Rapid Fermentation Process
for Dextrolactic Acid The submerged cultivation of Rhizopus oryzae in rotary aluminum fermenters under stated conditions results in the fermentation of 13 per cent glucose solutions in 30 to 35 hours, with 70 to 75 per cent yields of dextrolactic acid. A carbon balance shows that, in addition to lactic acid and alcohol, an unidentified soluble material is produced equivalent to 7.4 per cent of the glucose consumed. Possible industrial advantages of this process are discussed.
G. E. WARD, L. B. LOCKWOOD, B. TABENKIN, AND P. A. WELLS Bureau of Chemistry and Soils, U. S. Department of Agriculture, Washington, D. C.
T
HE production of dextrolactic acid [also known as sarcolactic acid or I(+)lactic acid] by Rhizopus oryzm
when cultivated on the surface of glucose nutrient solutions was the subject of previous communications from this division (6, 9). Surface cultivation gave 65 to 67 per cent yields of lactic acid in an incubation period of approximately 2 weeks. The present paper describes a process which involves the cultivation of this fungus in a submerged condition, whereby 13 per cent glucose solutions are fermented in 30 to 35 hours, with 70 to 75 per cent yields of d-lactic acid. This work is dependent on the use of the previously described rotary aluminum fermenters (4) which have also been employed as culture vessels to bring about the extremely rapid conversion of glucose to gluconic acid, using the organism Aspergillus niger (6, l l ) ,and of sorbitol to sorbose, using the organism Acetobacter suboxydans (12).
The germination medium had the following composition, in grams: 110 Commercial glucose 2.0 Urea MgSO4.7HzO 0.25 Distilled water t o make 1000 oc.
Materials and Methods The organism used in these studies was the previously described strain of Rhizopus oryzue Went and Geerligs (6), which was one of the best d-lactic said producers observed in the surface-growth studies. Although it is usually cultured on the surface of unagitated a e d i a , its growth and biochemical activity are enharxed when it is cultivated submerged under the favorable conditions described below. The culture of the organism in this work proceeded through the following three stages: ( a ) production ol' spores by growing on moist white bread for about 2 weeks, (b) germination of the spores in a glass bottle shaken during a period of 24 hours, and (c) the main fermentation, conducted in the rotary aluminum fermenters. The moist white bread cultures (one-half slice of bread in 200-cc. Erlenmeyer flasks) were inoculated from agar slants or from other bread cultures. After 5 to 6 days sporulation was profuse, and a t the age of about 2 weeks a portion of these spores was aseptically transferred to 50-75 cc. of sterile water and shaken to form a suspension, and a sample was removed to determine the spore concentration by means of a cytometer. A volume containing 420 million spores was then uised to inoculate 1.5 liters of germination medium. Usually this number of spores could be obtained from 5 to 10 cc. of spore suspension, and one bread culture would yield 5 to 10 billion spores.
KHzPO4 ZnSOa.7HzO CaCOa
0.60 0.088 10.0
All of these components were sterilized together. The glucose was a commercial grade and contained 91.5 per cent glucose, 8.0 per cent moisture, and 0.4 per cent dextrins. The other reagents were of c. P. quality. The spores were germinated in a 4-liter glass bottle which was provided with an outlet tube and was mechanically shaken in a manner analogous to that described previously for the gluconic acid fermentation (11). After 24 hours of shaking at 30" C . the spores had germinated] and 250-cc. aliquots, which contained material derived from 70 million spores, were used to inoculate 3-liter portions of nutrient solution of the following composition, in grams: Commercial glucose Urea MgS04.7HzO
150 2.0 0.25
Distilled water to make 1000 cc.
KHzPO4 ZnS04.7HsO Ootadec 1 alcohol (dissolved in 1.7 co.
0.60
0.044 0.03
ethyl alaohol)
Two hundred grams of calcium carbonate were sterilized separately and added to the 3-liter portion at the time of inoculation. The octadecyl alcohol prevented excessive foaming during the fermentation. After inoculation from the shaken bottle, the fermentation medium was transferred to the rotary aluminum fermenters] which were operated at 35" C., a gage pressure of 5 pounds per square inch (0.35 kg. per sq. cm.), a rotation rate of 13 r. p. m., and an aeration rate of 150 cc. per minute, measured as exit gas. As in the previously described firocesses conducted in this apparatus] the sterile air entered at one end of the fermenter and was brought into intimate contact with
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INDUSTRIAL AND ENGINEERING CHEMISTRY
the solution by the constant spilling of the medium from the buckets and baffles in t h e revolving fermenter. The progress of the fermentation was followed by analysis for reducing sugar (8). The final liquors were analyzed for reducing sugar, calcium in solution, ethyl alcohol ( I ) , and lactic acid ( 2 ) . Calcium was p r e c i p i t a t e d as t h e o x a l a t e , which was subsequently titrated with potassium permanganate in the usual manner. In the case of experiment B, which shows the carbon balance, wet comFIGURE 1. COURSEOF A TYPICAL d-LACTIC ACID SUBMERGED bustion analysis for carFERMENTAT!ON IWDUCED BY bon was performed on Rhzzopus oryzaa the solution a t the beginning of the fermentation and also on the final diluted liquors. Th-e method was that of Friedemann and Kendall (S), modified by the use of a sintered glass absorber as described by Wells, May, and Senseman ( I O ) . No samples were taken during the fermentation period, the progress of the reaction being indicated by a duplicate fermenter. This obviated the necessity for making complicated corrections for solution withdrawn during the course of the reaction. At the end of the fermentation the contents were drained from the fermenter, which was washed. The fungus growth, plus adhering calcium carbonate, was separated by filtration through cheesecloth and was washed repeatedly until the filtrates showed the absence of reducing sugar and calcium ion. All filtrates were combined and mixed, and the volume was determined; then the suspended calcium carbonate was removed by filtration through paper. Final analyses were made on the diluted solution. The mycelium, plus adhering calcium carbonate, was dried a t 60" to 80' C., and a gross weight was obtained. The mass was then ground and an aliquot was ashed. The ash was taken up in dilute hydrochloric acid, and its calcium content determined by oxalate precipitation and potassium permanganate titration. The calcium value was taken to be a measure of the calcium carbonate adhering to the mycelium and allowed the calculation of a net mycelium weight. Assumption that the ash consisted almost entirely of calcium oxide was not valid, for an appreciable quantity of phosphate was found to be present, owing to the fact that the phosphate of the medium was concentrated in the mycelium during the fermentation. Since the mycelium could not be freed of adhering calcium carbonate, an accurate determination of the carbon content of the fungus growth was not convenient. The value expressed in Table I is an approximation, based on the fact that a number of mycelia of various fungus species examined by this division and by Raistrick (7) contain about 50 per cent carbon. The carbon dioxide evolved was absorbed in 40 per cent potassium hydroxide solution through which the exit gas was passed before it reached the flowmeter. The alkaline solution was examined according to a modified Winkler method ( I O ) . The carbon dioxide thus found was obviously a measure of the sum of the carbon dioxide evolved by the
VOL. 30, NO. 11
metabolic processes of the organism and the carbon dioxide resulting from the neutralization by calcium carbonate of the organic acids produced during the fermentation. The carbon dioxide derived from calcium carbonate was calculated from the calcium content of the solution, and a metabolic carbon dioxide measurement was obtained by difference between this value and the total carbon dioxide.
Results of Typica1 Fermentation The course of a typical fermentation is shown in Figure 1 (experiment A ) . Only slight sugar utilization occurred during the first few hours, but after 15 hours the fermentation progressed rapidly, glucose consumption reaching a rate of 0.72 gram per 100 cc. per hour during the 21-35 hour period. The analytical data for experiment A are as follows, in grams per 100 cc.: Original glucose concentration Glucose consumed Ethyl alcohol produced Calcium in solution Lactio acid equivalent t o dissolved calcium Lactio acid found by analysis Acidity due to lactic acid Yield of d-lactic acid, baskpon glucose consumed, %
13 3 12 8
0.62
2.245 10 12 9 66 9.5 4 75.5
The fate of the fermented glucose is shown by data for experiment B. Since analyses were performed on diluted final liquors, data cannot be presented in the form used for experiment A but are summarized in Tables I and 11. Table I shows the fate of the fermented glucose and represents an over-all carbon balance. Table I1 shows the distribution of the carbon in the fermented liquor and is illuminating because it indicates the presence of an appreciable quantity of unidentified material in solution. As a product of the reaction, this material must also be considered in Table 1. TABLE I. CARBON BALAXCE (EXPERIMENT B) Weight of Compound Grams Glucose consumed Products: Lactic acid Ethyl alcohol Carbon dioxide Mycelium Unidentified compounds in soln. (Table 11) a
Carbon Equivalent Grams
383
153
265 23.4a 40.3 18.0
106 12 2 11.0 9.0
Unknown
Fermented Glucose Going to the Product
% .. 69.3 8.0 7.2
5.9
11.3
-
7.4 ~
Total 149.5 97.8 Corrected for alcohol added as solvent for antifoam agent.
TABLE11. CARBON DISTRIBUTION IN FERMENTED LIQUOR (EXPERIMENT B) Weight of Compound Grams Carbon in solution Lactic acid Ethyl alcohol Residual glucose (from C o reduction) Unidentified substances
...
Carbon Equivalent Grams
Unknown
%
145.5
...
106
73.0 9.8
265 27.4 34.8
Carbon in Solution
14.3
13.9
Y.5
-
__
134.2 11 3
92 3
~
145.5
7.7
__
100 0
In experiment B comparison of total acid as derived from the calcium in solution with the lactic acid determination shows that 93 per cent of the acidity is due to lactic acid. This fermentation was terminated a t 30.5 hours.
Discussion of Results The data cited for experiments A and B are typical of results obtained when the lactic acid fermentation is conducted in the described manner; the yields of lactic acid are consist'
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INDUSTRIAL AND ENGINEERING CHEMISTRY
ently 70 to 75 per cent, based on glucose consumed, in a fermentation period of 30 to 35 hours. Although in the two examples cited, the fermentation was terminated before all the glucose was consumed, unpublished data have shown that the reaction proceeds a t essentially the same rate until a reducing sugar value equivalent to 0.06 per cent glucose is obtained, and that no decrease in this value occurs even though the reaction time is extended several hours. It is not known whether this reducing material is glucose, a substance formed during fermentation, or an impurity introduced with the commercial glucose. The latter possibility seems probable, for it is known that cruder corn sugar products, such as "Hydrol," contain a large fraction of nonfermentable reducing materials, and it is possible that a small quantity of these are still present in the commercial crystalline sugar. Comparison of calcium and lactic acid determinations shows that 93 to 95 per cent of the acid'ity is due to lactic acid. The thorough examination of large quantities of culture liquors has shown the presence of traces of other acids, such as fumaric and succinic, in quantities too small to be demonstrated directly on the liquor. This circumstance does not interfere with the recovery of a high-purity dextrolactic acid from the liquor. The carbon balance is of considerable practical importance in indicating the general direction to be taken if increased yields are to be obtained. Such increased efficiency can result only a t the expense of ethyl alcohol, carbon dioxide, unidentified material in solution, or fungus growth. Decrease in ethyl alcohol would in all probability be accompanied by a corresponding decrease in carbon dioxide, which, according to knowledge of the mechanisni of alcoholic fermentation, is evolved in equimolar proportion to the alcohol : CsHnOs-+2C2HjOH
+ 2C02
If the 8.0 per cent of glucose converted to alcohol could be entirely eliminated, a decrease of 4.0 per cent of glucose converted to carbon dioxide might be expected, with a resultant possible 12.0 per cent increase in yield of lactic acid. Efforts to decrease the quantity of mycelium or the unidentified constituents might also lead to improved yields. Although the use of more concentrated sugar solutions in this process might appear to be advantageous with respect t o economical use of time and equipment, such procedure is prevented by the limited solubility of calcium d-lactate a t the temperature a t which the fermentation is conducted. I n earlier work 15 per cent glucose solutions were used, but subsequent improvements in the yield led to the crystallization of calcium-d-lactate within the fermenter, the contents setting to a solid white mass before all the sugar was fermented. When this occurs, fermentation practically ceases, and t h e vessel must be heated to liquefy the contents so that they may be drained out, T o overcome this difficulty, the glucose concentration was reduced to about 13 per cent. However,
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even this concentration may be slightly too high, for crystallization still occurs occasionally. I n the course of bringing this process to its present state, a mass of evidence has been accumulated concerning the effect of many of the possible variations in composition of the medium, in operation of the fermenters, and in general procedure. A discussion of these factors is reserved for future publication. Although the conditions outlined here are the best yet determined, the authors have reason to believe that the conditions for maximum yield have not yet been obtained. Work is being continued on'this aspect of the problem. It is of industrial significance that the use of low concentrations of a simple, pure, cheap, organic compound (urea) for a nitrogen source allows the preparation of a medium which, after controlled sterilization, has no more color than an aqueous glucose solution. I n addition, the fermented liquors are characterized by a faint, agreeable, esterlike odor and by the absence of volatile acids. These circumstances facilitate the recovery of colorless lactate preparations of high purity. Such recovery is a matter of some difficulty when dealing with bacterial lactic acid fermentation liquors, because of the presence of large quantities of crude nitrogenous materials which are required by the organisms. Other indicated advantages of the mold process are: ( a ) the ease of recovery of salts of d-lactic acid, (b) the rapidity of the fermentation, ( e ) the provision of a ready source of the physiologically important d-lactic acid, and (d) the provision of a source of material for the preparation of crystalline d-lactic acid (melting point 52.8" C.).
Literature Cited (1) Assoc. Official Agr. Chem., Methods of Analysis, 4th ed., p. 163 ( 1935). (2) Friedemann, T. E., and Graeser, J. B., J. B i d . Chem., 100, 291308 (1933). (3) Friedemann, T. E., and Kendall, A. I., Ibid., 82,45 (1929). (4) Herrick, H. T., Hellbach, R., and May, 0. E., IND.E N G CHEW, 27, 681-3 (1935). (5) Lockwood, L. B., Ward, G. E., and May, 0. E., J. Agr. Research, 53, 849-57 (1936). (6) Moyer, A. J., Wells, P. A , , Stubbs, J. J., Herrick, H. T., and May, 0. E., IND.ENG.CHEM.,29, 777-81 (1937). (7) Raistrick, H., and collaborators, Trans. Roll. SOC. (London), B220, 1 (1931). (8) Shaffer, P. A., and Hartmann, A. F., J. Biol. Chem., 45, 365 (1921). (9) Ward, G. E., Lockwood, L. B., May, 0. E., and Herrick, H. T., J . Am. Chem. SOC.,58, 1286-8 (1936). (10) Wells, P. A., May, 0. E., and Senseman, C. E., IND.ENG. Cmmf., Anal. Ed., 6 , 369-70 (1934). (11) Wells, P. A., Moyer, A. J., Stubbs, J. J., Herrick, H. T., and May, 0. E., IXD.EKG. CHEM.,29, 653-6 (1937). (12) Wells, P. A., Stubbs, J. J., Lockwood, L. B., and Roe, E. T., Ibid., 29, 1385-8 (1937). RECEIVEDMay 19, 1938. Contribution 286 from the Industrial Farm Products Research Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture.
