FERMENTATION m K V l D PERLMAN, WILLIAM E. BROWN, and SYLVAN B. LEE SOUIBB INSTITUTE FOR M E D I C A L RESEARCH, N E W BRUNSWICK, N. J.
During the past year, antibiotics were again responsible for a large expansion of the fermentation industry, Present production capacity for antibiotics is so large that a situation of oversupply, particularly of penicillin and streptomycin may be attained during 1952. Antibiotics and vitamin B12 made very significant progress as animal feed supplements. Additional citric acid production plants have been completed and are in successful operation. Dextran appears to have a firm position as a plasma volume expander and is being produced b y several companies. The ethyl alcohol and acetone-butyl alcohol fermentation industries face a serious economic situation caured b y lowered demand, high raw material costs, and the low production costs attained by manufacturers using synthetic processes.
EVIEWS of fermentation in this series (S40-$42, 366) have
R
emphasized present industrial fermentations and newer products which may be produced on a commercial scale by microorganisms. This review will be divided into three major sections: economics of the ferment,ation industries; industrial fermentation processes, and fermentation as a unit process (2.41, $42). Several publications have appeared during the past year which deal with various portions of the fermentation industry (51, 153, 173, 381, 638,523,~07,413).The revien. by Langlylrlre, Smythe, and Perlman (238)covers the practiral problems of the fermentation industry and has placed emphasis on the production of ena p e s for industrial uses, Raper (323) has contributed an exDecade of iintibiotics in America.” cellent review entit,led Kluvver ($6’1) has reviewed microbial metabolism and its industrial application. Govindarajan (165) has published a review of the progress of the fermentation industry in India, a n d Verona (407)has published a textbook in Italy dealing w i t h industrial microbiology. ~I
with demand and this has resulted in marked price reductions. It appears that in the future those companies with the most efficient product’ion processes and marketing facilities will be the major producers of these drugs. Decreasing prices and intensified sales promotion indicate that with Chloromycetin (chloramphenicol ), aureomycin, and terramycin, a similar situation is developing. In the fermentation production of ethyl and butyl alcohols, there is also an acute economic situation caused primarily by high costs of rav- materials, principally molasses and grains, thereby making it increasingly difficult t o compete with the low cost synthetic processes in a market where supply is greater than demand. I t appears that the citric acid fermentation industry may become more competitive with the advent of new producers. For several years ene company has been producing 95% or more of the tot’alcitric acid in this country. In considering the economic status of the fermentation industry in the United States, it should be pointed out that most companies reported high earnings in 1951 and that 1952 will most likely reflect the economic trends which became evident a t the end of 1951. E X P A N S I O N OF P R O D U C T I O N FACILITIES
ECONOMICS OF THE FERMENTATION INDUSTRIES During the past year, the economic picture has been critical in many of the fermentation industries; therefore, it seems desirable t o devote a portion of this review t o a discussion of some of the prime factors i n v o l v e d i n t h e present economic situation. Enlarged production f a c i l i t i e s a n d improved processes have resulted in vastly increased production of penicillin and streptomycin throughout t h e world. Supply i n t h e United States is apparently nearing an equal status
Group of Ten Gallon Pilot Plant Fermentors
1996
Antibiotics. During the past year, nearly all of the domestic producers of antibiotics h a v e c o m p l e t e d major expansions of their production facilities or are doing so a t the present time (45, 48, 68, 68, 74, 75, 78, 80, 88-84, 86). The greater portion of the expansion, which vi11 cost approximately $45,000,000 a n d was authorized b y Defense Production Authority through “Certificates of Kecessity,” was earmarked for p e n i c i l l i n and streptomycin production. However, major inc r e a s e s in manufacturing facilities for production of the newer antibiotics are also included (60). -4ntibiotic p r o d u e t i o n facilities in foreign countries are expanding rapidly and the competition from
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INDUSTRIAL AND ENGINEERING CHEMISTRY
these plants is undoubtedly in part responsible for the lowered prices in this country. During the past period, new plants have been completed, existing plants have been enlarged, or plans for additional plants have been announced in the following countries : Spain (69, 85), Germany, Italy, France (69), Great Britain (66), India (67, 63), Austria (76), Australia (81, 86), Argentina (“7, and Thailand (90). Dextran. The production of dextran for use as a blood plasma expander has received increased attention m d several companies in the United States and abroad have announced plans for the construction of plants (6R,78, 418). At present dextran for use as a plasma extender is of chief interest to the armed services. However, industrial concerns which produce dextran are of the opinion t h a t there will be a sizable civilian market. Citric Acid. Citric acid production facilities are being expanded and new producers are entering the field in this country and abroad. Charles Pfizer & Co., the leader in this industry, is greatly expanding its plant capacity and is reported to be using both the submerged and surface culture processes (69, 79). Miles Laboratories has completed its new plant and is in production (3&). The Staufler Chemical Co. announced plans to have a plant for the production of citric acid from beet molasses completed by the end of 1951 (83)and other firms in this country are reported to be interested in citric acid production (77). Abroad, a new plant is being built to supply citric acid for Canada (71) and a plant in Argentina established in 1945 has expanded to produce 40,000 pounds per month (367). QUANTITIES OF F E R M E N T A T I O N PRODUCTS PRODUCED
Chemicals. Ethyl alcohol production for industrial uses continued t o increase during 1951 but the proportion of the total produced by fermentation continued t o decline as in previous years (349). It is estimated t h a t of a total of approximately 450,000,000 proof gallons produced during the fiscal year ending June 30, 1951, no more than 40 t o 45% was produced by the fermentation industry. This was the result of the constant expansion of synthetic production facilities by the petrochemical industries. Production costs from synthetic processes are appreciably lower than by fermentation. The higher cost of the fermentation product resulted from increased prices of grain and molasses and the relative unavailability of these raw materials because of increased use in livestock feeds. This, along with decreased selling prices, has caused the shutdown of some fermentation plants and others are operating on a very narrow profit margin. One large plant on the West Coast which uses molasses has recently discontinued production after several decades of continuous operation. At the present time, the price of blackstrap molasses appears to be on the downward trend from a peak of 35 to 40 cent’s per gallon. However, the selling price of alcohol is decreasing at a more rapid rate and thus it appears that the alcohol fermentation industry profitwise may be a marginal business for a period of time. Significant quantities of ethyl alcohol (approximately 120,000,000gallons) for use in production of rubberlike polymers have been imported from France and other foreign countries where fermentation production costs are lower than in the United States (88). Interest in ethyl alcohol for fuel from the fermentation of farm wastes has also decreased. The pilot plant a t the Government’s Northern Regional Research Laboratory has discontinued these studies. The story of the production of n-butyl alcohol and acetone by fermentation is identical to that of ethyl alcohol. High raw material costs for fermentation and the expansion of plants using synthetic processes, capable of lower production costs, have also resulted in marginal profits in this fermentation industry. For example, it has been reported (66) that in April 1951 the selling price of n-butyl alcohol produced by the fermentation of molasses was 28 to 35 cents per pound while that produced
1997
by the synthetic process sold for l i to 18 cents per pound. Approximately 15 to 20% of the 150,000,000 pounds of n-butyl alcohol produced during 1951 was manufactured by the fermentation industries. Citric acid production for 1951 has been estimated to be approximately 50,000 pounds. While the price of 27.5 cents per pound r e w i n e d firm during 1950, it seems quite possible that this price will decrease as additional fermentation plants attain full scale operation. Although total production figures are not available for riboflavin, this product is being produced in ever-increasing quantities from both synthetic and fermentation processes. Riboflavin is used in the pure and concentrated forms, respectively, for human and animal consumption. While data are not available regarding the relative amounts produced by fermentation and synthesis or the relative costs of production by the processes, it is believed that in this case the fermentation process should be able to compete quite successfully with the synthetic chemical process. Vitamin B I ~one , of the newest products to be produced on a large scale by the fermentation industry, currently enjoys a very favorable position as i t is used in appreciable quantities in the pure or concentrated forms for human nutrition and therapy and in the crude form for the nutrition of farm animals. The price of the purified material dropped from $595 t o $350 per gram during 1951, reflecting improved production processes and a competitive sales situation. The market for animal feed, where B I serves ~ as the&,nimal protein factor” and replaces expensive animal proteins in the ration, has increased tremendously during the last year. This use shows considerable promise for future expansion of fermentation production unless other sources of vitamin BIZ,such as treated wastes from sewage disposal plants (271),prove to be practical sources of BIZfor animal feeds. Antibiotics. The volume of antibiotic production increased at an accelerated rate during 1951. While production figures for the individual broad spsctrum antibiotics, Chloromycetin (Chloramphenicol), aureomycin, and terramycin, are not available, output is increasing rapidly and most recent estimates place the total domestic production at approximately 12,000,000 grams per month. Chloromycetin and aureomycin are being produced a t approximately equal volumes with terramycin a close third (186).
The production figures for penicillin and streptomycin (Table I ) reflect the relative expansion in volume of the antibiotic industry during 1951. ~
Table
I.
Production of Penicillin
Penicillin Billion unitsa Pounds Streptomycin Billion unitaa Pounds
and
Streptomycin (421)
1949
1950
1951
133,464 176,400
222,305 293,800
324,293 428,500
83,700 184,200
92,447 203,800
159,494 350,000
a Conversion figures were derived by assuming 0.6 microgram per unit for penicillin and 1.0 microgram per unit for streptomycin. Streptomycin figure includes 144,344 billion units dihydrostreptomycin.
Penicillin production in 1951 increased approximately 45% over 1950 and i t has been estimated that when current industrial expansion is completed at the end of 1952, this country’s production capacity will be approximately 40 to 45 X 10I2 units per month or the equivalert of a potential annual capacity of 480 to 540 X 10l2units (136). It is doubtful that this production will be attained in 1952 owing to the recent decline in penicillin sales prices. This indicates that the supply of penicillin is becoming greater than the demand and several producers have al-
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INDUSTRIAL AND ENGINEERING CHEMISTRY
ready curtailed or ceased production. During the period April 1951 t o May 1952, the wholesale price per 100,000 units of procaine penicillin G in bulk quantities decreased fron 5.5 cents to approximately 1.9 cents. Apparent factors in the price decline were the reduced stockpiling by the armed services and the relatively static export market which amounted t o 31% of the production in 1950 (91,000 pounds) and 25% in 1951 (117,000 pounds). The future of the penicillin industry appears rather indeterminate a t the present time and it seems probable that among the factors which will be of importance in determining which producers will continue profitable operations arc: Quality of product Cost of production Cost of packaging Sales promotion and retail sales outlets Specialty formulations Specialty penicillin derivatives
*
I n general, the streptomycin production and economic situation is very similar to penicillin. Streptomycin production in 1951 increased approximately 70% over 1950 and it is estimated that it is currently being produced a t a rate of 20 t o 25 million grams per month, or at an annual rate of 240 to 300 million grams. If this production rate is maintained, further price declines will probably occur. Streptomycin exports decreased from 72% of total production in 1950 (146,736 pounds) to 58% in 1951 (203,000 pounds). Another factor which must be considered in connection with the streptomycin picture is the possible effect of isonicotinic acid hydrazide, the new synthetic antituberculosis drug. This drug, which was just released for distribution and sale, may have adverse effects on streptomycin sales However, a t the moment, the leading tuberculosis clinicians seem to be of the opinion that streptomycin and isonicotinic acid hydrazide may be used jointly for the treatment of this disease. The supply of broad spectrum antibiotics has not yet equaled worldwide demand and the competitive situation is not as acute &s with penicillin and streptomycin.
INDUSTRIAL FERMENTATION PROCESSES Except where other\yise stated, progress in the various fermentation processes prior to 1951 has been reviewed (140-842, 366) and only the recent developments are considered in this survey. SOLVENTS
Ethyl Alcohol. The competetive situation in the production of industrial ethyl alcohol in the United States has been described and evaluated in detail (60). The volume of fermentation production from such carbohydrate sources as wood hydrolyzates, sulfite waste liquor, and surplus farm produce (for example, potatoes) has not amounted to a significant fraction of the total production and competition from synthetic chemical processes largely precludes the use of these materials except in unusual circumstances (228). 4 similar study of industrial ethyl alcohol production in Canada indicated that production from grain accounted for nearly all that is produced in that country (39, 259). However, some plants used sulfite waste liquor as the raw material. Dawson ( I I S ) , who has reviewed the production of industrial alcohol from molasses, stated that batch rather than continuous processes are used in Great Britain. Re-use of the yeast (Boinot procees) has found favor and although some savings resulted from this type of operation, it appears t h a t the cost of production of fermentation alcohol will continue to depend to a very large extent on the cost of molasses. The use of fungal amylases instead of malt in the production of ethyl alcohol from grain has been mentioned in earlier reviews in
Vol. 44, No. 9
this series. A rather complete outline of the development of the process, including a summary of its advantages over the malt process as well as equipment requirements, has been presented by Jackson et al. (20W). A successful trial run using the process of the Northern Regional Research Laboratory has been made in a fermentation plant equipped with 100,000-gallon fermentors (YO). Under optimum conditions, a reduction in manufacturing cost of approximately 3 to 4 cents per gallon of neutral spirits may be obtained if fungal amylase is used instead of malt. However, apparently the cost of new equipment was not charged against this reduction in cost, and the real advantage may be less. Problems in the preparation of submerged fungal amylase have been summarized and fermentation conditions leading to elaboration of a-amylase and maltase have been studied (109). The use of fungal amylase as a malt replacement has also heen considered in connection with the fermentation of potato mashes (329) and grains other than corn (109, 936). While a culture of Aspergillus niger has been used as amylase source in the Northern Regional Research Laboratory process and the Seagram continuous process (129), Aspergillus oryzae, Rhizopus boulard, and Rhizopus delemar have also been used in large-scale equipment (185). Ethyl alcohol produced by means of processes using fungal amylase must be sold as industrial alcohol and not as beverage alcohol. Pan et al. (698) observed that hydrolysis of starch by a fungal amylase preparation was speeded when the maltose formed was removed by yeast fermentation. The production of ethyl alcohol from wastes of the food, paper, and other industries has received continued attention. Wastes from fruit canneries were fermented efficiently ( 2 )as were potato residues from starch plants (286). Acclimatization of the yeast t o inhibitory materials present in these wastes, as has been the practice in the fermentation of sulfite waste liquor (214),mag be necessary for efficient operation. Increased fermentation rates have been obtained by addition of such materials as water-insoluble, inactivated sludge ( $ 7 2 ) and malt sprouts extract (3555). In the latter case, concentrate5 of the fat-soluble, unsaponifiable fraction of malt sprouts contained most of the activity, The effect of vitamin deficiencies on glucose fermentation by yeasts has also received attention (191). The interpretation of the data indicated that biotin, pyridoxine, and pantothenic acid were indirectly involved in fermentation while nicotinic acid and thiamine were directly involved. Butyl Alcohol and Acetone. The volume of butyl alcohol and acetone produced by fermentation processes in the United States has declined sharply during the past few years. Hoaever, fermentation processes are being used in other countries, and a number of reports which discussed various aspects have appeared in the recent literature. +4list of more than 250 publications which appeared during the past 50 years is Ztvailable ( 2 3 3 ) a i is a summary of contemporary thought on the biochemical mechanism of these fermentations (149). One report suggested the use of monospore isolates (188) in order to obtain uniform performance. Nitrogen sources used in Clostridium acetobutylicum fermentations were cottonseed oil meal, copra, soybean cakc, casein, fish protein, zein, gluten, yeast protein, and gelatin ($88, 401). Cassava was suggested as a possible carbohydrate source (401). Fermentation rates were reported t o be stimulated by the addition of liver, rice, bran, a-alanine, a-methyl phenethylamine sulfate, or p-amino benzoic acid (289). The use of castor oil as a supplement (178181) permitted the fermentation of molasses and corn or sweet potato mashes having carbohydrate contents as high as 15%. It was claimed that the oil absorbed the solvents as they are produced and thereby reduced the inhibitory effect. Other studies suggested t h a t with certain cultures, when high carbohydrate concentrations were used, best results were obtained when fermentation temperatures were maintained at 33' C. ($89). Vergnaud (406) claimed that additions of acid t o control the p H of the fermenting mash resulted in higher solvent yields.
September 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
ORGANIC ACIDS
Citric Acid. That the problems associated with the mycological production of citric acid by the submerged culture process are being solved is evidenced by the three new plants which are in o eration. I n confirmation of earlier studies by Sakaguchi a n d g a b a (346), Moyer (276) has observed that the low yields obtained in molasses media were substantially increased when methyl alcohol was added as a supplement. Methanol or other substances including ethyl iodide and thiourea acted indirectly on the fermentation by sequestering certain of the metallic ions (348). Ferricyanide has also been studied and appears to be as practical in submerged as in surface culture fermentations (98, 260). The role of metallic ions in the production of citric acid by Aspergillus niger appears to be associated in part with degradation of the citric acid formed (94, 306,332, 348). The prepence of certain fatty acids in the medium including butyric, capric, and caprylic acids was claimed to reduce the yield of citric acid (297). Studies on the mechanism of citric acid formation by A. niger have suggested that in surface and submerged culture the carbon from fixed carbon dioxide accounts for less than 10% of the carbon in the citric acid formed (48,246,261). The position of the fixed carbon dioxide was accounted for in part by recycling. It was demonstrated that acetate may be a building block for citric acid formation (42, 245). Searches for precursors of citric acid have been made by following acid synthesis after the addition of known metabolites and metabolic poisons (4, 97, 111, 344-346). It was reported that aconitic acid will accumulate in quantity when methylene blue is added ta citric acid fermentations (347). Shu and Thorn (364)have constructed oxidation-reduction and carbon balances for the metabolism of sucrose by A. niqer (Wis. 72-4) grown in submerged culture.
Lactic Acid. All of the lactic acid produced for the food industries is prepared by microbiological processes. Lactic acid used in chemical industries is mainly the synthetic product. Few reports have become available in recent years concerning factors affecting microbiological production of lactic acid. Studies on lactic acid production by the genus Rhizopus, while of little interest industrially due to the low yields, indicated some of the pathways of mold metabolism. Apparently yields are dependent on the efficiency of aeration and p H control (41, 341, 342). Highest yields were obtained when the pH was maintained a t neutrality (343). Lactic acid was formed by Rhizopus nigricans when grown on either glucose xylose. or acetate, but Rhizopus oryzae formed lactic acid oniy from glucose and formed fumaric acid when grown on xylose and acetate (218).
Itaconic Acid. Because of its possible use in vinyl plasticizers (63)interest in itaconic acid has continued. The effects of changing fermentation conditions on the production of itaconic acid by Aspergillus terreus NRRL 1960 has been studied in 20-liter fermentors (281). Yields of 50%, baqed on the fermented glucose, have been obtained. These yields are somewhat higher than those obtained in fermentations of unrefined cane sugar by the surface culture process (423) where it was found that a substance in molasses ash stimulated acid formation,
Gluconic, Ketogluconic, a-Ketoglutaric and Related Acids.
A study of conditions affecting production of gluconic acid by bacterin (Pseudomonas s p . ) indicated that high aeration along with pH 3.8 to 5.5, resulted in weight yields of 101.7% based on the glucose (211). Of forty bacteria tested, Pseudomonas glum i c u m and Acetobacter suboxtjdans oxidized glucose to gluconic acid, while Pseudomonas fEuorescens and Serratia marcescens oxidized gluconic acid to 2-ketogluconic acid (196). These activities were partly correlated with the glucose-oxidase content of the cells (198). Blom et al. (SO,31) have studied conditions which affected the production of gluconic acid by Aspergillus niger grown in submerged culture in ilot plant fermentors. Maximum efficiency resulted when a 3 0 g glucose medium was used, provided the acid formed was continuously neutralized, Gluconic acid has also been identified as a metabolic product in the metabolism of fat by A. niger (358). Glucose was converted by Pseudomonas fluorescens and Serratia marcescens to 2-keto luconic acid with yields of about 75y0 of theory based on the &wose fermented (197). Yields of 90 to 95% have been obtained in .Cyanococcus chromospirans fermentations (377). 2-Ketoglucon1cacid was also found as a metabolic product of Acetobacter orleanense, Acetobacter pasteuri-
1999
anum, Acetobacter acetigenum, and Acetobacter turbidans, while 5-ketogluconic acid waR produced by A. acetigenum, A. orleanense, A. viscosum, A. kutzingianum, A . pasteurianum, and A. turbidans (232). A. melanogenum cultures formed gluconic and 5-ketogluconic from maltose (138), while certain Pseudomonas strains formed maltobionic and lactobionic acids from maltose and lactose, respectively, in yields of 60 to 90% (882). It was claimed that in a 17-day fermentation, Pseudomonas Jluorescens oxidized 5-ketogluconic acid t o tartaric acid with a yield of 25% (251). Other studies have reported @-ketoglutaric acid as a metabolite of Penicillium chrysogenum (186) and Aspergillus niger (416). The latter mold also formed pyruvic and dimethyl pyruvic acids (319).
Acetic Acid. The production of acetic acid from ethanol is one of the oldest known fermentations; however, to gain increased efficiency, there are still many problems t o be solved pertaining t o enzymology and equipment (166). Hromatka and Ebner (193-195) have considered the role of oxygen in the submerged culture process for production of acetic acid. When the oxygen content of the air was reduced to 4.5%, the fermentation ceased because of the accumulation of large amounts of acetaldehyde. Allgeier et al. (8) have considered the effect of the mineral content of dilution water on the operation of the generator. Comparison of a number of water samples demonstrated that, in general, the more desirable waters had low solids, low hardness, and low chloride content. I n all cases, treated waters-for example, those that have undergone chemical precipitation, aeration, and chlorination-were better than well waters. Yields were lowered approximately 8% in media prepared with untreated water which analyzed for total hardness in the range of 175 to 600 p.p.m. DEXTRAN AND OTHER POLYSACCHARIDES
The background and some of the problems involved in production of dextran for use as a blood plasma expander were mentioned in the previous review (84%). Of great interest is the recent report by Hehre (174)who found that a Streptococcus studied in the authors' laboratory was capable of producing a polysaccharide with a molecular weight of 70,000, the most favored size. He reported yields of 40 grams of dextran from 200 grams of sucrose and only small quantities of larger and smaller polysaccharides were found. This fermentation could largely eliminate the fractionation necessary for the preparation of plasma volume expander from larger polysaccharides produced by Leuconostoc sp. (131, 379). Koepsell and Tsuchiya (226) confirmed and extended the previous work of Hehre (176)when they demonstrated that cell-free preparations of Leuconostoc mesenteroides were able to form polysaccharides from sucrose. These workers have studied the effect of pH, temperature, and method of preparing the enzymatic extracts on the formation of dextrans by cell-free extracts (227, 400). The dextran sucrase from one strain of L. mesenteroides produced an essentially linear polymer, while that from another strain produced a highly branched polymer. Several new oligosaccharides were formed when glucose, maltose, and isomaltose were added along with sucrose in amounts equimolar to the sucrose. Leucrose, a fructose-glucose disaccharide, and panose, a trisaccharide, have been produced in quantity. Other developments include studies of the hydrolysis of dextran by enzyme preparations from bacteria and molds (393,428). The general problems in dextran production have been briefly summarized and literature reviews prepared (176,203). Studies of the nutritional requirements for dextran production by and for the growth of L. mesenteroides have also continued (376, 429) with the general conclusion that the requirements are not necessarily related. Dextran production by certain Acetobacter species has also been studied. Acetobacter capsulatum, when grown on dextrin, formed a polymer of D-glucopyranose units linked principally in the one to six positions (177). A soluble enzyme system, dextran-dextrinase, from A . capsulatum, synthesized, in vitro, serologically active dextran from dextrin. Commercial grades of maltose yielded small amounts of dextran, while no dextran was
2000
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
formed from dextrin-free maltose. This enzyme also synthesized dextran from molecules as small as amylotetrose. The Armed Services Medical Procurement Agency, for which most of the dextran blood plasma expander has been produced, has set minimum standards and specifications similar to existing standards for blood plasma. I n addition to sterility and freedom from metals and pyrogens, dextran solutions must pass antigenicity tests in rabbit,, and anaphylactic tests in guinea pigs (73). Hydrodextran, produced by catalytic hydrogenation, was suggested to be more desirable lhan dextran because of its greater stability when boiled in alkaline solutions (446). MICROBIOLOGICALOXIDATION OF STEROIDS
The realization of the importance in chemotherapy of cortisone ( 11-dehydrel’i-hydroxycorticosterone) and hydrocortisone ( 17-
hydroxycorticosterone) has stimulated the search for synthetic methods which would be less costly than the current chemical syntheses which use bile acids as the raw material. One of tho more costly operations in chemical synthesis of cortisone is the introduction of oxygen in the eleven position of the steroid nucleus. Colingaworth et al. (105) have reported that in Streptomyces jraddae fermentations, 17-hydroxy-11-desoxycorticosterone was apparently converted to a substance having 17-hydroxycorticosterone activity. Peterson and Murray (312) obscrvcd that Rhizopus urrhizus and other Mucorales sp. oxidized progesterone to 11,a-hydroxyprogesteroneand a dihydroxyproge*terone. These microbiological modifications eliminate several steps in the chemical synthesis of cortisone. These processes appeared so promising that coiistruction of a $3,300,000 production plant WM planned (62, 9.2, 126). Apparently microorganisms are capable of fornling a variety of oxidation products from progesterone including 16,whydroxyprogesterone; pregnanol-16,a-dione-3,20; and dihydroxyprogesterone (309). Fried et al. (140) have reported that a number of Aspergilli including Aspergillus niger converted progesterone t o 11,aprogesterone and 17-hydroxy-11-desoxycorticosterone to 17hydroxy-11,a-hydroxycorticosterone.They converted the latter substance to cortisone by chemical oxidation, and thus demonstrated that a practical process has been developed for the production of cortisone using a microbiological step. VITAMINS
Vitamin Biz. Studies of the chemistry and production of the vitamin Bla complex have continued in a number of laboratories. Several reviews of the early chemical developments have been published (,%’OfJ, 371, 396). A number of papers have discussed the relationships of vitamin BU to BIZ,,, Bm, and B m (107, 116, 207, 208, 369, 370, 430) and it appears that vitamins BIZ,, BIX, and BlZd we identical. Some actinomycetes and bacteria produce a form of vitamin Bl2 (perhaps the hydroxo form) capable of forming stable cyanide and dicyanide complexes. Another form produced by microorganisms was named “pseudo vitamin B12.” This WM the product of an anaerobic fermentation and had no activity in the chick growth test but stimulated the growth of vitamin BIZ requiring Lactobacilli. This bacterial growth factor differs from cyano-cobalamin in that it has adenine instead of the 5,6-dimethylbensimidazolemoiety (117). The search for microorganisnis producing vitamin BIZ nientioned in the previous review (242) has continued. Nearly all of the actinomycetes and some of the bacteria were found to produce vitamin Blz-like activity (37, 350). The highest yields reported were in the range of 8 mg. per liter of fermented broth (552). Gelection of actinomycete cultures after exposure to lethal agenta has given strains which produce larger amounts of vitamin Blz (566). While actinomycete cultures seem to have found widespread use for production of vitamin BIZ,there has also heen interest in certain cultures of Propionibacterium (164).
Vol. 44, No. 9
Sewagc wastes froin the activated sludgc process appeared to contain as much as 4 p.p.m. of vitamin BIZ, presumably produced by microorganisms (125,190,271). A number of reports discussed the fermentation aspects of the production of vitamin B12 and associated factors, Hall et al. (161) have summarized their studies with an actinomycete classified as Streptomyces olivaceus. They reported that this culture grew well and produced vitamin B12 and related factors on media containing soybean meal, glucose, distillers solubles, and salts. Yields of 3 p.p.m. have been obtained in a 3-day fermentation in laboratory scale equipment (73, 525). This organism is reported to be in use in a number of commercial processes producing animal feed supplement*. Garey et al. (146) reported than an actinomycet’egave yields of 4 p.p.m. in laboratory equipment when grown in a medium containing soybean meal, animal stick liquor, distillers solubles, glucose, and salts. These investigators found t.hat divided additions of cobalt salts were advisable because the addition of 25 p.p.m. cobalt initially inhibited growth and Biz production. This observation was also made with Streptomyces griseus (318, 434). Studies employing Coeoindicated that only 7 t o 17% of the cobalt added eventually appeared in the vitamin Biz (333). Other studies were concerned with factors affecting production of vitamin BIZcomplex by Streptomyces aureofacim and S. gi,iseus (330, 331, $91). The major portion of the vitamin x t i v i t y was found loosely bound t o the cells of several actinomycetes and bacteria and was freed by treatment with a.cid, salts, sonic disintegration, or heating in aqueous media (287, 391). Hodge et ul. (187) have given further details concerning the production of vitamin Blz by a mixed anaerobic fermentation. Pseudomonas sp., Streptococcus bovis, Aerobactw sp., Proteus vulgaris, and Clostridium putrificum were grown together in a medium containing soybean mesl and carbohydrate. A4nother process which received industrial attention (67) and which was described earlier (148) used a strain of Bacillus megatheriutn grown in an intensely aerated molasses media. (147). Riboflavin. While microbiologically produced riboflavin has accounted for a substantial portion of the riboflavin manufactured, only a limited number of reports describing technical aspect,s of this biosynthetic process have appeared during the past year. The nutritional requirements for growth and for riboflavin production by Erewzotheciu?n ashbyii, the organism most Lvidely used for riboflavin production, have been studied a t length. Best results have been observed in media which contained inositol, biotin, and thiamin added either as pure materials or in natural substances (231, 278-275). Synthetic media (386) have heen used as well as those containing such nutrients aF potato starch, ground malt, and \%-heatflour (171,388). Contamiriat.ion of E. ashbyii fermentations with bacteria has resulted in drastic reduction in riboflavin yields. Some control of these contaminants may be achieved by inclusion of antibiotics such as penicillin in the media (184), but such practices are poor substitutes for aseptic fermentation techniques. Pridham (317) has described the production of riboflavin by .Ishbya gossypii in fermentations periodically supplemented with glucose. Maximum yields were 1.8 mg. per ml., somewhat Ion-er than the maximal yields reported for E. ashbyii. I n addition to riboflavin, significant quantities of vitamin BIZ, Lactobacillus bulgaiicus factor, and other growth factors were produced in A . gossypii fermentations (568). Media containing stillage and animal proteins such as meat stick have been recommended (568, $90). Best results were obtained when “flash sterilization” techniques were used in the preparation of the media (313,368). While the two related fungi mentioned above have apparently accounted for production of nearly all of the biosynthetically produced riboflavin, other microorganisms have been studied. Inositol was required for maximum production by Clostridium acetobutylicurn grown in synthetic media (RIO), along with pantothenate, p-amhobensoate, pyridoxine, niacin, biotin, and folic
September 1952
INDUSTRJAL AND ENGINEERING CHEMISTRY
acid. Cereal mashes containing wheat flour have besn found suitable for riboflavin production by Cl. acetobutylicum cultures (943). The literature prior to 1951 concerned with riboflavin production by bacteria, fungi, and yeast was recently summarized by Pridham (316). Special attention was given to medium composition, strain and culture selection, and fermentation techniques. Other Vitamins and Growth Factors. While the isolation of biocytin from yeast was announced some years ago, a description of the isolation and the elucidation of its structure has recently become available (437). Biocytin has been shown to be E-Nbiotinyl-2-lysine and presumably accounts for the bound biotin in yeast (433,438). The sources and occurrence of the Lactobacillus bulgaricus factor (LBF) have been mentioned in previous reviews. Recent reports indicated that this factor is one of the many conjugated forms of pantothenic acid related to Coenzyme A (34,949). It has been produced by cultures of Bacillus subtilis (408), Ashbya gossypii (368))and probably Streptomyces fradiae (166). The studies on release of pantothenic acid activity from yeast cells are of interest in connection with the occurrence of LBF or pantotheirie in yeast (136). Lipoic acid, one of a group of growth factors found in yeast, liver, and other natural materials and required for the growth of certain strains of Corynebacterium and Streptococci, has been isolated in crystalline form (169, 325, 336). It apparently is related to or identical with the factor named “protogen-B,” required for growth of Tetrahymena gel& (314,367). In former years, many of the new growth factors were isolated or prepared as concentrates from liver or yeast. More recently fermentation liquors from various cultures have been looked upon as sources of new factors. An example was the observation that coprogen, a growth factor required for the coprophilic fungus Philobolus kleinii, was found in many fungi and bacteria (182). Coprogen is an iron-containing organic complex, and presumably is the “dung” factor required for the growth cycle of Philobolus sp. ENZYMES OF INDUSTRIAL INTEREST
During the past year, several reviews have been concerned with the properties and production of enzymes. The encyclopedic treatise edited by Sumner and Myrback (383) ha8 considered the properties of a number of enzymes and enzyme systems. Smythe (372) has discussed the microbiological production of enzymes and their industrial applications. H e included carbohydrases, proteases, lipases, and pectinases in his survey. Horwood (199) has covered the same general subject with respect to the technology in Great Britain. Many of the recent literature reports were concerned with production of amylases, but little information has become available regarding industrial practices. However, a few patents have been issued which presumably reflect industrial techniques. The submerged culture process has found favor for growing Bacillus subtilis and Bacillus mesentem’cus (189, 374)) and fungi (108) although the surface culture method, employing bran or other grain products, is still used (dO4,960,360). Increased production of bacterial amylase was achieved by the addition of tannin to the medium (996),control of the inorganic content of the medium (9662),or control of the carbohydrate content (424). Addition of‘ aluminum powder to media used for growth of Rhizopus bmlard, Rhizopus delemar, or Aspergillus oryzae resulted in increased production of enzymatic activity (183). Composition of media with respect t o nitrogenous constituents and p H control was important for satisfactory amylase production when cultures of aspergilli were used (10.9, 143,363). The characteristics of extracellular proteolytic enzymes from actinomycetes and molds have been surveyed with particular attention to the effect of fermentation conditions and composition
2001
of medium on production of enzymatic activity (118, 119, 263). It has been reported that certain actinomycetes produce substantial quantities of extracellular proteolytic enzymes when grown in submerged culture (963). A similar survey of fungi indicated that when grown on bran, members of the Aspergillus JEavus-oryzae group produced more extracellular proteinase than did other species (268). Lipases produced by fungi have received some attention (39(?-32@). Preliminary studies suggested that crude fungal extracts contained a mixture of enzymes (361). An industrial process for lipase production used Aspergillus luchuensis grown on a moist grain medium (378). Neuberg and Mandl (283) have described the preparation of D-and h i m i n o acids from D,L mixtures by enzymatic resolution. Acyl derivatives of D,camino acids are asymmetrically cleaved by commercially available acylases found in mold preparations. Optically pure hamino acids and D-acyl amino acids were obtained in almost quantitative yields. Attention has been given t o the use of mixtures of streptokinase and streptodornase t o disintegrate dead tissue and blood clots. Apparently certain cultures of Streptococci produce larger quantities of these enzymes. The development of this process has reached the pilot plant (69). MICROORGANISMS FOR FOOD AND FEED
Yeasts. The production of food yeast as a method of utilizing dilute carbohydraiqcontaining wastes has received continued attention. Wiley et al. (431) have grown Torula utilis in sulfite waste liquor media in a 20,000-gallon fermentor. The liquor, containing 1.5% sugar solids (80% hexose, 2001, pentose), was fed continuously and the average holding time was 4 hours. It was suggested that certain variants of yeast may be more adaptable t o this medium than others which will not tolerate sulfur dioxide (364). Distillers slop has also served as a medium for T . utilis (304, 410), while Saccharomyces fragilis has been proposed for disposal of whey and milk (316). The dissolved solids in spent grain liquor, a brewery waste, have been used in media for the growth of Saccharomyces cerevisiae (248). Growth of yeast8 on such media has resulted in reduction of approximately 60% of the biological oxygen demand, an important factor in considering the economics of this method for waste disposal. A number of problems associated with growth of bakers’ yeast on a large scale, using molasses as the major nutrient (47) have been reviewed by Garey (146). The effect of aeration methods and the role of oxygen in growth of yeast has receivcd continued study with indications that an exponential increase in yeast multiplication takes place only when nutritional requirements and aeration are adequhte (205, 425). Bioassays (using LactobaciZli as test organisms) of raw materials for the B vitamins required by yeasts were shown t o be of little value in predicting the usefulness of these raw materials in the manufacture of baker’s yeast (426, 427). While blends of materials seemed necessary t o give best results, high test cane molasses even in admixture with other molasses was undesirable, and mixtures of beet and refiners molasses were preferred. Methods of flocculation of yeast have been summarized (36) and conditions promoting preparation of easily plasmolized yeast (236) have been surveyed, as well as other studies of processihg techniques in preparation of finished yeast products (234). Algae. Cook (106) has described the chemical engineering problems encountered in large-scale culture of algae using Chlorella pvrenoidosa. Laboratory experiments in glass columns have been carried out using irradiation by both sunlight and artificial lights. Yields of 0.4 gram dry weight per liter per 24 hours have been obtained, with a protein content of about 50% of the cell weight. By restricting the nitrogen content of the medium, the fat content of the cells was increased to 80% of the dry weight (66,106, 900). When chhlorella vulgaris was grown
2002
INDUSTRIAL AND ENGINEERING CHEMISTRY
a t 30' C. in light (200 foot-candles) on a 1%glucose medium, 83 to 88% of the glucose carbon was converted to cellular material. Lower yields were obtained when other carbohydrates were used (280). Synthesis of Fats. Synthesis of fats by a number of fungi has been surveyed and mycelia of Aspergillus Jlavipes was found t o contain 40% of the dry weight as lipid (432). Fusaria have been found to convert carbohydrate t o fat with conversion coefficients ranging from 5 t o 11.5 (620). The fat content of Rhodotorula cells increased to 39% of the dry-cell weight when the incubation period was lengthened to 63 hours (219). The temperature of incubation affected lipid synthesis by Peniczllium chrysogenum when grown on Czapek-Dox medium (199) and probably affects synthesis by other microorganisms. Lundin (665, 266) has indicated that fat synthesis by microorganism^ has little chance of industrial exploitation a t present. Antibiotic Feed Supplements. As mentioned in the pievious review (84%'),the use of antibiotics as supplements in the preparation of rations for poultry, hogs, and cattle has opened new markets for these fermentation products. While a discussion of the formulation of poultry and livestock rations is outside the scope of this review, it is of interest to observe present practices in the use of fermentation products as feed supplements. Cuthbertson (110) has summarized much of the available literature on the role of antibiotics in nutrition. He has emphasized that in many instances the greatest benefit of antibiotic supplementation has been noted when the poultry and animals are housed in areas previously used for the same purpose (99). Studies of comparative growth responses of chicks t o detergents, germicides, and penicillin have suggested that best results were obtained when penicillin was used (980). Certain fatty acid derivatives have been effective in promoting hog growth, being more effective than aureomycin. The mode of action of the growth promotion by surface-active agents was not understood (91). Aureomycin has found use as a feed supplement in rations fed poults (9, 17, 26'7, 367), pigs (33, 40, 244, 409, 416), and calves (399). It retarded the growth of lambs (102) and was found to reduce hatchability of eggs when fed t o hens (160). Penicillin has been judged to be as effective as any of the antibiotic feed supplements used in promotion of the growth of chicks (100), poults (432), and swine (375), while streptomycin seemed less useful for these purposes (882, 384). Bacitracin implanted subcutaneously (a new method of administration) has been reported to be an ekfective growth promoting agent for pigs (65). ANTIBIOTICS
The search for new antibiotics has continued in many laborai tories throughout the world. To a large extent the major effort has been directed toward finding therapeutic agents for the treatment of tuberculosis and viral diseases. In addition t o aureomycin, terramycin, and Chloromycetin, other antibiotics are sought which will combat infectious diseases not effectively treated by these antibiotics and which will have lesser side reactions. The major antibiotics, penicillin and streptomycin, are manufactured by a large number of industrial concerns. Bacitracin, polymyxin, tyrothricin, and gramicidin have limited use as therapeutic agents; consequently there are only a few industrial producers. The production of the broad spectrum antibiotics, aureomycin, Chloromycetin, and terramycin, is closely controlled by product patents owned by individual companies. Neomycin is a newcomer t o the manufacturing field and has found use against gram-negative pathogens. Penicillin. The total production of penicillin in the United States during 1951 was approximately 4570 greater than in 1950 and almost 150% greater than in 1949. This continued increase in production ivm influenced by the demands caused by the war
Vol. 44, No. 9
emergency, and by the use of penicillin as an antibiotic feed supplement. Improved submerged culture techniques for penicillin production were illustrated in a pictured flow sheet of the penicillin plant operated by Cutter Laboratories (48). Erdmann (154) published a review which discussed the synthesis of penicillin by various microorganisms. Patents have been issued in the United States (199) and Swltzerland (%70)which cover a semicontinuous process for the production of penicillin. The technique involved the periodlc removal of about 20% of the fermented broth followed by replacement with fresh medium. Fermented broth was removed and increments of fresh medium were added as long as the fermentation continued a t the maximum rate. The isolation of mutant strains is one of the more promising methods increasing industrial production. Arima (10-12) reported investigations concerned with the search for highelyielding mutants of penicillin-producing molds. A pigmentless mutant of Penicillium chrpoqenum &I 7 6 , obtained by treatment with N-mustard, was reported (IS). Further studies have been completed in which a number oi different medium constituents have been compared for use in penicillin production. Parsons (302,303) developed an improved cornsteep liquor which supported increased penicillin yields The cornsteep was treated with an aluminum salt which precipitated phytic acid as the aluminum-magnesium salt. After removing the precipitate, the liquor was concentrated t o 30 O Be. and the separated solids were dkcarded. PYIarkov and Bogdanov ($58) prepared a satisfactory medium by steeping crushed soybeans in water. Addition of sodium chloride t o the filtrate resulted in a complete medium. Bar (20, 21) reported that brewing yeast and whey were satisfactory as media for grobl t h and penicillin production. Lucerne has been proposed a- 3. nitrogen source in a medium formulated by Lulla (253). Chllstensen (96) claimed that media containing animal glands such as the pancreas, small intestine, and stomach gave 1000 to 2000 units per ml in a 36-hour fermentation with Penzcdlium chrysoqenum Q176. It was claimed, in a Japanese patent, that the addition to the medium of an alkaloid such as quinine, hcroili. or berberine increased penicillin production (142). The incorporation of vegetable and animals oils in the medium t o stimulate penicillin production has been reported by Goldschmidt and Koffler (160). I t was recently reported, however, that soybean oil concentrations of 0.2% or less, added to shaken flask cultures after inoculation, markedly reduced the yield of penicillin (641 ). If the oil was added a t an earlier stage, penicillin production was stimulated, whereas if it was added a t the end of the fermentation, little effect was noted. The effects were more pronounced in a cornsteep liquor medium than in a semisynthetic medium. Later studies (440)indicated that the inhibitory effect of the oil was associated with the accumulation of ammonia nitrogen in the filtrate. Colingsworth (104) has claimed that the addition of 0.5 to 2.0% lard or corn oil t o the medium before inoculation increased final potencies from 800 units per ml. to 1300 units ppr ml. Kato (215) studied penicillin production in shaken flask cultures wing a semisynthetic medium. The yields obtained on the reciprocating shaker were dependent upon: ( a ) the composition of the medium, ( 6 ) the volume of medium in the flask, and ( c ) the fihaker stroke length. When the shaker stroke increased from 4.5 cm. t o 6.5 em., increased penicillin production up to 100 units per ml. was obtained only when the concentration of the medium ingredients was increased. Calam et al. (38) used 2-liter bolt head flasks and Kilner jars t o study the effect of agitation and oxygen transfer on the growth of P. chryscgenum and the production of penicillin. In the bolt head fermentor, the rate of oxygen transfer t o the liquid limited the metabolic activities of the mold, whereas in the Kilner jars some unknown reaction rate was limiting. High vields of penicillin did not
September 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
necessarily correlate with respiration rate and oxygen supply. It was further demonstrated in a study of fermentations grown at different temperatures that growth of mycelium, rate of respiration, and rate of penicillin production were semi-independent of each other. The types and quantity of penicillin produced during the fermentation is dependent on the use of penicillin precursors and has been the subject of a large number of investigations over the past years. Yasuda and Enomoto (442, 443) reported that total penicillin production was stimulated in shaken flask culture by the addition of phenylacetic acid shortly after inoculation or 48 hours later. Inhibition of penicillin production was noted when soybean oil and sodium phenylacetate were added simultaneously during the fermentation. Kotake et al. (230) found that 4 to 5 additions of 0.008 t o 0.25% phenylacetic acid per addition was the most effective technique for shaken flasks. They also noted that a single addition of 0.3% of this precursor gave maximum yields in tank fermentations. In studies with 4000- and 40,000-liter fermentations, the medium composition had a marked effect upon the choice of method of phenylacetic acid addition. Maximum penicillin titers of 1400 units per ml., 85 to 95% of which was penicillin G, were obtained by adding a total of 0.4 to 0.6% phenylacetic acid in 6 to 7 equal portions. Yasuda et al. (44.3) demonstrated that mother liquors from the extraction processes can be used effectively as benzylpenicillin precursors. Optimum efficiency was obtained when this waste material was added together with small quantities of phenylacetic acid. The Occurrence of ortho-hydroxyphenylacetic acid as a metabolic product in the penicillin fermentation, presumably when phenylacetic acid was used as penicillin G precursor, has been studied by King and Hambley (217) and Nishida (284). The presence of this metabolite may necessitate revised extraction procedures. Saunders et al. (351) reported the production, isolation, and oharacteriaation of the phenylmercaptomethyl-, p-bromophenylmercaptomethyl-, benzylmercaptomethyl-, and p-hydroxyphenylmercaptomethyl-penicillins. I n crystalline forms these penicillins had potencies ranging from 1900 t o 3400 units per mg. A study of specific enzyme systems of Penicillin chrysoqenum has revealed some information which may be of immediate application. The presence of “penicillin-amidase,” an enzyme which hydrolyzed benzylpenicillin with the formation of phenylacetic acid and “penicin,” was reported present in the mycelium of P . chrysogenum Q176 by Sakaguchi and Murao (349). The conditions for maximum rates of activity included a temperature in the range of 35” t o 38’ C. and a p H between 7.6 and 8.0. This enzymatic activity may account for the rapid deterioration of the penicillin titer in fermentations of advanced age. A coincidence not understood was the elaboration of phosphatase by P . chrysogenum Q176 a t the period of maximum penicillin produc tion (340). Streptomycin. As stated previously, the total streptomycin production in the United States rose from about 92,000,000 grams in 1950 t o over 159,000,000 grams in 1951. Waksman (414) and Emery (13.9) have recently reviewed the production, properties, and uses of streptomycin. Dulaney has demonstrated that artificially induced mutants give greatly increased fermentation yields. Dulaney (197) first claimed the isolation of a mutant strain, Streptomyces griseus var. Dulaney L-118, which was capable of producing 800 t o 1000 units per ml. in a soybean meal medium and which was resistant t o an initial streptomycin concentration of a t least 600 units per ml. Dulaney (128) has subsequently patented a second mutant designated 8. griseus albus mutant (Dulaney 2-38). This strain was selected by means of ultraviolet and x-ray treatment of the Dulaney L-118 strain and its progeny. This new variant was capable of producing 2000 units per ml. in a fermentation time of 110 hours. The Dulaney 2-38 strain
2003
differed from the L-118 culture in: ( a )temperature requirements; ( b ) fermentation time; ( c ) optimal pH; ( d ) horsepower requirements from mechanical agitation; and (e) its inability to produce vitamin BIZ. The failure of this strain to produce vitamin Ble is undesirable because this vitamin is frequently recovered as a by-product of the streptomycin fermentation. Many investigations have been concerned with the develop ment of cheaper media which give increased yields. Baron (28) was granted a British patent for a synthetic medium containing cerelose, ammonium nitrate, sodium citrate, and other salts. This medium gave streptomycin yields of 300 t o 500 units per ml. when a selected strain was used. Patents were issued for a fermentation medium containing nitrogen sources such as soybean meal and distillers solubles, and carbohydrate sources such as dextrose or hydro1 (965, 869). Various reports have indicated the following nitrogenous materials are satisfactory: fish meal, with or without autolyzed yeast, yeast extract (121), distillers solubles supplemented with sodium nitrate plus an organic source of nitrogen (2696), solids from the penicillin fermentation ( l a $ ) , and soybean flour plus corn steep liquor (3%”). Bennett (29) treated corn steep liquor with alumina-silica clays and thereby removed histaminelike materials which had frequently been found as impurities in the final product. A less expensive medium for the growth of inoculum was reported by Colingsworth (103). This medium contained the solubles from the molasses ethyl alcohol fermentation and waa claimed t o be a “growth-promoting and streptomycin-productionstimulating factor.” Thornberry and Shanahan (3996) found that soybean and peanut meals contain mineral nutrients which favor streptomycin production. Upon fractionation they found that, in the presence of proteins, the minerals were partially unavailable for antibiotic production. Substances inhibitory t o streptomycin production were present in peanut meal but not in soybean meal. Takeda et al. (16, 389) found that metallic iron inhibited streptomycin production and that phosphate relieved this inhibition. Further studies in shaken flask fermentations demonstrated the extent of corrosion on various steels and the successful inhibition of this corrosion by tribasic potassium phosphate. Chesters and Rolinson (96), who studied the effect of trace elements in surface culture, reported that zinc and copper were essential both for growth and antibiotic production. Animal and vegetable oils were used as replacements for glucose with no reduction of antibiotic yield (310). Best results were obtained when the substitution was made on a caloric rather than a weight basis. Jackson and Milner (123) claimed a n improved process for the production of streptomycin in which the culture liquor was drawn off and fresh medium introduced to the mycelium. Interest in hydroxystreptomycin, which was f i s t reported in 1950 (88, 167), continued throughout the past year. Benedict and coworkers (27) published a detailed morphological and physiological description of Streptomyces griseocarnew, which produces this antibiotic. Studies in 20-liter fermentors indicated that yields of 340 units per ml. could be obtained on a medium containing soybean meal, distillers dried solubles, sodium chloride, glucose, and calcium carbonate. Factors which were studied in lo-, 20-, and 600-liter fermentations included the effects of: air flow, temperature, initial p H of medium, age and amount of inocula, and rate of agitation. Peterson, Hanes, and Sylvester (312) demonstrated that optimal production of hydroxystreptomycin in shaken flasks by Streptomyces NA 432-MI could be readily duplicated in 30-liter stirred jars and in 400-gallon tanks. Maximal yields of 150 to 200 units per ml. were obtained on media containing soybean meal and glucose. A study of the chemical changes during fermentation indicated that biosynthesis of t h e antibiotic occurred after the period of rapid mycelium formation. The biosynthesis was also characterized by rapid sugar utilization and by marked
2004
IN D U S T R I A L A N D E N G IN E E R I N G C H E M I S T R Y
increase in total soluble nitrogen. The culture of Styeeptomyces used by Peterson and his associates was identified as a strain of
S. griseocarneus (168). Phage infections may have a great effect on fermentation yields and consequently studies have continued with emphasis on phage multiplication. Walton (419) observed t h a t M n + + and Mg++ ions supported the growth of S. griseus actinophage S-1. Both ions were apparently required as absorption cofactors. In contrast, Na+, K+, and N&+ cations completely inhibited multiplication. Perfman et d.(308) reported that the multiplication of phages infecting S. griseus may be limited by the addition of substances capable of sequestering C a + + ions. Citrate, oxalate, and phytate salts were effective in inhibiting actinophage without adversely affecting streptomycin production. The inhibition was reversed by the addition of metallic ions which formed complexes with the sequestering agents. A report of the actinophage infection of a Japanese manufacturing plant wm accompanied by a detailed study of the isolated particles (1). Broad Spectrum Antibiotics. The three broad spectrum antibiotics, aureomycin, terramycin, and Chloromycetin, are dealt with as a group because of their similarity. Their manufacture and sale are limited largely to individual companies by patents. All three are produced by Streptomyces s p . and are effective against rickettsiae, gram-negative, and gram-positive bacteria and are competitive because of their spectrum similarities., Chloromycetin has been identified chemically and can be synthesized. It is estimated that over 50% of the production of this antibiotic in the United States is accomplished by chemical syntheses. AUREOMYCIN.Production of aureomycin in Great Britain has been announced (64). This plant, which imports crude aureomycin from the United States for purification, has been reported capable of producing sufficient finished material t o supply all the requiremenkq of the United Kingdom. King (216) summarized the problems encountered in largescale production of this antibiotic including: Organization Engineering Chemistry Microbiology Process development arid maintenance Raw material supply Effluent disposal. Van Dyck and DeSomer (406) reported a medium containing peanut meal or peanuboil meal ea able of producing 1300 y per ml. A strain of S. aureofuciens, w%ch was isolated by process of natural selection and ultraviolet treatment, gave yields three . times greater than the parent. Dunitz and Robertson (129) have re orted the close relationship that exists between aureomycin a n i terramycin, and Pepinsky and Watanabe (506) have found the antibiotics to be isomorphs. The discovery and rapid development of terraTERRAMYCIN. mycin by Charles Pfizer & Co. has been mentioned in a previous review (242). Further accounts of this development have appeared during the past year (327, 382). Regna and his coworkers (328) described the isolation of terramycin from broths with titers of approximately 1000 7 per ml. The general production process used by Charles Pfizer & Co. has been described (827). The fermentation was conducted in 2000 and 25,000 gallon batches with a yield of 4.5 pounds of antibiotic per 1000 gallons of broth. Very little has been published during the CHLOROMPCETIN. ast year on We Chloromycetin (chloramphenicol) fermentation. %his may be partially explained by the fact that greater attention is being given to production by chemical synthesis (46). Alberti et ul. (5) described the synthesis from phenylaminopropanediol and thr&nitrophenylaminopropanediol.Gottlieb and Diamond (162)described a synthetic medium capable of producing yields of 100 to 120 y per ml. which contained glycerine, dl serine, sodium lactate, and inorganic salts. Agata et al. ( 3 ) reported broth potencies of 400 to 650 7 per ml. in 100liter fermentations with a medium containing peptone, starch, and inorganic salts. A British patent (299) described the production of Chloromycetin by fermentation with Streptomyces uenezuleae.
Bacitracin. The demand for bacitracin has steadily increased because of ita suitability for topical application and a.8 a n anti-
Vol. 44, No. 9
biotic feed supplement. h new plant for thc production of bacitracin was described in detail in a recent publication (201). The fermentation conditions for Bacillus subtilis Tracy grown in 24,000-gallon fermentors were a8 follows: temperature, 37” c . ; fermentation time, 24 to 72 hours; and a medium containing soybean meal or peanut granules, calcium carbonate, and starch. Darker (112) claimed yields of 46 units per ml. with a medium containing starch, soybean meal, calcium carbonate, and calcium lactate. Neomycin. Bulk production of neomycin was initiated in 1952 by Heyden Chemical Co. (61, 89). The therapeutic use of this antibiotic was initially confined t o topical application because of its toxicity. Reports now indicate t h a t neomycin is also proving satisfactory for use against intestinal infections and for sterilization of the abdominal cavity previous t o surgery (64). Several neomycins were apparently produced in, or derived from, fermentation broths. Swart et al. (386), who investigated the identity of the neomycin complex by means of countercurrent distribution, reported that neomycin A was a homogeneous substance. Neomycin B, on the other hand, was composed of a t least three similar compoundb. The clinical activities reported for neomycin were attributed t o the neomycin B complex. Dutcher et aE. (130) described a fraction, neomycin C, which differed from the previously described A and B. Neamine, an acid degradation product of neomycin which has antibacterial activity, was studied by Leach and Teeters (239). Polymyxins. During the past year there were few reports o n the polymyxins, the group of polypeptide antibiotics produccd by Bacillus polymyzu. Brownler ( 3 6 ) had previously published :I complete review. Of 27 strains of B. p o l y m y z u btudied in detail, 11 produced :tri antibiotic resembling polymyxin B or E, 3 were similar to D, :tiid 13 resembled A (279). Yonehara, Toyoma, and Sumiki (444) found that glucouc, sucrose, and soluble starch were equivalent carbon sources while lactose and fructose were inferior. Press yeast autolyzate, dry yeast, peanut cake, skim milk, “pupa” cake, and soybean meal were all satisfactory nitrogen sources. Miscellaneous Antibiotics. Previous reviews in this series have not attcmpted to cover antibiotics other than those of economic significance. Lee (242) departed from this procedure in 1951 and briefly mentioned those antibiotics of promise which had not reached commercial production. The following nntibiotics are in this clam VIomcIii, an antibiotic produced by two strains of Streptomijces, S. puniceus and S. Jloiidae, may prove useful in the treatment of tuberculosis (26, 192, 137, %”). .I detailed st,udy of the effect of the medium constituents upon viomycin production has been reported (101) FUMAGILLIN, a monobasic acid produced by Asperyillus f unia(latus, was reported to have amebicidal properties. McCowen et al. (264) reported i t to be extremely effective against amebiasis in rats, mice, and rabbits, and recent studies have &own that i t may be uscful in human therapy (216). In 1951, Waksman and his coworkers reported the ixolation of hHRLICIIIN, an antibiotic produccd by Streptomyces laueadulat. The antibiotic inhibited A and B influenza viruses and the infectious bronchitis virus in embryonic eggs (156). The discovcry of ohrlichin was considered significant because it was the first antibiotic reported which inhibited the true viruses. The ($oncentrate was ineffective against bacteria
FERMENTATION AS A UNIT PROCESS Consideration was given t o fermentation as a unit process in this series of reviews ( 2 @ 4 @ , 366). -4s these reviews elucidated the principles involved, and summarized the information available prior t o 1951, only those principles which have been emphasized in publications appearing during the paat year will be covered in this review.
September 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
THE MICROORGANISM
Selection of Cultures. Culture selection and culture maintenance are of major importance in the fermentation unit process. In many fermentations, for example, the microbiological production of vitamin BIZ(37, 562, 566), numerous cultures from many natural habitats have been screened in order to obtain those capable of producing larger amounts of the desired factor. In programs searching for cultures producing large amounts of vitamin B Lsome ~ success has been attained (146, 161, 569), while in other studies the results have not been so rewarding. In the latter cases, it has sometimes been found that: Laboratory techniques used were too rigid. Media selected for the growth of the microorganisms were not realistic with regard to composition and general suitability. Choice of microorganisms to be screened was not the best. The analytical methods used were not precise or sufficiently specific. The general practice, in searching for new cultures, is first to screen a large group of isolates from soil or other natural sources, using rather nonspecific and qualitative rather than quantitative techniques; then follow this program by selecting a few of the superior cultures and attempting t o improve them by selection of natural variants or induced mutants. Though a few surveys of taxonomically classified cultures have been attempted, study of the potentialities of this group appears to have been delayed or neglected (148, 196, 569). Nearly all of the antibiotic-producing actinomycetes of commercial interest may be new species (S93),and often species closely related morphologically to these antibiotic producers have had little, if any, ability t o produce the desired antibiotic. Morphology may have little relation t o physiological activities but the conditions of selection or screening may have a great effect. A culture selected for further study after growth on a medium containing complex proteinaceous materials, vitamins, growth factors, and carbohydrates may subsequently require these m&erials in order t o produce maximum amounts of the desired product. It has been repeatedly demonstrated that most cultures of microorganisms may have several closely related strains (18, 19, 45, 184, 260, 89.2),some of which have considerably different physiological characteristics. When such cultures are grown under proper conditions, certain elements of the population will outgrow the rest (19). If thisdominatingsegment of the population produces more of the desired substance, efforts are made t o encourage it and t o suppress the growth of less desirable elements. Yaturally, if the segment favored by the fermentation conditions or culture maintenance program does not produce the desired substance, efforts are made to alter the conditions t o allow the predominance of more desirable forms (960). This mixed strain theory perhaps may explain why certain laboratories have reported excellent results with a given culture, while others have been less successful. Experience with mixed culture fermenta1ions has shown how difficult it is t o control the process where two or more species are grown together (187, &6), and it is expected that it would be even more difficult t o control or balance the growth of rlosely related strains within a species because of the greater competition. The role of the microbial geneticist in increasing yields of aureomycin, penicillin, and streptomycin is becoming quite evident (18,393). I n these and other fermentations, natural variants (1.28, 188, 203, 562, 411, .$la), and variants selected after exposure of the culture to such lethal agents as x-rays ( I d , 128), ultraviolet light (11, 128, R%, 866),nitrogen mustard gas (13, I%), and bacteriophage (&‘O, 4W),have yielded strains which differ morphoIogically and physiologically from the parent culture (127,128). While there may be no limit to improvement of processes by strain selection, the growth requirements or fermentation conditions for the new culture may be so rigid that an impractical process will result. The advantage of an increase
2005
in yield of 10 t o 20% may be uneconomical if a more expensive medium or a more rigidly controlled process is required. Maintenance of Strain. The desired strain of the microorganism must be maintained in a manner conducive to the procurement of uniform results. Lyophilization or desiccation using soil or proteinaceous materials as supporting menstra, preservation on agar under mineral oil, or refrigerated storage of spore suspensions are popular techniques in many laboratories (258). While frequent transfer on one type of medium may lead t o selection of one segment of the population, infrequent transfer may also have this effect. Transfer on a number of media in rotation might complicate the population picture (422). For example, growth and metabolism of certain strains of Streptomyces griseus was inhibited by streptomycin (994). Infrequent transfer of these cultures or use of undesirable media may result in selection of the most antibiotic-resistant forms, but not necessarily the most productive strains (992). It is becoming more evident that the technical approach of the geneticist should be more extensively adapted t o microbiology in order t o interpret some of the changes which occur in the fermentations. A “stable” strain is necessary for efficient operation but i t is frequently difficult t o determine when stability has been accomplished. Preparation of Inoculum. The discussion on the importance of strain maintenance and stability leads directly into the problem of inoculum preparation. It is desirable from a n economic viewpoint to attain a minimum fermentation period for maximum yield in the large-scale fermentation equipment (258). Use of a large inoculum will help t o reduce the fermentation period. A small inoculum will need a longer growth period and also may permit the ratio of segments of the population t o vary. The use of inoculum media containing growth stimulatory substances not added t o the fermentation medium has found favor (51, 105, 141). Media favoring sporulation are not always useful for vegetative growth (247). In certain processes, it has been shown desirable t o collect the cells a t the end of the fermentation and reuse them in fresh media (214, 938). This has resulted in reduced fermentation cycle times and somewhat higher yields. A variation of this procedure is t o replace a portion of the culture liquid with fresh medium during the fermentation period (1.90, 125). Repeated transfer of streptomycin-producing cultures in the vegetative phme, without permitting sporulation, has usually resulted in lowered antibiotic production (4.39). This has not been observed in production of penicillin (919). SUBSTRATE
The types of raw materials used in fermentations, their availability and relative cost, as well as methods of prepamtion of media for fermentation of numerous carbohydrates and proteinaceous materials have received attention. The economics of the industry apparently limit the carbohydrate sources mainly to cane molasses, beet molasses, cereal grains, starch, lactose, glucose, and sulfite waste liquor, The principal nitrogenous materials are cornsteep ,liquor, soybean meal, packing house wastes, and fermentation residues. Strain specificity is important in the fermentation operations; media supporting growth and production of the desired chemical substance by one strain will not be satisfactory €or another. Carbohydrate Sources. The major product of the fermentation has determined the choice of carbohydrate especially if the product results from direct dissimilation of the carbohydrate. For example, in the ethyl alcohol fermentation the cost of the product is determined largely by t h e cost of the carbohydrate (228, 288). If the fermentation product is not the direct result of carbohydrate metabolism-for example, penicillin, streptdmycin, or vitamin Blz-the cost of the fermentation product is not so directly tied t o the carbohydrate cost. In Japan substrates such &s sucrose,
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INDUSTRIAL AND ENGINEERING CHEMISTRY
cassava, cane juice, and others (401) can be used as fermentation substrates for the production of butyl alcohol and acetone. I n Canada, on the other hand, where no synthetic ethyl alcohol is produced, molasses, grains, and sulfite waste liquor are used a8 substrates (39). While the metabolism of carbohydrates by the antibioticproducing actinomycetes has received little attention, the observations that antibiotic production is “high” or “low,” depending upon the carbohydrate used, indicate significant variations in usability. Thus, the observations that highest yields of aureomycin were obtained with sucrose and starch, lower titers with glucose, and very low titers with lactose and mannitol, may indicate that glucose is metabolized rapidly, sucrose and starch moderatelyfast, and lactose and mannitol not a t all (405). This apparent preference for starch or polymers of glucose, over the monosaccharide, may be overcome in certain instances by continuom addition of the glucose (317). The method of medium preparation-for example, the sterilization procedure-may affect the suitability of carbohydrates for fermentation (115,212), as does the eliinination of toxic materials (98, 393). Nitrogen Sources. Proteinaceous materials such as extracted seed meals, yeast, and concentrates such as distillers’ solubles, cornsteep liquor, fish stick liquor, and meat stick liquor are favored raw materials for many fermentations, particularly riboflavin, antibiotics, and vitamin BIZ(101, 146, 269, 286, 296, 303, 390, 394, 595, 397,402,404,406). A number of investigators have noted that with certain 8. griseus cultures, meat extract media will support higher streptomycin yields than media containing soybean meal, while with other cultures, the opposite was true (296, 402). This might be explained either on the basis of specific nutrient requirements for growth or to the presence of toxic materials in these products. The latter may be the more plausible explanation as 8.griseus will grow well and produce streptomycin on synthetic media containing glucose, salts, and glycine (287). The practice in many laboratories has been to use mixtures of natural materials in the medium, rather than a single substrate. Mixtures of ingredients might be expected t o offset some of the variations in composition of individual raw materials. For example, a proteinaceous mixture containing yeast extract, soybean meal, and Brewer’s yeast has been proposed for production of polymyxin (378)while a combination of cornsteep liquor, stillage, and animal stick liquor has been used for riboflavin (390). Other studies have indicated that yeast autolyzate, yeast, peanut cake, skim milk, or soybean meal used singly are sat,isfactory for the production of polymyxin (444). The specific growth requirements of the solvent-producing anaerobic bacteria may influence the choice of medium ingredients, An example was the report t h a t soybean meal media were preferred over those containing copra meal, cottonseed meal, or herring meal (178, 401). Another study suggested that soybean press cake and egg white protein were preferred t o casein, fish protein, ethyl alcohol-extracted soybean cake, zein, gluten, gelatin, and yeast protein ($89). Shu and Blackwood (ass, S65) have shown, contrary to an earlier report (398), that proteins and amino acids were not required as medium ingredients for production of amylolytic enzymes by Aspergillus niger. They reported t h a t the important factor was the adjustment of t h e proportions of ingredients so that the p H of the fermentation wm stabilized. This principle may also be operative in other fermentations such as penicillin, streptomycin, and neomycin where synthetic media have been proposed. Precursors. I n certain fermentations the addition of known chemical substances t o the fermentation medium results in the incorporation of these directly into the fermentation product. For example, the addition of phenacetyl derivatives to penicillin
VoL 44, No. 9
fermentations results in formation of benzyl penicillin. Recent studies (307)have mdicated that phenethylamine and phenacetamide are converted t o phenylacetic acid and the latter was used in the formation of penicillin G. Approviniately 10% of the phenylacetic acid added to the fermentation wafi incoi porated into the penicillin with the remainder being converted to benzoic acid and eventually to carbon d i o u i e (185, 307). Kluyver and van Zijp (225)have shown that homogentisic acid, quinone, and oxalic acid are formed from phenylacetic acid by Aspergillus niger. a-Hydroxyphenylacetic acid has been isolated from penicillin concentrates and may be a metabolic product of phenylacetic acid (227, 284). Phenacetylethanolamine [N(a-hydroxyethyl) a-toluamide] has received attention as a precursoi for benzylpenicillin (387, 442). It was utilized a t a slower ratc and only one tenth the normal quantity of phenylacetic acid ~ v a i needed. Inclusion of cobalt compounds in vitamin Biz production media resulted in incorporation of the cobalt into the vitamin BIZ (434). Other studies using Cow have demonstrated that only 7 t o 17% of the cobalt added t o the medium was incorporated (333). Cobalt ions are bacteriostatic and fungistatic and thus delayed addition ha3 been used (146)to overcome this effect. Precursors have been suggested for other fermentations including those producing Chloromycetin (151, COS), and streptomycin (6, 7). Examination of the data available suggests that only a small amount of the supplement added could have been incorporated into the antibiotic. It is possible that the action of these supplements is indirect and connected with the general metabolism of the actinomycete. This may also be the explanation of the observations by hIoyer that methanol additions t o citric acid fermentations resulted in increased acid productioii (276, S44, 346). Inorganic Nutrients. The role of inorganic trace materiale in the nutrition of microorganisms has been reviewed by Stiles (381), Wallace and associates (417), and Lilly and Barnett ($47). I n much of the literature summarized in these reviews, the effects of metallic ions on germinating spores or rapidly groxing cells has been measured. Studies with Memnonzelta echznata and A s p e r g i L lus niger (306)indicated that the young cells are much more scnsitive to the effects of cobalt and iron ions than thc “mature” cells. Accumulation of citric acid in A. niger fermentations occurred in the absence of iron, and when ferric ions were added, the accumulated acid was rapidly metabolized. The ferric ions can be sequestered by use of complex-forming agents such as ferricyanide (98). or oxalate (906), while cobalt can be sequestered by adding histidine to the medium (556). Reports from several laboratories indicate that addition of ferric ions to actinomycete fermentations resulted in reduced antibiotic production, This was true of fermentations producing aureomycin (405), luteomycin (167-169), Chloromycetin (291), and streptomycin (14-16, 162, 163). However, in synthetic media, traces of ionic iron were necessary for maximum production (22, 287), Asai et al. (16) suggested addition of phosphate to complex the iron but it has been reported according to others (310) that the presence of inorganic phosphate ions in the medium specifically inhibited formation of streptomycin. Alcohol production by Sterigmatocystis nigra and Aspergillus pavus was increased when iron and zinc were added to the medium (324). Zinc appeared to be required for growth of Aspergillus niger (94, 352) and for phosphatase formation by Penicill i u m chrysogenum (340).
Inorganic fractions in the ash from molasses appeared to increase production of itaconic acid (423) and 2,3-butylene glycol by a number of bacteria (23’8). I n the latter case sodium phoaphates could be used t o replace the ash fraction. ASEPTIC T E C H N I Q U E AND STERILIZATION
Aseptic Technique. Parker (300) has summarized the niajor factors in designing and operating a penicillin plant. These factors are: laboratory technique, plant construction, air sterilization, sterility check procedures, and other operating techniques. A differentia1,procedure for bacteriological studies in the yeast fermentation industry was described by Green and Gray (164). An antibiotic, Actidione, Tas included in the medium t o check
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yeast growth so t h a t the presence of bacterial contaminants could be more readily detected. Prevention of contamination in the riboflavin fermentation by the addition of penicillin or penicillin salts t o the culture medium was patented (124). Steriliza*ion of Air. A number of reports have appeared which are concerned with air filtration. Kluyver and Visser (884) compared the Folin bubbler, the Wheeler bubbler, the Moulton atomizer, and the capillary impinger to determine the number of microorganisms in air, The latter device, which retained over 99% of the microorganisms in the air, was reported to be the most satisfactory. The same investigators (994) devised a method for testing the filtration efficiency of cotton wool, stillite, and carbon, and satisfactory filters were designed using each material. The theory of air sterilization was reviewed by Schmitz (356). Cherry et al. (93,301)described experiments concerned with the application of air filtration t o industrial scale fermentations. Slag wool or glass wool, having a fiber diameter predominwitly below 6 ~ supplied , sterile air-provided a uniform packing of specified density was maintained. The filters were so designed that i t was impossible for air t o pass through less than a 3-inch layer of the filter medium and were large enough to handle the required volume of air at velocities less than 0.5 foot per second. The use of activated carbon for air sterilization was discussed briefly by Harris (166). Fifteen cubic feet of granular, active carbon of 10 t o 20 or 10 to 30 mesh were recommended for every 100 cubic feet per minute of air passed through the filter. Inskeep et al. (9001) reported that air passed through carbon-packed towers has to be heated above the dew point in order to ensure sterility. It was contended that without heating to 40' to 70' C., the carbon bed was eventually penetrated by airborne moisture droplets. These were formed as the result of expansion cooling of the air to its dew point when it had passed the inlet control valve. The use of the electrostatic precipitator as a method for obtaining large quantities of sterile air for fermentation processes and for sterile packaging areas was investigated by Munden (277). With two precipitators placed in series, 95 to 97% of the airborne microorganisms were retained. Absolute sterility apparently cannot be obtained by this procedure, Mellors (868) described methods of measuring bactericidal effects of ultraviolet light intensities. It was stated that the performance of ultraviolet for air sterilization can be improved by: precleaning of the air, humidity control, and by use of ducts having internal surfaces with good ultraviolet reflecting properties. Irradiation of air supplies with ultraviolet was thought useful only where complete sterilization was not required. Sterilization of Medium. The sterilization of the medium has been discussed in previous reviews (841, 849). Schmitz (366), who described the mechanics of continuous sterilization, claimed that this technique was desirable for large fermentor operation because the practice ( a ) reduced the thermal degradation of raw materials, ( b ) shortened the turnover phase of the fermentors, snd (c) effected a large heat economy. Pfeifer (313) studied, in pilot plant equipment, the application of a continuous sterilization procedure for several industrial fermentations. The importance of flash sterilization in t h e production of riboflavin by Ashbya gossypii was emphasized by Smiley et al. (368). High yields were obtained by flash sterilizing a t 280' to 290" F. for 3 minutes. FERMENTATION EOUIPMENT
In the two previous reviews Lee (141, 849) described equipment used in the fermentation industry. Recent articles have described plants used for the production of penicillin (47), bacitracin (901),and terramycin (237). No significant developments have been reported since fer-
2007
mentor design wm previously discussed (841, 849). The bacitracin plant described by Inskeep et al. (801) was designed t o provide remote coordinated actuation of almost every valve in the fermentation unit. It was assumed that this installation would decrease labor requirements and eliminate the possibility of contamination resulting from incorrect valve settings. The fermentors used were stainless steel vessels of welded construction with a capacity of 24,000 gallons. The fermentors had conical bottoms and the lower half of the fermentor was fitted with cooling coils. Sterile air was introduced through a "spider" type sparger situated near the bottom of the fermentor. No mechanical agitation was provided. The penicillin plant described in a recent report (47) had 5000gallon aerated, agitated fermentors. The materials of construction were not specified. Parker (300) described another penicillin plant with particular emphasis on design of equipment for aseptic operation. Essential features listed were: 1. Absence of direct connection between sterile and nonsterile units of the system 2. Welded construction 3. Joints of high quality finish, employing rubber a8 the sealing material 4. Careful selection of valve types 5 . Maintenance of all parts of the system under positive pressure after sterilization 6. The fitting of each section of the system for independent sterilization 7. The possible maintenance of contamination sites under continuous steam pressure when not in use. Lumb and Fawcett (264) described an improved laboratorytype fermentor having a capacity of 9 liters. The battery of 48 vessels was split into 4 frames, each holding 4 units of 3 fermentors. The stirrers were driven by means of flexible couplings from an overhead shaft thereby eliminating the problem of belt slippage. The sparger was a 8/r&ch bore pipe while the agitator was of the link or paddle blade design. All metallic parts were made of stainless steel. Heatley (179)designed a versatile arrangement for the aseptic removal of samples from a pure culture fermentation. This device, of glass and rubber construction, was also recommended for the addition of sterile solutions or inocula to the fermentation vessel. Beesch described a metallic device which could be used for the same purposes (96). The mechanics of fermentor mixers were discussed b y Boutros and Rushton (32). It was stated that the industrial fermentore require mechanical agitators of 100 t o 200 horsepower or larger. Design and construction of the mixer t o give reliability in operation a t a reasonable cost was stated to require a balanced design for the whole unit. Important features listed were: types of drives; gear ratios; begring mountings for the mixer shaft; mixer shaft design; stuffig boxes and steady bearings; and impeller. design. Gaskets made from Teflon were reporbed to be giving outstanding r f o r m a n c e in a domestic antibiotic plant (4) Teflon, . whic is a tetrafluoroethylene resin, was stated to be chemically inert and heat- and pressure-resistant, thus making it ideally suited to conditions encountered in the equipment used in the fermentation industry. THE FERMENTATION
The fermentation operation may be conducted by one of three basic processes: the batch process; the continuous process, single vessel system (180); and the continuous process, multiple vesseE system (114). Schmitz (366)compared the three processes OD t h e basis of cycle characteristics and operational costs. The cycle of the batch process was divided into three phases: the turnover' phase; the reaction lag phase; and the finishing phase. Maximal production was obtained only if the broth was harvested a t the point of the maximal over-all reaction rate. In the continuous systems a fourth phase was described. During this period the fermentor was supplied sterile medium and fermented broth was removed continuously. The point at which the continuous phase should be initiated to give maximum production was defined. The two basic disadvantages of any continuous process were stated to be associated with culturedegeneration and contamination. A detailed method for the estimation of costs on the various
I N D U S T R I A L A N D ENGINEERIhTG CHEMISTRY
2008
fermentation systems was also described by Ychniitz (356). It was demonstrated that the cost of a continuous fermentation may run higher than a series of batch cycles over the same time period. However, the cost per unit weight of product was highest with the batch process. Temperature. White and Munns (@6, 437) investigated the effect of temperatures upon the growth and metabolic activity of yeast under aerated conditions. They found that the rate of growth increased with increasing temperature u p t o 36" C. At higher temperatures the growth rate fell off markedly. The total rate of sugar utilization increased almost linearly between 20" C. and 40' C. It was stated that a greater conversion efficiency of sugai to alcohol could be expected at 36" t o 40' C. Calam et al. (38) studied the effect of temperature upon various metabolic functions of P. chrysogmnum in submerged culture. The rate of mycelium production increased directly with temperatures t o 30" C. and then fell sharply. The respiration rate reached a maximum point a t about 22 O C. but did not decrease until the temperature reached 29" C. Finally, the rate of penicillin production incremed with temperatures up to approximately 25" C. and then decreased. Aeration-Agitation. Previous reviews (241, 642) discussed in detail the present understanding of the interrelated effects of agitation and aeration upon aerobic fermentations. Gaden (1.44) has since outlined the general theory of interphase mass transfei to metabolizing cells m applied t o liquid-gas systems. The prediction and attainment of large-scale performance from pilot plant data is one of the fundamental problems of industrial fermentation development. Rushton (337) reviewed the application of mixing to fermentation processes and stated t h a t only the papers of Bartholemew et al. (23, 34) gave data suitable for 8cale-up and engineering purposes. Karow et al. (209) have since reported that the primary scale-up factor wm the rate a t which oxygen waa supplied from the gas t o the liquid phase. This factor was conveniently determined by measuring the rate of oxidation of a C u + + catalyzed sulfite-water system. Comparisons were made between 5-liter, 200-gallon, 10,000-gallon, and 15,000-gallon fermentors. Rushton (334-336, 338) pointed out that, in general, mixing prohleme can be studied in the pilot plant and the data used for prediction of large-scale performance. He stated that pilot plant mixing equipment could be scaled up t o give predictable results only when surface vortexing was eliminated by the use o! baffles and where rate coefficients could be determined for a series of impeller speeds or Reynolds numbers. It was further pointed out that equal power per unit volume could very often be misleading. The desirability of maintaining systems in geonwtrical similitude was emphasized. Calam et al. (58) studicd the effects of agitation and aeration on the production of penicillin, the rate of respiration, and the rate of growth of Penicillin chrysoqaum in two types of laboratory fermentors. The results indicated that there was no rigid relationship between the penicillin-producing mechanism and the other biological processes. The data showed that the achievement of apparently satisfactory rates of respiration and supply of oxygen were not necessarily accompanied by a high yield of penicillin. The effect of aeration on acetic acid production was investigaLed by Hromatka and Ebner (194). They recommended that fermentation vessels be furnished with 1.8 times the theoretical supply of air. Studies with pure oxygen and a 60 to 40 mixture of oxygen and nitrogen in a submerged fermentation showed a depression of acetic acid production accompanied by the formation of large quantities of acetaldehyde (196).
BIBLIOGRAPHY (1) Abe, Y., Shiotsu, S.,and Endo, T., J . Antibiotics (Japan),5,84 (1952).
(2) Adams, S. L., W a s h . Stale CoZZ. Bull., 2 0 7 , l (1950).
VOl. 44, No. 9
Agata, B.,Shihata, T., Ueno, T., and Nahzawa, K . , .I. A n t i biotics (Japan),4, Suppl. A, 44 (1951). Akabori, S., Uehara, K., and Suda, H., Svnapoaiu or6 E ? a z ~ m e Chemistry (Japan),1 , 7 0 (1949). Alberti, C . O.,Camerino, B., and Vercellone, h.,R e p f . Proc. I d t h I n t e m . Congr. Chem., p. 285 (1951).
Allen, W. F., U. S. Patent 2,596,971 (May 20, 1952). Ibid., 2,596,972 (May 20, 1952). Allgeier, R. J., Wisthoff, R. T., and Hildebrandt,, F. M., IND. EN+.CHEM., 44,669 (1962). Almquist, H. J., and Merritt, J. B., Poultrg Sci., 30, 312 (1951). Arima, K., J . Antibiotics ( J a p a n ) , 4 , 232 (1951). Ibid., p. 277. Ibid., p, 281. Arima, K., and -4be, S., J . Antibiotics ( J a p a n ) ,4, 342 (1961). Asai, T., Iwamoto, H., and Takahashi, T., J. Bntibiotics (Japun),3 , Suppl. B, 31 (1950). hsai, T., Takahashi, T., Iwamoto, H., and Shimabara, K., J . Antibiotics ( J a p a n ) ,3, Suppl. B, 25 (1950). Ibid., 4 , Suppl. A, 7 (1951). Atkinson, R. L., and Couch, J. R., J . Nutrition, 44, 249 (1951). Backus, E. J., Trans. N . Y . Acad. Sci., 12 (Ser. 111,270 (1950). Backus, M. P., presented before Microbiological Section, Ro-
tanical Society of America (Dee. 30, 1949). Bilr, F., P h a m a z i e , 1 , 32 (1946). Ibid., p. 198. Baron, A. L., Brit. Pat.ent 639,893 (July 5, 1950). Bartholemew,W.H., Karom, E. O., and Sfat, M. K . , IND.ENG. CHEM., 42,1827 (1950). Bartholemew, W.H., Karow, E. O., Sfat, M. It,, arid Kilhelm, R. H.. Ihid., p. 1810. t., Ehrlich, J., Mold, J. D., Penuer, M. A , , aiid Smith, R. M., Ame7. Rev. Tuberc., 6 3 , 4 (1951). Beesch, S. C.,U. 9. Patent 2,566,306 (Sept. 4, 19,51). Benedict, R. G., Lindenfelser,L. A., Stodola, E'. H., and 'l'raufler, D. H., J. Bact., 62,487 (1951). Benedict, R. G., Stodola, F. H., Shotwell, 0. L., Borud, -4. M., and Lindenfelser,L. A., Science, 112,77 (1950). Bennett, R. E,, U. S. Patent 2,576,513 (Nov. 27, 1951). Blom, R. H., Pfeifer, V. F., Moyer, A. J., Traufler, D. H., Conway, H. F., Crooker, C. K., Fanson, R. E., and Hannibal, D. V., IND.ENG.CHEM.,44,435 (1952). Blom, R. H., Sohns, V. E., and Moyer, A. J., 1;. S. Patent 2,594,283 (April 29, 1952). Boutros, R. D., and Rushton, J . H., presented before the Division of Agricultural and Food Chemistry, 121st Meeting of the AM.CHEhr. SOL, Milwaukee, wis. Briggs, J. E., and Beeson, C. M., J. Animal Sci., 10, 820 11951).
Brbwn, G . M., and Snell, E. E., Proc. Soc. E x p t l , B i d . Med., 77,138 (1951).
Brownlee, G., Symposia of Soc. f o r Exptl. Biol., 3, 81 (1949). Bunker, H. J., Food, 20,363 (1951). Burton, M. D., and Lochhead. A . G., C m . J . Bot., 29, 352 (1951).
Calam, C. T., Driver, N.,and Howerr, H H., J Applzed Chem. (London),1 , 2 0 9 (1951). Can. C h a . Process Inds., 35,1004 (1951). Carpenter, L. E., J. Aninial Sci., 10, 657 (1951). Carson, S. F., Foster, J. W., Jefferson, W. F>., Phares, E. F., and Anthony, D. S., Arch. Biochem. Biophys., 33, 448 (1951). Carson, S. F., Mosbaoh, E. H., and Phares, E. F., J. Huct., 62, 235 (1951).
Catcheside, D. G., "The Genetics of JIici oorganisms," London, Pitmann and Sons, 1951. Chem. Eng., 57,131 (Febuary 1950) Ibid., 5 8 , 7 0 , 7 4 (April 1951) Ihid., p. 200 (April 1951). Ibid., p. 67 (May 1951). Ibid., p. 174 (June 1951). Ibid., p. 238 (July 1951). Ibid., pp. 59,175,185 (Febuuiy 1952). Chem. Eng. News, 29,1190 (1981). Ibid., p. 1279. Ibid., p. 1308. Ibid., p. 1332. Ibid., p. 1364. Ibid., p. 1810. Ibid., p. 1889. Ibid., p. 2056. Ibid.,P. 2197. (60) Ihid., i.4932. (61) Ihid., 30,770 (1952) (62) Ibid., p. 1355. (63) Ibid., p. 2232. (64) Ibid., p. 2275.
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(65) Ibid., p. 2371. (66) Ibid., D. 2424. Ibid.,p. 2631. Chem. I d s . W e e k , 68, No. 1, 5 Chem. Inds. Week, 68,No. 1,20. Ibid., No. 3,19. Ibid., No. 10,6. Ibid.. No. 14, 14. Ibid., No. 14,22. Ibid., No. 16, 29. Ibid., No. 19,6. Chem. Week, 68, No. 23, 15. Ibid., No. 24,20. E d . , 69, No. 1 , 6 . Ibid., No. 1,33. IM., No. 6, 15. Ibid., No. 10,16. Ibid., No. 12,20. I b X , No. 15,20. Ibid., No. 16,7. Ibid., No. 16,19. Ibid., No. 18, 19. Ibid., No. 24, 17. Ibid., 70, No. 3,59. Ibid., No. 8,32. Ibid., No. 10,41. Ibid., N o . 13, 35. Ibid., No. 15, 25. Cherry, G. B., McCann, E. P., and Parker, A,, J . Applied Chem., 1, 103 (1951). Chesters, C. G. C., and Rolinson, G. N., J . Gen. Microbial. 5,
553 (1951). Ibid., p. 559.
Christensen, H., Swiss Patent 276,854 (Nov. 1, 1951). Chugtai, iM.I. D., and Walker, T. K., Biochem. J., 48, 524 (1951). Clement, M. T., Can. J . Technol., 30,82 (1952). Coates, M. E., Dickinson, C. D., Harrison, G. F., Kon, S. K., Porter, J. W. G., Cummings, 8. H., and Cuthbertson, W. F. J., J . Sck. Food Agr., 3, 43 (1952). Coatcs, M. E., Harrison, G. F., Kon, S. K., Porter, J. W. Go, and Thompson, 8. Y., Chemistry & I n d u s t r y , 1952,149. Coffey, G. L., Oyaas, J. E., and Ehrlich, J., Antibiotics and Chemotherapg, I, 203 (1951). Colby, R. W.. Rau, F. A., and Dunn, R. C., Proc. SOC.Exptl. Bid. Med., 75,234 (1950). . Colingsworth, D. R., U. S. Patent 2,504,067 (April 11, 1950). Colingsworth, D. R., Ibid., 2,538,721 (Jan. 16, 1951). Colingsworth, D. R., Brunner, M. P., and Haines, R. J., J . Am. Chem. Soc., 74,2381 (1952). Cook, P. M.. IND. ENO.C H E M . , 2385 ~ ~ , (1951). CooIey, G., Ellis, B., Petrow, V., Beaven, G. H., Holiday, E. R.. and Johnson, E. A., J . Pharm. Phannacol., 3,607 (1951). Corman, J., U. S. Patent 2,577,078 (June 19,1951). Corman, J., and Tsuchiya, H. M., Cereal Chem., 28,280 (1951). Cuthbertson, W. F. J., J . Sci. Food Agr., 3,49 (1952). Damondaran, M., and Rangachari, P. N., EnzymoZogia, 15, 83 (1951). Darker, G. D.. U. S. Patent 2,567,698 (Sept. 11, 1952). Dawson, E. R., Recent Advanres i n the Fermentation Industries, 1949,62 (1950). DeBecze, G., and Rosenblatt, M., A m , Brewer, 76, 11 (1943). Deckenbrock, W., and Sprick, M.,Branntweinurirt., 4, 115 (1950). Diehl, H., Vanderhaar, R. W., and Sealock, R. R., Iowa State Coll. J . Sci., 25,19 (1951). Dion, H. W., Calkins, D. G.,and Pfiffner, J. J., J . Am. Chem, Soc., 74,1108 (1952). Dion, W. M., Can. J . Research, C28, 577 (1950) (119) Ibid., p. 586. (120) Distillers Co., LM., Brit. Patent 660,145 (Oct. 31, 1951). (121) Distillers Co., LM., Jackson, C. J., Coppock, P. D., and Kelly, B. K., Ibicd., 660,203 (Oct. 31, 1951). (122) Distillers Co., Ltd., Jackson, C. J., and Caran, H. S., Brit. Pat. 649,818 (Jan. 31, 1951). (123) Distillers Co., Ltd., Jackson, C. J. and Milner, J., Brit. Patent 644,078 (Oct. 4, 1950). (124) Distillers Co., Ltd., Tozer, H., and Speedie, J. D., Ibid., 650,600 (Feb. 28,1951). (125) Drug Trade News, March 21,1952. (126) Ibid., April 14, 1952. (127) Dulaney, E. L., U. S. Patent 2,545,572 (March 20, 1951). (128) Ibid., 2,571,693 (Oct. 16,1951). (129) Dunitz, J. D., and Robertson, J. H., J . Am. Chem. Soc., 74, 1108 (1952).
2009
Dutcher, J. D., Hosansky, N., Donin, M. N., and Wintersteiner, O., J.Am. C h m . SOC.,73,1384 (1951). East Anglia Chemical Co., Ltd., Brit. Patent 650,783 (Marah 7,1951). Ehrlich, J., Smith, R. M., Penner, M. A., Anderson, L. E., and Bratton, A. C., Amer. Rev. Tubern., 63, 7 (1951). Emery, W. B., Chemistry & Industly, 1951,289. Erdmann, W., Apoth.-Ztg., 3, 154 (1951). Farrer, K. T. H., Australian J . Exptl. Biol. Med. Ai.,29, 285 (1951). FDC Reports, 14-W10 (April 5, 1952). Finlay, A. C., et al., Amer Rev. Tuberc., 63, 1 (1951). Foda, I, O., and Vaughn, R., Bo&. Proc., 140 (1950). Foster, J. W., and McDaniel, L. E., U. S. Patent 2,584,009 (Feb. 11,1952). Fried, J., Herte, J. E., Thoma, R. W., Gerke, J. R., Donin, M. N., and Perlman, D., J . Am. Chem. SOC.,74 (I 952). Fujii, S., and Toda, T., J . AntzXotics ( J a p u n ) ,4, 163 (1951). Fujimasa, S., Koyama, Y., Tsubata, M., and Kuiomwa, A., Japan. Patent 174,816 (Feb. 27, 1948). Fukumbara, T., Yoshida, H., and Shibuya, M., J . Agr. C h a , SOC.Japan. 24,133 (1951). Gaden, E. L., Jr., presented before the Division of Agricultural( and Food Chemistry, 121st Meeting of the AM. CHEM.SOC., Milwaukee, Wis. Garey, J. C., presented before the Allegheny Section of t h e Society of American Bacteriologists (March 22, 1952). Garey, J. C., Downing, J. F., and Stark, W. H., presented before the Division of Agricultural and Food Chemistry, 119th Meeting of the AM.CHEM.SOC., Cleveland, Ohio. Garibaldi, J. A,, Ijichi, K., Lewis, J. C., and McGinnis, J., U. S. Patent 2,576,932 (Dec. 4, 1951). Garibaldi, J. A., Ijichi, K., Snell, N. S., and Lewis, J. C., presented before the Division of Agricultural and Food Chemistry, 117th Meeting of the AM.CHEM.SOC.,Philadelphia, Pa. Gill, E., Recent Advances in the Fermentation Industries, 1949, 69 (1950). Goldschmidt, M. C., and Koffler, H., IND.ENG.CHEM.,42, 1819 (1950). Gottlieb, D., presented before the meeting of the Mycological Society (Dec. 30,1949). Gottlieb, D., and Diamond, S., Bull. Torrey Botan. Club, 78, 56 (19.51). Go-indarajan, V. S., Ann. Rev. Biochem. and Allied Research India, 21, 64 (1950). Green, S . R., and Gray, P. P., Arch. Bwchem. and Bwphys., 32,59 (1951). Gregory, J. D., Novelli, G. D., and Lipmann, F., J . Am. Chem. Soc.. 74,854 (1952). . . Groupi, V., Frankel, J. W., Lechevalier, M. I?., and Waksman, S. A,, J . Immunol., 67,471 (1951). Grundy, W. E., Schenck, J. R., Clark, R. K., Hargie, M. P., Richards, R. K., and Sylvester, J. C., Arch. Biochem., 28, 150 (1950). Grundy, W. E., Whitman, A. L., Hanes, M. E., and Sylvester, J. C., Antibiotics and Chemotherapy, 1,307 (1951). Gunsalus, I. C., Struglia, L., and O'Kane, D. J., J . Biol. Chem., 194,859 (1952). Halick, J. V., and Couch, J. R., Proc. Soc. Exptl. Biol. Med., 76, 58 (1951). Hall, H. H., Benjamin, J. C., Wieser, C. F., and Tsuchiya, H. M., presented before the Division of Agricultural and Food Chemistry, 119th Meeting AM. CHEM.SOC., Cleveland, Ohio. Handa, Y . , Uchiyama, K., Hosaka, M., Nakamura, Y.,Sakaguchi, Y., Takiiawa, J., and Harada, T., J . Antibiotdca (Japun),2,76 (1950). Handa, Y,, Uchiyama, K., Hosaka, M., Nakamura, Y . , Sakaguchi, Y., Takizawa, J., Iwamoto, H., and Shimabara, K., Ibid., 3 (Suppl. B ) , 25 (1950). Hargrove, R. E., and Leviton, A,, Bact. Proc., 21 (1952). Harris, J. B., Brewers Digest (February 1952), Harvard, P., and Picard, J., French Patent 959,218 (March 24, 1950). Hata, T., Higuchi, T., Sano, Y., and Sawachi, K., J . Antibiotics ( J a p u n ) , 3,307 (1950). Hata, T., Higuchi, T., Sano, Y . , and Sawachi, K., Kitasato Arch. Exptl. Med., 22, 229 (1949). Tbid., p. 279. Hatsuda, Y . , and Yoneshiga, K., Japan. Patent 176,477 (July 8,1948). Heatley, N. G., J . Gen. MicroMol., 4,410 (1950). Hehre, E. J., Adv. in Enwmol., 11.297 (1951). Hehre, E. J., Bact. Proc., 23 (1952). Hehre, E. J., J . Biol. Chena., 163,221 (1946)
.----,-
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
2010
(176) Hehre, E. J., “Phosphorus Metabolism,” Vol. I, 48, Baltimore, Johns Hopkins Press, 1951. (177) Hehre, E. J., and Hamilton, D. M., J. BbE. Chem., 192, 161 (I- 951 - - .,).. (178) Hemmi. F., and Tomoeda, M., J . Agr. Chem. Soc. J a p a n , 19, 559 (1943). (179) Ibid., 21,6 (1946). (180) Ibid., p. 19. (181) Ibid., 22, 1 (1948). (182) Hesseltine, C. W., Pidachs, C., Whitehill, A. R., Bohonos, N., Hutchings. B. L., and Williams. J. H.. J . Am. Chem. SOC..74. 1362 (1962). (183) Hildebrandt, F. M., and Erb, N. M., U. S Patent 2,556,084 (July 5,1951). (184) I-Iinshelwood, C., Nature, 167, 666 (1951). (185) Hockenhull, D. J. D., Walker, A. D., Wilken, G. D., and Winder, F. G., Biochem. J . , 50,605 (1952). (186) Hockenhull, D. J. D., Wilken, G. D., and Whder, F. G., Nature, 168,1043 (1951). (187) Hodge, H. M., Hanson, C. I., and Allgeier, R. J., IND.EXG. CHEM.,44,132 (1952). (188) Hongo. M., J . Agr. Chem. SOC.J a p a n , 24, 119 (1951). (189) Hoogerheide, J. C., and Laughery, E. G., U. S. Patent 2,549,465 (April 17, 1951). (190) Hoover, S. R., Jasewicz, L., Pepinsky, J. B , and Porges, N., Sewage and Wustes, 24,38 (1952). (191) Hopkins, R. H., and Pennington, R. J., J . G a . Microbiol., 4, 171 (1951). (192) Horwood, M., Chemistry &Industry, 62 (1952). (193) Hromatka, O., and Ebner, H., Enzymologia, 13, 369 (1949). (194) Ibid.. 14.96 (1950). ii95j Ibid., 15;57 (1951). (196) Ideda, Y., J . Agr. Chem. SOC.J a p a n , 24, 51 (1950). i197) Ibid.. a.56. ii98j Ibid.; i.147. 22, 192 (1950). (199) Imai, Y., J . Japan. Biochem. SOC., i200) IND.ENG.CHEM.,43, 11A (May 1951). (201) Inskeep, G. C., Bennett, R. E., Dudley, J. F., and Shephard, M. W., IND. ENG.CHEM.,43,1488 (1951). (202) Jackson, R. W., Blom, R. H., and Tsuchiya, H. M., Recent Advances in the Fermentation Industries, 1949, 93 (1950). (203) Jeanes, A., U . S . Dept. Agr. BUT.Agr. I n d . Chem. AIC-288 (1950). (204) Jeffreys, G. A,, U. S. Patent 2,505,360 (April 25, 1950). (205) Joslyn, M. A., iMycopath. et illycol. Appl., 5 , 260 (1951). (206) Jukes, T. H., and Btokstad, E. L. R., Vitamins and Hormones, 9 , 1 (1951). (207) Kacska, E. A,, Denkewalter, R. B., Holland, A,, and Folkers, K. A,, J . Am. Chem. SOC.,73,335 (1951). (208) Kacska, E. A., Wolf, D. E., Kuehl, F. A., and Folkers, K. A , , Science, 112,354 (1950). (209) Karow, E. O., Sfat, M. R., and Bartholomew, W. €I., presented before the Division of Agricultural and Food Chemistry, 121st Meeting of the AM. CHEX.SOC., Milwaukee, TVis. (210) Katagiri, H., Symposia o n Enzyme Chem. ( J a p a n ) ,6, 1 (1951). (211) Katagiri, H., and Itagaki, K., Bull. Inst. Chem. Res., 22, 94 (1950). (212) Kato. K.. J . Antibiotics ( J a p a n ) ,3,791 (1950). i2i3) Ibid., 5,32 (1952). (214) Keussler, H. von, Swiss Patent 264,588 (Jan. 16, 1950). (215) Killough, J. H., Magill, G. B., and Smith, K. C., Science, 115, 71 (1952). ~ - (216) King, A. J., Abst. Proc., IdthIntern. COWT.of Chem., 210 (1951). (217) King, N. K., and Hambley, A. N., Australian Chem. Inst. J . & Proc., 17,403 (1950). (218) Kitahara, K., and Fukui, S., J . Ferm. Technol., 28, 333 (1950). and Bass, K., Chem. Listy, 45, 303 (1951). (219) Kleinseller, -4,, (220) Kleinzeller, A., and Skoda, J., Ibid., 44, 184 (1950). (221) Kluyver, A. J., Chemistry &Industry, 1952, 136. (222) Kluyver, A. J., DeLey, J., and Rijven, A., Antonie van L e a uenhoek. J . Microbiol. S e d . , 16, l (1950). (223) Kluyver, A. J., and van Zijp, J. C. M., Ibid., 17, 315 (1951). (224) Kluyver, A. J., and Visser, J., Ibid., 16,299,311 (1950). (225) Knight, S. G., M o b , W. H., and Frasier, W. C., J . Dairy Research, 33,929 (1960). (226) Koepsell, H. J., and Tsuchiya, H. M., J . Bact., 63,293 (1952). (227) Koepsell, H. J., Tsuchiya, H. M., Hellman, N. N., Kasenko, A., Sharpe, E. S., Hoffman, C. A., and Jackson, R. W., Bact. Proc., 23 (1952). (228) Kolaohov, P., and Nicholson. L. W., Econ. Botanu. 5. 60 (1951). (229) Kolachov, P., Smith, L. A,, and Willkie, H. F., Rev. fermentations et inds. aliment, 5,154 (1950).
.
I
Vol. 44, No. 9
(230) Kotake, Y., Sasai, A,, Koaze, Y., and Saito, Y., J . Antibiotics ( J a p a n ) ,4,621 (1951). (231) Kozema, T., J . Ferm. Tech., 28, 120 (1950). (232) Kulka, D., Hall, A. N., and Walker, T. K., Nature, 167, 905 (1951). (233) Kuo, C. L., Taiwan Sugar J . Quart., 3,225 (1950). (234) Lacroix. H. v., Ger. Patent. 802,333 (Feb. 8,1951). (235) Ibid., 803.593 (April 5, 1951). (236) Lampe, B., Branntweinwirt., 4,152 (1950). (237) Lancet, 1951, No. 6661, 952. (238) Langlykke, -4.,F., Smythe, C. V., and Perlman, D., “The Enzymes,” 2, p. 1180, New York, Academic Press, edited by J. B. Sumner and K. Myrback, 1952. (239) Leach, B. E., and Teeters, C. M., J . Am. Chem. Soc., 73, 2794 (1951). (240) Lee, S. B., IND. ENG.CHEX.,41,1868 (1949). (241) Ibid., 42,1672 (1950). (242) Ibid.,43,1948 (1951). (243) Legg, D. A., and Beesch, S. C., U. S. Patent 2,581,419 (Jan. 8, 1951). (244) Leply, K. C., Catron. D. V., and Culbertson, C. C., J . Animal Sci., 9,608 (1950). (245) Lewis, K. F., and Weinhouse, S., J . Am. Chem. Soc., 73, 2500 (1951). (246) Ibid.. a.2906. (247j Lilly, V-G., and Barnett, H. L., “The Physiology of Fungi,” Xew York, MoGraw-Hill Book Co., 1951. (248) Lin, P. W., Purdue Univ. Eng. Bull. Ezt. Ser., 72, 181 (1950). (249) Little, P. A., and Oleson, J. J., J . Bact., 61,737 (1951). (250) Lockwood, L. B., presented before the Division of Mycology, K.Y. Academyof Sciences (April 25,1952). (251) Lockwood, L. B., and Nelson, G. E. N., U. S. Patent 2,559,650 (1951). (252) Lulla, B. S.,Biochim. et Biphys. Acta, 7, 244 (1951). (253) Lulla, B. S., Research, 3,581 (1950). (254) Lumb, M.,and Fawcett, R., J . A p p l . Chem. (London),S u p p l . , 2, s. 94 (1951). (255) Lundin, H., J.Inst. Brewing, 56,17 (1950). (256) Lundin, H., Suomen Kemistilehti, 23A, 23 (1950). (257) Mallea, 0. S., Industria g quimica ( B u e n o s Aires), 12, 264, 282 (1950). (258) Markov, N., and Bogdanov. I., A n n u a k m i u . So$a, Faci~lte Med., 28,295 (1948,1949). (259) Marks, B., Can. Chem. Process Inds., 35, 910 (1951). (260) Martin, S.M., and Waters, TV. R., Bact. Proc., 24 (1952). (261) RIartin, S.>I., and Wilson, P.T.,Arch. Riochem. Biophys., 32, 150 (1951). (262) Maxwell, M. E., AustrnZin,ri J . A p p l i e d Sci., 1, 348 (1950). (263) McConnell, W. B., Can. J . Research, 28,600 (1950). (264) MoCowen, M. C., Callender, M, E., and Lawlis, J. F., Jr., Science, 113,202 (1951). (265) MeDaniel, L. E., U. S. Patent 2,538,942 (Jan. 23, 1951). (266) Ibid., 21538,943(Jan. 23, 1951). (267) McGinnis, J., Stern, J . R., Wilcox, R. A., and Carver, J. S., Poultry Sci., 30,492 (1951). (268) Mellors, H., J. A p p l i e d Chen. (London),2,68 (1952). (269) Merck & Co., Inc., Brit. Patent 650,187 (Feb. 14, 1951). (270) Merck & Co., Inc.. Swiss Patent 275,274 (Aug. 16, 1951). (271) Miner, C. S.,and Wolnak, B., presented before the Division of Agricultural and Food Chemistry, 121st Meeting of the -4x.CHEM.SOC., Milwaukee, Wis. (272) Miner, C. S., and Wolnak, B., U. S. Patent 2,544,273 (March 6. 1951). (273) MiAoura,’K., J. Ferin. Technol., 28,125 (1950). (274) Ibid., p. 186. (275) Ibid., p. 308. (276) Moyer, A. J., Bact. Proc., 26 (1951). (277) Munden, D. L., J . Applied Chem. (London), 2, 65 (1952). (278) Murphy, D., Stranks, D. W.,and Harmsen, G. W.,Can. J . Techno?., F29,131 (1951). (279) Nash, H. A., and Reynolds, D. C., J . Bact., 61,98 (1951). (280) Seish, A. C.. Can. J . Botany, 29,68 (1951). (281) Nelson, G. E. N., Traufler, D. H., Kelley, S. E., and Lockwood, L. B., IKD. ENG.G H E M . , ~1166 ~ , (1952). (282) Nesheim, R. O., and Johnson, B. C., Proc. SOC.Exptl. BWZ. Med., 75,709,710 (1950). (283) Neuberg, C., and Mandl, I., Enzymologia, 14, 128 (1950). (284) Nishida, T., J . Antibiotics ( J a p a n ) . 4, 299 (1951). (285) Xolmander, R. N., Kgl. Lantbruksakad. Tid., 90, 395 (1951). (286) N. V. Konigklylcke Nederlandsche Gist-en Spiritusfabriek, Dutch Patent 65,936 (Yo\-. 15, 1951). (287) O’Brien, E., Wagman, CT. H., and Perlman, D., Bact. Proc., 25 (1952). (288) Oda, M., J . Agr. Chem. SOC.J a p a n , 21,15 (1946). (289) Ibid., 22,3 (1948). (290) Oda, M.,and Yamaguohi, S.,Ibid., p. 67.
September 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
(291) Ogata, K., J. Antibiotics ( J a p a n ) ,3,512 (1950). (292) Ogata, K., Shibato, M., Ueno, T., and Nakazawa, K., Ibid., 4, Suppl. A , 44 (1951). (293) Ogino, S., Ibid.,4 (Suppl. A ) (1951). (294) Oginsky, E., Smith, P. H., and Umbreit, W. W., J . Bact., 61, 639 (1951). (295) Ohki, S., and Okano, K., Japan. Patent 176,676 (Sept. 20, 1948). (296) Oyake, S., J . Antibiotics ( J a p a n ) , 3,463 (1950). (297) Ozaki, A., and Baba, S., J . Agr. Chem. SOC.J a p a n , 19. 339 (1947). Pan, S. C., Andreasen, A. A., and Kolachov, P., Arch. Biochem., 30,6 (1951). Parke, Davis & Co., Brit. Patent 651,174 (March 14, 1951). Parker, A,, Symposium on Recent Advances in Fermentation Industries, 1949, 138 (1950). Parker, A., and Cherry, G. B., Chemistry & Industry, 1950,806. Parsons, G. W., U. S.Patent2,515,157 ( J u l y l l , 1950). Ibid.,2,568,360 (Sept. 18,1951). Pattie, E. C., U. S. Patent 2,567,258 (Sept. 11, 1951). Pe’pinsky, R., and Watanabe, T., Science, 115,541 (1952). Perlman, D., Am. J. Botany, 38,652 (1951). Perlman, D.,presented before the Division of Agricultural and Food Chemistry, 119th Meeting of the AM. CHEM.SOC., Cleveland, Ohio. Perlman, D., Langlykke, A. F., and Rothberg, H. D., J . Bact., 61,135 (1951). Perlman, D., Titus, E. 0.. and Fried, J., J . Am. Chem. SOC., 74,2126 (1952). Perlman, D., and Wagman, G. H., J . Bact., 63,253 (1952). Peterson, M. H., Hanes, M. E., and Sylvester, J. C., IND. ENG. CHEM.,44,231 (1951). Peterson, D. H., and Murray, H. C., J. Am. Chem. SOC.,74, 1871 (1952). Pfeifer. V. F.. presented before the Division of Agricultural and Food Chemistry, 121st Meeting of the AM. C ~ E MSOC., . Milwaukee, Wis. Pierce, J. V., Macchi, M. E., Hoffmann, C. E., Fong, C. T. O., Stokstad, E. L. R., and Jukes, T. H., J . Am. Chem. SOC., 73, 5919 (1951). Porges, N., Pepinsky, J. B., and Jasewicz, L., J . Dairy Sci., 34, 615 (1951). Pridham, T. G., Econ. Botany, 6, 185 (1952). Pridham, T. G., U. S. Patent 2,578,738 (Dec. 18, 1951). Principe, P. A., and Thornberry, H. H., Phytopathology, 42, 123 (1952). --, Ramachandran, K., and Walker, T. K., Arch, Biochem. Biophys., 31,224 (1951). Ramakrishman, C. V., I n d i a n Soap J., 16,288 (1951). Ramakrishman, C. V., Science, 113,125 (1951). Ramakrishman. C. V., and Banerjee. B. N.. Ezperientia. 7, 434 (1951). Raper, K. B., Mycologia, 44,l (1952). Raveux, R., Compt. rend., 231, 1006 (1950). Reed, L. J., DeBusk, B. G., Johnston, P. M., and Getzendaner, M. E., J . Biol. Chem., 192,851 (1951). Reed, L. J., Getzendaner, M. E., DeBusk,.B. G., and Struglia, L. J., Ibid., 194,849 (1952) Reeves, R. V., Chem. Eng., 59,145 (1952). Regna, P. P., Solomons, I. A,, Murai, K., Timreck, A. E., Brunings, J. K. and Lazier, W. A., J . Am. Chem. Soc., 73, 4211 (1951). Reindel, F., Frey, A., and Lother, E., Branntweinwirt., 4, 179 (1950). Rickes, E. L., and Wood, T. R., Belg. Patent 490,106 (July 30,1949). Rickes, E. L., and Wood, T. R., Cuban Patent 24,342 (Nov. 2, 1951). Rolinson, G. N., Nature, 168, 210 (1951). Rosenblum, C., and Woodbury, D. T., Science, 113,21 (1951). Rushton. J. H.. Chem. Ena. 47.485 (1951). - Prowess. , . (335) Ibid., 48,33 (1952). (336) Ihid., p. 95. (337) Rushton, J. H., IND. ENQ.CHEM.,43,111 (1950). (338) Ibid., 44,88 (1952). (339) Rusoff, L. L., Davis, A. V., and Alford, J. A., J . Nutrition, 45, 289 (1951). (340) Sadasivan, V., Arch. Biochem., 28,100 (1950). (341) Sakaguchi, K., Asai, T.. and Hongo, M., J . Agr. Chem SOC. J a p a n , 19,222 (1943). (342) Jbid.. 22,36 (1948). (343) Sakaguchi, K., Asai, T., and Unekata, H., Ibid., 18, 793 (1942). (344) Sakawchi, K., and Baba, 8.,Ibid., 18.405 (1942). (345) Ibid., p. 619. (346) Ibid.,p. 1033.
.-
Ibid., p. 1127.
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Sakaguchi. K.. Baba. 8.. and Takahashi. M.. Ibid.., 19.. 347 (1942). Sakaguchi, K., and Murao, S., Ibid., 23,411 (1950). Sakai, H I and Tsunoda, J., J . Antibiotics (Japan),4, 352 (1951). Saunders, A. P., DeRose, A. F.. MacCorquodale, D. W., Sylvester, J. C., and Weston, A. W., J . Biol. Chem., 189, 479 (1951). Saunders, A. P., Otto, R. H., and Sylvester, J. C., preselited before the Division of Agricultural and Food Chemistry, 119th Meeting, AM.CHEM.SOC.,Cleveland, Ohio. Scalf, R. E., and Stier, T. J. B., Trans. Kentucky Acud. Sci., 13,69 (1950). Scardovi, V., Ann. Microbiol., 4,131 (1951). Schade, A. L.,J. Bact., 58,811 (1949). Schmitz, A. J., Rept. Proc., 18th Intern. Congr. Chem. (1951). Seaman, G. R., Proc. SOC.Exptl. Biol.Med., 79, 158 (1952). Seliber, G. L., and Galovkina, N. A., Microbiologia, 20, 20 (1951). Sen, H. D., Ann. Rev. Biochem. and Allied Research I n d i a , 19, 61 11948). Sen: H. D:, and Kapoor, B. D., Proc. Sugar Technol. Assoc. India,19,41 (1950). Shipe, W. F., Arch. Biochem., 30,165 (1951). Shu, P., Can. J . Botany, 30,331 (1952). Shu, P., and Blackwood, A. C., Ibid., 29, 113 (1951). Shu, P., and Thorn, J. A,, Ibid., 30, 252 (1952). Shull, G. M., and Routein, J. B., presented before the Division of Agricultural and Food Chemistry, 119th Meeting, AM. CHEM.SOC., Cleveland, Ohio. Silcox, H. E., and Lee, S. B., IND. ENQ.CHEM.,40,1602 (1948). Slinger, S. J., Gartley, K. M., Pepper, W. F., and Hill, D. C., J . Nutrition, 43,345 (1951). Smiley, K. L., Sobolov, M., Austin, F. S., Rrtsmussen, R. A,, Smith, M. B. Van Lanen, J. M., Stone, L., and Boruff, C. S., IND. ENG.CHEM..43.1380 (1951). Smith, E. L., Biochem. i.,50, Lxvi’(1952). Ibid,, xxxvii (1952). Smith, E. L., Nutrition Abstracts & Revs., 20, 795, (1951). Smythe, C. V., Econ. Botany, 5,126 (1951). Smythe, C. V., and Drake, B. B.. U. S. Patent 2.480.090 (AuB. 23,1949). Smythe, C. V., Drake, B. B., and Neubeck, E. C., Ibid., 2,530,210 (Nov. 14, 1950). Speer, V. C., Madlock, H. M., Cuff,P. W. W., and Catron, D. V., Antibiotics and Chemotherapy, 1,41 (1951). Spendlove, J. C., and Stahly, G. L., Bact. Proc., 24 (1952) Spolek pro chemickau a hutni vyrobu narodni Podnik, Brit, Patent 659,947 (Oct. 31, 1951). Stansly, P. G., Shepard, R. G., and Winterbottom, R., U. S. Patent 2,599,950 (June 10,1952). Stavely, H. E., and Toops, E. E., Federation Proc., 10, 251 (1951). Stern, J. R., Gutierrez, J. C., and McGinnis, J., presented before the Division of Agricultural and Food Chemistry, 121st Meeting of the AM.CREM.SOC., Buffalo, N. Y . Stiles, W., “Trace Elements in Plants and Animals” (2nd ed.), Cambridge, University Press, 1951. Stock, F. J., Drug & CosmeticInd., 68, 176 (1951). Sumner, J. B., and Myrback, K., “The Enzymes,” New York, Academic Press, 1951-52. Sunde, M. L., Waibel, P. E., Cravens, Pf. W., and Elvehjem, C. A., Poultry Sci., 30, 668 (1951). Swart, E. A,, Lechevalier, H. A., and Waksman, S. A., J . Am. Chem. Soc., 73,3253 (1951). Tabenkin, B., Brit. Patent 651,671 (July 16, 1947). Taira, T., Yamatodani, S., Fujii, S., Komatsu, H., and Takomato, I., J . Antibiotics ( J a p a n ) ,4,167 (1951). Takata, R., and Kimura, T., J . Ferm. Technol., 28, 103 (1950). Takeda, K., andsuzuki, W., J . Antibiotics ( J a p a n ) ,2,88 (1949). Tanner, F. W., U. S. Patent App. 26,233, 08.Gaz. 633, 1298 (1950). Tarr, H. L. A,, Can. J . Technol., 29,391 (1951). Tatum, E. L., Bact. Proc., 11 (1952). Thierfelder, K., German Patent 805,347 (May 17, 1951). Thorn, J. A., and Haskins, R. H., Can. J . Botany, 29, 403 (1951). Thornberry, H. H., and Shanahan, A. J., Arch. Biochem. Biophys., 33,459 (1951). Todd, A. R., Chemistry & I n d u s t r y , 1951, 967. Trussel, P. C., U. 8. Patent 2,541,726 (Feb. 13,1951). Tsuchiya, H. M., Corman, J., and Koepsell, H. J., Cereal Chem., 27,322 (1950). Tsuohiya, H. M., Jeanes, A. R., Brecker, H. M., and Wilham, C. A,, Rept. Proc. 18th Intern. Congr. of Chem.. 107 (1951).
INDUSTRIAL AND ENGINEERING CHEMISTRY
2012
(400) Tsuchiya, H. M.,Koepsell. H. J., Corman, J., Bryant. G., and Jackson, R. W., Bact. Proc., 22 (1952). (401) Tsuchiya, M., J . Chem. SOC.J a p a n , 20,587 (1944). (402) Umesawa, H., Hayono, S., Ogata, Y., and Okami, Y., J . A n t i biotics (Japan), ( S u p p l . B ) , 3, 1 (1950). (403) Umesawa, H.,and Maeda, K., J . Antibiotics (Japan),2, 41 (1 950). (404) Umesawa, H., Osato, T., Utahara, R., Yagashita, K., and Okami, Y., J . Antibiotics (Japan),( S u p p l . B ) ,3, 16 (1950). (405) Van Dyok, P., and DeSomer, P., Antibiotics and Clkemotherap?~, 2,184 (1952). (406) Vergnaud, P., U.S. Patent 2,541,581(Feb. 13,1951). (407) Verona, O.,“The Microbiology of Fermentations and Indus-
trial hlicrobiology,” Pirense, Casa ed. Dr. Luiji Maori, 1950. (408) Vitucci, J. C., Bohonos, IS., Wieland, 0. P., Lefemine, D. V., and Hutchings, B. I,., Arc?k. Biochem. Biophys., 34, 409 (1951). (409) Vohs, R.L.,Maddock, I€. M., Catron, D. V., and Culbertson, C . C., J . A n i m a l Sci., 10,42 (1951). (410) Voss, J. G.,U. S. Patent 2,567,257(Sept. 11, 1951). (411) Wakaki, S.,J . Antibiotics (Ja.pan),4, 479 (1951). (412) Ibicl., p. 545. (413) Waksman, S. A,, Antibiotics and Chemotherapy, 1, 1 (1951). (414) Waksman, S.A.,J . Hist. Med. Allied Sci., 6, 318 (1951). (415) Walker, T. K.,Hall, A. N., and Hopton, J. W., Nature, 168, 1042 (1951). (416) Wallace, H.D., Ney, W. A, and Cunha, T. J., Proc. SOC.Esptl. B i d . Med., 78,807 (1951). (417) Wallace, T., “Trace Elements in Plant Physiology,” Chroriica Botanica Co., 1950. (418) Wall St. J. (April 29,1952). (419) Walton, R. B., Antibiotics aikd Chemotherapy, 1, 518 (1951). (420) Weindling, R.,and Kapros, C., Bact. Proc. 48 (1951). (421) Weirich, L.,U. S. Dept. Commerce Chemicals Division, personal communication (1952). (422) Westerdyk, J., Antonio van L,eeuwenhoek. J . Microbid. S e d . 12,223 (1947). (423) Westhuisen, G. C. A . vander, Spruit, J. P., and Sephton, H. H.,J . Applied Cheni. (Londoa),1, 356 (1951).
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(424) Wholan, W. J., and Naer, H., B m h e m . J . , 48,416 (1951). (425) White, J., Wallerstein Labs. Comnmuns., 14, 199 (1951). (426) White, J.,and Munns, D. J., ,J. I n s t . Brewing, 56,141 (1950). (427) Ibid., p. 194. (428)Whiteside-Carlson. V., and Carlson, W. W.,Science, 115, 43 (1952). (429) Whiteside-Carlson, V., uud Itosano, C. L., J . Bact., 62, 583 (1951). (430) Wijmenga, H.G.,Veer, W.L. C., and Lens, J., Biocham. et Biophys. Acta, 6,239 (1950). (431) Wiley, A. J., Holdenby, J. &I., Hughes, L. P., and Inskeep, G. C . , I N D . E N G . C H E1702 ~ , ~ (1951). ~, (432) \liilson, J. E.,Nature, 169,715 (1952). (433) Wolf, D. E., Valiant, J., and Folkers, K. A., J . Am. Chem. Soc., 73,4142 (1951). (434) Wood, T.R.,and Hendlin, D., U. S. Patent 2,595,499(May 6, 1952). (435) Woodbine, M.,Gregory, hl. E., and Walker, T. K., J . Ezptl. Botany, 2,204 (1951). (436) Woodruff, H. B., U. S . Patent 2,685,713(Feb. 12, 1952). (437) Wright, L. D., Cresson, E. L., Skeggs, €I. R., Peck, R. L., Wolf, D. E., Wood, T. R.. Valiant, d., and Folkers, K. A., Science, 114,635 (1951). (438) Wright, L.D., Skeggs, H . H..and Cresson, E. L., J . Am. Chem. Soc., 73,4144 (1951). (439) Yagishita, K.,and Umeeawa, H., J . Antzbiotics ( J a p a n ) , ( S u p p l . B ) , 3,lO (1950). (440) Yasuda, H., Enomoto, K., and Skamoto, A., J . Antibiotics ( J a p a n ) ,4,493(1951). (441)Yasuda, H., Hori, H., Yamazaki, K., and Misoguchi, S., Ibid., 4,433 (1951). (442) Yasuda, H., Yamasaki, K., and Misoguchi, S., Ibid., 4, 618 (1951). (443) Yasuda, S., Enomoto, K., Yamasaki, K., and hlisoguchi, S., Ibid., 4, 173 (1951). (444) Yonehara, H., Toyoma, T., and Sumiki, Y., Ibid., 4, 16 (1951). (445) Yuill, J. L.,Biochem. J., 49,xix (1951). (446) Zief, M., and Stevens, J. R., J. Am. Chem. SOC.,74,2126 (1952). REOEIVED for review J d Y 7,1952. ACCEPTED July 9,1952.
Friedel-Crafts Reactions .
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NATIONAL PRODUCTION AUTHORITY, WASHINGTON, D. C.
SAMUEL B. DETWILER, Jr., BUREAU OF AGRICULTURAL AND INDUSTRIAL CHEMISTRY, U. S. DEPARTMENT OF AGRICULTURE, WASHINGTON, D. C.
Friedel-Crafts reactions continue to hold the interest of many investigators and familiar names appear in the current literature on the subject. In contrast to previous years, there i s a dearth of contributions from the petroleum industry to the patent and technical literature on FriedelCrafts catalyst and mechanism of reaction. Current research, which is largely of a fundamental character, confirms accepted concepts regarding the formation of ionized complexes and halogen exchange between the catalyst and organic reactants.
T
HE Friedel-Crafts reaction continues to be an interesting and fruitful field for research and development. Last year, the reports in this annual review emphasized theory of reaction, thermodynamics, and the preparation of chlorohydrocarbons. I n this review, new developments relate t o the exchange of chlorine from solid aluminum chloride, and further confirmation is obtained t h a t the Friedel-Crafts reaction is an electrophyllic transformation in which the role of the catalyst (aluminum chloride, ferric chloride, or sulfuric acid) is limited t o the production Of the cation, Xf, in the general reaction: ATH
+ X + e ATX + H +
where Ar is a n aromatic group and X + is a cation such as D+, CHa+, or CH&O+. The current literature shows a marked reduction in the number of patents pertaining t o the Friedel-Crafts reaction and catalysts therefor. Instead, there are a number of papers of Russian origin dealing with Radzivanovskiy aluminum chloride prepared from aluminum chips and hydrogen chloride.
MECHANISM OF REACTION Reactions of alld exchange of hydrocarbons in the presence of acid catalysts appear as particular caSeS of the Friedel-Crafts reaction, Chiurdoglu and Fierens that it is permissible to attribute such chemical transformations to the mechanism bJr Fairbrother for alkylations (11) and acylstions (12) which is exemplified by reactions of benzene:
CeH6 f A + S PhA
+ H+
