Citric Acid Production by Submerged Fermentation This study
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points out what factors affect the fermentation and how
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shows how factors can be balanced to obtain satisfactory f ermentation
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shows degree of control required
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gives range of conditions useful with different samples of molasses
DURWC
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the past decade considerable progress has been made toward solving the problems of utilization of crude substrates for citric acid production by submerged fermentation. Studies have been made on the use of corn sirup (8, 77), starchy materials (7), cane molasses (3, 7, 9), and beet molasses (2, 72, 73) as substrates, using ion exchange resins, charcoal, ferrocyanide, and other reagents, such as methanol, for substrate treatment. Although this work has dealt mainly with small scale equipment, some larger scale studies have been reported. Schweiger and Snell (8) described the fermentation of crude substrates in 275-gallon fermenters. Buelow and Johnson (7), using an adaptation of the procedure of Shu and Johnson (70), studied the aeration requirements of 100-liter fermentations in 50-gallon tanks. The following report summarizes results of studies on citric acid production by submerged fermentation of beet molasses, carried out at the National Research Laboratories of Canada. Details of equipment design, fermentation procedures, and experimental data have been published (4, 6, 72, 73). The fermentation procedure was basically that described by Martin and Waters (6). Ferrocyanide-treated beet
molasses mash was “fermented” in columnar glass fermenters, the inoculum being in the form of discrete pellets. The culture was aerated far the first 24 hours and then oxygenated (pure oxygen) until fermentation was complete. Glass fermenters having operating volumes of 2.5 liters (6) and 40 liters (72) were used. Factors Affecting Development of Inoculum Early work indicated that yields of citric acid on the order of 65 to 70% conversion of available sugar to anhydrous citric acid could be obtained in a short time by using a pellet type of inoculum. However, the variation, both within and between runs, was considerable and many interrelated factors appeared to be involved. Because the amount and type of inoculum used appeared to affect yield and considerable variation was evident in the inocula, it seemed essential to have a standard seed type before beginning an investigation of the fermentation itself. First, the objective had to be defined-i.e., what characterbtics should be sought. The prime characteristic of an inoculum must be its ability to give reproducibly good yields of citric acid. I t should develop in a convenient time (18 to 24 hours) and there should be some rapid means of assessing its fermentation potential. As it was felt that a change in morphology was simply a manifestation of a change in physiology, it was hoped that physiological uniformity could be achieved through maintenance of a uniform morphological type. A complete range of morphological types of mold growth had been observed during the preparation of inocula, ranging from a filamentous type showing abundant “slushy” growth to a very retarded pellet type showing very scant growth. An intermediate type was selected as the objective because it had given good fermentations, it developed in about 20 hours, direct counts could be made to determine pellet density, and the degree of uniformity could be rapidly assessed under low power magnification. The individual pellets were 0.2 to 0.5 mm. in diameter with a dense center composed of mycelium and enmeshed precipitated matter. The lateral hyphae were short, thickened,
vacuolated, and granular, with clublike branches. This type of inoculum was not necessarily optimum for the fermentation, but rather was selected because it appeared to meet requirements. The effect of various factors on the production of the standard seed type was studied. Of the three variables studied intensively, p H appeared to have the most pronounced effect on the morphology and rate of development of the mold. The useful range of p H was narrow (6.0 to 7.0) and common to all samples of molasses tested. Increase in p H from 6.0 to 7.0 decreased the rate of pellet development. Too low a ferrocyanide level resulted in excessive growth and a loss of the characteristic “pathological” morphology; too high a level retarded growth excessively. The effects of variation in p H or ferrocyanide level could be counteracted by adjustment in the spore inoculum level. Variation in mashing procedure, incubation temperature, and degree of aeration also affected the rate and type of development. I t was shown that all factors were interrelated and, within limited ranges, a change in one could be counteracted by appropriate change in another. After a standard predictable seed type, uniform in morphological development, had been produced, it remained to show that this inoculum possessed the required fermentative properties. Thus, after the best fermentation conditions were determined as nearly as possible, tests were run to assess the inoculum. Twenty-three individual fermentations gave an average yield of 85.470 conversion of available sugar to total acid expressed as citric acid (standard
Standard pellet-type mold ( I 3) VOL. 49, NO. 8
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Effect of inoculum level (pellets per liter X 103) on citric acid production in small fermenters with Chatham 1951 molasses ( 7 2)
deviation 0.4) and 68.5% conversion of available sugar to anhydrous citric acid (standard deviation 0.4) in 116 hours. Factors Affecting Fermentation
With the reasonable assurance that fermentation effects could be separated from inoculum effects, an investigation of some of the many factors affecting the fermentation was begun using 2.5liter fermenters. The fermentation was highly sensitive to sterilization conditions (72). Although sterilization a t 15 pounds per square inch gage resulted in better yields than sterilization a t atmospheric pressure, increasing the pressure from 15 to 22 pounds per square inch gage was without effect. The rate of cooling of the mash was also important, slow cooling giving better results than rapid cooling. This was probably the effect of ferrocyanide reaction time, as in all these trials ferrocyanide was added at the beginning of the cooling period. Increasing the sterilization time a t 15 pounds per square inch gage from 45 to 90 minutes resulted in large pellets of more variable size and somewhat reduced the yields. Under standard conditions of test the best combination of yield and production rate was obtained a t an inoculum level of about 280,000 pellets per liter, although a t 120,000 pellets per liter the initial production rate was only slightly lower and the final yield was the same. Below 120,000 pellets per liter, both yield and rate dropped off. At 360,000 pellets per liter the initial production rate was most rapid, but this rate was not maintained because the density of pellets in the fermenter became so great that both aeration and agitation were greatly reduced. The yield of acid increased sharply (approximately 5070) as the gas flow rate was increased from 200 to 300 ml. per fermenter per minute. Over the
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range 300 to 500 ml. per minute per fermenter the increase in yield was approximately 12y0and from 500 to 1200 ml. per minute per fermenter was approximately 8%. The optimum temperature was 31 " C. Although the initial rate of acid production was higher at 35" C., the final yield was about 15y0 lower; at 25" C. the rate of production was lower and the yield a t 116 hours was reduced by approximately 22%. The ratio of citric to total acid decreased with increasing temperature from 89% a t 25" and to Y7Y0 at 35" C. Highest yields and production rates were obtained when the initial reaction of the mash was adjusted to pH 7.0. When the initial reaction of the mash was p H 6.0 or 8.0 the rate of production was reduced and the final yield was about 870 lower. Addition of phosphate, as originally recommended, proved to be undesirable, for addition of as little as 0.00570 potassium acid phosphate reduced the average yield by about 4oY0 and increased the variability of the fermentation. Addition of phosphate markedly reduced fermentation time. Phosphate also affected the production of acids other than citric. In the absence of added phosphate, oxalic acid was the major secondary acid, accounting for about 12% of the total acidity developed. With 0.05% added phosphate only trace amounts of oxalate were produced and the major secondary acids were gluconic and 5-ketogluconic acids. The action of ferrocyanide in the treatment of beet molasses has generally been attributed to the removal of trace metals. However, observations made during studies of inoculum production, suggested that some other effect might also be involved. T o learn more about the action of ferrocyanide, its effect on growth and acid production was studied in a highly refined synthetic medium. The growth process was found to be extremely sensitive to ferrocyanide, whereas the sequence of reactions leading to acid production was relatively insensitive. Although the inhibition of growth was reversed by the addition of trace metals, the data strongly suggested that ferrocyanide did exert a direct toxic effect. The highest yields of acid were obtained in the presence of minute amounts of free ferrocyanide. In the fermentation of molasses mashes yields decreased sharply on either side of the optimum range of ferrocyanide concentration and this optimum varied from batch to batch of molasses (72). At the time of inoculation a minimum of about 100 p.p.m. of free ferrocyanide was required for good fermentation. With some samples of molasses the tolerated range of free ferrocyanide was narrow; with others, broad.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Scale-Up of Fermentation
After the best fermentation conditions and the sensitivity of the fermentation to a number of variables had been determined in small fermenters, the effect of an increase in scale (16 to 1) was studied. Although the range over which the variables were tested was not as great as in the small fermenters, the characteristics of the two pieces of equipment were similar and information gained in the small fermenters was directly applicable to the large fermenters (72). However, reproducibility of the fermentation was greater in the large fermenters. Relatively high yields were obtained in tests with low inoculum levels, presumably because there was less tendency for larger than normal pellets to pack in the larger fermenter. When the gas flow rate was scaled u p from the small fermenters on a crosssection area basis, the turbulence developed in the mash was appreciably less and this was reflected in lowered yields. Minimum satisfactory gas flow rates in the two fermenters were approximately in the same ratio as the mash volumes. Acknowledgment
The author wishes to acknowledge the contributions of C. P. Lentz, Robert Steel, and W. R. Waters to the work reported here. Literature Cited
(1) Buelow, G. H., Johnson, M. J., IND. ENG. CHEM. 4.4, 2945-6 (1952).
(2) Clkmeni9 M. T., Can.9. Technoi.30, 82-8 (1952). (3) Karow, E. O., Waksman, S. A, IND.ENG.CHEM.39, 821-5 (1947). (4) Martin, S. M., Can. J. Microbiol. 1, 644-52 (1955). ( 5 ) Martin, S . M.; Steel, R., Ibid., 1, 470-2 (1955). (6) Martin, S. M.; Waters, W. R.,IND. ENG.CHEM.44,2229-33 (1952). ( 7 ) My:w;?* J., Agpl. Microbiol. 1, 7-13 ( I7JJ J.
(8) Schweiger, L. B,, Snell, R. L., U. S. Patent 2,476,159 (July 12, 1949). ( 9 ) Shu, P., Ph.D. thesis, University of
Wisconsin, 1947. (10) Shu, P., Johnson, M. J., IND.ENG. CHEM.40, 1202-5 (1948). (11) Snell, R. L., Schweiger, L. B., U. S. Patent 2,492,667 (Dec. 27, 1949). (12) Steel, R., Lentz, C. P., Martin, S. M., Can. J. Microbiol. 1, 299-31 1 (1955). (13) Steel, R., Martin, S. M., Lentz, C. P., .Did., 1, 150-7 (1954). RECEIVED for review November 20, 1956 ACCEPTEDMay 22, 1957 Division of Agricultural and Food Chemistry, Fermentation Subdivision, Symposium on Fermentation Process and Equipment Design, 130th Meeting, ACS, Atlantic City, N. J., September 1956. Contribution from Division of Applied Biology, National Research Laboratories, NRC No. 4380.
