.
• the column has become relatively saturated with lactic acid, as can be determined for example by monitoring the influent and effluent pH of the broth, it can be replaced with another column containing fresh or regenerated polymer. This cycle can be repeated to provide a process which is highly productive and efficient for lactic acid. For example, in preferred processes lactic acid productivities ranging from about 1 up to about 4 g/L/hr have been achieved to date.
• a plurality of i.e.
• the pH and lactic acid content of the fermenting medium can be maintained at acceptable levels for growth-related lactic acid production (e.g. pH above about 4 for the lactic acid bacteria) without addition of further neutralizing agents.
• these fermentations have resulted in high cell densities and provided efficient conversion of the carbon source to the desired lactic acid product (e.g. about 60% or more of the carbon substrate converted to free lactic acid) .
• lactic acid can be recovered using a suitable desorbing agent.
• suitable desorbing agents will include for example polar organic solvents such as alcohols (e.g. methanol) as well as hot water.
• the acid can be isolated and worked up in a conventional manner. For instance, lactic acid can be concentrated by evaporation, distillation, or any other suitable means known in the art.
• FIG. 1 The fermentation apparatus used for extractive fermentation processes described in Examples which follow is shown diagrammatically in Figure 1.
• a fermentor having a capacity of 5 L working volume was equipped with a pH meter and a nitrogen sparge.
• the fermentor was connected to an acrylic PELLICON Cassette System (Model XX42 ASY60) available from Millipore Corporation of Bedford, Massachusetts. Cell filtration was achieved with a PELLICON
• Cassette membrane (Millipore HVLP000C5) having a pore size of 0.45 ⁇ m and a filtration area of 0.46 m 2. This membrane is stable at pH's as low as 2.
• XX8000000 variable speed pump circulated the fermentation broth from the fermentor and provided tangential flow across the membrane.
• the retentate from the cell filtration system was pumped back into the fermentor.
• the filtrate was circulated into one of two resin columns equipped with water jackets for cooling. Prior to use, each column was sterilized with methanol, and the methanol then eluted with sterile water. Each column measured 2.5 cm in diameter and was 60 cm long, and each was packed with 65 grams of ReillexTM402 resin.
• the columns exited into a single line which circulated back into the fermentor. This line was equipped with in-line pH probe, a flow meter and a sampling port.
• Example 1 In the extractive fermentation apparatus described in Example 1, an extractive fermentation was conducted using Lactobacillus delbrueckii NRRL-B-445 obtained from the United States Department of Agriculture, Peoria, Illinois. The organism was maintained at 4°C on agar slants.
• the fermentation medium used was as follows, with anhydrous glucose (SIGMA) serving as a carbohydrate source and yeast extract (DIFCO) providing a nitrogen source:
• SIGMA anhydrous glucose
• DIFCO yeast extract
• the initial working volume was 1.1 L, and fermentation was carried out with nitrogen sparging and at a temperature of 42°C. Initially, the fermentation was allowed to proceed without circulating the broth out of the fermentor. During this initial stage, the fermentation and buildup of lactic acid was allowed to proceed for about 5 hours at which point the broth pH had reached about 5.5. Thereafter, the pH was maintained at 5.5 for approximately 6 hours by the automatic addition of 4.16 N NaOH as necessary. 72 mL of NaOH were added over this 6 hour period, corresponding to the formation of 24.8 g/L of sodium lactate in the broth which acted effectively as a buffer in the system. A high cell density was also achieved. Following this initial buildup, the pH control was released by inactivating the NaOH pump.
• the pH of the broth dropped to about 4.3 and then the drop began to tail off.
• the cell filtration system was activated, recycling the retentate back into the fermentor, and feeding the cell-free broth into one of the two resin columns (the other was shut off) at a rate of about 220 mL/hr.
• the pH of the broth in the fermentor increased. This indicated that the resin was removing lactic acid faster than it was being formed in the fermentation broth.
• the resin became loaded with lactic acid its absorptive capacity decreased, which was evidenced by a renewed downtrend in the pH beginning about 7 hours after the first resin column was brought online.
• Lactic acid was eluted from the resin columns by a slow feed of methanol.
• entrapped glucose, lactate salt and lactic acid were first eluted before the eluted volume reached 115 mL. After that volume, lactic acid was eluted, reaching a maximum concentration of 48.4 g/L and totalling 11.4 g.
• glucose was not eluted because the column was removed after glucose was completely consumed in the fermentation. The concentration of lactic acid for this column reached 64 g/L and totalled 13.3 g.
• Figure 2 is a graphical representation of the respective pH's of the fermenting medium and column effluent broth versus time (hrs) .
• Figure 3 is a graphical representation of the cell density and the glucose and total lactic acid concentration (free acid + lactate) in the fermentation medium as a function of time (hrs).
• Two additional extractive fermentations were conducted analogous to that described above. In these additional runs, the amount of polymer vs. fermentation working volume was increased (about 242 g resin/L and 333 g resin/L, respectively), and productivities of 2.1 g/L/hr and 3.8 g/L/hr, respectively, were obtained. Excellent results are also obtained where the REILLEXTM 402 polymer is replaced with REILLEXTM 425 polymer.
• An aqueous solution was prepared by heating 300 mL of water to 40°C, adding 0.6 g of polyvinylalcohol (Airvol 205), and heating the slurry until it became clear.
• An organic phase was prepared containing 5 g of high purity divinylbenzene, 46 g of 4-vinylpyridine, and 0.5 g of Vazo 52.
• the aqueous solution was cooled to room temperature, added to a round-bottom flask, and stirred at a moderate rate under a nitrogen atmosphere.
• the organic phase was added to the stirred solution and the reaction mixture heated to about 50-55°C with continued stirring for 16 hours. After the reaction mixture was cooled, the gel beads were filtered, washed with water and then methanol, and dried overnight at room temperature.
• Example 2 is repeated, except that an 8% crosslinked gel poly-4-vinylpyridine resin prepared as in Example 4 is used for the adsorption step.
• the 8% crosslinked material demonstrates superior adsorption capacity as a greater amount of lactic acid is adsorbed on the first column. This can remove the need for replacing the first resin column with a second fresh or regenerated resin column, or at least advantageously decreases the frequency at which this operation must be performed.
• lactic acid can be eluted from the column with methanol.
• Example 2 is again repeated, except using AmberlystTM A-21 resin instead of the polyvinylpyridine resin.
• the lactic acid-loaded columns are rinsed with water, and lactic acid is desorbed with aqueous 5% NH_ to give aqueous solutions of ammonium lactate.
• lactic acid can be removed from the A-21 resin by diplacement on the resin with stronger acid, e.g. 5% aqueous sulfuric acid or hydrochloric acid.
• the stronger acid displaces the lactic acid with high efficacy while itself binding strongly to the resin.
• By monitoring the presence of the stronger acid in the effluent a majority of the lactic acid can be recovered without any substantial contamination with sulfuric or hydrochloric acid.
• the apparatus employed in this example is illustrated in Figure 5.
• a Rotating Biological Contactor (RBC) 1 was coupled to a resin column 2 loaded with Reillex 425 polymer, available from Reilly Industries, Inc., Indianapolis, Indiana.
• the RBC 1 generally comprises a fixed drum housing 3 in which a series of vertical disks 4 are mounted on a rotating horizontal shaft 5. The disks 4 are about half submerged when the RBC 1 is filled to the normal working level.
• the housing 3 has a medium inlet 6 and a medium outlet 7 positioned below the working level of the fermentation medium.
• the housing 3 also has an air inlet 8 and an air outlet 9.
• the rotating horizontal shaft 5 was driven by motor 10 at a speed of twenty-five revolutions per minute. For these experiments, the housing 3 was -ten cm in diameter and -thirty cm in length.
• the polypropylene disks 4 are -nine and one-half cm in diameter. Growth of Fungus
• the growth medium contained glucose (100 g/1), urea (2.0 g/1), MgS0 4 *7H 2 0 (0.25 g/1), KH 2 P0 4 (0.60 g/1) and ZnSO *7H_0 (0.088 g/1). This growth medium was
• the RBC 1 was thereafter operated at 25 rpm
• the non-growth medium comprised the following: glucose (100 g/1); MgS0 4 *7H 2 0 (0.25 g/1), KH 2 P0 4 (0.60 g/1) and eCl 3 (0.05 g/1).
• the RBC 1 was then operated at 25 rpm at room temperature while maintaining an air flow through the RBC of about 2 liters per minute.
• the fermentation medium was circulated from RBC 1 through resin column 2 via medium outlet 7 and medium inlet 6. Circulation of the fermentation medium was at a rate of about 2 liters per hour, and the medium was filtered as it exited medium outlet 7.
• Table 1 The results of this run are set forth in Table 1.
• This extractive fermentation provided the production of substantially L+ lactic acid coupled to its removal from the fermenting medium to remove product inhibition. As can be seen, glucose consumption (and lactic acid production) continued for at least about a day. Additionally, only trace amounts of lactic acid were found in the medium over this period due to adsorption of the lactic acid on the resin column.

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