April, 1945
INDUSTRIAL AND ENGINEERING CHEMISTRY
405
benzenes. With o-dichlorobenzenethe catalytic action was shown to occur even at room temperature.
DDT. Since this temperature is above that ordinarily encountered under normal outdoor conditions, decomposition effected by an accessory material under the accelerated test should be viewed &s indicating storage tests at working temperatures.
ACKNOWLEDGMENT
The fertilizer materials were supplied by the Bureau of Plant Industry, Soils, and Agricultural Engineering, through the courtesy of J. 0. Hardesty.
Solutions of DDT. For this series of tests 2 grams of recrystallized D D T and 2 grams of the solvent were placed in the U-tube. After the temperature of the tube and its contents had come up to that of the oil bath, 2-3 mg. of anhydrous ferric chloride were dropped down the inlet arm of the U-tube. The tube was then shaken gently to ensure good mixing with the catalyst. The results (Table 11) show that most of the solvents used with DDT have a marked inhibiting action toward the catalytic decomposition reaction. The notable exceptions are the nitro- and chloro-
LITERATURE CITED
(1) Annand, J . Econ. Entomol., 37, 125 (1944). (2) Fleck and Haller, J . Am. Chem. Soc., 66,2095 (1944). (3) Roark and McIndoo, I?. S. Dept. Agr., Bur. Entomol. Plant Quarantine. E-631.Dee.. 1944. (4) Zeker, Ber.,' 7, 1180 (1874); Brand and Busse-Sundermann. Ibid., 75,1819 (1942).
FERMENTATION PROCESS FOR ITACONIC ACID LEWIS B. LOCKWOOD AND GEORGE E. WARD Northern Regional Research Laboratory,
U. S . Department of Agriculture, Peoria, Ill.
A fermentation process for the production of itaconic acid, based on the cultivation of a superior strain of Aspergillus terreus (NRRL 1960) on the surface of glucose nutrient media, has been developed and operated on a semipilot plant scale. Itaconic acid yields in excess of 30 grams per 100 grams of glucose supplied are obtained in 12 days. The major portion of itaconic acid produced can be recovered by crystallization after concentrating and cooling the filtered liquors. The fermentation is resistant to contamination, since the nutrient medium is maintained at a low pH level throughout the culture period.
R
ESEARCH on the production of itaconic acid (methylene succinic acid), a potential raw material for resins of the methacrylate type, was undertaken as a means of increasing the industrial utilization of agricultural products. Itaconic acid was first reported as a product of mold metabolism by Kinoshita (3) who obtained it from cultures of Aspergillus itaconicus. Calam, Oxford, and Raistrick (1) reported obtaining small quantities of this acid from one strain of Aspergillus terreus, and preliminary investigations conducted by Moyer and Coghill (6) confirmed the suitability of Aspergillus terreus for bringing about this reaction, The present paper describes the production of itaconic acid from glucose on a semipilot plant scale, using the organism and conditions found best in this Laboratory. The organism was a strain of Aspergillus terreus isolated by Kenneth B. Raper from a soil sample obtained from San Antonio, Texas. It was one of the best itaconic acid-producing strains found in preliminary investigations. The organism is carried in this Laboratory's culture collection as Aspergillus terreus (NRRL 1960) and is maintained in stock culture on Czapek-Dox solution agar as cited by Thom and Church ( 7 ) . To obtain frangible spore-bearing material from Aspergillus terreus to be used for the inoculation of production cultures, a medium of the following composition was employed: Glucose monohydrate (commercial), grams 275 5 NaNOa, g r a m MgSOc7Hz0, gram 0.024 KC1, gram 0.005 H I P O I , gram 0.003 Concd. corn steep liquora, ml. 0.5 a A commercial by-producc of the corn wet-milling industries. I t contains approximately 50% total solida, and is a rich source of mineral nutrients and protein degradation products. ~~
Distilled water was added to bring the medium to 1000 ml. Sterile 50-ml. portions in 200-ml. Erlenmeyer flasks were heavily seeded with spores obtained from a IO-day-old slant culture. On the liquid medium, a good crop of spores was obtained after 5-day incubation a t 30" C. One flask culture is sufficient to inoculate 100 liters of nutrient solution; appropriate portions were used to inoculate the 12-liter quantities of medium used in each of the seven fermentation pans. The fermentation was conducted in shallow aluminum pans, 22 X 36 x 2 inches, in a cabinet described by Ward, Lockwood, May, and Herrick (8) for the cultivation of molds on the surface of solutions. The pans were sterilized with flowing steam for 3 hours, then cooled under a slight pressure of sterile air. The inoculated solutions then were blown into the pans through sterile glass tubes inserted through the front of the cabinet. Twelve liters of medium were placed in each pan. The fermentation solution had the following composition: Glucose monohydrate (commercial), grams MgS04.7H~0,g r a m NHdi01, gram NaC1, gram ZnS04.7H90, gram Nitric acid (sp. gr. 1.42), ml. Concd. corn steep liquor, ml.
165 4.4 2.5 0.4 0.0044
1.60
4.0
and distilled water was added to bring the volume to 1000 ml. The initial pH was approximately 2.0. Throughout the 12-day fermentation period the tier of cultures was aerated a t the rate of 5 liters of humidified air per minute. The entering air was sterilized by passage through a cotton filter. The considerable heat evolved during the course of the fermentation was dissipated by the circulation of cold water through coils installed in the cabinet. The temperature was maintained a t 30' to 32' C. by a thermostatically operated solenoid valve controlling the flow of water to the coils. At harvest the solutions were drained from the pans, and the pans and mycelia were washed with small quantities of cold water. Main filtrates and washings from each panwere combined, and appropriate samples were taken for analysis. Glucose determinations were made by the method of Shaffer and Hartmann (6). Itaconic acid was deterrdined by the bromination method of Koppeschaar ( 4 ) , as modified in this Laboratory by Friedkin ( 2 ) . /
406
INDUSTRIAL AND ENGINEERING CHEMISTRY
The combined liquors from each pan were evaporated on a steam bath, with constant stirring, to a volume of approximately one liter; a slurry of fine crystals of itaconic acid resulted. The agitated mass was cooled to room temperature, and the crystals were separated in a perforated basket centrifuge and washed with cold water. The resultant product, consisting of almost pure itaconic acid, was very light in color. I n some cases it was possible to obtain a second crop of itaconic acid crystals after further evaporation of the supernatant liquor, which was dark brown and low in viscosity. R E S U L T S OF FERMENTATIONS
Data from two groups of fermentations are presented to illustrate typical results. Table I A shows data obtained when the fermentation is conducted under the most favorable conditions yet found. I t is apparent that analytical yields representing approximately 50% of theory, based on glucose consumed, and in excess of 25% on a weight basis, are obtained. The theoretical yield is assumed to be one mole of itaconic acid per mole of glucose consumed, although there is no proof of this relation as yet.
. k I D FROM GLCcosE TABLE I. PRODLJC‘TIOXO F ITACOKTC
A . Typical Pan Fermentations Pan1 Pan2
B. Duplicate Pan Fermentations a t Initial Glucose Concus. of: 1 0 % 15% 20% 25%
Initial glucose, grams (calcd. to anhydrous basis) 1800 1800 1200 1800 2400 3000 Glucose consumed, grams 1583 1539 1128 1565 1617 1810 602 242 561 577 568 Itaconic acid produced, grams 569 Yield of itaconic acid based on glucose consumedn, % 49.9 54.3 29.8 4 9 . 7 4 9 . 5 4 3 . 5 Itaconic acid recovered 6 y crystallization, grams 453 490 192 436 457 429 79.4 77.8 79.2 75.2 79.7 81.4 Recovery efficiency % 1 6 . 0 24 2 1 9 . 0 1 4 . 3 27.2 Recovery weight yikldb, % 25.2 Nycelial weight, grams 222 144 223 233 245 277 a Based on assumption t h a t 1 mole of glucose should yield 1 mole of itaconic acid. b Ratio of grams itaconic acid recovered t o g r a m glucose supplied (anhydrous basis).
Data obtained in similar experiments have indicated the presence of appreciable quantities of neutral nonreducing materials in the fermented liquor. Also, in numerous experiments a small quantity of acidic material other than itaconic acid has been found. Analyses of the exhaust gases have shown a carbon dioxide content of about 1% and an oxygen content of 20.5% which indicates that the oxygen supply has been in excess of the requirements of the cultures. I n recovery studies approximately 20% of the itaconic acid produced was left in the mother liquors after the crystallized itaconic acid had been removed. Preliminary experiments have indicated that much of this acid can be recovered from the residual sirup by extraction with organic solvents, such as n-butanol. Using a 1%aqueous solution of itaconic acid a t 28” C., a water/ butanol distribution ratio of 0.469 was obtained. Some variation from this value was observed when residual fermentation sirups
Vol. 37, No. 4
were extracted. The itaconic acid crystallized readily from the butanol upon concentration. Analysis by alkali titration and by bromination methods of the first crop of itaconic acid crystals obtained from the evaporated liquors usually indicated purities of more than 97%; analysis of the second crop of crystals generally indicated purities greater than 90%. Table IB presents the results of the fermentation of solutions of different initial glucose concentrations. It is apparent that yields of itaconic acid and fermentation efficiencies are low when the initial glucose concentration is 10%. The efficiency of conversion of glucose to itaconic acid is considerably higher in solutions of 15% initial glucose concentration, but no significant improvement is found at the 20 or 25% concentration levels. At the latter concentrations the residual glucose content of the solutions, after fermentation, is disproportionately large. This residual sugar represents a loss in over-all operating efficiency and also interferes with the recovery of the product, since it has not been found possible to obtain a second crop of crystals by further evaporating the mother liquor obtained from the initial crystallization, These and similar data suggest that glucose solutions of approximately 15% Concentration are most desirable for itaconic acid production. The process described here is believed to offer a practical means of producing itaconic acid from cheap abundant materials. This compound has heretofore been produced from the more expensive raw material, citric acid. The quality of the crude product obtained by the fermentation process suggests that further purification may be unnecessary prior to the esterification step in resin manufacture. The raw materials cost would be about 22 cents per pound of itaconic acid recovered; this estimate is based on the assumptioii of a 25% recovery yield and a cost of 5.3 cents per pound of glucose (anhydrous basis), 2.5 cents per pound of corn steep liquor, and 0.4 cent for other nutrient salts required to produce one pound of itaconic acid. The fermentation process is readily conducted, and is not easily susceptible t o contamination, since the nutrient medium is maintained throughout the culture period a t a pH level too low for the growth of most organisms. LITERATURE C I T E D
(1) Calam, C. T., Oxford, -4.E., and Raistrick, H., Biochem. J . , 33
1488 (1939). (2) Friedkin, M., t o be published. (3) Kinoshita, K., Botan. Mag., 45, 60 (1931); Acta Phgtochim. (Tokyo), 5 , 271 (1931). (4) Koppesohaar, W. F., Z . anal. Chem., 15, 233 (1876). ( 5 ) Moyer, A. J., and Coghill, R. D., to be published. (6) Shaffer, P. A., and Hartmann, A. F., J. Biol. Chem., 45, 965 (1921). (7) Thom, C., and Church, &I., “The Aspergilli”, p. 39 (1926). (8) Ward, G. E., Lockwood, L. B., May, 0. E., and Herrick, H. T,, ISD. ENG.CHEM.,27, 318 (1935). PRESENTED by title before t h e Division of Agricultural and Food Chemistry at the 108th Meeting of t h e AMERICAN CHEMICAL SOCIETYin Xew York, N. Y .
