ButyLAcetonic Fermentation of Arabinose and Other Sugars T
H E commercial utilization L. A- UNDERKOFLER AND 3.E. HUNTER, JRpractically normal. Ratios for of the butyl-acetonic ferIowa State College, Arnes, I o w a f o u r a r a b i n o s e fermentations -L mentation process has rewere also given. The averages sulted in a considerable amount of for the results of these authors research work, both practical and theoretical. are also included in Table I. Here again the ratio for An interesting phenomenon connected with this ferarabinose is about 50: 40: 10 instead of the usual 60: 30: 10. mentation is the fact that 5-carbon sugars, or pentoses, as Underkofler, Christensen, and Fulmer (8) showed that the well as 6-carb0n carbohydrates, are fermented to form the butyl-acetonic fermentation of the 5-carbon sugar, xylose, same substances-namely, butanol, acetone, ethanol, cardiffers in solvent ratio, as a function of time, from that of bon dioxide, and hydrogen as principal products, and small the 6-carbon carbohydrates (Table 11). With the latter amounts of butyric acid and acetic acid as minor products. (dextrose, levulose, and sucrose) the normal ratios of butaGood yields of the solvents are obtained from the pentoses nol, acetone, and ethanol are maintained throughout the in the presence of certain protein supplements although, in course of the fermentations, except that the percentages of general, fermentation of these carbohydrates is slower and butanol may be a little higher a t the beginning of the ferless complete than for the hexoses, and increased acid formation is noted. The fact that the same neutral products are obtained from both the pentoses and hexoses, usually in TABLEI. AVERAGESOLVENT RATIOS FOR THE BUTYL-ACETONIC about the same ratio, has offered a perplexing problem to FERMENTATION O F SUGARS those interested in the mechanism of the fermentation. No. of I n the early history of the fermentation, only natural raw Fer-Av. Solvent Ratiomenta- Buta- AceEthamaterials, such as corn, other grains, and potatoes, were inInvestigators Sugar tions nol tone nol vestigated. The first studies of the action of the butylPeterson, Fred, and Schmidta Glucose 2 59 31 10 Xylose 3 61 29 10 acetone organism on pure sugars in artificial media were reArabinose 2 47 43 10 ported by Robinson (6) and by Speakman (7). Each of Johnson, Peterson, and Fred Glucose 18 60 28 12 Arabinose 4 48 39 13 these authors reported xylose and arabinose to be fermenta Ratios calculated from the data, assuming 10 per cent ethanol. able, but placed both of these sugars in the group characterized as giving abnormal fermentations in the peptonesalts media used. I n other words, this was the group in TABLE 11. SOLVENT RATIOSFOR CARBOHYDRATES which utilization of carbohydrate was incomplete, yields of Time, Butanol'"-%AcetoneEthanolsolvents were poor, and acidity curves were obtained which Hr. D L Su X D L Su X D L S u X reached high values without showing the characteristic .. 22 24 .. .. 7 13 24 71 63 .. break or drop in acidity after the peak of the fermentation. 31 64 62 * . . . 26 25 .. .. 10 13 .. 60 22 27 . . 29 .. 25 12 40 66 13 ..1 46 Quantitative determinations of the solvents were not made 23 26 26 24 14 46 63 55 73 31 19 45 55 63 59 67 40 25 24 30 28 12 17 3 32 by either of these authors. 65 60 61 65 54 25 23 14 16 28 29 7 17 7%
81 65 61 100 .. 118 131 62 58 168 a D = dextrose;
.. .. .. .. ..
PETERSON, Fred, and Schmidt (6) reported a more extensive investigation of the butyl-acetonic fermentation of the pentoses. These workers were not able to confirm the abnormal behavior noted by Robinson and by Speakman. They found that in the fermentation of xylose and arabinose, when either yeast water or Robinson's peptone-salts solution was used to furnish nutrients, the same products were formed and in essentially the same quantities as from glucose. The rate of fermentation was somewhat slower for the pentoses, but practically all of the sugars were completely utilized in 72 hours. Slightly less solvents and slightly more volatile acids were produced from the pentoses. The solvent ratios were not calculated by Peterson, Fred, and Schmidt; but these ratios can be calculated from their data and from the assumption that the proportion of ethanol in the mixed solvents is 10 per cent, which is known from other work to be approximately true. These calculated ratios are given in Table I. The ratios for glucose and xylose are practically the same and represent the normal ratio-i. e., 60 parts butanol, 30 parts acetone, and 10 parts ethanolwhereas arabinose shows a considerably higher proportion of acetone and a lower ratio of alcohol. Johnson, Peterson, and Fred (4) gave the solvent ratios for eighteen glucose fermentations and found them to be
67 62 58 65 64 L
7 %
55 25 24 54 .. 59 60 26 25 60 levulose; Su =
.. .. ..
-
.. ..
10 29 30 15 3 30 31 8 31 29 11 28 31 9 ii 4 32 30 4 sucrose; X = xylose.
.. .. .. .. ..
15 15 12 12 10
mentations and ethanol a little lower. The solvent ratio for sucrose is slightly abnormal in that a lower proportion of ethanol is formed, but the ratio is nearly the same throughout the fermentation. With xylose, as the fermentation proceeds, the proportion of butanol increases, that of acetone remains nearly constant, and that of ethanol decreases, until at the end of the fermentation the ratio of solvents is normal. The medium used for each of these fermentations contained, per 100 cc., 1.0 gram of corn gluten meal, 0.25 gram of monopotassium phosphate, and the weight of carbohydrate equivalent in carbon content to 6.25 grams of anhydrous glucose. TO EXTEND the studies of the butyl-acetonic fermentation of the pentoses, 2-arabinose was prepared from mesquite gum by the method of Anderson and Sands (I) and subjected to the butyl-acetonic fermentation in the same semisynthetic medium previously employed for xylose. The culture used was that which had been isolated and improved by Under480
APRIL, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
kofler, Christensen, and Fulmer (8), and employed in the previous studies. Duplicate flasks containing about 3100 cc. of medium were inoculated from the fourth active transfer of the culture in corn mash. Samples were taken from the flasks a t the time intervals indicated in Table I11 and analyzed for the solvents according to the methods of Christensen and Fulmer (2). As shown in Table 111, during the course of the butyl-acetonic fermentation of arabinose the proportions of butanol and acetone both increased, whereas that of ethanol decreased markedly. The solvent ratio a t the end of the fermentation was approximately 50:40:10 instead of the usual 60:30:10. For comparison, the results for previous xylose and glucose fermentations are also included in Table 111. Thus, it is found that the course of the fermentation, as well as the final ratio of products, for arabinose differs from that for xylose and likewise from that for the hexose sugars.
TABLEHI. COMPARISON OF SOLVENT RATIOSFROM BUTYLACETONIC FERMENTATIONS OF ARABINOSE, XYLOSE,AND DEXTROSE Time, Hr. 31 35 40 45 50 55 60 65 70 81 86 95 117 131 140 168 a
Butanol“? X D 64
7%
A
..
41 48 42 44
..
.. ..
..
29 31
66 63
40
63
..
.. ..
50 49 48
54
..
60
55
.. .. 59
65
60 52 50
51
..
.. .. .. 62 .. ..
..
60 60
..
2% Agtone-D ..
33 33 38 39
..
38 40 40 30 38 40
..
39
..
.. ..
26
..
-%
A
..
26 19 20 17
EthanolX D 10
.. ..
25 24
22 23
is
25
29
..
25
30
..
25
.... ..
ii
15
..
..
12 12
10
..
io
..
29
28 30
26
..
..
12 11 12 10 10
..
46 45
12 14
32
12
.. .. 17 ..
.. ..
..
..
14
io .. .. ..
9
A = arabinose, X = xylose, D = dextrose.
Speakman (7) advanced the theory that the differences observed in the butyl-acetonic fermentation of various sugars may be due to differences in molecular configuration, particularly as regards the number of adjacent hydroxyl groups in the sugar molecule. I n view of later results obtained by other workers with sugar fermentations, it is doubtful whether Speakman’s theory is of any value. Johnson, Peterson, and Fred (4) suggested that the differences in ratios of the various oxidized and reduced products which they obtained in fermentations of glucose, mannitol, calcium gluconate, and arabinose by Clostridium acetohutylicum were related to the degree of oxidation of the compound fermented. However, since arabinose, which has the same degree of oxidation as glucose, gave a n “oxidized” type of fermentation, these workers postulated a different fermentation mechanism for pentoses from that for hexoses. These authors do not seem to have recognized that solvent ratios obtained from the two isomeric aldopentoses, arabinose and xylose, differ so markedly; the ratio from xylose is the same as from glucose. O F ALL the carbohydrates which have been subjected to the butyl-acetonic fermentation, and for which data on solvent ratios are available, arabinose alone gives a decidedly different final ratio of solvents. No acceptable explanation has yet been advanced for this difference. However, of all these carbohydrates, only arabinose possesses the levo configuration; all the others belong to the dextro series. It was deemed of interest, then, to try other sugars which belong to the levo series. Such carbohydrates are not readily obtainable, but the ketose hexose, Z-sorbose, has recently become available
481
in quantities as a result of the methods developed by Fulmer, Dunning, Guymon, and Underkofler (3) for producing this sugar from sorbitol by fermentation. Repeated attempts to ferment sorbose in the same semisynthetic medium employed with the other sugars have invariably failed. It can be concluded that sorbose is not fermentable by the butyl-acetone organism, a t least in this medium which has been successfully used with all the sugars previously tried. As mentioned above, levulose, the only other ketose hexose used, was successfully fermented to give normal yields of solvents in the usual ratio. Hence, it has been shown that, of the two sugars belonging to the levo series which were subjected to the action of the butyl-acetone organism, arabinose is readily fermented but yields an abnormal ratio of solvents, whereas sorbose is not attacked by the butyl-acetone organism. Further work, employing carbohydrates of both the dextro and levo series, to determine the effect of configuration of the carbohydrate on the solvent ratio is in progress.
Literature Cited (1) Anderson, E., and Sands, L., Org. Syntheses, 8, 18 (1928). (2) Christensen, L. M., and Fulmer, E. I., IND.ENG.CHEM.,Anal. Ed., 7, 180 (1935). (3) Fulmer, E. I., Dunning, J. W., Guymon, J. F., and Underkofler, L. A., J. Am. Chem. Soc., 58, 1012 (1936). (4) Johnson, M. L., Peterson, W. H., and Fred, E. B., S. B i d . Chem., 91, 569 (1931). ( 5 ) Peterson, W. H., Fred, E. B., and Schmidt, E. G., Ibid., 60, 627 (1924). (6) Robinson, G. C.,Ibid., 53, 125 (1922). (7) Speakman, H.B.,Ibid., 58, 395 (1923). (8) Underkofler, L. A.,Christensen, L. M., and Fulmer, E. I., IND. ENQ.CHEM.,28,350 (1936). RBCEIVED November 11, 1937. Presented before the Division of Biochemistry and Nutrition a t the 14th Midwest Regional Meeting of the American Chemical Society, Omaha, Nebr., April 28 to May 1, 1937.
Man-Made Molecules SIR: I n order to correct any erroneous impressions that may be engendered by certain statements occurring in the paper on “Man-Made Molecules” by Thomas Midgley, Jr. [IND.ENQ. CHEM.,30,120-2 (1938)],it might be well to point out the following facts: Cellulose acetate is not the parent material of viscose rayon and cellophane as stated in Table I. Both of these products are regenerated cellulose, whereas cellulose acetate is an ester of cellulose and undergoes no chemical change during fabrication. Rayon yarn and protective wrapping sheets made from cellulose acetate differ widely from the corresponding regenerated cellulose products in properties and uses. On page 122, column 1, line 13, in referring to recent trends in automobile design, the statement is made: “The glass in the body . . . contains an internal layer of a transparent synthetic resin. Inside the car the steering wheel is made of, and much decorative finishing is accomplished by, other synthetic resins.” It is well known that practically all of the safety glass used in motor cars for the past five years and a t present utilizes a specially purified and plasticized cellulose acetate as the central bonding medium. Moreover, the new steering wheels and most of the decorative finishing referred t o are also made from cellulose acetate. This material, although a plastic, is usually not considered to be a “synthetic resin” in the conventional sense of the term. JOHN R. CALDWELL 1314 OAK STREET, KINGSPORT, TUNN. January 15, 1938
