mz
FERMENTATION SYLVAN
B. LEE, COMMERCIAL
SOLVENTS CORPORATION, TERRE HAUTE, IND.
P
Holding at the latter temperature and pH for 8 hours during which time the im urities of the "thick mash" settle at the base of the settling t a n i (during this period there is considerable conversion of SucrOSe to hexoses) Disposal of the sediment Dilution to a "thin mash" having a Brix density of 20" to 23" Fermentation in closed-type agitated fermentors having recording thermometers and pH indicators A 10 to 15% volume of inoculum was used; the total mask1 fermented was added in three increments, and fermentation time was 30 to 36 hours
REVIOUS reviews of fermentation (143, 266) have covered chiefly those aspects of industria] fermentation research, development, and production which can be used in an attempt to formulate a fermentation unit process. As has been pointed out (ICs),it is too large a task to cover all fermentation literature. The previous reviews, covering the period 1940-1949, have been confined to literature dealing with present industrial fermentation processes and other processes developed sufficiently so that large scale production could be attained when economics permit. This review is divided into two sections. The &-st section contains a discussion of recent literature on individual fermentstion processes. The second section contains a discussion of various factors regarded of prime importance in the consideration of fermentation as a Unit process, and this discussion is based On select references from the first section Of this review and from previous reviews (149, %W). During the past year, others have reviewed various aspects of industrial fermentations (87, 211, 9%).
.
INDUSTRIAL F E R M E N T A T I O N PROCESSES This section of the review is concerned with the recent literature on various large scale fermentation processes and other fermentations which appear to have industrial possibilities. In general, the discussion is confined strictly to fermentation and, with the exception of newer fermentations, little attention is given to the separation, purification and uses of fermentation products. ETHYL ALCOHOL
Previous reviews (111, ISO, 149, 223, 956) have covered the technological advances in ethyl alcohol fermentation up to approximately the beginning of 1949. This review presents only recent developments not already covered. The total production of industrial alcohol has risen gradually during the last 3 years, but the fermentation industry produced a smaller percentage of the total in 1949 (916).
Recent Alcohol Production (375) 1947
Total produotion4 Produced by fermentation, % Production expressed gallons. a
a8
149,400,000
55
1948 171,800,000 64
1949 187,500,000
52
gallons (95 % alcohol) t o the nearest 100,000
During the past year molasses continued to be the chief raw material for the production of alcohol by fermentation. Rao et al. ($29) reported a new yeast, used in the molasses alcohol fermentation in India, which gave an alcohol concentration of 12 to 15% in the fermented mash. They claimed that in two distilleries this process resulted in a 50% reduction in cost. Arroyo (16)described a fermentation technique for producing alcohol from blackstrap molasses. Through the use of this technique he claimed that 50 to 100% more molasses could be processed, resulting in a corresponding increase in production. The method consisted of: Addition of proper quantities of ammonium sulfate and calcium superphosphate to a "thick mash" which has a Brix density of 55"t? 60" Heating the '(thick mash" to 80' c. a t pH 4.5 to 5.2
Depending on the quality of the blackstrap molasses used, the fermented beers had alcohol concentrations in the range of 10 to 13.5% by volume. N~ data were given as to the efficiencyof carbohydrate conversion. owen(198) studied the A~~~~~ process and found that the clarified mash fermented more slowly than either the sludge or the untreated molssses, Be attributed the slower fermentation of the clarified mash to the removal of colloids. Erb and Zerban (74)described a method for detecting abnormal molasses. They also described a method for calculating probable yields from normal molasses. Sattler and Zerban ( i 4 6 ) discussed unfermentable reducing substances in molasses. They stated that about 10% of the reducing power of unfermentable substances in cane molasses was due to volatile constituents, such as hydroxymethylfurfuraI, acetoin, levulinic acid, and formic acid, which were decomposition products of the sugars. There were considerable quantities of melanoidin compounds in the molasses which yielded reducing sugar on mild acid hydrolysis, Owen (I97) studied the deterioration of blackstrap molasses during storage and suggested that this might have been due to a reaction of amin:, acids with sugar to form N-glycosides. Reich (238)described a method for removing calcium from molasses and other fermentable materials. During the past year the use of several starchy raw materials for alcohol fermentation has also been described. Mande el al. (167) described the use of Bassia flowers as the raw material. These flowers, which are found in large quantities in certain provinces in India, contain a high concentration of reducing sugars. A aO% solution of macerated flowers, extracted for 30 minutes a t 180" F., gave a fermentation efficiency of 98.3%. This corresponded to a yield of 5.32 gallons of 95% alcohol per 100 pounds of dry flowers. Ammonium sulfate and ammonium phosphate were satisfactory nitrogen sources; urea was unsatisfactory. A description of the process, with flow diagram, was presented for a distillery having a daily capacity of 2000 gallons of 95% alcohol. The cost was estimated a t 37 cents per gallon. Banzon, Fulmer, and Underkofler (I8)described the production of ethyl alcohol from cassava roots which are available in large quantities in the Philippines and South America. Acid hydrolysis of the starch resulted in a yield of 98.8% of the theoretical reducing sugars, but the alcohol yield on this material was only 69% of the theoretical. Using dilute sulfuric acid for thinning and an enzyme preparation for liquefaction and conversion, the following percentages of the theoretical yields of alcohol were obtained: malt, 67.2%; mold bran, 80.5%; Rapidase plus malt, 75.8%; and Rapidase plus mold bran, 87.231,. Teixeira, Andreasen, and Kolachov (684) also discussed the alcoholic fermentation of the cassava root and demonstrated that superior yields were obtained when the starch was converted with a fungal enzyme prepared in submerged culture from A . niger. Using various starch converting agents the yields, expressed in
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INDUSTRIAL AND ENGINEERING CHEMISTRY
September 1950
proof gallons per 100 pounds of wet meal, were aa follows: A . niger, 11.6; 7% barley malt, 9.2; and 16% corn malt, 10.8. Malchenko (166) described a mashing process for starchy substances which was carried out in coils. The mash was fermented under carbon dioxide pressure and carbon dioxide was also used for agitation. In another patent Malchenko (166) described a stepwise process using malt for saccharification and for conducting the fermentation continuously in two connected vats; maltose was fermented in the first vat, dextrins in the second. Tucker and Balls (993)described a process for extracting diastase from wheat. This process, which was used during World War I1 when wheat was a raw material for alcohol production, consisted of treating the uncooked wheat mash with an aqueous neutralized solution of sodium sulfite, and holding at 45' C. for 1 hour. The supernatant liquid, containing the extracted diastase, was siphoned off and was used to aid in converting the wheat starch after the grain was cooked. This process resulted-in a great malt economy, and the fermentation residue could be dried to a nonhygroscopic material. Wallerstein (813) claimed a faster fermentation and higher yields in the alcoholic fermentation of rye or wheat mashes by the addition of 1 to 27&, based on the grain weight, of kaolin, bleaching earth, fuller's earth or bentonite. Charcoal and synthetic adsorbents failed to give similar results. Barreto (20) claimed that the addition to the mash of small quantities of polychlorophenol inhibited bacterial contamination and resulted in increases in ethyl alcohol yields of as much au 12%. It was recommended that all fermentation plant e q u i p ment be washed regularly with a hot solution of sodium pentachlorophenate. Gai (93)claimed similar beneficial effects with 0.002% pentachlorophenol added at a low pH. r
Continuous Fermentation. Further attention has been iven t o continuous methods for alcoholic fermentation. Kan 7133) stated that with corn or wheat raw materials, the alcohol yield depended largely on cooking temperature and fermentation time. The cooking temperature and fermentation times for corn were 365 O F. and 45 to 65 hours, respectively, and for wheat, 368" F. and 50 to 70 hours. Adsma (3)has demonstrated, on a laboratory scale, a rapid continuous fermentation for pear apple, and cherry wastes. Adams ( 4 ) described a method for predicting cycle times for continuous fermentations. The cycle time is that time needed to replace completely the original fermentation medium. The length of the continuous fermentation cycle at any specific yeast population can be calculated from batch growth curves. By drawing tangents to the growth curve at desired levela i t was possible to calculate the theoretical cycles by dividing the total yeaat population, at the point to which the tangent waa drawn by the slope of the tangent. The method was a plicable to all media tested except those which had a nutritionsf deficiency. Andreasen (10) reviewed the fermentation methods used in a modern distillery. Microbial Amylase. Microbial amyleses are gaining frvor ~d converting agents for starchy raw materials used in ethyl alaohol fermentation. Although these amylases have not a8 yet gained wide industrial acceptance, it is reported that they are being used in a few large plants for the production of neutral spirits. ITOWever, their general use in the distilled beverage industry appears quite possible. Certainly, from an economic viewpoint, microbial amylase preparations are to be desired. It has been demonstrated by Le Mense et al. (1@) that such preparations effected a saving of 2.4 to 3.6cents per gallon of 190 proof alcohol. The group at Joseph E. Seagram & Sons, who have extensively studied microbial amylases, have reported further work, Lippa et al. (168) reported that crude culture filtrates of Aspergillw niger N. R. R. L. 337 produced dextrins, maltose, and glucose from gelatinized Cornstarch. The relative proportions of these products depended on tempertaure and substrate concentration. Starch hydrolysis proceeded stepwise; &-amylase hydrolyzed starch to dextrins and maltose; limit dextrinase hydrolyzed the dextrins to maltose and glucose; maltase hydrolyzed maltose to
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glucose. The effect of trace elementa on this stepwise hydrolytic process was given, Pan, Andreasen, and Kolachov (201) reported that the fermentation of corn mashes, hydrolyzed by barley malt or submerged cultures of fungal amylase, consisted of a rapid initial phase and a slow secondary phase. In the secondary phase, dextrin slowly underwent hydrolysis and fermentation. When such fungal amylases were used in combination with yeast, they gave a rapid fermentation of the dextrins and they considered that this rapid fermentation was more closely allied to the maltase activity of the culture than with a-amylase or limit dextrinase. As mentioned previously in this review, submerged fungal amylase (284) and mold bran amylase (18) have been used successfully for the conversion of cassava starch. Soriano and Trucco (963)reported the successful use of mold bran in the alcoholic fermentation of corn in South America. Tsuchiya et al. ( W )reported further work at the Northern Regional Research Laboratory on the production of fungal amylase in submerged culture. They have found, contrary to previous results, that if the pH was not allowed to drop below 4.0, calcium carbonate was not needed in the medium. A pH lower than this figure markedly curtailed the production of a-amylase, whereas the effect on maltase production was not so great. By using the proper balance of corn and distiller's thin stillage, enzyme yields were substantially increased. Gates and Kneen (98) reported a selective fermentation technique for evaluating the maltase activity of mold brans. Some mold brans had low amylase activity and high maltase activity. However, molds of high amylase activity did not always have high malthe activity. Blackwood and McCoy (34) described the properties of crude amylase preparations from several Bacillus and Clostridium species. Amylases from aerobic cultures gave high dextrinizing, liquefying, and saccharifying powers over a wide range of p H with optima in the range of 6.2 to 6.6. Anaerobic cultures, with optima in the range of pH 5.0 to 6.0,gave high activities over narrower pH limits. I n general, the starch conversion characteristics of amylase preparations from Bacillus subtilis and Clostridium species were similar whereas those from Bacillus polymyxa h d a higher saccharogenic activity. Rao and Sreenivasaya (.%XI) reported the effect of nitrogen sources on the formation of diastatic enzymes by microorganisms. ACETONE AND BUTYL ALCOHOL
The total United States production figures for n-butyl alcohol and acetone for the past 3 years are given in the following table. Production of Butyl Alcohol and Acetone (315) Thousands of Pounds
Butyl alcohol Produced by fermentation Acetone Total Fermentation % by fermentation
1947
1948
1949
140,000
140,200
120,300
397,200 40,000 10
470,600 26,900
5.8
413,100
Not available Not available
Tsuchiya et al. (292) described a process for producing butyl alcohol, acetone, and ethyl alcohol using crude pentoses as the principal source9 of carbohydrate. The process consisted essentially of preparing a mash containing crude pentose sugars which were derived by the acid hydrolysis of corncobs, oat hulls, bagasse, or flax shives. The acid hydrolyzate was neutralized with lime t o p H 6.0 to 7.0 at a temperature of 270Oto 280" F. Finely divided iron was added to remove or inactivate copper; the mash was then fortified with essential nutrients, such as ammonium sulfate, ammonium phosphate, and corn steep liquor, and was fermented with a strain of acetone-butyl alcohol bacterium. The chief advantage of the process was that the hot lime neutralization
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INDUSTRIAL AND ENGINEERING CHEMISTRY
plus the iron treatment, both of which removed toxic materials, permitted the pentoses in the hydrolyzate to be fermented satisfactorily. Weizmann (817)claimed that molasses was improved as a raw material for the acetonebutyl alcohol fermentation if it was given a pretreatment consisting of dialysis through parchment or collodion. In another process Weizmann (316) claimed the molasses-butyl alcohol fermentation was more rapid and gave higher yields if the fermentation wm started with a quantity of rice bran having fermentable carbohydrates equal to 0.2 to 2.0 times the sugar content of the molasses to be fermented. Yota et al. (333) described a process which utilized the spent liquor from an original acetone-butyl alcohol fermentation. Ammonia was added to the spent liquors to neutralize the organic acids, followed by the addition of a nonnitrogenous carbohydrate such as dried sweet potatoes. The chief advantage claimed was the saving of nutrient. Ono (195) claimed that the addition of guano to a sucrosesoybean mash ensured the success of the acetone-butyl alcohol fermentation and gave a rapid fermentation with desirable solvent ratio. Reizmann (318, 319) claimed that the addition of 0.001 to 0.1 part of paminophenylalanine or 0.1 to 0.2 part of p-aminobenzoic acid per 1000 parts of fermentable carbohydrate gave markedly increased yields of acetone and butyl alcohol. Gavronsky (99)described a method for conducting the acetonebutyl alcohol fermentation which did not require preliminary sterilization of the mash, A sterile concentrated carbohydrate mash was introduced to an intermediate vessel, inoculated with the acetone-butyl alcohol bacterium and allowed to fei-ment for a period of at least 1 hour. Then further increments of mash were added to the intermediate vessel. After sufficient inoculum had been obtained, it was transferred to the final fermentor which was gradually filled with nonsterile mash as the fermentation proceeded. The large quantities of inoculum caused the final fermentation to proceed so rapidly that bacteriophage and other contaminants did not have time to hinder the fermentation. Yasuda (331) described an interesting method for treating acetone-butyl alcohol bacteria. Although the details of the technique and results obtained are not available to the reviewer, the process consisted of: (1) heating the culture to 100" C. to destroy vegetative cells; (2) preserving the remaining spores a t -10' C; (3) treating the spores in vacuo by using 50,000 volts and 0.002 ampere of direct current; and (4) transferring the culture to sterile media in a fermentor. This fermentor contained a watertight glass tube filled with luminous silica gel through which high voltage direct current was passed. P,B-BUTANEDIOL
Currently there is little work being done on the 2,a-butanediol fermentation as the process is not being operated commercially in this country. The technologyof the fermentation, as discussed in revious reviews (69, 111,143, 856, 995) has been developed suflcientl so that if a large scale use for 2,3-butanediol is developed, it coudbe produced in large uantities a t low cost. Blackwood et al. (35)descr?bed the construction and operation of a pilot plant in which whole wheat mashes were successfully fermented by Aerobacillus polymyxa. A yield of 8.9 pounds of I-2,3-butanediol and 5.9 pounds of ethyl alcohol per bushel of wheat was obtained. This represents a fermentation efficiency of 90%. A 15% mash containing calcium carbonate was fermented a t 32' C. The use of agitation and reduced pressure increased fermentation efficiencies but reduced the ratio of 2,3-butanediol to ethyl alcohol in the finished fermentation. The fermentation was highly susceptible to contamination. Neish (189), using partition chromatography, studied t.he acids produced in the 2,a-butanediol fermentation. Tomkins, Scott, and Simpson (289) discussed pilot plant studies on the fermentation and recovery of 2,3-butanediol. They used Aerobacillus polyrnyxa to ferment barley mashes. Under laboratory conditions a 12.5% mash concentration was optimal although a higher concentration appeared acceptable in the pilot plant. Based on 1947 prices, the authors estimated a cost for 2-
Vol. 42, No. 9
2,3-butanediol of approximately 20 atid 18 cents per pound from wheat and barley, respectively. CITRIC ACID
Previous reviews (66, 130, 143, 856, 801) have covered the developments in the citric acid fermentation up to approximately 1949. Recent publications continue the emphasis on the development of submerged fermentation techniques for producing this acid and give special attention to the effect of trace mineral elements. Although actual production figures are not available, it is estimated that the annual production of citric acid in the United States is approximately 30,000,000 pounds. Recently, several patents (858, 968, 35'9) have been granted to the Miles Laboratories relating to various aspects of the submerged production of citric acid. Schweiger and Snell (869)d e scribed several variations of a submerged production technique. An example, showing the essential steps of their process, is as follows: the medium consisted of ammonium carbonate, monobasic potassium phosphate, magnesium sulfate heptahydrate, decationized raw sugar, decationized well water, and 500 p.p.m. of morpholine. The mash, with pH adjusted with hydrochloric acid to 2.5, was sterilized for 20 minutes a t 10 pounds per square inch gage steam pressure, and was then fermented with a special strain of A. niger. The fermentation was carried out in a glass column having an inside diameter of 3 inches and length of 48 inches. Sterile air was used for both aeration and agitation. After a fermentation period of approximately 9 days at 30" to 32' C., the citric acid yield, based on initial sugar in the medium, was 79.9%. The morpholine in the medium was claimed: to increase the efficiency of the fermentation; to stimulate spore germination and thereby give a faster rate of citric acid production in the early stages; and to have a stabilizing effect on the physiological characteristics of the fungus, thereby permitting better replication of results. Woodward et aE. (389) described a process for conditioning invert molasses used in the citric acid fermentation by A . niger; this consisted of diluting the molasses with 3 volumes of water, then decationizing the molasses by twice passing it through a cation-exchange resin.operating on the H-cycle. By this process the iron content of the molasses was reduced to within the range of 2 to 4 p.p.m. After the removal of iron, which was accomplished largely in the first decationization, sufficient zinc was added so that 10 to 30 parts of the zinc were present for each part of iron in the medium. The total zinc concentration did not exceed 150 p.p.m. By this process, citric acid yields of 75 to 77%, based on the total sugar in the medium, were obtained in 5 to 8 days with a final citric acid concentration in this medium of 15 to 18%. Snell and Schweiger (268) described a process for producing citric acid with a strain of A. niger in which the cellular morphology of the organism was controlled during fermentation by means of initial adjustment of the nutrient balance. Sufficient nitrogen, in the form of ammonium carbonate, was added for cell synthesis and the iron concentration was maintained below 1.0 p.p.m. These conditions induced a fungus cell structure characterized by : Abnormally short, stubby, forked, bulbous mycelium Numerous swollen, oval- to s herical-shaped cells well distributed throughout the mycelia? structure Mycelial structures all showing granulation and numerous vacuoles or refractile bodies Absence of normal reproductive bodies (vesicles or sterigmata) Formation of compact colonies less than 0.5 mm. across and averaging 0.1 mm.
It was claimed that cornstarch hydrolyzate was a suitable carbohydrate source. Perlman and Woodruff (808) discussed various effects of iron and other elements in the nutrition of A . m g e r in citric acid fer-
September 1950
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
mentation. Gluconic acid was reported as a by-product of certain strains of this fungus. Tomlinson et al. (890) discussed the effect of zinc, iron, copper, and manganese on the production of citric acid by A . niger in surface cultures. Zinc and iron were found to be necessary components of a sucrose-salts medium for maximum citric acid production when the fungus was grown from conidia in surface culture. Small quantities of manganese and copper were also essential, but all of t h e e elements were detrimental in excessive quantities. The quantities of iron, copper, and manganese needed for acceptable growth were higher than needed for citric acid formation. Gluconic acid waa reported as a by-product of certain strains of this fungus. Foster (87) pointed out that the response to various tram elements will depend on the strain of the fungus. Martin and Wilson (IYO), who used carbon dioxide containing C14, reported that less than 10% of the carbon of the citric aoid formed during the fermentation came from assimilated carbon dioxide. This finding should be of value in connection with aeration studies in the submerged citric acid fermentation. A few reports in foreign journals have dealt with citric acid production in surface culture. Bernhauer et a2. ( 3 1 ) stated that the most favorable medium for the citric acid fermentation of beet molasses in surface culture contained 15% sugar and 0.02% potassium ferro- or ferricyanide at pH 5.8. Excess iron caused slow fermentation. No test could be found that would indicate the suitability of a given molasses for the fermentative production of citric acid. Garcia et al. (86)described a process for producing citric acid in which fresh bagasse was chopped into 0.5-inch pieces and impregnated with molasses containing nutrient salts. After seeding with mold spores, the mass was spread on wire pans in a 2-inch layer. The authors claimed acceptable yields in a 40-hour fermentation. Erkama et al. ( 7 6 ) reported that intense aeration of a surface culture of A . niger nearly trebled the oxalic acid formation while decreasing the citric acid production to one-fourth. The authors discussed the significance of the Wood-Werkman reaction and stated that little citric acid was formed under conditions where no fixation of carbon dioxide occurred. Erkama and HItgerstrand (76)reported that, in surface cultures on a medium free of iron, normal citric acid yields could be obtained a t high oxidation-reduction potentials. OTHER ORGANIC ACIDS
Little work has been reported recently on the fermentative production of other organic acids (b6,14J1866). Lactic Acid. The total production of lactic acid in 1949 in the United States was approximately 8,000,000pounds. Needle and Aries (188) discussed the technical and economic aspects of the manufacture and use of lactic acid. These writers believe that, if lactio acid can be produced at a cost in the range of 8 t o 10 cents per pound, there is a potential annual market of about 200,000,000 pounds. The chief markets would be for the manufacture of alkyd, acrylic, and other resina. This low production cost for lactic acid is not impossible at the current cost of blackstrap molasses which is in the range of 5 to 7 cents per gallon. Narula and Chawla (187) reported a process for producing lactic acid in a molasses-calcium carbonate medium having a reducing sugar content above 40%. They used Lactoban'llua delbnickii in agitated culture at a temperature of 46' C. The lactic acid reached a concentration of 25% in the finished fermentation. Polti (%%I) reported the production of lactic acid by Lactobacillus sili in molasses solutious containing 10 to 12% sugar fortified with nitrogen and phosphorus salts and calcium carbonate. Yields of 90% were obtained in 8 to 10 days at a temperature of 40' C.
1675
Terui (,986) claimed a superior lactic acid fermentation of various carbohydrates if E. coli or A . aerogenes were added to the medium, thereby eliminating the necessity for adding inorganic salts. Kojic Acid. No further work on the kojic acid fermentation hrta been reported during the p a t year. AB pointed out previously (143)this compound could be produced readily on a large acale if industrial uma are developed. Campbell et al. (46) reported work on the preparation of several derivatives of kojic acid. Mehlig and Shepherd (173) reported the use of this acid in the determination of iron in ores. Itaconic Acid. Lockwood and Moyer (166) obtained s patent on the production of itaconic acid. They improved the yield by maintaining the aerated and agitated cultures of A . terreua at pH 1.4 to 2.8 by the use of nontoxic mineral acids or the ammonium salts of these acids. Several other patents on the production of itaconic acid have been issued recently (7,818, ,938). Ambler et al. ( 7 ) and Roberta et al. (&%) reported methods for producing itaconic acid by chemical rather than fermentative processes. Nelson et a2. (NO), using A . tmreus in %liter stainless steel fermentors, reported itaconic acid yields, based on weight of glucose supplied, that exceeded 50%. The initial sugar concentration was 0% in a corn-steep liquor medium in which the pH during the fermentation was maintained a t 1.8 to 1.9. Other conditions coneidered optimal were: (1) aeration at the rate of one thirtieth volume of air per volume of medium per minute; (2) 1% inoculum; (3) a constant supply of available nitrogen in the form of ammonium sulfate; (4) the use of sulfuric rather than nitric acid for pH adjustments; and ( 5 )the use of 0.5% magnesium sulfate heptahydrate. Miscellaneous Acids. Koepsell and Stodola (166) reported the oxidation of gluconic acid to 2-ketogluconic, a-ketoglutaric, pyruvic, and succinic acids by Pseudomonasfluorescene. Lockwood (164)obtained a patent on the production of pentoic acids from pentoses using several species of Psewlomonas. Weismann ( 6 0 )described a bacterial process for producing acetic, propionic and butyric acids using cultures isolated from roots of beets and carrots. A mold fermentation process has been described (116) in which the mycelium was grown submerged adhering to pieces of limestone. Rhizo us japonicus, in a medium containing 10% glucose, gave 60% yiefds of fumaric acid in 4 days a t 30' C. Lockwood and Stodola (166)described a rocess for preparing bionic acids, such as maltobionic and lacto!ionic acids, dwectly from the respective disaccharides without first hydrolyzing the disaccharide. This was accomplished by growing various Pseudomonas species under 30 ounds air pressure and at a temperature of 25 C. in an aerateimedium containing the carbohydrate, urea, corn-steep liquor, and salts. MICROBIOLOGICAL PRODUCTION OF VITAMINS
The production of vitamins and vitamin-rich food and feed products by microorganisms is assuming ever-increasing prominence in the fermentation industry. Those processes of greatest economic importance are the production of: Riboflavin b bacteria and yeasts Vitamin BI*Ey several bacteria and actinomycetes Erogosterol, the precursor of Vitamin D, by yeasts and potentially, a t least, by several molds Vitamin B' (thiamine) in the form thiamine-rich yeast Both the academic and industrial aspects of vitamin synthesis by microorganism have been recently reviewed (138, 146,899). Riboflavin. Riboflavin, both in the pure form and as riboflavin-rich concentrates, is being used in increasing quantities in the United States for the fortification of foods and feeds. The primary food use is in the fortification of white flour, but in the feed industry its primary use is in poultry rations. Because of their content of other growth factors and vitamins, riboflavin concentrates produced by fermentation appear to have some advantage over the synthetic material for aniinal feeds.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
During the past year several additional patents have been issued covering the fermentative production of riboflavin by Eremothecium ashbyii. This is the chief organism now being used for large scale production of riboflavin. A British patent specification (69) covers a variety of raw materials suitable for the production of riboflavin by E . ashbyii. Essential ingredients of the media were: proteinaceous materials such as distillers’ solubles, skim milk, whey, corn-steep water, cereal grains, seeds of leguminous plants, peptone, egg albumin, tankage, fish meal, and meat scraps; carbohydrates such as glucose, sucrose, and mannose; and lecithin or lipides from vegetable oils. The prescribed fermentation conditions were as follows: . a pH range of 4.5 to 9.0; aeration and agitation during the fermentation period; a temperature within the range of 23‘ to 34” C.; and a fermentation period of 50 to 90 hours. The final “beer” was heated to 60” to 120” C. for approximately 1 hour to liberate the riboflavin from the fungal cells; then the riboflavin was extracted and purified. Stiles obtained a United States patent (273) covering essentially the same process. The yields reported were 500 y riboflavin per ml. in the final ferment. Martin (169) was granted a patent for the production of riboflavin by E. ashbyii in which a porous solid material such as oat hulls, corn bran, or wheat bran was impregnated with an aqeuous nutrient medium. The inoculated mash was incubated with continuous aeration and agitation. Phelps (214, 215) obtained two patents on processes for producing riboflavin with E. ashbyii. One process (214) consisted essentially of the use of casein, malt extract, and sugar whereas the other process (216) was based on the use of casein, malt extract, sugar, and the glyceride of a fatty acid. Yields as high as 1295 y/mL were claimed. Tabenkin (281) described a process for producing riboflavin with E. ashbyii in a synthetic medium containing assimilable dorms of carbon such as sucrose, phosphorus, sulfur, zinc, potassium, and ammonium nitrogen. Riboflavin yields greater than 700 y per ml. were claimed. In another patent (239), when the medium consisted essentially of lentils, sucrose and ammonium salts of organic acids, E. ashbyii yielded 2480 y per ml. during a 5 to 7 day fermentation. In this agitated and aerated submerged fermentation, the pH was controlled by adding increments of carbohydrates such as glucose or molasses. Microbiological and chemical processes for recovering riboflavin from E. ashbyii fermentations were described in two British patents (67, 58). Leviton (149) described a process for improving the riboflavin yields in the acetone-butyl alcohol fermentation with Clostridium acetobutylicum. This process consisted of adding to the fermentation, catalase or yeast which, it was claimed, permitted the presence of higher concentrations of iron in the fermentation medium without effecton the production of riboflavin. Leviton suggested that hydrogen peroxide was decomposed by such additives and that the destructive effect of hydrogen peroxide and ferrous iron on riboflavin was thereby overcome. Further work has been reported on the production of riboflavin by other yeasts. Levine et aE. (148) reported that on a simple synthetic medium Cana’ida guilliermondia and Candida fEareri yielded 175 y per ml. and 567 y per ml., respectively. Concentrates having as high as 97,000 y per gram of solids were prepared from a liquid medium fermented with Candida jlareri. The function and effects of various mineral elements and vitamins in the synthetic medium were discussed. Tanner et al. (283) reported riboflavin yields of 500 to 600 y per ml. when Ashbya gossypii was grown in media containing materials of animal origin, such as stick liquor, tankage, or meat scraps together with carbohydrate sources such as glucose, sucrose, or maltose. Maximal yields were obtained when using small quantities of young inocula, efficient aeration, and minimal sterilization time.
Vol. 42, No. 9
I n order to obtain engineering data for the construction of a large plant and to estimate production costs, Pfeifer et al. (212) studied the production of riboflavin by A . gossypii in a large pilot plant operation. They reported that a t broth potencies of 500 to 600 y per nil., a dried product with a vitamin content of 2.5% could be produced at a cost of 3.75 cents per gram of riboflavin. Pridham and Raper (a26) reported that riboflavin yields of 1200 y per ml. were obtained regularly with A. gossypii. Under certain controlled conditions, where glucose was fed to the fermenting cultures, peak yields of 1760 y per ml. were obtained. Vitamin B12 and Atlimal Protein Factor. As indicated previously (143) the production o f Blz and BIz-like compounds, in crystalline or concentrate forms and in animal protein factor (APF) feed supplements, is rapidly becoming a major fermentation industry. This development has been stimulated by the potentially large markets for vitamin Biz in human nutrition, especially for the cure of Addisonian pernicious anemia (258, 264, 321) and for the normal growth of children (523). The APF roducts have great possibilities as supplements in animal feeds 106, 161, 196) particularly to increase growth and egg hnt(a1lability in poultry (191, 2U4) and in general to replace expensive animal proteins. At the present time the evidence indicates that vitamin B12and APF are closely related. This vitamin may be present in such , and Btnb (216) in animal protein :tnd modified forms as B I ~(152) also in fermentation broths. Full biological activity of these forms ma be attained if they can be converted to vitamin BL2. This wou& aid markedly in explaining the current discrepancies between microbiological and animal growth assays for the presence of vitamin Bl2 and APF in many materials. Vitamin BIZand APF can be synthesized by many microorganisms. The streptomycetes are excellent producers and Streptomyces griseus and aureofaciens, used for the streptomycin and aureomycin fermentations, respectively, are probably being used as sources of pure B1*and APF (255, 274, 276). Stokstad et al. (976) reported a nonmotile rod-shaped microorganism isolated from hen feces which produces APF as well as an anti ernicious anemis factor. This process was further described by j e t t y and Matrishin (81U),and one organism so isolated was identified as Flavobacterium solare. Ansbacher and Hill ( 1 4 ) referred to the fermentative production of APF, and McGinnis (164) stated that a number of BIZproducin organisms have been isolated from soil. Garibafdi et al. (97) reported a process for producing B12with Bacillus megatherium in a medium which contained sucrose and inorganic salts. Ten per cent sugar waa utilized in a 12-hour fermentation. The yield of B I was ~ 0.8 mg. per liter. A large fermentation company has announced tonnage production of APF as a primary fermentation product (62). The development of industrial fermentation processes for producing BI2 and APF would be aided if a microbiological assay were available which would correlate with chick assays. Many investigatora have recently reported microbiological assay procedures for these materials (47, 60,56, 90,I.%?, IUS, 326).
P
MICROORGANISMS FOR FOOD AND FEED
The use of microorganisms as food and feed is gaining innre attention throughout the world (300,332). Yeasts are a piiint: example. They are grown under special conditions for baking purposes; they are recovered from brewing operations and debittered for food and feed; and they are grown on a variety of waste products from industrial processes. About 15,000 tons of dry yeast are produced annually and if the price were competitive with other feed supplements, i t has been estimated that several hundred thousand tons could be consumed annually by the calf and poultry feed industry. There is little doubt that yeast and other microorganisms are far more efficient converters of plant materials into nutritious human food than any of the animals which now compose guch a large portion of the human diet. Unacceptable flavor and texture have, in the past, limited the use of yeast as food for humans. The use of microorganisms in foods to improve flavor-for example, the milk cheeses of Europe and the soybean products of Asia-is an ancient practice. However, direct use of microbial cells as food has gained favor only during the last 30 to 40 years. Yin (332) in his discussion of “Microbial Farming” cited sev-
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era1 advantages of the acreage-scale growth of microorganisms for food. These were as follows: The quantity and quality of nutrients in our usual foods can be fully supplied through the growth of microorganisms. Microbes are more efficient than higher animals in converting material and energy into human food. They require less space and labor than other forms of plant and animal life to produce the =me quantity of food. They are capable of fast growth and quick turnover. They thrive readily in an artificial environment and therefore are more suited to human control. Recently the yeasts as well as other forms of microorganisms such as algae snd mushroom fungi have been studied and proposed for large scale production for special food purposes, Yeast. White (394) has reviewed several aspects of yeast production. !l'orcdopsis utilis and numerous other yeasts can be grown to give a food having approximately 45% protein (838). Fermentor Area of Microbiological Pilot Plant, Commercial Solvents, Terre Haute, Ind Endomycea vernalis and Rhodotorula g r a d i s have been grown under commercially feasible conditions to contain as high as 00% fat (332), with a yield of fat equal to 16 pounds per Agarwal and Peterson (6) reported that the nonsugar carbon 100 pounds of sugar consumed. of cane and beet molasses was utilized during the growth of In this country, yeasts are being produced on E large soale many food yeasts. It was stated that 7.4 to 28.2% of the nonprimarily for baking purposes. Recently, variow types of sugar carbon was utilized by Saccharomyces cerevisiae, Torulopsic Torula have been grown, chiefly in continuous operations, on ulilis, and Candida arboreu. T. utilis utilized nonsugar carbon, molasses, fruit wastes, sulfite liquor wastes, and hydrolyzed wood other than alcohol, in the presence of sugar. The other yeasts sugars for direct use as supplements in animal feeds (19). An used only sugar so long as it waa present in the medium. extensive study has been made of the economics of the production Chang and Peterson (61)reported a study of the factors affectof fodder yeast from sulfite liquor ( 2 @ ) . ing the biotin content of yeasts grown in beet and cane molasses A patent has been issued which describes the growth of yeast media. In fermentations which gave yeast yields, based on sugar diimtly on particles of cereal grains coated with yeast wort fermented, of 50% or greater and in which 90% of the sugar in the (127). medium was fermented, the biotin content of 22 yeasts ranged Fk,wmqvist (249) described a process for producing Torula from 0.23to 5.27 y per gram in Mason City molasses medium yeast in which still residues, resulting from the alcoholic fermentaIn Hawaiian molasses medium the biotin content ranged from tion of sulfite waste liquor, were fortified with molasses. In this 1.1 to 7.6 y per gram. A strain of S. cerevisiae,.which required process, the pentoses remaining in the still residues were utilized biotin, absorbed as much as 214 y in synthetic media. However, for yeast propagation. biotin uptake could not be correlated with biotin requirementa. Hanson et al. (104) have obtained a patent for producing yeast Other Microorqanisrns. There are several recent re o r b and in a whey medium. Increased yields were obtained when the proposals concernmg the uses of microorganisms other t t a n yeast whey was sterilized for short periods of time a t low temperatures as source8 of food and feed. and a pH of 1.5 to 3.5, by adjustment to pH 5.0 to 8.0 before Yin (839)stated that Chlorella vulgaris, a unicellular green a1 a, inoculation. could be grown in mass culture as a source of carbohydrate for humans. Yin calculated that in 8 days an acre of landfyowing During recent years several investigators have described this organism would roduce approximately 4 tons of ry maapparatus and aonditions for the continuous production of yeast terial, one half of whic! would be carbohydrate. The comparable (149, 266). Stier et al. (972)described a glass apparatus for the figure for corn would be 1 to 1.5 tons in a growing period of 90 continuous cultivation of yeast under anaerobic conditions. Pure days. On this bask, Yin claimed the alga would be 25 to 30 times as roductive 88 corn. nitrogen was used for maintaining anaerobic conditions and for another calculation (63) it was estimated that continuous transferring fresh medium to the fermentation vessel. With this tank cultures of algae would give 130 times the protein yield obequipment, it was possible to produce, under anaerobic conditions, tainable from soybeans. successive crops of yeaata exhibiting stable fermentative charVinson (300) proposed the symbiotic growth of algae and yeasts thereby usin the algae to convert the sun's energy into acteristics. carbohydrate and &e yeash to convert the carbohydrate into Hatch and Hammond (107)described an apparatus for growing a nutritious food prpduct. yeast and other aerobic organisms. This apparatus had an agitaThe most extenslve summary of the possibilities of large scale tor placed a t the bottom of an inner chamber, within the fergrowth of algae was presented by Meier (176)who discussed several aspects of mass production of Chlorella. He suggested mentor proper, which was driven by a motor connected to a shaft that the process may be usable in the near future in highly entering through the bottom of the fermentor. This equipment lated areas of the world such as Israel &ndJapan, where had the ability of simultaneously agitating the liquid and dispersland is at a premum. Ifutchens (Ibf) &scussed factors favorable ing air into a central portion of the medium, thereby supplying to the mass culture of Chilomonas paramecium. Bernhauer and co-workers (99,30) discussed the effect of osvgen for the growth of aerobic organisms.
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numerous factors on the synthesis of protein and fat by various molds in submerged culture. Recently Humfeld (119) and Humfeld and Sugihara (190) demonstrated the production of mushroom mycelium in submerged fermentation. Using various strains of Arguricus campestris, the chief mushroom produced and sold in this count they were able to produce mycelium which gave excellent mux: mom flavor. At the present time, the product of this process shows good commercial prospects for use where mushroom flavor is desired in soups, sauces, and the like. The mycelium was reported to have good nutritive properties. ANTIBIOTICS
During the past year the production of antibiotics has continued to rise rapidly. In addition to the increased production of the older commercial antibiotics, penicillin and streptomycin, newer antibiotics such as aureomycin, bacitracin, and chloromycetin are now being produced in substantial quantities. The figures for the production of penicillin and streptomycin in this country are available. Production of Penicillin end Streptomycin (375) Penicillin Billion units' Grsma Pounds
1947
1948
1949
41.426 24,856,000 55,000
95,855 57,513,000 126,700
133,464 80,078,000 176,400
Streptomycin Billion units Grams" Pounds
9,700 9,675,919 21,000
37,700 37,709,180 83,000
83,700 83,699,137 184.200
a Figures supplied by U. 6.Department of Commerce: conversion fiprea were derived by assuming 0.6 y per unit for penicillin and 1.0 y per unit for streptomycin.
Penicillin production in this country is becoming extremely sompetitive as evidenced by the marked reduction in sale price of this antibiotic. During the past year, the manufacturerJs price for penicillin dropped to approximately 4.0 cents per 100,000 units in bulk quantities. It is obvious that only those f i m s which have ample technical staffs to improve their processes continuously will survive profitably in the penicillin business. Recent developments in antibiotics have been the subject of several reviews (40, 64, 148, 956, 802). Fortune et al. (86) described a pilot plant for the development of antibiotics. Rivett et al. (9%) described a versatile laboratory fermentor for fermentation studies. Lee and McDaniel (144) discussed some fermentation aspects of the large scale production of antibiotics. Penicillin. Although the price of penicillin has decreased markedly during 1949, the large increase in production has maintained i t in a top position in terms of total value of product produced. It is believed that a large share of the increased production during the past year was due primarily to increases in efficiency of operation by existing producers, rather than to increases in size or number of penicillin plants. Another point of interest is the fact that several United States firma are aiding foreign concerns in the production of penicillin. It is fully expected that, due to reduction of exports, foreign output will eventually limit the production of penicillin in this country. Further increases in penicillin production efficiency will most likely accrue from improved strains of Penicillium chrysogenum, superior methods for inoculum development, and improved fermentation conditions. Reese et al. (%?,!?)reported mutants of P . chrysogenum which gave 50% higher penicillin yields than the parent strain Q-176. Ultraviolet irradiation gave rise to a greater number of mutants than N-mustard treatment. The authors claimed that strain Q-176 of Backus, Stauffer, and Johnson ( 1 7 ) retained its penicillin producing capacities even when repeatedly subcultured on agar media.
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Vander Brook and Savage (298) described a process for using a Waring blendor to fragment the mycelium of P. chrysogenum. The inventor claimed that in their process a lesser quantity of inoculum was needed. Using a staining procedure involving 2,3,5-triphenyltetrazolium chloride, Fred and Knight (9,!?)determined the viability and age of penicillin-producing cultures. Jarvis and Johnson (126) discussed the mineral nutrition of P . chrysogenum Q-176. The iron, phosphorus, and sulfur requirements for penicillin production were, respectivclj, 20, 2, and 1.5 times higher than needed for normal growth. Magnesium requirements were the same for both growth and penicillin production. Potassium deficiencies increased and iron deficiencies decreased the fermentation period. The presence of chloride in the inoculum w a ~detrimental to subsequent penicillin production. There are several recent reports claiming improved media and fermentation conditions for the production of penicillin. Some of these reports, along with several patents, have been published previously and will not be discussed in detail. Moyer (183)stated the types and quantities of carbon and protein sources desirable for penicillin production. Another patent (186)claimed superior penicillin production from corn-steep liquor, which had been adjusted to pH 3.0 to 3.7, 1 week after the liquor was drawn from the steep tanks. Moyer (189) described a semicontinuous process for penicillin production. Calam and Hockenhull (43)discussed the nutrition of P . notatum grown on the surface of synthetic media. Perlman (905) reported a number of materials which were able to replace corn-steep liquor for penicillin production. Such materials were cottonseed meal, linseed-oil meal, coconut-oil meal, sardine meal, peanutsil meal, mustard flour, and soybean-oil meal. Moyer (181 ) reported that polysaccharides were advantageous in penicillin production as they aided in maintaining rapid prcduction for a longer period of time. Lactose, starch, raffinose, melibiose, sucrose, and insulin were examples of acceptable polysaccharides. Hockenhull (114) discussed the role of sulfur in penicillin production. Brown and Peterson (41) described the results of a series of studies on the production of penicillin by P . chrysogenum Q-176. They used two types of 30-liter fermentors: a standard type having agitator, aerator, and automatic pH control equipment and a Waldhof-type fitted with agitator, aerator, and the central draft tube. Careful studies of aeration, agitation, pH, medium composition, precursor addition, and antifoam agents resulted in yields of 1900 units per ml. in the standard fermentor and 2100 units per ml. in the Waldhof-type fermentor. The penicillin produced in these fermentors was over 97% G. These are the highest penicillin yields reported in laboratory fermentors. Behrens ($4) recently reviewed the subject of precursors in the penicillin fermentation. Several additional patents concerning precursors have been issued during the past year. One patent (26) listed several groups of chemical compounds which could be used for producing high yields of various types of penicillins. Another patent (26)claimed the use of phenacetyl amino acid esters as penicillin precursors. Cartland et al. (49) claimed an improved process for producing penicillin X in which parahydroxyphenylacetic acid is used as the precursor. Smith et al. (960) listed a series of penicillin precursors which gave acceptable yields in synthetic media. Streptomycin. The production of streptomycin in the United States in 1949, approximately 84,000,000 grams, was more than twice that produced in 1948. I t seems probable that, due to the increased production facilities in foreign countries, the 1950 production will not show a similar increase. Streptomycin producers in this country, like the manufacturers of penicillin, are aiding foreign companies with their production problem. However, if new uses for streptomycin are developed, the production in 1950 may 8hOW an increase comparable with that in 1949.
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I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
It appears that, in streptomycin production as in penicillin, future increases in production efficiency will depend largely on the development of higher yielding strains, superior production media, and generally improved fermentation conditions. It should be pointed out that during 1949, dihydrostreptomycin was the chief form marketed. It is claimed that this form, which is produced by the catalytic reduction of streptomycin, has a lower toxicity and gives less pain on injection than streptomycin. The development of higher yielding strains of Streptomyces griseus has been accomplished by methods identical with those used for Penicillium chrysogenum. Savage (848)reported yields greater than loo0 7 per ml. from mutant strains of Waksman's original culture of S. griseus. However, few of these new strains have proved sufficiently stable for use in production. Savage reported that less than 0.1% of these high yielding mutants had acceptable stability, and that the teat media had a marked effect on the results obtained. Dulaney and co-workers (79)reported ultraviolet and (CICH2CHn)3N-induced mutants of S. griseus which gave more than three times the yield of the parent strain. Various characteristics of mutant colonies were described, none of which could be correlated completely with streptomycin production. Wakman and Harris (806)reported on the streptomycin-producing capacity of various strains of S. griseus. Studies of medium constituents preferable for Streptomycin production have been reported by Dulaney (71). Pentoses were found to be poor, maltose was the best of the disaccharides, starch and dextrin were ,acceptable, and glucose and mannose were the most satisfactory. Mannitol was acceptable, and none of the organic acids supported high streptomycin yields. A medium containing mannose and Gproline gave yields of 900 y per ml. High yields of streptomycin have been obtained using media containing a soybean material such as low fat soybean flour (16). A synthetic medium for the production of streptomycin has been described (37). It has been found that when the final ferment WM acidified to pH 1.5 to 2.0 and held for a time at 45" to 65' C., considerable streptomycin was liberated (266). Streptomycin B, mannosidostreptomycin, is produced in appreciable quantities in most streptomycin fermentations. A method (141) has been described for converting this compound, or the dihydro derivative of this compound, into the more active forms of the antibiotic-namely, streptomycin and dihydrostreptomycin. These conversions were accomplished by adding the mannosido derivative to growing cultures or cell-free extracts of Streptomyces griseus. The ability of the microorganisms to bring about these conversions suggests the possibility that the mannosidostreptomycin may be an intermediate in the formation of streptomycin. Another incidence of actinophage has been reported (8.40) along with a discussion of the properties of the phage and its effect un streptomycin produotion. Mehnert (174)has described the instrumentation used in a large streptomycin production plant to control such vital factors aa temperature, pressure, and aeration. Chloromycetin. During the past year, chloromycetin (chloramphenicol) has firmlj established itself as a valuable antibiotic. Because it is produced entirely by one company, the total production figures are not available. However, it is being produced in appreciable quantities and is generally available to the medical profession where i t is the drug of choice for the treatment of certain diseases. Reports covering the discovery, production, assay, and purification Procedures and the potential usefulness of chloromycetin in the treatment of disease have been included in a previow review (143). Ehrlich and co-workers (78)were granted a patent for the production of chloromycetin by Streptontycea venemelae. The medium of choice contained glycerol, peptone, dried distillers'
1679
solubles, and sodium chloride. In 70 to 80 hours a t a tempere ture of 24' to 25" C., yields above 130 y per ml. were obtained in submerged aerated and agitated fermentations. Bartz (23)reported further details of the fermentation conditions for producing chloromycetin and also described the method for extracting the antibiotic. Oyaas, Ehrlich, and Smith (199)have discussed biochemical changes which occur during the chloromycetin fermentation. Japanese workers have described a strain of Streptomyces which produced an antibiotic identical with chloromycetin (294). The large scale fermentation and purification techniques used by Parke, Davis & Co. for the production of chloromycetin have been described in detail by Olive (193) and by Mohrhoff and Mogerman (179). Chloromycetin is the first antibiotic which has been produced economically on a large scale by chemical synthesis. At the present time, both chemical and biological production processes are being used (198). The structure and synthesis of chloromycetin have been described (60,167,168, 231). Aureomycin. Aureomycin, an antibiotic produced by Streptcmyces aureofaciem, has gained full status during the past 2 years as a valuable agent for the treatment of many diseases. Rose and Kneeland (941) after reviewing the literature on aureomycin, concluded that this agent approximates the aggregate effects of sulfonamides, penicillin, and streptomycin, and hag, in addition, antirickettsial and antiviral properties. It should be pointed out, however, that it is the drug of choice in only a few of the diseases for which it is effective. It is especially valuable because of its activity against some microorganisms which are resistant to penicillin and other antibiotics (900). Because it i s being produced by one firm only, Lederle Laboratories, Incorporated, the United States Department of Commerce has not released the total production figures. The quantity being produced is appreciable and it is available for general distribution to the medical profession. A previous review (143) covered the composition of production media, assay procedures, chemical properties, and the diseases in which the drug is effective. Lesser (147) has reviewed the aureomycin literature. A recent patent issued t o Duggar (70) gave further details of the production and properties of aureomycin. 8. aureofaciens was grown in media containing carbon sources such as starches, sucrose, glucose, or maltose; proteinaceous materials such as corn-steep liquor, casein, fish meal, soybean meal, meat extracts, or liver cake. Other nitrogen sources which were acceptable included amino acids, urea, nitrates, and ammonium compounds. The fermentation was carried out at a temperature of 26" to 28" C., at a pH of 0.0 to 7.0,and the fermentation period was 24 to 48 hours. No fermentation yields were given. Bacitracin. Bacitracin, an antibiotic produced by Bacillus subtilis Tracy, hss become a prominent antibiotic, especially for topical application in the treatment of many bacterial infections. Total production figures are not available from the United States Department of Commerce as it is made chiefly by one manufacturer. The volume of production is appreciable, and it is available for general use by the medical profession. Submerged fermentation processes have replaced the eurface culture method first used for the production of this drug. Production media of choice contain various plant protein materials such as soybean med. Dextrose, sucrose, and starch are suitable carbohydrate sources. Titers of more than 100 units per ml. have been reported (168)in such media, in fermentation periods of 36 to 48 hours. Bacitracin can be extracted by n-butylalcohol (11, 188, 168) and purified by fractional precipitation with ammonium sulfste (101). The amino acid composition of this polypeptide has been determined, The composition is significantly different from other and wlytxptide antibiotics such as gramicidin,. tyrocidine, gramicidin8 (82,69). A recent report showed the high efficiency of bacitracin in the
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
treatment of furuncles, carbuncles, chronic osteomyelitis impetigo, ulcers, sties, and infected operative wounds (176). &her us@ were in the treatment of nasal and pharyngeal infections. Nephrotoxicity, affecting mainly the tubular epithelium, limits the parenteral use of bacitracin to investigational studies, using certain lots having low mouse toxicity (177).
Neomycin. Waksman and Lechevalier (310) first reported this new antibiotic, which is produced by submerged cultivation of Streptomyces fradiae. Assays of approximately 240 units per ml. were obtained in media containing peptones, meat extract, glucose or starch, and sodium chloride (278, 311 ). Close control of the zinc content of the medium resulted in assays of 500 to 1000 units per ml. The inclusion of calcium carbonate was of some advantage. The most favorable pH range was from 7.0 to 8.0. The aeration requirement was somewhat less than that used in the production of streptomycin or streptothricin. Neomycin can be assayed by modifications of the agar-diffusion or cup methods (278) or by the agar-dilution method. A number of recovery methods have been investigated (278, 310), some of which are similar to methods employed for streptomycin. Neomycin has been described as an antibiotic complex ($02, 678). 8.fradiae forms an antifungal agent, fradicin (278, 279), in addition to the neomycin complex. Peck et al. (809) reported the isolation of neomycin A. The potency of the neomycin A hydrochloride was 1700 ‘‘neomycin units” per mg. The neomycin generally employed in clinical studies has been referred to either as neomycin or as neomycin B. The clinically-used material has not as yet been described as a pure compound. The sulfate of neomycin R has been prepared with potencies of approximately 220 units per mg. The antibiotic spectrum of neomycin is quite broad. Activity has been demonstrated against Gram-positive bacteria, including the acid-fast forms, and against Gram-negative bacteria and actinomyceten (304, S09). It is active against streptomycinresistant organisms (134, f 35, 304,306). Microorganisms do not as readily develop resistance to neomycin as to streptomycin (309, 811). Other favorable reports of studies in vivo have appeared (18,82, 113). Recently an antifungal property of neomycin was reported (45) in which activity was demonstrated against the causative agents of some deep mycoses, such as Histoplasma capsulatum. Some renal damage has been reported (134, lS5, 251) following extended parenteral neomycin treatment. Whether or not this toxicity is caused by an impurity or by neomycin itself is not yet known. Polymyxin. The polymyxins, a group of antibiotics produced by Bacillus polymyxa, were discovered simultaneously by two t e a m of workers in this country (27, 267) and by a group of English investigators (6),who referred to their material as aerosporin. The work on polymyxin has been reviewed briefly (148). Recently a series of papers appeared which resulted from a Wonference on Antibiotics Produced by Bacillus polymyxa” (160). Benedict and Stodola (28)studied the antibiotic production by 35 strains of B . potymyxa in various media and under different conditions. Soybean and peanut meal were found to be superior to yeast extract as protein sources and more suited economically for large scale production. Ultraviolet irradiation produced higher yielding mutants from one of four strains irradiated. Yields as high as 1290 units per ml. were reported. Porter et al. (221) reported further studies on the production and purification of polymyxins. Several polymyxins (A, B, C, D, and E) have been described from different strains of the organism. They are differentiated by their respective rates of movement on paper strip chromatographs and by their amino acid composition (131). All of the polymyxins are characterized by their action against a wide variety of Gram-negative bacteria. Long (159) stated that polymyxin was effective for treating severe infections caused by
Vol. 42, No. 9
Ps. aeruginosa, H . pertussis, H . influenzae, E. coli, and A. aerogenes, but that, to date, its renal toxicity precludes its use as a general therapeutic agent. This group of antibiotics has not, as yet, been produced on a large scale. It is possible that polympins may eventually be used in appreciable quantities for special uses where toxicity is not a major factor. Subtilin. The general conditions for the production of subtilin by Bacillus subtilis have been described (184). Media containing carbohydrate, organic, and inorganic nitrogen and mineral elements gave satisfactory production in surface (150)and submerged (277) laboratory fermentations. Good yields were obtained in a 200-liter submerged fermentation, utilizing media containing asparagus butt juice or beet molasses. The highest yield, 460 mg. per liter in an 11- to 1% hour fermentation period, w a produced in a medium containing sucrose, yeast extract, citrate, and mineral salta (96). The nutritional requirements of the subtilin-producing microorganism have been investigated extensively (78-80). Zinc was essential for growth and subtilin production (81). Microbiological aasay methods involving turbidimetric (161) and diffusion plate (116, 325) techniques have been reported. Methods for isolation and purification of subtilin are likewise available (84, 168). Also reported arc the chemical properties of subtilin and derivatives (48, 67). Subtilin has a broad antihiotic spectrum covering Gram-positive and acid-fast bacteria (845). It has also been reported to be effective against influenza A and Newcastle viruses (9&). No significant clinical studies have been reported. Of po&sible economic importance is the use of subtilin in food preservation. The combination of 10 p.p.m. of subtilin and a heating time of 10 minutes at 100” C. has been shown to protect canned peas, asparagus, corn, green beans, peeled tomatoes, tomato juice, and milk against microbial spoilage (8, 9). T rothricin. T rothricin wm the first antibiotic discovered in txis country w&ch has been used in the medical profession. This antibiotic was first reported by DuBos (68) as a result of his studies on the destruction of virulent pathogenic microorganisms in soil. The previous work on tyrothricin has been reviewed by Massey (171) and Rivoal (837). Tyrothricin, which is produced by an aerobic spore-forming organism, Bacillus brevis, has tu.0 components: gramicidin, which is active chiefly against Grampositive bacteria; and tyrocidine, which is less active a ainst Gram-positive bacteria, but is also active against some dramnegative pathogens. During the past year, ,patents have been issued covering various aspects of tyrothricin production. MitchelI (178) described fermentation conditions suitable for large production. Bacillus brevis was grown submerged in an agitated and aerated medium having as the chief component stillage residues from the ethyl alcohol fermentation of cereal grains. Three grams of tyrothricin per liter were obtained in a 6 to 8 day fermentation at a temperature of 30Oto 40 O C. Baron (19)described the production of tyrothricin in a synthetic medium containing mineral salts, sodium glutamate, and thiourea. Tyrothricin is effective especially where pneumococcal, streptococcal or staphylococcal organism are the infecting agents; but because of its high toxicity, including hemolytic effects, when applied either intradermally or intraveneously, its use is limited to surface applications. For this reason it has never been produced in large quantities in this country. Terramych. Terramycin, produced by an actinomycete designated as Streptomyces rimosus, is the most recent antibiotic reported to have acceptable therapeutic attributes (8.5). Teyramycin is similar to aureomycin and chloromycetin in its antibacterial action. It is reported to be effective against Gramnegative and Gram-positive pathogenic bacteria and may also have significant antirickettsial and antiviral properties. It is especially effective against those experimental infections in mice caused by Streptococcus hemolyticus, Diplococcus pneumoniae, Klebsiella pneumoniae, and Salmonel’la typhosa. Terramycin has a low degree of toxicity in animals and may be administered by either the oral or parenteral routes. This antibiotic is being produced by Chas. Pfizer and Company.
September 19SO
INDUSTRIAL AND ENGINEERING CHEMISTRY
FERMENTATION AS A UNIT PROCESS A study of fermentation as a unit process is limited to qualitative considerations. However, as quantitative principles of fermentation become better understood, the development of new processes will be much easier. In previous reviews (145, 366) certain factors have been discussed in relation to the unit process concept. Such factors are: the microorganism, the substrate, trace materials, sterilization, equipment, and the fermentation itself. The object of this portion of the review is to select literature from this and previous reviews for a discussion of these unit process factors. In many oases, several references could be cited to illustrate the points in the discussion, but this would necessitate the citation of many references previously covered and is deemed unnecessary. Quite often the same references are used in the discussion of several factors. THE MICROOROANlSM
Fermentation processes are composed of two closely related and overlapping parts. The first part consists of the growth of the microorganism which provides the enzyme system or “catalyst” needed to carry out the second part-the formation of the product. The microorganism used must satisfactorily fulfill these functions. To do this, it must be stable, capable of fast and abrmdant growth, and possess the ability to give uniform yields in an economical substrate under standard conditions. The microorganism must also be able to tolerate appreciable concentrations of the product formed. This last property can frequently be developed by strain selection. This has been done for yeasts used in the ethyl alcohol fermentation. In contrast, development of high butyl alcohol tolerance by CZ.acetobutylicum has not been accomplished to any great degree. The method of handling the parent or stock-culture is the h t important step. Usually, the preferred method is to carry the culture in the inactive state such as: in freeze-dried sand or soil cultures; in lyophylized suspension of spores or vegetative cells; in the dried state on the sporulation medium such as wheat bran; or on silica gel and other media. GrenfeIl et al. (103)carry stock cultures of P . chrysogenum on soil. Some workers (163,864, 506) have demonstrated that regular transfer on laboratory media has a deleterious effect on the fermentation properties of cultures. In general, although it has been claimed that repeated transfer on an agar medium does not affect the penicillin producing capacity of P . chrysogenum (33$),the usual practice is to carry stock-cultures in the dormant condition on inert materials. The next important step in the process is to prepare actively growing cultures, first in the laboratory and then in the plant, for the final production step. Microorganism transferred repeatedly in these early stages may, by selection or mutation, give rise to progeny which will give lower yields in the h a 1 fermentation. However, there are examples of adaptation, during inoculum development, of the microorganism to the production medium thereby obtaining a superior result in the final fermentation. An example of this has been reported by Peterson et al. ($09) who obtained higher yields of yeast when the culture was acclimatized to a wood sugar medium. It is generally advisable to use the minimal number of intermediate steps, from the stock-culture to the h a 1 fermentation, consistent with producing the proper age and quantity of inoculum. The optimal requirements pertaining to age and quantity of inoculum will vary for different fermentations. Tanner et al. (383)reported that 0.5 to 1.0% by volume of a 24-hour inoculum of A . gossypii gave superior yields of riboflavin. Lee and McDaniel (I&) reported that 10% by volume of a young inoculum gave acceptable results in the penicillin and streptomycin fermentations. Vander Brook and Savage (898) claimed that the fragmentation of the mycelium ok Penicillium chrysogenum with high
1681
speed agitation, resulted in higher penicillin yields from smaller quantities of inoculum. In the 2,bbutanediol fermentation using Aerobacillus polymyxa (53) it has been reported that, as the quantity of inoculum was increased, the initial fermentation rate increased along with the number of organisms, but the fermentations all finished a t the same time and gave similar yields. In general, a young inoculum is desirable and the quantity desired varies with the fermentation process.
A General criticism of the fermentation literature is the inexact descnption of the inoculum used and the conditions under which it is grown. Usually, the age of the inoculum is expressed in terms of the length of the growth riod in a stated medium. The y t i t y of inoculum is expresseEs per cent by volume used in t e final fermentation. It is suggested that the state of the inoculum be ex ressed in t e r m of both age and of growth stage as percentage ormaximal growth attainable, based on mycelial weight, under the given conditions. For example, if maximal mycelial growth is found to be 20 mg. r ml. and the inoculum a t the time of use shows 5 mg. per m r then the growth stage at that time can be stated to be 25% of maximal. It is also suggested that quantity of inoculum should be expressed in terms of percentage of peak mycelial weight attainable in the final fermentation. For example, if this latter value is 20 m per ml., and the inoculum employed amounts to 0.5 mg. per mf based on h a 1 fermentor volume, then the per cent inoculum is 2.5. Instead of mycelial weight values, cell counts can be employed where appro riate. Toennies and G a l t n t (887)have studied the precision of bacterial 4rowth. They arrived a t some conclusions which are of interest In large scale fermentation work-namely: (1) the rate of growth of cultures produced under standard conditions from refrigerated inocula decreased as the duration of the refrigeration was increased; (2) when organisms from freshly grown cultures were used as the inocula, the ensuing growth is the same under standard conditions, re ardless of the age of the parent cultures of those inocula. Altiough these investigators worked with Stre tococcus faecalis, it is believed their results are generally ap lcable. Etudies of strain selection and induced mutation have made the chief contributions to the advancement of industrial fermentation processes. This has been particularly true during the past decade in connection with antibiotic fermentations. Techniques for inducin mutation, such as treatment with ultraviolet light, x-rays, and $-mustard, have paid large dividends. Examples of succewful induced mutations in the penicillin fermentation are the reDemerec (6‘4), and Foster (88). Similar sults of Backus et al. (I?), results in the stre tomycin fermentation have been reported by Savage (348) and Sulaney et al. (78).
.
SUBSTRATE
The medium is equal in importance to the microorganism in the development of an industrial fermentation process. It ia undoubtedly true that numerous potential microbial products have never been discovered, or produced on a commercial scale, simply because the proper combinations of microorganism and substrate have never been attained. This fact must be kept constantly in mind in all fermentation research and development work. Because of the host of possible combinations of microorganisms and substrates which might be investigated, such development work may readily be classed as an artful science. Function. The medium has a twofold function in a fermentation process. It must provide conditions both for satisfactory growth of the microorganism and for synthesis of the desired product by the preformed enzyme system in the microorganism. Quite frequently, the medium suitable for growth is unsatisfactory for the production of tbe product. Shu and Johnson (864) have shown, for example, that small quantities of manganese in the sporulation medium markedly reduce the citric acid formed by A . niger in the subsequent fermentation. I n the fumaric acid fermentation (SOS),a medium Containing zinc waa essential during the growth stage, and a second medium containing iron was used during the final fermentation. The use of different media for growth and for production has also been successful in the citric acid fermentation (880,307,308).
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INDUSTRIAL AND ENGINEERING CHEMISTRY
The medium on which the inoculum is grown may affect the ratio of fermentation products produced. McCoy (163) succeeded in changing the butyl alcohol-acetone ratio by serially transferring the bacterial culture on a liver infusion medium. Yarmola (330) obtained similar results in the acetone-ethyl alcohol fermentation by varying the sugar concentration. Types of Raw Materials. No attempt will be made in this review to give a comprehensive list of raw materials used in various industrial fermentation processes. In general, the processes may be divided into two groups. The first group uses raw materials composed primarily of carbohydrates such m molasses, corn, rye, wheat, potatoes, sulfite waste liquors, and wood sugars. The carbohydrates are converted, through a series of reactions, directly to the finished fermentation product. Fermentation processes using this class of raw materials are ethyl alcohol, acetone-butyl alcohol 2,3-butanediol, citric acid, other organic acids, and yeast. The second group of fermentation processes use a wider variety of raw materials in the medium. Proteinaceous materials derived from both animal and vegetable sources are major constituenta of the media and the carbohydrates present are used primarily for growth rather than for conversion into end products. Proteinaceous materials for the various fermentations in the second group may be summarized as follows (143, 266): 1. RIBOFLAVIN. Distillers’ solubles, casein, corn-steep liquor, cereal grains, legumes, peptone, egg albumin, tankage, fish meal, meat scraps (69) wheat bran, corn bran, oat hulls ( I N ) , lentils (839), and stick ]liquor (283). 2. PENICILLIN. Corn-steep liquor (184, I N ) , cottonseed meal (91) linseed-oil meal, coconut-oil meal, sardine meal, peanut-oil meai, mustard flour, and soybean-oil meal (206). 3. STREPTOYYCIN. Meat extract, peptone, corn-steep liquor ( S I $ ) , whole-wheat bran (287, 228), yeast (146), and soybean meal (226). 4. CHLOROMYCETIN. Dried fermentation by-products, tanka e, and other meat industry by-products (MI), peptone, and distiflers’ solubles (73). 5. AUREOMYCIN. Corn-steep liquor casein, fish meal, soybean meal, meat extract, and liver cake (70). The sources of carbon in these fermentations are usually carbohydrates which are available in large quantities a t fairly low cost. Examples of such carbon sources are: glucose, sucrose, lactose, maltose, malt extract, molasses, sulfite waste liquors, glycerol, vegetable oils, and glycerides (148, 2.56). Synthetic Media. Synthetic media have been described for nearly every industrial fermentation process (1.68, 266). Such media are not used in large scale production in some cases due to higher cost but primarily to the lower Sields obtained. The lower yields are due to the lack of sufficient information concerning the proper quantities and balance of growth factors, vitamins, and other trace materials required for fermentation. When more information becomes available on the biochemical mechanism of fermentations, synthetic media may be used on a large scale. They would be advantageous, particularly in the producticn of antibiotics, because they would simplify the problem of extracting and purifying the product. Precursors. The user of precursors in fermentation media h w proved beneficial. The outstanding example is the use of various synthetic chemical compounds in the penicillin fermentation. Such additions have resulted in vast increases in the commercial production of penicillin G. For example, the penicillin G content in fermentation broths has been raised from 12 to about 100yo through the use of such precursors (110). The general subject of penicillin precursors has been covered in this and other reviews (94,143, 966). The synthesis of vitamins by microorganismsis aided markedly, in certain instances, by the inclusion in the medium of component portions of the vitamin molecule (299). Mannosidostreptomycin, which is found in appreciable quantities in the streptomycin fermentation, can be converted to
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streptomycin by growing cultures and cell-free extracts of Stmptomyces griseus (141). This suggests that mannosidostreptomycin may be an intermediate in the formation of streptomycin. The addition of cobalt, a component of the vitamin B I mole ~ cule, to fermentation media has been shown to increase the concentration of BIZand BIJike compounds (208). Miscellaneous Additions. Frequently, materials added to fermentations have a beneficial effect which may not be explained on the basis of their nutrient properties or their ability to act M precursors. An example of this effect is the addition of morpholine to media used for the production of citric acid by A . niger (2661). The morpholine is claimed to stimulate spore germination, stabilize the physiological characteristics of the fungus, and increase its efficiency in converting carbohydrates to citric acid. Another example is the addition of inert materials such as kaolin, fuller’s earth, or bentonite to the ethyl alcohol fermentation (313)thereby obtaining an increase in fermentation rate. Weizmann (318) claimed beneficial effects, in the acetonebutyl alcohol fermentation, resulting from the addition of small quantities of paminophenylalanine or p-aminobenzoic acid. Pretreatment of Raw Materials. Many raw materials which are by-product wastes of other industries are, when properly pretreated, suitable for fermentation processes. Sulfite waste liquors are acceptable when sulfur dioxide is removed by steam stripping followed by neutralization with lime (106). Molasses has been made suitable for the citric acid fermentation by decationizing with resins (389) and by treatment with potassium ferrocyanide (207). In pentose solutions, resulting from acid hydrolyzed corncobs, oat hulls, and bagasse, the copper toxicity can be removed by treatment with finely divided iron, thereby making such hydrolyzates suitable substrates for the acetone-butyl alcohol fermentation (292). Reducing agents such as sodium bisulfite, sodium sulfite, and sulfur dioxide have been used to reduce the toxicity of acid hydrolyzed materials (36). The detrimental effects of iron on the production of riboflavin by bacteria producing acetone and butyl alcohol may be overcome by adding proper quantities of 2,2’-bipyridine (109) and by including yeast and catalase in the medium (149). A process has been described for manufacturing a special corn-steep liquor which gives improved yields of penicillin (186). TRACE MATERIALS
Any discussion of the effeats of trace materials must involve the consideration both of substances which are stimulatory and those which are inhibitory. Substances in the stimulatory group consist of vitamins, growth factors, amino acids, and various inorganic elements which are e8sential in some processes. Industrial fermentation literature concerned with the necessity and role of vitamins and growth factors is limited. This is due to the use, in these fermentations, of a wide variety of complex organic materials of natural origin which have high contents of these materials. Most media used in current industrial fermentation processes have been developed by trial and error with economics, rather than a fundamental knowledge of microbial nutrition, as the chief guide in the selection of materials. Two lines of fundamental research currently being pursued will eventually supply the neceasary know-how for a more scientific approach to design and development of production media. These are studies in synthetio media of the nutrition of these microorganism and studies of the biochemical mechanisms involved in the synthesis of fermentation products. At present, yields in synthetic media ape generally lower than in media containing heterogeneous natural materials. However, one example of an exception to this rule is the work of Olson and Johnson (194) who obtained yields of yeast in a synthetic medium comparable to the usual yields in natural media, The use of such substances as molasses, corn-steep liquor, bean
September 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
meals, and meat by-products, which are rich in vitamins and growth factors, largely eliminates the necessity for discussion of these factors in connection with present industrial fermentation processes. Recent reviews of this subject have been previously cited (138, 299). The following discussion will be confined to other organic and inorganic substances which have exhibited stimulatory or inhibitory effects. Stimulatory Materials. There are many examples of the stimulating effects of trace materials in fermentation processes, but little is known regarding the mechanism of such stimulations. Waksman (303) claimed that zinc, manganese, and copper are essential for the growth of molds producing fumaric acid, whereas iron is essential in the replacement medium in which the fumaric acid is produced. In synthetic media, carefully controlled concentrations of zinc and iron are essential for high yields of citric acid (266),but this effect is not directly on the growth of the mold. Similar results have been obtained by Tomlinson et aE. ($90)who found that iron, copper, zinc, and manganese were essential for normal citric acid yields. The quantities needed for growth were higher than for citric acid production. Some microorganisms used for the large scale production of riboflavin are also stimulated by proper concentrations of certain elements. Iron, at a concentration of 0.5 to 4.5 p.p.m. favors the formation of riboflavin by the acetone-butyl alcohol bacterium in a whey medium (322). The concentration of manganese and magnesium also had a marked effect on the quantity of riboilavin produced (217-219). Candida g u i l l i e r d i a requires manganese, copper, zinc, molybdenum, and iron for the production of riboflavin in a synthetic medium (42). The production of penicillin by surface cultures on synthetic media is favored by iron, copper, zinc, manganese, and cobalt (43). Iron, copper, and magnesium are essential for the pro. duction of penicillin in submerged cultures (126'). The ash of corn-steep liquor increases penicillin production in a synthetic medium (137). The ash can be replaced by iron and soluble phosphates. Trace elements have a stimulating effect in other fermentation processes. Iron, copper, and zinc are essential for maximal yeast yields in synthetic media (194);zinc is essential for the production of subtilin (81);and cobalt is essential for the production of vitamin B12 and Ble-like compounds (108, 210). Other compounds have been shown to have specific effects in particular fermentations. Amino acids such as lrproline (71), Ircystine (269),and Irlysine (288) have been shown to have favorable effects on fermentation yields and on microbial growth and maintenance. Ethylamine is reported to have a favorable effect on penicillin yields (43) and morpholine is claimed to influence favorably the citric acid fermentation (262). Inhibitory Materials. Often the toxicity of trace materids is important in industrial fermentations because they show their effects in production media containing complex natural materials. Many elements which are essential in small quantities are toxic in large quantities Iron is chiefly toxic in the citric acid (206, 207, 266, 290, 329) and in some riboflavin fermentations (148, 149, 172, 282, 814). Small amounts of copper have been shown to be toxic in the penicillin (I&"), acetone-butyl alcohol (292), citric acid (&90),and riboflavin fermentations (314). Other common elements which have been reported to be toxic in trace amounts are lead, nickel, zinc, cobalt, and chromium. Inhibitory materials may be effective in the inoculum development stages. The presence of highly polymerized desoxyribonucleic acid is capable of bringing about changes in microbial types (38,162). Dhalanine is reported capable of preventing spore formation by certain bacteria (89). Manganese, which aids the sporulation of A. niger, reduces the yields of citric acid if it is present in the sporulation medium in sufficient quantity (264). Peptone, a constituent of many media, may contain materials which are toxic to the growth of microorganisms (260).
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STERILIZATION
Proper sterilization methods and a clear concept of aseptic operating techniques are vital to successful large scale fermentation operations. The degree of asepsis and the ease of attaining it may vary somewhat with the particular process, but in all cases strictly aseptic methods are advantageous. In certain fermentations, especially those conducted a t or near a neutral pH, contamination difficulties are much greater. This is due chiefly to the fact that bacteria, which are the chief contaminants, grow more readily at pH values approaching neutrality. Fermentations conducted under acid conditions, such as the ethyl alpohol and especially the organic acid fermentations, are much less susceptible to oontamination. In an aerobic process, the necessity for furnishing sterile air adds another hazard to large scale operations. Antibiotic fermentations and especially the riboflavin fermentations by yeasts are examples of those most difficult to conduct on a large scale. Antibiotic processes are somewhat self-protected once they have attained a satisfactory start. This discussion will deal with sterilization of the medium, sterilization of process equipment, and sterilization of air. The industrial fermentation literature does not contain a great deal of specific information on these three phases of sterilization. Sterilization of Media. Media are usually sterilized by heat. Steam pressure in the range of 15 pounds per square inch gage (120' C.) commonly used in the laboratory are also used in plant operations. However, satisfactory results can be obtained at lower temperatures, using a longer holding time, or at higher temperatures, using a shorter holding time. An example of the latter is the continuous cooking process reported by the group at Joseph E. Seagram & Sons (94, 297). The chief factor in choosing sterilizing conditions is the relative sensitivity of the medium to heat. Several reports are available which describe the destruction by heat of the nutritional qualities of naturalmaterials. Lankford and Lacy (142) reported the destruction of cystine and other amino acids essential for microbial growth, when they are sterilized in a medium containing glucose. Toennies and Gallant (287)stated that heat sterilization of the mediup adversely affects the subsequent growth of bacteria. Evans m d Butts (77) found that when soybean meal is heated alone, few of the amino acids are destroyed. However, when heated with sucrose in the medium, over 40% of the diamino acids, lysine and arginine, are destroyed. This destruction is caused chiefly by the reaction of free amino groups with sucrose. Other workers have shown that methionine is readily destroyed when heated in the presence of glucose, but it is not affected in the presence of starch or dextrin (102,117). Deseive (66)reported that the production of riboflavin by Eremothecium aahhyii is dependent on a heat labile factor which is destroyed by extended Sterilization of the medium. Tanner et al. (283)obtained riboflavin yields with Ashbqla goesypii of 248 y per ml. when the medium was autoclaved for 90 minutes and 648 y per ml. when autoclaved for 15 minutes. Bactericidal substances have been extracted from glucose solutions which have been heated at high temperatures for extended periods (180). Melanoidin compounds are formed by the reaction of amino acids and sugars at high temperatures (140). It is evident that the chief effect of heat is in causing unfavorable reactions between oarbohydrates and the free amino acids or the free amino groups in proteins. Thus, it is obvious that in many large scde fermentation operations where the media contain sugars and proteins, best results will be obtained either by using short sterilization periods or by individually sterilizing the sugars and proteinaceous materials. A general criticism of reports regarding conditions of sterilization is that oiily the holding period at the desired temperature is stated. No mention is made of the time involved either in reaching that temperature or in cooling to noncritical temperatures. This factor may be especially important when operating on a large scale.
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INDUSTRIAL AND E N G I N E E R I N G CHEMISTRY
Sterilization of Equipment. The fermentors and the process pipe lines in a fermentation plant are kept sterile by the use of steam at all critical points. Lee and McDaniel(14) stressed the necessity for steam locks and steam seals on all lines leading to and from the fermentors and on all other accessory equipment. In addition, all transfer lines for inoculum, raw materials, and fermented broths should be kept a t sterilizing temperatures at all times except when being used for such transfers. Many fermentation plants have installed thermometers or other temperature indicating equipment a t critical points to ensure the maintenance of sterilizing temperatures. The sterile process lines should contain a minimum number of pumps, valves, or other equipment difficult to sterilize. Special equipment such as steam-eketed valves and pumps with sterile packings offer definite advantages. Sterilization of Air. Large uantities of sterile air are needed
for antibiotic, riboflavin, an1 other aerobic processes. The literature contains little information concerning positive methods for sterilizing the air used in large scale processes. Stark and Pohler (968)have reviewed the methods for sterilizing air for industrial fermentations. These workers particularly emphasized a method involving the compression of air under adiabatic conditions, They claimed that special compressors, or ordinary piston-type compressors with the cooling units removed, will give sterile air by virtue of the high temperatures following compression. Another method of sterilizing air by heat involves pasing it through a tubular network which is heated by oil or gas burners. Instantaneous exposure to a temperature in the neighborhood of 500' C. is generally considered ample for air steriIiaation. In general, complete sterilization by heat alone is too costly in large scale operations. Numerous other methods or combinations of methods have been used successfully. These include : exposure to electronic precipitation or ultraviolet light; filtration through columns packed with cotton, activated carbon, slag wool, or glass wool; and scrubbing with caustic, acids, or disinfectant solutions. In general, the most commonly used method is that of filters alone or in combination with other techniques-for example, electronic precipitation plus filtration through columns of oarbon or glass wool. The fermentation industry is in need of sound scientific data relating to the efficiency of various air sterilization methods which could be used in the design of this type equipment. Terjesen and Cherry (985) have reported methods for evaluating such filtering materials and report favorable results with slag wool. FERMENTATION EQUIPMENT
There have been numerous descriptions of fermentors and auxiliary fermentation equipment. However, during the period covered by this review and previous reviews (143, 966)in which the unit process aspects of fermentation have been given chief consideration, the aerobic fermentation processes have received greatest attention. Therefore, this discussion will be confined to equipment needed for aerobic processes. For information on the equipment used in the older aerobic processes and for anaerobic processes, the reader is referred to the textbook by Prescott and Dunn (924). Industrial plants for the production of penicillin (44), streptomycin ($22), and chloromycetin (193) have been described. Petty (811) has reviewed the equipment and techniques used in antibiotic production. Several workers have described pilot plants for aerobic fermentations (86, 100, 183, 146, 870, $71). No consideration is given here to many types of equipment proposed for continuous fermentations. This technique has been applied chiefly to the alcohol fermentation and has been discussed previously. Fermentor. The fermentor is a tank, which usudly has a depth to diameter ratio of 2: 1, constructed of a metal which is not toxic to the particular fermentation. Carbon steel and stainless steel are the most common materials of construction, although Inconel, a nickel-chromium alley, has been used in one fermentation plant (195). In general, it is highly desirable to use a noncorrosive metal as such fermentors are much easier to keep clean and free from contamination.
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Fermentors must be suitably equipped so they can be cooled readily after sterilization and so that the fermentation can be maintained at the proper temperature. For this purpose, water is passed through external spray rings, internal coils, or jackets. Although all three are satisfactory, jacketed vessels have certain advantages. They give better temperature control than spray rings and do not leak as readily as internal coils. It is essential that all areas on a fermentor which may contact nonsterile areas should be protected against the entrance of contamination. Steam seals are commonly used for this purpose on all lines leading to and from the fermentor and on all other critical points such as light and sight glasses, manholes and around the agitator shaft at its point of entrance to the tank. Agitators. Fermentors used for aerobic processes, especially those processes involving molds and actinomycetes, are usually equipped with mechanical agitators, although some producers have had good results using aeration aa the sole source of agitation. The size, shape, and type agitator blades vary considerably with the shape of the fermentor and with the process operated. It is reported (911) that a large portion of the antibiotic industry in this country has standardized on the use of open back-slope bladed turbines. The size and number of such agitators will depend on the horsepower input required per unit volume of fermenting mash. The horsepower supplied varies from 0.3 to 2.0 per 100 gallons (211) depending on the operating technique and experience of the individual company. The agitator design will also be affected by internal cooling coils, baffles, spargers, and other installations in the fermentor (32). Defoamer. The foaming problem is mast acute in aerobic fermentations owing to the necessity for aeration and agitation. Defoamer materials of various types, such as animal and vegetable oils, petroleum oils, and various synthetic compounds (129) are commonly used. Equipment must be supplied in the plant for sterilizing such defoamers and for aseptically adding small quantities to the fermentors. The addition of defoamers may be automatically controlled (270). Various types of mechanical foam-breakers have been described (66, 96, 118,253).
Air Spargers. Tae types of air spargers used will also vary with the technique and experience of the manufacturer. Where a high degree of agitation is used, the spargers may be relatively simple in design and have a low capacity for dispersing air. I n mold fermentations, growing mycelium will frequently plug spargers having small orifices. de Becze and Liebmann (63)reviewed the various types of air spargers and mechanical agitation systems. Open pipes or perforated ring-spargers are most commonly used a t the present time. Achorn and Schwab (1) described an interesting method for aerating fermentations which consisted of passing air through an orifice at acoustic velocities. This technique produced small air bubbles in the liquid, many of which were below 10 microns in diameter. I t also produced violent agitation of the liquid. These workers reported their method to be superior to other types of spargers such as carbon, Aloxite, fritted glass, ring, dishpan, and sintered stainless steel. Valves. The type valve used and the proper maintenance of valves are vital factors in large scale fermentation operations. The valve types commonly used are gate, globe, plug cock, and diaphragm. Each of these has certain advantages and disadvantages. Gate and globe valves are usually cheaper and more easily maintained, but are objectionable because of the presence of acking which is difficult to sterilize. Multiple-port plug cocf valves are advantageous for simplifying the piping system in plants and are convenient to operate. Their disadvantages are the difficulties of maintaining them in a leakproof condition and of sterilizing the lubricating material. Diaphragm valves are easy to sterilize, but maintenance of the diaphragm is costly. Most fermentation plants use more than one type valve, each type. being used at points where their advantages are a t the maximum. The ideal valve should be relatively inexpensive, easy to sterilize, easy to maintain in a leakproof condition, and convenient to operate; none are available at present which incorporate all these desired features. Air Sterilization. Techniques and equipment for sterilizing air have been discussed previously. Filters, containing glass wool or
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INDUSTRIAL AND ENGINEERING CHEMISTRY
carbon, are constructed of either carbon steel or stainless steel. Data are needed which can be used for the design of such filters. The empiricism can be removed from filter design if an understanding is gained of the bed depth-diameter ratio of the filtering agent needed to remove all microorganisms from known volumes of air at known velocities. Terjesen and Cherry (986)reported this type data for slag wool and glass wool filters. Petty ($11) stated that a t reasonable velocities, a 4-foot packing depth will provide sterile air for periods up to 100 hours. Instrumentation. Instrumuntation has become a major factor in the design and operation of fermentation plants. It is essential for best results that such fermentation factors as temperature, airflow, air pressure, and pH be closely controlled. The instrumentation used in one large antibiotic plant has been described (174). Automatic controller-recorders were used to regulate temperature, airflow, and air pressure. For uniform fermentation yields, temperature should be maintained within * 0.5" to 1.0' C. of the desired temperature and airflow should be controlled within * 5 to 10%. Olive (193) described a pH recorder used in a fermentation plant. Broth is continuously withdrawn from the fermentor by an Oliver diaphragm pump, circulated to a pH recording instrument and then returned to the fermentor. Brown and Peterson (41 ) obtained excellent penicillin yields in laboratory fermentors by using automatic pH control. Breeze (39) described an electronic apparatus for precision pH control of fermentations and other biological systems. Proper pH control is a vital factor in all industrial fermentation processes. It is possible that, in the near future, fermentation plants will be designed and instrumented so that, like oil refineries and other industrial plants, they can be completely operated and controlled from a central point. Seed Fermentors. In general, the previous discussion of fermentors and auxiliary equipment also applies to seed fermentors. Most fermentation plants have seed fermentors of ample size and number to supply inoculum of proper quantity and age to the final fermentation. Five to ten per cent by volume of a "physiologically young" inoculum is generally preferable. Special equipment may be used for facilitating the inoculation of the smallest seed fermentor with laboratory cultures. Examples of such inoculating devices are the removable pipe-cap type described by Petty (911)and the rubber diaphragm type described by Olive (193). It is desirable, in order to facilitate strict asepsis in initial operations, that the first plant seed stage be located in a specially conditioned area removed from the remainder of the plant (179, 911). THE FERMENTATION
Industrial fermentation processes consist of the production of pure chemicals and food or feed substances by the growth and chemical reactions of microorganisms. The large number of fermentation processes being operated currently make it necessary to consider general factors which are of importance in all processes. The previous discussions in this review have all dealt with factors which contribute toward the success of the final fermentation. The purpose of this section is to place greater emphasis on certain critical fermentation factors. pH. pH is one of these critical factors. In order to obtain peak yields the pH must be controlled during one or more phases of all fermentations. The chief methods used for controlling pH are: the use of buffers; the selection of nutrients which are utilized a t rates such that the ions remaining in the medium stabilize the pH; and the addition of acids or alkalies during the fermentation. Buffers such as phosphates and citrates are commonly used in microbiological media, but the concentrations required are
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usually too high to be practical in industrial fermentations. Calcium carbonate serves a similar purpose and is frequently used. The control of pH through the choice of ingredients, which have anions and cations utilized at different rates thereby stabilizing the pK, has been applied in many fermentations. An excellcnt example of this method is the work of Jarvis and Johnson (195). They used this approach in designing a synthetic medium for penicillin production. Their medium contained lactose, glucosc, ammonium acetate, ammonium lactate, and salts. During the fist phase, pH 6.8 favored growth, and during the sccond phase maximum penicillin was produced at pH 7.3. The pH during the growth phase was regulated by simultaneous use of glucose, ammonium, and acetate ions. During the productioii phase, the lactate ion was metabolized slightly faster than lactose and the ammonium ion, thereby permitting control at a higher pH level. In practical penicillin production media, pH may be controlled through the use of varying concentrations of glucose, lactose, corn-steep liquor, and calcium carbonate. The control of pH by adding acids or alkalies has been uved in many fermentations. Brown and Peterson (41), using either manual or automatic addition of acids and alkalies, reported penicillin yields of 1900 to 2100 units per ml. in a medium containing lactose, glucose, corn-steep liquor, calcium carbonate, and phenyl acetate. The optimum pH for penicillin production was 7.15.
Other examples of the necessity for pH control and automatic equipment for such control have been cited in this and previous reviews (149, 956'). Temperature. Temperature is another factor which has a great effect on fermentation yields and must be closely controlled. Usually a microorganism will produce the desired products over a relatively wide temperature range-for example, many patents claim ranges as great as 10 to 15 ' C. However, most processes have an optimal range which is relatively narrow. I t has been pointed out previously in this review that, for greatest uniformity of yields, control limits of * 0.5" to 1.0"C. are desirable. The use of significantly different temperatures during different phases of the fermentation-for example, the growth and production phases-is not commonly used in industrial processes. I t seems likely that in some cases this technique could be used advantageously. Kovats (139) reported favorable results in the citric acid fermentation with A . niger as the result of using a growth temperature of 28" C. during the early period of the fermentation and a temperature of 20' C. during the later stages. Aeration-Agitation. In aerobic fermentations, there is present a three-phase system consisting of liquid, gas, and suspended solids. Gas absorption and transfer within the liquid phase must be considered, as these factors will dictate the aeration-agitation requirements. An understanding of several fundamental principles is needed to remove the empiricism from the methods commonly used for determining optimum aeration and agitation. For example, a clear picture is needed of the effect of aeration and agitation on rates of oxygen transfer from gas, through the intermediate phases, to the specific enzyme surface within the cell and a knowledge of how these diffusion rates are correlated with optimal yields. The fermentation literature presents several methods for expressing the degree of aeration. Examples are: O
The volume of air per volume of medium per unit time (967) The volume of air per unit time (270) The volume of air per unit weight of sugar consumed (88) The sparger area per volume of medium at a given volume of air per unit time (298) The volume of air per ram of organism per unit time (88) The volume of air perailogram of organism harvested (943) In few cases, is information given concerning the method of. dispersing the air in the medium, the degree of dispersion, the degree of agitation, or the dissolved oxygen concentration in the medium. Knowledge of thwe factors would simplify the problem
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of transposing laboratory and pilot plant yields into large scale operations. Cooper, Fernstrom, and Miller ( 6 1 ) reported that the variables sf importance in the transfer of oxygen to a liquid are the area of the gas-liquid interface, temperature, time of contact, concentration difference, and intensity of agitation. A sulfite oxidation method was used to measure aeration efficiency. Bartholomew et al. ( 2 2 ) and Hixson and Gaden (112) reported the results of studies concerned with oxygen transfer from the gas phase into the medium and from the midium to the cell. Equations have been derived which express oxygen transfer. I t was found ($2) that the important transfer resistances are in the liquid film of the gas-liquid interface and a t the cell boundary. It has been shown (112) that physical absorption of oxygen into the medium is a function of the design and operation of the equipment and of the physical properties of the medium. The oxygen absorption coefficient was correlated with aeration rates for any system of agitation and air dispersal. Such correlations may be used to recommend aeration systems for different types of processes. An important tool in these studia was the use of the dropping mercury electrode for determining dissolved oxygen (2666, 8 7 ) , Studies of this type will aid markedly in gaining a clearer view of the principles and methods of aerationagitation. The aeration problem in the penicillin fermentation was approached from another angle (270). The evolution of carbon dioxide by P.chrysogenum was shown to be a function of aeration. Assuming one molecule of oxygen is absorbed per molecule of carbon dioxide evolved, the amount of oxygen actually removed from the air was shown to be small-in the range of 1%. The minimum supply of air which was needed to keep respiration rates at a maximum was demonstrated. Olson and Johnson (194) used the sulfite oxidation technique (61) to compare the aeration efficiencies of shake flasks and a laboratory fermentor. These workers obtained excellent correlation between yeast yields in shake flasks and in fermentors at similar aeration efficiencies, as determined by oxygen absorption coefficients. Another interesting feature of the work of Olson and Johnson is that, at high aeration efficiencies, yeast yields were actually decreased. Starks and Koffler (269) discussed the solution of oxygen in shaken flasks; foam formation interfered with aeration efficiencies. The effect of pressure cannot be ignored. Stefaniak et al. ( 2 T l ) reported that air pressures in the range of 2 to 20 pounds per square inch gage had no effect on the penicillin fermentation but that a pressure of 40 pounds per square inch gage caused a definite reduction in yield. This decrease was not due to increased tension of carbon dioxide. Adams and Leslie (2) reported that increased tensions of carbon dioxide were detrimental to the fermentation of carbohydrates by Aerobm'llus polymyza and Aerobacter aerogenes. This effect could be overcome and fermentation times could be reduced from 96 to 48 hours by fermenting under reduced pressure. In aerobic processes the degree of agitation is closely related to the aeration needed. When mechanical agitators are used, the quantity of air needed will decrease as the agitation is increased. This will be especially true when the critical dissolved oxygen concentration needed for maximum growth and respiration is low. With yeast, for example, it has been shown (327) that the respiration rate was independent of oxygen concentration down to low concentrations. Petty (211) expressed the degree of mechanical agitation in terms of horsepower input per unit volume of fermenting liquid. He reported, as pointed out previously in this review, that the penicillin industry is using mechanical agitation which supplies 0.3 to 2.0 hp. per 100 gallons. This wide range .of horsepower utilization is apparently due to such factors as aeration technique, quantities of air used, strain of culture, medium, and fermentor dimensions. It is apparent that these approaches to the problem of aeration
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and agitation have already contributed a great deal toward eliminating the trial and error technique. Pure Culture Fermentation. Up to the present time, the fermentation industry has not developed a strictly pure culture technique for large scale production. From a practical viewpoint, however, the techniques and equipment now in use are sufficiently acceptable so that contamination is becoming a factor of lesser importance. In the antibiotic fermentation industry, for example, i t has been reported (144) that over one thousand consecutive production batches have been completed without a loss due to contamination. It is estimated that in most fermentation industries, less than 2% of the total production batches are lost due to contamination. When a pure culture technique is developed, it is possible that continuous fermentation processes will be used more widely in the industry. This will be especially true in those fermentation processes where the particular microorganism used is sufficiently stable and rugged, so that it will give high yields on repeated transfer in the vegetative state. Microbial phages are contaminating agents of major importance which must be given consideration in industrial fermentations These virus agents, which are pathogenic to microorganisms, have in some cases been so effective, especially when first encountored in industrial fermentation plants, that industrial concerns have been virtually bankrupt before the problem was solved. Bacteriophage has been a constant menace to the acetone-butyl alcohol industry for many years. However, the development of techniques for readily identifying an outbreak of phage in the plant, along with rapid and efficient methods for producing phageresistant cultures, has enabled the acetone-butyl alcohol fermentation industry to operate with slight losses due to this agent. In recent years phage outbreaks have been reported in other industrial fermentations. An actinophage capable of attacking Streptomyces griseus has been found in several plants producing streptomycin. Some of these attacks have been reported (234, $47, 328). A bacteriophage for Aerobacillus polymyxa has also been reported (224). Both S. griseus and A . polymysa were readily made resistant to the respective phage strains. There are innumerable phages which will attack a rather wide variety of organisms. Thus, it is necessary in industrial plants to combat the entrance of phage into the production process. Generally, the sources of phage infection are identical with the sources of bacterial contamination. Improved equipment, operating procedures, and asepsis used to combat bacterial contamination will also reduce the incidence of phage contamination (192, 224). An interesting angle, in this connection, is the patent granted to Gavronsky (99) who claimed that, through the use of large volumes of inoculum, the acetone-butyl alcohol fermentation could be conducted under conditions where mash sterilization is not required. Uniformity of Yields. I n some industrial fermentations, such as the ethyl alcohol and lactic acid processes, carbohydratesin the medium are converted directly, through a series of biochemical reactions, to the final product. In such fermentations a theoretical yield can be calculated and thus the efficiency and uniformity of the production operation can be gaged by how nearly the yields approach the theoretical. In antibiotic and vitamin fermentation processes, theoretical yields cannot be calculated, because the products are derived from more than one component of the substrate and beaanse the biochemical mechanisms used in the formation of these producta have not been determined. In these processes, there is no gage which can be used to evaluate the processes from the viewpoint of maximum efficiency or competitive efficiency. Quite often, comparison with the yields obtained in the laboratory or pilot plant, where presumably operations can be more closely controlled, are used to evaluate the efficiency of the results in the production unit. The disturbing fact concerning such a comparison is that
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I N D U S T R I A L A N D E N G I1T E E R I N G C H E M I S T R Y
plant yields may exceed those obtained in the laboratory or pilot plant. Frequently, in large scale fermentation operations, and especially in antibiotic and vitamin fermentations, there is a wide variation in yields from one batch to another or from one period to another. ‘(Biologicalvariation” is a phrase commonly used to explain such lack of uniformity. In other words, the inherent variability of the microorganism is offered as the explanation for poor uniformity. However, if the facts are closely examined, lack of proper control may be the more likely explanation. The philosophy that “the bug is never wrong” should be as applicable in large scale operations as it is in the laboratory. Batch to batch variation in antibiotic production operations has been discussed (I&) together with those conditions which must be satisfied to obtain uniform results. Such conditions are: Maintenance of the culture at peak efficiency at all stages in the laboratory and in the plant (103) Procurement of uniform growth at all stages of inoculum development Use of inoculum of proper age and in roper quantity Proper pretesting of all raw mate$als in the laboratory or pilot plant Uniform time and temperature of sterilization Close control of such factors as temperature, pH, and airflow Sensitive methods for quickly detecting contamination, especially in the inoculum development stages Regular inspection of fermentor equipment, especially valves, air spargers, and agitators Choice of an age for harvesting at which the average rate of increase of product is in the phase of negative acceleration. When all of these factors are properly controlled, it is possible
to obtain results, in a batch process, in which the maximum deviation from the mean is * 10 to 15%. ACKNOWLEDGMENT The author wishes to acknowledge the assistance of R. E. Bennett, R. J. Hickey, and G. A. Snyder, who contributed the reviews of bacitracin, neomycin, and subtilin, respectively. The author also wishes to express his grateful appreciation to G. C. M. Harris for his many valuable criticisms and suggestions during the preparation of the manuscript.
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(2) Adams, G. A., and Leslie, J. D., Can. J . Research, F24, 107 (1946). (3)Adams, S. L., FoodPacker, 30,26-7,64-5 (1949). (4)Adams, S. L., IND.ENQ.CEEM.,42, 1815 (1950). (5) Agarwal, P. N., and Peterson, W. H., Arch. Biochem., 20, 59 (1949). (6) Ainsworth, G. C., Brown, A. N., and Brqwnlee, G.,Nature, 160, 263 (1947). (7) Ambler, J. A., and Curl, A. L., U. S. Patent 2,448,506(Sept. 7, 1948). (8) Andersen, A. A., and Michener, H. D., ppaented before the Meeting of the Society of American Bacteriologists, Baltimore, Md. (May 1950). (9) Andersen, A. A,, and Michener, H.D., Food Technol., 3, 1815 (1950). (LO) Andreasen, A. A,, Am. Brewer, 82,30-2,37,74,76,78(1949). (11) Anker, H. S.,Johnson, B. A,, Goldberg, J., and Meleney, F. L., J . Bact., 55,249 (1948). (12) Anon., IND.ENQ.CEEM..41, No. 5, M A (1949). (13) Anon., Loncet, 256,290 (1949). (14) Ansbacher, S.,and Hill, H. H., presented before the Division of Agricultural and Food Chemistry, 116th Meeting,AMERICAN CEEMICAL SOCIETY, Atlantio City, N. J. (15) Arroyo, Rafael, Sugar J., 11, 5-12 (1949). (16) Ayerst, McKenns, and Harrison Ltd., Brit. Patent 616,381 (Jan. 19,1949). (17) B a c k , M. P., Stsuffer, J. F., and Johnson, M. J., J . Am. Chem. SOC.,68, 152 (1946). (18) Banzon, J., N m e r , E. I., and Underkofler, L. A., Iowa Slate Coll. J. Sci., 23, 219 (1949). (19)Baron, A. L., U. S. Patent 2,482,832(Sept. 27,1949). (20) Barreto, A., Ibid., 2,473,530 (June 21, 1949).
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(21) Barry, G. T.,Gregory, J. D., and Craig, L. C., J. Biol. Chem., 175,485(1948). (22)Bartholomew, W.H.,Karow, E. O., Sfat, M. R., and Wilhelm, R. H., IND.ENQ.CHEM.,42, 1801 (1950). (23) Bartz, Q.R.,U. S. Patent 2,483,871(Oct. 4, 1949). (24)Behrens, 0. K.,(Clarke, H. T., et al.) “Chemistry of Penicillin,” p, 657,Princeton, N. J. Princeton University Press, 1949. (25) Behrens, 0. K., U. S. Patent 2,463,939(March 8, 1949). (26) Behrens, 0.K., Corse, J. W., Jones, R. G., and Soper, Q. F., U. S. Patent 2,479,295(Aug. 16,1949). (27) Benediet, R. G.,and Langlykke, A. F., J . Bact., 54,24 (1947). (28)Benedict, R. G.,and Stodola, F. H., Ann. N. Y . Acad. Sci., 51, 866 (1949). (29) Bernhauer, K., Niethammer, A,, and Rauch, J., Biochem. Z., 319.,94 (1948). (30) Bernhauer, K., and Rauch, J., Ibid., 319, 77 (1948). (31) Bernhauer, K., Rauch, J., and Grow, G., Ibid., 319,499(1949). (32)Bissell, E. S., H e w , H. C., Everett, H. J., and Rushton, J. H., Chem. Eng. Profless, 43,649 (1947). (33) Blaokwood, A. C., and Ledingham, G. A., Can. J . Research, F25, 180 (1947). (34) Blackwood, A. C.,and McCoy, E., presented before the Meeting of the Sooiety of American Bacteriologists, Cincinnati, Ohio (May 1949). (35) Blackwood, A. C., Wheat, J. A,, Leslie, J. D., Ledingham, G.A., and Simpson, F. J., Can. J . Research, P27, 199 (1949). (36)Block, S. S., U. 5. Patent 2,474,139 (June 21, 1949). (37) BootsPureDrug Co. Ltd., Brit. Patent 616,102(Jan. 17,1949). (38) Braun, W.,Bact. Revs., 11, 75 (1947). (39) Breere, J. E., Proc. Natl. Electronics Cbnf., 4,451 (1948). (40) Brian, P. W., Chemistry & Industw, 1949, 391. (41)Brown, W. E., and Peterson, W. H., IND.ENQ.CHEM.,42, 1769 (1950). (42) Burkholder, P. R., U. 5. Patent 2,363,227 (Nov. 21, 1944). (43) Calam, C. T.,and Hockenhull, D. J. D., J. Om. Microbial., 3, 19 (1949). (44)Callaham, J. R., Chem. & Met. Eng., 51,94(1944). (45) Campbell, C. C., Saslow, S., and Strong, 8. H., presented before the Meeting of the Society of American Bacteriologists, Baltimore, Md. (May 1950). (46)Campbell, K. N., Ackerman, J. F., and Campbell, B. K., J . Org. Chm., 15,221 (1950). (47) Capps, B. F., Hobbs, N. L., Fox, S. H., J. Biol. Chm., 178,517 (1949). (48)Carson, J, F., Jansen, E. F., and Lewis, J. C., J . Am. Chem. SOC..71. 2318 (1949). (49) Cartland, G. F., Hainee, W. J., and Bohonos, N., U. 6.Patent 2,487,018(Nov. 1, 1949). (50) Caswell, M. C., Koditschek, L. K., and Hendlin, D., J . Biol. C h a . , 180, 125 (1949). (61) Chang, Wei-Shen, and Peterson, W. H., J. Bact., 58, 33 (1949). (52) C h m . Eng. New8,27,2848(1949). (53) C h m . I d . , 66, 181 (1950). (54) Clarke, H.T., Johnson, J. R., and Robinson, Sir R., “Chemistry of Penicillin,” Princeton, N. J., Princeton University Press, 1949. (55) Cochrane, V. W.,Econ. Botany, 2, 145 (1948). (56) Cohen, I. R,, and Bennett, R. E., presented before the Division of Agricultural and Food Chemistry, 117th Meeting, AMERICAN CHEMICAL SOCIETY, Philadelphia, Pa. (57) Commercial Solvents Corp., Brit. Patent 021,469 (April 11, 1949). (58)Ibid., 621,552. (59)Zbid., 623,082 (May 11, 1949). (60) Controulis, J., Rebstock, M. C.,and Crooks, H. M., Jr., J. Am. Chem. SOC.,71,2483(1949). (61) Cooper, C. M., Fernstrom, G. A., and Miller, S. A., IND.ENQ. CAEM.,36,504 (1944). (62) Craig, L. C.,Gregory, J. D., and Barry, 0. T., J . Clin. Invest.. 28, 1014 (1949). (63)De Becae, G.,and Liebmann, A. J., IND.ENQ.CHEM.,36,882 (1944). (64)Demerec, M., U. 8. Patent 2,445,748 (July 27, 1948) (65) Denhard, H. W., U. S. Patent 2,490,421(Dec. 6, 1949). (06) Deseive, E.,Milchwissenachaft, 2,141-9 (1947). (67) Dimick, I(. P.,Alderton, G., Lewis, J. C., Lightbody, H. D., and Fevold, H. L., Arch. Biochm.. 15,1 (1947). (68) DuBos, R.J., BUZZ.N . Y . A d . Med., 17,405 (1941). (69) Dudykiva, H.B., iUikrobiob&a, 18,181 (1949). (70) Duggar, B. M., U. 8. Patent 2,482,055(Sept. 13,1949). (71) Dulaney, E.L.,Mycologia, XU,l(1949). (72) Dulaney, E. L., Ruger, M., and Hlavac, C., IW., XLI,388 ’ (1949). (78) Ehrlich, J., Smith, R. M., and Penner, M. A., U. 8. Patent 2,483,892(Oot. 4, 1949).
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D. B., Seeley, D. B., Shull, G. M., Sobin, B. A., Solomons, I. A., Vinson, J. W., and Kane, J. H., Science, 111.85 (1950). (86) Fortune, W. B., McCormick, S. L., Rhodehamel, H. W., and Stefaniak, J. J., IND.ENQ.CHEM.,42, 191 (1950). (87) Foster, J. W., “Chemical Activities of Fungi,” New York, Academic Press, Inc., 1949. (88) Foster, J. W., U. 8. Patent 2,458,495 (Jan. 11, 1949). (89) Foster, J. W., and Heiligman, F., Meeting of Society of American Bacteriologists, Cincinnati, Ohio (May 1949). (90) Foster, J. W., Lally, J. A., and Woodruff, H. B., Science, 110, 507 (1949). (91) Foster, J. W., Woodruff,H. B., Perlman, D., McDaniel, L. E., Wilker, B. L., and Hendlin, D., J. Bact., 51, 695 (1946). (92) Fred, R. B., and Knight, S. G., Snence, 109, 169 (1949). brasil. agron., 8, 377 (1945). (93) Gai, A. F., Bol. SOC. (94) Gallsgher, F. H., Bilford. H. R., Stark, W. H., and Kolachov, P. J., IND. ENQ.CHEM.,34, 1395 (1942). (95) Garcia, R. F., Colon, I. A,, and Fortis, R. C., Rev. Azucar, (rumto R ~ c o )2,, 102-7 (1948). (96) Garibaldi, J. A., and Feeney, R.E., IND. ENQ.CHEM.,41, 432 (1949). (97) Garibaldi, J. A., Ijichi, K., Snell, N. S., and Lewis, J. C., pre-
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September 1950
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(169) Martin, J., U.8.Patent 2,491,927(Dec. 20,1949). (170) Martin, S, M., and Wilson, P. W., presented before the 49th General Meeting of the Society of American Bacteriologists, Cincinnati, Ohio (May 1949). (171) Maaeey, F. C., Penn. Med. J.,49,1090 (1946). (172) Meade, R. E.,Pollard, H.L., and Rodgers, N. E., U. 8. Patent 2,433,063(Dec. 23,1947). (173) Mehlig, J. P., and Shepherd, M. J., Jr., Anal. Chem., 21, 642 (1949). (174) Mehnert, E. S., T a u h Technology, 1,3 (1949). (175) Meier, R. L., Chem. Enq. News, 27,3112 (1949). (176) Meleney, F. L., and Johnson, B., J. Am. Med.ArSQC.,133, 675 (1947). (177) Michie, A. J., Zintel, H. A., Me, R. A., Ravdin, I. S., and R a g i , M.,Surgeru, 26, 626 (1949). (178) Mitchell, W.R., U. S. Patent 2,465,388(March 29,1949). (179) Mohrhoff, W. H., and Mogerman, W. D., Process I d . Quart., 12,l (1949). (180) Monodolfo, H., and Hounie, E., Farmalecta (Busnos Aires), 3, 35 (1948). (181) Moyer, A. J., British Patent 618,416(Feb. 22,1949). (182)Ibid., 624,411 (June 8, 1949). (183) Moyer, A. J., U. 8. Patent 2,476,107(July 12, 1949). (184) Moyer, A.J., and Coghill, R. D., J . Baot., 51,67(1946). (185) Ibid., p. 79. (186) Moyer, W.W.,U.8.Patent 2,477,763(Aug. 2,1949). (187) NaruIa, B. L., and Chawla, B. R., Indian Sugar, 11,116 (1948). (188) Needle, H. C., and Aries, R. S., presented before the Division of Sugar Chemistry & Technology, 116th Meeting, AMERICAN CHEMOAL SOCIETY, Atlantic City, New Jersey. (189) Neieh, A. C.,Can. J. Research, 27B,6 (1949). (190) Nelson, G. E.N., Traufler, D. H., Kelley, 8.E., and Lockwood, L. B., presented before the Division of Agricultural and Food Chemistry, 116th Meeting, AMERICAN CHEMICALS O C I ~ , Atlantic City, N. J. (191) Nichol, C. A., Robblee, A. R., Cravens, W. W., and Elvehjem, C. A., J,Bwl. Chem., 177,631 (1949). (192) Nordsiek, F. W.,Technol. Rev., 47,171 (1946). (193) Olive, T.R., Chcm. Eng., 56, 107,172 (1949). (194) Olson, B. H., and Johnson, M. J., J. Bact., 57,235 (1949). (195) Ono,H., Japanese Patent 155,713 (March 30,1943). (196)Ott, W. H., Rich-, E. L., and Wood, T. R., J. Biol. Chem.,174, 1047 (1948). (197) Owen, W. L., Intern. Sugar J., 51, 81-4, 100-111 (1949). (198) Owen, W. L.,Sugar, 43,364 (1948). (199)Oyaas, J. E.,Ehrlich, J., and Smith, R. M., IND.ENQ.CHEM., 42, 1775 (1960). (200) Paine, T.F., Jr., Collins, H. S., and Finland, M., J. Bad., 56, 489 (1948). (201) Pan, 8. C., Andreasen, A. A,, and Kolachov, P., IND.ENQ. CHEM.,42,1783 (1950). (202) Peck, R. L.. Hoffhine, C. E., Jr., Gale, P., and Folkers, K., J. Am. Chem. SOC.,71,2690(1949). (203) Peeler, H. T., Yacowitz, H., and Norris, L. C., Proc. SOC. Exptl. Biol. Med.,72,615 (1949). (204) Pensack, J. M., Bethke, R. M., and Kennard, D. C., Poultry IS&, 28,398 (1949). (206) Perlman, D., Bull. Torrey Botan. CZub, 76, 79 (1949). (200) Perlman, D., Dorrell, W. W., and Johnson, M. J., Arch. Biochem., 11,131 (1946). (207) Perlman, D.,E t a , D. A,, and Peterson, W. H., Ibid., 11, 123 (1946). (208) Perlman, D., and Woodruff, H. B., Rept. Proc. 4th Intern. C q . Mfcrobiol., 1947,646 (1949). (209) Peterson, W.H.,Snell, J. F., and Frazier, W. C., IND. ENO. CHEM.,37,30(1945). (210) Petty, M. A,, and Matrishin, M., presented before the Meeting of the Society of American Bacteriologists, Cincinnati, Ohio (May 1949). (211) Petty, R.D., Chem.Znds., 66, 184 (1950). (212) Pfeifer, V. F., Tanner, F. W., Jr., Vojnovich, C., and Traufler, D. H., IND. ENO.CHEM.,42, 1776 (1950). (213) Pflzer. Charles & Company. . . Brit. Patent 602,866 (June 4, 1948). (214) Phelps, A. S.,U. S. Patent 2,473,817(June 21,1949). (216)Ibid.. 2,473,818. (216) Pierce, J. V.,Page, A. C., Stockstad, E. L. R., and Jukes, T. H., J . Am. Chsm. Boo., 71,2952 (1949). (217)Pollard, H. L., Rodgers, N. E., and Meade, R. E., U.8. Patent 2,449,140(Sept. 14,1948). (218)Ibid., 2,449,141. (219) Ibid., 2,449,143. (220) Polti, I., Ann. Mdcrobiol. (Milan),3, 101 (1943). (221) Porter, J. N., Broschard, R., Krupka, G., Little, P., and Zellat, J. S., Ann. N. Y. A d . S&., 51,857 (1949). (222) Porter, R. W.,C h . Eng., 53, 142 (1946).
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(302) Waksman, S. A., “Streptomycin-Its Nature and Practical Applications,” Baltimore, Md., Williams and Wilkins Company, 1949. (303) Waksman, S. A., U. S. Patent 2,326,986(Aug. 17,1943). (304) Waksman, S.A.,Frankel, J., and Graessle, O., J. Bact., 58,229 (1949). SOC.Ezptl. Bid. Med., (305) Waksman, S.A,, and Harris, D. A., PTOC. 71,232(1949). (306) Waksman, 5. A., Hutchison, D., and Katz, E., Am. Rea. Tzrbero., 60, 78 (1949). (307) Waksman, S. A.,and Karow, E. O., IND. ENQ.CHEM.,39,821 (1947). (308) Waksman, S. A., and Karow, E. O., U. S. Patent 2,394,031 (Feb. 5,1946). (309) Waksman, 9. A., Ka& E., and Lechevalier, H. A., presented before the Society of American Baoteriologists, Baltimore, Md. (May 1950). (310) Waksman, S. A,, and Lechevalier, H. A., Science, 109, 305 (1949). (311) Waksman, S. A., Lechevalier, H. A., and Harris, D. A., J, Clin. Inueat., 28,934 (1949). (312) Waksman, S. A.,and Schatz, A. U., U. S. Patent 2,449,866 (Sept. 21,1948). (313) Wallerstein, L. W.,I W . , 2,476,785(July 19, 1949). (314) Walton, M.T.,Ibid., 2,368,074(Jan. 23,1946). (316) Weirich, L., U. 8. Dept. Commerce, Chemicals Division, personal communication (1950). (316) Weizmann, C., Brit. Patent 572,637 (Oct. 17, 1945). (317)Ibid., 572,641. (318)Ibid., 572,763(Oat. 23, 1945). (319) Ibid., 573,216(Nov. 12,1945). (320)I W . , 573,930(Dec. 13,1945). (321)West, R.,Science, 107,398 (1948). (322) Western Condensipg Company, Brit. Patent 602,029 (May 19, 1948). (323) Wetzel, N.,Fargo, W. C., Smith, I. H., and Helikson, J., Science, 110,661(1949). (324) White, J., Am. Brewer, 81,21 (1948). (325)Wilson, R.H., Humphreys, E. M., Reynolds, D. M., and Lewis, J. C., PTOC. SOC.Exptl. Biol. Med., 71,700 (1949). (326) Winsten, W. A., and Eigen, E., J. B i d . Chem., 177,989 (1949). (327) Winder, R. J., J . CeZlvlar Comp. Physiol., 17,263 (1941). (328)Woodruff, H.B., Nunheimer, T. D., and Lee, S. B., J. Bud., 54, 535 (1947). (329) Woodward, J. C.,Snell, R. L., and Nicholls, R. S., U.S. Patent 2,492,673(Dec. 27,1949). (330) Yarmola, Q. A.,Mikrobiologiya, 17,471 (1948). (331)Yasuda, Sa’rae, Japan. Patent 172,304(Feb. 8,1946). (332)Yin. H. C., Econ. Botany, 3,184 (1949). (333) Yots, H., et al., Japan. Patent 172,287 (Feb. 8, 1946). RECEIVBD July 1, 1950.
Friedel- Craf t s Reactions mg PHILIP H. GROGGINS and SAMUEL B. DETWILER, JR.,
BUREAU OF
AGRICULTURAL AND INDUSTRIAL CHEMISTRY, U. S. DEPARTMENT OF AGRICULTURE, WASHINGTON, D. C.
HE Friedel-Crafts reaction continues to be a fruitful field
T
for research and development. During the past year a number of new syntheses have been reported. Studies on the mechanism of reaction continue to throw new light on the fundamental concepts of reactions catalyzed by metal halides. Earlier studies by the senior reviewer (IO, 11, 88), involving the use of carboxylic acids and their anhydrides, have been ekewhere re-examined. Reports of reactions involving the preparation of halogeno derivatives have been numerous, and some show potential industrial application.
ACYLATION The Friedel-Crafts condensation of succinic anhydride and 1,2-diphenylethane was shown by Nicholas and Smith (83) to result in the formation of l,Zbis(,9-carboxypropionyIphenyI)ethane :
CoHsCHzCH2CsHs+ (cacoho AlCl: HOOCCHiCOCsHiCH~Cltr2C~H~COCH,COOH Malinovskil and Kislova (U) have extended earlier work of Newton and Groggins (81) on the condensation of carboxylic acids with aryl compounds to yield alkyl aryl ketones. It WBB found that butyric acid and toluene react in the presence of aluminum chloride to give a 35% yield of 4methylbutyrophenone. Isovaleric acid reacts similarly, giving a 39% yield of pCHaCaH&OCH&H(CH&. The authors suggest that the use of acids is more economical than that of acyl halides or anhydrides. This conclusion, however, would apply only to reactions with acids-for example, terephthalic acid-that are converted to acid chlorides or anhydrides with difficulty. By the use of melting point curves, Baddeley (1) has estimated
