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INDUSTRIAL AND ENGINEERING CHEMISTRY
the char for a sufficient length of time. By control of recirculation the gases to the stack could be brought down to any desired temperature due to the cooling action of the damp char as i t entered the drum, and the stack temperature could easily be held down t o 140’ F. with very little heat loss. The use of an air lift for bone char has been the only method used in Australian refineries for many years. Laboratory experiments in blowing char through glass tubes showed no abrasion if vertical pipes were used, but the least bend caused rapid abrasion both of the pipe and the char. Raising the char tn the desired height can best be accomplished in two steps wherel)y the char can be cooled to any desired temperature and all the heat returned to the combustion chamber.
THE fuel used was so-called furnace oil, which cost at that time about 7.8 cents per gallon. Fuel costs were found to be the same per pound of char treated in this internally fired kiln as they were in the norrnal type of kiln in which buckwheat coal was burned. If other cheaper fuel was used, either crude oil or natural gas, the fuel saving would be considerable; and since the control [ i f temperature proved not to be as critical as was at first supposed, a n ordinary type of burner with simple control apparatus could be satisfactorily used. It was found easy to hook the various units of the apparatus together in such a fashion that if the fire went out or any single step was interrupted for any reason, all the units stopped. This worked very well and merely required that some one should determine what was wrong and again start the units in proper sequence. No continual attention was needed and the labor cost was consequently small.
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The initial cost of this improved kiln with all its accessories was ahout equal to the average cost of upkeep for 2 years of two ordinary retort kilns which together have an equal capacity. This indicates a greatly reduced overhead cost for this department. The space required was one floor in height with a small hopper beneath the floor level to receive the reactivated char and discharge i t in turn into the bottom of t h e air-lift tube. Three floors are required for the ordinary vertical retort kiln with the necessary dryer above and cooler pipes below. The plant-scale unit was operated intermittently for a year and a half on low-grade char. A mass of operating data and many analyses were made of the char before and after treatment, as well as many comparative tests to ’indicate variations in the decolorizing ability of the char compared with that produced in the usual type of kiln and in the Weinrich apparatus. In no case were variations in results from the different methods of treatment greater than differences in day-to-day samples of material from any one method of treatment. It was not possible to segregate the treated char at that time, and the whole factory was dismantled before experiments could be made on new char. It is unfortunate that the work was terminated, but the process is not so radically different that one could expect abnormal results in a short period of time. The prospective large savings in operating and overhead costs make the possible use of the process attractive.
Literature Cited (1) Rav, A. B., Chem. & Met. Eng., 28, 977 (1923). (2) Rice, E. W., U. S. Patent 1,758,202 (May 13, 1930).
SORBOSE FROM SORBITOL Semiplant-Scale Production by Acetobacter suboxvdans J
P. A. WELLS, L. B. LOCKWOOD, J. J. STUBBS,AND E. T. ROE U.S. Department of Agriculture, Washington, D. C.
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N A PREVIOUS communication ( 3 ) studies on I-sorbose
production from sorbitol by submerged growths of Acetobacter suboxydans were described. Rotary drum fermenters were employed as culture vessels, since in a n earlier study of a similar oxidative fermentation process this type of equipment had proved to be satisfactory in providing the necessary conditions of agitation and aeration ( 2 ) . A consideration of the various factors involved in adapting the laboratory-scale sorbose process to pilot-plant scale suggested the desirability of further study. The object was to simplify the procedure and to effect certain economies of operation which, although of relativdy minor consideration in the laboratory, would be of importance for commercial use of the process. This paper reports the use of concentrated corn steep liquor as a nutrient material for the fermentation, a simplified method for large-scale inoculum preparation, further results on the effeck of sorbitol concen-
N. PORGES AND E. A. GASTROCK U. S. Agricultural By-products Laboratory, Iowa State College, Ames, Iowa
tration, and the adaptation of the process to semiplant-scale operation.
Materials and Methods (1) LABORATORY-SCALE EXPERIMENTS.The equipment, methods of analysis, materials employed, organism and culture methods were, in general, the same as those previously described ( 3 ) . Ten per cent sorbitol solutions were used in all cases
unless stated otherwise. I n experiments requiring small rotary fermenters, an air flow of 375 cc. per minute er liter of solut.ion, an air pressure of 30 pounds per square incff (2.11 kg per sq. cm.), a temperature of 30’ C., and a rotation rate of 13 r. p. m. were used sinre these conditions were found in the earlier work t o be most satisfactory for sorbose production. T h e concentrated corn steep liquor employed as a nutrient substitute for dried yeast extract was obtained from the A. E. Staley Manufacturing Company, and was known as “Yeast Compound”. Analysis supplied by the manufacturer showed that this product contains on the average 55-60 per cent solids, of which the protein content is 45 per cent, mineral matter, 18
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Continuation of previous studies on 2sorbose production from d-sorbitol by submerged growths of Acetobacter suboxydans revealed that concentrated corn steep liquor was a satisfactory nutrient to substitute for dried yeast extract although its use necessitated the addition of an antifoam agent, octadecyl alcohol. In the presence of a slight excess of calcium carbonate which afforded satisfactory pH control, dsorbitol in concentrations up to 30 per cent was rapidly oxidized t o Z-sorbose. Pilotplant-scale results revealed no essential differences from those obtained on a laboratory scale. When 20 per cent sorbitol solutions were employed under the most favorable conditions, the yields of sorbose obtained in fermentation periods of 25 hours on both laboratory and pilot-plant scale were in excess of 90 per cent. Recovery yields of crystalline sorbose approximated 70 per cent.
per cent, and carbohydrates, 38 per cent. The highly acidic steep liquor was neutralized before use by the addition of the calculated quantity of U. S. P. grade calcium carbonate. The amount of calcium carbonate required was determined by titration of a weighed sample of the steep liquor with standard alkali to about pH 5, using Bromocresol Green as an outside indicator. Octadecyl alcohol (Eastman Kodak Company's technical grade) in the amount of 0.3 gram per liter of solution was added to the culture medium before sterilization to prevent frothing during the fermentation. SEMIPLANT-SCALE EXPERIMENTS. These experiments were made at the United States Agricultural By-Products Laboratory, where the large rotary fermenter ( 1 ) was located. The sorbitol sirup was a technical grade and differed on1 slightly in composition from that used previously (3). I% contained 74.9 per cent sorbitol, 2.8 glucose, 0.9 sodium sulfate, and 21.4 water. The concentrated corn steep liquor which was added in the amount of 3 grams per liter was a separate lot of the "Yeast Compound". Calcium carbonate was added in an amount sufficient to maintain the pH within the range 4.2 to 6.4. The total amount required was determined by the acidity of the steep liquor and the amount of glucose in the sorbitol sirup. Preliminary experiments showed that both the hard and softened tap water were satisfactory for these fermentations, and the latter was used throughout the ex eriments. The pressure and rate of air flow were the same as for the laboratory experiments given above. A rotation rate of 9.5 r. p. m. was employed since studies on gluconic acid production showed that this rate gives results comparable to those a t 13 r. p. m. with the smallscale fermenters. The temperature of the fermenting solution was maintained automatically a t 30" C. (1). The c u h r e solutions were prepared by addition of the water, sorbitol sirup, steep liquor, calcium carbonate, and octadecyl alcohol to the fermenter; after the fermenter was rotated for about 10 minutes, the solution and apparatus were sterilized by heating a t 105110' C. for 75 minutes. Heating was effected by passing steam through aluminum tubing attached to the interior surface of the fermenter. During the subsequent cooling to 30" C. positive air pressure was maintained in the drum to inhibit entrance of contaminants. To the sterile culture medium (approximately 530 liters) were added 24 liters of a previously developed inoculum, the preparation of which is described later in this paper. The fermenter was then placed in operation, and the course of the reaction was followed by the removal of samples a t intervals for sorbose analysis. SORBOSE RECOVERY. When the fermentation was completed, the solution was discharged from the fermenter through a pipe line into several small aluminum tanks, decolorizing carbon
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and Filter-Gel were added, and the mixture was filtered through a filter press. The clear colorless filtrate was concentrated in a copper still under a vacuum of 22-28 inches (560-710 mm.) of mercury at about 60" C. The warm concentrated solution, which already contained a large amount of crystalline sorbose, was removed from the still and allowed to stand overnight at about 15" C. to effect further crystallization. The mass was then centrifuged, washed with ice water, and dried. The mother liquor and washings were combined and treated in the above manner t o obtain a second crop of crystals. Recovery yields, based on the sorbose in solution, approximated 70 per cent.
Nutrient Studies I n the work described previously (3) 5 grams of dried yeast extract per liter were employed as the sole nutrient substance, b u t because of the relatively high cost of this material a n effort was made to find an inexpensive substitute in the large-scale operation. Of a number of substances tried,-corn A-CORN STEEP LIOUOR 0.3 V O U
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4 6 8 IO 12 I 4 16 FERMENTATION PERIOD IN HOURS
FIGURE 1. COMPARISON OF CORN AND DRIEDYEAST STEEPLIQUOR EXTRACT AS NUTRIENTS IN SORBOSE FERMENTATION
steep liquor appeared to be best; i t caused excessive frothing of the culture solution, but this disadvantage was overcome by the addition of a small amount of octadecyl alcohol to the medium. Additional experiments using gas washing bottles as culture vessels indicated that a steep liquor concentration of 3 grams per liter was satisfactory. I n order to confirm these results and to test them under conditions similar t o those to be employed in the large-scale experimental work, a comparison of yeast extract and corn steep liquor as nutrients for the sorbose fermentation was made with small rotary fermenters as culture vessels. The initial results were not entirely satisfactory, the rate of sorbose fermentation being somewhat slower with steep liquor than with yeast extract. Routine measurements of the p H samples removed for analysis at intervals during the fermentation revealed what proved to be the cause of this difference. The p H of the yeast extract medium was initially in the range 4.8-5.0, and during the first few hours of fermentation i t decreased to a fairly constant value of about 4.2; with steep liquor from an initial p H of 5.0-5.2 the value decreased to 3.5-3.7 However, when the p H of the steep liquor medium was maintained above 4.2 by the addition of calcium carbonate, the rate of sorbose formation was essentially the same for both nutrient materials. Typical results are given in Figure 1. The agreement was close throughout the fermentation period of 16 hours during which over 90 per cent of the sorbitol was converted to sorbose. These results showed that under proper conditions corn steep liquor could be satisfactorily employed as the sole
INDUSTRIAL AND EN(3INEERING CHEMISTRY
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nutrient for the sorbose fermentation. This item of cost can thus be reduced to a negligible factor.
Effect of Sorbitol Concentration Previously it was reported that solutions in which the initial concentration of sorbitol was 30 per cent were unsatisfactorily fermented, whereas with concentrations up to 20 per cent high yields of sorbose were obtained. The results of studies on steep liquor as a nutrient in relation to pH suggested that a slight but important difference in pH might be
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SORBITOL CONCENTRATION A - I O PER CENT 8-15 PERCENT
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glass gas washing bottles, such a method was adapted t o large scale use. The procedure developed was as follows: Cultures were prepared by flooding, with a suspension of bacteria from 48-hour test tube slants, the surface of 50 ml. of medium (2 per cent agar, 5 per cent sorbitol, 0.5 per cent yeast extract) contained in Kolle flasks. After incubation for 3 days a t 30' C., 50 ml. of sterile water were added to each Kolle flask, and the agar surface was scraped. The resulting suspension from a single Kolle flask was transferred to a wide-mouth 9-liter Pyrex bottle which contained 6 liters of sterile culture solution. This culture solution contained, in grams per liter: sorbitol, 100; Difco yeast extract, 5 ; octadecyl alcohol, 0.5. The 9-liter Pyrex bottle was fitted with a rubber stopper through which was inserted a sintered glass distribution tube (Jena, type 33c porosity No. 0) extending close to the bottom of the bottle. The outer end of the tube was connected to a source of sterile air, the flow of which was controlled by a needle valve. A second tube inserted through the stopper provided an exit for the gas which was measured by means of a flowmeter. After inoculation, sterile air was passed through the culture solution a t the rate of 12 liters per minute. The relatively large flow of air was necessary to provide sufficient aeration and agitation of the solution. Analyses for acid and sorbose content and p H measurements were made on samples removed through a glass tube inserted through the stopper and extending into the solution. This preparation was used t o inoculate the fermentation solution a t the end of 48 hours, a t which time the sorbose concentration usually ranged from 6 to 7 per cent. These inoculum preparations were highly active, and when tested on a small scale in the proportion of one part to thirty-
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FIGURE2. EFFECTOF SORBITOL CONCENTRATION ON THE PRODUCTION OF SORBOSE BY SUBMERGED GROWTHSOF Acetobacter suboxydans the cause of failure to ferment solutions of 30 per cent sorbitol concentration satisfactorily. Although no pH data were recorded in this earlier study, it seemed probable that decreases in pH had occurred with the use of higher sorbitol concentrations due to the formation of larger amounts of gluconic acid from the glucose present in the commercial sorbitol sirup. Glucose is readily oxidized to gluconic acid by Acetobacter suboxydans under the conditions used. Studies on the effect of pH on sorbitol fermentation had shown a critical range a t pH 3.8-4.0. Accordingly an experiment was carried out (Figure 2) in which sorbitol a t initial concentrations varying from 10 to 35 per cent was fermented in the presence of a small excess of calcium carbonate over that required to neutralize the corn steep liquor and the gluconic acid formed from the small amount of glucose in the sorbitol sirup. Under the conditions employed, sorbitol in various concentrations up to 30 per cent was readily oxidized to sorbose, while a t 35 per cent concentration oxidation was negligible. From the standpoint of economical sorbose production, the ability of the organism to effect the rapid oxidation of sorbitol in a concentration of 30 per cent is distinctly advantageous.
Semiplant-Scale Operation INOCULUM PREPARATION. I n the previous report (3) a method for large-scale inoculum preparation was described which involved the use of small laboratory-scale rotary fermenters. Simplification of this operation in the process was considered desirable, and since highly active inoculum for the earlier experimental work had been obtained with Jena-
FERMENTATION
PERIOD IN HOURS
FIGURE 3. TYPICALPILOT-PLANTON THE PRODUCTION SCALERESULTS O F kSORBOSE FROM &SORBITOL two parts of fermentation solution, rapid oxidation of the sorbitol to sorbose resulted. I n the experimental work on the semiplant scale, 24 liters of inoculum prepared in the above manner were used for inoculating a 530-liter batch of culture medium. The results obtained throughout all of this work showed that such preparations possessed a uniformly high degree of activity. The adaptation of the laboratory LARGE-SCALE RESULTS. procedure to large-scale operation was somewhat simplified by previous experience obtained in making a similar study of gluconic acid production from glucose by mold fermentation (1). As mentioned previously, the most satisfactory conditions of aeration and agitation found for the large-scale gluconic acid process were also used for the sorbose experiments, since the optimal conditions were similar for both
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INDUSTRIAL AND ENGINEERING CHEMISTRY
fermentations on a small scale ( 2 , 3 ) . The experimental work was limited to five separate runs which were planned primarily to demonstrate the applicability of the laboratory procedure under pilot-plant conditions. The results given in Figure 3 show the progress of sorbose formation for two fermentations in which the initial sorbitol concentrations were 10 and 20 per cent. Comparable smallscale results (curves A and C, Figure 2) show that fermentations proceeded a t the same rate on both the laboratory and semiplant scale. The conversion to sorbose was completed in 16 and 25 hours when the sorbitol concentrations were 10 and 20 per cent, respectively. PILOT-PLANT-SCALE RESULTS ON THE PROTABLE 1. TYPICAL DUCTION O F &SORBOSE F R O M d-SORBITOL BY A . suboxydans
Weipht yield based on sorbose recovered, %
As Figure 2 shows, the initial lag period was increased approximately 8 hours when the sorbitol concentration was increased from 10 to 30 per cent. This suggested t h a t oxidation of sorbitol in high concentrations could be carried out in a shorter period of time by using a low concentration until vigorous fermentation had started and then raising the content t o the desired level by the addition of sterile sorbitol sirup. An experiment of this kind was made in which the initial sorbitol content was 10 per cent. When the sorbitol concentration had decreased to approximately 8 per cent, sterile sorbitol sirup was added to make the total concentration of sorbitol and sorbose equivalent t o an initial concentration of 28.6 per cent sorbitol. The highest sorbose concentration (26.6 per cent) was attained in 29 hours as compared to 34 hours for the same sorbose content when the initial sorbitol value was 30 per cent (curve E, Figure 2). The difference in time cannot be considered of any practical advantage in view of the increased difficulties of operation. Sorbose recovery and yield figures for this experiment are given in Table I. I n view of certain advantages t o be gained from semicontinuous operation of the process, the feasibility of such a procedure on a pilot-plant scale was investigated. The plan was simply to remove a portion of actively fermenting nutrient solution from one run and to use this as inoculum for a succeeding fermentation. The first fermentation was made in the usual manner from 10 per cent sorbitol with inoculum prepared in large bottles as described earlier in this paper. When the sorbose concentration reached 7.5 per cent, an appropriate amount of solution was withdrawn under sterile conditions from the large fermenter; during the time required to complete the first run and to prepare and sterilize the solution for the second fermentation, the transferred medium was kept under conditions similar t o those used for inoculum preparation in bottles. It was then used t o inoculate the charge for the second run. An additional period of 5 hours was required for completion of the second fermentation over t h a t required for the first one. The use of such a procedure for semicontinuous operation would be simplified greatly when two or more fermenters are employed. This would allow the transfer of inoculum directly to the succeeding batch to be fermented and thus minimize the danger of contamination by other organisms. This latter difficulty probably would represent the limiting factor on the extent t o which successive transfers could be made, although degenerative changes of the organism might also occur which would necessitate starting again with freshly prepared inoculum.
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Summary 1. Concentrated corn steep liquor in the amount of 3 grams per liter was found t o be a satisfactory and inexpensive nutrient for the sorbose fermentation. Although the use of this nutrient caused excessive frothing of the culture solution, this objection was overcome by the addition of a small quantity of an antifoam agent (octadecyl alcohol) to the medium. 2. Under comparable conditions no differences in the rate of sorbose formation were observed throughout the p H range 4.2 t o 6.4. Satisfactory p H control was obtained by the addition to the fermentation solution of a slight excess of calcium carbonate over t h a t required t o neutralize the highly acidic corn steep liquor and the gluconic acid produced from the small amount of glucose present in the sorbitol sirup. 3. Sorbitol in concentrations up to 30 per cent was rapidly and effciently oxidized to 1-sorbose. For 10, 15, 20, 25, and 30 per cent sorbitol solutions practically quantitative conversion to 1-sorbose occurred during fermentation periods of 13.5, 17, 24, 30.5, and 45 hours, respectively. 4. A simplified method for the preparation of uniform and highly active inoculum was developed. The method was found suitable for moderately large-scale operation of the process and required only glass apparatus which is readily available and relatively inexpensive. 5. The results obtained in pilot-plant-scale adaptation of the sorbose process were essentially the same as those on the laboratory scale.
Acknowledgment The writers wish t o express their appreciation for assistance rendered by T. F. Clark during the pilot-plant studies.
Literature Cited (1) Gastrock, E. A,, Porges, N., Wells, P. A., and Moyer, A. J., IND.ENG.CHEM.,30, 782-9 (1938). (2) Wells, P. A., Moyer, A. J., Stubbs, J. J., Herrick, H. T., and May, 0. E., Ibid., 29, 653-6 (1937). (3) . . Wells. P. A,. Stubbs. J. J., Lockwood, L. B., and Roe, E. T., Ibid., 29, '1385-8 (1937):
