INDUSTRIAL AND ENGINEERING CHEMISTRY
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Vol. 40, No. 7
ACKNOWLEDGMENT
LITERATURE CITED
The authors wish to thank Sedgev,-ick C. Rogers and Albert ~ l of the i Forest ~ products ~ Laboratory staff for the assistance given in this work. On behalf of the Forest Products Laboratory the authors wish t o express their appreciation to George M. Pohler of the Vulcan copper and supply Company, Cineinn&, Ohio, and E. ~ ~ holm of the Puget Sound pulp mill for suggestions and counsel on the continuous operations of a n alcohol plant. Valuable counsel was also received from IT', H. Peterson and Elizabeth McCoy of the University of Wisconsin.
(1) Fritzweiler and Rookstroh, 2. Spiritusind., 59, 229-30 (1936). ( 2 ) Harris, E. E., and Beglinger, E., IND. ENG.C H E ~ 3~8., ,8 9 0 (1946). (3) Harris, E. E., Hajny, G. J., Hannan, Martha, L.. and Rogers, S., Ibid., 3 3 , 8 9 6 (1946). (4) Kinnard, V., Foreign, Commerce Weekly, 17, 12 (Oct. 14, 1944). ( 5 ) Leonard, R., and Hajny, G. J., 1 % ~ .EXG.CHEM.,37, 390 (1945). S n e k and Frazier, Ibid., 37, 30 (1945). 1 (6)~ Peterson, (7) Saeman, J. F., Locke, E. G., and Dickerman, G. K., FIAT Final Report 499 (November 1945). RECEIVED December 7, 1946. Presented before the Division of Sugar Chemistry a t the 110th Meeting of the AYERICAK CHEMICAL SOCIETY, Chicago. 111.
Fodder Yeast from Wood Hvdrolvzates and Still Residues J
J
ELWIN E. HARRIS, JEROME F. SAERIAN, RALPH R . MARQUARDT, MARTHA L. HANNAN, AKD SEDGWICK C. ROGERS Forest Products Laboratory, Mudison, Wis.
T
HE possibility of proA strain of Torula yeast has been grown on wood hydrolyhydrolyzates if they were 'ducing fodder yeast from zates and on the nonfermentable sugars remaining after diluted t o approximately 1yo nwod as a means of supplyalcoholic fermentation of w-ood hydrolyzates. The most reducing sugar and if certain rapid conditions for continuous propagation were at pH growth factors were present. ing protein supplements for cattle feed has created un6 when 4.6% sugar solution was fed into the fermentor, The production of yeast from usual interest among cattle which required a throughput time of about 2.5 hours and such wood sugars has advanproducers, especially in forest 200 to 250 cubic feet of air per pound of yeast produced. tages over alcohol production in that practically all areas in which such producthe sugars and some of the tion would provide a n opporacidic materials are utilized. This ability to use organic subtunity to obtain locally the protein feed t h a t must now be shipped stances other than glucose has led to a n interest in yeast produdinto such areas. tion from the sugars in sulfite-pulp waste liquors, in cannery As part of a program to learn the best ways t o produce alcohol waste liquors, and in food-processing by-products. a n d yeast from wood, the Office of Production Research and The possibility of removing carbohydrates from the effluents Development of the War Production Board in 1943 financed produced by industries processing carbohydrate materials also research on the production of fodder yeast from wood sugar a t the Forest Products Laboratory, Madison, M'is. (6). Such fodder has made the growing of yeast on such wastes attractive. Many industries have spent large sums of money on sewage-disposal or food yeast is a combination of high-quality protein with plants without a n y return in order t o decrease stream pollution. vitamins of the B complex. I n these respects it is similar to The growing of yeast on the effluents from such industries would brewer's or baker's yeast. It has a n advantage over the latter reduce stream pollution and at the same time provide another in t h a t i t grows more readily and, when used as food, has a better source of income. flavor. Food yeast is of special value t o people whose diet is. deficient in protein, as in nations cut off from normal supplies of PRODUCTION OF SUGAR FROM WOOD WASTE meat. Nutritional studies may also show food yeast to be a The development of satisfactory conditions for the fermentavaluable supplement t o protein food for humans, livestock, and tion of wood sugars, and of conditions for hydrolysis which would poultry in localities where other protein sources are abundant. give a product that fermented more readily, has been studied at Yeast protein may be produced rapidly at high efficiency. the Forest Products Laboratory along with the production of Yields of yeast, containing 50Yc protein, mere from 40 to 60% yeast on wood sugars. of the sugar used for their production. I n Germany during The production of total sugar from wood used for these experiTT'orld JTar 11, food yeast aided in kceping the people of the ments has been described recently (8, 3 ) . The process consists Reich well fed. I n 1944 the production of food yeast was almost of pumping a stream of o.5rO sulfuric acid through a charge of 9000 tons per year in ekisting German plants, and other plants sawdust, shavings, or chipped wood waste a t temperatures rangwere in the process of construction. This food yeast was proing from 150" to 180" C. The resulting acid sugar solutions are duced from sugars derived from wood either by acid hydrolysis neutralized with lime to approximately p H 5 , cooled, and filtered. or during the sulfite pulping of wood (9). The sugars are present in about 5% concentration and are a The British Government, during T o r l d War 11, carried out mixture of pentoses and hexoses. Sugars from a softwood such research on methods for the production of food yeast from waste as Douglas fir are about 82%',fermentable t o alcohol, while those molasges. A plant for the production of food yeast is being from hardwoods such as red oak are about 63y0fermentable. constructed in Jamaica as a means of using molasses and as a Still residues used in these experiments were also obtained from eource of needed food for the people in tropical countries ( 1 ) . wood. The sugars produced by the hydrolysis of wood were I n the early research work at the Forest Products Laboratory subjected t o alcoholic fermentation by Torula utilis (4). The (6),it was found that Torula utilis yeast could be grown on wood
July 1948
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INDUSTRIAL AND ENGINEERING CHEMISTRY
alcohol was removed by distillation. The residual liquor contained xylose and other nonfermentable materials. The total reducing material content of these liquors calculated as glucose was about 0.9%. The actual sugar content was 0.5 t o 0.6%. PREPARATION OF INOCULUM
The yeast inoculum was prepared by transferring a small amount of yeast from a glucose malt-sprout agar slant t o 30 ml. of glucose malt-sprout liquid medium in a n 8-inch test tube. This culture was placed in a shaker (see Figure 5 , I ) and incubated at 30' C. for 24 hours, after which the contents of the tube were placed in 150 ml. of glucose medium in a 500-ml. Erlenmeyer flask. The cultures were again shaken for 24 hours at 30' C. Yeast from this flask was used to inoculate wood-sugar solution. METHODS OF SAMPLING AND ANALYSIS
The utilization of the sugar and the production of yeast and alcohol were followed in both the batch and continuous yeast propagation. Samples were taken directly from the propagator t o avoid variations. For sugar analysis, the yeast was removed in a bottle centrifuge and the liquor filtered. The Brix was measured and sugar determined by the Shaffer and Somogyi method ( 8 , l O ) . Yeast volume was obtained by centrifuging a 10-ml. sample in a graduated centrifuge tube for 5 minutes. Samples for yield of yeast were obtained by placing 10 ml. of the substrate with the yeast i n a 5 by 0.825-inch culture tube, weighing the tube with contents, and then centrifuging. The supernatant liquor was decanted off. Water was added to the remaining contents of the tube, which were stirred with a glass rod and recentrifuged. The liquid was again decanted off, and the yeast residue was dried for 24 hours a t 100" C. in the tube, which was then weighed again with contents. Nitrogen determinations were made on the same sample by the Kjeldahl method. BATCH PROPAGATION OF YEAST
The wood-sugar solution obtained by wood hydrolysis is sterile and contains only small amounts of the inorganic salts required for yeast production. Nitrogen in the form of urea, ammonium sulfate, and ammonium phosphate was added to the feed liquor to produce from the sugar a 50% yield of yeast with a 50% protein content. The solutions contained small amounts of phosphate, but the amounts were increased by the addition of the ammonium phosphate. Because in the German operation (9) it was customary to add potassium and magnesium salts, these were added without determining the actual need for them. The acidity of the solution was adjusted t o between p H 4.5 and 5. Because of the rise in p H due to the utilization of organic acids during yeast growth, it was necessary to add acid from time to time to keep the acidity in the range pH 5 to 5.5. STANDARD FERMENTOR. For batch propagation of yeast by stand&rd procedure for yeast production (Figure l), 25 gallons of nutrient salts containing 1 to 2y0 wood sugar were
Figure 1. Standard Fermentor for Propagation of Yeast
placed in a 66-gallon tank. The temperature was brought t o 31" C. and the yeast added. The pumps for agitation were started, and then the valves were opened for the introduction of air. Air at a rate of 1 to 2 cubic feet per minute was introduced through porous, fused ball spargers in the bottom of the tank. Foaming was so great that i t was necessary to cut the rate to about 1 cubic foot per minute and add antifoam a t frequent intervals. After about 8 hours, the tendency to excessive foaming became less and the rate could be increased t o 2 cubic feet per minute.
TABLE 11. GROWING OF YEASTON DILUTEDWOODHYDROLYZATE I N 66-GALLON STANDARD BATCHFERMENTOR Sugar Series No.
Concentration, %
1 2 3 4
0 94 12 15 2.0
TABLE I. EXPERIMENTAL CONDITIONS FOR G R ~ W I NYEAST Q ON DILUTED WOODHYDROLYZATE IN STANDARD BATCHFERMENTOR
(Figure 1) Equipment. Single 66-gallon tank. Circulation. Centrifugal pump, 15 gallons per minute. Spargers. Fused porous balls or cloth to bottom of tank. Reducing sugar concentration variable. 1 to 2%. Initial pH. 4.5 to 5 . Initial yeast count. 100,000,000 cells. Temperature.. 30' to 31' C. Nutrients. Based on sugar content. Air. 0.5 t o 1 cubic foot per cubic foot of medium per minute. Time required. 18 to 20 hours. Volume of liquid. 30 gallons. Antifoam agent. Oleic acid. Temperature control. Water for cooling. Electric heater controlled by bimetallic temperature controller.
(Values represent averages of a series) Air, Cu. Ft. Sugar
a
Air, Cu. Ft. per iMinute
1 1 1 1 Yield of alcohol, 12.7%.
2 2 25 2
per Pound of Yeast
1500 1200 1100 1250
Dry-Yeast Yield on
Used,
Total Sugar,
% 94 92 89 84
% 45 0 43 0 39 0 25 Oa
0 0 0 0
TABLE 111. GROWING OF YEASTON RESIDUES FROX ALCOHOLIC FERMENTATION IN GA GALLON STANDARD BATCHFERMENTOR a
5 6
7 8
(Values rewesent averages of a series)
0.71 0.81 0.67 1.10
2.0 2.5 2.5 2.5
1550 1830 1950 1820
75 76 76 65
'
125.0 116.0 130.0 100.0
a Yield corrected for material which came down with yeast, so values given are for yeast with 50% protein.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Table I gives the conditions and Table I1 the results of growing yeast on diluted wood hydrolyzate. Table I11 gives the results of growing yeast on the residues from alcoholic fermentation. I n the case of the residues from alcoholic fermentation, the loss of volatile acid from the solution during the growth of yeast must account for the increased yield of yeast from these residues.
Vol. 40, No. 7
vias found to be adaptable for use with full-strength wood hydrolyzates, whereas other fermentors when operating on wood hydrolyzates required dilution of the sugar. Continuous feed was controlled by a proportioning pump, and continuous discharge by a standpipe slightly higher than the draft tube. The capacity of the tank to the top of t,he draft tube was 34 liters. As the fermentor filled, it overflowed down the standpipe.
SPECIAL FERMENTOR. ' For batch propagation in a 20-gallon open tank with a draft tube and a propeller agitator, one series of experiments was made without additional air; and another series with additional air introduced through porous, fused ball spargers. I n each case, 6 gallons of diluted wood sugar with nutrient were added to the fermentor. The p H was adjusted to about p H 5. At a temperature of 31 O C. the yeast was added. The stirrer whipped air into the liquid and forced the liquid up the sides and over the edges of the draft tube.
The yeast propagation was started by placing 2 liters of a solution containing about 100,000,000 yeast cells per ml. in t,he fermentor. The spinner was started, and the diluted wood hydrolyzate, cont'aining about 2 . 5 7 , reducing sugar and nutrients, was introduced a t a rat,e of 2 liters per hour, and air at the rate of 0.5 cubic foot per minute. In about 5 hours, the spent media and yeast began to overflow through the standpipe. Yeast was centrifuged from the liquor and reburned to the fermentor until its content was about 7% wet yeast by volume or 1 3 % dry yeast. The feed was then changed to full-strength wood hydrolyzate (4.5 t,o 5% concentration) and the air to 0.75 cubic foot per minute, as measured by a dry gas meter. For full-strength wood TABLEIV. EXPERIMENTAL COXDITIOKS FOR GROWING YEAST hydrolyzate, equilibrium conditions maintained a met-yeast conON DILUTED WOODHYDROLYZATE AND ON RESIDUAL SUGARS t.ent of 7 to 10%. For the growing of yeast on the sugar residues FROM ALCOHOLIC FERMENTATION I N SPECI.4L BATCHFERMENTOR from alcoholic ferment'ation of wood hydrolyzate, equilibrium conditions maintained a wet-yeast content of 4 to 694, and on sulfite Equipment. Single 20-gallon open tank. liquor, of 6 to 9%. The still bottoms also contained some darkCirculation. Propeller-type stirrer at 1780 r.p.m. through draft tube. colored material precipitated from the solution on aeration, but Spargers. Fused porous balls in bottom of tank. the values for yield were corrected to give yields of yeast with Reducing sugar. Total hydrolyzate, 1 t o 2 % ; still residues, 0.5 to 50yGprotein. 1.0%. Initial pH. 4.5 to 5 . Initial yeast count. 100,000,000 cells. Table VI! gives the conditions for propagation and Table VII Temperature. 30Oto 31' C. the values for production of Torula yeast by continuous propagaAir. One-half to 1 cubic foot per cubic foot of medium per minute. tion. Protein content was obtained by multiplying the nitrogen Time. 18 to 20 hours. content by 6.38. Volume of liquid. 8 to 9 gallons. Antifoam agent. Oleic acid. DISCUSSION
GROWTH OF YEASTON DILUTED WOODHYDROLYZATE The growth of Torula yeast on wood-sugar solutions in conSUGAR RESIDUES FROM ALCOHOLIC FERMENT.4TION O F ventional equipment is handicapped by inhibiting properties WOODSUGARIN SPECIALBATCHFERMENTOR of the solution, and by the tendency to foam excessively, which (Values are averages of a series of experiments) limits the amount of air that can be introduced. For yeast DryYeast production in the conventional-type fermentor, it was necessary Yield Sugar Air, Based to dilute the wood hydrolyzate to about 170reducing sugar in Concen- Cu. Ft. Sugar on order to obtain satisfactory yeast growth. Continuous feeding Series tration, per Used, Total NO. Type of Medium To JIinute To Sugar, yo of full-strength wood-sugar solutions in the standard fermentor X-1 Diluted hydrolyzate 2.19 0 50 25 has not been practical. At any concentration of sugar, the X-L Diluted hydrolyzate with foaming tendency made it impractical to introduce more than Aloxite spargers 1.4 0.5 86 35 X-3 Alcoholic fermentation about 0.5 cubic foot of air per cubic foot of medium until a part residues 0.77 0 80 lOOa X-4 Alcoholic fermentation of the sugar \vas utilized. It was necessary to use large amounts residues with fused por0.93 15 80 llOa of antifoam to keep the foaming under control. These condiO U R ball spargers tions limited the rate of production per fermentor. The air 5 Yield corrected for material which collected with yeast. consumption per pound of yeast produced was of the same magnitude as that required in small equipment for growth of brewer's and baker's yeast. Table IV gives the conditions and Table V the average results of series of experiments with and without additional air on diluted wood-sugar solutions and on the residual sugars from alcoholic TABLEVI. EXPERIMENTAL COXDITIONSFOR COXTIXUOUS fermentation. GROWING O F YEAST O S W O O D HYDROLYZATE, O N SUGAR RESIThis special type of propagator had a n advantage over the DCES FROM ALCOHOLIC FERMENTATION O F \VOOD SGGlRS, AND O S usual type in that better control of foaming was obtained; but SULFITELIQXORS because some air was introduced by the action of the stirrer, it Equipment. Single open tank with 34-liter fermentation capacity was not possible to determine the amount of air used for the (Figure 2 ) . propagation of yeast. Circulation. Motor-driven spinner which draws liquid down a draft tube and forces it up the sides. Spinner, 6 inches in diameter; speed, 1100 r.p.m. CONTIhUOUS PROPAGATION OF YEAST Spargers. Air introduced through spinner. Reducing sugar. Total hydrolyzate, 4.5 t.o 5 7 0 ; still residues, 0.5 to The third type of equipment used for the study of yeast 1.0%; sulfite liquor, 1.66%. propagation was a modification of the Waldhof fermentor used in Initial pH. Of feed, 4 . 2 to 4.5; of fermentor, 4 . 7 to 6.6. Germany for the growth of food yeast from the sugars in sulfiteInitial yeast count. 100,000,000 cells. Average yeast count under pulping waste liquor (Figure 2 ) . I n this equipment ( 7 ) , the equilibrium condition with 4.5y0 reducing sugar, 700,000,000. Temperature. 30" to 32' C. agitator and aerator are combined in a spinner which introduces Air, 1 to 1.5 cubic feet per cubic foot of medium per minute. the air from rapidly revolving tubes and forces the liquid up, Time. Continuous; holdup time to 2 t o 4 hours. around, and over a draft tube where the foam gives up its gases Antifoam agent. None. as it falls down the tube. This equipment is especially valuable Temperature control. Cooling coil. Electric heater with bimetallic temperature controller. where foaming is of importance. In work on wood sugars obtained from either hydrolysis or sulfite liquor, this fermentor TABLE V.
AND ON
July 1948
INDUSTRIAL AND ENGINEERING CHEMISTRY
1223
The sugar residues from alcoholic TABLE VII. CONTINUOUS PROPAGATION OF Torula utilis ON WOODHYDROLYZATE, fermentation, which were . principally ON SUGARRESJDUESFROM ALCOHOLIC FERMENTATION OF WOODSUGARS,AND pentoses, had less tendency t o foam, so ON SULFITELIQUOR that i t was possible by the use of antiDry-Yeast foam t o introduce about 0.75 cubic Yield Protein Rate Sugar Air Air, Reducing Based on Content foot of air per cubic foot of medium. of ConcenCu. $t. Cu. Ft. Sugar Total of Series Feed, tration, per per Pound Used, Sugar, Yeast, Yeast growth mas slow, however, and No. Liters/Hr. % pH Minute of Yeast % % % 18 hours were required t o utilize the Wood Hydrolyzate sugars. While the sugars were being 9 3.0 4.5 4.7 0.f5 415 88.4 36.4 52.0 used, organic acids were also being used, 10 2 0 4.5 5.0 0.75 630 92.0 35.9 52.3 11 4.0 4.5 5.0 0.75 313 86.5 36.0 52.7 so that it was necessary to add sulfuric 12 4.5 4.5 5.0 0.75 275 85.3 36.5 54.4 acid from time to time to hold the 13 5.0 4.5 5.0 0.75 296 83.1 30.5 52.5 14 3 0 4.6 6.6 0.75 295 93.8 49.7 58.6 acidity between p H 5 and p H 5.5. An 15 4.0 4.6 6.0 0.75 210 93.4 52.6 57.3 16 4 5 4.6 6.0 0.75 212 93.2 examination of the solutions remaining 46.5 56.5 17 5.0 4.6 6.0 0.75 203 91.1 43.4 52.5 after 75% of the reducing material had 18 5.0 4.6 5.5 0.75 208 90.4 42.5 51.9 been utilized by the yeast, showed that Fermentation Residues there were no sugars present that re19 3.0 0.94 5.4 0.60 60.0 1100 52.0' 50.0 20 4.0 0.91 5.5 0.60 55.0 950 47.0" 50.0 sponded t o an osazone test. It was concluded that about 25% of the reSulfite Liquor ducing properties of these solutions was 21 2.0 1.66 5.0 0.50 1040 79.0 39.2 52.7 22 3.0 1.66 5.0 0.50 695 78.0 39.0 52.5 due t o materials other than sugar. 23 4.0 1.66 5.0 0.50 540 75.0 37.6 52.0 24 5.0 1.66 5.0 0.50 550 59.0 29.6 51.0 This observation has previously been made on small scale experiments at " Values corrected t o product with 50% protein. the Forest Products Laboratory, reported by Kurth (5). The amount of air required for yeast production on the fermentation residues was 25 to 50% greater than that required was compensated for by the short holdup time required. Sterile conditions were not used, and at p H 5.0 there was no evidence of for diluted wood hydrolyzate. contamination over a period of about 4 months' operation. Batch yeast production in a fermentor with a draft tube and a There was some evidence of contamination a t pH 6 t o 6.6, which propeller agitator which caused the liquor to flow down the draft tube, made possible the control of foaming. Without additional could be overcome by changing back t o p H 5. Growth rates and air, the sugar was about 50% utilized in 18 hours. It required yields of yeast were higher a t p H 6 than at p H 5. It is believed t h a t enclosing the fermentor would make i t possible t o grow about 48 hours to bring about the same degree of utilization as Torula yeast at p H 6 without trouble from contamination. that obtained when air was introduced through spargers. Continuous propagation of Torula yeast in a modified Waldhof fermentor appeared to give the most satisfactory operation. ACKNOWLEDGMENT Yeasts produced in this equipment were cleaner, no antifoam On behalf of the Forest Products Laboratory the authors agent was required, air needed could be supplied by an ordinary wish t o express their appreciation t o the Vulcan Copper and blower, and the air requirements were less. AS the propagation Supply Company, Cincinnati, Ohio, for counsel and for supplying was carried out entirely in foam condition, the liquor contained funds for a part of this research; t o R. Hatch of the Weyerin the fermentor was about one third of its liquid capacity. This haeuser Lumber Company, Longview, Wash., for information on the special fermentor used in a portion of this research; and t o W. H. Peterson, University of Wisconsin, for counsel. Acknpwledgment is also made to J. L. Bubl, A. A. Kline, and J. E. Koehler, of the laboratory staff, for the assistance given by them in this work. LITERATURE CITED
(1.) Colonial Food Yeast, Ltd., Food Yeast, Colonial Development
and Welfare Act (Great Britain), 1940. (2) Harris, E. E., and Beglinger, E., IND.ENQ. CHEM.,38, 890
(1946). (3) Harris, E. E., Beglinger, E.; Hajny, G., and Sherrard, E. C., Ibid., 37, 12 (1945). \ - - - - I
(4) Harris, E. E., Hannan, M. L., Marguardt, R. R., and Bubl, J. L., Ibid., 40, 216 (1948). (5) Kurth, E. F., I b i d . , 38, 204 (1946). (6) Peterson, W. H., Snell, V. F., and Frazier, W. C., Ibid., 37, 30 (1945). (7) Saeman, J. I?., "Experimental Fermenta with Good FoamControl Properties," U. s. Forest Products Laboratory Report, 1946. (8) Saeman, J. F., Harris, E. E., and Kline, A. A.,IND.ENQ.CHEM., ANAL.ED.,17, 95 (1945). (9) Saeman, J. F., Locke, E. G., and Dickerman, G. K., "Production of Wood Sugar in Germany and Its Conversion t o Yeast and Alcohol," FIAT Rept. 499 (1945). (10) Schaffer, R. A., and Somogyi, N. J., J. BioZ. Chem., 100, 695 (1933). Figure 2.
Special Fermentor for Propagation of Yeast
Modification of Waldhof fermentor used i n Germany
RECBIVED December 7, 1946. Presented before the Division of Sugar ChemCHEMICAL SOCIETY, Chicago, istry at the 110th Meeting of the AMERICAN Ill.
