Feed Yeast and Industrial Alcohol ARTHUR J. NOLTE, HARRY W. VON LOESECICE, AND GEORGE N. PULLEY Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Winter Haven, Fla.
H E drying of citrus waste, which consists of the peel, rag, and seeds from canning plants, for the production of cattle feed has become an important industry in Florida. Prior to dehydration, the ground waste is limed and pressed to remove as much water as possible and thus decrease the load on the dryers, and to prevent ekcessive “balling” of the material during passage through the dryers. The press juice obtained not only represents a loss of nutrients but, because of its character, constitutes a public health nuisance. The nature of the press juice (Table I) prohibits economic treatment by regular sanitary engineering practice. At present the waste is disposed of by any one of the following methods: trucking to some remote spot, running into deep wells, flooding waste lands or ponding (Figures 1and a), or concentrating and adding to the feed. All of these methods have serious objections. Trucking is expensive; running into deep wells is a public health hazard, especially in Florida where the water supply is obtained from deep wells; location of plants is not always convenient for flooding waste lands or ponding, and this practice also encourages the breeding of flies and mosquitoes unless the land is disked after application; concentration is not only expensive but yields a concentrate of extremely bitter taste because of the presence of naringin. The quantity of press juice obtained amounts roughly t o 1800 gallons for every ton of dry feed produced. During the 194041 season, 35,000 tons of dry feed were made in Florida. The discarded juice obtained in producing this feed represents a waste of nearly 36,703,000 pounds of sugar (sucrose and reducing sugars), 2,490,000 pounds of protein, and nearly 3,800,000 pounds of pectin. It is unlikely, however, that any of these materials could be economically recovered as such, but their value could be indirectly utilized for the production
T
A method is described for the production of feed yeast from the press juice of cannery waste dehydration plants. An average yield of 46 per cent of dry yeast, based on the total sugar content of the press juice, was obtained. T . utilis was found to be the most suitable of the yeasts tried for protein synthesis from the waste juice of dehydration plants. Alcohol can be obtained from the press juice with an average yield of 91 per cent of the theoretical. Estimated raw material cost is less than half that of blaclrstrap molasses.
of other substances of industrial importance. Thus, the sugar content of the press juice might be fermented with suitable organisms to yield industrial alcohol, feed yeast, or lactic or butyric acid. This paper deals with the possibilities of making the two former substances from the press juice. Neither the production of yeast nor alcohol will entirely solve the waste problem, but the effluents obtained from these two processes have a lower biochemical oxygen demand (B. 0. D.) than the original press juice. Whereas the 5-day B. 0. D. of the original liquor generally runs between 40,000 and 65,000 p. p. m., the spent liquor obtained in the production of yeast of 1 per cent original total sugars content varied from 2628 to 10,000p. p. m., depending upon the number of times the spent liquor was used in the process. Although these biochemical oxygen demands are high when compared to domestic sewage, they could be treated in accordance with present-day engineering practice.
Preparation of Feed Yeast Eleven years ago Frey (3)said: “Yeast offers an unlimited supply of high-quality protein which may be invaluable to a
F I G C R E1 . P O N D I N G O F \T‘ASTE WATERSFROM CITRUSDRYING PLANTS T h e waters are allowed t o settle a short time in each basin and from basin t o basin. T h e sludge settling from t h e waters is shoveled out and trucked away.
.,
r *
from Citrus-Waste Press Juice
..
OF WASTELANDSWITH WASTEWATERSFROM CITRUSDRYING PLANTS FIQURE 2. FLOODINQ
The soil becomes clogged unless the land is disked.
nation in a case of a national emergency." It would thus seem that the problem is one of present economic importance. Torula utiZis was selected as the organism because it propagates rapidly with ammonium sulfate as a nutrient and produces little alcohol. For biological protein synthesis it has been found the most suitable of several microorganisms tried on glucose and wood-sugar wort (4). This observation was confirmed for citrus waste juice also (Table I). Torula utilis has the ability to assimilate pentoses (8) and to synthesize vitamin B complex (6), nicotinic acid, and glutathione ( 1 ) from sugar and mineral salts in the nutritive medium. All of these factors make it desirable as a stock feed yeast.
I*
TABLE GATED FROM
OF Toruza AND OmER ynAsTs PRESSJUICE OF CITRUSDEHYDRATION PLANTS (ONE PERCBNTSUGARCONTENT)
Dry Yeast Based on T o h Protein in Hours Rewired Sugar in Press Yeast (N X for Complete Juice, % 6.25), % Propagation Kind of Yeast Torula utilis 46.0 55.28 8.0 8.5 42.5 52 20 Bakers' Distillers', (a PO ular2372 brand) 5. apirrtus vini. 37.6 50.20 14.0 Wine, No. 2338 S. cllipsoidcus Hansena 36.4 55.80 10.0 Beer (from Fls.brewery) 35.3 53.00 12.0 0 Obtained from the American Type Culture Collection.
80.
PREPARATION OF STOCK YEAST. Sterile malt wort containing approximately 6 per cent of total carbohydrates was inoculated with a pure culture of T. utilis grown on Difco wort agar. The inoculated wort was incubated for about 2 days a t 24" C. (75' F.). This starter was then used to inoculate the press juice which had been prepared by screening, maintaining a t boiling temperature for about 5 minutes, filtering clear with filtering aid, and diluting with tap water until the total sugars amounted to about 1.0 per cent. To the cooled (30" C. or 86" F.) solution thus prepared, 4 per cent by volume of the starter was added. As already pointed out, nutrient salts are also necessary, and therefore trisodium phosphate and ammonium sulfate were added at the rate of 9 per cent, based on the total sugar content of the batch. This amount of trisodium phosphate meets the requirements for phosphate, but the ammonium sulfate added does not satisfy the nitrogen requirements and additional ammonium sulfate must be added during the course of propagation. It was found, however, that if the entire quantity was added a t the start, excessive foaming resulted during aeration, propagation was retarded, and yields were low. Additional quantities of ammonium sulfate were therefore added a t the rate of 1.75 per cent (based on the weight of the total sugar content of the
TABLE 11. APPROXIMATE COMPOSITION OF PRESSJUICEFROM CITRUSDEHYDRATION PLANTS (Figures represent seasonal average of ten analyses of press juice from one plant; composition may vary somewhat from plant t o plant ) Constituent Maximum Minimum Average 6.4 5.4 5.7 at 17,50 c. 12.6 6.1 10.1 Total solids % 11.61 5.64 8.93 94.36 88.39 91.07 % 3.09 1.20 2.40 6.81 2.82 4.23 % 5.58 4.08 6.63 Protein fN 6.25), 7 0.59 0.40 0.47 0.88 0.27 0.66 ~ ~ ~ ~ & & ( $ 'ppt')* $l 0.42 0.23 0.31 0.94 0.43 0.72 g:;d2cid,. as citric, .% 0.30 0.15 0.21 Volatile acld, as acetlc, % 0.78 0.01 0.14 0.00 0.22 0.39 &%~Al?i~$~$ voi. 0.58 0.12 0.23
Table I1 shows that the total sugar content of the press juice varies from about 4 to as high as 8.5 per cent. Attempts
& l$g:
to propagate T. utiZis in these concentrations were not satisfactory. The maximum concentration of sugar was about 1.8 per cent, and even a t this concentration yields were lower than when more dilute solutions were used, and time of propagation was lengthened (Figure 3). The press juice contains insufficient nitrogen and phosphates for the needs of the yeast, and it was therefore necessary to add nutrient salts to supply these deficiencies. It was also found desirable to maintain the pH within certain limits by the use of sodium carbonate.
~$~$.,",',.~ k
'
, 671
INDUSTRIAL AND ENGINEERING CHEMISTRY
672
TABLE 111. RESULTS OBTAINED IN
FOR
~ i Yeast, ~ ~yo by, Volume Hoursla 2 3 4 5 Startb 1 . 0 1 C 1.4 2.0 2.6 2.8 3.0 ;f 6 3.2 7 3.5 7.5 4 . 0 9
::
..
1.0 1.4 1.6 2.0 2.4 2.6 3.0 3.2 3.4 4.0
Dryyeastg, %
1.0 1.4 1.8 2.4 3.0 3.2 3.5 4.2
1 . 0 6.25 1 . 4 5.66 1 . 6 4.26 ... 3.75 3 . 0 2 : k 4.85 3 . 5 3.0 4.70 4.0 3.2 4.82 4.2 2 . 8 4.70 4.58 1.0 1.4 1.8
. . . . . .
2 5.62 5.35 4.64 4.67 5.05 5.15 5.20 4.86 4.70
3
4
5.30 6.56 4.99 5.66 4.74 4.83 4.65 4 . 4 6 4:53 5 . 1 4 5.35 5.18 5.25 4.95 5.14
5 '
44.8
44.8
46.6
44.3
JCICEINSTIL~D OF bJ-4TCR
Formaldehyde KO., Cc. 0.1 iV Titratable Acidity. Co. 0.1 X NaOH/100 Cc. XaOH/100 Cc. 10 2 3 4 5 ' l a 2 3 4 5
6.63 6.00 5.02 4 : b 4.90 5.10 5.15
. . . . . .
14.1 12.4 12.4 8.6 8.2 9.4 7.0 3.8 3.0
. . . . . . . . . . . . . . . . . .
14.4 14.0 16.0 12.4 15.8 11.7 8.9 1.7
16.7 16.2 14.3 16.0 16.3 15.5 13.2 9.6 8.7 4.4
14.4 14.0 14.3 8:4 7.6 3.6 2.4
14.8 5 . 1 14.7 7 . 0 14.0 9.6 10.F 13:O 4 . 7 11.4 5 . 5 10.5 5,O 11.4 4 . 8
. . . . . .
. . . . . . .
8.3 8.8 13.3 10.8 8.7 7.3 5.6 6.6 4.8 6 . 5 . 5.8
11.0 11.7 9.4 12.8 10.8 6.6 5.6 5.6
8.8 9.8 14.4 l3:6 6.8 6.8 6.8
Temperature, 2 3 4
la
9 . 4 86 8 . 8 86 15.6 87 86 1 5 : l 86 1 3 . 8 86 7 . 9 88 6 . 4 88
83 83 84 83 85 86 86 87
83 84 84 85 86 87 88 90
F. 5
84 84 84
85 85
8s 89 90 89
86
85
88 86 86
.. .. .. .. .. .. .. .. ii :: .. .. .. ..
34.5
Number of times spent liquor was used. 7.3 grams ammonium sulfate 6.3 trisodium phosphate added. C 1.5 grams ammonium sulfate added. d 3.0 grams sodium carbonate 1.5 ammonium sulfate added. a b
USING WASTE
-
PH l a
Torula 2Ltili.S YEAST DILUTION
THE PROCESS O F PROPAGATING
Vol. 34, No. 6
e
+ +
5.0 grams sodium carbonatc
+ 1.5 ammonium sulfate added
f 1.5 grams ammonium sulfate added. 9
Based on total sugar content of pressed watcr.
batch) at the end of the first hour and for the succeeding three hours, No further addition was required. The addition of sodium carbonate was found desirable to maintain the pH between 4.4 and 6.5. It was not generally necessary to add sodium carbonate until after the second hour. The batch was
violently aerated with filtered air, and the temperature niaintained between 29" and 30" C. (84" and 86" F.). When the solution had a total sugar content of about 1.0per cent, propagation was completed in about 8 hours. The solution was immediately centrifuged by means of a supercentrifuge and the yea& sludge mashed with water. The yeast thus obtained represented stock yeast used for starting larger batches. For the preparation of large batches (8 to 33 liters of diluted press juice), butt'er tubs mere used as containers. They were fitted ./-with false bottoms of bleached sheeting which dispersed the air in small bubbles. The air was filtered by passing through a cotton plug. Xutrient salts m-ere added in the same quantities as described for the preparation of stock yeast. The amount of stock yeast (containing 25 per cent of solids) added amounted to 50 per cent, based on the \ weight of the total sugar content of the \, diluted press juice. It was found desirable to follow the course \ of propagation by hourly readings of the amount of yeast present (as per cent by \ volume), pH, titratable acidity, and formaldehyde number, In Figure 3 are plotted the yeast content, formaldehyde number, and pH of the citrus waste juice in the process of propagation of Torula utilis. The results represent averages of 8-liter batches of press juice from ten runs containing 1.0 per cent and ten runs containing 1.8 per cent total sugars. The yields from the juice containing 1.8per cent total sugars ranged from 37 to 47.7 per cent' of dry yeast (148 t,o 1.90.8 per cent wet yeast containing 25 per cent solids), based --3on the total sugar content of the juice. Yields ------- pH OF PRESS UQUOR OF 1.0 PERCENT TOTAL SUGARS CONTENT pH OF PRESS LIQUOR OF 1,8 PERCENT TOTAL SUGARS CONTENT 2 from press juice containing 1.0 per cent total I I I I I I I I I I I sugars ranged from 44.3 to 48.0 per cent of dry yeast, (177 to 191.9 per cent $-et yeast containing 25 per cent solids) based on the total sugar content of the juice. The use of a press juice containing not more than 1.0 per Nutrients Added, Grams cent total sugars seems the most desirable Nutrient Start 1 hr. 2 hr. 3 hr. 4 hr. because propagation is completed more 8 Liters Press Liquor, 1,870T o t a l Sugars Content (N&)zSOa 12.2 3.0 3.0 3.0 3.0 quickly and yields are higher. XaaPOI. l0HzO 1.3 ... ... ... ... SPENT-YEAST LIQUORFOR DILUTIOP;.If NazCOa. lOHzO ... ... 4.4 5.0 ... spent liquor mere used for dilut'ing the press 8 Liters Press Liquor, 1% Total Sugars Content (NH42SO4 7.3 1.5 1.5 1.5 1.5 juice, it would mean a saving of water. To deNaaPOi. 10H20 6 3 ... ... ... ... termine what action such a procedure would NaKOs 10H20 ... ... 3.0 5 0 ... \&
7
-
INDUSTRIAL AND ENGINEERING CHEMISTRY
June, 1942
have on the propagation of feed yeast, the spent yeast-water was employed for dilution (Table 111). After four uses, the spent yeast-water showed a tendency to decrease the yield. Repeated use of spent yeast-water also increased the 5-day B. 0. D. of the water, and this would complicate suitable disposition. ACTIONOF OTHER NUTRIENT SALTSAND INORGANIC MATERIALS. Attempts were made to find other nutrient and inorganic materials t o accelerate propagation. Potassium and magnesium sulfates, alone or in combination, and calcium carbonate failed to show any advantage. Ammonium acid phosphate with ammonium hydroxide and ammonium sulfate showed no significant advantage over ammonium sulfate with trisodium phosphate and sodium carbonate. Since the latter three chemicals are the least expensive, their use would be preferable. COMPOSITION OF DRYYEAST. A composite was made of the yeast from ten runs. The sample was dried a t 100" C. (212" F.) and then allowed to come to equilibrium in the air. All constituents, with the exception of fat, were determined by official methods. Fat was determined according to Staiger (6). The results are given in Table IV. TABLEIV. COMPOSITION OF DRYYEASTAKD ANALYSIS OF ASH Dry Yeast Moisture Protein ( N X 6.25) Crude fat Glycogen Cellulose, gum, etc. Ash
Per Cent 1.29 55.28 4.51 12.78 18.06 8.08
Ash Phosphorus pentoxide Magnesium oxide Calcium oxide Silicon dioxide Sulfur trioxide Chlorine Iron oxide Sodium and potassium oxides
Per Cent 45.75 4.95 1.26 2.58 7.79 0.22 0.40 37.05
Beside the use of the yeast in feeds, its utilization as a plastic may have possibilities, and this phase is being further investigated.
Alcohol from Press Juice In the production of alcohol from the press juice, foui different races of yeast were used. The press juice was screened but not filtered, and was then inoculated with 4 per cent by volume of starter. During fermentation the temperaturetYas maintained between 24" and 29" C. (75" and 84" F.), and the run was completed in 72 hours in accordance with the internal revenue laws.
673
TABLEVI. . YIELDOF ALCOHOL FROM PRESS JUICEWHENSUGAR Is INCREASED CONTENT Total Sugars,
%
Organism No.
Vol., %
by
% of. Theoretical Yield
4.98 6.65 6.65 5.43 5.92
88.9 88.8 88.8 97.7 91.0
A1coho1 PH
Concentrated Press Juice 4098 6'. ellipsoideus, vm. Steinberg
9.24 12.39 12.39 9.24 10.82
37
Average
4.0 3.9 3.9 4.0 4.0
Press Juice to Which Blackstrap Was Added
Average
9.27
4.0
5.45
93.3
To increase the yield of alcohol, it was found necessary to concentrate the press juice or add blackstrap molasses until the sugar content amounted to 10-12 per cent. The cost of concentration of the press juice to the desired sugar content would be about one third as much as if blackstrap were added. The data for the production of alcohol from concentrated press juice and from press juice to which blackstrap was added are given in Table VI. The data indicate that about 25 gallons of press juice were required to yield one gallon of 190 proof alcohol. As a byproduct about 12.5 ounces of dry yeast, which could be used as a stock feed, would be obtained per gallon of alcohol produced. The problem of the economic disposal of the spent wort, although not so difficult as that of the original press juice, would be of some moment. PRODUCTION COSTS. The production cost per gallon of 190 proof alcohol manufactured from the press juice as compared with the cost of manufacture from blackstrap molasses is estimated as follows: Press Liquor Raw material" $0.058 Labor, steam, chemiqals, power, water, 0 055 interest, depreciation, taxes Total 0 . 1 1 3
-
Blackstrap Molasses $0 125
0 055 0 180
In the case of press juice, the cost includes hauling. These figures which are manufacturing costs, do not include denaturing, sales and freighi costs. 5
Acknowledgment TABLEV. YIELD OF ALCOHOL FROM ORIQINAL PRESS JUICE c
Vol. acid as aceti: in original,
pH 5.6 Vol. acid, as acetic Alcohol after ferby mentation, volume,
%
%
0.56 0.46 0.73 0.50 0.78 0.67 0 32 0.31
1.34 2 15 1.48 2.09 1.41 1.88 1.41 1.75
% of theoretical yield 32.4 46 8 35.8 45.5 34.2 41.0 34,2 38.2
Average 0 21 0.54 a From American Type Culture Collection. b Isolated from Florida Valencia oranges.
1.68
38.5
Organism No, 2372 S. spiritus winia 4134 S. salc8a 376 4098 S ellipsoideus, var. Steinberg5
%
0.27 0.15 0.27 0.15 0.27 0.15 0.27 0.15
The results in Table V show that larger yields were obtained when the p H of the press juice was approximately 4.0, and that the quantity of alcohol obtained was too small for economic recovery* The pH Of the juice was adjusted by the addition of either sulfuric or hydrochloric acid.
pH 3.9
Vol. acid, as acetia after fermentation,
Alcohol by volume,
0.20 0.02 0.23 0.02 0.16 0.02 0.12 0.01
2.98 3.83 2.60 3.90 2.98 4.04 3 54 3.97
%
%
% of
theoretical yield 72.3 83.4 60.6 85 0 72.3
The writers are indebted to the Dow Chemical Company for the subculture of Torula utilis used in this study.
Literature Cited
88.1 85.8 86.4
(1) Fink and Just, Biochem. Z., 303. 234 (1939). (2) Frey, IND.ENG.CHEM.,22, 0.10 3.47 79.2 1161 (1930). (3) Lechner, Biochem. Z., 304, 84-9 (1940). (4) Luers and Morika, Z. Spiritusind, 59, 383-4, 386-7 (1936). (5) Scheunert, Wagner, Fink, and Krebs, Biochem. Z.,302, 1-11 (6) Staiger, (1939). Brennerei
z t ~ 47, . , 44 (1930).
CONTRIBUTION 61 from the Agricultural Chemical Research Division, Bureau of Agricultural Chemistry and Engineering.
