1290
INDUSTRIAL AND ENGINEERING CHEMISTRY
The danger of loss of available plant food in the ammoniation of high-analysis mixtures containing double superphosphate is therefore greater than for low-analysis mixtures unless means are taken to cool the mixture rapidly as in the process of granulation. The heat developed in ammoniation is sufficient to granulate and dry double superphosphate and also certain mixtures that can be granulated with a low moisture content, but some additional heat is necessary to granulate and dry ordinary superphosphate and mixed fertilizers that require a relatively high moisture content for satisfactory granulation.
Literature Cited (1) Bassett, 2.anorg. Chem., 53, 34 (1907); 59, 1 (1908). (2) Beeson, IND.ENG. CHBM.,29, 705 (1937). (3) Beeson and Ross, Ibid., 26, 992 (1934).
VOL. 29, NO. 11
(4) Biohowsky and Rossini, “Thermochemistry of Chemical Substances,” New York, Reinhold Publishing Corp., 1936. (5) Clark, J. them,, 35, 1232 (1931). (6) D,, pont de N ~E, I., and ~ co.,‘~ d ~ ~ ~~~ i ~ ~~ ~ ~ -, May, 1933. (7) Hill and Hendricks, IND. ENG.CBEM.,28,440 (1936).
i:j
~
~~$d$~;~~~b~.(~~~; z ~ ~
f
197 (1937). (10) Luckmann, Kunstd-iLnger u. Leim, 33, 131, 163,200 (1936). (11) Lundstrom and Whittaker, IND. ENG.CHEM.,29, 61 (1937). (12) MacIntire, Hardin, Oldham, and Hammond, Ibid., 29, 758 (1937). (13) Marley, proc. sot. (London), 45, 591 (1933). (14) R~~~ and Hardesty, Commercial Fertilizer Yearbook. D. 28 (1937). (15) Ross, Jacob, and Beeson, S. Assoc. Oficial Agr. Chem., 15, 227 (1932). ENG.CHEM.,27, 562 (1935). (16) White, Hardesty, and Ross, IND. RBCEIVEXI July 30, 1937.
Oat Hull Utilization by Fermentation L. A. UNDERKOFLER, ELLIS I. FULMER, AND MORTON M. RAYMAN Iowa State College, Ames, Iowa
ELLULOSIC materials constitute a large proportion of the agricultural and trade wastes, including wood waste, oat hulls, cottonseed hulls, corncobs, peanut husks, and others. The profitable disposal of such materials constitutes an industrial problem of considerable economic importance. Recent developments along this line have been the hydrolytic conversion of the pentosans of oat hulls into furfural (8) and the production of crystalline xylose by the hydrolysis of cotton seed hulls (6,I2). One of the most promising possibilities for the industrial utilization of wood waste is the Bergius process, (a) for the conversion of wood to carbohydrates, using concentrated hydrochloric acid. Probably the largest outlet for the crude sugar produced in this way will be in the fermentation industry for the production of industrial alcohol. Bergius mentions the well-known fact, however, that the xylose present in the mixed sugar solutions obtained in the process is not fermented by yeast. It might be desirable, therefore, to subject the wood to a prelirriinarymild acid hydrolysis, thus removing the xylose for use in a n appropriate fermentation process, and then to treat the residual cellulosic material by the Bergius process. The fermentative utilization of cellulosic materials, especially the pentosans, was recently reviewed by Fulmer (6). The purpose of the present paper is to present the results of an investigation to determine the best conditions and methods for the preparation and subsequent fermentation, by the butyl-acetone organism (Clostridiumacetobutylicum, r), of acid hydrolyzates from oat hulls. Oat hulls were selected as a representative cellulosic waste because of their uniformity in composition and ready availability in Iowa. The oat hulls were kindly furnished by The Quaker Oats Company. The butylacetonic fermentation is, next to the alcoholic fermentation, the most important industrial fermentative process. In this fermentation are produced solvents, in the approximate ratio
C
Butyl-Acetonic Fermentation of the Acid Hydrolyzate of 60 parts butyl alcohol, 30 parts acetone, and 10 parts ethyl alcohol, along with large quantities of carbon dioxide and hydrogen. Starch from corn has been used most extensively in this fermentation on the industrial scale, although various investigators have shown that, in the presence of suitable nutrients, other carbohydrates, including most of the simple sugars, are fermented by C1. acetobutylicum. More recently molasses has become an important industrial raw material for the production of butyl alcohol and acetone; a different organism has been used, however, from the one mentioned here. Robinson (11) and Speakman (Id)found that xylose and arabinose were fermented by the butyl-acetone organism; Peterson, Fred, and Schmidt (IO)reported a more detailed investigation of the butyl-acetonic fermentation of pentoses. Hydrolyzates from cottonseed hulls, peanut husks, corncobs, and sawdust from five different kinds of wood were fermented with C1. acetobutylicum by Weinstein and Rettger (17). None of these workers attempted t o determine optimum conditions or to use commercially feasible concentrations of carbohydrate. Hence, preliminary to the more exhaustive investigation of the possibility of utilizing xylose-containing hydrolysates in the butyl-acetonic fermentation, a study was made of the fermentation of pure xylose. The results were published by Underkofler, Christensen, and Fulmer (16). Data were given on the influence of physical and chemical conditions and the treatment of the cultures to bring about maximum yields of solvents from xylose. Using the optimum conditions, final yields and ratios of solvents from a semisynthetic medium containing 6.25 grams xylose and 1gram corn gluten meal per 100 cc. were practically the same as from corn mash. It is interesting to note that, although as much as 80 per cent of the usual corn mash could be replaced by glucose and 90 per cent by starch (the yields of solvents remaining normal), only 40 per cent replacement of the corn mash was possible with pure xy-
NOVEMBER, 1937
INDUSTRIAL AND ENGINEERING CHEMISTRY
lose if full yields were to be obtained. The failure of complete fermentation in the case of higher replacements may be due in part to insufficient nutrients, but it is more probable that the high buffering action of the products of the proteolysis of the mash is the essential factor. The fact that xylose required a higher concentration of corn gluten meal than did glucose in order to obtain maximum solvent yields from the semisynthetic medium would also tend to confirm this theory. The difference in the behavior of glucose and xylose has not yet been fully explained. It should be recalled, however, that xylose is a five-carbon compound and it is not surprising to find certain differences between the fermentation of this sugar and the six-carbon carbohydrates. For example, although the solvent ratios produced by the fermentation of starch, maltose, SUcrose, dextrose, and levulose did not vary greatly during t h e course of the fermentation, those of xylose differed markedly. With the latter sugar, as the fermentation proceeded, the ratio of the butyl alcohol increased, acetone was constant, and ethyl alcohol decreased. For the hydrolysis of pentosan-containing materials, sulfuric acid has commonly been employed, followed by neutralization with lime or calcium carbonate. Since calcium sulfate is harmful to some fermentations although not to all, it has been thought desirable to avoid the use of sulfuric acid as a hydrolyzing agent in these cases. I n a communication by Bryner, Christensen, and Fulmer (3) data were presented on the optimum conditions for the production of reducing sugars b y the hydrolysis of oat hulls, using hydrochloric acid. For most of the experimental work to be reported in this paper a hydrochloric acid hydrolyzate was used. I n this connection, the concentration of acid required for the maximum production of reducing sugars from the oat hulls during hydrolysis a t a given temperature does not necessarily give a hydrolyzate which produces the maximum yield of fermentation products. This is due to the fact that the use of the optimum concentration for the production of reducing sugars may also lead t o the formation of furfural or other materials which are toxic to the fermenting organism.
Preparation of Hydrolyzate The concentrations of acids employed in preparing the hydrolyzates for the fermentation experiments were those which had been found by preliminary tests to be optimum, a t the temperature used, in order t o obtain a Concentration of about 4 per cent reducing sugars in the hydrolyzates. For hydrochloric and nitric acids the proper concentration was found to be 0.08 normal, and for sulfuric acid 0.16 normal. The hydrolysis of the oat hulls was carried out as follows: Into each of four 6-liter Erlenmeyer flasks were placed 600 grams of air-dried oat hulls and 3600 cc. of acid. After standing overnight, the flasks were heated in the autoclave at 20 pounds per square inch (1.4 kg. per sq. cm.) steam ressure for 60 minutes. A t the end of the hour the steam was s i u t off, and compressed air was introduced at the same pressure to prevent the liquid in the flasks from boiling over because of a too rapid drop in pressure. The contents of the flasks were then cooled below the atmospheric boiling point by gradual1 lowering the pressure during the course of 20 minutes and finaEy, after removal from the autoclave, cooled thoroughly in running water. The hydrolyzate was separated from the solid oat-hull residue by filtering through cheesecloth su ported on a large Buchner funnel, the flasks and residue being aioowed to drain overnight. The acid filtrate was then neutralized with calcium carbonate and allowed t o stand an hour or so until the solid material, mainly lignin, had settled. The clear supernatant liquor was siphoned off, adjusted to a H of approximately 6.0, heated to boiling, and filtered. The Rtrate to be used for fermentation studies was measured into individual Erlenmeyer flasks and sterilized at 10 pounds per square inch (0.7 kg. per sq. cm.) steam pressure for 30 minutes. After cooling it was brought t o incubator temperature (37" C.) before being added t o the supplementary media.
1291
When oat hulls are treated with proper concentrations of dilute hydrochloric, sulfuric, or nitric acids under suitable conditions, hydrolyzates are obtained which contain reducing sugars, largely pentoses. When added to corn mash, the reducing sugars of the hydrolyzates are fermented by the butyl-acetone organism, giving good yields of butyl alcohol, acetone, and ethyl alcohol in the normal ratios. Relatively high proportions of corn mash are required for optimum fermentation of the hydrolyzates. The procedure offers possibilities for the butylacetonic fermentation of pentoses from the acid hydrolysis of wood and other cellulosic materials besides oat hulls.
The concentration of reducing sugars in the a t r a t e was in each case determined by the Shaffer-Hartmann method (IS). The proportion of pentose in one of the hydrolyzates was determined by distillation with hydrochloric acid according t o the methods of the Association of Official Agricultural Chemists ( I ) , and the furfural formed was estimated by precipitation with diphenylthiobarbituric acid (16). The results of this procedure indicated that practically the entire reducing sugar content of the hydrolyzate was xylose.
Preparation of Fermentation Media Since corn mash is the most favorable medium for development, of the butyl-acetone organism, the experimental fermentation media were prepared by mixing hydrolyzate with corn mash. Ground yellow corn, in quantities calculated to make a total carbohydrate content equivalent to 6.5 per cent corn mash (on the dry basis ) when the hydrolyzate liquor was added, was mixed with tap water in 4-liter Erlenmeyer flasks and then steamed for 45 minutes. The amount of water used in each case was sufficient so that when mixed with hydrolyzate the final volume of medium in each experimental flask would be about 3000 cc. The flasks were plugged with cotton, capped, and sterilized at 20 pounds steam pressure for 2 hours. After cooling the sterilized mash, it was allowed to come to incubator temperature (37' C.). The requisite amount of sterile hydrolyzate at 37" C.was added asepticall to the individual flasks of corn mash. Each &sk of experimental medium was inoculated with 100 cc. of an active 20-24 hour culture of Clostridium acetobutylicum from a 6.5 per cent corn mash. The culture used was one which had been originally isolated in the laboratories of biophysical chemistry at Iowa State College. It was handled in the manner usual for butyl-acetone cultures and as outlined in the previous communication (16). After completion of the experimental fermentations (3 to 7 days) two 250-cc. aliquots of the beer were measured from each flask and distilled after adding a little solid calcium carbonate t o neutralize acids. In each case 100 cc. of distillate were collected, and the distillates were analyzed for solvents by the method of Christensen and Fulmer (4). The results in the tables are the average values for the duplicate determinations.
Results Several factors were studied inan effort to determine the optimum conditions for securing maximum yields of solvents from the hydrolysates. It was a t first believed that the fermentation of xylose might proceed more effectively after the culture had multiplied in a favorable medium for a time and had produced a large number of organisms. Hence, experiments were made in which the xylose hydrolyzate was added to
INDUSTRIAL AND ENGINEERING CHEMISTRY
1292
the inoculated corn mash a t varying times during the course of the growth of the culture. It was found that there was no advantage in delaying transfusion of the hydrolyzate until after the inoculation, and a distinct disadvantage was discovered if the hydrolyzate was transfused during the first 14 hours of the fermentation. Hence in all subsequent experiments the hydrolyzate was mixed with the corn mash a t the time of inoculation. The effect of the concentration of xylose in the medium was studied by a systematic replacement of corn by hydrolyzate liquors in such a manner that the total concentration of carbohydrate was constant in all flasks (equivalent to 6.5 per cent corn mash). Fermentation tests with such replacement series, using hydrochloric acid hydrolyzate, were repeated many times, and typical results are given in Table I. It is evident that normal yields of solvents were obtained when not more than 40 per cent of the corn mash had been replaced by hydrolyzate. Replacement of more than this percentage of the corn gave incomplete fermentations which were very slow and sluggish, The solvent ratios were practically constant for the entire series of fermentations. These results are exactly analogous to those previously reported (16) for experiments in which starch of corn mash was replaced in series by pure xylose. TABLEI. SOLVENT YIELDSFROM ACIDHYDROLYZATES Corn Meal Replaced Per Cent
0 10 20 30
40 50 60
n
10 20 30 40 50 60 0
10 20 30 40 50 Q
b
Total Solvent Yield Wt. % of car-
-----Solvent B
Ratioa-A
E
bohydrate 0.08 N Hydrochloric Acid 33.4 34.4 31.0 30.0 30 7 19 2 2 7
63 67 58 59 66 57
..
0.16 N Sulfuric Acid 34 0 58
34 2 56 32 3 60 30 7 60 23.26 65 30 5 55 6 9 53 0.08 N Nitric Acid 34.0 68 32.4 54 32.4 56 18.8 62 17 0 58 4.9 49
25 27 33 32 33 33
12 6 9 9 11
29 31 33 33 34 35 28
13 13 7 7 10 19
29 31 31 27 29 24
13 15 13 11 13 27
..
io
..
1
B = butyl alcohol, A = acetone, E = ethyl alcohol. Abnormal fermentation.
I n view of the fact that sulfuric acid is cheaper than hydrochloric, experiments were undertaken to determine whether sulfuric acid hydrolyzates could be used for the butyl-acetonic fermentation, Table I gives the results of a series of typical fermentations in which the corn mash was replaced by oat hull hydrolyzate, prepared by the use of sulfuric acid. The data show that fermentations with the sulfuric acid hydrolyzate are as good as those with the hydrochloric acid hydrolyzate. Replacement of 50 per cent of the corn by this hydrolyzate gave the normal solvent yield and ratio. There was practically no fermentation in the case of 60 per cent replacement. The fermentation in which 40 per cent of the corn was replaced by the hydrolyzate was, however, abnormal in all respects. Such abnormal fermentations occasionally occur even in the most favorable media; no satisfactory explanation has been advanced for such anomalous behavior. Since the presence of nitrates is advantageous in some fermentations, this factor was tested in the butyl-acetonic fermentation by using a nitric acid hydrolyzate. Fermentation tests were again run in the manner already described. Results for the single series run are given in Table I. The fer-
VOL. 29, NO. 11
mentations were quite active, and yields were normal for 10 and 20 per cent replacements but were only a little better than half normal for the 30 and 40 per cent replacements. Practically no fermentation occurred in the case of 50 per cent replacement.
Conclusions The hydrolyzates obtained by treating oat hulls with dilute mineral acids are fermentable but require the presence of considerable corn mash in order to give full and normal yields of solvents. The hydrolyzates prepared by the use of hydrochloric acid and sulfuric acid give somewhat better results than those prepared with nitric acid. About 40 to 50 per cent of the carbohydrates in 6.5 per cent corn mash can be replaced by hydrochloric or sulfuric acid hydrolyzates from oat hulls without decreasing the normal solvent yields. Evidently, reasonable concentrations of calcium sulfate do not exert the inhibiting effect on the butyl-acetonic fermentation that is so marked in yeast fermentations. On the industrial scale either sulfuric or hydrochloric acids could be used; the choice would depend upon the relative cost of the two acids. Although this would appear to give the sulfuric acid the advantage, it must be remembered that the normal concentration of this acid necessary to give the same degree of hydrolysis was found to be twice that of the hydrochloric acid. A procedure such as that developed here on the laboratory scale-that is, the addition of hydrolyzates t o corn mash or other nutrient material, should offer possibilities in the butyl-acetonic or other fermentations of pentoses resulting from the acid hydrolysis of other cellulosic materials, as well as of oat hulls. During the mild acid hydrolysis of the oat hulls as carried out in these experiments, only the easily hydrolyzable pentosans are removed. The residue is not charred, and hence waste materials of this type might well serve as an excellent source of both lignin and cellulose. The lignin is readily removed by ammonia (@,and almost pure cellulose remains. The resulting product could then be hydrolyzed by the Bergius process, subjected to a thermophilic or other fermentation process employing cellulose, or employed as such in the many uses for cellulose.
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 2nd ed., 1925. (2) Bergius, F., IND. ENO.CHEW,29, 247 (1937). (3) Bryner, L. C., Christensen, L. M., and Fulmer, E . I., Ibid., 28, 206 (1936). (4) Christensen, L.M., and Fulmei, E . I., IND. ENQ.CHEM., Anal. Ed., 7, 180 (1935). (5) Fulmer, E. I., IND. ENO.CHEM.,28, 778 (1936). (6) Hall, W. L., Slater, C. S., and Acree, S. F., Bur. Standards S. Research, 4, 329 (1930). (7) McCoy, E . , Fred, E. B., Peterson, W. H., and Hastings, E. G . , J. Infectious Diseases,39,457 (1926). (8) Peters, F. N., IND. ENQ.CHEX.,28, 765 (1936). (9) Peterson, C. J., and Hixon, R. M., Iowa State Coll. J . Sci., 7, 2 5 (1932). (10) Peterson, W. H., Fred, E. B., and Schmidt, E. G., J. Bid. Chem., 60, 627 (1924). (11) Robinson, G. C., Ibid.,53, 125 (1922). (12) Schreiber. W. T.,Geib. N. Y.. Wingfield. B., and Acree, S. F., IND. ENQ.CHEW.,22,497 (1930). (13) Shaffer, P. A., and Hartmann, A. F., S. Biol. Chem., 45, 365 (1921). (14) Speakman, H. B., Ibid., 58, 395 (1923). (15) Tischenko, V. E., and Koshkein, N. V., J. Applied Chem. (U. S . S . R.), 7, 1307 (1934). (16) Underkofler, L. A,, Christensen, L. M . , and Fulmer, E. I., IND.ENO.CHEM.,28,350 (1936). (17) Weinstein, L., and Rettger, L. F., J. Bact., 25,201 (1933). ,
I
/
RECEIVED June 2, 1937. Paper presented before the Division of Celluloae Chemistry a t the 93rd Meeting of the American Chemical Society, Chapel Hill, N. C., April 12 to 15, 1937. 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.
I
