A PROCESS FOR THE PRODUCTION OF SUCCINIC ACID BY ANAEROBIC FERMENTATION

Abstract: An improved process for the production of succinic acid by fermentation of carbohydrates by anaerobic bacteria. The fermentation is conducted in an aqueous medium containing carbon dioxide. The pH of the medium is maintained between about 5.8 and about 6.8.

Description: This invention relates to an improved method for the production of succinic acid by a fermentation process conducted in the presence of carbon dioxide. Succinic acid and its derivatives are widely used as specialty chemicals for applications in foods, pharmaceuticals, and cosmetics. Furthermore, succinic acid is a valuable 4-carbon intermediate useful for the production of 1,4-butanediol, tetrahydrofuran, and gamma -butyrolactone. Although the succinate ion is a common intermediate in the metabolic pathway of many organisms, there are no examples of any fermentation that produces succinate in large amounts or with high yields. For example, succinate is a key intermediate for anaerobic fermentations by propionate-producing bacteria, but it is only produced in low yields and in low concentrations. Succinate is also produced by anaerobic rumen bacteria. These bacteria include Bacteroides ruminicola (hereafter written B . ruminicola ) whose growth and metabolism is described by Howlett, et al, Applied Environ. Microbiol. , 32 , 274-283 (1976) and Bacteroides amylophilus (hereafter written B . amylophilus ) whose culture and growth are described by Caldwell, et al, J. Bacteriol. , 98 , 668-676 (1969) and by Hamlin, et al, J. Bacteriol. , 72 , 548-554 (1956). Although the rumen bacteria give higher yields of succinate than do the propionate-producing bacteria, the reported fermentations were run in very dilute solutions and gave a variety of products in generally low yields. Moreover, the rumen organisms tend to lyse after a comparatively short fermentation time, thereby, leading to unstable fermentations. In 1961, Anderson and Ordal isolated a facultative anaerobe, Cytophaga succinicans , which produced succinate, acetate, and formate from dextrose with fixation of carbon dioxide, J. Bact. , 81 , 139 (1961). However, this organism produced succinate in such low concentrations that it would not be economically feasible to recover succinic acid from the fermentation medium. Similar results were observed with the Bacteroides fragilis obtained from the gastrointestinal tract, Caspari, et al, Arch Microbiol. , 135 , 16-24 (1983). We have now discovered a method for the anaerobic fermentation of carbohydrates to succinate in high yields and with high productivity. Furthermore, the succinate is produced in sufficiently high concentration in the fermentation medium to permit economical recovery of succinic acid. In accordance with this invention, there is provided an improved fermentation process for the production of succinic acid in high yield by the fermentation of carbohydrates with a succinate-producing bacterium. The improved process comprises conducting the fermentation in an aqueous fermentation medium containing carbohydrates assimilable by the bacterium, nutrients required for growth of the bacterium, and dissolved carbon dioxide, wherein the initial carbohydrate concentration on the medium is between about 20 g/l and about 100 g/l, and the pH of the medium is maintained between about 5.8 and 6.8 during the fermentation. The fermentation process of this invention involves the fermentation of carbohydrates by a succinate-producing bacterium to form succinic acid and lesser amounts of other materials. In general, any strain of bacterium which forms primarily succinate can be employed. A useful strain for the practice of this invention is the strain of Anaerobiospirillum succiniciproducens (hereafter written A . succiniciproducens ) which was originally deposited in the American Type Culture Collection, Rockville, Maryland, as ATCC 29305. It has been redeposited under the provisions of the Budapest Treaty as ATCC 53488, and is freely available. The process of this invention was also followed using strains of the rumen bacteria, B . amylophilus and B . ruminicola . Although the yields of succinate obtained with these microorganisms were lower than those obtained using A . succiniciproducens , it is evident that the process can be used with a number of succinate-producing bacteria. The carbohydrate used in the practice of this invention can be any carbohydrate that is fermented by the strain of bacterium used. For A . succiniciproducens , these carbohydrate sources include dextrose, sucrose, fructose, lactose, soluble starches, and corn syrups. The fermentation is conducted in an aqueous medium containing dissolved carbon dioxide. Nutrients and other growth factors needed for growth and reproduction of the microorganism employed are also added to the medium. The concentration of carbohydrate in the medium is between about 20 g/l and about 100 g/l, preferably between about 40 g/l and about 80 g/l. Carbohydrate concentrations above about 100 g/l give solutions with such high osmotic pressures that the organisms do not grow. Although the organisms will grow in solutions containing less than 20 g carbohydrate per liter, the concentration of product is so low that its recovery is not practical. Carbon dioxide can be supplied to the fermentation medium in various ways. The medium can be sparged with CO₂ gas. The fermentation can be run in a pressurized reactor which contains carbon dioxide at superatmospheric pressure. The CO₂ can be mixed with other gases as long as the gases employed do not interfere with the growth and metabolism of the organism employed. Carbon dioxide can also be supplied to the fermentation medium by the addition of carbonates or bicarbonates which generate this gas under the conditions of the fermentation. In order to obtain good production of succinate, the pH of the medium is maintained in the range of from about 5.8 to about 6.8. At higher pH values, the main product is lactate rather than succinate, while at lower pH values, the fermentation is inhibited. The pH is conveniently maintained by the addition of alkaline carbonates, alkaline earth hydroxides, or mixtures thereof. When calcium hydroxide is used to maintain the pH, the succinate formed is converted to sparingly-soluble calcium succinate which is precipitated from the reaction mixture, thereby, simplifying recovery and further purification of the product. The fermentation process of this invention is carried out at a temperature between about 20°C and about 49°C. Optimum growth of the A . succiniciproducens organism is about 39°C. Since this is a strict anaerobe, fermentations using the organism are carried out under anaerobic conditions in a medium which has been sterilized by heat or other means well known in the fermentation art. The following examples further describe the embodiments of this invention. All parts are by weight and all percentages are by weight unless expressly stated to be otherwise. Products of the fermentation were determined using high-performance liquid chromatography (HPLC). Components were analyzed chromatographically by elution with 0.006 N H₂SO₄ from a cation-exchange resin in the hydrogen form. The eluted components were detected by means of a differential refractometer, plotted on a recorder and quantified using an electronic intergrator. The area under the curve, which represents the concentration of each component, is reported as a percentage of the total area. The general procedure is that given in "Analysis of Carbohydrate Mixtures by Liquid Chromatography", Am. Soc. Brew. Chem. Proc. , 1973, pp. 43-46. The separations were made on a 1-foot HPX-87 column in the hydrogen form, available from Bio-Rad Laboratories, Richmond, California. The carbohydrate was determined by a YSI dextrose analyzer (Yellow Springs Instrument Company, Yellow Springs, Ohio) using standard methods. It was reported as grams per liter of dextrose. EXAMPLE 1 A . succiniciproducens , ATCC 53488 (ATCC 29305), was grown and maintained under strict anaerobic conditions. Anaerobic conditions were established by the use of a cysteine·HCl-Na₂S·9H₂O reducing agent and an anaerobic glove box containing an atmosphere of 5% H₂, 5% CO₂, and 90% N₂. Seed cultures of A . succiniciproducens were grown in medium of the following composition: dextrose - 20 g/l, polypeptone - 10 g/l, yeast extract - 5 g/l, K₂HPO₄ - 3 g/l, NaCl - 1 g/l, (NH₄)₂SO₄ - 1 g/l, MgCl₂·6H₂O - 0.2 g/l, CaCl₂·2H₂O - 0.2 g/l. To 100 ml of heat-sterilized medium in a 125-ml Erlenmeyer flask in an anaerobic glove box was added 1.0 ml of 0.03 M Na₂CO₃ followed by 0.15 ml of 0.18 M H₂SO₄. A 0.5 ml solution of the following reducing agent was added: Cysteine·HCl - 0.25 g/l, Na₂S·9H₂O - 0.25 g/l. After 20 minutes, the reduced flasks were inoculated with frozen stock cultures (-70°C) of A . succiniciproducens . The seed flasks were incubated at 39°C overnight. Seed culture (50 ml) from the seed flask was used to inoculate 1 liter of fermentation medium in a standard 1.4-liter New Brunswick Bioflo, Model C-30, reactor. The fermentation medium had the following composition (g/l): Dextrose 50 Polypeptone 10 Yeast Extract 5 K₂HPO₄ 1.0 NH₄Cl 0.4 CaCl₂·2H₂O 0.2 MgCl₂·6H₂O 0.2 FeSO₄·7H₂O 1 ppm (as Fe) Before inoculation, the medium was autoclaved for 20 minutes, 15 psi at 121°C. The reactor was cooled without agitation and a CO₂ sparge was utilized (10 ml/min) throughout cooling and temperature adjustment to 39°C. Ten milliliters of a sterilized 3 M Na₂CO₃ solution was added followed by 1.5 ml of concentrated H₂SO₄ to adjust pH of the reactor to 6.8. A 5-ml aliquot of a reducing solution (cysteine·HCl-Na₂S·9H₂O) was added and the fermentor was allowed to agitate at 200 rpm for 20 minutes before inoculation. The temperature was controlled at 39°C, the mixture was agitated at 200 rpm, and a 10 ml/min CO₂ sparge was employed throughout the fermentation. The pH of the fermentation was allowed to drop to 6.4 and was maintained at 6.4 by addition of a sterile solution of 2 M Na₂CO₃. Fermentation was rapid with A . succiniciproducens establishing a high cell population within 16 hours. The cells were motile at the beginning (0-24 hours) and then became nonmotile towards the end. The fermentation lasted 38 hours at which time there was <0.5 g/l dextrose remaining in the reactor. A total of 215 ml of 2 M Na₂CO₃ was added for pH control during the course of the fermentation. The fermentation results are shown in Table I. Comparative Test 1 The procedure of Example 1 was followed except that a nitrogen gas sparge was substituted for the CO₂ sparge. The reactor was reduced with a 5-ml aliquot of reducing agent, cysteine·HCl-Na₂S·9H₂O, before inoculation. A 10-ml aliquot of 3 M Na₂CO₃ followed by 1.5 ml concentrated H₂SO₄ was added to the reactor. (Addition of carbonate was necessary to initiate growth.) The pH of the fermentation was controlled at 6.4 with the addition of 4 N NaOH. The cells of A . succiniciproducens became swollen and the fermentation was sluggish. The fermentation was stopped after 40 hours and the products were analyzed. The results given in Table II show that the fermentation run without dissolved carbon dioxide produces very little succinic acid and a number of by-products. EXAMPLE 2 The general procedure of Example 1 was modified as follows. The iron salt was omitted from the medium and 0.5 g/l of NaCl was added. The CO₂ sparge was stopped after inoculation; so the only source of CO₂ in the fermentation was from the 3 M Na₂CO₃ added for pH control. The pH was maintained at pH 6.4 ± 0.1. This fermentation, without a CO₂ sparge, did not initiate as rapidly as the fermentation conducted with a CO₂ sparge. Analyses of products after a 40-hour fermentation are given in Table III. EXAMPLE 3 Three fermentations were run using the general procedure of Example 2 except that the pHs of these fermentations were controlled at 5.9 ± 0.1; 6.8 ± 0.1; and 7.2 ± 0.1, respectively. The results of these experiments are given in Table IV. When the fermentation was run at pH 5.5 ± 0.1, the fermentation was very slow and a low concentration of succinate was produced. These results together with the results of Example 2 indicate that the production of succinate proceeds best in a medium where the pH is maintained between about 5.8 and about 6.8, preferably between about 5.8 and about 6.5. EXAMPLE 4 The general procedure of Example 1 was followed except that the pH was maintained at 6.4 by the addition of a 25% slurry of Ca(OH)₂ in water, and the fermentation temperature was 37°C. The fermentation proceeded more rapidly than did the fermentation of Example 1 where the pH was maintained by addition of Na₂CO₃ solution. At 21 hours from startup, there was a sudden solidification of reactor contents. The solid was separated and the contents of both the solid and solution were analyzed. About 99% of the dextrose had been consumed. The yield of products is given in Table V. These results show that the production of succinic acid occurs rapidly when the fermentation is run in the presence of calcium ion and indicate that the acid can be separated from the fermentation medium as the sparingly-soluble calcium salt. Since calcium succinate monohydrate is less soluble at higher temperatures, more product can be recovered from the fermentation broth by heating it to 80-90°C and recovering the resulting precipitate. EXAMPLE 5 The general procedure of Example 1 was followed using a strain of rumen bacterium, B . amylophilus (available from the Deutsche Sammlung von Microorganismen, Grisebachstr. 8, 34 Gottingen, Federal Republic of Germany, as DSM 1361), using the following medium in which the concentrations are given in grams per liter unless otherwise indicated: K₂HPO₄ 0.9 KH₂PO₄ 0.9 NaCl 1.8 (NH₄)₂SO₄ 1.8 CaCl₂·2H₂O 0.18 MgSO₄·7H₂O 0.4 FeSO₄·7H₂O 2 ppm (as Fe) Maltose·H₂O 50 Resazurin 1 ml Cysteine·HCl·Na₂S·9H₂O 5 ml The addition of maltose was necessary because B . amylophilus does not utilize glucose. The fermentation proceeded very rapidly and was completed within 21 hours. Cells of B . amylophilus began to lyse after 12 hours as indicated by a decline in optical density and cell mass in the fermentation. Analyses of the products given in Table VI indicate that this strain of rumen bacterium produces succinic acid under the conditions of this invention. EXAMPLE 6 The general procedure of Example 1 was followed except that the microorganism used was a strain of the rumen bacterium, B . ruminicola , available from the American Type Culture Collection, Rockville, Maryland, as ATCC 19188. The results given in Table VII show that this strain of rumen bacterium, like that of Example 4, also produces succinic acid by the process of this invention. Succinate yields obtained from these bacteria using the process of this invention are much higher than yields obtained from these bacteria by previously-reported processes. However, succinate yields from rumen bacteria are lower than those obtained with A . succiniciproducens . EXAMPLE 7 A series of fermentations was run in 160-ml serum bottles using 25 ml of a fermentation medium similar to that in Example 1. The concentration of dextrose was 38.2 g/l and the fermentation medium also contained 30 g/l of MgCO₃. The pH of the medium was adjusted to 6.8 and the headspace in the bottles was filled with carbon dioxide at various pressures. At the end of 44 hours, the pressure was measured in the vessels and the contents of the aqueous medium were analyzed. In each flask, the yield of lactate was about 5% and the yield of acetate was about 23%. The yields of succinate at various pressures are given in Table VIII. They demonstrate that the yield of succinate increases as the pressure of the carbon dioxide gas in equilibrium with the fermentation increases above atmospheric pressure. Thus, it is apparent that there has been provided, in accordance with the invention, an improved fermentation process for the production of succinic acid. While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to include all such alternatives, modifications, and variations as set forth within the spirit and scope of the appended claims.

Claims: A fermentation process for the production of succinic acid in high yield which comprises fermenting a carbohydrate with a succinate-producing bacterium in an aqueous fermentation medium containing carbohydrates assimilable by said bacterium, other nutrients required for the growth of said bacterium, and dissolved carbon dioxide, wherein the initial carbohydrate concentration in the medium is between about 20 g/l and about 100 g/l, and the pH of the medium is maintained between about 5.8 and 6.8 during the fermentation and wherein sufficient calcium ion is added to fermentation medium to give a precipitate of calcium succinate which is then separated from said fermentation medium. The process of claim 1 in which the calcium ion is added to the fermentation medium in the form of calcium hydroxide. The process of claim 1 wherein the initial concentration of carbohydrate in the medium is between about 40 g/l and about 80 g/l. The process of claim 1 wherein fermentation medium is sparged with carbon dioxide during the fermentation process. The process of claim 1 wherein the fermentation medium is in equilibrium with carbon dioxide gas above atmospheric pressure. The process of claim 1 wherein the fermentation is run as a continuous process.

Inventors: DATTA, RATHIN, LEMME, CHRISTOPHER J.
Applicants: MICHIGAN BIOTECHNOLOGY INSTITUTE
Application year: 1987
Main classification: C12P7
IPC codes: C12R1, C12P7