GLUCONIC ACID PRODUCTION Repeated Recovery and Re-use of Submerged Aspergillus niger by Filtration N. PORGES, T. F. CLARK, AND S. I. ARONOVSKY In the semicontinuous method of producing gluconic acid from glucose by A . niger, the mycelia were recovered by pressure filtration and re-used in nine successive fermentations of media containing 16 grams of glucose per 100 cc. In the fermentation of a solution containing 16 grams of glucose and about 2.6 grams of calcium carbonate per 100 CC., the uncombined gluconic acid is present in sufficient concentration to prevent the formation of calcium gluconate crystals which tend to mat with the mycelial growth and thus hinder filtration. This filtration permits the utilization of a full charge of fresh medium of higher glucose concentration than was feasible with the flotation method previously reported. In the cases where the free acid concentration was low, centrifugal filtration was the more efficient method of removing the mycelia from the fermented liquor.
Agricultural By-products Laboratory', Ames, Iowa
sterilizer was made from a 12-quart (11.4-liter) aluminum domestic-type cooker. The original cover was replaced by an aluminum plate provided with the following fittings and accessories: inlet A and outlet B for an aluminum coil t o permit rapid heating and cooling by steam and water, packing gland C for the agitator shaft, agitator motor D,pressure gage F, thermometer well G, air inlet H , and charging port and air vent J . The speed of the agitator was regulated by a rheostat control, E, for the motor. K was a leaf-type pressure filter, L a rotary fermenter, and R a drain cock. With the use of this apparatus, nutrient sugar solutions were heated for 15 minutes a t 121" C. without apparent caramelization.
General Technique
Eight liters of solution containing the materials for 9.6 liters of fermentation medium (9,4) were charged into the cooker. While the solution was agitated continuously steam was circulated through the aluminum coil and was regulated by valves in the inlet and outlet ( A and B, Figure 1). The contents of the sterilizer were maintained a t 121" C. under 15 pounds per square inch (1 kg. per sq. cm.) steam pressure for 15 minutes and then cooled rapidly to 30' C . by passing cold water through the coil. Positive pressure was maintained within the cooker by sterile compressed air during the cooling period and until the solution was transferred to the fermenter. The necessary amount of calcium carbonate to furnish 2.65 grams per 100 cc. and the remaining 1.6 liters of water were placed in flasks and sterilized separately. The preparation of the inoculum (8, 4) and the charging of the fermenter for the initial fermentation were described previously (3). Upon completion of the fermentation, the fermented liquor was passed through the sterilized pipe line, &, to the previously washed and steam-sterilized aluminum fllter. The clear filtrate was recovered a t outlet P. The fungal growth retained within the filter unit was washed back into the fermenter in the following manner: By proper control of the valves the filter was filled with sterile solution. The solution in the filter was then agitated by compressed air which was exhausted from the top of the filter and through the fermenter. The agitation of the solution in the filter was continued a few moments, and then the solution with the suspended fungal growth was backwashed through the bottom of the filter into the fermenter. This procedure of loosening the fungus from the filter cloths and passing the solution into the fermenter was repeated four or five times. The separately sterilized calcium carbonate and water were made into a slurry and passed into the fermenter through its charging inlet ( 3 ) . Upon completion of charging, the fermenter was disconnected from the filtration assembly 1 Established by the Bureau of Agricultural Chemistry and Engineering, U. 5.Department of Agriculture, in oooperation with the Iowa State College. and fermentation was resumed. 1065
H E semicontinuous proceas previously reported (6) for the biological production of gluconic acid showed that a single culture of AspergiZlus niger, strain 67, could be used repeatedly under agitation, aeration, and pressure in rotary aluminum fermenters. However, because of the flotation technique, only 80 per cent of the fermented liquor was removed a t the completion of fermentation; the remaining 20 per cent, which contained the greater portion of the active fungal growth, was retained within the fermenter. The present paper reports investigations on the semicontinuous process in which the total volume of fermented liquor is removed a t the termination of each fermentation of a series. The liquor is separated from the mycelial growth in a specially designed filter; and the retained mycelia are returned to the fermenter as inoculum for the next fermentation in the series. The construction of a laboratory-size, leaf-type pressure filter of aluminum (2) was undertaken after a number of tests conducted in the previously described large rotary fermenter (9,6) in conjunction with an iron filter gave unsatisfactory results. Evidently the inhibiting effect was caused by the iron and other metals dissolved by the action of gluconic acid and retained by the mycelia, with which these contaminating substances were introduced into the fermentation solution. Aluminum had been previously shown to be nontoxic to the organism, noninhibitory to the gluconic acid fermentation, and not seriously attacked by the organic acids formed in the course of fermentation previously (3). The assembly for laboratory studies employing the small aluminum filter (equipped with 24 X 110 mesh wire cloth) and a small rotary drum fermenter is shown in Figure 1. The solutions were sterilized in a modified pressure cooker and were transferred by means of sterile compressed air. The
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mental data are presented in Table I. Since the results obtained with the small drums can be translated Fermentation No. 1 2 3 4 5 6 7 8 9 directly to the large fermenter holdRefined corn sugar ing a charge of 530 liters (2, 6), charged, kg. 1.575 1.575 1.680 1.680 1.680 1.680 1.680 1.680 1.680 calculations were made o n t h e At the Beginning of Fermentation basis of eleven fermentations in Glucose, g./lOO cc. 16.3 14.2 15.4 15.5 15.4 15.4 15.1 15 3 15.7 the latter for comparison with the Gluconic acid, g./ previously reported yields (6). The 100 cc.) ; : ; ; : ; ; : ; ;:; PH over-all time required for the fer0.12 0.52 0.58 0.49 0.41 0.49 0.40 0.29 0.39 Fungus, g./l.C mentations on the large scale, usAt the Termination of Fermentation ing filtration for the recovery of the Age hr. 25.5 22.0 20.0 19.0 19.7 20.5 20.3 17.0 16.5 mycelia, would approximate 234 Residual glucose, 1.1 1.3 1.7 1.5 1.8 1.7 1.6 1.4 1.1 g./100 00. hours, divided somewhat as follows : Gluconic acid, g./ 100 cc.6 10.5 10.1 10.3 10.5 10.1 10.5 10.5 10.2 12.6 6 hours for preparation of the first Free gluconic acid. charge, 25 hours for the initial ferg . / l O O cc.d 5.9 5.5 6.3 6.9 6.4 6.4 6.0 6.5 4.1 Av.. glucose conver194 hours for the ten mentation, Elon, &/loo cc./ 0.82 0.89 0.67 0.67 0.69 0.68 0.74 0.59 hr. 0.60 succeeding fermentations, and the re8 . 5 3 . 2 3 . 2 3 . 3 8 . 1 3 . 2 3 . 1 3 . 3 3 . 3 PH maining 9 hours for separation and 0.38 0.46 0.50 0.67 0.48 0.49 0.77 0.61 1.25 Fungus, g./l.C recovery. The 5830 liters of solua Inocula for all fermentations, exclusive of t h e in/tial, were fungi retained in aluminum filter and then tion containing 1016 kg. of glucose returned t o t h e fermenter; filtration and transfer time required about 0.75 hour. b Calculated from calcium determinations. would yield approximately 942 kg. Dried a t 80' C. d Determined by titration with standard NaOH. of gluconic acid, hence the production per fermenter per hour would be about 4.02 kg. ?'he yields per fermenter per hour in the singlebatch and flotation methods were 3.18 and 3.46 kg., respecSolutions Containing 16 Grams of Glucose tively, for liquors of approximately the same acid concenper 100 Cc. tration (6). Figure 2 presents the results of a series of nine fermentaThis added advantage may be attributed mainly to the tions inoculated with repeatedly used mycelia separated by practically complete removal of the fermented liquor a t the filtration. The separation of the fungal growth from the end of each fermentation, which permitted the use of a full solution by filtration was not difficult since the final composicharge of fresh medium and not just 80 per cent of the charge tion of the fermented liquors showed a relatively high amount as prescribed in the flotation method. The glucose content of free acid which favored filtration, Earlier studies had of the solution at the beginning of each fermentation was shown that amounts of calcium carbonate in excess of 2.65 approximately that of the prepared solution and was not grams per 100 cc. in a solution containing approximately 15 diluted as in the flotation method by the 20 per cent of liquor grams of gluoose per 100 cc. favored the formation of calcium remaining in the fermenter. It is obvious that the filtration gluconate crystals which tended to retard fermentation (2, 4). method requires fewer fermentations, less time, and hence The presence of relatively large amounts of free acid, although favoring good separation of the growth, retarded its activity before all the glucose had been utilized as shown by curves 3,4, 6, and 8 in Figure 2; the remaining fermentations were usually terminated before such retardation became evident. At this point the p H of the solution was about 3.2 and the free acid concentration approximated 6.2 grams per 100 cc. Under these conditions the filtered liquor at the end of the fermentation contained about 1.5 grams of glucose and 11.8 grams of calcium gluconate per 100 cc. in addition to the free acid. These values, calculated to glucose equivalents, total more than the 16 grams of glucose originally present, an increase in concentration evidently caused by evaporation of liquor during fermentation. The amounts of mycelia returned to the fermenter for each succeeding fermentation averaged about 75 per cent of the amounts present at the end of the preceding fermentations. (Mycelia weights were estimated after treating 100-cc. portions of the solutions with 15 to 25 cc. of concentrated hydrochloric acid, filtering through tared paper, washing, and drying a t 80" C.) The use of a wire cloth of mesh finer than 24 X 110 may retain a greater amount of the smaller strands of mycelia. The semicontinuous production of gluconic acid in a solution of 16 grams of glucose per 100 cc. was satisfactory with the mycelia recovered by the filtration method, although it had been previously reported that the fermentation of solutions to yield 15 grams of gluconic acid per 100 cc. by the FIGURE 1. LABORATORY EQUIPMENT FOR PRODUCIKG GLUmultibatchmethodusing the flotation technique had no decided CONIC ACID BY A SE\fICONTIKUOUS kIETIToD I N t y H I C H THE advantages over the single-batch method ( 5 ) . The experiFUXGAL GROWTHS A R E RECOVERED BY FILTRATION TABLE I,
SEMICONTINUOUS PRODUCTION OF GLUCONIC ACIDFROM BY THE A . niger" RECOVERED BY FILTRATION
;:7"
C
GLUCOSE
;:; ;::
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0:
015
812 g 9 2 6
a3 0
0 TIME OF GLUCOSE IN FIGURE 2. DECREASE
THE
IN
HOURS
SEMICONTINUOUS CONVERSION TO GLUCONIC ACIDBY A . niger EACH FERMENTATION IN THE SERIES
The total contents of the fermenter were filtered after each fermentation.
less manipulation for the production of similar yields of calcium gluconate.
Solutions Containing 12 Grams of Glucose per 100 cc. Previous studies on the multibatch fermentations with recovery of mycelia by flotation showed that solutions prepared to yield 12 grams of gluconic acid per 100 cc. gave the highest rate of acid production per fermenter per hour (6). The retention of 20 per cent of the fermented liquor in the fermenter diluted the fresh charge so that the glucose content was approximately 9 grams per 100 cc. a t the start of each fermentation. Such diluting effect was avoided in a series using filtration for the separation of the fermented liquor at the end of each fermentation and, as in the case of solutions containing 16 grams per 100 cc., these results were calculated on the basis of the large fermenter. On the basis of eleven fermentations on a large scale, using filtration, the over-all time required would approximate 180 hours, divided as follows: 6 hours for preparation of the first charge, 20 hours for the initial fermentation, 145 hours for the ten succeeding fermentations, and the remaining 9 hours for separation and recharging. During this time 762 kg. of glucose would form approximately 745 kg. of gluconic acid and thus produce 4.13 kg. per hour, as compared with the 4.68 kg. reported for the flotation method (6).
Centrifugal Filtration In the series using a concentration of 12 grams of glucose per 100 cc., separation of the liquor from the fungal growth became difficult after the third fermentation, owing to the impervious nature of the filter cake resulting from the accumulated mycelial growth and calcium gluconate crystals. This undesirable crystallization of calcium gluconate could have been avoided by decreasing the amount of calcium carbonate to increase the degree of acidity of the fermented liquors below pH 3.7, the observed value. This step was not carried out since it was desired to have the composition of the final liquor approximate that obtained by the flotation technique (6). Separation of the liquor from the impervious residual mass was accomplished in a stainless-steel basket-type centrifuge. The material retained within a canvas sack in the centrifuge was returned to the fermenter in the form of a slurry made with sterilized nutrient solution. While chance microbial contamination occurred during the process of separation with this particular centrifuge, it apparently caused no interference with gluconic acid production under the conditions of this experiment. It is believed
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TIMEREQUIRED FOR
Mycelia retained in the filter was re-used as inoculum.
that contamination difficulties with this method might seriously interfere with the yields in other fermentations and in other environments, and may be overcome by suitable means, such as housing an enclosed centrifuge in a small chamber similar to a culture transfer room. The equipment could then be sterilized readily by flowing steam and thus permit the recovery and return of the growth without exposure to contamination.
Summary and Conclusions Studies on the multibatch or semicontinuous method for producing gluconic acid by submerged growths of Aspergillus niger showed that mycelia recovered for re-use by filtration satisfactorily fermented a solution containing 16 grams of glucose per 100 cc. Equipment and technique for the recovery of mycelia were developed, which made possible the removal of practically all of the fermented liquor and permitted the use of a full charge a t the beginning of each fermentation. Although gluconic acid production from a solution of 16 grams of glucose per 100 cc. proceeds a t a lower rate than from a solution of 12 grams per 100 CC., the more concentrated solution offers certain advantages: The growth is readily recovered for re-use by simple filtration because the higher concentration of free gluconic acid tends to prevent the formation of gluconate crystals which might form a mat with the fungal growth; the use of the higher concentration of glucose requires fewer fermentations to give the same amount of acid; and the processing of the final liquor is facilitated because of the decreased volumes of liquor involved. The over-all rate of fermentation and therefore the amounts of acid produced per fermenter per hour may be enhanced through the utilization of a filter and equipment designed specifically for work of this nature. Such a design should permit greater recovery and facilitate the return of the mycelia to the fermenter.
Literature Cited (1) Clark, T. F.,Porges, N., and Aronovsky, S. I., IND.ENG.CHEM., Anal. Ed., 12, 755-7 (1940). (2) Gastrock, E. A., Porges, N., Wells, P. A., and Moyer, A. J., IND. ENG.CHEM.,30,782-9 (1938). (3) Herrick, H.T.,Hellbach, R . , and May, 0. E., Ibid., 27, 681-3 (1935). (4) Moyer, A. J., m'ells, P. A., Stubbs, J. J., Herrick, H. T., and May, 0.E.,Ibid., 29,777-81 (1937). (5) Porges, N., Clark, T. F., and Gastrock, E. A., Zbid., 32, 107-11 (1940). (6) Wells, P. A., Lynch, D. F. J., Herrick, H. T., and May, 0. E., Chem. & Met. Eng., 44, 188-90 (1937).
