SYLVAN B. LEE MERCK & C O M P A N Y , INC., R A H W A Y ,
N. J.
PREVIOUS review of fermentation (186) covered industrial fermentation research, development, and production since 1940. Other review articles have appeared recently which have emphasized various aspects of industrial fermentation. Johnson (101) reviewed the progress of individual industrial fermentations. Boruff and Van Lanen (3.4)reviewed the production of fermentation industries during TTorld War 11. Porter (160) presented a summary of recent advances in fermentation, and Hildebrandt (94) discussed the wartime technological advances in the acetone-butyl alcohol, ethyl alcohol, and 2,3butylene glycol fermentations. Recent advances in antibiotic research have been reviewed by Benedict and Langlykke (M), Regna (160), and Bailey and Cavallito ( 8 ) . Engels (66) has described the industrial development of antibiotics. Since it is too large a task to cover all phases of progress in fermentation science, as in the previous review (leg),emphasis will be placed on the industrial aspects of fermentation with particular attention to those factors which contribute toward the establishment of a firmer unit process. This review is concerned primarily with the postwar period and is intended to supplement the previous review (182).
ETHYL ALCOHOL The chief technological advances in the ethyl alcohol fermentation science during the war years have been adequately reviewed (94, 101, 160, 18W). These wartime developments may be summarized as follows: 1. Alcohol from fermentation of wood sugar and improved methods for saccharification of wood 2. Alcohol from the fermentation of waste sulfite liquors 3. Alcohol from wheat 4. Alcohol from other raw materials such as Irish and sweet potatoes, milo, and various waste products 5 . Use of mold amylase for converting starches 6. Continuous cooking and mashing of starchy raw materials 7. Flash conversion by amylolytic agents 8. Continuous yeasting 9. Advances in the design and operation of continuous fermentation units
Although the quantity of industrial alcohol produced in this country has suffered a sharp decline since the wartime peak of nearly 600,000,000 gallons (95% alcohol) in 1945 (84),it remains the largest fermentation industry in terms of tonnage of material produced. Postwar Production of Alcohol (762) 194G
Total productiona Produced by fermentation, % Q
183,000,000 62
1947
149,400,000 55
1948
176,800,000
64
Production expressed as gallons (95% alcohol) to t h c nearest 100,000
gallons.
The bulk of industrial alcohol produced by fermentation during these years has again, as before the war, come from molasses. Haywood, Emerson, and Davis (93) have discussed the typical problems of a molasses industrial alcohol unit converting to wartime raw materials and reconverting to molasses. Erb and Zerban (67) have demonstrated that acid hydrolysis of unfermented substances in blackstrap molasses fermentation
residue will give reducing sugars and thereby more potential alcohol production. The hydrolysis must, be carefully conbrolled with regard to acidity and concentration, as the reducing sugars formed are readily destroyed. It has been established (144) that each lot of molasses ha,s an optimum pH for best alcohol yields. Fifty pcr cent of the Cuban m.olasses tested required a n initial p H value of 4.0 to 4.5; 22y0 fermented best at p E 4.0 to 4.24 and 28% at pI-1 5.0 or above. Legg (113) claims that the fermentation efficiency of offgrade molasses can be increased by using an inocuIum having various proportions of grain and niolasses fermenting yeast strains. Legg suggests this may be due to greater resistance of one yeast strain to toxic factors or greater ability t o utilize certain polysaccharides present. During the war years, considerable attention was given to various techniques for converting starchy raw materials 'co reducing sugars which are fermentable by yeasts. Dworschack and Burdiclr (59) described a malt saccharification process which uses a plurality of cooking and premalting stages followed by a final cooking a t 160 C. and cooling to 60" to 75' C. before final saccharification. This treatment makes possible 85% conversion of carbohydrate within 1 to 3 hours and yields a sugar solution that is readily filtered and sterilized before fermentation. Wickerham (219) described a process for simultaxieously saccharifying and ferment,ing starchy materials to ethyl alcohol by the sgmbiotio action of two strains of yeasts-namely, Endonzycopsis jibuliger var. hordei and Saccharomyces cerevisiae. A ferment,ation efficiency of 78.3% is claimed as compared to 80.5% for the malt control. Several processes involving acid hydrolysis techniques for converting starchy raw materials have been reported. Hayek and Shriner (9%') have reported a series of studies using sulfurous acid for starch hydrolysis. IZuf et al. (169) reported a continuous process for both acid hydrolysis and fermentation of the resulting mashes in a 12-hour period with a fermentation efficiency of 87%. However, to obtain yields comparable to present COMmercial methods which employ barley malt, it was necessary to supplement acid hydrolyzed mash with such materials as wheat bran, mold bran, Mylase, Rhozyn-le S, or submerged mold cultures before initiating fermentation. This process has the advantages of fermenting a sterile mash in which the starch is completely hydrolyzed. Dunning and Lathrop (58) described a process for saccharification of plant materials such as corncobs, sugar-cane bagasse, cottonseed and oat hulls, and cereal straws to five- and sixcarbon sugars 'by a progressive degradation rising sulfuric acid of varying concentrations and temperatures. The acid hydrolyzing steps are used intermittently on the resulting solid from the previous step by (a) drying the separated residues having entrained free sulfuric acid and ( b ) mechanical mastication a t substantial pressures. The authors claim they procure readily fcrnicntable solutions in which the dextrose yjelt-ls from the hydrolytic procedures are 85 to 90% of theory. In a shudy concerned with the conventional malt conversion process for ethyl alcohol fermentation, Van Lanen et al. (210) reported that the use of (1) proteolytic enzymes such as papain and ficin, (2) protein hydrolyxates such as casein hydrolyzate, (3) vitamins and growth factors, (4)availabie organic nitrogen such as urea, and ( 5 ) culture filtrates from Bacillus subtilis, will serve to reduce t,he normal amount of barley malt to 60% and
2860
September 1949
INDUSTRIAL AND ENGINEERING CHEMISTRY
1869 tails of a practical mold amylase for es at the Northern culture ( A .niger),
,a t the time of calculation, the mold t. This represents a possible saving cents per gallon of 190 proof alcohol coho1 produced by fermentation at the asses, the large potential use of mold #
It was necessary t o “ada hydrolyzates b y serial transfer before sati obtained.
for yeast growth. y.
.
MICROBIAL AMYLASE
The technology of the use of microorganisms for
1914 Takamine (198) reported a successful use of mold amylase in beverage alcohol fermentation but the distillers have been redue. t o the belief t h a t it may result in urrent processes for producing mold overcome previous objections as it is ie now being used in a t least one disore bushels of grab per day for the from bacteria and yeast have been reffer solution and found that the superior t o barley malt. I n vised a technique for producing thin stillage. T h e medium was
(94) and Underkofler et al. ($08)’ w
pointed out that Takamine iirst the manufacture of commercial di
This was one of the first reports on
tion of the mold
a
c
Corman and Langlykke (48) studied the types of amylase in various fungal preparations and found that alcohol yields correlated more closely with the potency of the glucogenic enzyme system than with a-amylase. The or-amylase liquefied the starch and converted i t t o dextrins and maltose while the glucogenic enzyme hydrolyzed maltose, dextrins, and apparently starch itself to glucose. Le Mense et al. (114) studied the ability of more than thirtyfive fungi to produce fungal amylase under submerged aerobic conditions. Several species of Aspergilli ( A . wentii, A . oryzae, A , alliaceus, and A . niger) were superior t o Rhizopus, Mucor, Penicillium, and Monilia for producing dextrhizing enzymes. A strain of A . niger (NRRL 377), having both dextrinizing and saccharifying ensymes, grown on a medium consisting of thin stillage, corn meal, and calcium carbonate under aerated submerged conditions gave culture liquors which were satisfactory replacements for distiller’s malt in the alcoholic fermentation of corn. Reese et al. (169) have reported the details of a rapid fermentation method for evaluating the ability of fungal amylase preparations to saccharify grain mashes. Adams et al. (1) reported that distiller’s dried solubles are superior t o thin stillage as a substrate for submerged amylase production. The characterizing features of this process. are the use of (1) calcium carbonate for neutralization, (2) 24-hour submerged culture stages prior t o final propagation, ( 3 ) 0.6% mycelial transfer for initiating submerged growth, and (4)conversion temperature of 145’ F. This process resulted in increased yields of 0.2 t o 0.3 proof gallon per bushel of grain when completely replacing malt with submerged amylase.
CONTINUOUS FERMENTATION
During recent years considerable attention has been given to continuous fermentation processes and from the viewpoint of a unit process it appears worth while t o summarize recent work. The ethyl alcohol fermentation is the more suitable commercial process for adaptation to continuous operation; consequently it has received greater emphasis. Earlier work on continuous fermentation, which has been reviewed by DeBecze and Rosenblatt (60), has given valuable information concerning successful continuous operation. The results obtained during the last few years have contributed further toward adapting the alcohol fermentation to various substrates on a continuous basis. Much work on the continuous fermentation of malt-hydrolyzed grain and other starchy raw materials has been done by the research department of Joseph E. Seagram & Sons. Stark, Kolachov, and Willkie (194)reported a method of continuously cooking grain based on the introduction of high pressure steam into mash using a mixing jet which discharged into a relatively small retention tank. This process replaced the bulky cookers with motor-driven stirrers used in orthodox procedures and greatly reduced the time and cost for the construction of grain alcohol plants. The malting operation has also been done on a continuous basis (30,86)using a flash conversion procedure. A process for the continuous production of yeast from grain has been described (209). The feasibility of operating a continuous grain alcohol fermentation process using malt conversion is questionable due to the possible contamination introduced with the barley malt.
1870
INDUSTRIAL AkD ENGIN&EhING CHEMISTRY
Vol. 41, No. 9
Seed Fermentor Area, Antibiotic Fermentation Plant, Merek & Company, Inc., Elkton, Va.
Recent work using submerged mold amylase preparations ( I , 58, 1161,will aid in eliminating contamination hazards from the continuous processes since such amylase preparations can be produced which are free of bacterial or other contaminants. Altsheller et al. ( 4 ) described a continuous two-bushel per day alcohol unit which used sulfuric acid hydrolyses of grain mashes followed by continuous fermentation utilizing two successive vessels and a total fermentation time of 11 hours. Alcohol (190 proof) is produced in a single column built for both stripping and rectifying operations. The entire unit is fully instrumented and is automatic in operation. Ruf et al. (169) reported further work on continuous fermentation of acid-hydrolyzed grain mashes including successful pilot plant operation without apparent yeast degeneration. By adding certain supplements t o these mashes, discussed previously in this review, i t was possible to complete the fermentation cycle in 12 hours with plant efficiencies of 87%. Roeckeler (31) reported that grezter p~oductionof alcohol and distillers residues are obtainable from a given fermentor if the toxic effect of the alcohol on the yeast is reduced by continuously removing the alcohol. This is accomplished by continuously distilling under mild conditions, so as not to kill the yeast, by withdrawing fermenting liquor which is run through an alcohol stripping column operating under reduced pressure and returning She partially stripped liquor to the fermentor. Bilford et al. (38)described successful continuous laboratory
fermentations using molasses and established the relation between yeast cell count and fermentation rate. The authors suggested that, this technique could be applied t>o large scale operations. Victorero (df1 j described tl fermenting appara,tus designed primarily for the continuous alcoholic fermentation of molasses which consists of several partitions diridiiig the vessel into a succession of superimposed fermentation compartments, each compartment having a,n aperature for permitting restricted upward movement of the mash. Fermented “beer” is continuously withdrawn from t>hetop and fresh mash continuously a8ddeda t the bottom. The inventor claims the apparatus has been used on a large scale and has operated continuously for periods of several weeks with minimum of attention and control work. Owen (I,@) has described a somewhat similar appa,rabus for continuous molasses fermentations using a glass column having six decks. I n this apparatus the mash is fed continuously into the top of the column and flows from upper to lorn-er decks. At the time of his report the author had not tested his apparatus in a. large scale operation. Harris et al. (89) stated that wood hydrolyzates can be fermented continuously with Torula utilis. Sulfite waste liquors have been fermented continuously on a plant scale (37, 6Y, 103). This process has a fermentation time of 20 hours and the yeast is re-used thereby largely eliminating fermentation lag time and the use of carbohydrates to develop yeast cells.
INDUSTRIKL
September 1949
HEMISTRY
ACETONE-BUTYL A L C O H O L Developments in the acetone-butyl alcohol fermentation during World War 11 and early postwar years have been reviewed (94, 101,182). The total United States production figures for butyl alcohol and acetone from fermentation processes for the postwar years 'are given in the following table.
Production of Butyl Alcohol and Acetone (762) 1946 Butyl alcohol Fermentation Other proce8ses
Total Acetone Fermentation Synthetic
Total
r
-i
Thousands of Pounds 1947
1948
126,200 104,300 230,500
140,100 116,000 256,100
140,200 , Not available Not available
37,400 298,100 335,500
40,000 357.200 897,200
Not available Not available 469,900
Leonard et al. (118)reported a successful butyl alcohol-acetone fermentation of wood sugar using Clostridium butylicum. The wood species as well as the conditions of acid hydrolysis greatly affected the case of fermentability of the sugar solutions. Solutions having as high as 3% sugar were fermented successfully giving solvent yields ranging from 24 to 38% of the sugar fermented. Langlykke et al. (111) reported successful butyl alcohol fermentations of xylose saccharification liquors from corncobs using a selected strain of butyl alcohol bacteria. The liquors were treated with powdered iron or activated charcoal prior t o fermentation. The powdered iron served to remove copper from acid hydrolyzates in copper-bearing equipment and made the medium more favorable for the fermentation from the Viewpoint of oxidation-reduction potentials. Fermentation of xylose liquors gave solvent yields and ratio of products similar t o fermentation in glticose and molasses media. Success was achieved in fermenting 5 t o 6% sugar solutions when the xylose exceeded 50 t o 70% of the total sugar in the medium. Boehm, Hall, and MacDonald (SX) described a process for improving sugar solutions from wood liquors for the butyl alcohol fermentation using sulfide precipitation and treatment with activated carbon. Bekhtereva (84) described the effect of an acid-forming organism on the acetonebutyl alcohol fermentation. Maravall ( 1 2 7 ) and Sebek (177) discussed several aspects of the mechanism of the acetone-butyl fermentation. Schachermayr (17'6) discussed the acetone-butyl fermentation of corn press juice. As the press juice concentration was raised from 3 to 11%,the yield of solvents became less and fermentation time was lengthened. The relative amounts of solvents produced were 65 to 71% butyl alcohol, 23 to 27% acetone, and 5 t o 8% ethyl alcohol. Beesch (14) described a process wherein a new organism Clostridum saccharo acetoperbutylicum is capable of fermenting blackstrap molasses and starches. A mash having 6% sugar is fortified with ammonium sulfate, ammonium hydroxide, and calcium carbonate, and fermented in temperature and time ranges of 28' to 35" C. and 40 to 72 hours, respectively. The solvent yields are claimed to be between 28 to 31% based on sugar fermented and contain 70 to 75% butyl alcohol, 20 to 25% acetone, and 5% ethyl alcohol. Carnarius (39) described equipment which can be used for continuously sterilizing mashes for the acetone-butyl alcohol and other fermentations.
P,3-BUTANEDIOL At the present time there is little commercial interest in 2,3butanediol (2,Sbutylene glycol). The technology of fermentation processes involving Aerobacter aerogelaes and Aerobacillus
1871
polymyza for the manufacture of the compound was well developed during the war years (94, 160, 182, 906). The research during this period was prompted by the potential use of thie compound for making 1-&butadiene. However, the possible use of Z,&butanediol in other products (41, 4.8, 106, 133, 156) such as antifreezes, solvents, plastics, resins, humectants, pharmaceuticals, and coatings gave hope for large scale postwar operations. Since wartime secrecy orders have been lifted, certain phases of this development have been published. ID addition to work previously reviewed, Kooi, Fulmer, and Underkofler (107) have reported a process for the fermentation of cornstarch with Aerobacillus polymyza. It seems possible that the production and sale of 2,3-butanediol may in the future come into favor as did primary butyl alcohol from the acetone-butyl alcohol fermentation after World War I. Liebmann stated (12s) that if demand can be developed to warrant production of the 2,3-butanediol on a sufficiently large scale, the cost of production may be 10 to 12 cents per pound or less.
CITRIC A C I D Developments in the citric acid fermentation industry up to 1947 have been reviewed (101, 189, 81s). Recently Cochrane (43)has published a general review of the commercial production of acids by fungi. The emphasis of citric acid research and development is on submerged fermentation technique since such a process should have certain advantages over the shallow pan technique used a t the present time. Szucs (197) was granted a patent for a submerged citric acid fermentation process in which low concentrations of dry skim milk solids were used in an otherwise synthetic medium having refined sucrose and mineral salts. A strain of Aspergillus niger was grown in this medium at 18' to 25' C. under aerated and agitated submerged conditions giving yields as high as 90% in 9 days. Shu and Johnson (179)studied the effects of the sporulation medium on citric acid production by A. niger in submerged cultures. The addition of manganese and Trommer malt extract accelerated spore formation whereas increasing the concentration of monobasic potassium phosphate, ammonium nitrate, and zinc retarded spore formation. Manganese and malt extract in the sporulation medium reduced the yields of citric acid in subsequent submerged fermentations but did not decrease yields in surface culture fermentations. In a later report Shu and Johnson (181) described the results of submerged culture fermentations with Aspergillus niger in shake flasks on a synthetic! medium. Citric acid yields were 72 grams per 100 grams of sucrose. Fermentation times were 7 and 12 days for sucrose concentration of 140 and 260 grams per liter, respectively. The optimum medium conditions for shake flask fermentations include potassium dihydrogen phosphate above 1 gram per liter, magnesium sulfate heptahydrate above 0.25 grams per liter, iron concentration of 1 mg. per liter, 2.5 grams per liter of ammonium nitrate, and initial p H between 2.2 and 4.2. The interdependence of medium constituents in submerged citric acid production has been studied by Shu and Johnson (180). An aluminum hydroxide coprecipitation method was shown to be satisfactory in the purification of commercial glucose for submerged citric fermentation. After this treatment, the addition of 0.3 mg. of zinc and 1.3 mg. of iron per liter supported a yield of 67 grams citric acid per 100 grams of glucose in a 14% sugar solution in 9 days. Five essential ingredients were used in the medium: ammonium nitrate, magnesium sulfate heptahydrate, potassfum dihydrogen phosphate, zinc, and iron, of which the latter three effected citric acid yields by some means other than their effect on growth. Manganese ions and high con-
INDUSTRIAL AND ENGINEERING CHEMISTRY
1872
centrations of iron and zinc ions gave lesser reduction in citric acid yields on a phosphate-free medium. Groot ( 8 H ) obtained a patent for citric acid production with A . niger based on the use of water extracts of white or red beets or turnips in a 14% sugar solution having potassium dihydrogen phosphate, ammonium nitrate, and magnesium sulfate heptahydrate. The citric acid yields were low. Kovats (110) reported that the output of citric acid by A . niger is increased by using growth temperatures of 28 C. during the early period of the fermentation, then dropping the temperature t o 20 C. during the later stages of fermentation. The author claims there is no increase in over-all fermentation time using this technique. Kovats (109) also reported the effect of various samples of beetsugar molasses on cit,rir-acid production in t2hesubmerged process. O
OTHER ORGANIC ACIDS A recent rBsum6 of the commercial production of acids by fungi has been published (@). LACTIC ACID
Annual production uf lactic acid in t,his country during l94G, 1947, and 1948 was betveen 6,000,000 and 6,500,000 pounds, all of which was produced by fermentation processes. Most of the recent work on the lactic acid fermentation has been done in other countries and reported in foreign journals. In this country, Leonard, Peterson, and Johnson (117) have reported extensive investigations of sulfite waste liquor as a subst,rate for lactic acid fermentation. A production medium containing sulfite liquor, previously steam-stripped and having the sulfite precipitated at, pH 8.6, malt sprouts, and calcium carbonate was inoculated with a strain of Lactobacillus pentosus grown in a medium containing malt sprouts and blackstrap molasses. The fermentation rcquired 40 to 48 hours at a temperat,ure of 30" C.; during this time the pK was controlled by adding calcium carbonate or calcium hydroxide. Two thousand gallons of sulfite waste liquor corresponding to a ton of pulp made from spruce wood, yielded 205 pounds of lactic acid and 75 pounds of acetic acid after recovery. The authors described an extract,ion and purification process using solvents such as amyl alcohols and isophorone which yielded 90% pure lactic acid. Costs €or raw nnateria,ls, with no credit for sulfite waste liquor, mere estimated at 2.0 to 3.5 cents per pound of acid depending on whether the lignin residue was used for fuel. Blaisten ( 2 9 ) fermented pafiteurized molasses media containing 15% reducing sugars plus malt sprouts, with a strain of a sporeforming lactic organism. The fermentation time was 4 days #during which time calcium carbonate was added to maintain pH 6.0. Yields of 80% n-lactic acid were obtained based on sugar utilized. PerqUin (146)reported that hydrolyzates of keratin-containing compounds were beneficial to lactic acid fermentations due to the ,quantities of cysteine, lysine, tyrosine, and phenylalanine present. Xakharov asd Fedorova (286 j reported favorable la8ctic acid fermentations using barley shoots as the rlut,rient source, Zakharova and Utenkov (227) reported successful continuous lact,ic acid fermentation using Lactobacillus delbruckii i n a fermentation unit consisting of a series of connected fermentors. The use of a mixed culture of a Lactobacillus and tt Mycoderina was used by Nilsson (159) t.o ferment whey. Fermentations of whey and acid hydrolyzed wheat starch with Lactobacillus bulgar.icus have been described (196). KOJIC ACID
Kojic acid is produced by many Jnolds of the Aspergillus genus in acceptable yields. This compound, yith its many reactive chemical groups, is extremely interesting but to date it has no large scale use. A comprehensive review of the fermentative production and recovery of kojic acid as well as its
Vol. 41, No. 9
structure and chemical reactions has been published (9). This is another compound which could be produced economically in a large scale fermentation process if there were a demand for the product. ITACONIC ACID
Little information on the itaconic acid fermentation has appeared recently. Yuill (236) described a new species of Aspergillus which produced both itaconic and kojic acids. The most extensive recent report on itaconic acid was that by Lockwood and Nelson (194). These workers, from the Xorthern Regional Research Laboratory where a greater poytion of the work on itaconic mid fermentation has been done, report further results using Aspergillus terreus in submerged agitated cultures. Maximum production of the acid by this organism requires rigid pH control in the range of 1.8 t o 1.9. Ma,gnesium sulfate is essential whereas zinc sulfate is not. Sodium chloridc oauses greater mycelial growth and less acid. Superior yields of itaconic acid are obtained when the inoculum is -very small and the maximum sugar concentration in the medium is not abovc 6%. Thus far there is no large-scale use for itaconic acid. a-KETOGLUTARIC ACID
Lockwood and StodoIu (126) described a process for making a-ketoglutaric acid hy growing a bacterium of the genus Pseudomoms with agitation and aeration in a medium containing a soluble gluconate salt and a neutralizing agent. The fermentation is continued until the 2-ketogluconic acid, u-hiah is formed first, is converted furbher t o a-ketoglutaric acid. GLUCONIC ACID
Blaisten ( 8 9 ) obtained 65% yields of calcium gluconate in 20 hours with Aspergillus niger in an aerated and agitated fermentation medium containing acid-hydrolyzed cornstarch as tfhe carbon source. I-KETOGLUCONIC
Blaisten (29)obtained 80% yields of 2-ketogluconic acid in 10 to 20% glucose solutions using a strain of Pseudomonas fiuore IXo::bsm., 10,
365 (1946).
(125) Lockwood, 1,. R., and Sjtodol.~.F'. IT., TJ. S. FatenG 2,443,919 (June 22. 1948).
the Antibiotic Sy~ilpobi~:n, before t h e Diviaion of Biological I C A S CEEILfICAXI SOCIETY,
Chicago, 12. (12'3) Mead, T . E.. Jtack, M. V., and Smith, E. L., U. 8 . Pattlnt 2,451,633 (Oct 19, 1948). (130) tMeade, 12. E , Pollard, 13. L., and Xodgors, N. E., Ibid,, 2,433,063 (Drc. 23, 1947). (131) Moyer, A. J., U. S. Patent 2,442,141 ( M a y 25, 1948). (132) I b i d . , 2,443,989 (June 22, 1948). (133) Moyer, A. J., and Cogliill, R.13.. J . Bact., 53, 329 (1947). and Gpsok, M.I,., LT. S. Patent 2,448,6380 (GrpC. (134) Myers, It. P., 7, 1948). (135) National Institute of Ilealth. Bethesda, Md., Antibiotic Study Section. Antibiotics Substances-Their Biological and Chemical Properties (February 1948). (136) Neijh, A. C., and Maodonald, F. J., Can. J. Research, 2SB. 70 (1947). (137) Nichol, C . A,, Dietrich, L. S., Cravens, W.W., and Elvehjem, C. A., PTOC. Soc. ExptE. Bid. Med., 70, 40 (1949). (138) Niohol, C. A,, Robblee, A. R., Cravens, W. W., and Elvehjenl, C . A., J. Biol. Chem., 170, 419 (1947). (139) Ivilsson, O., Svenska Mejeritid., 40, 207-10 (1948) (140) Nord, F. F.,U. S. Patent 2,450,055 (Sept. 28, 1948). (141) Olson, B. H., and Johnson, ,M.J., J . Buct., 57, 235 (1949). (142) Owen, W.L.,Sugar, 43, 36 (1948). (143) Pearl, I. A., Chem. Eng. A'ews, 26, 2950 (1948). (144) Perdomo. E. V., Mem. asoc. tbcwicos azucar. Cubu, 20, 467-73 (1946). (145) Perlman, D., and Langlykke, A. F., J . Am.. Chem. Soc., 70, 3968 (1948). (146) Perquin, L. H. C., Dutch Patent 58,548 (Nov. 15, 1946). (147) Pollard, H. L., Rodgers, N. E., and Mea,de, R.E., U.X. Patent 2,449,140 (Sept. 14, 1948). (148) Zbid., 2,449,141, (149) Zbid., 2,449,143. (150) Porter, R. W., C'fkem. Eng., 54, No. 12, 141 (1947). (151) Povolotskaya, K. L., and Skorobogatova, E. P., Biokhimiya, 12, 268 (1947). (152) Price, C. W., Randall, W. A , and Welch, H., Ann. N . Y . Arad. A%
