IMPROVED FERMENTATION PROCESS FOR CARBOXYLIC ACIDS

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to a fermentation process for the production of certain carboxylic acids. More particularly, the invention concerns a method for improving the yield of certain carboxylic acids in Rhizopus cultures by limiting the amount of oxygen during the acid-production stage of the fermentation.

Background References

Production of certain carboxylic acids, e.g., fumaric acid, by fungi of the Rhizopus has been a subject of several patents and other contributions to the technical literature. Rhodes et al., Appl . Microbiol. , 1_ , 74-80 (1959), describe a series of fermentation experiments in which optimal conditions were sought for maximizing fumaric acid yields in Rhizopus arrhizus cultures. In a later publication, Appl. Microbiol, . 10, 9-15 (1962), Rhodes et al . describe preferred conditions for producing fumaric acid by fermentation of Rhizopus arrhizus in 20 liter

fermenters. In particular, use of CaC0 ₃ to neutralize the resulting carboxylic acid as it is formed is disclosed. Waksman, U.S. Pat. No. 2,326,986, describes a method for producing fumaric acid by fermentation of Rhizopus nigricans, or other fungi of the order Mucorales, in the presence of zinc and iron salts. Kane, et al., U.S. Pat. No. 2,327,191, disclose a process for producing fumaric acid by submerged aerobic fermentation of Rhizopus nigricans. Lubowitz, et al., U.S. Pat. No. 2,861,922, disclose use of nickel salts to promote fumaric acid production by Rhizopus fungi. La Roe, U.S. Pat. No. 2,912,363, describes improvements in fumaric acid yields by Rhizopus and related fungi which are attributable to limiting trie concentration of nitrogen sources in culture media.

A number of patents and publications disclose that fats, fatty acids, or their derivatives can be added to microbial fermentation media as supplemental carbon sources, or as yield promoters in fermentation processes for producing glutamic acid and certain antibiotics. For example, U.K. Pat". Nos. 679,087 and 700,316 disclose the use of fats or fatty acids as carbon sources or supplements. Goldberg and Stieglitz, U.S. Pat. No. 4,564,594, disclose the use of fatty acid esters or triglycerides as culture medium additives to enhance the rate of production of carboxylic acids, especially fumaric acid.

Fermentation processes, especially those employing inexpensive and abundant carbon sources derived from biomass, offer alternative sources of supply of commercially important organic acids. Such organic acids include fumaric acid, which is utilized by the plastics industry in the manufacture of polyester and alkyd resins; lactic and malic acids,

which are utilized by the food industry; and succinic acid, which is consumed in the manufacture of pharmaceuticals, plastics, and protective coatings. Thus, improved fermentation processes for producing these compounds are desirable.

SUMMARY OF THE INVENTION

The present invention provides an improvement in a fermentation process for producing carboxylic acids selected from the group consisting of fumaric acid, succinic acid, malic acid, and mixtures thereof, the improvement comprising growing fungi of genus Rhizopus in a culture medium with controlled dissolved oxygen levels in which the dissolved oxygen concentration is maintained between 80 and 100% for the cell-growth phase and 30-80% for the acid-production phase.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an improved process for producing fumaric acid, succinic acid, malic acid or mixtures of these carboxylic acids by growth of fungi of the genus Rhizopus. The invention is characterized by a process improvement comprising limiting the dissolved oxygen levels and maintaining the dissolved oxygen concentration between 80 and 100% for the initial (i.e., cell-growth) phase and between 30 and 80% for the subsequent acid-production phase. Yields of these carboxylic acids, particularly fumaric acid, are enhanced when Rhizopus cultures are grown under conditions of controlled oxygen concentrations, especially when the concentration of oxygen is limited to less than about 80% during the acid-production phase. The increased yields of carboxylic acid production observed using the process of the invention are believed to be

attributable to the regulation of the flow of glucose towards reaction with calcium carbonate rather than with oxygen under conditions of controlled oxygen availability.

Microorganisms Suitable fungal species for the process of the invention are fungi of the genus Rhizopus. Examples of suitable species include Rhizopus arrhizus, R. oryzae, and R. nigricans. Because of observed higher productivity, R. arrhizus is a preferred species. R. arrhizus NRRL 1526, a strain described by Rhodes, et al., Appl. Microbiol., 2 74-80 (1959), is a particularly preferred microorganism for the process of the present invention.

Culture Media

Various media formulations known to be suitable for Rhizopus fermentation can be employed in the process of the invention. Generally, a suitable medium will provide at least one carbon source, a nitrogen source and inorganic salts.

A suitable carbon source consists of an organic source of carbon such as glucose, sucrose, xylose, fructose, invert sugar, maltose, invert high test molasses, syrups, or various starches, grains, malted grains, cereal products or other materials containing any of the foregoing substances. Glucose is a preferred organic carbon source. When glucose is employed as an organic carbon source, it is preferred to use from about 10 to about 16 g glucose per 100 mL of medium.

During the acid-production stage of the fermentation, a suitable carbon source consists of an organic source of carbon such as defined above and an

inorganic carbonate salt. A preferred inorganic carbonate salt is calcium carbonate.

Suitable nitrogen sources include such organic and inorganic sources as urea, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium nitrate, ammonium biphosphate, asparagine and protein hydrolyzates, e.g. casein hydrolyzate and whey hydrolyzate. Of the foregoing, urea and ammonium sulfate are preferred.

To optimize yields of organic acids, particularly fumaric acid, available nitrogen in Rhizopus cultures should be limited to a range of about 10 to about 30 mmol available nitrogen per liter medium. Therefore, when ammonium sulfate is employed as a nitrogen source, it is preferred to employ from about 0.06 to about 0.18 g ammonium sulfate per 100 mL of medium.

The inorganic salts added to Rhizopus culture media should include sources of phosphate, sulfur, iron, magne.sium, and zinc. Suitable sources of phosphate include monobasic or dibasic potassium phosphate, monobasic or dibasic sodium phosphate, ammonium biphosphate or mixtures thereof. Suitable inorganic salts employed in the fermentation include zinc sulfate, iron salts such as ferric tartrate or ferric chloride, and magnesium sulfate. Suitable sources of sulfur include ammonium sulfate, zinc sulfate and magnesium sulfate.

In a preferred fermentation medium, the carbon sources are glucose and calcium carbonate, the nitrogen source is ammonium sulfate, and potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, ferric chloride, and corn steep liquor or biotin are present.

In the preferred embodiment of this invention, the dissolved oxygen concentration is

maintained between about 80% and 100% during the cell-growth phase of the fermentation. The preferred dissolved oxygen concentration during the acid-production phase is between about 30% and about 80%.

The cell-growth stage occurs during the initial phase of the fermentation, lasting from about 18 to about 28 h. The subsequent acid-production phase is allowed to proceed for a time sufficient to obtain optimum yields of the desired carboxylic acids. Times of about 3 to about 6 days are preferred. It should be appreciated that fermentation times are influenced to a significant degree by temperature, concentration and choice of microorganisms, nutrient concentration, and other factors known to those skilled in the art.

To realize the benefits of this invention, it is necessary to add calcium carbonate to the culture medium used during the acid-production stage of the fermentation. Calcium carbonate may also be added to the culture medium used during the cell-growth stage. The advantages of using calcium carbonate to neutralize the solution during fermentation are well-known in the art. However, it has not previously been recognized that the carbonate portion of CaC0 ₃ can also provide a source of carbon and oxygen atoms for the increased production of fumaric acid under conditions of limited oxygen availability. When oxygen levels are high during the acid-production phase, one molecule of glucose is converted to one molecule of fumarate, as shown in Equation 1. When oxygen levels are limited, one molecule of glucose is converted to two molecules of fumarate, as shown in Equation 2.

^C ₆ ^H i ₂ ° ₆ ^{+ Ca C0} ₃ + ° ₂ ^→ CaC ₄ H ₂ 0 ₄ + 5H ₂ 0 +

3CO, ( Eq . 1 )

^C ₆ ^H i ₂ ° ₆ ^{+ 2 a C0} ₃ ^~ * 2CaC ₄ H ₂ 0 ₄ + 4H ₂ 0

( Eq . 2 )

Process Conditions Fermentation is conducted at a temperature of about 25°C to about 35°C, preferably about 33°C to about 35°C. A major product of the fermentation is usually fumaric acid, although succinic acid, malic acid, and other monocarboxylic and dicarboxylic acids can also be produced.

Production rates and yields in individual experiments can be significantly affected by such unpreditable factors as strain deterioration and adventitious impurities in media formulations and culture equipment. Accordingly, culture conditions should be continuously monitored and the productivity of fungal strains frequently checked.

After the process of this invention has gone to completion, the desired acid can be collected in pure form by conventional methods. Calcium salts of product acids can be converted to free carboxylic acids by acidification with mineral acid. Fungal mycelia and insoluble CaS0 ₄ can be removed by heating media to 80°C to 100°C for a brief period, followed by filtration. The resulting product acids can be recovered by crystallization.

The concentration of the microorganism required to practice the process of the invention efficiently is preferably about 0.1 to about 10 percent based on the volume of the reaction medium. Too little microorganism will cause a decrease in fermentation rate, and too large a quantity of

microorganism will result in excess mycelium growth and decreased yields of acid relative to the amount of carbohydrate consumed.

The pH of the process medium is determined by the presence of excess calcium carbonate and is not maintained at a constant value during the fermentation. Generally, the pH of cultures grown in accordance with the process of the present invention should be maintained from about 4 to about 8, preferably from about 5 to about 7.

The following are illustrative examples of the invention in which all parts and percentages are by weight, and all degrees are Celsius unless otherwise noted.

GENERAL METHODS

Culture Maintenance

Rhizopus arrhizus NRRL 1526 was preserved by adding 1 mL of spore suspension to 16 x 150 mm test tubes, each containing 1 mL of 0.02M phosphate buffer, pH 7.0, in 0.1% Tween 80 and 20% glycerol. The tubes were stored frozen at -70°C.

Culture Media The culture media used in the Examples were adapted from those previously described by Rhodes et al., Appl. Microbiol., 1_, 74-80 (1959), and are set forth in Table 1 below. All media were sterilized by autoclaving. Medium A, below, was used in Rhizopus spore development. Medium B was employed for Rhizopus vegetative seed fermentation, and Medium C was used for production or organic acids in aerobic fermentation experiments. The media were sterilized

by autoclaving at 120°C and 20 psig for 20 min. Calcium carbonate was sterilized dry inside the flasks for 24 h or as a 50% slurry in the fermenter for 1 h. The nitrogen? source, ammonium sulfate or urea, was sterilized separately from other media components by autoclaving, and added aseptically to media mixtures prior to inoculation.

Preparation of Inoculum

Working spore slants were prepared by adding 5 L of sterile- 0.05M sorbitan monooleate, to 1 mL of frozen spore solution and shaking well. One mL of the resulting spore suspension was plated on a Medium A agar slant which was incubated at 32° for 5-7 days.

Vegetative mycelial seed cultures were prepared by resuspending spores from 5-7 day medium A agar slants in 30 mL of 0.05M phosphate buffer, pH 6.8, containing 0.1% of polyoxyethylene sorbitan monooleate. The resulting spore suspensions were transferred to sterile Medium B, and incubated with agitation at 32° for 18-24 h. The resulting cell growth is referred to as "standard inoculum".

Fermentation

Fermentations were carried out in 3 L (2.5 L working volume) bioengineering glass fermenters (Model KLF 2000). All fermenters were equipped with automatic temperature controls, pH probes, pO ^* probes, sampling tubes, inoculation ports, and a control shaft with 2 disc turbine impellers (0.6 mm diameter) .

Glucose (325 g) and 1-1.5 L of water were placed in the fermenter and sterilized at 121°C for 20 in. Ammonium sulfate (4.5 g) and 50 mL of water were autoclaved at 121°C for 20 min. KH ₂ P0 ₄ (0.75 g), MgS0 ₄ .7H ₂ 0 (1.0 g), ZnSO„.7H ₂ 0) (O.llg), FeCl ₃ .6H ₂ 0 (0.01875 g), tartaric acid (0.01875 g), corn steep liquor (1.25 mL) and 300 mL of water were autoclaved at 121°C for 20 min. Calcium carbonate (200 g) was dry autoclaved for 24 h. After the glucose was sterilized and the temperature of the

fermenter decreased to 60-70°C, the calcium carbonate, ammonium sulfate and other salt solutions were transferred to the fermenter using sterile technique. Sterile distilled water was added to bring the final volume of the solution to 2.5 L.

Production of fumaric acid was initiated by the addition of 5% (v/v) of a standard inoculum of Rhizopus arrhizus NRRL 1526 to the fermenter. The temperature was maintained at 34°C. The aeration rate was 0.5 v/v and the dissolved oxygen concentration was controlled by the stirring rate which was maintained between 200 and 800 rpm.

Analysis Fumarate, succinate, alate and α-ketoglutarate were analyzed as their methyl esters. Properly diluted samples (0.1 mL) were mixed with 0.05 mL of concentrated H 2,SO4, and 0.25 L of methanol and incubated at 60°C for 1 h in sealed autosampler vials. Water (0.25 mL) and chloroform (0.75 mL) were injected into the vials after cooling and the vials shaken to facilitate the extraction of methyl esters into the chloroform layer. For each sample, a 3 _/ iL chloroform sample was then injected into a Hewlett-Packard 5880A gas chromatograph equipped with a Hewlett-Packard 7672A automatic liquid sampler. The following chromatographic conditions were used: 10% SP 2340 on chromosorb 100 w/a W column (1.8 x 0.32 cm); helium as carrier gas; injection port, 200°C; f.i.d., 250°C; oven, 2.5 min at 100°C with a temperature increase of 20°/min to 180° for 8.0 min.

EXAMPLE 1 This example illustrates the effect of maintaining very low dissolved oxygen concentrations (less than 5%) during the acid-production stage of the fermentation:

EXAMPLE 2 This example illustrates the effects of maintaining a low dissolved oxygen concentration (approximately 10%) during the acid-production stage.

α-Keto- glutaric Total

Acid Acid

(g/L) (g/L)

EXAMPLE 3 This example illustrates the effects of maintaining a moderate concentration of dissolved oxygen (approximately 40%) during the acid- production stage of the fermentation.

87.0 122 9.2 11.8 17.7 160 91.0 126 9.1 12.9 18.0 166

EXAMPLE 4 This example illustrates the effects of maintaining a moderate dissolved oxygen concentration (approximately 60%) during the acid-production phase of the fermentation.

α-Keto- glutaric Total

Acid Acid

(g/L) (g/L)

0 0 1.0 0 0 0.5 0 0 30.8 0 0 35.8 6 8 56.3

14 109.3 16 125.8 17 142.8 17 143.5 17 137.6 18 157.6

EXAMPLE 5 This example illustrates the effects of maintaining a moderately high dissolved oxygen concentration (approximately 80%) during the acid-production phase of the fermentation.

EXAMPLE 6 This example illustrates effects of maintaining high concentrations of dissolved oxygen

(approximately 100%) during the acid-production phase of the fermentation.

The above results show that controlling oxygen concentration during the acid production phase increases the rates of formation of fumaric acid where the dissolved oxygen concentration is limited within a 30 to 80% of saturation range. Although preferred embodiments of the invention have been illustrated and described hereinabove, it is to be understood that there is no intent to limit the invention to the precise constructions herein disclosed, and it is to be further understood that the right is reserved to all changes and modifications coming within the scope of the invention as defined in the appended claims.