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constituents, such as aliphatic acids and phenols, provided such substances are in hydrocarbon Oil to d u t i o n s deviating from ideality in the requisite manner* Alcohols mag be recovered from hydrocarbon fractions through treatment with metallic sodium. Esters may be removed by saponification of the fraction; the alcohol remaining with the hydrocarbons may be removed as already described. In a great many cases, substances normally insoluble in hydrocarbon oil may be rendered soluble by formation of derivatives; for instance, the lower aliphatic alcohols and acids may be esterified. Obviously, other carrying agents may be used; if nitrogen bases are employed, hydrocarbons may be separated by this method. The special advantages accruing from the application of amplified distillation to the separation of very small samples have already been discussed. The Union Oil Company of California obligingly furnished the hydrocarbon oil used in this investigation.
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Literature Cited (1) Beatty and Calingaert, IND. ENG.CKEM.,26, 504 (1934). (2) Benedetti-Pichler and Schneider, 2. anal. Chem., 86, 69 (1931). (3) Bruun and Hicks-Bruun, BUT.Standards J. Research, 5, 933 (1930). (4) Cassel, Chem.-Ztg., 53,479 (1929). (5) Chamot and Mason, Handbook of Chemical Microscopy, Vol. 11, p. 17, New York, John Wiley & Sons, 1931. (6) Emich, Monatsh., 53-54, 329 (1929). (7) Felsing and Thomas, IND.ENG.CHEW,21, 1269 (1929). (8) Jones, J. Chem. Soc., 33, 175 (1878). (9) Rosanoff and Easley, J . Am. Chem. Soc., 31, 953 (1909). (10) Schleiermacher, Ber., 24, 944 (1891) (11) Trew, Trans.Faradav SOC.,28, 509 (1932). (12) Weissenberger and Schuster, J. prakt. Chem., 113, 180 (1926). (13) Weissenberger et al., Monatsh., 46, 47, 57, 157, 167, 291 (1925). R ~ C ~ I VNovember ED 29, 1935. Presented before the Division of Petroleum Chemistry a t the 90th Meeting of the Amerioan Chemical Society, San Francisco, Calif., August 19 t o 23, 1935.
PRODUCTION OF ETHANOL BY THERMOPHILIC FERMENTATION O F CELLULOSE M. K.VELDHUIS, L. M. CHRISTENSEN, AND ELLIS I. FULMER Iowa State College, Ames, Iowa
O C
ONSIDERABLE attention has been paid to the commercial production of combustible gases and of acetic acid by the direct fermentation of cellulosic materials. Occasional reference has been made in the literature to the simultaneous formation of ethanol in these fermentations, but practically no systematic work has been reported on the production of this compound. It is apparent that a method which would give considerable yields of ethanol by the direct fermentation of cellulose would be of value in the utilization of agricultural products in the production of industrial chemicals, and might possess some advantages over the indirect method involving preliminary saccharification of the cellulose. Since the work here reported is primarily concerned with the factors involved in the formation of ethanol by the thermophilic fermentation of cellulose, the literature references will be almost entirely confined to those which mention ethanol as a product. The general literature on the topic of the fermentation of cellulose is voluminous and the reader is referred for details to the quite complete bibliography given by Simola (9). The first mention of the thermophilic fermentation of cellulose was made by MacFayden and Bloxall in 1899. Woodman (15) and Woodman and Stewart (16,17) concluded that the thermophilic cellulose fermenters play an impoftant part in the utilization of cellulose by ruminants. Khoutrine (a) found bacteria in the intestinal flora of man which would bring about the thermophilic fermentation of cellulose, and claimed to have isolated a pure culture which she named Bacillus celluloseae dissotuens. This culture grew more slowly
than did the crude culture and fermented only cellulose among the carbohydrates tested. The optimum temperature range was 35" to 51" C. but the culture would withstand 61" C . The products included hydrogen, carbon dioxide, ethanol, and volatile acids. The yield of ethanol was about 11 per cent of the cellulose fermented. Lymn and Langwell (5) claimed to have isolated a pure culture by plating. The optimum temperature range was 60" to 68" C . Alcohol yields of 27 per cent were obtained from sulfite pulp, 8.3 per cept from filter paper, and 15.5 per cent from xylose from rice straw. Langwell(4) reported yields as high as 7.75 per cent ethanol and 40 per cent acetic acid from 6.2 per cent corncob medium. The temperature employed was 60" C . and the p H was adjusted to a value of 6.0. The alcohol yield was increased while the yield of acetic acid was decreased by aeration during fermentation. Viljoen, Fred, and Peterson ( l a ) employed a medium consisting of 2.0 grams microcosmic salt, 1.0 gram monopotassium phosphate, 0.3 gram peptone, and 15 grams cellulose per 1000 cc. of tap water. Excess calcium carbonate was used as neutralizing agent. At 65" C. they obtained 70 to 95 per cent decomposition of the cellulose. Of the cellulose decomposed, 50 to 55 per cent was obtained as acetic acid and 5 to 25 per cent as ethanol. Snieszko (lo),using a culture which he believed to be pure, obtained yields of about 50 per cent acetic acid and 13 per cent of ethanol per 100 grams of cellulose fermented. Tomoda (11, 19) obtained the following yields from a 2 per cent filter paper medium: 15 to 17 per cent ethanol, 21 to 26 per cent acetic acid, 6 to 8 per cent butyric acid, 0.5 to 1.5 per cent lactic acid, 18 to 19 per cent
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OF INCUBATION TIMEAND TEMPERATURE TABLEI. INFLUENCE UPON YIELDS OF ALCOHOLS AND ACIDS
~i~~ of Fermentation Hours 24 48 72 96 144 192 264 360
Total Alcohol Yield per 100 G. Cellulose Added, % 500 c. 550 C. 600 C. 650 c.
Total Volatile Acid Yield per 100 G . Cellulose Added, % 500 c. 550 C. 60° C. 650 c.
0.13 0.58 1.40 2.77 8,99 13.81 14.67 14.72
0.81 2.41 4.74 1i:i5 17.08 22.22 26.41
0.16 0.85 3.69 9.69 15,77 18.67 22.03 17.90
0.17 1.80
l0:97
13.23 12.77 12.87 13.10
0.37 1.86 2.48 2.86 3,67 3.26 2.56
..
0.81 3.42 7.04 2i162 25.44 26.75 27.15
...
0.81 5.81 11.61
6.38 16.04
31.68 34.15 34.15
28.31 29.16 29.15
ii:;: gi:?; a
carbon dioxide, and 0.2 to 0.5 per cent hydrogen. No methane was found.
Preparation of Cultures Mixed cultures capable of fermenting cellulose a t thermophilic temperatures are readily obtained from nature. The feces of animals, particularly ruminants, and cellulosic materials undergoing decomposition under natural conditions are generally good sources of such cultures. Attempts to separate these mixtures have always yielded cultures of markedly less vigor than the original, and it is probable that the thermophilic fermentation of cellulose to yield acids and alcohols is an example of synergistic action by several bacterial species. The culture used in this study was obtained by introducing horse manure into a medium containing 3 grams of pulped filter paper, 0.25 gram of ammonium chloride]0.25 gram of dipotassium phosphate, and 4 grams of calcium carbonate in 100 cc. of tap water. Vigorous fermentation became evident after 4 days of incubation at 60' C., and the culture was then transferred every 6 days in the same medium. Transfer was made from a hot to a cold flask since this procedure seemed to yield a stronger culture than that obtained by transfer to a flask at 60" C. Since the medium was difficult to pipet, inoculations were made by means of a graduated cylinder. The inoculation ratio used was 1 to 15.
Method of Analysis Alcohols were estimated in the distillate from a 300-cc. sample adjusted to pH 7.5. This distillate was made acid to Congo red and redistilled to eliminate ammonia, which was sometimes present in the first distillate, and the alcohols were estimated by the method described by Christensen and Fulmer ( I A ) . The distillates from a number of the better fermentations were combined and the alcohols concentrated by redistillation and salting out with anhydrous potassium carbonate. The anhydrous alcohols were carefully fractionated and the components identified by measurement of physical properties and as the 3.5-dinitrobenzoates. The alcohols produced were ethanol and n-butanol. Volatile acids were recovered from the sample from which the alcohols had been removed by steam distillation after acidification. The acids were titrated and the ratio of acetic to butyric estimated by the methods of Virtanen and Pulkki (Id) and that of Fyleman (2). Besides acetic and n-butyric, traces of higher volatile acids were also found but were not identified. Residual cellulose was estimated by the A. 0. A. C. method for crude fiber (I). Reducing sugars were sometimes present in the fermented medium and were estimated by the method of Shaffer and Hartmann (7). Gases were analyzed in a williams apparatus after colledtion over saturated sodium chloride solution. Carbon dioxide and hydrogen, with occasional traces of methane, were produced.
Influence of Temperature of Fermentation The data in Table I show the marked influence of the temperature of incubation upon the ratio of alcohols to acids produced. I n this investigation the medium was the same as that used to carry the cultures, and the flasks were shaken several times daily to facilitate the neutralization of the acids by the calcium carbonate. The most favorable temperature for alcohol production was 55" C., and the best yields of acids were obtained a t 60" C. Within this range the decomposition
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of cellulose in 360 hours was 99 per cent, but a t 50" C. only 92 per cent of the cellulose was consumed and a t 65" C. only 63 per cent. The fermentation started more rapidly a t the higher temperatures, and it was thought that it might be possible to carry the flasks a t 60" to 65" C. during the first 2 days, dropping t o 55" C.for the balance of the incubation period, and in this way shorten the time of fermentation. The results were the Same as though the higher temperature had been used during the entire incubation period.
Influence of pH of Fermenting Medium I n this series the medium was the same as that previously employed except that the calcium carbonate was eliminated and the pH adjusted to the indicated value twice daily by means of sodium carbonate or hydrochloric acid solution. The flasks were incubated 8 days a t 55" C. The data are shown in Table 11. The alcohol yield was greatest when the reaction was adjusted to pH 7.75-8.00; the acid yield was greatest when the reaction was adjusted to pH 7.50-7.75. The decomposition of cellulose was nearly constant over the range of p H 7.50-8.00 but decreased markedly with more alkaline reactions.
Using mixed cultures obtainable from natural sources, the proportion of ethanol in the products of fermentation of cellulose at thermophilic temperatures is dependent upon the composition of the medium and the temperature employed. With the cultures used, the best alcohol yields were obtained at 55" C.; the acid yield was greatest at 60" C. The optimum pH for alcohol production is probably about 7.5; for acid production a value of about 7.2 is preferable. With adjustment twice daily, the best value for alcohol production was pH 7.75-8.00; for acid production, adjustment twice daily to pH 7.50-7.15 was preferable. The best medium used in this series contained 0.25 gram KzHP04.3HzO ; 0.30 gram ammonium chloride, 0.50 gram peptone, 2.0 to 5.0 grams cellulose (pulped filter paper), and 100 cc. tap water. The water used must not contain more than 0.015 gram calcium sulfate per 100 cc. The presence of even very low concentration of heavy metals may be undesirable. Under the best conditions yields of 26 per cent ethanol and 24 per cent acetic acid were obtained, on the basis of the cellulose added. n-Butanol and n-butyric acid were also identified among the products of fermentation.
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TABLE 11. INFLUENCE OF PH OF FERMENTING MEDIUM Yield per 100 Grams Cellulose Added Total Cellulose volatile decomacid posed
Total alcohol
PH 5.00 5.50 6.00 6.50 6.75 7.00 7.25 7.60 7.75 8.00 8.50 9.00
%
%
%
0.20 0.27 1.33
0.67 1.33 2.00 2.33 5.00 8.67 18.00 24.33 23.67 22.67
5.33 5.33 11.00 12.67 21.00 46.33 81.33 93.33 91.67 9 2 . QO 76.67 18.67
...
4.03 8.80 15.67 21.25 20.77 22.53 19.23 0.23
...
1.00
The reaction obtained by the use of excess calcium carbonate is rarely more alkaline than pH 7.0, yet the alcohol yields obtained are nearly as great as those a t pH 7.75 with sodium carbonate neutralization. Neutralization only twice daily does not maintain the indicated reaction; thus the flask adjusted to pH 7.75 usually showed a reaction of pH 6.95, 12 hours after neutralization, so that the “average reaction” was only about pH 7.35. This does not, however, fully account for the yields obtained with calcium carbonate neutralization. Calcium carbonate, even the reagent grade, contains impurities of which some may be beneficial.
Influence of Cellulose Concentration Using the medium previously described and employing calcium carbonate for neutralization of the acids produced, the cellulose added was varied from 0.5 to 6.0 grams per 100 cc. Within the range of 2.0 to 5.0 grams per 100 cc. the yield of alcohols and acids and the decomposition of the cellulose, in terms of cellulose added, were independent of the cellulose concentration. Below 2.0 grams per 100 cc. the yields of alcohols were less but there was little change in cellulose decomposition; above 5.0 grams per 100 cc. there was a decrease in the decomposition of cellulose as well as in the yields of acid and alcohols. It is probable that the physical character of the medium is of greater influence than is the concentration of the products of fermentation in establishing the upper limit of cellulose concentration. Langwell (4) noted that very thick mashes could not successfully be employed in this type of fermentation.
Development of the Medium The medium used in the preceding investigations was formulated upon the basis of a few preliminary trials. In the following experiments, the concentration of nutrients was varied in an attempt to improve the yields, particularly of alcohols. Detailed data are given only for the effect of am-
PL.o-1
OF
SHARPLES
SOLVESTS CORPOR 1TIOS
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monium chloride among the salts tested. Similar data were obtained for the other salts discussed. The ammonium chloride concentration was varied in a medium containing 0.25 gram of potassium dihydrogen phosphate and 3.0 grams of cellulose per 100 cc. An excess of calcium carbonate (3.3 grams per 100 cc.) was used for neutralizing the acids produced. The flasks were incubated 8 days at 55” C. As shown in Table 111, the optimum concentration of ammonium chloride, both from the standpoint of yield of alcohol and acid and that of degree of cellulose decomposition was about 0.30 gram per 100 cc. It is significant that the optimum calculated by the formula developed by Sherwood and Fulmer (8) for the influence of temperature upon the optimum ammonium chloride concentration for growth of Saccharomyces cerevisiae gives a value of 0.264 gram per 100 cc. a t 55’ C. TABLE
111. INFLUENCE OF AMMONIUM CHLORIDE
CONCENTRATION
Yield per 100 Grams Cellulose Added Total Cellulose Total volatile deoomalcohol acid posed
Ammonium Chloride G . / l 0 0 cc. 0.05 0.10 0.15 0.20 0,26 0.30 0.40 0.50 0.60
%
%
%
2.97 6.67 12.53 17.33 16.93 17.77 17.27 14.77 14.60
8.33 13.67 15.33 18.00 18.33 20.00 19.33 20.67 16.33
29.0 52.7 70.7 82.3 87.0 89.7 88.7 85.3 80.7
When the concentration of phosphate (KzHP04.3HzO) was varied within the limits of 0.016 and 0.600 gram per 100 cc., there was no well-defined optimum but the yields of products and decomposition of cellulose were greatest between 0.200 and 0.400 gram of KzHP04.3HzO per 100 cc. Probably 0.25 gram per 100 cc. might be regarded as the most desirable concentration, taking into account both yields of products and the rate of fermentation. Because of the value of phosphates as buffers, this optimum is somewhat dependent upon the methods of neutralization. It was repeatedly observed that cultures did not grow so well in media prepared with distilled water as in those made with tap water. The tap water contained 0.0125 gram of calcium sulfate per 100 cc., and it was thought that this was the only salt present in sufficient concentration to be of importance. It had also been noted that distilled water redistilled in glass was better than the original distilled water from a tin-lined still and block tin condenser and pipes; this behavior indicates that very low concentrations of tin, and possibly other metals, may have a marked action upon this fermentation. It was found that calcium sulfate in concentrations above 0.015 gram per 100 cc. greatly depressed the yield of both ~
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INDUSTRIAL AND ENGINEERING CHEMISTRY
alcohol and acid. There was a distinct optimum a t about 0.012 gram per 100 cc. or 50 cc. of saturated solution per 800 cc. of medium. That is, the tap water used contained nearly the optimum calcium sulfate concentration. From the media prepared with a saturated calcium sulfate solution (containing 0.17 gram per 100 cc.), the yield of alcohol was only 0.2 per cent of the cellulose added, and that of volatile acids was 3.0 per cent. A marked odor of hydrogen sulfide was observed when the medium was more than one-fourth saturated with calcium sulfate. The addition of magnesium chloride (MgC126HzO) to the medium prepared with distilled water had no effect upon yields of alcohol or acid or upon the decomposition of cellulose within the range of 0 to 0.55 gram per 100 cc. The addition of ferric chloride (FeCl3.6H20) to the same medium lowered the yields and the decomposition of cellulose when more than 0.10 gram per 100 cc. was added. Lower concentrations had no effect. Langwell (4) noted that cellulose fermentations were abnormal when conducted in iron fermenters; this difficulty was overcome by using aluminum fermenters. Table IV shows the results obtained when varying amounts of peptone were added to the medium containing 0.25 gram of ammonium chloride, 0.25 gram of dipotassium phosphate, and 3.0 grams of cellulose per 100 cc. of tap water. Incubation was at 55" C. for 8 days and the fermentations were neutralized twice daily to pH 7.5 with standard sodium hydroxide solution. The addition of 0.2 to 0.5 gram of peptone produced marked increase in yields of alcohol and acid and in decomposition of cellulose. TABLEIV.
EFFECT OF PEPTONE
Peptone, g . / l O O cc. 0.00 0.25 Ethanol, g./100 g. cellulose added 19.67 23.33 0.21 n-Butanol, g./IOO g. cellulose added 0.27 Volatile acids, g . / l O O g. cellulose added 17.67 21.67 Cellulose consumed, g . / l O O g. cellulose added 71.00 78.00
0.50 25.33 0.38 23.67 90 .OO
1.00 26.00 0.45
26.67 91.00
Effect of Aeration during Fermentation Several experiments were carried out to test the effect of a&rationduring fermentation. In every case aeration lowered the yield of alcohol and occasionally lowered the yield of acid and the decomposition of cellulose. The gases leaving the flasks were scrubbed with sulfuric acid so that the effect was entirely due to the increased oxygen tension in the medium. T h e best yields of alcohols and generally the best acid yields were obtained from fermentations in large flasks with small surface volume ratio, and it may be concluded that strictly anaerobic conditions are the most desirable.
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yields of alcohol is as shown in Table V. I n addition to the neutral volatile products listed, a small amount of a material giving a strong iodoform reaction and distilling over at around 50" C. was also noted. Calculated as acetone, the yield was 0.135 per cent of the added cellulose. The amount present was so small that positive identification was not made.
TABLEV. PRODUCTS OF FERMENTATION UNDER CONDITIONS FAVORABLE TO ALCOHOL FORMATION G./100 G: Added Cellulose 26.60 0.36 0.28
Carbon dioxide Hydrogen ,Methane Acetic acid 24.40 %-But rio and higher volatile aci& 1.22 Nonvolatile acid as lactic 0.24
G 100G.
idded Cellulose Ethanol 26.30 %-Butanol 0.72 Reducing sugars aa glucose 0.71 Gums and pigment'" 12.85 Residual cellulose 3.22 Unaccounted for 3.10
a A considerable amount of material, found in the determination of crude fiber, which was soluble i n the acid b u t insoluble i n the base.
It was assumed that the nonvolatile acid was lactic but positive identification was not made. This product has been identified in similar fermentations by other workers (6).
Literature Cited (1) Assoc. Official Agr. Chem., Methods of Analysis, 3rd ed., p. 2x0. _ _ , i~m n-. ~ (1A) Christensen, L. M., and Fulmer, E. I., IND. ENG.CHEM.,Anal Ed., 7, 180 (1935). Fyleman, E., J . SOC.Chem. Ind., 43,142T (1924). Khouvine, Y . ,Ann. inst. Pasteur, 37, 711 (1923). Langwell, H., J . SOC.Chem. I n d . , 51, 988 (1932). Lymn, A. H., and Langwell, H., Ibid., 42, 279T (1923). Scott, S. W., Fred, E. B., and Peterson, 1' . H., IXD. ENG. CHEM.,22, 731 (1930). Shaffer, P. A., and Hartmann, A. F., J . Bid. Chem., 45, 365 (1920). Sherwood, F. F., and Fulmer, E. I., J. Phys. Chem., 30, 738 (1926). Simola, P. E., Ann. Acad. Sci. Fennicae, 34, No. 1, A1-91; N o . 6, 1-115 (1931). Snieszko, S., Zentbl. Bakt. Parasitenk., 11,88, 403 (1933). Tomoda, Y . , J . SOC.Chem. I n d . J a p a n , Suppl. Vol. 35, 534B (19.32).
\----I-
Ibid., 36,436B (1933). Viljoen, J. A., Fred, E. B., and Peterson, W. H., J . Aar. Research, 16, 1 (1926). Virtanen, A. I., and Pulkki, L., J. Am. Chern. SOC.,50, 3138 (1928). Woodman, H. E., J . Agr. Sci., 17,333 (1927). Woodman, H. E., and Stewart, J., Ibid., 18,713 (1928). Ibid., 22,527 (1932).
Ratio of Products The balance of products from a fermentation carried out under the conditions favorable to the formation of good
RBCEIVED October 15, 1935.
This investigation was supported in part by a grant from the Industrial Science Research funds of the Iowa State College for the study of the fermentative utilization of agricultural products.
Here amyl alcohol, amyl aoetate, and their derivatives are synthesized.
