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INDUSTRIAL AND ENGINEERING CHEMISTRY
resolution of the apparatus and using as specimens pure triesters of glycerol, which are known to give linseed oil its drying properties, more detailed information of the linoxyn structure will undoubtedly be uncovered.
Acknowledgment The author wishes to acknowledge gratefully the many helpful suggestions given during the course of this work by Professors B. E. Warren and Arthur C. Hardy, and to thank the International Printing Ink Company for their courtesy in supplying specimens.
653
Literature Cited (1) Bradley, T. F., ISD. ENG.CHEM., 29, 440 (1937). (2) D’Ans, Angew. Chem., 41, 1193 (1928). (3) Eibner, “ D a s Oeltrocknen,” Berlin, Allgemeinen Industrieverlag, 1928. (4) Germer, L. H . , Rev. Sci. Instruments, 6, 138 (1935). (5) Morrell and Wood, “Chemistry of Drying Oils,” pp. 47, 85, N e w York, D. Van Nostrand Co., 1925. (6) Murkon, C. A . , Phil. Mag., [7] 17, 201 (1934). (7) Thomson, G. P., Proc. Roy. SOC.(London), A133, 19 (1931). (8) Yearian and Howe, Rev. Sci. Instruments, 7, 26 (1936). RECEIVBD March 6, 1937.
GLUCONIC ACID PRODUCTION Effect of Pressure, Air Flow, and Agitation on Gluconic Acid
Production by Submerged Mold Growths P. A. WELLS, A. J. MOYER, J. J. STUBBS, H. T. HERRICK, AND 0. E. MAY Industrial Farm Products Research Division, Bureau of Chemistry and Soils, Washington, D. C.
A
PREVIOUS communication from this division ( 3 ) showed that the production of gluconic acid from glucose by submerged mold growths under increased air pressure greatly reduces the time required to complete the fermentation. This work was carried out on a laboratory scale in special glass apparatus. More recently a new type of rotary drum fermenter for carrying out submerged mold fermentations under pressure was described (1). The development of the rotary drum fermenter for this purpose was the result of continued effort to find the type of equipment best suited for conducting such a process on a commercial scale. The preliminary results presented a t that time on the production of gluconic acid showed that this type of equipment possessed decided advantages over shallow-pan vessels used for this process ( 2 ) . Table I presents some results previously obtained on the production of gluconic acid by different methode. These
TABLE
I.
Type o$ Fermentation
OF GLCCONICACID B Y DIFFERENT results show the advantages of the submerged growth type of METHODS OF MOLDFERMENTATIOW
PRODUCTION
Organism
Fermentation Vessel
Yield of dcid (Theo-
retical)
% Surface Submerged (pressure)
A study of the effect of air flow, agitation¶ and air pressure on the production of gluconic acid from glucose by submerged mold growths in rotary aluminum drum fermenters has revealed that the fermentation rate is, to a large extent, dependent on the proper adjustment of these factors. Under the best conditions found, using 15 per cent glucose solutions, yields of gluconic acid in excess of 84 per cent based on the glucose available and 97 per cent based on the glucose consumed, were obtained in 18 hours from the time of inoculation with germinated spores of Aspergillus niger. The fermentations were carried out in a type of vessel which, it is anticipated, can be easily adapted to large-scale operation.
Penicillium luteum p u r purogenum Penicillium
Shallow pan (aluminum)
57.4
11
Glass bottle
80.4
8
80.0
2.2
(sintered glass false bottom) Submerged PeniciZZium Rotary drum chrysogenum (aluminum) (pressure) a Ueing 20 per cent glucose solutions. ehrysogenun
Fermentation Period Days
fermentation over the surface type. A much greater decrease in time required is obtained when the submerged fermentation is carried out in a rotary drum fermenter instead of in the glass culture vessels used on a laboratory scale. The experimental work on this problem was continued, with the result that the average time required to ferment a 15 per cent glucose solution completely to gluconic acid was reduced to about 35 hours. However, the results obtained from week to week, while satisfactory from the standpoint of yields obtained and time required, were not consistent. Moreover, it was concluded that for commercial-scale operation, the organ-
INDUSTRIAL AND ENGINEERING CHEMISTRY
654
ism Penicillium chrysogenum would be unsatisfactory because it does not readily produce the large quantities of spores necessary for inoculation purposes. Another gluconic-acidforming organism, Aspergillus niger (strain 67 of this laboratory) was therefore selected because it produced spores readily and seemed to have other desirable characteristics. A few preliminary runs with this organism showed that uniform fermentations could be obtained with little difficulty.
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The spore-bearing mycelia from seven flasks (total area, 285 sq. cm.) were added to a 4-liter bottle containing 1500 ml. of a previously sterilized nutrient solution of the following composition: (NHa)zHPO4 KHzPOa MgSOa.7HzO
1.035 grams 0.450 gram 0.375 gram
Peptone Glucose Beer Water to make
0.225 grams 183 grams 100 ml. 1500 ml.
The bottle was stoppered with a rubber stopper through which was inserted a large glass tube bent in such a manner as to prevent the loss of solution during subsequent shaking. This glass tube, which provided access of air for the fungus, was protected at the outer end by sterile cotton. The bottle was vigorously shaken for several minutes to disperse the spores throughout the solution as thoroughly as possible, placed in a horizontal position on a mechanical shaker, and gently agitated back and forth for 46 hours. Three hundred milliliters of this inoculum were used for inoculating each drum. Some variation in the percentage of spores germinated was observed from run to run, but uniform results were obtained with inoculum so prepared. Further work revealed a method of preparing an inoculum which is somewhat better adapted to commercial practice (4). The course of the fermentation was followed by removing samples at intervals for glucose and acid analysis. The fermentation was usually terminated when the crystallization of calcium gluconate occurred, even though some glucose remained unchanged for the reason that the semisolid mass thus formed slows, or in some cases completely stops, the fermentation. All of the drums of any one run were stopped at the same time in order to obtain comparable final yields. Yields were calculated as the ratio of glucose used to produce gluconic acid, to glucose consumed, and to glucose available in the culture solution. The total glucose available was the sum of the glucose originally weighed out plus that contained in the inoculum.
AGE IN HOURS
FIGURE 1. EFFECTOF RATE OF ROTATION OF FERMENTER ON FERMENTATION OF GLUCOSE TO GLUCONIC ACID BY Aspergillus niger
The present report deals with the effect of air flow, air pressure, and the speed of rotation of the drum fermenter (agitation) on the fermentation of glucose to gluconic acid by Aspergillus niger, strain 67.
Experimental Procedure The operation of the rotary drum has been adequately described ( I ) , and the analytical methods used were the same as those previously employed ( 3 ) . Commercial glucose, containing approximately 91.5 per cent glucose and 8 per cent water, was used in all experiments for the culture solutions which were made u p to contain approximately 15 per cent of pure glucose. The actual concentrations were then accurately determined in samples taken from the drums after inoculation and thorough mixing of the solutions. I n all cases the volume of solution added to each fermenter was 3200 ml. The fermentation solution contained the following nutrients : KHzPOI MgSOa.7Hz0 (NHdzHPOd
0.188 gram per liter 0.156 gram per liter 0 . 3 8 8 gram per liter
One hundred grams of sterile calcium carbonate were added to the nutrient solution for each drum a t the time of inoculation. The spores were obtained by cultivating the organism for 6 to 7 days on a nutrient solution of the following composition per liter: MgSOa.7HzO KH2P04 "aN08
0.060 gram 0 , 0 7 2 gram 0 , 4 5 0 gram
Glucose Beer
9 1 . 5 grams 60 ml.
Erlenmeyer flasks (capacity, 200 ml.) were used as culture vessels; each contained 50 ml. of solution. The inoculum for the drum fermenters was prepared from the spores by germinating them in the following manner:
I,
#I
,.
,I
.. I,
4, IO
AGE IN HOURS
FIGURE 2. EFFECTOF AERATION ON FERMENTATION OF GLUCOSETO GLUCONIC ACID BY Aspergillus niger
The curves in Figures 1, 2, and 3 show the results of analysis for glucose in samples taken a t intervals during the runs. The corresponding results obtained for acid analysis are not shown, since in all cases they reveal the same relationships.
Effect of Agitation The effect of agitation on the rate of fermentation, as measured by the decrease in the glucose concentrations, was studied by varying the speed of rotation of the fermenters. Adequate provision for such variations in the speed had been made in the set-up by means of pulleys. A gage pressure of 30 pounds per square inch (2.11 kg. per sq. em.), an air flow of 400 ml. per minute, a volume of 3200 ml., and a temperature of 30" C. were used in this study; 496 grams of glucose were available, and the drum circumference was 2.36 feet (71.9 cm.). The results obtained are shown in Figure 1 and
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655
decrease of the glucose concentration during this period was 1.5 per cent per hour. The initial lag period in this experiment was approximately 2 hours longer than that which usually occurred (Figure 2). This explains the difference in yields obtained a t 30 pounds per square inch (2.11 kg. per sq. cm.) gage pressure (Table IV) and those shown in Table 111,under the same conditions of air flow and rate of rotation. This difference with respect to time amounts to about 2 hours, which is not unusual in such a biochemical process. A gage pressure higher than 45 pounds per square inch was considered unsafe with the equipment available. Moreover, some difficulty in preventing leaks was experienced a t the highest pressures employed. A gage pressure of 30 pounds TABLE 11. EFFECTOF ROTATION RATEOF DRUMFERMENTERSper square inch is most practical on the basis of these results. ON FERMENTATION OF GLUCOSE TO GLUCONIC ACIDBY A s p e r -
Table 11. The yield of gluconic acid obtained a t 4.5 r. p. m. (peripheral speed 10.6 feet, or 3.2 meters, per minute) was more than doubled by increasing the speed to 10.2 r. p. m. The yield of acid based on the glucose consumed was also increased, indicating a more efficient utilization of glucose for acid formation. A further increase in speed was apparently without effect, as indicated by the results obtained a t 13 r. p. m. Moreover, a t speeds higher than 13 r. p. m. excessive frothing occurs which, in some cases, may clog the exit pipe. S o trouble of this kind was experienced a t a speed of 13 r. p. m., and this rate was used in studying the effect of air flow and pressure.
g i l l u s niger
Discussion of Results
F ~ ~~l~~~~~ G ~ ~ ~ $ iGluconic c Acid Yield Based on: Speed
R. p . m. 4 5
mentation ConPeriod sumed Hours Grams 26
208
Produced Grams 203 254
Glucose consumed
Glucose available
%
%
89.7 93.0
37.7 47.7
Effect of Air Flow The effect of air flow on the fermentation rate was next studied. The results are shown in Figure 2 and Table 111. A gage pressure of 30 pounds per square inch and a speed of 13 r. p. m. were used with all the drums; other conditions were 3200-ml. volume, 30" C., 18-hour fermentation period, and 495 grams of available glucose. The effect of greatly increasing the oxygen supply is shown by the curves of Figure 2 and in the final yields of gluconic acid obtained a t 18 hours (Table 111). It is apparent that an increase of air flow over 1200 ml. per minute is without any significant effect in reducing the fermentation period. The fermentation rates a t 400 and 800 ml. per minute are not appreciably lnwer than those a t 1200 and 1600 ml. per minute. In practice the shorter fermentation period, which can be obtained with a ~ justify the added cost of supplying high air flow, W O U Ihardly the larger amount of air required. TABLE 111. EFFECT OF AERATION ON FERMENTATION OF GLUCOSE TO GLUCONIC ACIDBY A s p e r g i l l u s niger Air Flow iMl./min.
Glucose Consumed Grams
~ l Acid Produced Grams
200 400 800 1200 1600
248 310 349 410 413
269 317 360 420 429
Gluconic Acid ~ ~ Yield ~Based on: ~ Glucose Glucose consumed available
% 99.5 93.7 94.7 94.2 95.4
% 49.8 58.8 67.8 77.9 79.5
Effect of Air Pressure The effect of air pressure on the fermentation rate was next studied, using an air flow of 1200 ml. per minute, a speed of 13 r. p. m., a volume of 3200 ml., a temperature of 30" C., and a fermentation period of 18 hours; 495 grams of glucose were available. The remarkable effect of increased air pressure on the fermentation is shown by the curves in Figure 3 and by the final yields in Table IV. At 45 pounds gage pressure (3.16 kg. per sq. cm.) a yield of 84.2 per cent of gluconic acid was obtained in 18 hours, as compared to 32.1 per cent a t 5 pounds gage pressure (0.35 kg. per sq. cm.) during the same period. For this same drum a t 45 pounds gage pressure the fastest fermentation rate ever encountered in this work OGcurred between the 12- and 18-hour period. The average
The three factors which condition the oxygen supply for the fungus-air flow, agitation, and pressure-have a pronounced effect on the fermentation rate. These three closely related factors determine to a large extent the speed and efficiency of the process. In order to obtain the best result (highesd yield in- the shortest time), it is essential that all three factors be p r o p e r l y adjusted to meet the oxygen requirements of the f e r m e n t i n g o r g a n i s m . This is strikingly illustrated by the results shown in curves A and D , F i g u r e 3 . This experiment was carried out using an air supply a n d a s p e e d of r o t a t i o n of the ferm e n t e r which were known to be favorable for acid formaR.P M 13' tion; y e t w i t h low p r e s s u r e (curve A ) the fermentation lagged far behind that o b t a i n e d when the AGE IN HOURS was greatly i pressure ~ FIGURE 3. EFFECTOF AIR PRESSURE increased (curve D ) . ON FERMENTATION OF GLUCOSETO GLUCONIC ACIDBY Aspergillus niger Such a rapid fermentation has a great many advantages over those which require more time. For example, ~ e n i i i ~ i u rpurpurogenum, n a-contaminating organism which was known to have a pronounced adverse effect on acid formation when the fermentation period exceeded 48 hours, had no such influence when the run was completed in 20 hours. It may thus be possible to carry out this fermentation on a large scale without sterilization of either the equipment or the nutrient solution, which is an important cost item in any such process. TABLEIV. EFFECTOF AIR PRESSURE ON FERMENTATION OF GLUCOSE TO GLUCONIC ACIDBY A s p e r g i l l u s niger ~ l Gluconic ~ Acid ~ Yield Based ~ on: ~ Glucose Acid Glucose Glucose Gage Pressure Consumed Produced consumed available Lb./sq. in. ( k g . / s q . cm.) Grams Grams % %
5 15 30 45
(0.35) (1.05) (2.11) (3.16)
178 257 336 429
173 258 351 454
89.1 92.1 96.0 97.1
32;l 47.9 65.1 84.2
i
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656
The adaptation of these results to large-scale operation should not prove difficult. Other conditions being equal, the most important factor is the oxygen supply available to the organism. This supply is conditioned by the air pressure, the air flow through the fermenter, and the degree of agitation of the solution. I t is probable that the latter, because of the different dimensional relationships involved in the use of large equipment, may prove to be more adequately expressed in terms of peripheral speed rather than in revolutions of the fermenter. The construction of a much larger fermenter, similar in principle to the small fermenters used in this work, has now been completed. This equipment will make possible a study of the various factors involved in this process, on a scale
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closely approaching that which would be used in commercial operation.
Literature Cited (1) Herrick, H. T.,Hellbach, R., and May, 0. E., IND.ENG.CHEM., 27. 681-3 (1935). (2) May; 0. E., Herrick, H. T., Moyer, A. J., and Hellbach, R., Ibid.. 21, 1198-1203 (1929). (3) May, 0. E.,Herrick, H. T., Mouer. . . A. J., and Wells, P. A.. Ibid.. 26, 575-8 (1934). (4) Moyer, A. J., Wells, P. A,, Stubbs, J. J., Herrick, H. T., and
May, 0. E., Ibid., to be published.
RECEIVEDApril 8, 1937. Presented before the Division of Industrial and Engineering Chemistry at t h e 93rd -Meeting of t h e American Chemical Society, Chapel Hill, N. C . , April 12 to 15, 1037. This paper is Contribution 273 from the Industrial Farm Products Research Division.
COMPOSITION OF ZINC YELLOW A. A. BRIZZOLARA, R. R. DENSLOW, AND S. W. RUMBEL Krebs Pigment & Color Corporation, Newark, N. J.
T
HE wide use of zinc yellow Eight typical samples of zinc yellow amount of free zinc oxide. AIby paint manufacturers were analyzed ; the indicated that though chemical specifications and the growing appreciafor pigment colors are recognized this pigment has the composition Kz0.- to be of less tion of its value in rust-inhibitthan practical 4Zn0.4Cr03.3HzO- The results, which tests related to actual use, the ing paint formulations havemade have been submitted to the National general importance of the subthe question of its chemical comBureau of Standards, are discussed in ject, as well as the interest of the position a matter of considerable i n t e r e s t . The National their relation to those found in the literaNational Bureau of Standards, Bureau of Standards is interested was sufficient to justify a rather in finding out how much zinc turem A system Of is e x t e n s i v e analytical study. yellow is present in rust-inhibiThe results have been reported tive paints submitted under a specification calling for a definite to the bureau, where they are now under consideration, and amount of zinc yellow; one reason for carrying out the present are reviewed briefly together with the analytical methods study was to develop data of possible interest to the bureau which are believed to be most reliable for the purpose. in this connection. Table I shows the composition of eight typical samples Pigment manufacturers have long known that the product of zinc yellow, representing the products of three American is a more or less complex salt containing not only zinc and pigment manufacturers. Although careful evaluation would chromium but also substantial amounts of potassium. It is indicate certain differences in pigment properties, tinting likewise basic in character. Simply mixing solutions of a strength, etc., all were quite similar and would be considered zinc salt and a soluble chromate results in a product of low typical examples of this pigment. tinting strength and generally poor tinctorial properties, The generally close agreement between the samples in lacking the clean green tone of the commercial products respect to composition is striking, particularly when the available on the American market. However, the formulas difficulty of some of the separations is considered and the additional probability of differences in manufacturing procreported in the early literature are conflicting and of doubtful significance. esses. The figures point to the (‘oxide formula,” K2O.An interesting paper by Ellis, Fox, and Hirst ( 1 ) which 4Zn0.4CrOs.3H~0,with a fraction of the chromate replaced appeared some years ago represents the first important by an equivalent amount of sulfate or basic sulfate. It differs attempt to determine the quantitative composition of the from the one suggested by Ellis et al., in that the latter contains a higher proportion of zinc, corresponding to an addipigment. The authors pointed out the importance of suitable analytical methods and indicated the possibility of tional mole of zinc hydroxide. What is designated as ‘(combined water” is apparently serious error in estimating any component “by difference.” On the basis of their experiments, they concluded that the very firmly held. Drying a t 110’ C. gave a loss in the range of 0.1 to 0.2 per cent, and a much higher temperature average product was 5Zn0.4Cr03.K20.4HzO but suggested that too much importance should not be attached to this is needed to effect the removal of the 6 to 7 per cent reported in Table I. stoichiometric ratio. This caution was probably justified Tests on the alkali metal residues indicated very little in view of the rather wide variations in composition between sodium to be present. Sample A, for example, contained the samples which they analyzed. only 0.51 per cent, undoubtedly an impurity. Preliminary analytical work in the present writers’ laboratory on various American zinc yellows indicated that Analytical Methods they are lower in zinc content (or more specifically, in the REAGENTS. The reagents used were sulfuric acid ( 6 N ) , ratio of zinc oxide to chromic oxide) than those reported by Ellis et al., suggesting that the latter might contain a certain hydrochloric acid (concentrated), potassium ferrocyanide,
