INDUSTRIAL A N D ENGINEERING CHEMISTRY
July, 1923
729
Propionic Acid and Ketones from Whey' By E. 0. Whittier and J. M. Sherman RESEARCH LABORATORIES, DAIRYDIVISION, DEPARTMENT OF AGRICULTURE, WASHINGTON, D. C.
E :;d:
L
The factors affecting the propionic fermentation of lactose have ACTOSE is a sub~ n ~ $ ~ ~ $ ~ d been inoestigated, and conditions determined whereby approximately 0.05 N sodium hydroxide. sbance of such slight 2.4 Ibs. of propionic acid and 1 Ib. of acetic acid may be obtained The ratio of propionic to utility a t present that acetic acid and the actual tremendous amounts are from 5 2bs. of lactose in 12 days' incubation. weight Of each acid present A mixed culture of Bacterium acidi propionici ( d ) and Lactorun to waste as whey. The were calculated from these bacillus casei, incubated at 30" C. for 3 days, is usedfor the inoculadata and ~~~l~~~ tables. demand for refined lactose The Duclaux method gave tion oj'the preoiously sterilized and buffered whey. fo? use in infant feeding is very consistent results; it is The mixture of propionate and acetate obtained may be either satisfied by an insignificant d'fficult to understand why conoerted into free acids and refined, or distilled to yield a mixture proportion of the amount investigators so frequently that could be produced. of acetone, methylethyl ketone, and diethyl ketone. attempt to modify it. The manager of a western creamery recently stated One of the chief objecthat his company alone would like a market for 5,000,000 Ibs. tions to almost any fermentation is the excessive time factor. of lactose annually. The cost of production of milk albumin, Preliminary experiments showed that a 5 per cent lactose for which a demand is developing, is bound UP with the prob- solution containing peptone and the propionic organism was lem of disposal of the crude lactose produced from the whey only about 10 per cent converted to volatile acid in 14 days. along with the albumin. Aerobic fermentations may be greatly accelerated by aeraThe results reported in this article are the outcome of one tion methods, but such a scheme is of no benefit in an anof several investigations undertaken in the laboratories of the aerobic process. D~~~~~~~~~~~~ OF M~~~ D~~~~~~~~ p~ v ~ ~ ~ e~ Dairy Division in an effort to devise a method for the commercial utilization of lactose. bacteria are quite sensitive to changes in hydrogen-ion concentration. To determine the most desirable pH value for PROPIONIC ACID the propionic organism, the series recorded in- Table I was The amount of propionic acid produced and marketed a t carried out. Organism 8 is Lactobacillus casei, an accelerator the present time is very small. The chief and probably sole of the growth of the propionic organism, 6.2, Each commercial source of this acid is the residue from the refining culture contained 2 g. calcium lactate and 1 g. peptone, and of acetic acid. Perfume manufacturers use practically all was incubated for 14 days. It will be noted that the amount that is consumed, converting it into various esters. The of acceleration caused by Lactobacillus casei is very small in use of ethyl propionate as a pyroxylin solvent has been pat- this series, especially in comparison with the acceleration ented,2and propionyl cellulose has been suggested as a possiindicated in the following tables. This is probably due to ble substitute for acetyl c e l l ~ l o s e . ~The wide temperature the fact that Lactobacillus casei does not grow well in the range in which propionic acid is fluid (-22" to 140" C.) medium used. indicates that it might advantageously replace acetic acid as a H-ION CONCENTRATIONS ON RATE OB solvent under some conditions. Development of a cheap TABLEI-EFFECT OF DIFFERENT PROPIONIC FERMENTATIOX source of propionic acid should considerably stimulate its Weight of Acid Produced use. Inoculating pH, Ratio of Mols. Propionic Propionic Acetic Previous work is summarized and a number of experiments hTo. Organisms Initial to Mols. Acetic G. G. About 0.05 g. are reported by Sherman and Shaw in an article appearing 1 62 D volatile acid 0.5599 0.2137 elsewhere. 2 62 6 2.01
+
EXPERIMENTAL-The
propionic organism used in this work was
Bacterium acidi ProPionici (d), isolated by Sherman in his work
on Swiss ~ h e e s eand , ~ commonly referred to in this laboratory as Culture 62. One hundred cubic centimeters of medium were used for each experiment, inoculated with 1 cc. of a 62 culture unless otherwise specified, and incubated at 30' C. A t the end of the incubation period the material wa$ transferred to a 100-cc. graduated flask, made up t o the mark, transferred t o a centrifuge tube without rinsing, and whirled for 5 min. A 50 or 75-cc. aliquot of the clear liquid was pipetted into a 500-cc. roundbottomed flask, made acid t o Congo red with 10 per cent sulfuric acid, diluted to a volume of about 250 cc., and steamdistilled a t approximately constant volume till 1000 cc. of distillate had been collected. An aliquot of the distillate was titrated with 0.05 N sodium hydroxide to determine total volatile acid present. A volume requiring approximately 28 cc. 0.05 N sodium hydroxide was made alkaline t o phenolphthalein and evaporated t o about 60 cc. It was then transferred to a 110-cc. graduated flask, made acid to Congo red with 10 per cent sulfuric acid, diluted to the mark, and transferred to a 200-cc. flask without rinsing. It was then slowly distilled, five 20-cc. 1 Presented before the Division of Industrial and Engineering Chemistry at the 64th Meeting of the American Chemical Society, Pittsburgh, Pa., September 4 to 8, 1922. 2 U. S. Patents 1,397,173 and 1,397,493 (1921). 8 Dubosr, Rev. prod. chim., 24 (1921), 499. 4 J. Bact., 6 (1921), 379.
3 4 5
62 62 62,8
7 8 5
2.05 2.33
6
62,8 62,8 62,8
6 7 8
2.11 2.04 2.40
7 8
0.6526 0.2582 0.5895 0,2048 About 0.04 e . volatile a c i Z 0.6028 0.2323 0.6621 0.2635 0.6324 0 2137
Growth of the propionic organism is practically nil a t the H-ion concentration represented by p H 5 . The so-called neutraf point, p H 7 , is the most desirable reaction for the growth of the organism, a change in p H in either direction retarding the production of acid. I n subsequent experiments, 5 g. of calcium carbonate were added to each 100 cc. culture to maintain the pH value a t approximately 7 , and the cultures were gently shaken every few days.
EFFECT OF ORGANISXS O N ID RATIO-In most of our experiments, a fairly constant ratio of 2 molecules of propionic acid t o 1 molecule of acetic acid was obtained, but frequently the ratio was noticeably disturbed. I n several cases this was attributed to contaminating organisms or t o accelerating organisms purposely introduced. To get a t the mechanism of this effect, cultures were made containing, instead of lactose, sodium propionate and acetate, and these were inoculated with organisms of several types. Culture 45.4 is an unidentified organism known to accelerate the
-
I N D UXTRIAL A N D E N G I N E Z R I A T G C H E M I S T R Y
730
propionic fermentation. The results after 19 days’ incubation are shown in Table IIe6 TABLE 11-EFFECTOF VARIOUS ORGANISMS ON PROPIONIC AND ACETICACIDS Ratio of Mols. Propionic t o Mols. hTo. INOCULATING ORGANISM Acetic Sterile, average of three 1.67 9 Bacillus subtilis 2.09 10 Proteus 2.57 3.50 11 Proteus vulgaris 5.67 12 45.4
Weight of Acid Found Acid Destroyed ProPropionic Acetic pionic Acetic G. G. % %
0.6637 0,6709 0.6596 0.6603 0.3399
0.3072 0.2606 0.2075 0.1528 0.0488
.0
li:2 32.6 50.4 46.4 84.4 0 0
Sherman and Shaw report Lactobacillus casei a s a more effective accelerator than 46.6. This may be due only to the destructive activity on both volatile acids produced. The results reported above show further t h a t organisms of the Proteus type may be used to purify propionic acid from acetic acid, though the time factor is considerable. With the idea of nullifying this time factor as much as possible, an attempt was made to carry on the fermentation and purification simultaneously. The results are shown in Table 111. Controls are included for comparison. Culture 62 is the propionic organism; S is Lactobacillus casei, used for accelerative purposes; 110 is Proteus vulgaris. The media contained 5 g. lactose, 5 g. calcium carbonate, and 1 g. yeast. TABLE 111-EFFECT
Inoculating No. Organisms
13 14 15 16 17 18 19 20 21
110
62 62,110 62,8 62,8,110 62 62,110 62,8 62,8,110
OB
PRESENCE OF Proteus Vulgaris
FERMENTATION
ON
PROPIOXIC
Weight of P e r cent Ratio of Acid Produced of TheoIncubating Mols. Pro- Proretical Yield Period pionic to pionic Acetic Pro: Days Mols. Acetic G. G. pionic Acetic FFormic and butyric acids only 30 18.1 25.8 0.4971 0.2859 1.41 30
30 30 33 44 44 44 44
1.50 2.47 2.09 1.40 1.56 2.72 2.12
1.3442 2.3517 2.3231 0.5799 1.4741 2.4387 2.3636
0,7266 0.7713 0.8988 0.3380 0.7643 0,7209 0.9020
65.5 69.5 80.8 30.5 68.9 8 9 . 0 64.9 86.3 81.3 49.1 85.8 84.8 21.2 53.8
It will be seen t h a t when used in combination with other organisms, Proteus vulgaris does not selectively destroy acetic acid. I n fact, when used with both the propionic organism and Laclobacillus casei, it appears to inhibit the destruction of acetic acid. Evidently, the effect a n organism will have in mixed culture cannot safely be predicted from its conduct in pure culture. If Proteus is to be used for elimination of acetic acid, it must be used by itself. Incidentally, it may be noted t h a t Proteus vulgaris of itself exerts an accelerative action on the propionic fermentation. COMPARISON OF VARIOUSSOURCESOF NITROGEN-TO compare the efficiency of 46.4 and Lactobacillus casei as accelerators, and to determine the relative utility of various sources of nitrogen, a series of experiments was run, part of the results from which are given in Table IV. A per cent of theory yield is calculated for the whey cultures, based on the assumption that 100 cc. of whey contains 5 g. of lactose. This is justifiable for purposes of comparison, and is very close to fact. The incubation period in the experiments reported was 42 days.
The table shows clearly t h a t the proteins of whey are more stimulating to the mixed cultures than either peptone or yeast. Since mixed cultures show considerable advantage over pure cultures for practical use, i t is fortunate t h a t fortification of the nitrogen source in whey is not necessary. The choice of nitrogen source appears consistently to influence the ratio of propionic to acetic acid, but since this should come about only through its influence on the destruction of the volatile acids first produced, and since all volatile acid decomposed appears t o be acetic, the effect may be disregarded for practical purposes. The difference i n the destructive activity of 46.4on volatile acids by itself and in mixed culture may be seen by a comparison of data in Tables I1 and IV. Table IV shows conclusively that an accelerating organism is highly desirable, t h a t Lactobacillus casei is a more effective accelerator than 45.4 and t h a t whey is better than an artificial medium for the activities of the propionic organism plus Lactobacillus casei. EFFECT OF LACTOsE-sherman and Shaw have shown t h a t about 95 per cent of the lactose fermented can be accounted for on the basis of the Fitz reaction6 as propionic and acetic acids. I n Tables I11 and IV, several runs occur in which yields of about 85 per cent have been obtained, the calculation being based on the total quantity of lactose present. Under these conditions, then, about 90 per cent of the lactose present was fermented, which is as much as may reasonably be expected. T o obtain this yield a t least a month’s incubation has been necessary. While this period is excessive for practical production from crude whey, it might not be impractical if a whey containing a high percentage of lactose could be used. To test t’his possibility, the series shown in Table V was run. Cultures were made u p with crude whey, and with the same whey to which 5 per cent and 10 per cent lactose had been added. TABLE V-EFFECT OR ADDEDLACTOSE ON PROPIONICFERXSNTATION OP
No. 31 32 33 34 35 36
6
U. S. Patent 1,450,392 (1923).
Whey Cc.
Lactose G.
100 100 100 100 100 100
0 5 10 0 5
10
WHEY
Ratio of Mols. InocuIncubating Propionic lating Period t o Mols. Organisms Days Acetic 9 2.00 62,8 9 2.33 62,8 9 2.35 62,8 623 14 2.18 62,8 14 2.12 62,8 14 1.97
Weight of Acid Produced Propionic Acetic G. G.
1.4695 1,4013 0,9863 2.3118 1.6113 1.2893
0.5878 0.4870 0.3396 0.8621 0.6066
0.5316
The results show that, not only was the additional quantity of lactose not converted t o volatile acids, but the increased concentration of lactose actually retarded the fermentation. EFFECTO F CONDITIONS I N CULTURES-In order to test the effect of conditions in the inoculating cultures upon the fermentation, both pure and mixed cultures of the propionic organism and Lactobacillus casei were grown for different periods before being used for inoculation of. test cultures of whey. The results are given in Table VI. HABITUATION OF PROPIONIC AND ORGANISMS TO EACHOTHER Ratio of Mols. Incubating Propionic Inoculating How Age Period t o Mols. Organisms Grown Days Days Acetic 1’7 62,1%8 Apart 3 7 1.82 2.08 62,1y08 Apart 3 7 7 2.78 62 8 Together 3 2.25 7 1% 62:8 Together 3 2.23 1% 62,1%8 Apart 13 7 13 7 2.12 1% Apart 2.13 1% 62,1%8 623 Together 13 7 2.33 1% 62,8 Together 13 7
TABLE VI-EFFECT
OF NITROGEN TABLE IV-RELATIVE UTILITY OF VARIOUS SOURCES
Per cent of Ratio of Weight of Theoretical InocuMols. Acid Produced Yield Nitrogen lating Propionic ProProOrgan- t o Mols. pionic Acetic Lactose Source Dionic Acetic G. G. Acetic isms -No. G. (1 G.) 3.17 1.3538 0.3466 49.4 31.2 62 22 pe@;ne 1.84 0.7081 0.3118 25.8 28.1 62 23 62 2.12 0.4891 0.1865 17.Sa 16.aa Whey 24 Whey 0.7763 0.1989 28.3 17.9 6 2 4 5 4 3 17 Peptone 25 62:45:4 1:70 0.9497 0.4522 34.7 40.7 Yeast 26 62,45.4 2.25 1.7612 0.6328 64.3‘ 57.OU Whey 27 Whey 62,8 2.08 1,7705 0.6913 64.6 62.3 5 Peptone 28 2.16 2.2041 0.8260 80.4 74.4 62,8 5 Yeast 29 1.95 2.4039 0.9998 87.8a 90.1“ 62,8 Whey 30 Whey I yield calculated for the whey CLiltures on the a Per cent of theorj assumption t h a t 100 cc. of whey contains 5 g. of lactose.
Vol. 16, No. 7
No.
37
% 40
41 42 43 44
OF
ACCELERATIVE Weight of Acid Produced Propionic Acetic G. G.
0.6573 0.2919 0.6560 0.2663 1.5063 0.4428 1.2760 0.4584 1,0527 0.3816 1.1232 1.3711 0.4288 0.5219 1.3083 0.4546
It appears to be advantageous to grow the inoculating organisms togetherj especially if they are to be used within 2 or 3 days. The difference in the potential activities of the 6
B e y . , 11 (1878),1890;12 (1879).474; 13 (1880),1306.
TABLEVII-EFFECT
No. 45 46 47 48 49 50 51 52 53 54 55
56
Whey cc. 100 95 90 80 100 95 90 80 100 95 90 80
731
INDUSTRIAL A N D ENGINEERING CHEMISTRY
July, 1923
I'noculation cc.
1 5 10 20
1
5 10 20 1 5 10 20
Incubating Period Days 3 3 3 3 6 6
6 6 9 9 9 9
AMOUNTOB INOCULATION ON PROPIONIC FERMENTATION Weight of Acid Produced Calculated t o 100 Cc. Weight of Acid Produced Ratio of Propionic Acetic Propionic Acetic Mols. Propionic G. G. G. G. t o Mols. Acetic Contaminated butyric acid present OF
2.23 2.22 2.50 2.01 2.46 2.53 2.36 1.90 2.15 2.26 3.60
0.8417 0.8397 0.9055 0.8414 1.3226 1.3135 1.3099 1 2980 1.6183 1.7403 1.8831
0.3053 0.3059 0.2943 0.3990 0.4366 0.4204 3.4487 0.5546 0.6041 0.6223 0.5881
0.8860 0.9330 1.1319 0.8414 1.3922 1.4593 1.6374 1.2980 1.7035 1.9336 2.3639
0.3213 0.3400 0.3679 0.3990 0.4595 0.4671 0.5609
0.5546
0.6359 0.6914 0.7351
32.2
34.1 41.3 30.7 50.8
53.3 59.7 47.4 62.1 70.6 85.9
29.0 30.6 33.2 30.6 41.4 42.1 50.5 50.0 57.3 62.3 66.2
These values are calculated for the whey cultures on the assumption t h a t 100 cc. of whey contain 5 g. of lactose.
cultures disappears largely after about 2 wks., the separately grown cultures becoming nearly as effective as those grown together. EFFECTOF VARYINGAMOUNTSOF INocuLura-With an organism growing so slowly as the propionic bacterium, the amount of inoculating medium should have considerable influence on the rate of fermentation, particularly in the first 10 days. Therefore, a series was run using various amounts of inoculum, other conditions being the most favorable which had been discovered in previous series. A mixed culture of the propionic organism and Lactobacillus casei was grown in whey for 11 days and then used for inoculation. The volatile acid in the inoculating culture a t the time of its use was determined, and corrections were applied to the results obtained. The values in each experiment were calculated to the basis of 100 cc. of whey for comparison. No correction could be determined which would account for the acids produced after inoculation from the lactose still present in the inoculating culture. However, since the quantities of unfermented lactose present in the amounts of inoculum used must have been rather small, the comparison of results from samples incubated the same length of time cannot be appreciably affected. The results are shown in Table VII. It is clear that 1 per cent inoculation is by no means sufficient to obtain the most rapid rate of fermentation. Increasing the amount of inoculation above 5 per cent shows only slight advantage. Five per cent is undoubtedly the most practical quantity t o use. While there are probably other effective means of accelerating the propionic fermentation, they are mostly factors that cannot be handled satisfactorily in the laboratory, but are bound up with the design and operation of large-scale equipment. Eventually, our minimum time of 12 days' incubation for an 85 per cent yield could probably be considerably diminished. With the development of a demand for propionic acid, it should be possible to meet it a t a reasonable cost by this process.
g
KETOXES There is a growing demand for solvents with the general properties of acetone, but with higher boiling points and lower solubility in water. For a long time acetone has been made on a large scale by the distillation of calcium acetate and of wood. Within recent years patents have been obtained for the catalytic conversion of acetic acid vapors into acetone.' These methods, applied to calcium propionate and propionic acid, respectively, yield diethyl ketone, b. p. 102.7' C . , slightly soluble in water. If a mixture of acetate and propionate, or of the acids, is used, the product contains acetone, methylethyl ketone, and diethyl ketone.* Methylethyl ketone boils at 81' C., and has a solubility in water intermediate between acetone and diethyl ketone. A mixture of ketones, 7 Brit. Patent 14,085 (1915); U. S. Patent 1,315,544 Patent, 1,315,525 (1919). * Schramm, Be?., 16 (1883). 1581.
evidently containing chiefly methylethyl ketone, is obtained from the distillation of the calcium salts extracted from wool s c o u r i n g ~ . ~This mixture has been recommended as a denaturant for ethyl alcohol. On the assumption that the volatile acids from the propionic fermentation exist in the proportion of 2 molecules of propionic to 1 of acetic-which they closely approximate-the limiting theoretical possibilities in the ratios of the constituents of the acetone oil obtained therefrom are 37.4 per cent diethyl ketone, 62.6 per cent methylethyl ketone, and no acetone, on the one hand, and 74.8 per cent diethyl ketone, no methylethyl ketone, and 25.2 per cent acetone, on the other. Some combination of values between these limits is to be expected. With the idea of determining roughly the possibilities of the ketone conversion as a means of utilizing the product of the propionic fermentation, several distillations of dried calcium salts were carried out. The products in each case gave nearly identical distillation curves. Several attempts t o determine the proportion of the components by chemical means failed to give satisfactory differentiation of the ketones. Finally, repeated fractionation through a narrow 2-ft. column of beads was resorted to. The final distribution of the fractions indicated approximately 20 per cent acetone, 40 per cent methylethyl ketone, and 40 per cent diethyl ketone. Because of incomplete conversion of the calcium salts due to lack of agitation in the retorts, and because of frequent cracking of the retorts, no reliable yield data were obtained. With continuous operation, there is no doubt that as satisfactory yields can be secured as are obtained in calcium acetate distillation. It is advisable to distil off the volatile acids from the fermentation mixture and to reconvert to calcium salts before the dry distillation. Distillation of the evaporated fermentation mixture without previous purification gives considerable quantities of evil smelling protein decomposition products that are difficult to eliminate satisfactorily in the subsequent fractionation.
(1919);
U. S.
A . and P. Buisine, Comp. rend., 128 (1889), 561.
Frank Cummings Cook Frank Cummings Cook, M.A., M.S., Yale; Ph.D., George Washington, died in Dallas, Texas, on June 19, after an operation for appendicitis. Since leaving Yale in 1904, Dr. Cook had been .connected with the Bureau of Chemistry, U. S. Department of Agriculture. At the time of his death, he was engaged in special research work on the control of insects infesting live stock, and was temporarily located a t Dallas, Texas. He was a member of the American Chemical Society, the Association of Official Agricultural Chemists, the Society of Biological Chemists, the Washington Academy of Sciences, and was a delegate to the International Congress of Applied Chemistry at Rome, 1906, and at London, 1909. He was the author of numerous papers and bulletins on food metabolism, enzymes, insecticides, fungicides, and related subjects. Dr. Cook had a host of friends in the chemical fraternity, by whom his untimely death will be keenly felt. He is survived by a wife and one son.
