d-Lactic Acid Fermentation of Jerusalem Artichokes A. A. ANDERSENI AND J. E. GREAVES Utah Agricultural Experiment Station, Logan, Utah HE Jerusalem artichoke, Jerusalem artichoke tubers are an ideal soiirce of of tubers. Reynolds and IT‘erkman (15) got relatively high nutrient for the lactic fermentation brought about H e l i a n t h u s tuberosua, yields of propionic and acetic sometimes called the by B. dextrolacticus. They furnish the growth requirements of the culture and also fermentable acids from artichokes with “wild sunflower”, is a native of Propionibacterium arabinosum. America and in many places carbohydrate. The addition of malt sprouts (or As far as can be determined grows as a hardy weed. The other growth-promoting material) usually added to from the literature no work has plant produces tubers which the lactic fermentation is omitted. Hydrolysis of the tuber material is necessary and been done on lactic acid fercontain starchlike substances, mentation using Jerusalem inulin and levulans, both of is readily accomplished by heating to 95’ C. for 1 artichokes. Lactic acid has which are easily hydrolyzed to hour at a pH of 2.0. The addition of glucose (or many uses, the principal ones levulose. The yield of tubers levulose) to a diluted hydrolyzate improves the being in the leather industry, and the sugar content vary quality of the final product without decreasing the in dyeing and finishing texgreatly with varieties. Shoerate of fermentation. tiles, in the production of The addition of ammonium sulfate and potasmaker (17) stated that the wild ethyl lactate and other chemisium dihydrogen phosphate to the medium is types could be listed by the cals, and in food and beveressential for a rapid and coxnplete fermentation. hundred thousands, the great age products. The lactic acid majority of which have not been The optimum concentration of ammonium sulfate of commerce generally is is 3.0 grams per liter and potassium dihydrogen cultivated by man, and that optically inactive. It is most of those cultivated have phosphate 0.50 gram per liter. preferable to the inactive or had little or no care. Little Aeration of the fermenting medium proved to be levo form when used in food plant breeding has been done necessary for a rapid and complete fermentation. and beverage products and to improve the yield of tubers Fourteen per cent sugar solutions were fermented poultry and stock foods, as it or their sugar content. Shoeto dextrolactic acid in 4 to 5 days with a yield of 92 is more readily assimilated than niaker (17) reported carbohyto 94 per cent. is the levo (6, 9). If d-lactic drate content ranging from acid could be obtained a t a 8.64 to 19.47 Der cent. Mcreasonable price, a large market probably would be created Glumphy and Eichinger (11) found levulose content as high for it. It has definite advantages over the organic acids now as 24 per cent and believe it possible by plant breeding to used in the beverage and pickling industries. develop a variety yielding 30 per cent levulose, with large yields per acre. Upward of 20 tons per acre have been reAt present the lactic acid produced in the United States is derived largely from the fermentation of refined corn sugar ported. The primary interest in the Jerusalem artichoke is for the with 1 to 3 per cent malt sprouts added to supply nutritive production of commercial levulose, a sugar which has specific requirements, and in smaller amounts from skim milk and advantages over sucrose or glucose. The main difficulty whey. I n the past only members of the genus Lactobacillus have been used in the production of commercial lactic acid. encountered in the manufacture of levulose-that of crystallization-is not met where fermentations are run directly on Werkman and Andersen in 1938 (26) reported a fermentation process for the conversion of glucose into d-lactic acid by a the hydrolyzed juice, and this is the method suggested in this new species of the genus Bacillus. They (5) described the paper for the production of d-lactic acid. organism and named it Bacillus dexti*olacticus. More reThe carbohydrates of the Jerusalem artichoke are not availcently Pan et al. (14) reported the production of d-lactic acid able to Clostridium acetobutylicum until hydrolyzed (16, 18, using strains of Lactobacillus and Bacillus. The latter were g4). The addition of corn or soybean meal is necessary for a not described or classified. good yield of solvents in butyl-acetonic fermentation (24, but Sacchetti (16) reported that artichokes gave a better Lactic acid has been reported by several investigators to be a product of mold fermentation. Kanel (10) was the yield of solvents than corn. Several investigators (1, 4, 5, 7 , first to report substantial quantities (40 per cent) of d-lactic 19, 20) have reported the use of Jerusalem artichokes in alcoholic fermentation, and a few semicommercial trials have acid produced by fungi. I n 1936 Ward et al. (22) reported been made. Alcohol factories in France have used some of several species of Rhizopus which were able to convert the crop ( 1 7 ) . Hydrolysis may or may not be beneficial, glucose to d-lactic acid with yields as high as 62 per cent. depending on the strain of yeast used. Amati (1) reported Later V7ard et al. ($5)obtained a 70-75 per cent conversion. alcohol yields of 8.1 to 9.3 per cent on the tubers. The Waksman and Foster (21) also reported lactic acid production Department of Scientific and Industrial Research of Great by species of Rhizopus. Britain (6) obtained 28.4 gallons of 95 per cent alcohol per ton This investigation has been concerned with the development of a fermentation process for the production of d-lactic 1 Present address, Western Regional Research Laboratory of the U. S. acid by B. deztrolacticus, using Jerusalem artichokes as a Department of Agriculture, Albany, Calif.
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FIGURE 1. EFFECT OF pH AND TEMPERATURE ON RATE OF HYDROLYSIS OF JERTJSALEM ARTICHOKES source of fermentable material and with supplying nutritive requirements for the bacterial culture.
Materials and Methods The Jerusalem artichokes used in this investigation were of the Mammoth French White variety obtained at Utah Agricultural Experiment Station farm. The bacterial culture used was Bacillus dextrolacticus Andersen and Werkman (5). Since this organism does not ferment inulin, i t is necessary to hydrolyze the material. A number of experiments were conducted to determine the pH, the time, and the temperature required. This was done as follows: The tubers were washed and ground. Portions of 250 grams were added to each of four flasks containing 500 ml. of water acidified with sulfuric acid to give a final pH of 1.5 in two of the flasks and 2.3 in the other two. One of each air was heated a t 84" C . and the other two were heated a t 95" for 75 minutes. Samples were taken at 15-minute intervals, hydrolysis was stopped in the samples as they were taken, and total reducing sugar determined. To develop a successful fermentation process it was necessary t o determine the essential constituents of the medium, their optimum concentration, and the conditions which give the most rapid fermentation with complete utilization of a constant amount of sugar (approximately 14 grams per 100 ml.). The procedure in general for setting up a fermentation wa.s as follows: The tubers were washed and ground and 2 parts water added. The pH was adjusted to 2.0 with sdfuric acid and the material heated one hour a t 95" C., neutralized with calcium carbonate, and filter-pressed through cloth. In making up the medium to be fermented this hydrolyzate plus additional sugar, calcium carbonate, and distilled water were sterilized in the fermentation flask. Ammonium sulfate and potassium dihydrogen phosphate were sterilized in a little water and added to the other constituents. Numerous experimental fermentations were carried out to determine essential constituents and the optimal concentration of each. All fermentations were conducted in 200 ml. of medium in 500-ml. Erlenmeyer flasks or 2000 ml. of medium in 3-liter Fernbach flasks. The inoculum for these experiments was 5 to 10 per cent, of a 24-hour culture in a medium similar to that being tested. An incubation tem erature of 47" to 50" C., which previous work (5) had shown to {e optimum for the culture, was used. Calcium carbonate was used to neutralize the acid produced and was kept in a suspension by a mechanical agitator. This imparted a whirling motion to the fermenting medium for 30 seconds every 7 minutes. Sugar was determined by the method of Munson and Walker ( I S ) . Lactic acid was determined by the. method of Friedemann and Graeser (8) after ethyl ether extraction. The course of the
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FIGURE2. ON
EFFECTOF TIME OF HYDROLYSIS RATEOF FERMENTATION
fermentations was followed by determining the amount of sugar present in the medium initially and periodically, and curves were drawn plotting rams of sugar per 100 ml. against time elapsed, Extrapolation o f curves beyond the final sugar determination has been made but need not be taken into account in arriving a t the conclusions drawn in each particular experiment. Ordinarily this type of extrapolating in lactic acid fermentations is unreliable because the fermentations usually slow up near the end; however, after running numerous fermentations with B. dextrolacticus, some of which were followed to completion and many to less than 0.2 gram sugar per 100 ml., it can be stated that in no instance has the rate of sugar utilization changed appreciably while the last gram or two of sugar per 100 ml. is being fermented. I n a number of fermentations when the sugar had all disappeared or nearly so, the fermented liquor was analyzed for lactic acid and the conversion factor calculated. Andersen and Werkman (a) reported that the type of acid produced by B. dextrotacticus under various conditions is constantly of the dextro type. Therefore it is considered unnecessary to report again on the type of acid produced by this organism.
Fermentation of Tuber Material The results of the hydrolysis experiments are indicated in Figure 1. The carbohydrates were quickly hydrolyzed at 95" C. at either p H 1.5 or 2.3. Hydrolysis was rapid also at 84"C. and p H 1.5, b u t at p H 2.3 and 84"C. considerable time was required. The curves indicate further that there was practically no decomposition of the sugar. Later in the investigation it was found that, although hydrolysis was complete in 20 minutes, a more rapid fermentation was obtained by heating the material at 95" C. for 60 minutes at p H 2.0 (Figure 2). The growth-promoting substances of Jerusalem artichokes are affected little during longer periods of hydrolysis at the temperature and p H employed. From these experiments the conditions for satisfactory hydrolysis were selected as p H 2.0 at 95" C. and a time of 60 minutes. These conditions are less severe than those employed by other investigators (1, 4, 84) who hydrolyzed the Jerusalem artichoke for fermentations. Lower pH or higher temperature or both have been used. Steam under 3-4 atmospheres has been employed with slight acidity, p H 3.5 ( I ) , but this is not so easily carried out on an industrial scale. Vadas (20) ground the tubers and allowed the inulase present to act
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FIGURE 4. EFFECTOF AMMOXIUM SULF-ATE ox R.ITE O F FERMENT.4TIoN
for 2 hours a t 56-60' C. This method probably has some advantages, and its application t o the lactic fermentation might well be investigated. The most satisfactory method for large-scale production is either extraction by battery diffusion of dried chips as described by McGlumphy et al. ( l a ) or the extraction of the fresh tubers, in either case to be followed by hydrolysis. The method employed in this work gives complete hydrolysis of the carbohydrates, with no significant destruction of the reducing sugar and a minimum
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destruction of grorvth factor. The method could easily be adapted to an industrial scale. The constituents of the medium and the concentrations which proved optinium are :
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Artichoke l i r d r o i y e a t e (diluted 1 21, ml ( N H ~ z S O Igrams , KHzP.04, g r a m Additional sugar (glucose or levulose), grain3 CaCOa, grams W a t e r , ml
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FIGURE 3. EFFECTO F DILUTIXG THE HYDROLYZATE o s RATEOF FERXENTATION
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5 . EFFECTOF PHOSPHATE CONCENTRATIOK ON RATEOF FERMENTATION
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FIGURE6. EFFECTOF AERATIOSON RATEOF' FERMEWPATION
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noms OF PROPORTION OF INOCULUM ON FIGURE 7. EFFECT FERMENTATION
This medium was used throughout the following experiments m-ith modification as indicated. It was found that ground Jerusalem artichokes diluted with two parts water for hydrolysis could be further diluted and still give rapid fermentation. As the material was diluted, sugar was added to maintain the concentration near 14 grams per 100 ml. The rate of sugar utilization was found to be slightly greater when the added sugar was levulose instead of glucose. In parallel fermentations 13.28 grams of levulose viere fermented, compared with 12.22 grams of glucose. The juice may be diluted as much as 1 to 12 before the rate of fermentation is greatly changed (Figure 3). The advantage of diluting the hydrolyzate and adding sugar is that a more nearly pure lactic acid or an edible grade can be produced without extraction or distillation. However, a technical or crude lactic acid can be produced by fermenting a hydrolyzate of 10 to 15 per cent sugar concentration. I n addition to the Jerusalem artichoke hydrolyzate, it has been found that ammonium sulfate and phosphate are essential for a satisfactory fermentation. The effect of ammonium sulfate on the rate of sugar utilization is shown in Figure 4. Fermentations were carried out containing 0.0, 1.0, 2.0, 3.0, and 4.0 grams ammonium sulfate per liter. As indicated by the curves, ammonium sulfate has a remarkable influence even a t low concentrations but has an optimum a t 3.0 grams per liter. The influence of various phosphate concentrations on the rate of fermentation is shown in Figure 5. The potassium dihydrogen phosphate was used in concentrations of 0.15, 0.30, 0.45, and 0.60 gram per liter. Of these concentrations 0.45 gram produced the best fermentation. Additional experiments using concentrations of 0.40, 0.45, and 0.50 gram indicated 0.50 gram per liter to the optimum. Since potassium dihydrogen phosphate and ammonium sulfate proved so important, it is probable that other fermentations, not investigated in this respect, may well be reconsidered. The presence of these inorganic compounds in the proper concentration makes it possible to reduce the &mount of nutrients and also to obtain a more rapid fermentation and a more nearly pure product.
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FIGURE8. EFFECT OF hfETHOD OF STERILIZATION ON RATEOF FERMENTATION The experimental fermentations described above were aerated. The effect of bubbling air slowly through the fermenting medium is shown in Figure 6 . Approximately 3 grams of sugar per 100 ml. were utilized in 100 hours without aeration, compared with approximately 11 grams when the fermenting medium was aerated; thus aeration is important. The role that air or oxygen plays has not been determined, but when yeast extract is used to furnish nutritive requirements of the organism, aeration is unnecessary; if aerated, the yield of lactic acid is somewhat reduced (2). When the inoculum was increased from 5 to 10 per cent, the time required for complete fermentation was shortened by 10 per cent (Figure 7). To make sure that the results of these experimental fermentations were not influenced by contamination, all constituents of the media were sterilized a t 15 pounds steam pressure for 15 minutes or longer, whereas in industries the medium is usually heated only to a boiling or pasteurizing temperature. Therefore an experiment was carried out to determine what effect the method of sterilization has on the rate of fermentation. It was found that the 15-minute boiling method gave complete fermentation of 14 grams sugar per 100 ml. in 25 per cent less time than 15 pounds of steam for 15 minutes (Figure 8). Lactic acid yields of 92.6 and 94.2 per cent of theoretical were obtained in two fermentations run according to optimum conditions outlined above, except that the medium was sterilized a t 15 pounds steam for 15 minutes. I n fermentation processes the raw material is of prime importance. It must be cheap, obtainable in sufficient quantities, and of such a nature that rapid and complete fermentation is possible with a high yield of the desired products. The Jerusalem artichoke fulfills these conditions ideally. A crop of 18 tons of tubers of 20 per cent carbohydrate would yield 7200 pounds of fermentable material per acre. The cost would be less than that of corn, much less than that of corn sugar, and about equal to that of blaokstrap molasses, which constitute the chief sources of raw material for fermentation processes a t present. Although glucose was added to these fermentations, levulose, the principal
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INDUSTRIAL AND ENGINEERING CHEMISTRY
sugar of the Jerusalem artichoke hydrolyzate, n-as equally as good if not better. Further investigation will undoubtedly shorn that the relatively expensive glucose can be replaced with a semipurified levulose sirup made from the Jerusalem artichoke. I n addition to being a cheap source of carbohydrate, the Jerusalem artichoke supplies the nutrient requirement of the bacterial culture and thereby substantially increases the economic efficiency of the process. It is essential in lactic acid production that all the sugar be utilized before the fermented liquor is processed. A small amount of residual sugar in the liquor would be caramelized in processing, and the product would be of inferior quality. The type of graph used in this report gives the amount of sugar present in the fermenting medium a t any particular time and shows how near or when the fermentation reaches completion. Hence it may be seen that the fermentation proceeds rapidly throughout the process until the sugar is completely utilized.
Literature Cited Amati, Agostino, Indzts saccar. ital., 31,411 (1938). Andersen, A. A,, thesis, Iowa State College, Ames, Iowa. Andersen, A. A., and Werkman, C. H., Iowa Slate Coll. J . Sci., 14, 187 (1940). (4) Asai, Tosinobu, J . A g r . Chem. SOC.J a p a n , 13,247 (1937). (5) Brit. Dept. of Sei. and Ind. Research, "Power Alcohol from Tuber and Root Crops in Great Britain", London, H . M. Stationery Office, 1926. Cori, C. C., and Cori, G. T., J . B i d . Chem., 81,389 (1929).
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(7) DuMont, H., J . Agr. (Barral), 12 (4), 384-8, 423-7 (1877). (8) Friedemann, F. R., and Graeser, J. B., J . Biol. Chem., 100,291 (1933). (9) Furth, O., and Engel, P., Biochem. Z.,229,381 (1930). (10) Kanel, E., Microbiology (U. S.S . R.),3,259 (1934). (11) McGlumphy, J. H., and Eichinger, J. W., J . Chem. Education, 10, 453 (1933). (12) MoGlumphy, J. H., Eichinger, J. R., Hixon, R. M.,and Buchanan, J. H., IND.ENG.CHEM.,23, 1202 (1931). (13) Munson, L. S., and Walker, P. H., J . Am. Chem. Soc., 28, 663 (1906). (14) Pan, S. C.,Peterson, W.H., and Johnson, 31. J., IND.ENG. CHEM.,32, 709 (1940). (15) Reynolds, H., and Werkman, C. H., Proc. I o b a A c n d . Sci., 41, 75 (1934). (16) Sacchetti, Mario, I n d u s . saccar ital., 32,294 (1939). (17) Shoemaker, D. N., U. S. Dept. Agr., Tech. Bull. 33 (1927). (18) Thaysen, A. C., and Green, B. M., J . I n s t . Brewing, 33, 236 (1927). (19) Cnderkofler, L. A., McPherson, W. K., and Fulmer, E. I., IND. ENQ.CHEM.,29, 1160 (1937). (20) Vadas, Rudolf, Chem.-Ztg., 58, 249 (1934). (21) Waksman, S. A., and Foster, J. W., J . A g r . Research, 57, 873 (1938). (22) Ward, G. E., Lockwood, L. B., May, 0. E., and Heirick, H. T., J . Am. Chem. Soc., 58, 1286 (1936). (23) Ward, G. E., Lockwood, L. B., Tabenkin, B., and Wells, P. A.. IND. ENG.CHEM.,30,1233 (1938). (24) Wendland, R. T., Iowa State Coll. J . Sci., 12,170 (1937). (25) Werkman, C. H., and Andersen, A. A., J . Bact, 35,69 (1938).
PUBLICATION approved by t h e
Director of t h e Utah Agricultural Experiment
S t a t i o n as Project 199.
Phase Equilibria in Hvdrocarbon Svsterns J
H. H. REAMER, R. H. OLDS, B. H. SAGE, AND W. N. L-4CEY
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Methane-Decane System'
California Institute of Technology, Pasadena, Calif.
H E volumetric and phase behavior of the methanedecane system has been studied only partially, although these coniponents are present in a large number of naturally occurring hydrocarbon systems. The specific volumes of several mixtures of methane and It-decane were studied earlier a t pressures up to 5000 pounds per square inch in the temperature interval between 70" and 250" F. ( 7 ) . These data serve to establish the behavior of the bubblepoint liquid throughout that range of pressures. The composition of the dew-point gas was studied a t pressures up to 2500 pounds per square inch a t loo", 160°, and 220" F. ( 3 ) . The volumetric behavior of methane has been established with adequate accuracy a t pressures up to 15,000 pounds per square inch a t temperatures between 32" and 400" F. ( 2 , 6). The specific volume of decane was studied as an incidental part of an earlier investigation a t pressures up t o 3000 pounds per square inch and for temperatures between 70" and 250" F. (7). The specific volume of the liquid phase a t atmospheric pressure and the normal boiling point for n-decane
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1 T h i s is the thirty-seventh paper in this series. Previous articles appeared d u r i n g 1934-40, inclusive, a n d in June, July, September, a n d October, 1942.
were determined by Shepard and co-workers ( 8 ) , while the vapor pressure was ascertained a t two temperatures by Young (9). These limited data did not suffice to establish the volumetric and phase behavior of decane or mixtures of it with methane throughout the range of conditions of interest in the prediction of properties of naturally occurring hydrocarbon mixtures.
Method I n principle the methods employed in the volumetric study involved the introduction of known weights of methane and decane into a stainless-steel chamber whose effective volume was systematically changed by the introduction and withdrawal of mercury. Equilibrium was attained by the use of a magnetically driven agitator located within the equilibrium vessel. The pressure was determined from the indication of a sensitive pressure balance (6) with an uncertainty of not more than 0.05 per cent, except a t pressures below 500 pounds per square inch where the uncertainty was possibly as large as 0.1 per cent. The total volume of the system was deter-
