September, 1944
INDUSTRIAL AND ENGINEERING CHEMISTRY
protein must be prepared from flakes from which the oil has been extracted with alcohol. The color of products prepared from this latter type of protein is, for all practical purposes, equivalent t o those prepared from casein, and the yellowish tinge is no longer a problem. Any cost estimate would be premature until certain other factors are known. Among these factors are yield of protein under optimum conditions, effects of water-flake ratio and temperature on the processing of the protein, and optimum conditions for drying both the protein and the mash. These problems are now being studied in this laboratory.
803
LITERATURE CITED
(1) Beckel, A. C., Bull, W. C., and Hopper, T. H., IND.ENG.CHEM. 34,973 (1942). (2) Boyer, R. A,, Ibid., 32,1549(1940). (3) Brother, G.H.,and McKinney, L. L., Ibid., 32, 1002 (1940). (4) McKinney, L. L.,Deanin, R., Babcock G., and Smith, A. K., Ibid., 35,905 (1943). ( 5 ) Nagle, R. H., Becker, H. C., and Nlilner, R. T.,Cereal Chem., 15, 463 (1938). ( 6 ) Smith, A. K., and Circle, S. J., IND. ENG.CHIW., 30, 1414 (1938). (7) Smith, A. K.,Circle, 5 . J., and Brother, G . H., J. Am. Chem. Soc., 60, 1316 (1938).
Production of 2,S-Butylene Glycol from DAVID PERLMAN College of Agriculture, University of Wisconsin, Madison, Wis.
WOOD HYDROLYZATES
Production of 2,3-butylene glycol from wood hydrolyzates by Aerobacter aerogenes has been investigated. Wood sugar solutions in concentration up to about 17% have been completely fermented. About 35% of the fermented sugar is converted into &%butyleneglycol, and a small amount of acetoin is also formed. Acclimatization of the Aerobacter aerogenes culture to the hydrolyzate simplifies the treatment of hydrolyzates for fermentation purposes.
T
HE conversion of wood waste into sugar by the Scholler
(9) and similar processes has been extensively investigated in recent years. I n these processes dilute sulfuric acid (0.2-1.0%) is percolated through wood chips at elevated temperatures (e.g., 160' C.). From a fermentation standpoint these hydrolyzates contain appreciable amounts of hexoses, pentoses, and acetic acid. The hard-wood hydrolyzates are higher in pentose concentrations than those from the softwoods. These sugar solutions have been used in Germany for the production of ethyl alcohol and of yeast. Sjolander et al. (6) reported on the fermentation of hydrolyzates from several kinds of wood by the butyl anaerobes, Clostridium jelsinium and Clostrid-
ium butylicum. Since 2,3-butylene glycol is formed by Aerobacter aerogenes from sugar solutions and hydrolyzed grain mashes, its production from wood hydrolyzates seemed possible. This organism will ferment pentoses as well as hexoses, both of which are found in wood hydrolyzates. Hydrolyzates from both hard- and softwoods were tested. They included white spruce, southern yellow pine, southern red oak, and Douglas fir. METHODS
The Aerobacter aerogenes culture was obtained from the Northern Regional Research Laboratory, and is designated in lheir collection as A . aerogenes No. 199. On a glucose-salts medium this culture produces mainly the rneso form of 2,3butylene glycol (6); it is presumed that the same form is obtained on the wood hydrolyzate medium. Other products formed during the fermentation of glucose by this organism include acetoin, ethyl alcohol, lactic acid, and succinic acid. The inoculum for the fermentations was grown (unless otherwise indicated) on a medium consisting of 0.5% corn-steep liquor, 0.5% calcium carbonate, and 5y0glucose. The inoculum was aerated continuously on a mechanical shaker for 12 to 16 hours before use. The inoculum size varied between 3 and 5y0 of the fermentation volume, The fermentations were conducted in cotton-plugged 500-ml.
Erlenmeyer flasks containing 100 cc. of medium. The media were prepared, divided among the flasks, and autoclaved for 30 minutes a t 120" C. After inoculation and addition of nutrients, the flasks were placed in a mechanical shaker and shaken continuously for the duration of fermentation. This shaker, placed in a 30" C. incubator, had a stroke of 3 inches and a frequency of 96 cycles per minute. The results obtained with shaken flask fermentations on a glucose-salts medium have been found t o be comparable with those obtained in 12-liter aerated fermentations and in pilot-plant operations. Urea, potassium phosphate, and calcium carbonate (as a slurry) were sterilized separately from the rest of the medium, and were added t o the fermentation medium at the time of inoculation. The levels of these compounds used in the following experiments were probably greatly in excess of those required. Preliminary experiments showed that 2 grams per liter of urea, 1 gram of potassium monobasic phosphate, and 3 grams of calcium carbonate are usually sufficient. When received, the liquor contained 3.0 to 6.0% reducing material as glucose and 0.4 to 0.3% organic acids (as acetic), and had a titratable acidity of 0.2 to 0.25 N (a corresponding p H of about 1.5). Wood sugar concentration was determined by analyses for reducing sugar (calculated as glucose) according to the method of Shaffer and Somogyi (4). The samples were heated with the sugar reagent for 30 minutessin order to oxidize a larger portion of the pentoses present. The sugar determinations were corrected for the presence of acetoin. 2,a-Butylene glycol was determined on the ether extract of the neutralized fermented medium according to the procedure outlined by Johnson ( I ) , and also by the "direct oxidation" procedure mentioned by him. The latter method was applied to a butyl alcohol extract of the fermented medium. Before butyl alcohol extraction, the medium was saturated with potassium carbonate. Acetoin was determined by a modification of the method of Langlykke (2). When the 2,3-butylene glycol values by these two methods had been corrected for the acetoin present in the samples, they were found to agree satisfactorily. I n no case did the acetoin formed account for more than 2% of the reducing sugar fermented, and i t was usually less than 1%. The yield of 2,3-butylene glycol in the tables is the weight yield-Le., grams of 2,a-butylene glycol plus grams of acetoin found in the sample, divided by the grams of reducing sugar present in the sample before fermentation. Both butylene glycol and acetoin are included in the yield data; for if the fermentation is continued too long, a considerable portion of the butylene glycol will be converted to acetoin. This culture has
INDUSTRIAL AND ENGINEERING CHEMISTRY
804
TABLE I. EFFECT O F TREATMESTa OF HYDROLYZATES ON 2,3-BUTYLENE GLYCOL AND ACETOINPRODUCTIOX
Wood
GLYCOL A N D ACETOIN PRODUCTION FermenYield of Original tation Sugar Fer- 2,3-Butylene G./100 Sugarbo. Time, Hr. mented, Yo Acetoin, Glycol and yo
supplement. 10Liter Grams/
Neutralization Treatruentb Southern yellow pine Southern red oak
,
Douglas fir White spruce
4.3 10.0 10.0 10.0 5.0 7.5 5.9 5.9
18 48 48 48 48 48 48 30
91.0 55.0 91.0 90.0 92.5 77.8
3.3 32.8
None None Difco yeast ext. Malt sproutfi None Sone None N a l t sprouts
38.8 37.2 26.6 30.0 24.5 31.7 37.7 36.9 31.7
None None None Difco yeast ext. Malt sprouts Kone Kone yeast Malt sprouts
39.8 16.5 33.0 35.2 28.5 26'.0
10.0
89.5
Alkaline Tveatment C Southern yellow pine Southern red oak White spruce
4.3 8.5 10.0 10.0 10.0 5.9 17.8 17.8 17.8
18 38 36 36 36 48 75 75 75
90.1 89.6 90.5 94.0 87.0 87.6 88.0 89.3 87.6
Nutrients added t o niedium per liter: 5 grams CaCOs, 4 grams urea, 2 grams KHaPOa b Hydrolyzate adjusted t o pH 6 with lime, filtered t o remove precipitated calcium sulfate. Hydrolyzate adjusted to pH 10.5 with lime and filtered; adjusted to p H 6 and filtered again.
been found to utilize acetic acid and it is probable that a small part of the butylene glycol formed from the wood hydrolyzate fermentation comes from this source. However, since the organic acid concentrat'ion is so small compared to the reducing sugar concentration in the medium, the most satisfactory method for expressing yield is to base it on the reducing sugar available in the medium. TREATMENT O F HYDROLYZATE
Before the wood sugar could be fermented, a preliminary treatment' of the hydrolyzates was necessary. Since the fermentation Proceeds best a t PH 6, it Was necessary to adjust all media to this reaction. Several methods of further treatment were tried, and
TABLE 11. FERMENTATION OF SOUTHERN YELLOW P I N E HYDROLYZATE" AT VARIOUSSUGAR CONCENTRATION s Initial Reducing Sugar. G./100 Cc.
4.0 6.0 8.0 10.0 12.0
'
,
Sugar Fermented,
%
Time of Fermentation, Hr.
Yields of 2,3-Butylene Glycol and Acetoin, yo
92.3 91.5 92.0 90.6 89.8
15 24 30 48 48
34.8 33.3 35.1 32.1 33.8
Vol. 36, No. 9
the following two seemed simplest: The first was a neutraliza,tion procedure in which the liquor as neutralized to pH 6 with lime slurry or alkali. When the lime slurry mas used, t,he precipitated calcium sulfate was removed by filtration before sterilization of the medium and subsequent fermentation. The second met,hod may be called an alkaline procedure in which the hydrolyzate wa.s adjusted t'o p H 10 or 11 with alkali or lime slurry and filtered. This alkaline solution was then adjusted to pH 6 with sulfuric acid and again filtered. Other methodk which were found to be less sat'isfactory included clarification of t,he liquor with charcoal at, various pH levels, and treatment with both acidic and basic exchange materials. A s Table I shows, hydrolyzates from various woods vary in ease of fermentability by this organism. In general it may bc, said that dilute sugar concentrations of most of the hydrolyzates may be fermented easily after treatment by the neutralizatiori procedure. However, recovery 'of 2,3-butjylene glycol from dilute solutions is impractical, and fermentation of more concentrated hydrolyzates was att'empted. These solutions were prepared from the dilute hydrolyzabes by concentration a t pH 3 in a vacuum still. As the sugar concentration was increased fermentation of several of the hydrolyxates treated by the neut,ralization method became more difficult. These difficulties were overcome by t'he addition of such substances as malt sprout's or yeast extract. However, the addition of these suhstances was not necessary when the solutions had been treated by the second method of treatment. Preliminary experiments with Douglas fir hydrolyzate (at a 10% reducing sugar level) indicated that the medium should be adjusted to between pH 9 and 10.5 wit,h alkali or lime for best results when the alkaline method of treatment was used. I n all these experiment;. the inoculum was grown on the glucose-steep water medium. Rather concentrated wood sugar solutions were fermented after treatment by the alkaline procedure. Table I1 gives data obtained from the fermentation of southern yellow pine hydrolyzate at various sugar concentrations. Similar result$ were obtained with other hydrolyzates, shown in part by Table I. ACCLI&fATIZATIOS O F CULTURE TO HYDROLYZATE
Experiments on the fermentation of hydrolyzed grain mashat by this culture indicated that if the culture were grown for a fexv transfers on the hydrolyzed grain mash, improved fermeutatiojlh resulted. This procedure ww repeated with several wood hydrolyzates wit'h favorable results. The advantages are shown in Table 111, where southern red oak was used as subst,rate. By this procedure the alkaline method of t'reating the hydrolyzate. which had been necessary when rather concentrated sugar soluLions were fermented without the addition of auxiliary substances, was eliminated and the neutralization method w-a,+ satisfactory. ACKNOWLEDGJIEKT
Hydrolyzate treated as follows: adjusted to p H 10.5 with lime, filtered; adjusted t o p H 6.0 with HzSOa, filtered; diluted t o appropriate sugar concentration. Nutrients added per liter: 3 grams KHapOa,3 grams urea, 5 grams CaCOs.
The advice and criticism received from Et. H. Leonard are gratefully acknowledged. The wood hydrolyzates used in thic investigation were obtained from the Forest Products Laboratory. Madison, Wis. The work was supported in part by a grant fromL the Heyden Chemical Corporation.
TABLE 111. ACCLIMATIZATION OF CULTURE TO SOUTHERS RED OAKHYDROLYZATE BY HXPEATEDTRAKSFER
LITERATURE CITED
Transfera Number
1 8 12 16
Fermentationb Time, Hr. 36 36 36 34
Sugar Fermented 27 76 90 93
Yields of 2,3-Butylene Glycol and Acetoin, yo 5.7 25.9 32.5 35.5
Transfer medium: 4% southern oak hydrolyzate (neutralized t o P H 6 and filtered), 0.2% KHaPOa, 0.4% urea, 0.5% CaCOs. b Fermentation medium: concentrated southern red oak hydrolyzate neutralized t o p H 6 with lime and filtered, diluted t o 10% reducing sugar: 0.3% IiHzPOa, 0.3% urea, and 0.5% CaCba added.
Johnson, hI. J., to be published. (2) Langlykke, A. F.,and Peterson, W.H., IND. ESG. CHEM.,AK.
