1078
INDUSTRIAL AND ENGINEERING CHEMISTRY
75 per cent sulfuric acid in the liquid phase was made a few years ago (S), and the value 34.15 kg.-cal. per mole of acid reacted at 60” C. to form butylsulfuric acid (as determined in the acid reaction product) was reported. Review of the data, however, shows that an assumption was made which probably is not justified, particularly in light of the above findings. It was assumed that all of the butenes reacting with 75 per cent sulfuric acid form butylsulfuric acid. The present investigation shows that acid extracts of the butenes prepared with 75 per cent acid would undoubtedly contain less than 30 per cent of the butenes as products of sulfation. The calculated heat value, therefore, is probably several hundred per cent too high.
Vol. 33, No. 8
Acknowledgment The writer tenders his thanks to the Standard Oil Development Company for permission to publish this work, to Ralph F. Howe for assisting in the analytical work involved here, and to others among his colleagues who offered kind criticism.
Literature Cited (1) Bacon, IND. ENG.CHEM., Anal. Ed., 1,89(1929).
(2) Brooks, IND. ENQ.CHEM.,27, 278 (1935). (3) Khokhlovkin et al., Sintet. Kauohulo, 1936,No.2, 12. (4) Ogg, J . Am. Chem. SOC.,61,1946 (1939). (5) Smith, Ibid., 61,254 (1939). (6) Suter and Oberg, Ibid., 56, 677 (1934).
Butvl-Acetonic Fermentation of Jerusalem Artichokes J
RAY T. WEiVDLAND, ELLIS I. FULMER, AND L. A. UNDERKOFLER Iowa State College, Ames, Iowa
HE so-called Jerusalem artichoke or girasole (Helianthus tuberosus) is a native plant of this country, and because of its ability to produce high yields of tubers, increasing attention has been given to its possible value as a new farm crop. B u t as repeatedly pointed out, this new crop can become profitable only when proper methods for its industrial utilization have been worked out. At present a farmer would be justified in raising only as many girasoles as he could use directly on the farm as feed for hogs or cattle. One suggested commercial outlet is the production of levulose. The artichoke tubers are rich in levulans which are easily hydrolyzed to levulose by mild acid treatment. The Sugar Section of the Sational Bureau of Standards (1, 6 ) has contributed to the development of this possible use. Independently a semicommercial plant, capable of producing 22 pounds of crystalline levulose per batch run, was developed and operated continuously by the Chemistry Department of Iowa State College in 1931 and 1932. A second large-scale use which has been contemplated is in the manufacture of ind, stria1 alcohol. The tubers have long been recognized as a possible raw material for this purpose and have actually been employed to a limited extent in Germany and France. A report, based on work a t Iowa State College, was published on the alcoholic fermentation of Jerusalem artichokes in 1937 (18). A fermentation process which is second only to alcoholic fermentation in industrial importance, is that in which butanol, acetone, and ethanol are produced. This process employs a bacterium now most generally designated as Clostridium acetobutylicum, the name proposed by McCoy, Fred, Peterson, and Hastings ( 7 ) . I n this butyl-acetonic fermentation the organism, acting on the usual raw materials, produces the sohents in the approximate ratio of 60 parts butanol, 30 parts acetone, and 10 parts ethanol, along with large amounts of carbon dioxide and hydrogen. The weight of gases formed is about one and a half times that of the sol-
T
vents, the carbon dioxide representing about 60 per cent of the gas by volume. Industrially the by-product gases are employed for the synthesis of methanol and for the production of solid carbon dioxide. In the commercial fermentation corn has been largely used as the raw material. Recently molasses has also become an important raw material for the production of the solvents mentioned, the process employing a different organism from that named above. I n view of the commercial importance of this fermentation, i t was believed that an exhaustive investigation of the butyl-acetonic fermentation of the Jerusalem artichoke should prove of considerable interest. The only investigators mho have reported attempts to subject Jerusalem artichokes to the butyl-acetonic fermentation have been Thaysen and Green ( I S ) and Reynolds and Werkman (11). The latter workers reported merely that “the carbohydrates of the artichoke tuber are not readily available to attack by C1. acetobutylicum”. Thaysen and Green employed fresh tubers. They were steamed until soft and the juice was removed by pressing. It was found that the sterilized juice did not ferment, but the juice fermented readily after acid hydrolysis. The juice was hydrolyzed by adding sulfuric acid to 0.2 per cent concentration and heating for 1 hour a t 15 pounds steam pressure; the acid was then neutralized by calcium carbonate. The hydrolyzed juice was diluted to several concentrations and inoculated with 2 per cent of fresh maize culture of Weizmann’s butyl-acetone organism. The results of the fermentations shon ed that the optimum concentration for best fermentation \\as about 3 per cent of total sugar. Yields of solvents dropped off rapidly, with sugar concentrations lower than 2 or higher than 4 per cent. The best yields obtained were in the neighborhood of 33 to 36 per cent of carbohydrate present. Fermentations were conducted on a semitechnical scale with properly diluted hydrolyzed artichoke mashes in 20- and 500-gallon tanks. The concentration of sugar before fermentation va-
August, 1941
INDUSTRIAL AND ENGINEERING CHEMISTRY
ried from 2.7 to 3.6 per cent, and yields of solvents per ton of fresh artichokes varied from 9.5 to 14.5 gallons, or from 28.9 t o 35.6 per cent of sugar fermented. Thaysen and Green concluded that, with due consideration paid to proper hydrolysis of the original carbohydrates and proper concentration of sugar in the mashes, "artichoke tubers may be successfully used for the production of +butyl alcohol and acetone, and may be expected t o yield 12 gallons of 'oil', or more, per ton of fresh tubers".
The levulans of the Jerusalem artichoke are converted to levulose by mild acid hydrolysis. After the acid is neutralized, the resulting hydrolyzate is a suitable raw material for fermentation by the butylacetone organism, Clostridium acetobutylicum. To secure maximum yields of products from the fermentations, it is necessary to add additional nutrients, in the form of corn meal or soybean meal, to the Jerusalem artichoke hydrolyzates. Hydrolyzates prepared from extracts of dried Jerusalem artichoke chips ferment somewhat more satisfactorily than hydrolyzed aqueous pulps of the dried chips or the fresh tubers. The solvents formed by the butyl-acetone organism from fermentations of Jerusalem artichoke extract hydrolyxates are identical with those from fermentations of corn mash and are produced in about the same ratio.
Materials and, Methods As raw material for most of the work reported in this aper the dried artichoke chips prepared by McGlumphy and &chin er and stored a t Iowa State College were employed. This ensure8 a uniform material of constant composition thrdughout the course of the investigations. However, fresh tubers were also used in certain cases as indicated. In the preparation of the media, when the levulans of the artichokes were hydrolyzed with acid, the method of McGlumphy, Eichinger, Hixon, and Buchanan (4, 8) was employed. An aqueous extract of the chips or a suspension of Y u n d chips or tuber pulp in water was suitably diluted to give a nal reducing sugar content in the nei hborhood of 5 per cent. Sufficient acid was added to give a H of 1.75 if hydrochloric acid was used, or a pH of 1.50 if sul&ic acid was employed, and hydrolysis was accomplished by heating for 1 hour at 80' C. The hydrolyzate was cooled somewhat and neutralized to a pH of 5.5-6.0; the adjustment was made by the addition of strong sodium hydroxide solution unless otherwise indicated. I n some of the earlier experiments calcium carbonate was employed, but subsequently it was found that higher and more uniform yields were obtained when sodium hydroxide was used for the neutralization. Many investi ators-Reilly, Hickinbottom, Henley, and Thaysen (IO), D o a e r (S), van der Lek (6),and othershave called attention to the fact that excess calcium carbonate depresses the yields of solvents obtained from the butyl-acetonic fermentation. The presence of calcium carbonate results in an accumulation of calcium salts of the acids formed and checks conversion of the acids into solvents. It was found that the treatment during hydro1 sis adequately sterilized the hydrolyzate so that it could be usedTdirectly in the preparation of the fermentation media, provided transfers of the hydrolyzate were made aseptically to the sterile fermentation
I079
flasks. Since levulose is not stable at temperatures above 80" C . (9) this procedure was used in most cases when the artichoke hydrolyzate was employed. The other constituents of the medium were sterilized separately in the fermentation flasks by autoclaving, and the sterile hydrolyzate was then added, with care against contamination, in the requisite amounts. All experimental media not containing hydrolyzates were sterilized in the autoclave for 1.5 to 2 hours at 15 to 20 pounds per square inch (1.0 to 1.4 kg. per sq. cm.) steam pressure in quantities of approximately 1600 ml. in 2-liter Erlenmeyer flasks unless otherwise indicated. The bacterial culture was an active commercial butyl-acetone culture and was handled in the customary manner as outlined by Underkofler, Christensen and Fulmer (16). The experimental media were inoculated &om vigorous 24hour subcultures (fourth or fifth transfer from the original spore culture) of the organism in corn mash, using 3 per cent of inoculum in each case. Incubation was at 37" C. When gassing had ceased (usually after 4 days of incubation) the solvents were distilled from a 300-ml. aliquot of the fermented beer, 100 ml. of distillate being collected. The solvent distillates were analyzed by the methods outlined by Christensen and Fulmer (2). In the tables the solvent yields are calculated as per cent of the glucose equivalent of carbohydrate in the mash. Analyses for the carbohydrate contents of the media or substrates were made by the Shaffer-Hartmann method (12). The polysaccharides were subjected to acid hydrolysis before the sugar determinations. All pH measurements were made by a quinhydrone electrode.
Fermentation of Chips Preliminary attempts were made to ferment a mash prepared directly from the ground, dried, Jerusalem artichoke chips by heating with water. The results showed that the artichokes cannot be fermented directly by the butyl-acetone organism and confirmed the results of the previous investigators. Somewhat better fermentations occurred after acid hydrolysis of the polysaccharides in the mash of artichoke chips. Typical results for some of the better fermentations made a t different times are given in Table I. Inspection of the data shows that the results were quite erratic. Other similar fermentations of hydrolyzed chips gave irregular results with even lower solvents yield. In general, all of the fermentations of hydrolyzed ground artichoke chips lacked the vigor characterizing the typical transformation of starch, from corn, into solvents. The yields of solvents, in terms of glucose equivalent, are normally 36 t o 40 per cent in corn mash fermentations. Occasional fermentations of the acidhydrolyzed chips gave yields of this order, but they were usually lower and at best the fermentations were sluggish. The solid material also caused some difficulty b y excessive heading.
TABLEI. YIELDS OF SOLVENTSFROM FERMENTATIONS OF HYDROLYZED GIRASOLE CHIPS Reducing Sugar Concn. in Mash, G./100 M1.
Volume of Medium, MI.
Total Solvent Yield, yo
CaCOa
3.98 3.98 3.90 5.05
1400 1400 1500 1600
35.6 31.0 22.1 21.3
HzS04
NaOH
3.90 4.90
1600 1600
36.3 31.0
HCI
CaCOs
4.45
1600
21.5
HC1
NaOH
5.00 5.75 3.80 4.60
2700 3000 1600 1600
31.2 30.5 38.2 29.9
Acid for, Hydrolysis
Neutralizing Agent
Ha604
As a result of the irregularities observed in attempting to ferment the hydrolyzed chips directly, i t was decided t o employ for future work a sirup prepared from the artichoke chips. The method of preparing the sirup was described in a previous communication (18). The unhydrolyzed artichoke
INDUSTRIAL AND ENGINEERING CHEMISTRY
1080
sirup was diluted t o form a series of solutions of total solids content ranging from 4 to 12 per cent. After sterilization for 1hour a t 10 pounds per square inch (0.7 kg. per sq. cm.) steam pressure, the solutions were inoculated and incubated. The fermentations resulting were irregular and ceased after about 24 hours, leaving liquids with sour odors. The solvents yield was inappreciable. Previous studies (16, 17) showed that certain unfavorable media may be fermented successfully by the butyl-acetone organism if considerable amounts of corn are added, confirming the prior observations of Weinstein and Rettger (19) and of Tsuchiya (15). This favorable substrate of corn serres for growth of the organisms, and the enzymes produced then dissimilate the total carbohydrates present. T o test this possibility for artichoke extracts, an experiment was planned involving a systematic replacement of corn by unhydrolyzed artichoke sirup in such a manner that the total concentration of carbohydrate was constant in all media. This was equivalent to a corn mash containing 7 per cent corn on the air-dry basis. The results of this fermentation series are given in Table 11. It is evident that the unhydrolyzed polysaccharides of the artichoke sirup were not fermented, but that the reducing sugar content was dissimilated. A series of fermentations was conducted in which corn mash was replaced by various amounts of the hydrolyzed artichoke sirup up to 70 per cent replacement. The results are plotted in Figure I, curve A . Owing to an error in making up the media, the total carbohydrate content of each fermentation flask was not identical and this accounts for the slight irregularity in the results. I n a subsequent series the corn mash was replaced by various amounts of the hydrolyzed sirup in 10 per cent increments from 0 to 100 per cent. Each medium of the series contained starch and levulose equivalent to 4.5 grams of reducing sugar per 100 ml. The solvents yields are plotted as curve B. Throughout this series, up to and including 80 per cent replacement, the average percentage of
While addition of corn meal in amounts sufficient to furnish a t least 20 per cent of the total carbohydrate gave satisfactory fermentation of the artichoke sirup hydrolyzate, it was desired to find a suitable nutrient which would regularly give satisfactory fermentations of the sirup as sole carbohydrate source. Since corn gluten meal had previously been found a satisfactory nutrient for sugar fermentations (16), it was tried with the artichoke sirup hydrolyzate. Suspensions of various quantities of corn gluten meal in 2 liters of water were sterilized in a series of 4-liter Erlenmeyer flasks. To each flask were then added 1000 ml. of sterile sirup hydrolyzate containing 114 grams of reducing sugar. The final concentration of sugar in each flask was therefore 3.8 per cent; the corn gluten meal content in the series ranged from 1 to 2 per cent. After inoculation, the resulting fermentations proved to be not very vigorous, and the final solvents yields amounted to less than 30 per cent. Repetition of the experiment confirmed the conclusion that media containing corn gluten fermented only slightly better than the sirup hydrolyzate alone. 40
$8 * o
Corn iMeal Replaced,
%
0 10 20 30 40 50
Available Carbohydratea. Grams 79.2 73.2 67.1 61.1 55.1 49.0
I
From artichokes, grams 36.5 33.2 30.3 28.0 25.2 22.8
Total Solvent Yield From F r o m available corn, carbohydrate, % of grams glucose equivalent 36.5 36.5 35.9 32.85 29,20 35.7 25.55 36.2 21.90 36.2 18.25 36.9
a T h e s u m of t h e starch in t h e corn plus t h e small amount of actual reducing sugar in t h e artichoke juice. The total carbohydrate content in each medium was 79.2 grams.
solvent yield from the total carbohydrate was 37 per cent of the glucose equivalent. Above 80 per cent replacement the yields diminished to 25 per cent in the medium containing no corn mash. From these results it appears that the essential nutrients for the butyl-acetonic fermentation of the hydrolyzed sirup from Jerusalem artichoke chips are supplied when corn mash sufficient to furnish about 20 per cent of the total available carbohydrate in the medium is used. An experiment to determine whether it was of advantage to add the hydrolyzed artichoke sirup to the sterilized corn mash before inoculating or during the course of the fermentation was tried. Yields of solvents were practically identical by both methods, averaging 34.1 and 34.4 per cent, respectively. With molasses Tsuchiya (15) apparently found the contrary-namely, that more sugar was transformed if the molasses was added to the corn mash during fermentation.
w-+=+= 60 40 REPLACEMENT OF CORN,
0
20
80
Io0
96
SOLVENTS YIELDFROM T W O SERIES OF FERMENTATIONS I N W H I C H HYDROLYZATES
FIGURE 1. FRON
TABLE11. SOLVENTS YIELD FROM MEDIA IN WHICH CORN WASREPLACED BY UNHYDROLYZED ARTICHOKE SIRUP
Vol. 33, No. 8
ARTICHOKE CHIP EXTRACTREPLACED CORNMEAL
ilnother nutrient investigated was soybean meal. This had previously been employed by Tsuchiya (14) as the source of protein in certain butyl-acetonic fermentations. Suspensions of various quantities of soybean meal in 150 ml. of water were sterilized in a series of 1-liter Erlenmeyer flasks. To each flask were then added 650 ml. of sterile artichoke sirup hydrolyzate containing 3.5 grams of sugar per 100 ml. The final concentration of sugar in each flask was therefore 3 per cent; the soybean meal content ranged from 0 to 4 per cent. The results of the fermentations of these media are given in Table 111. The data show that maximum yields of total solvents were produced from the sirup hydrolyzate when 0.95 t o 2.50 per cent of soybean meal was present t o furnish nutrients. TABLE111. C H I P SIRUP
SOLVEXTS Y I E L D FROM HYDROLYZED ARTICHOKE EJIPLOYIKG VARIOUS AMOCliTS O F SOYBEAN 1\IE.4L AS NUTRIENT
Soybean Meal Grams/lOO Mi. 0 0.37 0.62 0.95 1.50
T o t a l Solvent Yield, % 21.2 25.1 32.4 38.0 37.3
Soybean hieal,
Giams/lOO I11
1.88 2.50 3.10 3.70 Corn mnsh control
Total Solvent Yield, yo 38.2 38.0 36.9 35.7 40.6
To prove definitely that the solvents formed in the fermentation of the hydrolyzates from the artichoke chip sirup were the same as the products obtained in the industrial fermentation of starchy materials, three fermentations containing
August, 1941
INDUSTRIAL AND ENGINEERING CHEMISTRY
1081
drolyzed by the same treatment as used for the ground chips or sirup. The tubers could be used either sliced or after passing through a food chopper. I n the first attempt to ferment the fresh tubers after hydrolysis, with addition of corn meal, corn gluten meal, or soybean meal t o furnish nutrients, almost no solvents were obtained. The beers from these fermentations had sour odors and abnormally high titratable acidities. A second series of fermentations was conducted in which corn meal was replaced by carbohydrate equivalents of the fresh tuber hydrolyzate, in the same manner as previously used with the sirup from the dried chips. The results presented in Table V show that decreasing the proportion of corn resulted in decreased yields. The whole-tuber hydrolyzate was fermented, but not so satisfactorily as was the chip hyTABLEIV. RESCLTSOF BUTYL-ACETONIC FERMENTATIONS OF drolyzate previously used. Thaysen and Green ( I S ) obHYDROLYZED JERUSALEM ARTICHOKEMATERIALSWITH 0.9 GRAMOF SOYBEAN MEALPER 100 ML. TO FURNISH ADDITIONAL tained satisfactory fermentation of the hydrolyzed juice NUTRIENTS from fresh tubers. Hydrolyzates from two varieties of JeruVol. of Reducing salem artichokes were subsequently used in similar fermentaArtichoke Carbo-Solvent HydrolyVol. of hydrate Total Solvent Yield tion experiments. The hydrolyzates from tubers of the purple Ratio0 zate, Medium, Concn., artichoke gave slightly better solvent yields than did hydrolyMI. G./100 hI1. G./100 MI. % o f sugar B A E M1. zates from the tubers of the Mammoth French White variety 65 28 7 4.08 1.65 40.5 Ob 1,500 SOOc 1,500 3.02 1.22 40.4 68 32 10 although the difference was not of practical importance. The 39.9 59 33 8 10000 1,500 3.78 1.51 60 33 7 1.59 35.0 12OOc 1,500 4.54 dried chips previously employed had been prepared from the 19,000 5.12 1.78 34.8 58 29 13 1300d latter variety. Repeated experiments with both varieties a B = butanol A = acetone E = ethanol confirmed the finding that the hydrolyzates from the tubers b Corn mash odntrol (100grahs corn of 6l.i carbohydrate, as glucose). Hydrolyzed suspension of artichoke chips g.67 grams levulose per 100 did not ferment so readily as hydrolyzates of the extract from ml.) . dried artichoke chips. d Concentrated artiohoke sirup (76 grama levulose equivalent per 100 ml.).
approximately 19 liters of the hydrolyzed artichoke sirupsoybean mash were carried out a t different times in a 22-liter flask. From each of the fermentations the solvents were distilled and, by repeated salting out and redistillation, were separated from the water and dried. The mixed solvents were then subjected to fractional distillation, and the individual fractions were characterized by the preparation of solid derivatives. This fractionation of the solvents and their identification proved conclusively t h a t the products from these fermentations were the same as those obtained in the industrial fermentation of the usual raw materials.
~
C
The results of solvent determination by the usual analytical methods for one of the larger fermentations are included in Table IV; a recovery of 75 per cent of the total solvents based on this analysis was obtained by the fractionation. From the total solvent mixture, 120 grams of butanol and 55 grams of acetone were separated. This corresponds t o the usual 2:l ratio for these two products. I n addition t o the data for the above-mentioned large fermentation of the hydrolyzed artichoke sirup, the results for a series of fermentations of hydrolyzed ground artichoke chips employing 0.9 gram of soybean meal per 100 ml. of medium to furnish additional nutrbnts are also given in Table IV. The corn mash control gave a 40 per cent yield of solvents, and the yields from the best of the artichoke fermentations were practically the same. A yield of 40 per cent of solvents, based on sugar used, represents practically the theoretical
TABLE V. SOLVENTS YIELDFROM MEDIAIN WHICHCORNWAS REPLACED BY HYDROLYZED FRESH ARTICHOKE TUBER PULP Corn Meal Replaced, %
Carbohydrate Glucose) in Medium, Yo
(as
0 20 40 60
Total Solvent Yield, Yo 31.3 30.9 29.0 27.8 29.4 25.2
SO
100 ~
~~
conversion. The solvent ratios were normal. Yields were a little better from the chips when the sugar concentration was less than 4 grams per 100 ml. This corresponds with the optimum sugar concentration also found by Thaysen and Green (IS) for the hydrolyzate of the juice from fresh artichoke tubers.
Fermentation of Fresh Tubers Preliminary experiments with fresh Jerusalem artichoke tubers showed that the polysaccharides could readily be hy-
Aoknowledgment This work was supported in part by a grant from the Industrial Science Research Institute of Iowa State College for studies on the fermentative utilization of agricultural products. The authors wish to thank E. s. Haber and the Agronomy Department of Iowa State College for making available the fresh tubers used in this work, and Lu Cheng Hao for checking some of the experimental data.
Literature Cited (1) Bates, F., Natl. Bur. Standards, Letter Circ. 500 (1937). ( 2 ) Christensen, L. M., and Fulmer, E. I., IND. ENG.CHEM.,Anal. Ed., 7 , 180 (1935). (3) Donker, H. J. L., “Bijdrage t o t de Kennis der boterzuurbutylalooholen Acetongistingen”, Delft, Proefschrift Technische Hoogeschool, 1926. (4) Eichinger, J. W., McGlumphy, J. H., Buchanan, J. H., and ENG.CHEM., 24, 41 (1932). Hixon, R. M., IND. (5) Jackson, R. F., Silsbee, C. G., and Proffitt, M. J., Ibid., 16, 1250 (1924). ( 6 ) Lek, J. B. van der, “Onderzoekingen over de Butylalkoholgisting”, Delft, Proefschrift Technische Hoogeschool, 1930. (7) McCoy, E., Fred, E. B., Peterson, W. H., and Hastings, E. G., J. Infectious Diseases, 39, 457 (1926). (8) McGlumphy, J. H., Eichinger, J. W., Hixon, R. M.,and Buchanan, J. H., IND. ENG.CHEM.,23, 1202 (1931). (9) Mathews, J. A., and Jackson, R . F., Bur. Standards J. Research, 11, 619 (1933). (10) Reilly, J., Hickinbottom, W. J., Henley, F. R., and Thaysen, A. C., Biochem.J.,14, 229 (1920). (11) Reynolds, H., and Werkman, C. H., Proc. I o w a Acad. Sci., 41, 75 (1934). (12) Shaffer, P. A., and Hartmann, A. F., J . B i d . Chem., 45, 365 (1921). (13) Thaysen, A. C., and Green. B. M., J . I n s t . Brewing, 33, 238 (1927). (14) Tsuchiya, M., J. Agr. Chem. SOC.J a p a n , 8, 1209, 1267 (1932). (16) Ibid., 9, 783 (1933). (16) Underkofler, L. A., Christensen, L. M., and Fulmer, E. I.. IND. ENG.CHEM.,28, 351 (1936). (17) Underkofler, L. A., Fulmer, E. I., and Rayman, M. M., Ibid., 29. 1290 (1937). (18) Underkofler; L. A., McPherson, W. K., and Fulmer, E. I.,Ibid., 29, 1160 (1937). (19) Weinstein, L., and Rettger, L. F., J . Bact., 25, 201 (1933).
