OCTOBER,1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
Literature Cited (1) Campbell,
hf. R . ,
u. S.Geol. Survey, Professional
Paper 1OOA
(1922). (2) Dobbin, C. E., Ibid., Bull. 812, 1-55 (1930). (3) Eisner, A., and others, U. S. Bur. Mines, Rept. Inzestigations 3498 (1940). (4) Fisher, C . H., and others, I~w.EBG.CHEM.,31, 1155-61 (1939). ( 5 ) Hirst. L. L.. Storch. H. H.. Fisher. C. H.. and Surunk. G. C.. Ibid., 32, 864-71 (1940). (6) Hirst, L. L., and others, Ibid.,31, 869-77 (1939). ( 7 ) Keystone Coal Buyers' Manual, p. 226, New York, McGrawHill Book Co., 1939.
1379
(8) Leonard, -4.G., and others, N. Dak. Geol. Survev. Bull. 4. 240 (1925). (9) Starch, H. H., and Fieldner, A, c,, $fech. E ~ ~61, . ,605-11 (1939). (10) Storch, H. H., Hirst, L. L., Fisher, C. H., and Sprunk, G. C., U. S.Bur. Mines, Tech. Paper 622 (1940). (11) Storch, H . H., and others, IND.ENG.CHEM.,29, 1377-80 (1 937). snrvey, ~ ~ 2 341, 2 , 123-5~, (19~9). (12) ~ ~ J. f fA.,, U. s. PRESENTED before the Division of Gas and Fuel Chemiatry at the 99th Meeting of the American Chemical Society, Cincinnati, Ohio. Published (Not subject t o copyright.) hy permission of the Director, U. S. Bureau of Mines.
t
Fermentation of Concentrated Solutions of Glucose to Gluconic Acid Improved Process A. J. MOYER, E. J. UMBERGER, A
4J. J. ~ STUBBS ~
Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Washington, D. C.
A difficulty encountered in previously described processes for the fermentation of concentrated solutions of glucose-namely, the inhibition of the fermentation by precipitation of calcium gluconate or through injury to the fermenting organism by exposure to free gluconic acid-has been overcome by the addition of boron compounds during the fermentation to prevent the precoipitation of calcium gluconate. This use of boron compounds, such as boric acid or borax, with an excess of calcium carbonate permits the continuous neutralization of the gluconic acid formed. With an improved nutrient medium and an organism able to tolerate the amount of boron necessary, it is possible to ferment 20, 25, and 30 per cent solutions of glucose in half the time previously reported. Under these conditions and with a process made semicontinuous by re-use of the mycelium, it is possible to ferment 25 per cent solutions of glucose to gluconic acid every 24 hours. This method is applicable to gluconic acid fermentations by certain bacteria as well as by fungi.
HE presence of a neutralizing agent such as calcium
T
carbonate in the culture medium favors the fermentation of glucose to gluconic acid by molds (2, 10, 19). Previous data from this division (10, 18, 14, 17) have shown that the gluconic acid formed from 12-14 grams glucose per 100 cc. of culture medium can be completely neutralized by calcium carbonate without precipitation of calcium gluconate during the fermentation. Precipitation of calcium gluconate during the fermentation was associated with an immediate and marked inhibition of the fermentation. It was therefore necessary to conduct the later stage of the fermentation of more concentrated glucose solutions in an acid medium. There is considerable evidence to show that inhibition of the fermentative capacity of the fungus growth occurs after prolonged exposure to a n acid medium. It was shown (12) that prolonged exposure of the mycelium to free gluconic acid caused a marked increase in the lag period in subsequent fermentations when the mycelium was re-used as an inoculum. Also the decrease in the fermentation rate encountered during the final stage of the fermentation of 20-30 per cent solutions of glucose was believed (12, page 781) to be due to the inhibiting effect of an acid medium (pH 3.G3.5). This type of inhibition made i t impossible to ferment satisfactorily a medium containing 30.0 grams of glucose per 100 cc. It was apparent that if some way could be found to overcome these inhibitions to the fermentation, full advantage could be taken of the use of concentrated media. Such procedures might be of interest to industrial utilization of this fermentation process, where efficient use of time, equipment, and labor are important considerations. The solubility of calcium gluconate at 30' C. (the tempera-
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ture a t which the fermentations were conducted) is only about 4.0 grams per 100 cc. (4, 7 , 1 1 ) . This salt readily forms a supersaturated solution, which is responsible, a t least partially, for the f&ct that in the fermenters no precipitation occurred until a calcium gluconate concentration of 13-15 grams per 100 cc. was reached. TABLEI. PRECIPITATION OF PURE CALCIUM GLUCOXATE IN THE PRESENCE OF DIFFERENT AMOUNTSOF BORICACID Grams Ca Gluronatein 100 c c . 15 20 25
30
Boric Acid Added Boron, 3’% bprio p. p. m. acid 0 0
Ca Gluconate Ppt. 48 hr. 72 hr.
24 hr.
0
+++
++ +
+++ +
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Temporary Calcium Gluconate Stabilization A permanent stabilization of calcium gluconate did not appear to be necessary in the fermentation process. The object was to prevent precipitation of calcium gluconate during the actual fermentation by the addition of relatively small quantities of boric acid br borax. Too high a concentration of these boron compounds would interfere with the normal method of recovery of calcium gluconate by crystallization from an aqueous solution. In order t o determine the possibilities of obtaining a temporary stabilization, solutions having different concentrations of calcium gluconate and boric acid in Pyrex flasks were placed on a shaking machine operated a t 30’ C. The presence or absence of precipitated calcium gluconate was recorded on three successive days (Table I). The results show that a temporary inhibition of calcium gluconate precipitation can be obtained with small amounts of boron as compared with the amounts required for permanent solubility (1.0, 2.0, 3.0, and 4.0 grams boric acid per 100 cc. of 10, 20, 30, and 40 per cent solutions of calcium gluconate, respectively, 1).
Boron Tolerance by Molds The boric acid tolerance of several strains of A . niger,
The use of several compounds to prepare concentrated stable solutions of calcium gluconate has been reported (5, 4, 13, 16). Di Carli (4) found that 3.67 per cent of boron oxide increased the solubility of calcium gluconate a t 20” C. from 3 to 39 per cent. Austin ( 1 ) used about 4 per cent boric acid to prepare a stable 40 per cent solution of calcium gluconate for therapeutic use. The formation of calcium borogluconate has been described (9). Since boric acid is only slightly toxic to many molds, i t occurred to the authors that some boron compounds might be used successfully to prevent the precipitation of calcium gluconate during the fermentation. This paper describes a method for preventing precipitation of calcium gluconate by the addition of boron compounds during the submerged fermentation of concentrated solutions of glucose by Aspergillus niger.
capable of fermenting glucose to gluconic acid, was studied first in surface cultures with spore inoculations. The standard fermentation medium was employed, with and without calcium carbonate. The boric acid was added after sterilization and a few hours before inoculation. A comparison of the growth of two A . niger strains, under these conditions, is given in Table 11. These results show that A . niger 67, which had been employed in former investigations (5, 1.2, 14, l 7 ) , exhibited a marked inhibition of growth in the presence of more than 400 pa p. m. boron, while strain 3 grew well in the presence of 2000 p. p. m. boron. The boron tolerance was slightly greater for both organisms in the presence of calcium carbonate. Germinated spores similar to those used for inoculation of the fermentation solutions showed slightly more tolerance for boron. OF A . niger STRAINS 67 TABLE11. BORICACID TOLERANCE AND 3 AS SHOWN BY 3-DAY-OLD CULTURES INOCULATED WITH
SPORESa
Materials and Methods The fermentations were conducted in rotary aluminum drums which were previously described (6). The method for germinating the spores and growin the spore-bearing mycelium remained fundamentally unchanget ( I d ) . The composition of the fermentation medium was the same as that previously employed (1.2) except that the concentration of (NH&HPO, was increased to 0.50 gram per liter and 2-3 cc. of corn steep liquor, also known as “Yeast Compound” ( I 6 ) , was added per liter. The drums were operated in the same manner, except that the air flow was increased to 440 cc. per liter per minute during the fermentation to avoid any possibility of aeration being a limiting factor. It was necessary to add small amounts of an antifoam agent, octadecyl alcohol, at frequent intervals during the fermentation to prevent loss of drum contents.
Analytical Procedure Analytical methods were essentially the same as previously described (IO). The course of a fermentation was followed by analyzing samples withdrawn at intervals. Yield calculations were based upon the analysis of the final liquor for reducing sugar, for free gluconic acid, calcium in solution and boron. Levy and Doisy (8) pointed out that in the presence of large amounts of boron in the form of boric acid or borax, the amount of copper reduced by glucose is decreased, indicating an error in this method. However, with the low boron concentrations used in this work, the error is of minor importance. Correction was made for calcium taken into solution by the free boric acid added.
A . n;ger 67 Without CaCO1
Boron,
P. P.M.
a
With CaCOa
A . nine? 3 Without CaCOa
With CaCOa
5 represents size of mate in absence of boron. ~~~~
~~
A . niger 3 was selected for the fermentation studies with boron compounds, using the rotary aluminum drums. The first experiments in the rotary fermenters showed that boric acid when added a t time of inoculation inhibited the fermentation of glucose to gluconic acid. This decrease in the fermentation rate was completely eliminated by adding dry boric acid or borax when approximately 8.0 grams of glucose per 100 cc. had been consumed. In other experiments in which inoculation was made with ungerminated spores, best results were also obtained by adding the boron compound at the time indicated above. The boric acid added in the dry form does not cause a violent evolution of carbon dioxide and foaming of the drum contents, nor does it cause an appreciable change in volume of the fermentation solution.
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Table IV show the time required to ferment various amounts of glucose to gluconic acid, first with a deficiency of calcium carbonate without boron, and secondly with a slight excess of calcium carbonate with the proper amount of boron. i
TABLEIV. EFFECTOF DEFICIENCY AND EXCESSOF CALCIUM CARBONATE PLUSBORICACIDON THE FERMENTATION OF DIFFEREST CONCENTRATIONS OF GLUCOSE TO GLUCONIC ACIDB Y Aspergillus niger 3
Original Glucose Concn. per 100 Cc. Grams
20 25 30 35
FIGURE1. EFFECTOF DEFICIENCY OR EXCESSOF CALCIUM WITH AND WITHOUT BORIC ACIDON THE FERMENCARBONATE TATION OF GLUCOSE TO GLUCOXIC ACIDBY A . niger Deficiency of calcium carbonate, 2.34 grams per 100 cc.; A , B , and c, 20.0 grams glucose per 100 cc. a t start; D and E , 30.0grams glucose per 109 cc. at start
Deficiency of CaCOa (2.34Grams/lOO Cc.) N o Boric Acid Glucose conTime sumed/100 cc. Hour8 Qrarna 28 19.0 32 20.6 32 22.5 40 21.0
Excess of CaCOa Boric Arid Preaeit Glucose conTime sumed/100 ca. Hours Grams 17 19.5 25 24.7 31 29.6 40 32.0
A better comparison of the fermentation rates was obtained by following the course of the fermentation of glucose solutions having, first, a deficiency, and secondly, a slight excess of carbonate, as shown in Figure 1. The calcium carbonate was all dissolved a t 9 and 11hours, respectively, for fermentations B and D. I n each case the complete neutralization of the calcium carbonate was followed by a sharp increase in
Amount of Boron Compounds Required A series of fermentations was run to determine the amount of boron required to prevent precipitation of calcium gluconate during the fermentation of glucose in different concentrations with a slight excess of calcium carbonate (5.0 grams per liter over that required for neutralization of gluconic and boric acids). The amount of glucose consumed when precipitation of calcium gluconate occurred in the presence of different concentrations of boron is shown in Table 111. The results show that the amount of calcium gluconate which can be stabilized by 1500 to 2500 p. p. m. of boron is dependent to some extent upon the time required for the fermentation. The original concentration of glucose governs the time required for the fermentation; for example, 24 hours are required to ferment 20 grams of glucose in an original concentration of 35 grams per 100 cc., while a medium containing only 20 grams of glucose per 100 cc. can be fermented in 17 to 18 hours. The precipitation of calcium gluconate during the normal fermentation of 20, 25, 30 and 35 grams of glucose per 100 cc. can be prevented by 500, 1000, 1500, and 2500 p. p. m. boron, respectively.
FIGURE 2. CONVERSION OF GLUCOSE TO GLUCONIC ACIDBY A . niger 67 WITH DEFICIENT CALCIUM CARBONATE (5) AND BY A . niger 3 WITH BORICACIDAND EXCESSCALCIUM CARBONATE
the acidity of the fermentation solution-i. e., from pH 5.4 to 3.0. Under this acid condition the fermentation rate gradually decreased. Fermentation A had an excess of calcium One of the outstanding advantages resulting from the use carbonate but contained no boron, and a heavy precipitate of boron as a stabilizing agent is the decrease in time required of calcium gluconate occurred at 13-14 hours, followed by to ferment concentrated solutions of glucose. The data in complete cessation of the fermentation. Fermentations C and E had a slight excess of calcium carbonate with the proper amounts of boric acid (500 and 1500 p. p. m. boron) TABLE111. STABILIZING ACTIONOF BORICACIDON CALCIUM and showed only a slight increase in acidity of the media GLECONATE PRECIPITATION DURING THE FERMENTATIOX WITH throughout the fermentation period. The fermentation rate AN EXCESS OF CALCIUX CARBOKATE remained nearly constant until less than 0.5 gram of glucose Glucose Consumed Age of Fermentation Original Glucose Boron in 100 Cc. a t Pptn. at Pptn. of Ca per 100 cc. remained in the solution. in 100 Cc. Added of Ca Gluconate Gluconate Fermentations were conducted in the presence of borio Grams P . p . m. Grams Hours 20 0.0 13-14 13 acid with 20,25, and 30 grams glucose per 100 cc. with slightly 30 500 21-22 25 less calcium carbonate than that required for complete 30 1000 27-28 28 35 1500 30-31 38 neutralization of the gluconic acid formed. Exposure of 35 2000 33 42 35 2500 34.5 None at 45 the mycelium during the last 1 or 2 hours to free gluconic acid resulted in only a slight decrease in the fermentation rate.
Application of Boron Stabilization to Single Fermenter Process
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A comparison of these gluconic acid fermentations of concentrated solutions of glucose by A. niger 3 with fermentations reported with A . niger 67 ( 5 ) are of interest (Figure 2). The fermentations with A . niger 67 were made with a deficiency of calcium carbonate (2.6 grams per 100 cc.). Fermentations with A . niger 3, under optimum conditions here described, required about half as much time as A . niger 67 with corresponding glucose concentrations. This difference in time required is especially apparent in the fermentation of a medium containing originally 30.0 grams glucose per 100 cc. An original fermentation medium (5) containing 30.8 grams glucose per 100 cc. was fermented to a glucose concentration of 4.9 grams per 100 cc. in 75 hours by A. niger 67 (curve G, Figure 2), while A . niger 3, under improved fermentation conditions, fermented 29.5 grams glucose in 32 hours (curve E , Figure 2). These results show the advantage of using sufficient calcium carbonate to avoid the inhibiting action of free gluconic acid during the fermentation of concentrated solutions of glucose by A . niger 3.
Bacterial Fermentations Since some of the acetic acid bacteria are known to be capable of oxidizing glucose to gluconic acid, it was considered desirable to determine whether the boric acid stabilization procedure would be adaptable to a bacterial fermentation. It was convenient to test the method on a bacterium, Acetobacter suboxydans (16), which was being investigated by co-workers for the conversion of glucose to gluconic acid and thence to ketogluconic acid. A number of fermentations of a medium containing 20 grams glucose per 100 cc. with a slight excess of calcium carbonate were carried out in the rotary drum fermenters. A heavy precipitate of calcium gluconate occurred in the medium without boric acid, while there was no precipitation of calcium gluconate in the medium containing boric acid. Although in this particular case a precipitate of calcium 5-ketogluconate appeared in the fermenter containing the boric acid before all the glucose was consumed, it is only logical to believe that if other strains of Acetobacter were used which were not able to oxidize further the gluconic acid produced, results would be obtained entirely analogous to those found in the mold fermentation process. These results show that boric acid can be used to prevent precipitation of calcium gluconate in a bacterial conversion of glucose to gluconic acid and should be of general applicability to bacterial processes involving the formation of calcium gluconate.
re-use of the fungus growth, only when sufficient calcium carbonate was used to prevent inhibition of the fermentation rate which results from exposure to free gluconic acid. Four successive semicontinuous fermentations were made with repeated re-use of the mycelium of A . niger 3, with 25 grams glucose per 100 cc. having a slight excess of calcium carbonate and boric acid (1500 p. p. m. boron) present to prevent precipitation of calcium gluconate. The results of these fermentations are given in Figure 3. Fermentations A and A1 were conducted according to the standard method, using a germinated inoculum. An excess of calcium carbonate and boric acid was added to A , and an insufficient amount of calcium carbonate (2.34 grams per 100 cc.) to A I . It would have been impractical to try to re-use the mycelium in Al because of its reduced fermenting capacity a t the end of this fermentation. The excess calcium carbonate and mycelium were separated by filtration from the fermented liquor of A a t 25 hours and placed back in the fermenter with a fresh glucose medium (containing the same nutrients and calciunl carbonate as in A ) for fermentation B. This process was repeated to obtain fermentations C and D. The results show that a semicontinuous fermentation process can be conducted satisfactorily with an original glucose concentration of a t least 25 grams per 100 cc., with an excess of calcium carbonate and sufficient boric acid to prevent precipitation of the calcium gluconate formed. These four fermentations were completed in 100 hours, including time necessary for each filtration and recharge of the fermenter. Owing to inconveniences of recharging the drums before or after normal laboratory hours, no attempt was made to conduct a semicontinuous fermentation with other concentrations of glucose, although there is no doubt that a similar procedure could be used for the fermentation of higher or lower glucose concentrations.
Re-use of the Mycelium During the preparation of a germinated inoculum in the rotary aluminum fermenter (5, 12) there is a rapid conversion of glucose to gluconic acid. Aliquots of this medium containing the fungus growth are used to inoculate a standard fermentation medium. This method of inoculation acBually involves a re-use of the fungus growth. Studies have been made (5, 1%’)of the culture conditions which affect the fermenting capacity of this kind of inoculum. It was demonstrated (1%’)that an inoculum developed in a medium containing a slight excess of calcium carbonate (calcium carbonate 2.8 grams and glucose 9.1 grams per 100 cc.), to neutralize the gluconic acid formed, was far superior t o one developed in the same medium without calcium carbonate. The correlation between the acidity of the medium and the fermentation rate for A. niger 3, used in the fermentation of more concentrated solutions of glucose, has been presented (Figure 1). Therefore, it seemed logical to assume that high concentrations of glucose could be fermented rapidly by utilizing a process made semicontinuous through
2
o
8
16
24
0
a
18
24
0
now5
a
!e
24
o
8
16
24
ACID PRODUCTION FROM GLUFIGURE 3. GLUCONIC COSE BY REPEATED RE-USEOF A. niger 3 MYCELIUM
It is important to note that filtration of the excess calcium carbonate and mycelium was not conducted under strictly aseptic conditions. At the end of the fermentation, the pressure was rapidly reduced, and after approximately 20 minutes most of the mycelium was floating in the upper layer. The solution was withdrawn through a tube running from the vent on the bottom side of the drum to a cloth-covered sterilized Buchner funnel containing a cloth filter pad, fitted to a suction flask. This filtration was accomplished without any
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INDUSTRIAL AND ENGINEERING CHEMISTRY
special difficulty. A small quantity of mycelium adhering to the baffles and inner walls was not removed from the fermenter. The medium used for refilling the fermenter contained slightly more than 25 grams glucose per 100 cc., to compensate for the slight dilution encountered from the wet mycelium in the drum and from the pad of calcium carbonate and mycelium collected on the filter pad. During this filtration process there were opportunities for contamination by bacteria and other fungi. Examination of the fermentation medium (Figure 3, curve D) revealed no contamination. Apparently this fermentation is not very susceptible to contamination by most common bacteria and fungi.
Discussion Calculation of the exact yields of gluconic acid was not possible with these fermentations for the following reasons: (a) The frequent withdrawal of samples for analysis entailed small losses of the culture medium; ( b ) as a result of foaming, small amounts of the medium sometimes escaped through the air exit tube; and ( c ) there was a slight concentration of the medium due to evaporation caused by the flow of air through the fermenter. Yields based on glucose consumed as shown by analysis of the sample taken directly from the drums were slightly higher than theory. When reasonable allowance is made for these sources of error, a yield of gluconic acid approximately 95 per cent of theory is obtained, in agreement with the practically quantitative conversion of fermented glucose to gluconic acid noted in previous fermentation studies of this division (5, 12, 1 7 ) . The approximate halving of the time required for fermentation of concentrated solutions of glucose in the single fermenter process as compared with that reported in other investigations is in part due to factors other than continuous neutralization of the gluconic acid formed. First, it is believed that A . niger 3 is better adapted to this fermentation process than 8 . niger 67. Secondly, the use of steep liquor improved the nutritive \-slue of the fermentation medium and was therefore instrumental in decreasing the lag period a t the beginning of the fermentation. Recent investigations (14) have shown that more than 910 grams glucose per 100 cc. could not be used to advantage in a process made semicontinuous by the re-use of the mycelium of A. niger 67. This failure to obtain satisfactory results with more concentrated solutions of glucose was due in part to injury of the mycelium by prolonged exposure to free gluconic acid, or to the presence of calcium gluconate crystals, which inhibit the fermentation, It is believed that a semicontinuous process by means of which 25 grams glucose per 100 cc. can be fermented every 24 hours has a decided advantage over one which requires more frequent recharging of the fermenter with a medium containing a much lower concentration of glucose. KO attempt has been made in these studies to ascertain the feasibility of preparing a pure, crystalline calcium gluconate from the fermentation liquors containing boric acid. Since only a relatively small quantity of boric acid was used, to secure a temDorarv sta-
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The fermentation process described here would be directly applicable to the production of calcium gluconate preparations which are stabilized with boric acid. It is understood that a considerable quantity of such material is now marketed. The advantages of this modified fermentation process could also be used in the preparation of industrial grades of gluconic acid and its salts, which, it is hoped, will find extended use in the future. Summary 1. Precipitation of calcium gluconate during the fermentation of concentrated solutions of glucose, with a slight excess of calcium carbonate, can be prevented by the addition of boric acid or borax to the fermentation medium. 2. Aspergillus niger 3 was not injured by boric acid or borax, when added a t the proper stage of fermentation, in the concentrations necessary to prevent precipitation of calcium gluconate. 3. Single-drum fermentation of ’concentrated solutions of glucose can be made in approximately half the time previously reported by using A . niger 3 in a medium containing a slight excess of calcium carbonate by adding boric acid or borax during the fermentation to prevent precipitation of calcium gluconate and by making slight changes in the nutrients. 4. A semicontinuous process by which 25 grams glucose per 100 cc. can be fermented every 24 hours is made possible by re-use of t,he mycelium of A . niger 3 under the conditions described above. Literature Cited (1) Austin, J. &4.,U. S. Patent 2,007,786 (July, 1935). ( 2 ) Bernhauer, K., “Die oxidativen Gliwngen”, p. 17, Berlin, Julius Springer, 1932. ( 3 ) Christina, T., C . S.Patent 1.983,344 (Dee., 1934). (4) Di Calli. F., A n n . chim. applicata. 21, 44i-53 (1931). (5) Gastrock, E. 1., Porges, N., Wells, P. A,. and Moyer. A. J., ISD. E S G . CHEM., 30, 782, 789 (1938). (6) Herrick, H . T.. Hellbach, R.. and May, 0. E., Ibid., 27, 681-3 (1935). (7) Hornibrook, F. B . , Univ. Maryland, M.S.thesis, 1933. (81 Levy, ?d.,and Doisy, E. A , .I. Bioi. C‘hem., 77, 733-51 (1928). (9) MacPherson. H. T., and Stewart, .J.. Biochem. .J., 32, 76-S (1938). (10) May, 0 . E.. Herrick. H. T.. Moyer, J., and Kells, P. .I., I K D . ESG. C H E x f , , 26, 575-8 (1934). (11) May, 0. E., Keisbwg, 9. M.,and Herrick, H. T . , .I. W’ash. Acad. Sci.. 19. 443-7 (1923). (12) Moyer, A. J., Wells. P. .I.,Stuhhs. J. J., Hewick, H . T., and May, 0. E., Ihid., 29, 777-81 (1937). (13) Pasternack. R., and Giles, W.R . . IT S. Patent . 1,965,535 (.July. 1934). (14) Porges, S . ,Clark, T. E.. and Gastrock. E . .I., IXD.ENG.CHEY.. 32, 107-11 (1940). (15) Torigian, J., U. S. Patent 1,!306,666 (h[ay, 1933). (16) Wells. P. A,, Lockwood, L. B., Stubbs. ,J. J., Roe, E. T., Porges. N., and Gastrock, E. A , , ISD. ENG.CHEY.. 31,1518-21 (1939). Moyer, A. J., Stubbs. J. J., Herrick. H. T.. and (17) Wells, P. .1.. May, 0. E., Ihid., 29, 653-6 (1937). PRESENTED before t h e Division of Agricultural and Food Chemistry a t t h e 99th Meeting of t h e American Chemical Society, Cincinnati, Ohio.
