814
INDUSTRIAL AND ENGINEERING CHEMISTRY
When coagulating lime-softened water (Figure 3) the economy line indicates the value of using approximately as much silicate as copperas beyond a very small iron lead until a dosage of 3 p. p. m. copperas is reached; treatment for lower turbidities can be more economically selected by increasing only the silica sol and still retaining slightly over 3 p. p. m. copperas as a constant feed. The high indicated relative amount of silica here reverses the roles of main and auxiliary coagulants and gives more value to activated sodium silicate than do previous studies. The economy of high silica proportions can be appreciated from its power to produce very clear water. However, if high settled-water turbidities are tolerable, the limiting condition points to the economy of copperas alone. For inferior filtrate quality (Figure 4 ) copperas unaided is the economical choice up to 2 p. p. m., but for improved clarity activated sodium silicate is best added in the proportion of 2 p. p. m. for every 1 p. p. m. copperas above a 2 p. p. m. copperas level. Figure 4 indicates that economical silicate dosage approximately equals the copperas dose at and above the 4 p. p. m. level. From these observations it is evident that considerably more emphasis is due to activated silica sol in its joint performance with copperas than has been accorded heretofore. APPLICATION TO PLANT PRACTICE
Although these curves definitely demonstrate the economy of sodium silicate in conjunction with lime softening and final coagulation with copperas, application of the Baylis sol
Vol. 35, No. 1
to plant operation has been restricted at Carrollton to use as a palliative during periods of basin outage or obscure intervals of poorly coagulable turbidity, particularly in cold weather. When i t has been employed, the progress of enhanced floc formation produced by preceding 0.3 grain per gallon of copperas with 0.1 grain per gallon of silica could be followed as a front advancing through the course of an aroundthe-end mixing basin. However, a t the Algiers plant (capacity of 6 million gallons per day with limited mixing and settling facilities), serving a municipal division on the west bank of the river, adoption of silica sol as routine in connection with the lime-iron process has resulted in improved performance. LITERATURE CITED
(1) Baylis, J. R., J . Am. Water W o r k s Assoc., 29, 1355-96 (1937). ( 2 ) Baylis, J. R., Water W o r k s Eng., 90, 971-4, 1035-9, 1101-5 (1937). (3) Baylis, J. R., Water W o r k s & Sezmrage, 83,470-3 (1936). (4) Ibid., 84, 61-3 (1937). (5) Ibid., 84, 221-5 (1937). (6) Ibid., 85, 855-8 (1938). (7) Christenson, C. W., a n d Lavine, Irwin, Trans. Am. I n s t . Chem. E ~ Q ~ 36, s . ,71-90 (1940). (8) Graf, A. V., and Schworm, W. B., Water Works Eng., 90, 1514 (1937). (9) Hurwitr, E., and Williamson, F. M., Sswaaa W o r k s J . . 12. 562-70 (1940). (10) Lordley, H. E., and Smith, M. C., J . Am. Water W o r k s Assoc., 31, 2149 (1939). (11) Schworm, W. B., Rept. 24th Ann Missouri Water Sewage Conf., 10, 37-41 (1938).
FUNGAL AMYLASES AS SACCHARIFYING AGENTS IN THE
Alcoholic Fermentation of Corn LU CHENG HAO, ELLIS I. FULMER,
I
N TWO previous papers (3, 6 ) data were presented on
the use of mold amylase preparations in the saccharification of corn mash for alcoholic fermentation. The molds were cultured on wheat bran in rotating drums. I n a general review of the use of microbial amylases in the alcoholic fermentation, one of the authors ( 5 ) discussed briefly the relative merits of these materials and malt. The present paper describes a new and more efficient laboratory method for growing the molds and compares the efficiencies of the mold-bran preparations from twentyseven strains of molds, representing four genera, as saccharifying agents in the alcoholic fermentation of corn. FERMENTATION PROCEDURES
From the results of preliminary investigations, the folloiving standard procedures were adopted. The stock cultures of the molds were kept on wort-agar slants. For cultivating the molds in flasks, transfers were made from well sporulated cultures to wheat bran mashes. The latter were prepared by mixing equal weights of wheat bran and 0.3 N hydrochloric acid in Erlenmeyer flasks and sterilizing for 30 minutes a t 15 pounds per square inch (1 kg. per sq. cm.) steam pressure. The bran mashes were heavily inoculated from well sporulated stock cultures and the flasks, lying on their sides, were
AND L. A. UNDERKOFLER Iowa State College, Ames, Iowa
incubated a t 30" C. For most of the work here reported, 500-ml. Erlenmeyer flasks containing 25 grams of bran were employed, but it was found later that more rapid growth and sporulation are secured if 10 grams of bran are used in 250-ml. flasks. After abundant sporulation had taken place, the cultures were used as inoculum for larger batches of bran mash. The well sporulated mold cultures on the bran may be allowed to dry undisturbed in the flasks and kept a t incubator or room temperature for many months without loss of potency as inoculum. The mold amylase preparations were produced by growing the molds on the wheat bran mash in special 3-quart aluminum pots equipped for aeration. The apparatus is simply constructed (Figure 1); it is a modification of that employed by Beresford and Christensen ( d ) , and has several advantages over the drum method previously employed in these laboratories (3, 6 ) . It takes less space and requires no special mechanical devices. There is no disturbance of the mold mycelium during growth and more uniform aeration is obtained. The growth of the molds is more rapid, and the pot
July, 1943
INDUSTRIAL AND ENGINEERING CHEMISTRY
815
preparations lead t o more moval from the autoclave the consistent and higher yields pH is adjusted t o 4.5-5.0 Mold-bran preparations of twenty-seven of ethanol. using sodium hydroxide, sostrains of molds, representing four genera, The method of culturing dium carbonate, or calcium the molds in the pots is as carbonate; for most of the have been tested as saccharifying agents follows: The bran mash is work here reported sodium hyfor the alcoholic fermentation of corn. droxide was employed. The prepared by moistening 750 The molds were grown on wheat bran in grams of wheat bran with an mash (at 30' C.) is transferred special aluminum pots equipped for aeraequal weight of 0.3 A' hydroto a Whiz mixer or similar tion. The growth of the molds in the pots chloric acid. The wet bran is equipment, and the mold-bran packed into the pot and added in the form of a slurry was more rapid and uniform than in the in water. The mixture is agisterilized in the autoclave rotating drums previously employed, and tated for one minute, returned a t 15 pounds per square inch the saccharifying activities of the products t o the flask, and allowed to steam pressure for 30 minutes. were greater. Saccharification at 30' C. stand in the incubator a t The cooled mash is mixed was as satisfactory as at higher tempera30" C. for about an hour. with 5 to 10 grams of well The mash is then inoculated sporulated mold culture grown tures; in fact, with some molds higher alin flasks on wheat bran mash, by adding 20 ml. of a 24-hour cohol yields were consistently obtained at yeast culture (Saccharomyces and the inoculated material is Asperthe lower temperature. Strains of cerevisiae No. 43) grown on a packed firmly into the pot. gillus oryzae, Rhizopus delemar, and Rhizo10 per cent malt extract meThe pot is placed on a layer dium. After fermentation for p u s oryzae gave the best yields. However, of cotton batting and the 3 or 4 days, the fermented material incubated a t 30" C. A . oryzae was preferred for it showed the medium is transferred to a until the temperature rises to most consistent results and was the easiest Kjeldahl flask, and about 0.5 37-40' C. This temperature to handle. Under optimum conditions the gram of solid sodium carbonate is reached in about 8 hours alcohol yields obtained by using certain or a little solid calcium carbonand indicates rapid growth ate is added to neutralize strains of A . oryzae were about 95 per cent of the mold. The mass is the acids. The mixture is then aerated by passing air of theory. then distilled, and exactly through the pot a t a pres100 ml. of the distillate are sure of 0.3 t o 3 inches of collected in a volumetric flask. water, the flow of air being The specific gravity of the so regulated as t o maintain distillate (25"/25" C.) is determined by means of a a temperature below 45" C. After a,eration for 12 to 24 Chainomatic Westphal balance, and the alcohol content hours-the contents are removed, spread on paper, and read from an appropriate table. dried a t room temperature. The dried material is ground Corrections were made for the ethanol from the inoculum in a Wiley mill and is used as the saccharifying agent in and from the mold-bran. That is, the activities of the the fermentation tests. Such material is designated "moldvarious mold-bran preparations were compared on the basis bran". of the alcohol yields from the starch of the corn alone. The Yields of ethanol from fermentations of 20 per cent corn theoretical yield of ethanol was calculated from the well mash, saccharified with the various mold-bran preparations, known equations for the conversion of starch to sugar and the were employed as the index of their amylolytic effectiveness. latter t o ethanol by alcoholic fermentation. The authors believe (6) that the final yields of ethanol furnish I n a typical experiment the fermentation mash conthe only reliable means for comparing the usefulness of the tained 60.0 grams of corn with a starch content of 57.9 and saccharifying agents; Lintner numbers are wholly unreliable a moisture content of 12.5 per cent, 3.6 grams of mold-bran, in such comparisons. The data represent the averages of and 20 ml. of yeast culture as inoculum. After fermentation duplicate fermentations, and all were confirmed by repeated for 3 days the entire fermented mash (365 ml.) was subjected experiments. t o distillation and the first 100 ml. of distillate collected. The corn meal used in these investigations was prepared The specific gravity (25'/25") of the distillate was 0.9686, by grinding whole yellow corn, obtained in several lots a t corresponding to 19.77 grams of ethanol produced from the different times; moisture and starch analyses were carried corn, mold-bran, and inoculum, which represents 5.41 grams out on each lot. The starch analyses were made by the of ethanol per 100 ml. of final beer or 6.87 per cent by volume. official A. 0. A. C. diastase method with subsequent acid The ethanol derived from inoculum and mold-bran, as deterhydrolysis (I) ; the reducing sugars formed were determined mined from control fermentations containing malt extract by the modified Shaffer-Somogyi method developed in these and mold-bran, was as follows: 0.68 gram from the 20 ml. of laboratories (7), The average moisture content ranged inoculum and 3.6 x 0.045 = 0.16 gram from the mold-bran, from 11 to 14 per cent, and the starch content from 56 to or a total of 0.84 gram of ethanol. Therefore, the quantity 60 per cent. The wheat bran was purchased from a local of ethanol from the starch of the corn alone was 19.77 grain elevator. Blue Ribbon malt extract (Premier-Pabst 0.84 = 18.93 grams of ethanol. From the equation for the Corporation) was employed in the preparation of the wort alcoholic fermentation, for yeast culture. The method employed in the comparison of the different CBHiaOs HzO +2COz 2CzHbOH mold-bran preparations follows: 60.0 grams of corn meal are mixed with 300 ml. of 0.04 N hydrochloric acid in a 500-ml. it is evident that the complete fermentation of 162 grams of Erlenmeyer flask, and the starch is gelatinized by heating the starch yields 92 grams of ethanol; i. e., 1 gram of starch mash on a hot plate or over a small fla.me with occasional yields 0.568 gram of ethanol. I n the 60.0 grams of corn stirring to produce a uniform paste. The mash is then there were 60.0 X 0.579 = 34.74 grams of starch which would cooked in the autoclave a t 20 pounds per square inch (1.4 kg. per 6q. cm.) steam pressure for 30 minutes. Upon regive a theoretical yield of 34.74 X 0.568 = 19.72 grams of
+
+
I N D U S T R I A L AND E N G I N E E R I N G CHEMISTRY
816
Vol. 35, No. 1
for 1 hour as a t 55" for 1, 2, or 3 hours. I n commercial practice it may be advantageous to add the mold-bran a t 55" C. to lower the mash viscosity and then pump immediately through the mash coolers into the fermenters. Table I1 shows the effect upon alcohol yields of cooking the corn mash \vith various concentrations of hydrochloric acid for 30 minutes a t 20 pounds per square inch steam pressure. There is a definite optimum a t 0.04 N acid. There was a distinct difference in the consistency of the mash cooked with 0.02 N and with 0.04 i\; acid; t,he former was quite thick, the latter was very thin. The mashes cooked with concentrations of acid higher than 0.04 N were brovn in color, and the extent of caramelization increased with increasing acid concentration. The decrease in viscosity of the mash markedly increases the ease of handling and simplifies the procedure of saccharification. The mash can also be thinned by the prelimi. nary addition of mold-bran or of malt, but the use of acid gave much more uniform results. AMYLASE PREPARATIONS FROM DIFFERENT MOLDS
Figure 1.
Apparatus for Growing Mold
ethanol. The yield from the above fermentation was, therefore, 100 X (18.93/19.72) = 96.0 per cent of theory. It is evident from the above example that the activities of the various mold-bran preparations were compared on the basis of the alcohol yields from the starch of the corn alone, in terms of the percentage of theoretical yield of alcohol obtained. However, in order to make possible a more ready comparison of the results obtained in this study with industrial plant practice, the data of the above typical experiment may be used to calculate the results as commonly done in the distilling industry. Since 18.93 grams of ethanol were obtained from 60.0 grams of corn, this would represent 18.93 pounds of ethanol from 60.0 pounds of corn or 56 X (18.93/60.0) = 17.67 pounds of ethanol or 5.34 proof gallons of alcohol per bushel of corn as used. Since the corn as used contained 12.5 per cent moisture, 60.0 pounds would be equivalent to 60.0 X 0.875 = 52.5 pounds of moisture-free corn. The yield on the dry basis, therefore, was 100 X (18.93/52.5) = 36.06 pounds of ethanol or 10.9 proof gallons of alcohol per 100 pounds of dry corn, or was 36.06 X (56/ 100) = 20.19 pounds of ethanol or 6.10 proof gallons of alcohol per 56 pounds of dry corn. The standard procedures were based upon many preliminary experiments. For example, Table I gives data on the effect of saccharification temperature and time upon alcohol yields. It is evident that the alcohol yields are just as good, or better, when the conversion takes place at 30" C.
Twenty-seven representative strains of molds were chosen for detailed studies; four genera were included, Aspergillus, M u c o r , P e ni c i l l i um, and Rhizopus. The strains are listed in Table 111. With the exception of two unidentified black molds (probably species of R hi zopus) which were isolated in these laboratories, all cultures were obtained from carefully kept collections and are designated by the names under which they were received. Data on the alcohol yields from corn mash saccharified with representative mold-bran preparations are given in Table IV. All the strains of Aspergillus were very active; cultures of Aspergillus oryzae Nos. 2,38, and 40 were es-
TABLE I. EFFECTOF SACCHARIFICATION TENPERATURE AND TIMEON ALCOHOL YIELDS FROM CORN M A S H SACCHARIFIED B Y SEVERAL hf OLD-BRAN PREPARATIONS MoldBran, Preparation Aspergillus oryzae 2
Aspergillus oryzse 38
Rhizopus delemsr 34
Temp.,
C.
Time
Hour;
30 30 30 55 55 55 55 55 55
%o::
Alcohol Yield,
% of Theory 88.6 91.5 91.3 87.0 88.4 89.5
6 8
30 30 55 55
1
6 8
3 3
6
30 55
1 3
6
1
8
6
86.1
88.0 89.5 94.5 94.8 93.5 93.7
92.5 91.0
INDUSTRIAL AND ,ENGINEERING CHEMISTRY
July, 1943
pecially potent. Mucor rouxii and Mucor circinelloides proved to be as effective as the Aspergilli; M . javanicus was inferior to the other preparations. The two species of Penicillium were inferior to the Aspergilli, the P. chrysogenum being particularly poor. With the exception of one strain of R. oryzae, all of the strains of Rhizopus gave very active mold-bran preparations.
TABLEIV. ALCOHOL YIELDSFROM CORNMASHSACCHARIFIED WITH AMYLASE PREPARATIONS FROX STRAINS OF FOUR GENERA Mold and Culture No.
A. niger 1 A. niger 3
A. niger 63
TABLE11. EFFECTOF HYDROCHLORIC ACID CONCENTRATION USEDIN COOKINGCORNMASH ON ALCOHOL YIELDS (Cooked for 30 minutes at 20 pounds per square inch steam pressure; 8 per cent mold-bran prepared from Rhizopus deEemar No. 12) Normality Alcohol Yield of HC1 % of Theory' 86.5 0.005 0.01 86.7 0.02 87.6 0.04 93.5 0.08 90.0 0.16 88.8 0.32 86.8
817
Mold-Bran % of Corn) ASPERQILLUS 4 6 S 6 8 6
8 4 6 8 4 6 8 4 6 8 4 6 8 4 6 8
A. oryrae 2
A. oryrae 2 A. oryrae 38
A. oryrae 40 A. oryzae 42
Alcohol Yield % of Theory'
86.4 91.4 91.3 90.0
91.6 84.1 89.5 88.6 91.5 93.2 90.6 92.8 92.5 93.2 93.8 93.5 92.5 93.5 93.0 89.4 91.1 91.6
MUCOR
M. rouxii 4
EFFECT OF STORAGE. Studies were made of the effect of several variants in the handling of the mold-bran. Moldbran preparations from Aspergillus oryzae No. 38, added to the mash in the form of wet lumps, dry lumps, dry powder, and wet powder, showed no significant differences in alcohol yields. The effect of storage upon the activity of the dry powdered preparations is shown in Table V. It is evident that there is no significant deterioration during storage. I n order to avoid deterioration, however, it is advisable that
P.chrysogenum 7 P. purpurogenum 8
R. delemar 12
Lab. No. 1 2 3 4 7 8 11 12 13 14 15 16 17 18 19 20 21 32 33 34 35 38 40
42 67
KI Ka
Name Aspergillus niger
Source Botany Dept., Iowa State
Aspergillus oryzae Aspergillus niger Mucor rouxii Penicillium chrysogenum Penicillium purpurogenum Rhiwpus nigricans Rhizopus delemar Rhizopus delemar Rhizopus oryzae Rhiaopus oryzae Rhizopus oryzae Rhizopus oryzae Rhiaopus peka I Rhizopus tritici Mucor circinelloides Mucor javanicus Rhizopus oryzae Rhizopus oryzae Rhizopus delemar Rhizopus shanghaiensis Aspergillus oryzae Aspergillus oryzae Aspergillus oryzae Aspergillus niger Unidentified black mold Unidentified black mold
A. T. C. C.O, No. 4814 N.R. R. L.b, No. 3 A. T . C.C.. No.4855 Thom", No.5034.11 Thom, No. 413.2670 A. T. C.C., No. 1210 A. T.C.C., No. 4859 A. T. C.C.,No.4858 Lockwoodd, No.649 Lockwood, No.660 Lockwood, No.664 Lockwood, No. 704 Lockwood, No. 839 Lockwood, No.664 Lockwood, No. 840 Lockwood, No. 718 N.R. R. L., No.395 N.R. R. L., No. 1034 N R . R. L., No. 1472 N. R. R.L., No. 1518 Rohm and Haas', No 38 Rohm and Haas, No. 40 Rohm and Haae, No. 42 N.R. R. L., No. 67 Isolated in lab. Isolated in lab.
Collene ----__
a American Type Culture Collection, Georgetown Univ. Medical School, Washington, D. C. Northern Regional Research Laboratory, U. 8. Dept. Agr., Peoria, Ill. Charles Thom, Bur..of Agr. Chem. and Eng., U. S. Dept. Agr., Washington, D. C. L. B. Lockwood, Bur. of Agr. Chem. and Eng., U. 8 . Dept. Am., Washington, D. C. 6 Rohm and Haas Co., Bristol, Penna.
PBNICILLIUM 6 8 6
8
64.7 73.4 86.1 90.5
6
R. oryzae 14
6
R. oryrae 15 R. oryzae 16 R. oryrae 17 R. oryrae 32
6 8 6 8
92.5 93.9 92.6 93.7 93.8 93.8 93.4 90.6 90.9 92.0 93.5 85.2 90.5
6
71.6
8
84.0 85.0 90.6 93.0 92.8 93.5 94.0 81.0 89.5 86.9 91.0 90.8 89.6 88.5 90.3 90.3
R.delemar 34
MOLDSTESTED FOR AMYLASE PRODUCTION
6
8
87.5 92.7 91.0 92.8 72.8 82.8
RaIzoPue
R. delemar 13
TABLE111.
6 8 6 8
M.oiroinelloides 20 M. javanicus 21
8 6 8
4 6
8 8
4 6
R. oryzae 33 R. nigrioans 11
R. shanghaiensis 35
R. tritici 19 Unidentified K I Unidentified: KP
8 6
8 8 6 8
the moisture content be not above 12 per cent; in a few cases where the moisture content was from 15 to 17 per cent, spoilage occurred. EFFECT OF SECONDARY ADDITION OF MOLD-BRAN.If the amylase preparation were added a t intervals, in amounts which might maintain the concentration of the enzymes a t a level where possible synthesis of fermentable into nonfermentable sugars would not take place, it was thought that the alcohol yield would be improved. Data on this secondary addition of mold-bran are given in Table VI. Preliminary tests had shown that 2 per cent mold-bran was an adequate amount for the secondary addition and that the best time for its addition was between the twentieth and twenty-fourth hour of fermentation. The data show that the secondary addition leads to small but consistent increases in alcohol yields. The advantage decreases with increase of alcohol yield. It is probable that secondary addition is of
INDUSTRIAL AND ENGINEERING CHEMISTRY
818
TABLE V.
EFFECTOF STORAGE ON AMYLOLYTIC ACTIVITYOF MOLD-BRAN PREPARATIONS
Mold and Cult u r e No. A. oryzae 2 A. oryzae 2 A. oryzae 2 A. oryzae 2 A, oryzae 2 A. oryzae 2 A. oryzae 2 A. oryzae 2 A. oryzae 2 A . oryzae 38 R. oryzae 33
Pot Run
No. 17
15 13 8 8 3 1
1 Drum 00 33
~i~~ of Storage, Nonths 1 3 3.5 6 10 7.5 9 13 24 4.5 3
3ornof 6 6 8 8 8 8
S 8 6 6 8
Alcohol Yield, m o f o r y Stored Fresh92.5 94.2 93.3 90.6 91.0 92.2 90.6 91.0 91.5 93.5 93.0
92.8 94.0 93.2 90.2 90.2 90.5 91.0 91.0 92.0 93.8 93.5
Vol. 35, No. 7
the same fermentation series (Table VIII). The preparations all give yields of about 95 per cent of theory, and the results are remarkably uniform. It is unlikely that much better alcohol yields than those shown above may be expected under laboratory conditions since some of the carbohydrate must be utilized in building up the protoplasm of the yeast, some alcohol is lost by evaporation, and small amounts of by-products, such as glycerol, are always produced in the normal fermentation.
TABLEVII. ALCOHOLYIELDS FROM CORN MASHSACCH.4RIFIED ANYLASEPREPARATIONS FROM MOLDSGROWN ON BRAN, WITH AND WITHOUT MINERAL SALTS TABLE VI. ALCOHOLYIELDSFROM CORNMASHSACCHARIFIED WITH AMYLASEPREPARATIONS, WITH AND WITHOUT SECONDARY Mold and Culture Mold-Bran, -Uooho1 Yield, % Of Theory No. 70 of Corn With salts Without salts ADDITION WITH
Mold and Culture No.
Mold-bran* % of Corn 1 s t addition 2nd addition
Alcohol Yield % of Theor;
A. oryzae 2
4 6 8 4 6 8 4 6 8 4 6 8 4 6 8 4 6 8 4 6 8 4 6 8
A. oryzae 38 A. oryzae 40 R. delemar 12
R. delemar 13 R. delemar 34 R. oryzae 32
little or no value n hen the alcohol yields, without secondary addition, are already close to maximum. Tertiary addition of mold-bran n-as also tried-i. e., dividing the amount of mold-bran into three portions, and adding one portion before the fermentation starts and the other two portions at 20-24 hour intervals; a secondary addition of 10 ml. of active yeast culture was also made. Neither of these modifications nor a combination of them further improved alcohol yields. The effect of mixtures of various mold-bran preparations, representing different genera, was also studied. None of them, with or xithout secondary addition, gave higher alcohol yields than those obtained by using the corresponding amount of the better strain alone. EFFECT OF MINERAL SALTS. It is well known that traces of mineral salts stimulate the growth of molds. The salts recommended by Steinberg ( 4 ) were added t o the acid used in preparing the bran mash for the growth of the molds. To each liter of the dilute acid were added 0.000625 gram each of ferrous sulfate and zinc sulfate. The alcohol yields obtained from fermentations of mashes saccharified with these preparations are given in Table VII. The addition of the salts increases somewhat the amylolytic activity of the strains of Aspergillus oryzae but is disadvantageous with the representatives of Rhizopus. It is possible, however, that the latter might be stimulated by different concentrations of the salts. COMPARISON OF STRAINS.On the basis of the above data and other repeated experiments, certain strains of Aspergillus oryzae proved to be the most satisfactory molds. Although some of the species of Rh i z o p u s gave excellent results, the cultures of Aspergillus are much easier to handle. The Aspergilli produce more abundant sporulation, which facilitates groa th of the inoculum, makes possible a heavier inoculation of the mash, and thus minimizes danger of contamination. Moreover, the mycelium formed by the Aspergilli is more dense and makes the mold-bran easier t o handle. The three best strains of Aspergillus oryzae were selected, and two mold-bran preparations of each were compared in
R. oryzae 33
91.0 94.2 95.5 94.0 94.5 95.0 92.5 94.4 92.0 91.0 93.0 92.0 88.4 90.2 90.6 92.5 91.9 90.3 90.4 90.2 90.0 89.3 89.1 88.9
90.5 92.8 93.2 93.2 93.8 93.5 92.5 93.5 93.0
..
92.5 93.9
91:5
93.7 93.8 93.8 93.4 85.0 90.6 93.0 92.8 93.5 94.0
TABLEVIII. ALCOHOLYIELDS FROM CORNMASH SACCHARIFIED WITH DIFFERENT AMYLASEPREPARATIONS FROM STRAINS OF Aspergillus oryzae Culture No.
P o t Run
2
15
2
38
38
00
38
44
40
18
40
41
KO.
Mold-Bran, % of Corn
Alcohol Yield 70 of Theory'
6 8 6 8 6 8 6 8 6 8 6 8
95.7 96.0 94.3 95.0 95.5 96.0 95.0 96.0 94.5 95.5 96.3 95.5
ACKNOWLEDGMENT
This work was supported jointly by a Research Fellowship of The China Foundation for the Promotion of Education and Culture and funds from the Industrial Science Research Institute for studies on the fermentative utilization of agricultural products. LITERATURE CITED
(1) Assoc. of Official Agr. Chem., Methods of Analysis, 5th ed. (1940) (2) Beresford, H., and Christensen, L. M., Univ. Idaho Agi. Expt. S t a . , Bull. 421 (1941). (3) Sohoene, L., Fulmer, E. I., and Underkofler, L. A., IND.E m . CHEM.,32,544 (1940). (4) Steinberg, R. A,, Botan. Guz., 70, 466 (1920). (5) Underkofler, L. A., Brewers Digest, 17, No. 12, 29 (1942). (6) Underkofler, L. A., Fulmer, E. I., and Sohoene, L., IND.ENG. CHBM.,31, 734 (1939). (7) Underkofler, L. A., Guymon, J. F., Rayman, M. M., and Fulmer. E.I., Iowa State Coll. J . Sei., 17, 251 (1943). PRESENTED before t h e Division of Sugar Chemistry and Technology a t t h e 103rd Meeting of the AMERICAN CHEMICAL SOCIETY, Memphis, Tenn.
