FERMENTATION DAVID PERLMAN, ARTHUR E. TEMPEL, Jr., and WILLIAM E. BROWN SQUlBB INSTITUTE F O R M E D I C A L RESEARCH, N E W BRUNSWICK, N. J.
During the past year no significant expansion OF the fermentation industry occurred. Production of antibiotics did not increase significantly and a short period OF oversupply OF penicillin and streptomycin existed. Use OF antibiotics as animal feed supplements increased and a large fraction of the production of penicillin, bacitracin, aureomycin, and terramycin has been directed to the animal feed market, The ethyl alcohol and acetone-butyl alcohol Fermentation industries have benefited b y the lowered raw material costs, and the costs of the Fermentation products have met those OF the synthetic processes.
R
EVIEWS of fermentation in this series (IOA-IZA, 14A) have re-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 fermentation industries; industrial fermentation processes; and fermentation as a unit process. Several publications have appeared during the past year which have dealt with various portions of the fermentation industries (6A-8A, 1 S A j. Short summaries of the fermentation industries have been prepared by Aubel(1A) and Bolcato ( 2 A j, and a more complete and authoritative treatment has been prepared by Underkofler and Hickey and their associates ( 2 6 A ) . The series of papers concerned with microbial gron-th and its inhibition presented as part of the first international symposium on chemical microbiology contains discussions of aeration in submerged fermentations, recent advances in penicillin fermentation processes, and use of antibiotics as animal feed supplements ( I 7 A ) . Chain and others have contributed to a series of reviews on industrial microbiology including a general survey ( 6 A ) , a study of mycological production of citric acid (18A),antibiotic production ( 4 A ) ,and microbial syntheses (16A). Kluyver ( Q A )and Brian ( S A ) have included a general consideration of industrial microbiology in their discussions of various phases of microbiology.
E C O N O M K S OF T H E F E R M E N T A T I O N INDUSTRIES I n the previous review of this series (14A) a section was devoted to the economics of the fermentation industries. This policy has been continued this year as it reflects trends in industrial applications of fermentation processes, I n general it can be stated that the industry in the United States encountered a period of decreased earnings in 1952 despite the increased gross sales which were experienced by many companies. E X P A N S I O N O F P R O D U C T I O N FACILITIES
The $45,000,000 expansion mentioned in the previous review ( l 4 A ) which was authorized by the Defense Production Authority through “certificates of necessity” for the antibiotic industry in the United States has in large part been completed ( I S B , WIB). The goal for penicillin production was set by the Defense Production Authority a t 600 trillion units per year t o be reached by January 1, 1955. This compared with an industrial capacity of 250 trillion units on January 1, 1952. The goal, however, was reached by December 15, 1952 (SOB). It is quite apparent that the antibiotic industry has already expanded beyond current needs and is able to produce more material than required. Several producers are, or have been, operating on an intermittent production basis and i t was recently indicated t h a t one large producer has placed a new antibiotic plant on a stand-by basis for t h e year 1953.
Facilities for the production of a t least 1,0G0,000 t o 1,500,000 pirlts of dextran per year in the United States were completed in 1952 (21B). Current domestic production could reach 3,000,000 bottles (18B) in 1953. Facilities for the production of 40,000,000 proof gallons of ethyl alcohol for industrial purposes per year by synthetic processes will be completed in 1953 (19B, 22B). Acetonc will be manufactured t o the extent of 50,000,000 pounds per year as a by-product of the new process by which phenol is synthesized from cumene ( 1 6 3 ) . These facilities mill provide increasing competition for fermentation products. Two plants, one capable of producing 45,000 tons of dried yeast per year from sulfite waste liquor, t,he other with an annual capacity of 1000 tons, are reported to be close t o full-scale operation
(QB). Q U A N T I T I E S O F F E R M E N T A T I O N PRODUCTS PRODUCED
Solvents. A slight increase in production of ethyl alcohol for industrial uses was noted in the fiscal year ending June 30, 1952. Of the 467,000,000 proof gallons produced, not more than 40 to 50% was made by the fermentation industry. It is expected that the share of this group in domestic production will continue to decline as a result of ever increasing competition of synthetic facilities. The fermentation production capacity in the United States is now rated a t 200,000,000 proof gallons, while the synthetic processes will soon be capable of manufacturing 350 t o 400,000,000 proof gallons annually; 185,000,000 proof gallons from ethylene gas (S‘B), and the remainder mainly from ethyl sulfate. An advantage enjoyed by the synthetic producer is the Sthility to offer long term contracts to industrial alcohol conwmeis because of relatively stable raw material costs. On the othei liand, the fermentation producer can never be assured of the wppiy or the cost of his raw material one year in advance. Industrial alcohol prices dropped from 90 cents per pioof gallon early in 1952 to as low as 40 cents per proof gallon late in the year (4B) and by March 1953 ( 8 B )had recovered to 48 cents per proof gallon. The decline in alcohol prices in 1952 was brought about by several factors including decreased demands for synthetic rubber production, importation of stocks from France, and a general over supply on the market (97B). Prices firmed in 1953 as a result of increased government buying and the influence of rising costs for Cuban molasses. Unlike ethyl alcohol, the produetion of n-butyl alcohol has been relatively steady for the past 9 years, ranging from 116,000,000 to 153,000,000 pounds (19B).Although the estimated fermentation capacity is over 100,000,000 pounds, the fermentation share of the market has dropped precipitously from 112,000,000 pounds in 1944 t o 29,000,000 pounds in 1952. The loss of the market to synthetic producers has been attributed in large part to the extreme variability in price of the two raw materials used in fermentation-Le., corn and molasses. Two different prices have been set for n-butyl alcohol for several years depending on its source, with the higher priced material being the fermentation
1944
September 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
product. However, in 1952 fermentation producers met the synthetic price and n-butyl alcohol from both sources is currently listed a t 12 cents a pound. Acetone produced by fermentation is a by-product of the butyl alcohol fermentation and accounted for less than 4y0 of the total 560,000,000 pounds produced in this country in 1951 (29B). The quantity produced by fermentation decreased from 42,000,000 pounds in 1945 to 21,000,000 pounds in 1951 and its future will depend in large part upon the survival of the industrial production of butyl alcohol by fermentation process. Acids. Acetic acid, produced for human consumption in the form of vinegar, accounts for only a small fraction of the total acetic acid manufactured in this country. Annual production of acetic acid for vinegar was approximately 20,000,000 pounds for the years 1949, 1950, and 1951, and in 1952 was estimated a t 23,500,000 pounds (29B, SOB). Total production of acetic acid (100%) for 1951 was estimated a t 454,000,000 pounds. Lactic acid production in the United States has averaged approximately 5,000,000 pounds per year since 1948, including both edible and technical grades. In 1951, the last year for which Egures are available, 5,290,000 pounds were produced (29B). There are a t least four manufacturers of this product in the United States, all of whom employ fermentation processes using raw material such as starch, dextrose, whey, or molasses (22B). Annual production of citric a d d in the United States is estimated to exceed 50,000,000 pounds ( I B ) . No changes in the sales price of this compound have occurred during the past year.
1,
Steroids. A commercial process for the synthesis of cortisone which includes a fermentation step has been in operation a t the Upjohn Co. for approximately a year. This process has been competitive with the chemical synthesis bf cortisone from bile acids.
I
Vitamins. In 1951, 245,000 pounds of riboflavin were produced compared with production of 199,000 pounds in 1950 and 160,000 pounds in 1949 (29B). It is very likely that the production of feed grades, in particular, increased in 1952. Although the production is divided between synthetic and fermentation processes no figures are available for the relative amounts made by each. I n 1951 production of all forms of vitamin Bl2 exclusive of feed supplements totaled 84 pounds (29B). Process improvement during 1952 has resulted in the price of the purified material dropping from $350 to $295 per gram. The use of this substance as an animal feed supplement appears to be expanding. Vitamin B I ~ is now produced both as a by-product of several antibiotic processes and also as a primary fermentation product.
Dextran. Four companies in the United States with an annual production capacity of over 2,500,000 bottles a year are producing dextran for use as a plasma expander (7B). This production compares with anticipated annual requirements of 4,000,000 pints for civilian use. In addition there is an estimated demand of l pint per year for every man in the Armed Forces as well as a stockpile in all metropolitan areas of 1pint per person to meet the needs of civilian defense. Plants manufacturing dextran are also operating in Sweden, Great Britain, and South Africa (2B, 18B). Antibiotics. The total quantity of all antibiotics produced in the United States in 1951 for therapeutic purposes was 1,286,000 pounds. This included 625,000 pounds of penicillin salts; 354,000 pounds of streptomycin and its derivatives; and 307,000 pounds of all others (29B). An additional 236,000 pounds were produced for use in antibiotic feed supplements. During 1952 antibiotic production increased but not as rapidly as in the previous year. Production of penicillin for the years 1950, 1951, and 1952 was 223, 319, and 350 trillion units, respectively. Production of streptomycin for the same periods was 92,400, 159,500, and 175,100 kg. (2SB-WBB).
1945
Production figures for the other antibiotics are not available on an individual basis but combined they amounted to 220,000 pounds in 1950 and 307,000 pounds in 1951 (5’923). In May 1952 the estimated production of the three broad spectrum antibiotics, Chloromycetin, terramycin, and aureomycin, was estimated at 24 tons ( S I B ) which amountb to over 550,000 pounds on an annual basis. The industry was confronted with a series of competitive cuts in the selling price of penicillin and to a lesser extent streptomycin and the other antibiotics during 1952. This situation developed, with particular reference to penicillin, as a direct result of oversupply which in turn was attributed to overexpansion of plants since the outbreak of hostilities in Korea, During early 1953, sales of antibiotics were reported to be increasing but it is considered unlikely that prices will regain former levels. The decline in selling price and oversupply of penicillin and streptomycin has been attributed to decreased exports. However, this has not been substantiated for, although the quantity of penicillin exported fell from 3101, of total production in 1950 to 21% in 1952, the quantities involved were 69, 84, and 75 trillion units, respectively, for the 3 years 1950, 1951, 1952 (29B). A similar situation was found for streptomycin with exports for 1951 and 1952 amounting to 93,000 and 91,000 kg., respectively. Despite the fact that export volumes held relatively constant during 1952, dollar volumes did fall because of the reduced selling price of the product. The production of antibiotics has continued to expand in foreign countries. Increased production of penicillin in Russia ( 1 4 B )and Poland ( 6 B )has been announced and new plants for the manufacture of penicillin and/or streptomycin have been reported for Brazil (16B) and Sweden (6B). Antibiotic manufacturing has expanded in Great Britain where there are a t least six industrial concerns producing penicillin ( 1 l B ) . France no longer imports antibiotics ( I d B ) ; production in 1950 was listed a t 1.8 billion units of penicillin and about 1400 kg. of streptomycin. Japanese production increased from 116 kg. of streptomycin in 1950 to 2140 kg. in 1951 ( 2 4 B ) and from 7.5 trillion units of penicillin in 1950 (2SB) t o 16.3 trillion units in 1951 (25B). At least five producers of streptomycin and 25 of penicillin were listed. The market potential for antibiotics in the animal feed business is estimated a t $20,000,000 annually. In addition to aureomycin, terramycin, and bacitracin, three salts of penicillin are used for this purpose. These include the procaine-, and Gephenamineand the dibenzylethylenediamine-salts of benzylpenicillin. I n 1951, 236,000 pounds of antibiotics valued at $18,000,000 were produced for use in preparation of feed supplements (29B). There is every expectation that the volume of sales of antibiotics for use in feed supplementation will eventually exceed that used for medicinal purposes (26B). Several factors are now contributing to this trend. Recent cuts in the prices for feed grades of penicillin and bacitracin place the cost of antibiotic per ton of feed in the range of 60 to 75 cents. Changes in the Food, Drug and Cosmetic Act will allow freer use of antibiotics in feeds by eliminating the federal certification of preparations ( I 7 B ) . I n addition t o their use as growth stimulants antibiotics are now being employed in therapeutic amounts in animal feeds for the treatment of livestock ailments (26B).
INDUSTRIAL FERMENTATION PROCESSES Except where otherwise stated, progress in the various fermentation processes prior to 1952 has been reviewed (IOA-ldA, 1 4 A ) and only the more recent developments are considered in this survey. In the present review the discussion has been limited to fermentations that appear to be of industrial interest in this or other countries. Fermentations have also been included where biochemical processes, the process requirements, or products contain some element of novelty or scientific significance that yields greater understanding of basic factors in the field.
1946
INDUSTRIAL AND ENGINEERING CHEMISTRY
SOLVENTS
Acetone and Butyl Alcohol. While the volume of butyl alcohol produced during the past 10 years has ranged from approximately 150,000,000 pounds (in 1944 and 1951) t o 116,000,000 pounds in 1952, t h a t produced by fermentation processes has dropped from 112,000,000 pounds in 1944 t o 29,000,000 pounds in 1952. Acetone produced by the fermentation process has remained relatively constant during this period, averaging about 24,000,000 pounds (9%'). Beesch (8817, 39C) has combined personal industrial experience with data previously available in the literature to present a detailed summary of the production of acetone and butyl alcohol by fermentation. H e has included a discussion of fermentation of sugary mashes such as sucrose, beet molasses, citrus molasses, hydro], sulfite waste liquor, and inverted starches. A number of starch mashes including maize, kaffir corn, rye, wheat, rice, milo, cassava, and potatoes are also mentioned. Isolation and handling of cultures, plant operations and equipment, yields of products obtained, conditions of the process, and contamination problems are discussed in detail Matagrin (276C) has reviewed processes used in Central Europe and Great Britain. 2,3-Butylene Glycol. A number of reports have appealed during t h e past few years discussing microbiological conversion of carbohydrates t o 2,3-butylene glycol. However, owing t o the availability of the synthetic product (88C)the fermentation process has never been very seriously considered in the United States from a commercial viewpoint. The fermentation process has been operated in the Netherlands (%@C) and seriously considered in Canada. Freeman (246C) has summarized some of the problems encountered in fermenting cane and beet molasses mashes by Aerobacter aerogenes. Both batch and slow-feed techniques were used in his experiments. I n another report, addition of phosphate salts to beet molasses mashes resulted in higher yields and production rates by Aerobacillus polymyxa, Bacillus subtilis, and Seriatia nzarcescens strains (SObC, 387C). Yields of 143 to 187 pounds of 2,3-butylene glycol per 100 pounds of sugar were obtained when Pseudomonas hydrophzla or A. aerogenes was used to ferment beet molasses mashes in pilot plant as well as laboratory studies ( I s C , SO6C, 455C). Sulfite puIp waste liquor has also been fermented ( I Z S C , 3O4C) with yields equal t o those obtained when media containing equivalent quantities of glucose or cellulose hydrolyza,tes were used. Aerobacter cloacae has been reported to form 2,3-butylene glycol from d-glucosamine with a 17% weight yield (8'77C). Significant quantities of 2,3-butyIene glycol were formed by certain strains of Bacillus ceTeus but rates of glucose utilization and the yield of 2,3-butylene glycol formed were lower than found with other bacteria (423C). Crewther (106C-lOSC)has summarized studies on production of 2,3-butylene glycol by A. polymyxa strains when grown on 10 to 15% wheat mashes. These fermentations by cultures isolated in Australia resembled previously reported results with the exception that addition of growth factor sources was found to be necessary for rapid fermentations. Ethyl Alcohol. The competitive situation in the production of industrial ethyl alcohol in the United States mentioned in the last review ( l 4 A )has continued during the past 12 months. The volume of fermentation production from such carbohydrate sources as wood hydrolyzates, sulfite pulp waste liquor, and surplus farm produce has not amounted to a significant fraction of the total production (65C). Competition from synthetic chemical processes largely precludes the use of these materials except in unusual circumstances ( 2 4 9 C ) although reports on use of these materials in laboratory experiments still appear. Among the factors found to affect yield of alcohol in the fermentation of glucose were: sugar concentration, temperature of incubation, growth factors, and nitrogen sources, as well as yeast concentration (61C). Several studies emphasized the importance of the yeast concentration OR fermentation rate (167C, 436C).
VoI. 45, No. 9
&ohol tolerance of yeasts was increased by addition of thiamin t o the medium (861C). Nutritional requirements of brewer's yeasts have been reviewed by Thorne (QSOC), Barton-Wright (SSC), and others (S86C). Manufacture of ethyl alcohol from sweet potato starch has been studied extensively in Japan with yields approaching those obtained with starch from other sources (295C-297C). The amylo process in which the starchy substrate is hydrolyzed t o simple sugars by certain fungi (usually belonging to the genus R h i z o p u s ) has also been used in connection with the fermentation of sweet potatoes (dSbC, 325C) and tapioca starch (3WlC). Other studies have concerned fermentation of hydrolyzates of waste from soybean-protein manufacture ( W S C ) , wood hydrolyzates (180C, 218C, 37SC), fruit wastes (82QC),as well as sulfite waste liquor (411C, 427C', 469C). The use of fungal amylases instead of malt in the production of ethyl alcohol from grain has been mentioned in earlier reviews in this series. An evaluation of fungal amylases prepared from surface and submerged culture preparations has been reported by Pool and Underlrofler (341C, S4ZC). They obtained approxiniatelyequal yields of alcohol nhen either amylaee preparation wm used. h number of reports have dealt with the formation of unfermentable trisaccharides and polysaccharides during hydrolysis of starchy mashes by amylases from mold preparations (7C, 26C, 27C, 168C). Ethyl alcohol produced by means of processes using fungal amylases may be sold as industrial alcohol or as beverage alcohol. The deamination of amino acidP during the alcoholic fermentation has been the source of the alcohol found in fusel oil. The presence of 0.1 gram per liter of ammonium salts in the fermentation medium reduced the fusel oil formation to between 1 and 2 grams per kg. of fermented sugar (167C). Some of the fusel oil alcohols (amyl, isoamyl, and isobutyl alcohols) mere apparently derived from the yeast amino acids rather than the wort amino acids when the fermentation period was extended from 3 t o 7 days ( 7 S C ) . Glycerol. While all of the glycerol produced commercially in the United States has been made either as a by-product of the manufacture of soap or from propylene (298C), there has been a continuing interest in production of glycerol by microbiological processes, especially in Europe (405C). Practical and theoretical aspects of the various microbiological processes have been reviewed by Underkofler et al. (441C ) . Their experiments with the addition of magnesium sulfite and other slightly soluble sulfites to yeast fermentation mentioned in the patent literature (162C) are described in detail (44lC). Maximum yields obtained from glucose were of the order of 24 grams per 100 grams of glucose fermented, or about 46% of theory. Fermentation of molasses, saccharified starch, saccharified corn meal, and maltose under similar conditions resulted in about the same yields of glycerol based on carbohydrate content of the mashes (44%'). In other studies, yields from sulfite waste liquor were unsatisfactory (SOSC). Use of haloduric yeast cultures has been reported as helpful in increasing fermentation rates and glycerol production ( 4 7 4 C ) . A certain quantity of glycerol is formed during fermentation of sugary mashes by yeast. This has been reported to be independent of the sugar content of the mash (SOOC). Contamination of fermented mashes with Lactobacilli and other unidentified bacteria has often resulted in conversion of the glycerol to acrolein (291C, 379C). ORGANIC ACIDS
Acetic Acid. While the production of acetic acid from ethyl alcohol is one of the oldest known fermentations, there are still many problems t o be solved if highest process efficiency is t o be realized. The manufacture of vinegar by the Luxembourg or Michaelis method (using rotating drums) has been reviewed (86%') and appears t o be less efficient than the modified Fringa
September 1953
i
h
INDUSTRIAL AND ENGINEERING CHEMISTRY
generator process ( 1 4 C , 16C). The submerged culture technique has also been successfully used in an apparatus resembling a Waldhof-type fermentor ( 2 7 i C ) , as well as in a conventional aerated fermentor (2OIC, 2O2C). Studies conducted in the latter unit indicated that interruption of aeration for as little as 15 seconds resulted in marked inhibition and death of the Acetobacter. -4study of acetic acid bacteria suggested that the strains which tolerated high concentrations of ethyl alcohol and acetic acid were not necessarily the most desirable for fermentation processes. Most strains able to oxidize ethyl alcohol rapidly were usually not able to grow in 4% ethyl alcohol or acetic acid (269C, 27OC). Further study of the ability of Acetobacter suboxydans to oxidize various alcohols has indicated that while melibitol and maltitol were not altered, epimelibiitol was converted t o fructose and plantebiose ( i 4 7 C ) . It has been shown that the p H and carbohydrate present in the medium affect the vitamin requirements of A. suboxydans ( 1 7 4 C ) . When glucose was present in the media and the p H was above 5.0 t o 5.3, alanine was not essential, and niacin and valine were not required. When glycerol or sorbitol was substituted for the glucose a t p H 5.0 to 5.3 all three were rerequired, while if the pH was 4.6 valine was not essential. Citric Acid. While the mycological process for the production of citric acid by fermentation of molasses has been operated successfully on a commercial scale for more than 30 years, a number of basic changes have recently been suggested. Yuill ( 1 8 A ) has indicated a number of problems encountered in operating a surface culture process on a commercial scale including culture selection, maintenance of culture characteristics, medium preparation, and recovery of citric acid from fermented media. Moyer (SOOC, SOiC) has summarized studies on the effectiveness of alcohols in stimulating production of citric acid by A s pergilli and other species. This stimulation was observed when the cultures were grown on synthetic media as well as media containing crude carbohydrates. Apparently the alcohols acted as metabolic poisons in both surface and submerged culture processes preventing the metabolism of the carbohydrate to the “normal” products, carbon dioxide, and cell-substance. Addition of methanol t o a growing culture resulted in a shift from production of gluconic acid as major metabolic product, to production of citric acid. Moyer reported that experiments have been run successfully in pilot-plant as well as laboratory equipment. Yields of the order of 7001, (based on the weight of the glucose or carbohydrate fermented) were obtained as compared with less than half that efficiency obtained during fermentation of unsupplemented media. The addition of methanol was found to counteract the effects of metallic ions on the accumulation and metabolism of citric acid during the fermentation. An alternative to the use of these metabolic poisons has been the treatment of the media with ion exchange resins to remove the contaminating metallic ions which inhibit the accumulation of citric acid (99OC).
-4number of authors have mentioned the effect of aeration and medium composition on citric acid production by Aspergillus niger when grown in submerged culture. Yields of acid equivalent to 80% of the weight of the sucrose utilized were obtained in a 7day incubation period when A. niger (Wisconsin 72-4) was grown in a synthetic medium containing 15y0 sucrose. These fermentations were carried out in 50-gallon glass-lined tanks under conditions where the effective aeration rate (measured by sulfite oxidation in solution with viscosity of the order of I cp.) was between 0.9 and 3.5 m M of oxygen per liter per minute (64C). When this strain was grown on a medium containing ferrocyanide-treated beet molasses (120 grams of sucrose per liter) conversions as high as 72y0 were obtained in 70 hours in a laboratory size fermentation unit. Aeration with a mixture of air and oxygen was required to obtain this high conversion (275C). When less vigorous aeration was used, lower yields were obtained (1OOC). Conversions ranging from 30 to 80% of the sugar available were re-
1947
ported when other strains of A. niger and Aspergillus wentiz n-ere used in laboratory and production plant units in Russia (@IC). Study of citric acid production by strains of A. niger when grown in surface culture on media containing Brazilian molasses has indicated that metallic ions present in the molasses inhibit the accumulation of the desired acid during the fermentation period (119C). A surface culture process operated in Germany used a strain of A. niger which grew well a t 35’ C. on impure glucose media and produced moderately high yields of acid (259C). When a citric acid forming culture of A. niger was grown on a medium containing 140 grams of glucose per liter, gluconic, citric, and succinic acids were formed along with traces of oxalic acid (l75C). I n other studies it was found that in fermentation where large amounts of citric acid accumulated, citrate appeared to be formed by condensation of active acetate and a Cd-dicarboxylic acid. Oxalate appeared to be formed by splitting of oxalacetate or oxalsuccinate but not by direct oxidation of acetate (55C) contrary to another report (176C). Gluconic, Ketogluconic, a-Ketoglutaric, and Related Acids. Prescott et al. ( S 4 5 C ) have reviewed available information on the properties and production of gluconic acid, its salts, and d-glucono-&lactone. These substances which have been produced by bacterial or fungal fermentations have found use in the chemical industries as sequestering and catalytic agents. A submerged culture process fermentation has been designed by Blom et al. (5SC) and operated on a pilot-plant scale. The sodium gluconate produced was to be used in stabilization of waters where calcium carbonate and magnesium carbonate were present. A mash containing 35% of glucose was fermented by A. niger during a 40hour fermentation period. The sodium gluconate was recovered by drum-drying the fermented medium. Under these conditions the estimated factory cost was 11.78 cents per pound for a plant producing 3,000,000 pounds per year. Several alterations in this process have been suggested, including continuous addition of sodium hydroxide to neutralize the acid as it was formed (iO9C), as well as special techniques to be used in preparing the inoculum
(640 Production of 2-ketogluconic acid by fermentation of glucosecontaining media continued to receive attention although this process has not been operated on a commercial scale. Large quantities of 2-ketogluconic acid accumulated in fermentations when certain strains of Gluconobacter sp. were used, while little was found when other strains were studied ( 2 4 C ) . Strains of Cyanococcus chromospirans were also found efficient in oxidizing glucose to 2-ketogluconic acid and galactose to 2-ketogalactonic acid ( i S 7 C , 4OC). A process suggested for the production of succinic acid from glucose or starch involves microbial oxidation of glucose to 5-ketogluconic acid (Acetobacter sp. ) followed by oxidation with nitric acid to yield succinate ( 4 7 S C ) . Koepsell et al. ( S 4 4 C ) have summarized in some detail their studies on the oxidation of glucose to or-ketoglutarate by a certain strain of Pseudomonas Jluorescens and have indicated some of the conditions affecting this conversion. Itaconic Acid. Interest in production of itaconic acid has continued and a number of reports are available of operations on a pilot-plant scale. Conversions of the order of 50 t o 60% of sugar available have been obtained when Aspergillus terreus was grown on a 6% glucose medium for 48 t o 72 hours in submerged culture in a stainless steel fermentor ( 3 3 7 C ) . The itaconic acid was recovered from the fermented medium by filtration, followed by evaporation and crystallization. Estimated cost of producing this material on a commercial scale ranged from 28 to 31 cents per pound. Similar fermentation yields were obtained in studies conducted in smaller equipment using a medium containing more sugar and a longer fermentation period (311C). Itaconic acid is apparently formed during degradation of citric acid by this mold
(105C).
1948
INDUSTRIAL AND ENGINEERING CHEMISTRY
Lactic Acid. Although lactic acid has been produced in this country for more than 70 years on a commercial scale, few details have been revealed regarding present production methods. The report by Inskeep et al. (22B) described the process used a t the production unit of the American Maize-Products Co. in Hammond, Ind. Continuous neutralization of the acid produced in a medium containing 15% corn sugar, 10% calcium carbonate, 0,375y0 malt sprouts, 0.25% ammonium sulfate, and 74.375% water b y high-temperature strains of Lactobacillus delbruckii has been practiced. The calcium lactate was recovered by concentration, and then converted to lactic acid by treatment with sulfuric acid. Alt.ernatives to the use of malt sprouts, which were recently suggested in Japanese studies, include koji, rice bran, peanut oil cake, soybean cake, corn, and wheat bran ( S 2 6 C ) confirming earlier studies by Leonard et al. (266C). Certain Lactobacilli were found to convert starch directly t o lactic acid (238C) while in other processes the starch was digested with mold enzymes before fermentation (21C) by L. delbruckii. Addition of 1% ethyl alcohol to the medium was found t o increase the yields of lactic acid, and the tolerance of the microorganisms to lactic acid, as well as t o accelerate the fermentation (351 C, 3 7 0 C ) . Microbial production of D (-) lactic acid or L ( +) lactic acid may be obtained depending on the strain of bacteria used (61C) as reported many years ago by Tatum and Peterson (426C). Strains of Rhizopus sp. apparently produced only the D (-) form ( 2 3 7 C ) . The lactic acid racemate present in Lactobacillus p l a n t a r u m cells was obtained in cell-free form by grinding young cells with sand (2396). Montgomery and Ronca (298C) have summarized experiments studying production of lactic acid during the alkaline degradation of molasses. When Cuban blackstrap molasses was heated to 230" to 240' C. for 30 minutes, 1.2 equiv. of esterified lactic acid per mole of molasses were produced. A tubular reactor 10 feet long and 18 inches in diameter, operated continuously, could produce 5,000,000 pounds annually. Such an installation would be able t o replace a fermentation capacity of 100,000 gallons. VITAMINS
Riboflavin. While riboflavin produced by fermentation has accounted for a substantial portion of the riboflavin manufactured, only a few ieports describing technical aspects of this biosynthetic procese have appeared during the past year. Pridham's review (346C) of the biosynthetic processes has covered most of the literature available in 1951. H e emphasized the importance of culture maintenance and selection, medium composition, and fermentation operations in determining the efficiency of the biosynthetic processes. His studies on biosynthesis of riboflavin by A s h b y a gossypii suggested that increased vitamin production resulted when glucose was added intermittently to the fermentation (347C-34QC). Minimal heating of the medium during sterilization was necessary in order to obtain high yields (34%'). When A . gossypii was grown on media containing unhydrolyzed plant proteins instead of animal proteins, yields of the order of 1 gram per liter were obtained (40C). Plaut ( 3 4 0 C ) has studied the mechanism of riboflavin biosynthesis by A. gossypii. He found that while the carbon atoms derived from metabolized acetate were randomly distributed throughout the molecule, formate carbon entered the C-2 position and bicarbonate carbon only the C-4 position. Other studies indicated that the yeast will metabolize certain of the Krebs cycle intermediates, but the role of these metabolic products in riboflavin biosynthesis was not determined (289C). The biosynthesis of riboflavin by representatives of the related species, Eremothecium ashbyii, has also received attention. Yields of the order of 1 mg. per ml. were obtained when the culture was grown on a medium containing glucose, maltose, casein, salts, and growth factors (192C). It was apparent that inclusion of maltose in this and other media ( 2 5 3 3 resulted in increased vitamin
Vd. 45, No. 9
production. Other studies, concerned with the effect on riboflavin production of the presence or absence from the media of certain known growth factors or vitamins, were not too conclusive, a s the riboflavin yields were lower than n-as considered normal for this yeast (266C, 480C). Reports from Japan suggest that processes in which the yeast was grown on semisolid media were successfully operated. Soybean oil cake and other pressed seed cakes, rice and barley germ, and bran from different grains were used to support the growth of the microorganisms (M.4CJ41 7C4 2 0 C ) . Addition of sodium alginate to liquid media also appeared to improve vi$amin production ( 3 1 7 C ) . Riboflavin production by other yeasts has been studied in several laboratories. The yields obtained have been approximately 1 to 5y0 of those obtained with E. ashbyii. Myeocandida riboflavina (18C) and certain top yeast mutants (294C) appeared to produce more than other yeast cultures studied (346C) Vitamin Biz. Studies of the chemistry and production of the vitamin BIZcomplex have continued in a number of laboratories. Several reviews of the chemical developments have been published (111C, 391C, 392C) and it appears that a t least three forms of this vitamin are found in microbial products (122C, 659C, 389C, 390C, 392C). Frost concluded that there is some evidence that hydroxycobalamine rather than cyanocobalamine is the dominant form of vitamin BIZin liver extract (150C). It was shown that nearly all of the microorganisms used for bioassay of preparations containing vitamin B12 responded to pseudo vitamin BIZ,vitamin Blzr,and other factors which have not shown activity in animal tests (114C, 360C), a complication in a n already complicated bioassay (466C). Vitamin Blz containing CoB0and P32 has been prepared by biosynthetic methods and may be useful for analytical purposes (393C, 394C). A review of the literature available prior to 1952 concerning production of vitamin BIZ by microorganisms as well as its occurrence in plant tissues has been prepared by Darken (113C). In all experiments reported, the addition of ionized soluble cobalt salts to media used for growth of microorganisms resulted in a significant increase in quantity of vitamin Blz-like materials synthesized, as previously claimed by Wood (466C). Saunders et al. ( 3 7 1 C ) have reported on a survey of actinomycete cultures which produce vitamin BIZ and have concluded that considerable variation exists in the ability of representatives of this species to form vitamin Blz-like materials. A4similar conclusion was drawn regarding ability of Rhizobium sp. to form vitamin BIZ-like materials (67f2, 6"). Certain strains of propionic acid bacteria were found to produce relatively large quantities of vitamin BIZ with yields of the order of 3 mg. per liter of medium (300-hour fermentation cycle), Half that quantity was produced in shorter fermentation periods (d68C). Problems encountered in producing vitamin BIZ on a large scale by biosynthesis using Bacillus megatherium (N.R.R.L. B-938) have been summarized by Garibaldi et al. (1532). They included determination studies on effects of aeration rates, foaming, variation in medium composition, contamination, bacteriophage infections, and temperature control. This process has been carried out successfully under semisterile conditions using 20,000gallon fermentors and equipment originally designed for production of yeast. Many microorganisms absorb vitamin B12-like substances, and in some instances have been shown to hold considerable quantities (WQC,66C). This accounts for the vitamin BIZfound in algae (136C, 322C). Release of absorbed B12 may be accomplished by treatment with acid, hot water, or extraction with propyl alcohol ( 9 7 C , 141C, 424C). Other Vitamins and Growth Factors. While a large number of fungi and bacteria synthesize carotenoids and related pigments, no record of commercial exploitation is avitilable. Goodwin (163C) has summarized the literature available prior to 1951 concerning microbial synthesis of these pigments. Recent publications have indicated some of the mechanisms involved in syn-
September 1953
.L
INDUSTRIAL AND ENGINEERING CHEMISTRY
thesis of these carotenoids by bacteria, yeasts, and fungi (ISSCISSC). Maximum yields have been of the order of 10 mg. per liter of medium fermented. The structures of the pantothenic acid derivatives, coenzyme A and Lactobacallus bulgaricus factor, have recently been elucidated and the latter shown to be the disulfide of N (-pantotheny1)-8mercapto-ethylamine ( S I C , S99C, 4SSC). Media fermented by A. gossypii were used as a starting material for isolation studies (SSOC). Purified coenzyme A has also been prepared from yeast (S8C, 4SC). Ferrichrome, an organo-iron pigment isolated from the smut fungus, Ustilago sphaerogena (SO9C, SIOC), has been shown to be identical with coprogen, a growth factor required by Pzlobolus species ( 1 8 9 C , 19OC). This material was shown to have erythropoietic activity ( S 5 Q C ) . Large-scale preparation of adenosine triphosphate has been described ( S 5 4 C , S 9 8 C ) . Adenosine was incubated with plasmolyzed brewer’s lager yeast, inorganic phosphate, and glucose until conversion to adenosine triphosphate was practically complete. MICROORGANISMS FOR FOOD AND FEED
The possibilities of using microbial products as protein sources in the diet have continued to receive attention. Robinson (36SC) has reviewed some of the available literature and supplemented this with a summary of investigations in Germany. Molds and yeasts were grown there during World War I1 for use as protein supplements. Production reached 15,000 tons per year, far less than the goal of 130,000tons per year ( 4 S 6 C ) . Joslyn ( S S 7 C ) has commented on some of the problems encountered as follows: “To date no palatable, acceptable food or feed yeast has been produced. The efforts to make a desirable food yeast from agricultural and wood wastes might be successful, if present research shows what constituents in food yeast are undesirable and how these constituents are synthesized during the growing of the yeast. Another conceivable possibility is the upgrading of nonedible photosynthesized algal carbohydrates and protein into food for humans. Microbiological agents could convert these materials into suitable food for fish, which would then be edible protein for humans.” The efficiency of this ‘%wo-stage” process would probably be low, but still higher than conversion of ammonia nitrogen into meat obtahed by feeding ammoniated molasses or urea to ruminants. Algae. The literature on the cultivation of various species of Chlorella and other algae has been reviewed by Danckwerts and Sellers ( I I S C ) . Fowden 1 4 4 C ) has studied the effects of age on the bulk protein composition of Chlorella vulgaris and found the proteins similar in composition to those of brewer’s yeast, with the exception that the algae contained tryptophan and also more proline and arginine than the yeast. Syrett ( 4 1 S C , 4 1 S C ) has studied the assimilation of ammonia by nitrogenstarved cells of C. vulgaris, and has shown that the nitrogen was first converted to a-amino acids and then to basic amino acids. Light had little effect on ammonia uptake with these starved cells, but did affect normal cells. A study (W48C) on the efficiency of conversion of nitrogen to cells by Chlorella indicated that between 20 and 24y0 of the nitrogen of a medium containing nitrate was converted to cellular nitrogen. Continuous light favored cells of low nitrogen content, while alternating light and dark resulted in cells of high nitrogen content. Pirie ( S S 8 C ) has shown that production of Chlorella for food would be too expensive to compete with the present agricultural methods. Yields of 30 to 50 tons per acre per year were anticipated with high initial immtments in apparatus for temperature control and light. Mushrooms. The 10,000,000 pounds (dry matter) of mushrooms marketed in the United States annually have been produced by growth on flats in sheds. The submerged culture process was suggested sometime ago (204C, 206C) and has under-
(lac,
..
1949
gone further development. Nutrient requirements of Agaricus campestris when grown in submerged culture were studied, and i t was found that the flavor of the mycelium depended in part on the medium composition (SOSC). Only three of 52 cultures isolated from commercial mycelium grew on a simple synthetic medium. Wastes from the citrus, pear, apple, and asparagus canneries and other industries were found suitable as medium ingredients ( 2 0 7 C ) as was sulfite pulp waste liquor (9SC). It was estimated that a million pounds (25% dry matter) can be produced annually in a single 12,000-gallonfermentor ( 9 S C ) . The use of other varieties of mushrooms has been suggested (9SC) as their mycelium may be more nutritious (SOSC, S62C) and contain a more desirable flavor (9SC). Yeast. Dawson (11SC) has prepared a detailed summary of the cultivation and propagation of baker’s yeast. Medium composition, equipment used for propagation, separation and recovery of the yeast, and selection of yeast strains were mentioned in his report, Pyke (S6OC) has presented a few of the scientific principles used in the manufacture of baker’s yeast. He describes a good strain as one which may be produced in high yields, is easily centrifuged, has good “creaming” properties for dough, ferments sugar rapidly, and retains stability between production and use in baking. Recent practices in the panary fermentation have also been described (SS7C). Among the factors shown to affect conversion of medium carbohydrate to yeast cell material were p H of the medium ( 4 S l C ) and temperature of incubation ( 4 5 9 C ) . Optimal conditions varied somewhat with different strains of Saccharomyces cerevisiae. Production of fodder yeast from waste products has continued to receive attention. Use of sulfite pulp liquor as a major ingredient of the medium ( W S C , 47OC-47SC) has been studied in Japan. Investigators have reported that a strain of Mycotorula japonica converted approximately 63 % of the carbohydrate present to cell substance. This was a higher conversion rate than that obtained with the strains of Torula utilzs studied. T . utilis strains grew well on a medium containing hydrolyzed eucalyptus sawdust ( 1 S 8 C , 1S9C), pedia containing bagasse hydrolyzate ( 9 9 C ) , and media containing citrus press liquor ( 4 4 4 C ) . Relatively high conversion rates of fermentable sugars (hexoses and pentoses) to cell substance were obtained, Brewer’s yeast appeared to have higher nutritive value than T o r u l a in one study ( 1 9 1 C ) . Nearly all of the inorganic sulfur compounds present in media used to support the growth of Torula or Saccharomyces species was incorporated in amino acids in the cell wall (S6OC, 376C, 4 S l C ) , as shown by studies using radioactive sulfur. De Becee (118C) has shown the advantages of a mixed culture of Aerobacter cloacae and S. cerevisiae for use as a source of vitamins in animal feed supplements. Thaysen (4SSC) has summarized some of the economic factors involved in production of a food yeast from molasses. He indicated that 80% of the nitrogen added t o media used for the growth of T. utilis was converted to protein and the yeast contained 21.2 grams of protein per pound. The cost of this protein in the British West Indies was approximately 14 pence per pound. Meat in the same area cost approximately 11.5 times as much on a protein nitrogen basis but was more desired by the population. Production of a food yeast has also been started in South Africa. Cost of the product has been more than that of fish meal or soybeans and the material has not found wide use as an animal feed supplement. Bacteria. Processes for producing animal growth factors and feed supplements by growth of certain bacilli in nutrient media have been suggested (117C, 164C). I n some of the experiments 25 to 50% of the carbohydrate present in the medium was converted to cell material in a 6-to &hour fermentation when selected strains were grown on a 5y0 glucose-ammonia-salts medium in 20,000-liter, or smaller, fermentation units. Antibiotic Feed Supplements. As mentioned in previous re-
1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
views in this series, the use of antibiotics as supplements in the preparation of rations for poultry, hogs, and cattle has opened new markets for these fermentation products. A number of hypotheses t o explain this effect have been advanced (SOSC) and it seems likely that the antibiotics exert t,heir action by affecting the balance of the intestinal flora of the animals ( l l O C , 4OgC). Experiments with germ-free animals have shown that inclusion of antibiotics in the ration did not increase the growth rates of the species studied (S56C). n'either did feeding of cell material of nonantibiotic-producing actinomycetes (2Z9C), while antibiotics had no effect on the growth rates of chicks kept in sterile units until infected fecal contents were added to the ration (1OlC). The quality of meat produced n.hen pigs were fed rations containing antibiot,ics did not differ from that obtained from pigs not fed the supplement (6C). The increased growth rate was traced t o more efficient utilization of the ration and increased consuniption of feed (SOC, 62C, S61C). While apparently any one of a number of antibiotic supplements including penicillin, bacitracin, aureomycin, terramycin, and strept,omycin resulted in increased growth rates when fed to pigs, certain of these appeared to be more useful than others under specific conditions (168C, W B C , .409C).
Synthesis of Fats. Studies on the synthesis of fats by microorganisms have continued, although production on a comniercial scale has only been attempted during periods of extreme economic stress. Studies conducted in Japan ( S l l C ) , Turkey ( 3 1 8 C ) , Yugoslavia (5SC), and Czechoslovakia (SdC, 8.41C) have dealt with lipid production by yeasts and fungi. Highest yields reported (lipid per 100 grams of carbohydrate utilized) were obtained with Rhodotorula gracilis where t'he conversion rate v a s 27.4 (S18C). It was observed that the fat produced by E . gracilis was more saturated and of lower molecular weight when formed at higher incubation temperatures ( 3 4 C ) . DEXTRAN
The background and some of the prob,lems involved in production of dextran for use as a blood plasma expander were mentioned in previous reviews ( 1 2 ~ 4l ,4 A ) . The literature concerning the properties and chemistry of bacterial polysaccharides has been summarized by Stacey (403C-405'C), and Barker and Bourne ( S I C ) . Gropper et al. (I7OC) have reviem-ed the properties and potential use of a number of materials as blood plasma expanders Their survey included gum acacia, gelatin, oxypolygelatin, de.;tran, polyvinylpyrrolidone, isinglass, pectin, globin, casein digests, ascitic fluid, hemoglobin, polyvinyl alcohol, methylcellulose, animal blood products, polyglucose, and subsidon ( a rutin preparation). Dextran has been the only one of these blood plasma expanders which has been approved by the U. S. Food and Drug Administration ( 69C, 323C). This approval followed studies on the metabolism of Cl4-labeled dextran (377C). This mateiial was prepared by fermentation of uniformly labeled sucrose. K h e n this dextran was administered to animals all of the CL4was found in the excretory products. A4number of reports have dealt with the problems encountered and techniques used in commercial production of dextran. Details of the process used in the production unit operated by Commercial Solvents Corp. have been described by Bixler et al. ( 4 9 C ) and others ( 7 4 C ) with especial emphasis on techniques used in hydrolysis and fractionation of the dextran. The process used a t Dextran, Ltd., has also been described (2876). It differs from that used a t Commercial Solvents Corp. in length of fermentation time (48 hours vs. 8 hours) equipment size and design, strain of Leucoizostoc species used, and the use of ultrasonic treatment rather hhan acid hydrolysis to reduce the dextran polymer t o the desired size. The process used a t the production plant operated by the R. K. Laros Co. has utilized an enzymatic preparation from Leuconostoc mesenteroides culture rather than the whole culture to synthesize the dextran polymer ( 7 6 C ) . Other comn~ercial
Vol. 45, No. 9
scale operations in the United States have been started by the J. T. Baker Co. and the Dextran Corp. Both of these companies have used the whole culture process (7417). The details of several studies of the biosynthesis of dextran polymers have been mentioned in the previous reviews in this series. In a more recent st,udy (169C) nearly all of 55 strains of L. mesenteroides mere found to produce gums from sucrose, using only t'he glucose portion of the molecule for polymer synthesis. These stra,ins converted the fructose portion to mannitol ( 1 6 K \ . A strain of L. mesenteroides was grown on a sucrose-salts medium suppleniented with cornsteep liquor or malt sprouts in a process designed for production of dextran polymers for use as thickening agent in sucrose sirups. During the fermentation the pH wai controlled between 5.0 and 8.0 (128C). ICoepsell and Tsuchiya (246C, 246C, 4 S S C ) confirmed and extended the work of Hehre ( l 8 1 C , 1832, 184C) when they demonstrated t,hat cell-free preparations of L. mesenteroides were able to form polysaccharides from sucrose. Factors found to influenre dextran-sucrase production by L. mesenferoides (N.R.R.1,. B-512) )rere a source of nitrogen, satisfied by cornsteep liquor, optimum pH for enzyme production (found to be 6.7), and the optimum sucrose coiicent,ration (4SQC). It was difficult, t o separate the cells from the ferimrited medium when media containing more than 2Oj, sucrose were used. Certain cultures will produce dextrans &h molecular weight of 80,000 when grown under controlled conditions (182C). The conditions affecting synthesis of dextran by the dextransucrase found in the fermented media were st'udied in relation to size of polymer produced. When the enzyme was added to 70% sucro'e solut~ionsa large proportion of t'he polymers formed were of lon- molecular weight, while large size polymers (molecule iyeight, 400,OUU) were formed when the enzyme was added to n 10yGsucrose solution (437C, 438C). Addition ol a small amount of maltose, isomaltose, a-methylglucoside, or glucose to the sucro~e~olut~ioii was necessary to initiate chain formation. In contrast to normal dextran synthesis, the process in the presence of efficient alternate acceptors led to extensilre oligomccharide formation snd simultaneous growth of many dextran chains. The oligosaccharides produced appeared to comprise the series t.0 be expected from successive addition of glucose by a-l,6-glucopyranosidic linkage on the alternat'e acceptor (246C). Carlson et al. (71C, 7RC) have studied the dextran-sucrase from Leuconostoc d e z t r a n i c w n (strain elai). A large fract'ion of this enzyme apparently was associated with the bacterial cell, and no evidence of dialyzable coenzymes or met8allicion activators was found. iiddition of glucose or fruct,ose to the sucrose-enzyme mixture resulted in the inhibition of dextran synthesis. Several authors have described methods of cont~rolleddepolymerization of the dextran polymers formed by the above processes. These include thermal depolymerization (4O6C, 464C), alkaline degradation (11C), acid hydrolysis (@C), and sonic energy (887C). Enzymes from fungi also degrade a number of (!extrans ( f O C ,ROSC, S19C, @9C, 460C). Xliile a discussion of t h e chemical and physical properties as ne11 as proposed structures of dextrans is beyond the scope of this reviem-, niention of several of the recent reports may be in order. Shape a,nd size of the polymers have been determined by light sca,tteriiig photometry ( 4 S S C ) and methods utilizing centrifugation, viscosity, and osmotic pressure data have been reported (169C, 374C). Chemical characterization and classification of dextrans have been attempted ( S S C , S S l C , 222C). Sulfated dextrans have had potential use as anticoagulants (288C, %7C, 368C, 407C). The preparations have had approximately one sixth the activity of heparin (in vitro assay) with less toxicity. Toxicity was found t o be a function of molecular size. Sulfated dextrans have been proposed as alt'ernatives t o sulfaterl alginic acid, or sulfated polygalacturonic acid (IO.@, 256C, a88C). E N Z Y M E S OF INDUSTRIAL INTEREST
During the past pear a number of reports have emphasized the niultiplicity of enzymes produced simult8aneouslyby microorgan-
September 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
1951 t
i
isms. A filtrate from Aspergillus oryzae when studied by paper chromatography and filter paper electrophoresis was found to contain esterase, amylase, p-glucosidase, sucrase, protease, catalase, cellulase, and alkaline phosphatase, contrary to earlier beliefs that only protease was present in significant quantities (160C). It seems likely that similar results will be found on analyzing other culture filtrates by these new techniques. Release of enzymes from bacterial cells by bacteriophage treatment may also reveal the presence of hitherto undetected enzymes (380C). Amylases. A large number of papers have dealt with aspects of production of amylases from bacteria and fungi. Lockwood (261C) has summarized some of the industrial practices. Purification and isolation in crystalline form of bacterial a-amylase ( 1 7 I C , i72C), fungal amylase (66C, 140c, 448C), taka amylase (SC, SC),and limit dextrinase ( 4 4 3 3 have been accomplished recently, Characterization of bacterial saccharogenic amylase and bacterial liquefying amylase has been undertaken (161C) as have studies on the Schardinger dextrinogenase (376C) and p-galactosidase (l03C). The effect of fermentation conditions on amylase production by fungi has been the subject of many studies. Control of the p H of the growing culture was shown t o be the limiting factor in production of a-amylase, maltase, and limit dextrinase by A s pergillus niger (375C) and possibly other related fungi (1%6C, 240C, 386C). Comparative studies on the types of amylolytic enzymes produced by various fungi grown under the same conditions have shown the value of the A. oryzae (high amylase) and the A. niger (high maltase) types (342C), as well as the A. u s a m i i (low saccharification) type (SI4C). Blackwood (5OC) has found Bacillus subtilis filtrates effective on hydrolyzing barley gum. Purified amylases mayobe freed from contaminating pectinesterases by treatment with urea or thiourea, agents which do not affect the former but practically inactivate the latter (397C). Pence (327C) and Reed (3632) have discussed production and use of fungal amylases in the panary fermentation and other aspects of bread making. Proteases. Extracellular proteinase production by members of the Aspergillusflavus-oryzae group when grown on corn mealsoybean meal media was observed and certain straips were shown to be far superior to the average (121C, 127C). I n studies with A , orgzae, rate of utilization of carbohydrate components of a modified Raulin medium was correlated with protease production. Highest yields were obtained when the carbohydrate was slowly utilized (179C) although variants induced by ultraviolet irradiation varied from the behavior of the parent culture (280C). Pilot-plant investigations utilizing A. oryzae grown on bran have shown that high yields obtained depended on the strain used (278C, g8iC). Purification of the protease from Mortierella renispora was attempted by fractional precipitation with ammonium sulfate solutions. The specific activity of the material obtained compared favorably with trypsin (454C). Streptomyces albus was found to produce a proteinase capable of lysing group A hemolytic streptococci (262C, 2?63C). Other Enzymes. A combination of glucose oxidase and catalase obtained from A. niger has found wide use in prolonging shelf-life of dried eggs. The preparation removes free glucose from egg yolks and whites by oxidizing i t to gluconic acid, water, and oxygen (SC,83C, 94C, 261C). Use of streptodornase and streptokinase preparations from Streplococci to disintegrate dead tissue and blood clots has continued to receive attention. A number of production problems have been mentioned, including culture selection, purification of the enzymes, and effect of medium composition on enzyme yield (46C, 9Oc, 91c, I S i C , 961C, 58iC). Study of cellulose decomposition by fungi and bacteria has continued, with the eventual objective of determining the relationship of ennymatic action to deterioration of fabrics. While
Myrothecium verrucaria has been found to produce more of these enzymes than other organisms so far studied (372C, 468C), a number of other organisms produce significant quantities (364C, 356C). Several enzymes were found in the crude cellulase of M . . verrucaria (456C, 467C). These cellulases were inhibited by salts of heavy metals, oxidizing agents, and alkaline conditions. The inhibition by most of these agents was reversed by sulfhydryl compounds and other reducing agents (35C). A culture filtrate of A. oryzae was found to contain enzymes capable of depolymeriz- ’ ing carboxymethyl cellulose and splitting all a-glucosides tested. At least eight components were found in the a-glucosidase mixture (294C, II5C). The mechanism by which yeasts synthesize phenylacetyl carbinol from pyruvic acid and benzaldehyde was found t o be a dismutation of pyruvic acid t o lactic acid and an acetyl-coenzyme A complex, which then condenses with benzaldehyde (396C). This condensation was first described by Neuberg and Hirsch ( S i a C ) and attributed to the enzyme carboligase. Phenylacetyl carbinol has been used in the manufacture of ephedrine. MICROBIOLOGICAL OXIDATION OF STEROIDS
The preliminary reports (149C, 83sSC) of the use of microbiological processes to introduce oxygen in the 11-position of the steroid nucleus were mentioned in the previous review. More recent publications from the laboratories of the Upjohn Co. indicated some of the details of the processes using R h i z o p u s nigricans and R h i z o p u s arrhizus for production of 1l-a-hydroxyprogesterone from progesterone (3O7C, 333C). These organisms have also been found to convert 4,16-pregnane-3,20-dioneto 11-a-hydroxy,17-a-progesterone (284C), 17-~~-hydroxyprogesteroneto 6-p17-a-dihydroxyprogesteroneand ll-or,l7-a-dihydroxyprogesterone (R85C), desoxycorticosterone to 11-epicorticosterone and 6-phydroxy-11-desoxycorticosterone (134C), 6-dehydroprogesterone to 1I-a-hydroxy-6-dehydropogesterone (334C), and 17-whydroxy-11-desoxycorticosteroneto 11-a-hydroxy-17-hydroxycorticosterone, 11-a,17-a,2l-trihydroxy pregnane-3,20-dione, and 6-phydroxy-17-a-hydroxy-11-desoxycorticosterone(331C), and pregnane-3,20-dione, and allopregnane-3,20-dione to the 11-hydroxy derivatives (136C). While a number of these products are formed in only minor amounts during the microbiological conversion, changes in fermentation operations, use of washed cell or cell-free preparations or other species of Mucor, Rhizopus, or related genera gave indications of increasing the yields of these substances (307C). Reports from other laboratories (RsOC, 268C, 366C) have served to confirm these observations that R h i z o p u s sp. are capable of introducing oxygen into the 11-position of progesterone When these cultures were used the 11-a-hydroxy derivative was formed, which may also be formed chemically from 11-keto compounds by reduction with sodium (188C). This 11-a-hydroxy progesterone has been used as a starting material for the production of cortisone (967C) and 17-a-hydroxycorticosterone (957C). According to Mancera et al. their studies “appear t o make this combined microbiological-chemical route the best yet described synthesis of cortisone” with possible over-all yields from progesterone of the order of 20% (268C). While the ll-a-hydroxy17-a-hydroxy corticosterone (epi-hydrocortisone) formed by microbial oxidation from 17-a-hydroxy-11-desoxycorticosterone (Compound S) did not have hydrocortisone activity in in vivo tests (149C ISOC) this material has served as starting material for syntheiis of the 9-halo-hydrocortisone derivatives. These compounds have had higher activity than hydrocortisone in in vivo studies (i48C). Cunninghamella blakesleena has been found to convert ll-desoxy-17-a-hydroxycorticosterone to cortisone and 17-a-hydroxycorticosterone. Helicostylum piriforme converted this substrate to the 14hydroxy,*the 6-@-hydroxy,the 11-a-hydroxy, and the 8hydroxy derivatives (SO7C). Mucor griseocyanus and M u c o r parasiticus have also been found to convert progesterone and other steroids to the l4hydroxy derivatives (983C).
1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 45, No. 9
1
Certain of the androgens, including androstenedione, testosterone, and 17-methyltestosterone were oxidized by R. arrhizus, R. nigricans, and R. reflexus to the 6-&hydroxy, the ll-a-hydroxy, and sometimes the 6-6-11-a-dihydroxy derivatives ( I S S C ) . Pseudomonas cultures apparently tended to degrade testosterone completely when this was added to growing cultures, washed cells, or cell-free preparations as indicated by opening of A-ring of the steroid (SSSC, S69C, 4 H C ) . Microbial metabolism of cholesterol has continued to receive attention. The conversion of cholestenone has been brought about by a number of organisms following the early studies with Acetobacter xylinum ( 2 4 7 C ) , Azotobacter sp. and proactinomyces sp. (198C), and other organisms ( 2 R C ) . Studies vr-ith Flavobacterium maris have shown that the cholesterol v a s converted to cholestenone and the latter to other products with the rate of conversion depending on fermentation conditions ( 2 3 C , 45C, 4 4 5 C ) . When an unidentified organism was grovm on a medium which contained cholesterol, cholestenone was formed, while if the medium contained potassium cholesteryl sulfate the 7-keto derivative was formed. This organism converted potassium cholestanyl sulfate to a dicarboxylic acid (opening ring -4) (4IOC). A number of fungi and actinomycetes have been found to oxidize pregnanolone to progesterone ( S 2 8 C ) , a conversion first observed by Mamoli using Corynebacterium mediolanuin (a66C). ANTIBIOTICS
The number of antibiotics offered for sale for therapeutic use in the United States increases every year as a result of the continuing search for drugs with broader applications. h'early all of the antibiotics marketed in the United States are mentioned in this review although few reports on the production of these substances have been available. The properties of antibiotics produced by actinomycetes (432, I Z O C ) and fungi ( 5 8 C ) have been recently summarized. General reviews of technology ( 1 8 7 C ) , chemistry (bOQC),and production in the antibiotic field have also appeared ( 2 I I C , 231C, S42C, 468C). The antifungal antibiotics have similarly received considerable attention (45IC). Beyond the established uses for antibiotics in therapy of human and animal infectious diseases, as well as the stimulation of animal growth, new uses for these compounds have been suggested. Control of certain plant diseases by the use of antibiotics inhibiting the growth of phytopathogenic fungi (67'2, 451 C ) and bacteria ( @ I C ) is a strong possibility. Furthermore, penicillin, streptomycin, terramycin, and other antibiotics have been reported to stimulate plant growth and increase percentage of seed germination .iT-hen added t o percolate water a t concentrations as low as 1 p.p.m. (SOC, SI5C). The proposed use of antibiotics as food preservatives has been banned by the Food and Drug Administration (86C). I n making this ruling the possibilities of sensitization of the consumer as well as the emergence of pathogenic microorganisms resistant to these drugs were considered. Penicillin. Several general papers have covered the subject of penicillin production since the last review. The various phases of the subject, including chemical and biological properties, manufacture, formulations, and uses have been surveyed (SSC). Johnson, while reviewing recent advances in penicillin fermentation, pointed out t h a t technological knowledge unfortunately was still ahead of scientific understanding of the process ( 9 2 S C ) . Florey and Abraham (14SC) summarized the early development of penicillin production and clinical use carried out a t Oxford University. The very competitive price situation which developed in 1952 resulted in practically no increase in production of this antibiotic over that of 1951, while production in 1951 w m 45% greater than that of 1950. New formulations and the introduction of penicillins of unusual biological properties have recently resulted in increased sales. Repository salts which have been claimed to be
better than procaine penicillin, from the standpoint of blood levels and allergic response, have been released for medical use. These include the 1,2-diphenyl-2-methylaminoethanolsalt of benzylpenicillin (401C), the N,N'-dibenzylethylenediamine salt of benzylpenicillin (ISOC), and the hydriodide of the 2-diethyiaminoethylester of benzylpenicillin, which have been marketed as Compenamine, Bicillin, and Leocillin, respectively. Bicillin has properties which are claimed to render more effective therapy by the oral route and also is said to simplify the formulation of repository preparations (463C), while Leocillin has been reported to have the property of marked diffusibility into pulmonary tissues ( 2 2 3 2 ) . A method of producing penicillin 0, the biosynthetic penicillin which commands a portion of the penicillin market on the grounds that it shows reduced allergenic efferts associated with its use, has recently been described (41C). One of the major factors contributing to overproduction of penicillin G i n the United States in 1952 was the technological advance in recent years which has increased plant capacities far beyond the original expectations of the designer. Greater productivity has resulted from the introduction of strains producing higher yields of penicillin, the use of more suitable media, and a better understanding of effect of environmental conditions on antibiotic production. The basic medium used in the industry for penicillin production has contained lactose, cornsteep liquor, calcium carbonate, animal or vegetable oils, and salts. I n order to induce maximum productivity of benzylpenicillin, the use of a precursor has been also required. A number of substitutes for lactose and precursor, the most expensive items in this medium, have been investigated in recent years in an effort to reduce manufacturing costs. Descriptions of the facilities for industrial manufacture of penicillin in Great Britain, Germany, and France have indicated that processes used in these countries have been similar to those used in the United States ( 4 A , 89C, 367C). Continuous processes which have the advantage of economy of operation as compared with the batch processes have not apparently been used on a commercial scale to date, Kolachov and Schneider (Z50C) described laboratory scale equipment in which 600 units per ml. of penicillin were obtained in a continuous fermentation operation. Three variations of a rebatching technique using 2000-liter fermentors have also been reported (447C). With this technique claimed as most promising from the industrial standpoint, old fermentations were rejuvenated by the addition of broth from younger fermentations. The young fermentation was kept at constant volume by the addition of fresh medium, Further studies on isolation of strains of penicillin-producing fungi have shown that higher productivity will result from careful selection of cultures. Gattani (166C) has used uranium nitrate to induce mutation in two strains of Penicillium chrysogenum. Mutants were obtained which produced more antibiotic than the parent strains when grown in surface culture on CzapekDox medium. Mutants which did not produce a water-soluble yellow pigment were isolated from spore suapensions of P. chrysogenum Wisconsin Q176 irradiated with ultraviolet light ( I C , SSSC). These mutants produced as much as 2630 units per ml. when grown in submerged culture in shaken flask fermentations. Further studies illustrated the difficulty of obtaining stable mutants following irradiation with ultraviolet light (S84C). A series of mutants producing a brownish-black pigment when grown in submerged culture in a medium containing cornsteep liquor, lactose, and phenylacetic acid were described (SSZC). Peterson and his colleagues, a t the University of Wisconsin, have described the geneology and biochemical performance of the races of pigmentless mutants obtained from P. chrysogenum Q176 b y treatment with ultraviolet light and nitrogen mustard (27C, 336C). Various members of this series have been and are being used in industrial plants all over the world. Outstanding members of this series are strains P. chrysogenum Wisconsin 47-1564 and Wisconsin 49-133.
September 1953
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INDUSTRIAL AND ENGINEERING CHEMISTRY
A better understanding of the microbiology of the fermenta tion has resulted “fom several morphological and cytological studies made on P. chrysogenum growing in submerged culture. Camici et al. (7W)described the conditions in shaken flasks which led to the development of the “pellet” type and the ‘Lhomogeneous filamentous’’ type of mycelial growth. Factors affecting the type of growth were the numbers of germinating conidia, the composition of the medium, the shaking motion, and the ratio of volume of medium to flask size. They also reported that the organism undergoes autolysis in submerged culture with the formation of vacuoles as the first indication of cell degeneration. The behavior of the cell nuclei during germination of the conidia and during the subsequent growth of the mycelial hyphae has been described (W17C, 4SSC). Studies with synthetic media have indicated that maximum yields of penicillin could be obtained when glucose or sucrose was added continuously a t a fate of 0.03% per hour to the fermentation, replacing the lactose usually added initially (926C). Hosler and Johnson (199C)obtained titers as high as 1774 units per ml. in 114 hours in fermentations carried out in 30-liter fermentors. A basal synthetic medium containing ammonium sulfate and other salts was used, and glucose and potassium phenylacetate were fed continuously a t rates of o.0370 per hour and o.c@t2~0per hour, respectively. The p H of the fermentation was controlled in the optimum range 7.0 to 7.4 by the automatic addition of gaseous ammonia. Soltero and Johnson (400C) studied the effect on penicillin production of the replacement in synthetic media ot lactose with glucose and sucrose in shaken flask fermentations. Investigations on the use of precursors for penicillin formation have been concerned with both the biochemical mechanisms involved and with the superficial effect on the roduction of penicillin alone. Mortimer arid Johnson (999Cp studied the relationship between precursor structure and biosynthesis of penicillins and found that P. chrysogenum Wisconsin Q176 used most effectively carboxylic acids which were not substituted in the alpha position. It had been demonstrated by earlier investigators that the major portion of phenylacetic acid added t o the fermentation could not be accounted for by the penicillin formed or the residual precursor remaining in the broth (S88C). Hockenhull et al. (196C) suggested that phenylacetic acid was oxidized completely t o carbon dioxide by mycelial suspensions. Other studies (2OC) mentioned the relationship between production of penicillin and technique of phenylacetic acid addition t o the fermentation. It was found that antibiotic production was often suboptimal because insufficient precursor was available t o the organism. The studies of earlier investigators on possible precursors for the biosynthesis of benzylpenicillin have been extended. Twentyeight esters of phenylacetic acid were prepared and tested (414C) as precursors for the biosynthesis of benzylpenicillin. It was demonstrated in shaken flask fermentations that the higher alkyl esters and the di-esters of certain glycols were equivalent t o or better than 8-phenylethylamine. AdditionR of the diethylaminoethyl ester of either phenylacetic or phenylaceturic acid t o the fermentation resulted in increased yields of benzylpenicillin and decreased toxic effects of the precursor (2C,SC, 146C). The use of these latter precursors was also said t o assist in subsequent recovery procedures because of the low solubility of the esters in acid organic solvents.
A new type of penicillin, as yet uncharacterized, has been reported in fermentation broths of species of Cephalosporium (4C). Unfortunately, its future utility in human therapy appears limited because of its low activity against both Grampositive and Gramnegative organisms. Taira et al. (416C) have prepared a series of alkylmercaptomethyl penicillins biosynthetically. Upon isolation these penicillins were found to be heat-stable in aqueous solution and less toxic to animals than benzylpenicillin. Several reports in recent years have indicated that penicillin production was stimulated by the addition of animal or vegetable oils to the medium in small quantities. Hickey (19%’) claimed that the addition of oleic acid, or its nontoxic metal salts, in combination with phenylacetamide increased penicillin productivity.
1953
I n a series of detailed investigations, Japanese workers have reported on the use of various oils as defoamers and their effect upon various phases of the fermentation. It was found that 0.1 to 7.070 soybean oil and castor oil stimulated penicillin production when added to the fermentation during the fist 24 hours (gl,@C,914C). When added later in the fermentation period a t the same concentration, these same oils brought about abnormal fermentation condition-.@;., foaming, irregular p H changes, and mycelial lysis. The abnormalities varied with the type of oil and subsequent studies indicated that the fatty acids were the responsible agents (SlSC, 116C,2 l S C ) . I n a series of parallel investigations (476C-479C) it was demonstrated that the autolytic effect of soybean and other oils in later stages of the fermentation was accompanied by increased lipase activity and an accumulation of ammonia nitrogen in the filtrate. Mycelial autolysis was slowed by various techniques, including the addition of 0.1% boric acid or acetic acid, or by adjustment of the pH of the medium to between 4.5 and 5.0 before inoculation. These procedures apparently reduced lipase production or counteracted its activity once i t was formed. The oxidative metabolism of P . chrysogenum has attracted the attention of several groups. In studies on the oxidative metabolism of P. chrysogenum Wisconsin 48-701, it appeared that high aeration was not directly required for the production of penicillin, but rather for the formation of the enzyme mechanism necessary for subsequent penicillin formation (S66C).- Later studies suggested that carbohydrate served as the principal carbon source in submerged culture fermentations when relslr tively low aeration rates were used (S64C), while a t high aeration rates lard oil appeared to be utilized in preference to available carbohydrate. In a study of the influence of 18 carbon sources on growth, respiration, and penicillin formation, the maximum penicillin yields, which were obtained when lactose was fermented, were associated with low respiration rates (S16C). Fermentation studies using CI4-labeled substrates, including carbonate, formate, acetate, and lactate, demonstrated that carbon from carbonate was not incorporated into penicillin (S74C, 4SlC). However, the data collected were interpreted as suggesting that metabolic pathways by way of acetate may provide the starting point for penicillin biosynthesis. Degradation of radioactive benzylpenicillin indicated that CI4 from carboxy- and methyl-labeled acetate was found entirely in the 0-lactam-thiazolidine portion of the molecule. Carbon from the methyl group was found in the keto carbon of the B-lactam ring only to a limited extent, while the carboxyl carbon occurred both in the ketone carbon and the thiazolidine grouping. Other investigations have shown that the carbon dioxide fixed by P. chrysogenum was incorporated into amino acids of the mycelium (161C). Sulfur and nitrogen metabolism have been recurring subjects of study and it was demonstrated that sulfur provided as sulfate, cysteine, methionine, glutathione, cysteic acid, taurocholate, or choline sulfate was equally available for penicillin production (899C, 408C). No volatile sulfur compounds were detected during these investigations. Analyses for sulfhydryl groups in the filtrate and in the mycelium during fermentation have suggested that oxidized forms only were found in the filtrate (951C). Reduced forms were found in the mycelium although glutathione could not specifically be detected. Rao and Venkataraman (S61C)were able to show that the culture medium became relatively free of amino acids during the period of maximum rate of penicillin syntheses in cultures grown on synthetic media. Streptomycin. Despite the temporary reduction in output of streptomycin by many companies during the second half of the last year, approximately 385,000 pounds were produced in the United States in 1952. This figure represents an increase in production over 1951 of between 5 and lo%, and was attained even though the synthetic antitubercular drug, isonicotinic acid hydrazid, was introduced on the market. S. A. Waksman has described the historical background, pro-
INDUSTRIAL AND E N G I N E E R I N G C H E M I S T R Y
1954
duction, properties, and utilization of streptomycin in his address on the occasion of his acceptance of the 1952 Nobel prize for medicine (Q48C). I n addition a bibliography of all the literature on streptomycin through 1952 has appeared (449C). Industrial aspects of streptomycin were stressed by Emery ( I S S C ) who reviewed the uses, properties, and production, and by Campbell ( 4 A ) who presented a flow sheet for the manufacture of the antibiotic. Techniques used in the isolation of superior mutants for streptomycin production, utilizing treatments of 8. griseus with ultraviolet light, were described by Pittenger and McCoy (SSK’). The principal technique used was a n assay procedure by which a portion of the mutant colony was stamped out and transferred aseptically t o a fresh test plate, overlayed with an agar layer seeded with B. subtilis and incubated. I n a series of experiments, five of 4000 colonies selected gave yields in submerged culture exceeding that of the parent by more than twice the standard deviation. The mutagenic effects of potassium permanganate, hydrogen peroxide, and x-rays on S. qrzseus spores have also been studied (177C-179C). A sigmoid relationship in the survival curve was noticed when the chemical agents were used, while the x-ray results were interpreted as a single hit effect It was observed that most morphological mutants resulting from treatment of these agents did not produce streptomycin. The disastrous effects of phage infection on streptomycin production have been mentioned previously. A procedure was described by which strains of S. griseus resistant to actinophage were selected (286C, 4 6 7 C ) . I n a second instance 74 of 111 fermentations in one manufacturing plant were infected with a multivalent phage during a 3-m.eek period ( 7 2 C ) . Changes in the culture medium which resulted in enhanced streptomycin production have been reported. A combination of nitrogenous materials including cornsteep solids, wheat gluten, and distiller’s solubles was claimed t o stimulate streptomycin production by supplying an “activity factor” (32OC). In another study, addition o€ inositol and a mixture of monoamino carboxylic acids, obtained by the hydrolysis of wheat, corn, or soybean protein, to soybean meal resulted in increased streptomycin production when these supplements were added either I S C ) . Fish meal has also been individually or together proposed as a suitable nitrogen source for the production of this antibiotic (219C). Factors affecting streptomycin yields obtained with a medium containing peptone, meat extract, glucose, and sodium chloride were investigated using four physiologically different strains of S. griseus. Antibiotic production was markedly influenced b_y the choice of the strain, the concentration of meat extract in the medium, and the use of tap water (156C). Approximately 1200 units per ml. were obtained when a selected strain was grown on a medium containing 1% peptone, 0.25% meat extract, 1% glucose, and 0.5%sodium chloride. In the last review of this series, investigations on the production of hydroxystreptomycin by two different laboratory groups were reported. Although this antibiotic has been of no apparent value, therapeutically, a patent was recently issued describing a process for its manufacture (44C). A fermentation process in which S. griseus was grown on a synthetic medium containing glucose, phosphate buffer, ammonium nitrate, ammonium sulfate, sodium lactate, and other salts has recently been patented. Yields were of the order of 165 units per ml.(4288c, 429c).
(lac,
WIDE SPECTRUM ANTIBIOTICS
A new wide spectrum antibiotic, erythromycin, was introduced on the market in 1952. Actual figures for the total production of the wide spectrum antibiotics have been unavailable but it has been indicated that the total production for aureomycin, Chloromycetin, and terramycin reached 24 tons in May 1952 ( S Z B ) .
Vol. 45, No. 9
Sales of Chloromycetin dropped during 1952 as a direct consequence of reports that the antibiotic was responsible for aplastic anemia and other serious blood disorders ( 7 7 C , 82C). -in investigation into the situation by a committee of outstanding medical authorities selected by the National Research Council resulted in the issuance by the Food and Drug Administration of the following ruling: “The Administration has weighed the value of the drug against its capabilities for causing harm and has decided that it should continue t o be made available for careful use by the medical profession in those serious and sometimes fatal diseases in which its use is necessary’’ ( 7 Q C ) . Aureomycin. KO literature directly related t o aureomycin production has been available during the past year. Metabolic aspects of Streptomyces aureofaciens were studied with particular emphasis on the octanoxidase system (194C). In addition a patent was issued for an animal and poultry feed containing aureomycin mash but no claim was made for the growth-promoting characteristics of antibiotic in the feed (338C). Cblorotetracycline is the generic name recently assigned by the Food and Drug Administration for aureomycin (95Cl Chloromycetin. The initiation of production of Chloroniycetin by chemical synthesis in Brgentina was recently announced (86C). The processes used in production and therapeutic applications of this antibiotic were recently reviewed ($56C). Terramycin. Oxytetracycline is the generic name assigned by the World Health Organization and b y the American Medical Association Council on Pharmacy and Chemistry to terramycin (SSC). The chemical configuration of this antibiotic has been elucidated (196C) but it is unlikely that the antibiotic will ever be produced by means other than fermentation (782). Erythromycin. This material is a wide spectrum antibiotic of low toxicity produced by Streptomyces erytheus. The antihiotic has been found active against Gram-positive and Gramnegative bacteria, mycobacteria, Rickettsiae, and certain large viruses ( I 7 S C , 264C). Studies have indicatrd that it retains therapeutic value when administered orally ( 285C). The crystalline compound has the empirical formula C74-36 HBo-86 NII-la,a molecular weight of 725, and is an organic baqe (264C). M I S C E L L A N E O U S ANTIBIOTICS
During the past few years, several antibiotics have found limited therapeutic use and consequently have been produced in relatively small quantities. Among these are bacitracin, polymyxin, tyrothricin, nisin, neomycin, carbomycin, viomycin, fumagillin, rimocidin, and achromycin. Bacitracin. A polypeptide produced by Bacillus subtilis Tracy, has been manufactured by several companies in the United States. It has been used topically for the treatment of infections caused by Gram-positive bacteria. The industrial process for the production of bacitracin includes fermentation, acidification, filtration, and extraction with butanol (76C). A process has been described in which B. subtilis was grown under aerobic conditions on a soybean meal, starch, calcium carbonate, and calcium lactate medium t o give antibiotic yields as high as 92 units per ml. (SS5C). The carbon-nitrogen ratio in the medium has been reported t o determine the relative proportions of bacitracin and licheniformin found in the fermentation broth ( 4 A ) . It has been demonstrated t h a t crude bacitracin contains 10 polypeptide entities which can be separated from each other by countercurrent extraction ( S I S C ) . The amino acids present in the various peptides have been determined and studies on bacitracin A indicated that it is cyclic in nature ( 2 l g C , 54SC). Bacitracin A has been found to have a molecular weight close to 1500. The P o l y m y x i n s , a group of a t least five polypeptides produced by Bacillus polymyxa, Bacillus aerosporus, and Baczllus circulans, have been manufactured in relatively small quantities in the
September 1953
*
Y
INDUSTRIAL AND ENG INEERING CHEMISTRY
United States. A process has been described in which maximum yields of antibiotic were obtained when B. p o l y m y x a was grown in submerged aerated culture a t 22’ C. in a medium containing glucose, corn meal, yeast extract, and salts (SSSC). Another similar process has been reported for the production of these antibiotics using either B. p o l y m y x a or B. aerosporus (SOC). Thyrothricin. An antibacterial substance produced by Bacillus brevis Dubos contains a t least two antibiotically active components, Gramicidin and Tyrocidin. Among the .organisms that are inhibited by tyrothrycin are Gram-positive bacteria and t o a limited extent, some Gram-negative pathogens. I t s use, therapeutically, has been limited t o surface applications because of its high toxicity resulting from hemolytic effects. Production in this country has not been carried out on a large scale. Mitchell (W93C)described a process in which 1.2 t o 1.4 grams per liter of antibiotic substance were produced when B. brevis was grown in submerged and surface cultures on a medium containing 4% cornsteep liquor, 3% cerelose, 0.05% potassium dihydrogen phosphate, and 0.05% calcium carbonate. Investigations by Battersby and Craig (SSC, 37C) have shown that commercial grade tyrocidin contains three major components. Tyrocidine A has a molecular weight of 1270 and is a cyclic polypeptide containing the following amino acid residues: valine, tyrosine, proline, ornithine, glutamic acid, and aspartic acid. Nisin. This antibiotic is produced by certain streptococci, and has been demonstrated t o consist of four polypeptides of varying biological activity. These have been found t o be similar to subtilin in amino acid composition (48C). Both nisin and subtilin contain the amino acid 0-methyllenthionine (314C). The antibiotic has been shown to be active against Gram-positive organisms and has been recommended to reduce growth of’ undesirable bacteria in the making and processing of cheese (47C). Neomycin. An antibiotic produced by Streptomyces fradiae 3535, neomycin, has been manufactured in the United States and in Italy. A bibliography of all the literature appearing through 1952 concerned with production, properties, and uses of neomycin has recently been published (440C). The production of neomycin in synthetic media has been studied using media containing glutamic acid and salts. Antibiotic production was increased markedly when mannose, arabinose, sodium malate, or sodium citrate was included in the medium ( I a S C ) . Carbomycin. This antibiotic has been reported to be effective in inhibiting the growth of Gram-positive bacteria, Rickettsiae, and certain of the large viruses (83C, 42SC). The antibiotic, produced by an organism identified as Streptomyces halstedii, has been found to be of relatively low toxicity. Chemical studies indicate that the compound is basic, has the empirical formula C4*HGesNOls and that i t can be extracted from filtered broth with butanol or toluene (44SC). The potential role of this antibiotic in human therapy remains to be clarified. Viomycin. This antibiotic is produced by a t least three species of Streptomyces, including S. puniceus, S . floridiae, and S . vinaceus (832, 84C); i t has been reported to be effective in the treatment of tuberculosis. Although the toxicity of this drug has limited its use (87C), it may be effective for complementing streptomycin treatment (QSC). The antibiotic substance called vinactin has been found to be identical with viomycin (98C, .972C, $732, W8dC, 4S4C). A detailed study of the effect of medium constituents upon viomycin production by S. jforidiae has been reported (108C). Fumagillin. A monobasic acid produced by strains of AspergilEus f u m i g a t u s , fumagillin, has been described as the most promising of the antibiotics for use as a direct-acting amebacide ( I N ) . Asheshov anti colleagues (26C) have demonstrated that fumagillin and phagopedin sigma, an antiphage agent, are identical Rimocidin. An antiobiotic produced by Streptomycin rimosus, rimocidin, has been found in the fermentation broth along with
~
1955
terramycin. The antibiotic has been shown to inhibit pathogenic fungi (116C) and protozoa (37SC) but has no effect on bacteria. Achromycin. Produced by Streptomyces albo-niger, achromycin inhibited both Gram-positive and Gram-negative bacteria. It was reported t o be therapeutically active against T r y p a n o s o m a equiperdum infections (African sleeping sickness) in mice when administered orally or parenterally (344C, 81C).
FERMENTATION AS A UNIT PROCESS Consideration was given to fermentation as a unit process in this series of reviews (ZIA, IWA, 144). As many of the principles involved have been summarized in the previous report only those which have been emphasized in recent publications will be covered in this review. Methods of recovery of fermentation products and disposal of fermentation wastes have often been a deciding factor in determining the economic success of fermentation processes, and surveys of these aspects of fermentation operations have been included. THE MICROORGANISM
Selection of Cultures. Culture selection and culture maintenance are of major importance in the fermentation unit process. Most industrial processes which harness microorganisms for biochemical work presuppose the maintenance of a “pure-line” of the organism used. With molds, for instance, once a desirable strain has been isolated in pure culture it must be regularly subcultured, if only for the provision of the necessary quantities of inoculum for the day-to-day requirements of the process (1380). In practice a number of complications are often encountered. Continued subculture on the same type of medium, particularly a more “synthetic” medium, tends t o produce abnormal features and, what is more serious for the manufacturer, may lead to the loss of the very properties for which the original strain was chosen. A more subtle and fundamental cause of change is the inherent tendency of molds to mutate, or suddenly to throw out lLsports” (1890). These mutants may be classified as spontaneous mutants, induced mutants arising as a result of certain treatments or growth on certain media, or mutants arising from heterokaryosis. All of these variants are of concern to the operation of a successful fermentation. It has been evident for many years that cultures or strains of a given species vary considerably in their abilities to produce a given metabolic product. This ‘‘natural variation’’ has led to the large-scale screening of cultures isolated from diverse natural habitats for abilities to perform the desired reactions most efficiently. A number of reports appearing during the past year have indicated that such screening programs have been successfully operated for the selection of actinomycetes ( S 7 I C ) , and bacteria (16SC), for the production of vitamin BI2,of fungi for the production of proteinases (iW7C,WSOC), of bacteria producing 2-ketogluconic acid (S4C), of bacteria for the production of acetone and butyl alcohol from sugary and starchy mashes (SSC, SSC), of fungi-producing penicillin ( I C ) , and of actinomycetes producing streptomycin (130, 600). Other studies have shown that strains of propionobacteria (268C) and Rhizobiu (68C)vary considerably in ability to produce vitamin Bl2 when grown under certain conditions. A large group of dextran-producing cultures have been studied as a result of the interest in blood plasma expanders. Some of the Leuconostoc species form branched chain polymers while others do not (W9ZC). In some instances, cultures isolated from natural habitats grow poorly on the common laboratory media after several transfers. This has been noted with mushrooms (WOSC). Associative growth of microorganisms may overcome this condition, and has been practiced with lactic acid bacteria (8aD) and bacteria and yeast mixtures ( I I 8 C ) . Flocculence of yeasts, often thought to be the result sf asseciative bacterial growth, has re-
1956
INDUSTRIAL AND ENGINEERING CHEMISTRY
cently been attributed t o genetic characters ( l f D , 9 8 0 ) as well as ionogenk groups on the cell surface ( 6 9 0 , SOD;. The taxonomist classifying microorganism often has difficulty in grouping cultures with certain similarities which also have many differences. These difficulties have beenrecentlyemphasized with regard t o the taxonomy of yeasts by Wickerham (l%?D) and by Lodder and Kreger-van Rij ( 6 2 D ) , the actinomycetes by Duggar (SOD), and the lactic acid bacteria by Pederson ( 8 7 D ) . Brian has summarized techniques used in identification of common fungi ( 9 0 ) and Cowan has surveyed the present culture collection systems ( $ 3 0 ) . The role of the microbial geneticist in increasing yields of antibiotics is becoming quite evident. I n these and other fermentations natural variants (580, 3 4 6 6 , 349C, 103D) and variants selected after exposure of the culture to such lethal agents as x-rays (1D , 179C, 6 4 0 , 117 D ) , ultraviolet light ( 17C, SJQC, 383C, 3 8 4 C ) , nitrogen mustard gas ( S D , 3 5 D ) , and a number of inorganic chemicals including oxidizing agents ( 177C, l 7 8 C ) and manganous ions ( 2 5 0 , 2 6 0 , 1060) often produced more of the desired metabolite than did the parent culture, Colonial variation caused by manganese can be prevented by addition of metal complexing compounds to the media ( 1 9 D ) . Addition t o media of compounds containing radioactive sulfur has also resulted in mutations ( 4 8 0 ) as has addition of uranium salts (166C). Study of a number of agents used in culture selection programs suggests that mutations may be induced by exposure to ultraviolet and x-rays, or manganous salts, while those strains isolated by treatment with antibiotics or bacteriophage have probably been natural variants ( 8 0 ) . The mutants derived from Peniczllium chrysogenum (Wisconsin Q-176) which produce large amounts of penicillin and do not form significant quantities of the yellow pigments have found widespread use in penicillin manufacturing plants (1 7 C , 336C, 38SC, S84C). Several mutants were found to elaborate brown or black pigments instead of the yellow pigments normally associated with P. chrysogenum (1?C, S8ZC). In some instances it has been observed that the mutants or variants selected for production of certain fermentation products have nutritional requirements differing from those of the parent culture. This has been true in studies on production of streptomycin (165C), chloramphenicol ( 6 8 0 ) ,penicillin ( 1 7 C ) , and riboflavin (349C). 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 has found favor in certain laboratories. Proom (1010) has found that viability of the lyophilized cultures depends in part on the rate of freezing of the cell suspension prior to lyophilization as well as the storage processes used after lyophilization. Rapid freezing a t -35' C. was necessary if high viability was to be obtained in the lyophilized preparations. These observations have been confirmed in other studies (4OD, 7 2 D , 1 3 3 D ) . Suspension of the cells in proteinaceous materials ( 3 4 0 , I l d D ) , or sugar solutions ( 4 0 0 ) prior to lyophilization has been found to increase the viability of the dried material. This was observed in experiments using bacteria, fungi, yeasts, as well as bacteriophage ( 8 3 D ) . Cultures of microorganisms may also be preserved by covering the cells with paraffin or similar oils. Studies of viability of bacterial, fungal, and yeast cultures preserved on agar slants covered by a layer of paraffin oil have shown that over a 5- t o 6-year observation period this method of preserving microorganisms was in general quite satisfactory ( 3 9 D ) . No improvements in this technique have been made since it was first suggested ( 7 4 D , 800, 1180). Bacteriophage. Among other problems concerned with selection of cultures is that of susceptibility t o bacteriophage. A bacteriophage infection affecting a culture used in a fermentation process may cause complete disruption of operations. Beesch
Vol. 45, No. 9
reports (38C,39C) that, when a butanol-acetone fermentation was infected, the process started as usual with the organism multiplying in the mash and building up acid in characteristic fashion. Instead of passing the acidity peaks as usually observed, the organisms did not continue the process which normally resulted in solvent formation. Cultures resistant to phage infection were obtained by taking advantage of the fact that in every culture there were a few natural variants which were resistant to the bacteriophage. Only resistant cells survived when the culture was grown in the presence of the phage and susceptible cells v, ere eventually lysed. Phage infections of streptomycin-producing fermentations have been mentioned in earlier reviews in this series A recent report by Carvajal ( 1 3 0 )mentioned a multivalent phage in an infected streptomycin fermentation. Cells of S. grzseus, S. 012vaceus, S. viridans, and S. griseolus were lysed by phage preparations. Reduced yields of streptomycin and vitamin Blz were found in fermentations inoculated with resistant strains obtained after exposure of the parent culture to the phage, contrary to observations in another laboratory ( 4 6 7 C ) . I n anothw report the undesirable effect on antibiotic production was mentioned (124C). I t took nearly 4 months to regain normal streptomycin production in this manufacturing unit. Garibaldi et al. ( 1 6 S C ) found many bacteriophages which lysed the vitamin Blz-producing Baczllus megatherium culture. A number of resistant strains were obtained, and used in rotation in operations in a fermentation where infection hazards were high owing t o the plant engineering and design. It was thought that by alternating these cultures the potentiality of a new infection might be reduced. A number of reports have dealt with phages infecting Eschej ichia coli, and it is likely that phages infecting other genera n-111 have many similar properties ( 6 7 D ) . StudieJ on the chemistry of the E. coli phages (32D) have shown a high content of nucleic acid. A hitherto unreported nucleic acid, 5-hydroxymethyl cytosine, was isolated from one strain (1350). The E. colz phages require organic substances for multiplication not required by the host cell ( 3 8 0 ) as well as inorganic ions which are involved 111 adsorption of the phage on the host cells (1000). The calcium required for the adsorption has been replaced by strontium, barium, and manganese, but not by other ions. Complexing of the calcium by citrate, phytate, or other substances reduced phage iufection and did not affect antibiotic production in a study nith 8,griseus (90D). SUBSTRATE
The varieties of nutrients used in fermentation processes, their availability and relative cost, as well as methods of preparation of media for fermentation of numerous carbohydrates and proteinaceous materials have received attention in earlier reviews in this series. The economics of the industry apparently limit the carbohydrate sources mainly t o cane and beet molasses, cereal grains, lactose, glucose, and sulfite waste liquor. The principal nitrogenous materials used have included cornsteep liquor, soybean meal, packing house wastes, and fermentation residueq. Strain specificity has been important in selection of medium ingredients; media supporting growth and production of the desired chemical substance by one strain have not always been satisfactory for another. Carbohydrate and Energy 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 or the acetone-butyl alcohol fermentations, the cost of the product has been largely determined by the cost of the carbohydrate, molasses. During the past 5 years the cost of cane molasses has ranged from 4 cents t o 37 cents per gallon. Butyl alcohol produced by fermentation processes has had t o meet selling prices set by synthetic processes
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INDUSTRIAL AND ENGINEERING CHEMISTRY
(19B). It is not surprising that production of these products by fermentation processes has been economically uncertain. When the fermentation product has not been the direct result of carbohydrate metabolism-for example, antibiotic production-the cost of the fermentation product has not been directly tied to the cost of the carbohydrate component of the media. Substrates including grains, potatoes, cassava, cane juice, and others have been used in fermentation operations in Japan, Europe, and Canada where these materials are relatively cheap, while use of these substrates in the United States is practically precluded b y their relatively high cost. The important raw materials used in the production of lactic acid have been glucose prepared from starch by acid hydrolysis, whey, or cane molasses (EB).Processes used for hydrolysis of corn starch have been described by Van Patten and McIntosh (1860). A rather extensive study suggests that the carbohydrate content of corn varies significantly with the source as do other components ot the grain ( 11.20). Beet molasses has been produced on a relatively large scale in the United States with production of nearly 80,000,000 gallons in 1950 ( 7 6 0 ) . Production in Canada approached 40,000 tons in the same year (455C). Processing techniques for recovering sucrose from the beet sugar juice and production of beet molasses have been discussed by McGinnis ( 7 0 0 ) and Murdock (76D). Removal of dissolved inorganic ions has often been accomplished by passage of the juice over ion exchange resins ( 6 8 0 , 1020). Such treatment also removes a portion of the often desirable amino acids present in beet molasses ( 3 7 0 ) which have served very well as nitrogen sources for growth of organisms producing vitamin B12 (15SC), citric acid ($?‘6C, SOlC), and 2,3-butylene glycol (456C). Cane molasses either as invert or blackstrap molasses has been used in production of ethyl alcohol and the acetone-butyl alcohol processes. Invert or “high test” molasses is an evaporated sugar cane juice containing approximately 75% total invert sugar, 1%protein, and about 2% ash. Blackstrap molasses is the sirup left after recovery of the crystalline sugar from the concentrated juice of sugar cane and usually contains about 58% total invert sugar, 8% ash, and 2% protein (S9C, 4 6 0 ) . Among the amino acids found in cane juice and blackstrap molasses from Florida were asparagine, aspartic acid, glutamic acid, glutamine, glycine, alanine, valine, leucine, serine, tyrosine, and a-aminobutyric acid (57D). Stiles has recently described the production of ammoniated molasses for use as an animal-feed supplement ( l 2 0 D ) which may result in withdrawal of considerable quantities of molasses from possible use as fermentation substrates. Lactose, a sugar utilized slowly by penicillin-producing fungi, has found wide use in media used for production of penicillin. Soltero and Johnson ( 1 1 6 0 )have reported that the less expensive starch, glucose, or sucrose when intermittently fed to the fermentation, has given penicillin yields on synthetic medium equal to those obtained under the same conditions with lactose. Further studies in pilot plant equipment confirmed these laboratory results (199C). Lipids including lard oil, corn oil, soybean oil, and peanut oil have also been used t o replace the lactose component of media used for production of penicillin, without significant change in antibiotic production. Best results were obtained when the replacement was made on a less than an equal weight basis, but above a caloric equivalent (la&‘, 91D, 9 3 0 ) . Lipids have also been used as energy sources in fermentations producing neomycin (92D), and streptomycin ( 9 5 0 ) . Highest antibiotic yields were obtained when the lipid replaced the carbohydrate on a caloric basis. Lipids have often been used as antifoaming agents in penicillinproducing fermentations. Continuous addition of these oils in large quantities sometimes results in interruption of the normal penicillin production processes (8laC-214C). Inhibition of growth and eventual lysis were apparently associated with the
1957
formation of fatty acids during the metabolism of the lipids (816C, 616C). However, slow addition of lipids stimulated antibiotic production (193C, 476C, 477C-479C, 1S6D). The fungi have been found to metabolize lipids preferentially when high aeration rates were used and the organisms were grown on carbohydrate-lipid media (364C). Precursors. I n certain fermentations the addition of known chemical substances t o the fermentation medium results in thq; incorporation of these directly into the fermentation product. For example, the addition of phenacetyl derivatives t o penicillin fermentations results in formation of benzyl penicillin. A number of derivatives of phenylacetic acid have been used in studies on biosynthesis of benzyl penicillin, and highest conversions were obtained with those which were slowly metabolized. These included the diethylamino ethanol esters of phenylacetic acid and phenaceturic acid (2C, SC, 146C), as well as the higher alkyl esters and the diesters of certain glycols of phenylacetic acid (414C). Studies on the metabolism of phenylacetic acid by P. chrysogenum suggested benzaldehyde and benzoic acids as intermediates in the degradation to carbon dioxide (196C). A large number of acids have been found to serve as precursors for the biosynthesis of penicillin, entering the molecule in place of the benzyl group (299C). Alkylmercaptomethyl penicillin (penicillin 0) has been produced on a commercial scale following earlier studies on biosynthesis ( d l C). Difficulties in producing other alkylmercaptomethyl penicillins were encountered as the precursors appeared to inhibit growth as the size of the alkyl group increased (415C). Among the precursors suggested for the production of streptomycin have been inositol (192) and glycine ( l 2 l D ) . Glycine was not found to be a direct precursor in biosynthesis of streptomycin in studies using C14-labeled glycine ( 8 1 0 ) . Several techniques have been used in adding the steroids to fermentations in which progesterone was oxidized to 16-a-hydroxyprogesterone, 11-a-hydroxyprogesterone,and related compounds. I n one series of experiments the steroid was added to media containing lipids which served t o solubilize the steroid ( 9 4 0 ) . In another series, the steroid was added in acetone solution to the growing culture 24 hours after inoculation (S07C, S33C). Nitrogen Sources. Proteinaceous materials such as extracted seed meals, yeast, and concentrates such as distiller’s solubles, cornsteep liquor, fish stick liquid, and meat stick liquor have been favored raw materials for many fermentations, particularly those producing riboflavin, antibiotics, or vitamin Bl2 (12C, 102C, 1137, 21QC, SO2C, 307C, 460C). A number of reports have suggested that combinations of seed meals with amino acid-containing hydrolyzates or other proteinaceous materials have resulted in higher yields of streptomycin when added to the fermentation media ( I d C , S08C) than when the seed meals were used alone. Similar results were obtained with media based on fish meal (919C). Study of the composition of media previously proposed for use in production of riboflavin by Ashbya gossypii has indicated that inclusion of packing house waste products resulted in increased vitamin production (346C). More recent studies have shown that plant products will also stimulate production of this vitamin by this microorganism (40C), and that methods used in sterilization of the medium are quite important in determining ultimate riboflavin yields (970). “Flash” sterilization of the media was shown to result in highest vitamin yields, with significantly lower potencies being obtained when other methods were used. While “overcooking” of the medium has often resulted in marked reduction in riboflavin yields, such practices have seemed desirable in processes used for production of streptomycin (1842). The nitrogen content of the media used for the growth of Aerobacter aerogenes was found by Virtanen and Alonen to affect the nitrogen content of the cells ( 1 2 7 0 ) . Cells containing lLsub-
1958
INDUSTRIAL AND E N G I N E E R I N G C H E M I S T R Y
normal” quantities of nitrogen feimented glucose slowly, produced less lactic acid, and more succinic and acetic acids, than cells with “normalJ’ nitrogen content. It seems likely t h a t observations on the value of increasing or decreasing the nitrogen content of media used for production of penicillin ( 5 1 D ) or streptomycin (155C) may have been related t o the absorption of nitrogen b y the cells of the microorganisms. The value of media containing mixtures of a number of substances containing nitrogenous compounds--e g., seed meals, yeast products, by-products of packing houses-may have been related to the availability of the nitrogenous compounds present (102C). Synthetic media containing ammonia or nitrate nitrogen have been proposed for production of penicillin (199C, 1 1 5 0 ) , streptomycin (4S9C),and vitamin Blz(153C),as well as a number of other microbial products. T h a p e n (42?6C) has indicated that yeast grown for food purposes converted nearly 85% of the ammonia nitrogen added to protein nitrogen. I n nearly all of the experiments reported, yields of microbial products obtained M hen the cultures were grown on synthetic media were significantly lower than when the cultures were grown on media containing natural materials Inorganic Nutrients. Earlier reviews in this series have discussed the apparent effects of the addition of inorganic salts or trace materials on the produetion of fermentation products More recent reports indicate t h a t addition of iron or magnesium ions t o synthetic media or media containing meat extract resulted in increased streptomycin production (166C, 4%8C, 489C). Zinc and iron ions when added to media in trace amounts have been found to affect growth and acid production by the fungi producing citric acid ( 1 8 A ) or gluconic acid (54C). Healy et al. ( 4 1 0 ) have studied the trace metal content of several natural and synthetic media. Their studies suggest that metallic ions can be removed from water by distillation followed by passage over an ion exchange resin (mono-bed). Donald et al. (W9D) have also studied this problem, and have concluded t h a t the most efficient procedure consists of its removal of contaminating ions by formation of solvent-soluble chelation complexes. Moyer (SOOC, 3UlC) has reported t h a t the addition of methanol, ethanol, or propanol to media containing metallic ions (as contaminants in molasses or intentionally added) resulted in counteracting the inhibitory effects of these ions on citric acid production by Aspergillus n i p . A similar result was obtained when substances sequestering metallic ions mere added to synthetic media (88D).These studies suggest t h a t the metallic ions serve to increase metabolism of the citric acid formed by the mold. The change in ratio of the quantities of citric acid t o oxalic acid formed may be influenced by the metallic ioiis present ( U D ) , although citric acid and oxalic acid have been reported to be formed by different biosynthetic mechanisms (55C‘). Sporulation and changes in the growth cycle of microorganisms have been shown to be related to the metallic ion content of the media. The iron, copper, zinc, molybdenum, and manganese content of soil and natural products have been determined by the growth response of A . niger, under standardized conditions (79D). Cobalt has been found to stimulate sporulation in many Streptomyces species ( 4 9 0 ) . Inhibition of yeast growth b y cobalt and nickel ions has been reversed b y addition of magnesium ( 6 9 D ) . Uptake of cobalt by Proteus vulgaiis and potassium tellurite by Escherichia coli has been studied using radioactive isotopes (WID, 7 8 0 ) . S T E R I L I Z A T I O N TECHNIQUES
Sterilization of Medium. The sterilization of the medium has been discussed in previous reviews in this series. I n a recent conference on bio-engineering, the principles of industrial sterilization were discussed and interpreted as a statistical probability ( S S D ) . It was pointed out that medium sterilization \vas a race
Vol. 45, No. 9
between the sterility of the batch and the concomitant destruction of proteins and the generation of toxic substances. ReaIistically speaking, the degree of heating most beneficial t o the process could be defined as t h a t required t o render medium “commercially sterile.” Conversely, the possibility cxists that optimum periods of heat steiilization may be beyond the point defincd as commercially sterile. A variety of methods has b ~ e i suggested i for destroying microorganisms in a fermentation medium, including: ( a ) application of heat, ( b ) ultraviolet radiation, (c) mechanical removal of bacteria by filtration, ( d ) chemical sterilization, ( e ) irradiatioii with high energy Roentgen rays, cathode rays, or ultrashort electrical impulses, ( f ) high frequency sound waves, and (g) a combination of chemical disinfection and relatively mild heat treatment. The first method has been generally accepted as the method of choice (97D). The principal advantage of continuous over-batch sterilization with steam was the retention of the nutritive value of the sterile medium as demonstrated by Pfeifer when the effects of both types of medium sterilization on yields of riboflavin and vitamin B,z were compared (97D). Means have been found for evaluating the qualitative and quantitative effects of high energy cathode rays in food-irradiation sterilization utilizing methylene blue (36D). The destruction of the methylene blue by cathode lays in the range 2 to 16 in e.v. can be used for determining penetration indexes and comparing different radiation sources. Sterilization of Air. The sterilization of air in the fermentation industry has been discussed in previous reviews (11A, IWA, l 4 A ) . Studies of thc mechanisms of filtration of bacteria from air stieams was reported (47D), summarizing and critically analyzing the knowledge of these mechanisms. An analogy between filter operation and the general characteristics of adEorption beds and the possible application to filter design were given. The several mechanisms by which bacteria may impact has been reported by Humphrey (47D) to include direct interception, inertial impaction, settling, diffusion, electrostatic attraction, turbulence, and thermal diffusion forces. Of these, interception, inertial impaction, and electrostatic forces are the most effective means of collection when considering bacterial aerosols of a particle diameter of the order of 1 micron. Pilot-scale investigation has shown that slag wool or glass .rvool can be used t o produce sterile air by filtration in carefully specified conditions of fiber size, packing density and uniformity, and air velocity (180). Studies in full scale fermentation equipment have confirmed these results. FERMENTATION EQUIPMENT
A general discussion of equipment used in the fermentation industry has been presented in earlier reviews in this series (11-4, 129, 144). Descriptions of new equipment intexided for use in fermentation plants have appeared, and modifications of units previously used have frequently occurred. Publication of AOTT sheets of processes used in production of dextran (49C), lactic acid (22B), bacitracin (,$SO), streptomycin (4A, 13RC), penicillin (367C), and other fermentation products has sh0TT.n which equipment has been used for certain unit operations A fermentation unit operation which has received major emphasis in the field of equipment design has been t h a t of media sterilization. The advantages of continuous or “flash” sterilization over batch sterilization are numerous, including steam and cooling water economy, uniformity of heating and cooling, and minimizing substrate destruction while continuing to obtain sterile medium. Continuous cookers, for the preparation of mash in the alcohol fermentation, have been i n use and operated satisfactorily for a number of years i n distilleries throughout the country. These units viere equipped with triplex or centrifugal pumps supplying mash t o steam jet heaters, followed by coil holding sections, and a “flash” tank. Back pressure on these
September 1953
1.
INDUSTRIAL AND ENGINEERING CHEMISTRY
units was controlled a t the entrance to the “flash” tank. A heat exchanger, for cooling the medium, was installed at the discharge from the tank. Some bumping or vibration occurred in units where the triplex pump was used. I n considering a continuous sterilizer for use i n antibiotic fermentation or any fermentation requiring sterility new problems arose which had not been encountered in designing equipment for use in the alcoholic fermentation where semisterility has been acceptable (97D). As higher sterilization temperatures and pressures were required, the selection of a pump became a major problem. Positive displacement pumps with a low degree of slippage were required. Overdesign was necessary when selecting a centrifugal pump to operate under high back pressure. Some measure of success was obtained with progressing-cavity pumps, which eliminated the bumping action experienced with triplex pumps. Other problems in designing a continuous sterilization system were concerned with the nature of the medium. It has been impractical to use a “flash” tank when sterilizing media free of suspended solids because of excessive foaming; cooling under pressure has been used (Q?D). Studies on continuous feeding of nutrients to fermentations operated on laboratory or pilot-plant scale have introduced problems in materials handling because sterile liquid transfer was necessary. Various types of metering equipment for laboratory and pilot-plant use have been described which may be operated under sterile conditions and used for addition of aqueous and lipid solutions (17C, 840, 199C, 1150). Engineering investigations into pump designs applicable to sterile metered solutions transfer operations were required before these techniques could be applied to production equipment. Included among the pumps investigated were gear pumps, proportioning pumps, and diaphragm pumps. Dolman (880) discusses the principle of the fluid-operated diaphragm pump and has prepared a handy and definite classification of all types of pumps, with special emphasis on chemical pumps, Materials used in fabrication of valves designed to meet the demands of the chemical industry have been described with reference to protective linings (184D) and diaphragms for diaphragm valve assemblies. The practical limitations of diaphragm valves due to constant wear and destruction a t high temperatures were considered. An entire plant equipped with diaphragm valves i n fermentation and extraction areas was reportedly obtaining average diaphragm life of 3 months (MC). The trend has been toward the use of stainless steel in the selection of materials of construction i n most of the antibiotic fermentations. Sanitary stainless steel construction was used in fabrication of a fermentation plant designed for production of pyrogenfree dextran (49C). Iron and carbon steel equipment have not heen used in fermentors for the production of lactic acid because traces of iron have been difficult to remove from the lactic acid. Type 316 stainless steel heat transfer coils have been used in the wooden fermentors and processing tanks, t o minimize iron contamination (88B). Routine maintenance procedures were recommended for use on stainless steel, thereby preventing foreign matter deposition and electrolytic cell formation, and reducing corrosion (660). Previous reviews (11A, Z8A,14A) have discussed the appiication of fluid mixers t o fermentation operation. The primary scale-up factor was shown to be the rate a t which oxygen was supplied from the gas to the liquid phase (53D). Oxygen-absorption data obtained in 5- and 33-liter fermentors showed that in stirred gas-liquid containers of conventional design the primary locus of transfer was a t the sparger impeller. Most of the emphasis in agitation studies has been toward understanding the mechanics and principles of conventional type mixers. Methods used for determining agitation-aeration efficiencies in order t o scale-up to plant equipment have included measurement of oxygen absorption by following oxidation of sulfite sohtions (8OD). Practical studies indicated that the penicillin fermentation called
1959
for increased power levels and larger motors and shaft sizes were installed along the lines of conventional mixers. The location of the turbines and air sparger with respect to each other and the walls of the fermentor has been defined (1080, 1OQD) and by applying the theory of equipment similitude, relative results were obtained. The relationships between impeller horsepower input and air-flow, whereby horsepower consumption of a mixing impeller was markedly influenced by the air flow through the vessel, were discussed by Oldshue (850). The selection of a particular turbine design was, in most cases, not far the purpose of increasing fermentation productivity but to meet the physical conditions of the medium agitated (850). The ratio of turbine diameter t o tank diameter was perhaps more critical than turbine design so long as the shaft speed could be increased to a point where equal horsepower was introduced to the medium from the shaft, and critical speed zones did not interfere with equipment rigidity. The degree of baffling and the manner in which the air was introduced into the fermentor has continued t o receive attention. Data have been presented which indicate that the ring sparger was superior t o the open, centered-pipe sparger (850). The critical nature of the zone in which air was introduced into the fermentor was brought out by Karow et aE. (630). Automatic process control has been considered for use in fermentation manufacturing units (490). One of the problems in installing automatic control units in fermentations has been the selection of representative factors for control which are directly related t o yield of the fermentation product. Factors in which only a short lag period is encountered between adjustment and control of the fermentation will yield the best performance (710). Recent articles have described laboratory and pilot-plant scale fermentors for the batchwise and continuous production of antibiotics (199C, 250C, 65D),itaconic acid (337C), citric acid by submerged fermentation (64C, 275C), vinegar (ZdC, 15C), microbial protease (88IC),and vitamin Blz(153C). Descriptions of production plants, designed for the manufacture of acetone and butyl alcohol (38C, 39C), lactic acid (89B), penicillin (367C), streptomycin (QA),and dextran (49C) have also been made available. These reports have indicated some of the equipment which has been used in production of these products. A general review article describing a fermentation plant manufacturing antibiotics in Germany and including pictures of pilot plant and production equipment has been published (860). The Fermentation. AERATION-AGITATION. Previous reviews (18A, I4A) discussed the techniques which have been used for scale-up purposes a t the laboratory and pilot-plant stage and which supply information applicable to production equipment. The primary scale-up factor was identified as the rate a t which oxygen was supplied from the gas t o the liquid phase (630). This factor can be determined in a production unit by measuring the rate of oxidation of a Cu++-catalyzed sulfite-water system. A relationship was established between superficial air velocity and shaft horsepower input to the liquid. This relationship aided in the successful scale-up of penicillin fermentations without the necessity of measuring the rate of oxidation in the production fermentor (@8C). It has been estimated that the power-input to the liquid in flask fermentations placed on a reciprocating shaker was of the order of 0.1 hp. per 100 gallons (l84C). In another study it was observed that aeration efficiency (as measured by sulfite oxidation) increased from 0.2 mM of oxygen per liter per minute t o 5.3 mM when indented Erlenmeyer flasks were used instead of the smooth wall flasks ( 8 4 0 ) . The importance of maintaining continuous aeration in a fermenting medium was emphasized in the production of itaconic acid by Aspergillus terreus (SIZC). Studies carried out in 20liter fermentors indicated that interruption of the aeration for 20 minutes stopped the fermentation permanently. The relative efficiency of different aerating devices was studied
1960
INDUSTRIAL AND ENGINEERING CHEMISTRY
quantitatively and the merits of one such device, a “spargerless” fermentor, were reported (140). Such a n unbaffled fermentor, in which air was passed across the surface of a deeply vortexing fermentor, was said to have economy in power consumption, economy in air consumption, simplicity of construction, and absence of interference from foam formation. FERMENTATION CYCLES.Practically all of the descriptions of fermentation processes appearing during the past few years have dealt with operations on a batch arrangement. The fermentor has usually been filled with medium, sterilized, and then inoculated. The incubation period has been continued until the maxinium yield of desired fermentation product was reached. Donovick (124C) has shown that extension of incubation period to the time when the maximum yield was obtained has often been quite uneconomical and inefficient. I n many fermentation processes including those where antibiotics, riboflavin, vitamin BIQ, or citric acid has been the product of major interest, a plot of the biosynthesis of the product as a function of time forms a sigmoid curve. Both Donovick and Schmitz (1110) have indicated t h a t the end of the period of maximum efficiency of the process has usually been reached a t the inflection point of the curve, and continuation of the incubation period beyond this point may not result in as efficient a n economic process. While it has been obvious that no economic return has been forthcoming while the fermentor was being harvested, prepared for fermentation operations, or while the production curve was i n the “lag period” of the produetion curve, these portions of the fermentation cycle are very necessary, and have to be considered in fermentation operations. Study of data obtained in processes producing gluconic acid ( 5 3 3 , itaconic acid (337C), citric acid ( 6 4 C ) , penicillin (17‘3, and vitamin B,z (propionobacterium process) (26PC) suggests t h a t in each of these fermentations operated on a batch basis, the “lag phase” occupied 15 to 3ovOof the time necessary to reach maximum yield. It was also noted that the period of maximum rate of biosynthesis of the fermentation product usually ended at a point 10 t o 20 hours before the maximum yield was reached. It is possible that if these processes were expanded to a commercial scale without modification maximum economic return would not result. On the other hand, in the processes used for production of vitamin Blz (Bacillus megatherium) (16SC), citric acid (275C), vinegar ( 1 5 C ) , and dextran (4QC),the “lag phase’’ was considerably shorter or practically nonexistent. The time necessary t o obtain maximum yield practically coincided with the end of the period of maximum rate of production. I n studies on continuous and semicontinuous processes, the “lag phase” has been eliminated as soon as the continuous operation was started, and the process has been balanced so that maximum production rates were obtained, Continuous processes for production of penicillin have been studied on a pilot-plant scale (250C, 447C) and the data collected suggested that this type of operation might be economically feasible. A semicontinuous process, in which partially fermented media taken from the early part of the fermentation cycle were added t o fermenting media at the latter part of the cycle, was found to extend the productivity cycle (447C). According to recent reports i t has not been possible t o operate certain fermentations including those producing streptomycin ( I Z 4 C ) or acetone and butyl alcohol (SQC) on a continuous basis, The microorganisms used in the biosynthesis of these products apparently go through a definite sequence of reactions in synthesizing these products, and operation on a continuous basis interrupts the sequence. RECOVERY OF FERMENTATION PRODUCTS
Methods used in recovery of fermentation products from the fermented media have often determined the economic success or failure of t h e process, While no formal treatment of this phase of the fermentation unit process has been included i n earlier reviews i n this series ( I I A , 1gL4),some of the techniques used have
Vol. 45, No. 9
been mentioned in passing. The problems encountered in r e covery of fermentation products usually have included removal of the product from the fermented medium and purification and treatment of the crude extract. Limitations of space permit only a brief survey of some of the processes used in such recoveries a t this time. Solvents and Other Distillery Products. Products of the distilleries have been ethyl alcohol, carbon dioxide (recovered as dry ice), and the fusel oils, consisting mainly of propyl, butyl, and amyl alcohols. I n the recovery processes, following the yeast fermentation, the fermented medium is pumped t o a beer stripping still where steam is passed in a countercurrent fashion t o the fermentation medium ( 1 S l D ) . The bottoms from the beer still (stillage) are processed into animal feeds while the vapors are processed in a rectifying still for removal of fusel oils and further purification of the alcohol. The stillage containing 5 to 7% of solids, of which half are suspended solids, is pumped over shaker screens to remove a major portion of the solids. The screenings are then pressed and drum-dried to produce distiller’s dried grains. The liquid is concentrated i n a n evaporator t o between 25 and 35% solids and dried on drum driers to produce distiller’s dried solubles ( 4 0 , 7 5 0 ) . Another process modification (1SUD) for production of dried solubles utilizes centrifugation and evaporation steps, producing a slurry containing 35 to 507, of solids content prior to drying. Products recovered from the acetone-butyl alcohol fermentation include acetone, butyl alcohol, ethyl alcohol, hydrogen, carbon dioxide, and feeds. The fermented mash is pumped to a beer still; the acetone is recovered, and the ethyl alcohol and butyl alcohol are separated i n a rectifying tower (SSC, 5QC). Feed production is handled in a manner similar to the alcohol fermentation ( 4 0 ) . The hydrogen and carbon dioxide produced during the fermentation are collected, purified, and prepared for sale. The carbon dioxide produced as a by-product of the alcoholic and the acetone-butyl alcohol fermentations is usually of higher purity than that produced by other methods ( 7 D ) . Fermentation Acids. Several of the problems encountei ed in the recovery of citric acid from fermented media were described by Yuill (18A). An alternative to precipitation of the calcium salt, a procedure usually used ( S I D , 6 4 0 ) ,has been t h a t of direct crystallization of the citric acid from the filtered fermentation liquor. It was found that this procedure produced a pure hydrated citric acid, but the crystals were in a n orthorhombic form (“alpha” habit) rather than a crystal having the { 101 ] faces. The latter form is obtained when calcium citrate is treated with sulfuric acid, and the free acid subsequently crystallized from the calcium sulfat+free solution. A method suggested for recovery of sodium gluconate from fermented media includes press filtration to remove mycelium, followed by evaporation of the liquor to 45y0 solids, and subsequent drying on a drum dryer. The product is then pulverized i n a hammer mill and found to contain 95Y0 of sodium gluconate. An alternative process involves evaporation to 21% solids and crystallization to produce a product with a purity of 99% (53C). I n the recovery of lactic acid from fermented media, the proteinaceous material is coagulated with hydrated lime a t elevated temperatures. The calcium lactate solution is then decanted, bleached with activated carbon, filtered, and the liquor is concentrated in a single effect evaporator t o a point short of crystallization. Sulfuric acid is then added, the precipitated calcium sulfate is removed on a string filter, and the liquor is slurried with activated carbon. Following removal of the carbon by filtration through a plate and frame press, the filtrate is concentrated in single and double effect evaporators so that the lactic acid content increases from 8 to 52%. Heavy metal contaminants are removed by addition of sodium sulfide, and a third carbon bleach, filtration, concentration to 80% lactic acid, a fourth carbon treatment, and final filtration result in a waterclear solution of “edible grade” lactic acid (28B).
September 1993
a
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INDUSTRIAL AND ENGINEERING CHEMISTRY
A modified process has been suggested in which digestion with sulfuric acid, filtration, and concentration is followed by a methanol esterification. The methyl lactate solution is filtered, distilled, and hydrolyzed to liberate lactic acid in a pure form. The liberated lactic acid is concentrated by evaporation in vacuo. It has been suggested that chemically pure lactic acid may be prepared most efficiently and economically by this method, although the process has not been tried on a large scale (990). The steps involved in the recovery of itaconic acid from fermentation broth have included a filtration step to remove mycelium and an evaporation and crystallization step. The mother liquor can be recycled to the evaporator and crystallizer (557C) or extracted with butyl alcohol (610) to obtain maximum yields. Vitamins. On completion of a riboflavin fermentation, the mash is adjusted t o a pH of 5.5 prior t o heat treatment. The acidified mash is then heated and held at a temperature from 60' to 120" C., for B period of 0.5 to 2 hours t o remove the riboflavin in the cells of the fungus. The mash is then filtered and the riboflavin is recovered by adsorption on fuller's earth or adsorbent clay. The resulting adsorbate is then eluted with a solvent, the riboflavin is crystallized, filtered, and dried (90). An alternate method of recovery from the filtrate stage involves reduction to the leuco form, by inorganic reducing agents or bacterial enzymes, followed by extraction with butyl alcohol, oxidation, and crystallization (1190). I n the production of a feed material the fermented liquor can be concentrated directly to 27% solids and dried on a double drum dryer, or spray dried (96D). The recovery of vitamin Blz or vitamin Blz-like materials from bacterial use in feed supplements requires only a concentration of the fermentation medium to 15% solids, acidification, and either spray or drum drying (440). Since the cells contain 97% of the BIZ,collecting the cells by centrifugation, and drying only the solids removed by this treatment has been found to be practical (163C). Pure crystalline vitamin B12 can be recovered from fermented media by a process involving treatment of the filtered fermentation broths, or suitable concentrates, with a source of cyanide ions (1390). After completion of the cyanide reaction and removal of,the unreacted cyanide, the vitamin BU solution is extracted with benzyl alcohol, the extract dried in vacuo, and ether added to effect precipitation of crude vitamin Bl*. The precipitate is then dissolved in water and successively extracted with approximately equivalent portions of benzyl alcohol. The combined extracts are then dried and treated with ether to,effect precipitation of purified vitamin B12. This precipitate is dissolved in water and crystallized to obtain vitamin BIZof about 95% purity, which was reported to be preferable for clinical use. Antibiotics. One of the early extraction processes developed for recovery of penicillin, an acid soluble in a wide range of solvents, was the carbon adsorption process (1930). The fermented medium was filtered on a rotary vacuum filter and the crude liquor was slurried with activated carbon, upon which the penicillin was absorbed. Following absorption, the carbon slurry was filtered on a plate and frame filter, washed, and then eluted with acetone. The acetone was removed in a three-stage vacuum spray evaporator and the penicillin-rich acetone-free liquid from the third stage was extracted batch-wise with amyl acetate, followed by chloroform. The purified penicillin was fed to a flask concentrator and the concentrate and purified water solution were filtered through a bacteriological filter of the Seitz asbestos pad type to remove all bacteria. The resulting product was dried by lyophilization. The carbon, acetone, and batch solvent extraction process was later replaced by the continuous countercurrent solvent extraction method (160). I n this process penicillin is purified and extracted from the clear broth through a three-stage solvent extraction utilizing the Podbielniak extractor for the first two stages and completing the cycle in a batch extractor. The first solvent phase is treated with carbon to remove color components, and
1961
the penicillin is extracted into an aqueous buffer solution. Following the final batch extrmtion a conversion is made of the penicillin into either crystals of potassium penicillin or procaine penicillin. The dried crystals are ground, granulated, blended, or mixed with other ingredients for final pharmaceutical purposes. In order to meet sterility speciiications, the concentrate is sterilely filtered or heat-sterilized a t some point in the final processing. Recovery and purification of streptomycin salts from fermentation broths have been accomplished by a number of chemical process techniques. The procedures used when the antibiotic was first made available on a large scale involved: removal of the insoluble solids from the fermented medium by filtration; adsorption of the antibiotic on activated carbon; elution with an aqueous acid solvent (methanol) mixture; concentration of the eluate, followed by precipitation of the antibiotic by addition of acetone or chromatography of the concentrate; solution of the streptomycin salt, decolorization, and lyophilization ( 9 9 0 ) . A more recent report ( 4 A ) gives some of the modifications which have found favor. The fermented medium is treated with acid to release the streptomycin bound to the cells of the actinomycete. The cells are then removed by filtration, the filtrate is neutralized and passed over a cationic exchange resin (several types of resin have been used) (46D, 1960). The resin is then treated with acid, the released streptomycin salt is purified further, if necessary on formatioa of complexes ( 6 5 0 , 13701, and the purified material is crystallized. A solvent extraction procedure for purifying streptomycin was reported in which a water-immiscible, primary, liquid amine extracted streptomycin from aqueous solutions and did not extract many of the contaminating impurities (1040, 1060). Streptomycin and mannosido-streptomycin, antibiotics produced by certain cultures of Streptomyces griseus, have been separated by countercurrent d i e tribution in a system containing lauric acid in amyl alcohol and an aqueous phosphate borate buffer (840). A liquid-liquid extraction process has been reported for the recovery of bacitracin (490, 115D). The filtered broth containing the bacitracin was fed to a Podbielniak extractor where the bacitracin was extracted from the filtrate by the butyl alcohol. The butyl alcohol solution was fed semicontinuously to a glass lined concentrator equipped with a three-stage vacuum ejector, and the final aqueous concentrate was removed a t the end of the cycle. The aqueous concentrate was carbon-treated t o remove colors and odors, filtered to remove carbon, and Seitzfiltered to remove bacteria. The final bacteria-free concentrate was dried in bulk tray dryers and the dry powder was used in pharmaceutical formulations. A zeolitic resin adsorption process was developed in which the volume of the final aqueous solution, from which the purified bacitracin was to be recovered, was only one fifth of that of the solution obtained by use of the solvent extraction method (160). The advantages of the resin process over the solvent extraction method are: (a) an organic solvent is not needed, ( b ) the process is not affected by temperature variations, and (c) the product obtained is more potent and less toxic. WASTE DISPOSAL TECHNIQUES
It has been necessary for the fermentation industry to devise techniques for the disposal of wastes from the fermentation operations. While the reviews in these series (11A, 194, 14A) have mentioned some of the technical problems involved, no centralized discussion has been included. I n recent years sanitary boards of various municipalities, in conjunction with the industry, have placed emphasis upon elimination of sources of stream pollution; waste disposal has become part of the fermentation unit process. Where the principal fermentation product has enjoyed a wide profit margin, little attention has been given to the recovery of by-products from plant waste unless troublesome sanitation problems were experienced. The competitive situation in the production of many fermentation products to-
1962
INDUSTRIAL AND ENGINEERING CHEMISTRY
gether with the sanitation problems have combined t o intci est the fermentation industry in recovering soluble by-products from the fermented media. The bulk of the waste in the fermentation industry is organic in nature. The organic matter present in clean and polluted streams is oxidized by the dissolved oxygen in the water and by the microorganisms present, with the production of simpler compounds such as water, carbon dioxide, sulfates, and nitrates. When excessive loads of waste are discharged into a stream, the fish are killed, foul odors are given off as a result of anaerobic decomposition of the organic matter, and nuisance conditions are created. The means suggested for disposing of this waste. other than discharge directly into the streams, include: 1. 2. 3. 4.
Establishing a subsurface irrigation system. Lagooning or land disposal. Trucking to sandy areas. Concentration and incineration of the suspended and dissolved solids. 5 . Concentration and drum drying of solids, or spray drying of solids for feed production. 6. Aeration of the wastes utilizing trickling filters, biofilters, and sand filters. 7 . Digestion by fermentation procedures combined with one of the above procedures. h n o n g the factors which make the first three methods difficult to operate are (a)impervious soils which permit only slight percolation, ( b ) objectionable odors, and ( c ) high transportation costs. Odor problems often arise in the drum drying, spray drying, or incineration of fermentation wastes which prevent consideration of these methods in some heavily populated localities. Evaporation and drying have also been expensive operations. An illustration of the magnitude of the problems encountered is t h a t of an antibiotic producing plant where a million gallons of waste containing 4% suspended and dissolved solids (equivalent t o 160 tons) has t o be disposed of per week. This is in addition to wastes arising from other phases of the plant operations. Detailed discussions of waste disposal methods used in the fermentation industries have been prepared by Buswell ( 1 2 D j , Boruff (6D, 6 D ) , Mohlniann ( 7 3 0 ) , and Willlrie and Prochaska ( 1 3 i D ) ,with the latter including a description of equipment used in drying the solids and liquids found in distillery wastes for feed purposes. Distilleries. The stillage from the alcoholic fermentation of molasses mashes produces a high pollution load-a total solids content of 5y0,organic solids of 4’3& and a 5-day B.O.D. of 22,000 p.p.m. have been reported ( 4 0 ) . Usually these wastes have been concentrated t o a solids content of 50 t o 60% in multiple effect evaporators and used as a molasses substitute in cattle feeds, or completely dried and used in mixed feeds. From 6 t o 8 pounds of dried stillage have been recovered per wine gallon of 190 proof alcohol produced or per 2.4 gallons of molasses mashed, Similar practices have been used in grain distilleries (60). Stillage from the acetone-butyl alcohol fermentation of molasses is more dilute than alcoholic stillage because of the Ion-er sugar concentration fermented, but it still has a high pollution equivalent, with a 5-day B.O.D. ranging from 7000 to 11,000 p.p.m. While disposal of this material has been accomplished through anaerobic digestion processes, recent developments have indicated that it has value as a source of riboflavin, and has been used in poultry feeds. This material, known in the trade as “butyl fermentation solubles,” m’as introduced some years ago on the market with a riboflavin content of 4 0 per ~ gram and has since been enriched t o give concentrates assaying 8 mg. per gram. An average of 2 pounds of dried stillage has been usually recovered per pound of mixed solvents produced (or per 0.6 gal!on of molasses used (do). Yeast Production. Undiluted vaste from yeast plants using molasses as a substrate contains from 1 t o 3% tot41 solids (75%
VoL 45, No. 9
organic) and possesses a &day B.O.D. of i o 0 0 to 14,000 p.p ni depending on the materials and concentration of ingredients used in developing the yeast. The waste is too low in solids content and food or feed value t o justify recovery, and one disposai method installed at the Pekin plant of the Fleischmann Yeast Co. includes a settling basin t o remove filter aid followed by digestion tanks. The final effluent from the digesters has a B.O.D. or 1000 p p in.; it flows into a river. The process permits heavy loadings (0.1 pound of organic matter per cubic foot per day) and, therefore, large quantities of fuel gas (0.7 cubic foot per day per cubic foot of tank capacity) have been produced ( I I O D ) . I11 another installation, an anaerobic digester followed by a trickling filter plant mas successful in reducing the B.O.D. by 80 to 98% ( i 0 7 D1. Riboflavin Manufacture. The mash consisting of protein carbohydrate, and minerals is fermented by riboflavin-producing strains of Eremothecium ashbyii or Ashbya gossypii. At the end of the fermentation period the mold is killed and removed b j filtration. The riboflavin is recovered from the solution by precipitation and filtration. The remaining filtrate contains the unused nutrients together with the ethyl alcohol, acetic acid, eqters, and other water-soluble nonriboflavin by-products of the fermentation. The B.0 D. bas been of the order of 5000 t o 10,000 p.p.m. Knoedler and Babcock ( 5 6 0 ) have described a process where the filtrate and mycelium are mixed, spray-dried, and the recovered solids are incinerated. An alternative to this procedure is to dry the whole culture a t the end of the fermentation, and to direct the product to animal and poultry feeds (96D). Antibiotic Manufacture. Media used for the production of antibiotics usually contain cornsteep liquor (or other sources of protein), carbohydrate, and inorganic salts. At the end of the fermentation period, the mycelium has been removed by filtration, and the antibiotic is recovered from the filtrate by any one of a number of methods. The mycelium from the aureomycin and penicillin processes has found use as a n animal feed supplement and has been sold in the dry form (lOD, iSCD). The dried penicillium mycelium has also been digested in anaerobic digesters with a yield of 4000 liters of gas from 1000 kg. of organic substance (66D). Incineration of the dried mycelium from the aureomycin and streptomycin process has also been used to dispose of this material ( 5 6 0 , 99D). I n another process the slop5 from the still8 used in recovering the extraction solvents were added t o the mycelium, the mass was dried andincinerated (56D). Methods for the disposal of spent liquors from the antibiotic processing include aeration of the wastes with use of shallon trickling filters followed by deep trickling filtrations for disposal of wastes from penicillin processing (77D) and a similai combination for treatment of wastes from the processing of aurcornycin (IOD,1 H D ) and streptomycin ( 4 2 0 ) . A reduction of the B.O.D. from 1000 p.p,m. to 35 to 40 p.p.m. has been accomplished by these treatments. An alternative t o this method which has found use in treatment of aureomycin wastes has been t , ~use the liquid as nutrient for the growth of Torula utilis. The B.0.D was reduced by 50% in a 4 h o u r fermentation period (IOD).
BIBLIOGRAPHY G E N E R A L REFERENCES T O T H E F E R M E N T A T I O N INDUSTRIES
Aubel, E., “Les Fermentations,” Paris, Presses Universitaires de France, 1952. Bolcato, V., “La Chimica della Fermentaeioni,” Bologna, h-. Zanichelli, 1952. Brian, P. W., J . Roy. SOC.Arts, 101, 194 (1953). Campbell, A. H., Research, 6 , 42 (1953). Chain, E. B., Ibid., 5 , 567 (1952). Haehn, H., “Biochemie der Garungen,” Berlin, Walter de Gruyter & Co., 1952. Hartmann, G., “Die Drogen in der Spirituosen-Industrie,” Rerlin-Cha~lettenb~r~, Carl Knopphe, 1952.
September 1953
I N D U S T R I A L A N D E N GI N E E R I N G C H E M I S T R Y
(8A) Hauduory, P., “Techniques bacterioiogiques utilisBes pour l’iaolement la determination et la conservation des microorganisms,” Paris, Masson & Cie, 1952. (SA) Kluyver, A. J., Proc. Roy. Sac., B141, 147 (1953). (10A) Lee, S. B., IND. ENG.CHEM.,41,1868 (1949). (11A) Ibid., 42, 1672 (1950). (12A) Ibid., 43, 1948 (1951). (13A) Mey, H., “Handbuch der Maelzeri-Technologie,” Nuremburg, Verlag Hans Carl Briete Gasse, 1951. (14A) Perlman, D., Brown, W. E., and Lee, S. B., IND. ENG.CHEM., 44, 1996 (1952). (15A) Stacey, M., Research, 4, 159 (1953). (16A) Underkofler, L. A., and Hickey, R. J., “Industrial Fermentations,” New York, Chemical Publishing Co., Inc., 1953. (17A) World Health Organization, “Microbial Growth and Its Inhibition,” Monograph Series No. 10, Geneva, 1952. (18A) Yuill, J. L., Research, 6, 86 (1953). E C O N O M I C S O F THE F E R M E N T A T I O N INDUSTRIES cc
(1B) (2B) (3B) (4B) (5B) (6B) (7B) (8B) (9B) (10B) (11B)
Chem. Eng., 59, 107 (April, 1952). Chem. Eng. News,30, 3601 (1952). Ibid., p. 4594. Ibid., p. 4809. Ibid., p. 5452. Ibid., 31, 262 (1953). Ibid., p. 735. Ibid., p. 1063. Ibid., p. 1264. Ibid., p. 2363. Chemistry & I d u s t r y , C27, Supplement to December issue (1952). (12B) Chem. Week, 68 (Nov. 10, 1951). (13B) Ibid., 71, 26 (Nov. 22, 1952). (14B) Ibid., p. 13 (Dec. 6, 1952). (15B) Ibid., 72, 32 (March 28, 1953). (16B) Ibid., p. 30 (May 2, 1953). (17B) Ibid., p. 48 (May 9, 1953). (18B) Ibid., p. 50 (May 9, 1953). (19B) Ibid., p. 76 (May 16, 1953). (20B) F. D. C. Reports, March 7, 1953, Vol. 15, #4, W-13. (21B) IND.ENG.CHEM.,45, 33A, 39A (January 1953). (22B) Inskeep, G. C., Taylor, G. G., and Breitzke, T V . C., Ibid., 44, 1955 (1952). (23B) J . Antibiotics ( J a p a n ) , 4, 392 (1951). (24B) Ibid., 5, 332 (1952). (25B) Ibid., p. 348. (26B) Stenerson, H., Chem. Eng. News, 31, 1926 (1953). (27B) Sugar Reports U. S. Dept. Agr., Washington, D. C., KO.16-M (October, 1952). (28B) U. S. Tariff Commission Chemical Division, Facts for Industry, Ser. 6-2-107, March 4, 1953. (29B) U. S. Tariff Commission, Synthetic Organic Chemicals, U. S. Production and Sales, 1948-51. (30B) Waring, C. E., Chem. Eng. News, 31, 1964 (1953). (31B) Welch, H., Antibiotics & CLemothempy, 2, 279 (1952). INDUSTRIAL F E R M E N T A T I O N PROCESSES
(1C) Abe, S., Shiga, T., and Nakano, Y., J . Antibiotics ( J a p a n ) ,5,
433 (1952). (2C) Abilgaard-Elling, K., Brit. Patent 670,986 (April 30, 1952). (3C) Ibid., 670,987 (April 30, 1952). (4C) Abraham, E. P., Newton, G. G. F., Crawford, K., Burton, H. S., and Hale, C. W., Nature, 171, 343 (1953). (5C) Agr. Food Chem., 1, 129 (1953). (6C) Ibid., p. 193. (7C) Aiso, K., Shibasaki, K., and Yamanouchi, F., J. Ferm. Technol. ( J a p a n ) , 30, 316 (1952). (8C) Akabori, S., Hagihara, B., and Ikenaka, T., PTOC.J a p a n Acad., 27,350 (1951). (9C) Akabori, S., Ikenaka, T., and Hagihara, B., Symposia o n Enzyme Chem. ( J a p a n ) , 7, 107 (1952). (1OC) Aktiebolaget Pharmacia, Brit. Patent 675,025 (July 2, 1952). (llC) Ibid., 675,085 (July 2, 1952). (12C) Allen, W. F., (to A. E. Staley Mfg. Co.), U. S. Patent 2,596,971 (May 20, 1952). (13C) Ibid., 2,596,972 (May 20, 1952). (14C) Allgeier, R. J., Wisthoff, R. T., and Hildebrandt, F. M., IND. ENG.CHEM.,44, 669 (1952). (15C) Ibid., 45, 489 (1953). (16C) Anastassiadis, P. .4.,and Wheat, J. A., Can. J . Technol., 31, 1 (1953). (17C) Anderson, R. F., Whitmore, L. M., Jr., Brown, W. E., Peterson, W. H., Churchill, B. W., Roegner, F. R., Campbell,
1963
T. H., Backus, M. P., and Stauffer, J. F., IND.ENG.CHEM., 45, 768 (1953). ’ Anheuser-Busch, Inc., Brit. Patent 676,773 (Aug. 6, 1952). Antibiotics & Chemotherapy, 2, 462 (1952). Aoki, T., and Taniguchi, Y., J . Antibiotics ( J a p a n ) , 5, 151 (1952). Aries, R. S., and Needle, H. C., U. S. Patent 2,588,460 (March 11, 1953). Arnaudi, C., Esperientia, 7, 81 (1951). Arnaudi. C.. and Colla, C.. Proc. Second Intern. Conar. Biochem., 118 (1952). Asai, T., and Ikeda, Y . , J . Agr. Chem. SOC.( J a p a n ) , 22, 50 (1948). Asheshov, I. N., Strelitz, F., and Hall, E. A., Antibiotics & Chemotherapy, 2, 361 (1952). Aso, K., Shibasaki, K., and Yamanouchi, F., J . Ferm. Technol. ( J a p a n ) , 30, 311 (1952). Bacon, J. S. D., Biochem. J.,50, xviii (1952). Baddiley, J., Thain, E. M., Novelli, G. D., and Lipmann, F., Nature, 171, 76 (1953). Ballentine, R., presented before the Division of Biological CHEMIChemistry a t the 123rd Meeting of the AMERICAN CAL SOCIETY, Los Angeles, Calif. (1953). Barber, R. S., Braude, R., Kon, S. K., and Mitchell, K. G., Chemistry & Industry, 1952, 713. Barker, S. A., and Bourne, E. J., Quarterly Rev., 1, 56 (1953). Barker, S. A,, Bourne, E. J., Bruce, G. T., and Stacey, M., Chemistry &Industry, 1952, 1156. Barton-Wright, E. C., J . Inst. Brewing, 57, 415 (1951). Bass, A., and Hospodka, J., Chem. Listy, 46, 243 (1952). Basu, S. N., and Whitaker, D. R., Arch. Biochem. Biophys., 42, 12 (1953). Battersby, A. R., and Craig, L. C.,J . Am. Chem. Sac., 74, 4019 (1952). Ibid., p. 4023. Beesch, S. C.,Appl. Microbiol., 1, 85 (1953). Beesch, S. C., IND.ENG.CHEM.,44, 1677 (1952). Beesch, S. C., and Firman, M. C. (to Publicker Industries, Inc.), U. S. Patent 2,631,120 (March 10, 1953). Behrens, 0. K., Jones, R. G., Soper, Q. F., and Corse, J. W. (to Eli Lilly and Co.), Ibid., 2,623,876 (Dec. 30, 1952). Beinert, H., VonKorff, R. W., Green, D. E., Buyske, D. A , , Handschumacher, R. E., Higans, H., and Strong, F. M., J. Biol. Chem., 200, 385 (1953). Benediot, R. G., Botan. Rea., 19, 229 (1953). Benedict, R. G., and Stodola, F. H. (to U. S. Secy. Agriculture), U. S. Patent 2,617,755 (Nov. 11, 1952). Benetti-Treccani, R., Ann. microbiol., 5, 1 (1952). Bernheimer, A. W., Trans. N. Y . Acad. Sci. (Ser. 111, 14, 137 (1952). Berridge, N. J., Chemistry & Industry, 1953, 374. Berridge, N. J., Newton, G. G. F., and Abraham, E. P., Biochem. J. (London), 52, 529 (1952). Bixler, G. H., Hines, G. E., McGhee, R. M., and Shurter, R. A., IND. ENG.CHEM.,45, 692 (1953). Blackwood, A. C., Can. J . Botany, 30, 28 (1953). Blair, M. G., and Pigman, W., Arch. Biochem. Biophys., 42, 278 (1953). Blinc, M., Sloven. Akad. Znanosti Umetnosti, Razred Mat., Fie. Tehn. Vede, Class 111, Ser. A., Razprave, 111, 41 (1951). Blom, R. H., Pfeifer, V. F., Moyer, A. J., Traufler, D. H., Conway, H. F., Crocker, C. K., Parison, R. E., and Hannibal, D. V., IND. ENG.CHEM.,44, 435 (1952). Blom, R. H., Shons, V. E., and Moyer, A. J. (to U. S. Secy. Agriculture), U. 8 . Patent 2,594,283 (April 29, 1952). Bomstein, R. A., and Johnson, M. J., J . Bid. Chem., 198, 143 (1952). Bovard, F. C., Iowa State CoZZ. J . Sci., 27, 132 (1953). Brian, P. W., Ann. Appl. Biol., 39, 434 (1952). Brian, P. W., Bot. Rev., 17, 357 (1951). Brin, M., Olson, R. E., and Stare, F. J., Arch. Biochem. Biophgs., 39, 214 (1952). Brown, A. M., U. S. Patent 2,602,041 (July 1, 1952). Brown, G. IM., and Snell, E. E., J . Am. Chem. Soc., 75, 1691 (1953). Brown, P. B., Becker, D. E., Terrill, S. W., and Card, L. E., Arch. Biochem. Biophys., 41, 378 (1952). Brown, W. E., “Encyclopedia Chemical Technology,” 9, 922 (1952). Buelow, G. H., 4nd Johnson, M. J., IND.ENG. CHEM.,44, 2945 (1952). Bureau of Internal Revenue, Annual Report, 1952. Burkholder, P. R., Arch. Biochem. Biophys., 39, 322 (1952).
1964
INDUSTRIAL AND ENGINEERING CHEMISTRY
(67C) Burton, bI. O., and Lochhead, A. G., Can. J . Botany, 29, 352 (1951). (68C) Ibid., 30, 521 (1952). (69C) Business We&, No. 1192, 30 (July 5, 1952). (70C) Camici, L., Sermonti, G., and Chain, E. B., Bull. World Health Organization, 6 , 265 (1952). (71C) Carlson, W.W., Rosano, C. L., and Whiteside-Carlson, V , J . Bacteriol., 65, 136 (1953). (72C) Carvajal, F., Mycologia, 45, 209 (1953). (i3C) Castor, J. G. B., and Guymon, J. F., Science, 115,147 (1952). (i4C) Chem. Eng., 59, 215 (September 1952). (75C) Ibid., p. 240 (December 1952). (76C) Ibid., 60, 282 (June 1953). (77C) Chem. Eng. Y e w s , 30, 3065 (1952). (78C) Ibid., p. 3107. (79C) Ibid., p. 3377. (80C) Ibid., p. 3954. (81C) Ibid., p. 3956. (82C) Ibid., p, 4262. (83C) Ibid., 31, 772 (1953). (84C) Ibid., p. 828. (85C) Ibid., p. 976. (86C) Ibid., p. 1551. (87C) Ibid., p. 2182. (88C) Chem. I n d . Week, 6 8 , 17 (March 17, 1951). (89C) Chem. I n g . Tech., 24, No. 5, 277 (1953). (9OC) Chem. W e e k , 68, 22 (Oct. 6, 1951). (91C) Ibid., p. 29 (Oct. 20, 1951) (92C) Ibid., 72, 50 (March 28, 1953). (93C) Ibid., p. 76 (May 16, 1953). (94C) Ibid., p. 88 (May 16, 1953). (95C) Ibid., p. 12 (May 23, 1953). (96C) Ibid., p. 28 (June 10, 1953). (97C) Chiao, J. S., and Peterson, W. H., A p p l . Microbiol., 1, 42 (1953) (98C) CIBA, Ltd., Brit. Patent 651,269 (March 14,1951). (99C) Cid, A. R., Fernandee-Cano, L. H., and hlarquex, J. G., Rev. ceinc. apl. (Madrid), 5 , 403 (1951). (1OOC) Clement, M. T., Can. J . Technol., 30, 82 (1952). (101C) Coates, &I. E., Chemistrg &. Industry, 1953, 374. (102C) Coffey, G. L., Oyaas, J. E., and Ehrlich, J., Aiztibiotics & Chemotherapy, 1, 203 (1951). (103C) Cohn, M., and Torriani, A. M., Biochim. et Biophysica Acta, 10,280 (1953). (104C) Coleman, L. L., LlcCarty, L. P., Warner. D. T., Willy, R. F., and Flokstra, J. H., presented before the Division of Biological Chemistry at the 123rd Rleeting of the AMERICAN CHEniIcAL SOCIETY, Los Angeles, Calif. (1953). (105C) Corzo, R. H., and Tatum, E. L., Federation Proc., 12, 470 (1953). (106C) Crewther, W.G., Australian J . A p p l . Sci., 1, 437 (1950). (107C) Ibid., p. 447. (108C) Ibid., p. 468. (109C) Cracker, C. K., &Toyer, A. J., and Pfeifer, V. F. (to U. S, Secy. Agriculture), U. S. Patent 2,602,768 (July 7 , 1952). (llOC) Cuthbertson, W. F. J., Chemistry & Industry, 1952, 477. (111C) Ibid., 1953, 169. (112C) Danckwerts, P.V., and Sellers, E. S., Food, 21,459 (1952). (113C) Darken, M. A., Botan. Rev., 19,99 (1953). (114C) Davis, B. D., J . Bacteriol., 64, 432 (1952). (115C) Davisson, J. W.,Tanner, F. W.,Finlay, A. S..and Solomons, I. A., Antibiotics &. Chemotherapy, 1, 289 (1951). (116C) Dawson, E. R., Chemistry & Industry, 1952, 793. (117C) deBecze, G. I. (to Schenley Industries, Inc.), L-. S. Patent 2,626,868 (Jan. 27, 1953). (118C) Ibid., 2,636,823 (April 28, 1953). (119C) Delima, 0. B., Nato, B. M., and deMatos, -1.G., Anois soc. biol. Pernambuco, 9, 19 (1949). (12OC) Depois, R., Produits pharm., 7, 18 (1952). (121C) Dingle, J., and Solomons, G. L., J . AppZ. Chem. (London),2, 395 (1952). (122C) Dion, W.H., Calkins, D. G., and Pfiffner, J. J., J . Am. Chem. SOC.,74, 1108 (1952). (123C) Doi, S., J . Agr. Chem. SOC.( J a p a n ) , 19, 551 (1943). (124C) Donovick, R., presented a t the meeting of the American Institute of Chemical Engineers, New Brunswick, N. J., Dec. 9, 1932. (125C) Drews. B.. Soecht. H.. and Rothenbach. E.. Biochem. 2.. 332, 380 (1952). (126C) Dulmage, H. T., A p p l . Microbiol., 1, 103 (1953). (127C) Dworschack, R. G., Koepsell, H. J., and Lagoda, -1. A, Arch. Biochem. Biophys., 41, 48 (1953). (128C) East Anglia-Chemical Co. Ltd., Brit. Patent 657,420 (Sept. 19, 1951). (129C) Edelman, J., and Bealing, F. J., Biochem. J . , 53, ii (1953).
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(130C) Elias, W.,Price, 9.H., and Merrion, H. J., Antibiotics & Chemotherapy, 1, 491 (1951). (131C) Elliott, 8. D., Trans. N . Y . A m d . Sci. (Ser. 11), 14, 137 (1952). (132C) Emery, 1%’. B., Chemistry &Industry, 1952, 254. (133C) Eppstein, S.H., Meister, P. D., Leigh, H. M., Peterson, D. H., Murray, H. C., Reineke, L. AI., and Weintraub, -4., presented before the Division of Biological Chemistry at the 123rd Meeting of the AMERICAX CHEMICAL SOCIETY, Los Angeles, Calif. (1953). (134C) Eppstein, S. H., Ileister, P. D., Peterson, D. H., Murray, H. C., Leigh, H. >I,,Lyttle, D. A,, Reineke, L. M., and Weintraub, A., J . Am. Chem. Soc., 75, 408 (1953). (135C) Eppstein, S. H., Peterson, D. H., Leigh, H. M.,Murray, H. C., Weintraub, A , , Reineke, L. M., and Meister, P. D., Ihid., 75, 421 (1953). (136C) Ericson, L. E., Chemistry & Industry, 1952, 829. (137C) Ettel, V., Liebster, J., and Tadra, M., Chemicke Listy, 46,45 (1952).
(13%) Fernandez-Cano, L. H., and Cid, A. R., Rev. cienc. apZ. (Mad&), 6 , 37 (1952). (139C) Ibid., p. 124. (140C) Fischer, E. H., and deMontomollin. R., Helv. Chim.Acta, 34, 1987 (1951). (141C) Fischer, R. A., presented before the Division of Agricultural and Food Chemistry a t the 123rd Meeting of the AMERICAN CHEYICAL SOCIETY, Los Angeles, Calif. (1953). (142C) Florey, H. W., and Abraham, E. P., J . Hist. M e d . , 6 , 302 (1952). . , (143C) Fowden, L., Biochem. J . (London), 52, 310 (1952). (144C) Ibid., p. 355. (145C) Frederiksen, E. K., and Kielsen, E. J. (to rlmerican Cyanamid Co.), U. S. Patent 2,611,733 (Sept. 23, 1952). (146C) Freeman, G. G., Royal Inst. Chem., London, “Recent Advances in the Fermentation Industries,” p. 79 (1950). (147C) French, D., Suhadolnik, R. J., and Underkofler, L. A., Science, 117, 100 (1953). (148C) Fried, J., and Sabo, E. F., J . Bm. Chem. SOC., 75,2273 (1953). (149C) Fried, J., Thoma, R. W., Gerke, J. R.. Hcrz, J. E., and Donin, hl. K.,Ibid., 74, 3962 (1952). (150C) Frost, D. V., Fricke, H. H., and Spiuth, H. C., J . A*utritzon, 49, 107 (1953). (151C) Fukumoto, J., Yamamoto, T., and Ichikawa, K., Symposia o n Enzyme Chem. ( J a p a n ) , 7, 104 (1952). (152C) Fulmer, E. I., Underkofler, L. A , , and Hickey, R . J., U. S. Patent 2,388,840 (Kov. 13, 1945). (153C) Garibaldi, J. A , , Ijichi, K., Snell, 1;. S., and Lewis, J. C., IND.ENG.CHSM.,45, 838 (1953). (154C) Garibaldi, J. A., Ijichi, K., Sugihara, T. F., and Lewis, J. C., presented before the Division of Agricultural and rood Chemistry at the 123rd Meeting of the AMERICAN CHEMICAL SOCIETY, Los Angeles, Calif. (1953). (155C) Garner, H. R., Koffler, H., Tetrault, P.A., Fahmy, >‘ MalI., lett, N . V., Faust, R. A., Phillips, R. L., and Bohonos, W., Am. J . Botanu, 40, 289 (1953). (156C) Gattani, M .L., Science, 116, 596 (1952). (157C) Genevois, L., Inds. agr. et aliment. (Paris), 69,27 (1952). (158C) Gerard, W. E., Read, D. C., and Pensack, J. If.,presented before the Division of Agricultural and Food Chemistry at the 123rd Meeting of the AMERICAN CHEMICAL SOCIETY, Los Angeles, Calif. (1953). (159C) Giglio, D. &I., and McCleskey, C. S.,J . Bacteriol., 65, 75 11953). (l60C) Giilespie, J. M., Jermyn, IT.A,, and Koods, E. R., Nature, 169, 487 (1952). (161C) Gitterman, C. O., and Knight, 8. G., J . Bacteriol., 64, 223 (1952). (162C) Goodwin, T. W., Biochem. J . (London),53, 538 (1953). (163’2) Goodwin, T. W., Botan. Rev., 18, 291 (1952). (164C) Goodwin, T. W., and Griffiths, L. A , , Biochem. J., 52, 499 (1952). (165C) Goodwin, T. W., Jamekorn, hl.. and Willmer, J . S., Ibid., 53, 531 (1953). (166C) Goodwin, T. W., and Osman, H. G., Ibid., 53, 541 (1953). (167C) Gray, L., and Gaden, E. L., presented before the Division of Agricultural and Food Chemistry at the 122nd Meeting, AMERICAN CHEMICAL SOCIETY, Atlantic City, N. J. (1952). (168C) Green, S.R., and Stone, I., Wallerstein Labs. Communs., 15,
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(2230) Jensen, K. A., Diagsted, P. J., Moller, P., and Kiaer, I., Ugeslcrift Laeger, 112, 1075 (1950).
1965
(224’2) Jermyn, M. A., Australian J . Sci. Research, 5B, 409 (1952). (2250) Ibid., p. 433. (2260) Johnson, M. J., Bull. WorEd Health Organization, 6, 99 (1952). (2270) Joslyn, M. A., Agr. Food Chem., 1, 36 (1953). (228C) Jukes, T. H. (to American Cyanamid, Inc.), U. S. Patent 2,619,420 (Nov. 25, 1952). (229C) Jukes, T. H., presented at the meeting of the N. Y. Acad. Med., Dec. 10, 1952. (230C) Kahnt, F. W., Meystre, Ch., Neher, R., Vischer, E., and Wettstein, A., Experientia, 8, 422 (1952). (2310 Kane, J. H., Routien, J. B., English, A. R., and Schmita, A. J., Indian J. Pharm., 14, 7 (1952). (232C) Kawamura, S., J. Fermentation Assoc. (Japan), 7, 86 (1949). (2330) Kawamura, S.,J . SOC.Brewing (Japan), 46, 78 (1951). (234C) Kawano, Y., and Honda, S.,Japan. Patent 4100 (July 27, 1951). (2350) Keko, W. L., Bennett, R. E., and Arzberger, F. C. (to Commercial Solvents Corp.), U. S. Patent, 2,627,494 (Feb. 3, 1953). (236C) Kihara; Y.,Sato, T., and Yamaguchi, T., J . Agr. Chem. SOC. ( J a p a n ) , 22,45 (1948). (2370) Kitahara, K., and Fukui, S., Bull. Research, Inst. Food Sci., Kyoto Univ., No. 7, 1 (1951). (2380) Kitahara, K., and Ishida, H., Ibid., No. 2, 27 (1949). (239‘2) Kitahara. K.. Ubayashi. A.. and Fukui, S.,Enzymologia, 15, 259 (1952). (240C) Kleinaeller, A., and Lacko, L., Chem. Listy, 46, 679 (1952). (241C) Kleinzeller, A., and Malek, K., Ibid., 46, 674 (1952). (242’2) Klussendorf, R. C., Vet. Med., 47, 288 (1952). (243C) Kluyver, A. J., Chemistry & Industry, 1952, 136. (244C) Koepsell, H. J., Stodola, R. H., and Sharpe, E. S.,J . Am. Chem. SOC.,74, 5142 (1952). (2450) Koepsell, H. J., and Tsuchiya, H. M., J . Bacteriol., 63, 293 (1952). (2466) Koepsell, H. J., Tsuchiya, H. M., Hellman, W. W., Kazenko, A., Hoffman, C. A., Sharpe, E. S.,and Jackson, R. W., J . Biol. Chem., 200, 793 (1953). (247C) . . Koester. H.. Mamoli. L.. and Vercellone. A., U. S.Patent 2,236(574 ?April 1, 1941). (248C) Kok, B., Acta Botanica Neerlandica, 1, 444 (1952). (249C) Kolachov, P., and Nicholson, L. W., Econ. Botany, 5, 60 (19511. (250C) Kolachov, P., and Schneider, W. C. (to Jos. E. Seagram and Sons, Inc.), U. S.Patent 2,609,327 (Sept. 2, 1952). (251’2) Kotake, Y., Sosai, A., and Saito, Y., J . Antibiotics (Japan), 5, 496 (1952). (2526) Kunstmann, F. W., Pharm. Zentralhalle, 89, 259 (1950). (2530) Larsen. D. H . (to Commercial Solvents Corp.), U.S. Patent 2,615,829 (O&. 28, 1952). (2540) Laufer, L., Schwarz, D. R., and Stewart, E. D., Agr. Food Chem., 1, 91 (1953). (255C) Lee, J. (to Hoffmann-LaRoche, Inc.), U. S.Patent 2,599,564 (June 10, 1952). (2560 Leonard, R. H.. Peterson, W. H., and Johnson, M. J., IND. ENG.CHEM.,40, 57 (1948). (2570) Levin, R. H., Magerlein, B. J., McIntosh, A. V., Hange, A. R., Fonken, G. S.,Thompson, J. L., Searcy, A. M., Scheri, M. A., and Gutsell, E. S.,J . Am. Chem. Soc., 75,502 (1953). (258C) Leviton, A,, and Hargrove, R. E., IND. ENG.CHEM.,44, 2651 (1952). (259‘2) Lewis, U. J., Tappan, D. V., and Elvehjem, C. A., J . Biol. Chem., 199, 517 (1952). (260C) Livermore, A. H., Lindstrom, A., and Muecke, E., presented before the Division of Biological Chemistry a t the 123rd Meeting of the AMERICAN CHEMICAL SOCIETY, Los Angeles, Calif. (1953). (261C) Lockwood, L. B., Trans. N . Y . Acad. Sci. (Ser. I I ) , 15, 2 (1952). (2620) M k a r t y , M., J . Exptl. Med., 96, 555 (1952). (2630) Ibid., p. 569. (264C) McGuire, J. M., Bunch, R. L., Anderson, R. C., Boas, H. E., Flynn, E. H., Powell, H. M., and Smith, J. W., AntibiOtiCs & Chemotherapy, 2, 281 (1952). (265C) MaoLaren, J. A., J . Bacteriol., 63, 233 (1952). (2660) Mamoli, L., Ber., 71, 2701 (1938). (2670) Mancera, O., Ringold, H. J., Djerassi, C., Rosenkranz, G., and Sondheimer, F., J . Am. Chem. Soc., 75, 1286 (1953). (268C) Mancera, O., Zaffaroni, A., Rubin, B. A., Sondheimer, F., Rosenkrana, G., and Djerassi, C., Ibid., 74, 3711 (1952). (269C) Marcelli. E.. Riv. viticolt. e enol. (Conealiano), 5, 57 (1952). . . (270‘2) Ibid., p. ‘85. ’ (271C) Markhof, Th., and Markhof, G.,Austrian Patent 173,231 (Nov. 25, 1952). (272C) Marsh, J. S.,Mayer, R. L.,Mull, R. P., Schola, C. R., and Townley, R. W. (to CIBA), U. S.Patent 2,633,445 (March 31, 1953). ~I
1966
INDUSTRIAL AND ENGINEERING CHEMISTRY
S.,and Scholz, C . R., presented a t the Meeting of the Sorth Jersey Section of the ANERICANCREMICAL SOCIETY, Jan. 26, 1953. (274C) Martin, E., Berky, J., Godzesky, C., Miller, P., Tome, J., and Stone, R. W.,J . Biol.Chem., 203, 239 (1953). (275C) Martin, S. Xi., and Waters, W. R., IND.ENG.CHEW,44, 2229 (1952). (276C) Matagrin, A., I n d s . agr. et aliment. ( P a l i s ) , 69, 287 (1952). (277C) Mataushima, Y., and Shimagu, Y., Science, 115, 499 (1953). (278C) Maxwell, NI. E., Australian J . A p p l . Sei., 1, 348 (1950). (279C) hIaxwell,M.E.,Australia?aJ.Sci.Research, Ser.B,5,43 (1952). (280C) Ibid., p. 56. (281C) Maxwell, 31.E., Tech. Paper No. 1, Vi'ool Research Lab., C.S.I.R., Australia (1950). (282C) Slayer, R. L., Crane, C., DeBoer, C. J., Rnopha, E. A , , Marsh, 3. S., and Eisman, P. C., hbst. of 12th Intern. Congr. Chem., p. 283, 1951. (283C) SIeister, P. D., Eppstein, S. H., Peterson, D. H., Murray, Weintraub, A., and Reineke, L. A I . , H. C., Leigh, H. M., presented before Div. Biol. Chem., 123rd Meeting of the AXSRICAX CHEMICAL SOCIETY, Los Angeles, Calif. (1953). (284C) Meister, P. D., Peterson, D. H., Murray, H. C'., Eppstein, S. H., Reineke, L. A I . , Weintraub, &4., ard Leigh, TI. M., J . Am. Chem. SOC.,75, 55 (1953). (285C) hleister, P. D., Peterson, D. H., Murray, 13. C., Spero, G. B., Eppstein, S. IT., Weintraub, A., Reineke. L. M., and Leigh, H. XI,, Ibid., 75, 416 (1953). (286C) Merck & Co., Inc., Brit. Patent 6G7,lOS (Feb. 27, 1952). (2870) M f g . Chemist, 24, 49 (February 1952). (2880) Ibid., 55 (February 1952). (289C) Mickelson, M. N., and Schuler, AI, K., J . Bacteriol., 65, 297 (1953). (290C) Miles Laboratories, Inc., Brit. Patent 669,733 (-4pril 9, 1952). (291C) Mills, D. E., Baugh, 1%'. D., and Conner, H. A., presented before Div. Agr. and Food Chem. a t the 122nd Meeting of the AXERICAN CHEMICAL SOCIETY, Atlantic City, N. J. (1952). (292C) Miner, C. S.,and Dalton, N. N., "Glycerol," Xew York, Reinhold Pub. Corp. (1953). (293C) Mitchell, W. R. (to Commercial Solvents Corp.), U. S. Patent 2,602,043 (July 1, 1952). (294C) Mitra. K. K.. J . Sci.I n d . Research ( I n d i a ) . 11B. 109 (1952). i295Cj kIiyaji, N., J . Agr. Chem. SOC.(Japan),22, 78 (1948). (296C) Ibid., 79 (1948). (297C) Ibid., 80 (1948). (298C) Montgomery, R., and Ronca, R. A , , ISD. ENG.CHEM.,45, 1136 (1953). (2990) Mortimer, D. C., and Johnson, 31.J., J . Am. Chena. SOC., 74, 4098 (1952). (300C) Moyer, A. J., A p p l . Microbiol., 1, 1 (1952). (301C) Ibid., 1, 7 (1953). (302C) Moyer, W.W., and Allen, W. E'. (to A. E. Staley SIfg. Co.), U. S. Patent 2,597,019 (May 20, 1952). (303C) Murphy, D., Can. J . Technol., 29,471 (1951). (304C) Murphy, D., and Stranks, D. W., Ibid., 29, 413 (1951). (305C) Murphy, D., Stranks, D. W., and Harinsen, G. W., Ibid., 29, 131 (1951). (306C) Murphy, D., Watson, R. W., Murphy, D. R., and Barnwell, R., Ibid., 29, 375 (1951). (307C) Murray, H. C., and Peterson, D. H. (to Lpjohn Co.), U. S. Patent 2,602,769 (July 8, 1952). (308C) Nature, 170, 868 (1952). (309C) Neilands, 3. B., J . Am. Chem. Soc., 74, 4846 (1952). (31OC) Neilands, J. B., presented before Div. Biol. Chem., 123rd Meetling, AMERICANCHE~IICAL SOCIETY,Los Angeles, Calif. (1953). (311C) Kelson, G. E. N., Traufler, D. H., Kelley, S. E., and Lockwood, L. B., IND.ENO.CHEM.,44, 1166 (1952). (312C) Keuberg, C., and Hirsch, J., Biochem. Z., 115, 282 (1921). (313C) Newton, G. G. F., and Abraham, E. P., Biociiem. J . (London), 53, 597 (1953). (314C) Ibid., p. 604. (315C) Nickell, L. G., P r o c . Soc. Erptl. B i d . X e d . , 80, 615 (1952). (316C) Nielsen, N.,Proc. Second Intern. Congr. Biochem,.,p.109 (1952). (317C) Nishiyama, T., and Kozeki, T., Japan. Patent 2365 (May 18, 1951). (318C) Nokay, N . , Turk. Bull. Hug. E&. Biol.,11, 344 (1951). (319C) Nordstrom, L., and Hultin, E., Svensk Kern. Tidskr., 60, 283 (1948). (320C) Nunheimer. T. D. (to Wlerck gS Co., Inc.), U. S. Patent 2,628,931 (Feb. 17, 1953). (321C) Obara, I., J . Fermentation Technol. ( J a p a n ) , 26, 193 (1948). (322C) Office of Naval Research, European Scientific Notes, 6, 16 (Aug. 15, 1952). (323C) Oil, Paint Drug Reptr., 162, No. 2 , 34 (1952). (273C) Marsh, J.
Vol. 45, No. 9
(324'2) Okazaki, H., Svmposia on Enzyme Chem. ( J a p a n ) , 7 , 103 (1952). (3232) Ono, H., and Xsoni, A I . , J . Femneittation Assoc. (Japan), 7, 253 (1949). (326C) 0110,H., Murayama, K., and Suzuki, T., Ibid., 7, 76 (1950). (3270) Pence, J. W.,Agr. Food Chem., 1, 1517 (1953). (328C) Perlman, D., Science, 115, 529 (1952). (3290) Perret, C. J., J . Gen. iWicrobiol.,8 , 195 (1953). (330C) Peters, V. J., Brown, G. M., Williams, W. L.,and h e l l , E. E., J . Am. Chem. Soc., 75, 1688 (1953). (331C) Peterson, D. H., Eppstein, 8.€I., hleister, P. D., Magerlein, B. J., Murray, H. C., Leigh, H. M . , Weintraub, A., and Reineke, L. M., Ibid., 75, 412 (1953). 1332C) Peterson, D. H., and Murray, H. C., I b i d . , 74, 1871 (7952). (333C) Peterson, D. H., Murray, H. C., Eppstein, S.H., Reineke, L. XI., Weintraub, A., fiIeister, P. D., and Leigh, H. M., Ibid., 74, 5933 (1952). (334'2) Peterson, D. H., Nathan, A. H., Neister, P. D., Eppstcin, S. H., Murray, H. C., Weintraub, A., Reineke, L. M., and Leigh, H. M., Ibid., 75, 419 (1953). (335C) Peterson, W.H., Proc. Second Intern. Congr. Biochem., p. 110 (1952). (336C) Petty, M. A. (to American Cyanamid Co.), U. S. Patent 2,595,605 (May 6, 1952). (337C) Pfeifer, V. F., Vojnovich, C., and Heger, E. K., 1x1). ENG. CHEX.,44, 2975 (1952). (338C) Pirie, X. W., Chemistry & Industry, 1953,442. (3396) Pittenger, R. C., and McCoy, E., J . Bacteriol., 6 5 , 56 (1953). (340C) Plaut, G. W. E., Federation Prod., 12, 254 (1953). (341C) Pool, E. L., and Underkofler, L. A., A g r . Food Chem., 1, 87 (1953). (342C) Pool, E. L., and Underkofler, L. A., presented before Div. hgr. and Food Chem., 122nd Meeting, AMERICAN CHRMICAL SOCIETY, Atlantic City, N . J. (1952). (343C) Porath, J., Acta Chemica Scand., 6, 1237 (1952). (344C) Porter, J. N.,Hewitt, R. I., Hesseltine, C. W., Krupka, G., Lowery, J. A., Wallace, W. S., Bohonos, N., and Williams, J. H., Antibiotics & Chemotherapy, 2, 409 (1952). (3432) Prescott, F. J., Shaw, J. K., Bilello, J. P., and Cragwall, G. O., IND. ENG.CHEM.,45,338 (1953). (346C) Pridham, T. G., Econ. Botanu, 6, 185 (1952). (347C) Pridham, T. G. (to E. S. Secy. ilgriculture), U. S. Patent 2.578.738 (Dee. 18. 1951). (348C) Pridham, T. G., and Raper; K. B., Mycologia, 42, 602 (1950). (349C) Ibid., 44, 452 (1952). (350C) Pyke, "I,Chemistru & Industry, 1952, 1100. (351C) Rahn, O., Growth, 16, 59 (1952). (352C) Rao, P. L. N., and Venkataraman, R . , Experientia, 8, 350 (1952). (353C) Reed, G . , Trans. Am. Assoc. Cereal Chemists, 10, 21 (1952). (354C) Reese, E. T., Gilligan, W., and Norkrans, B., Physiol. Pluntarzm, 5, 379 (1952). (3556) Reese, E. T., and Levison, H. S., Ibid., 5, 345 (1952). (356C) Reyniers, J. A., Luokey, T. D., and Gordon, H. A,, presented at ineeting held at Univ. of Kotre Dame (June 4, 1952). (357C) Ricketts, C. R., Biochem. J . (London), 51, 129 (1952). (358C) Ricketts, C. R., and Waiton, IC. W., C'hemistru & Industru, 1952, 869. (359C) Ritter, H. B., Oleson, J. J., Hutchings, B. L., and Williams, 3. H., Arch. Biochem. and Biophys., 42, 475 (1953). (360'2) Robbins, W. J., Hervey, A,, and Stebbins, M. E., Nature, 170, 845 (1952). (361C) Robinson, K. L., Coey, W.E., arid Burnett, G. S., Chemistrg & Industry, 1952, 562. (362C) Robinson, R. F., Sei. Monthly, 75, 149 (1952). (363C) Rolinson, G. N., J . Gen. iMicrobioZ., 6, 336 (1952). (364C) Rolinson, G. N., and Lumb, M., Ibid., 8, 265 (1953). (365C) Romo, J., Rosenkranz, G., Djerassi, C., and Sondheimer, F., J . Am. Chem. Soc., 7 5 , 1272 (1953). (366C) Rosenstiel, P., Inst. Grand-Ducal Luxembourg, Sec. sci. nat., phvs., et math., Arch., 19, 157 (1950). (367C) Sainclivier, M., Bull. S O C . scientifique de Bretagne, T. X X V , 33 (1950). (36SC) Santer, M., and Ajl, S. J., J . B i d . Chem., 199, 85 (1952). (369C) Santer, XI., Ajl, S. J., and Turner, R. A,, Ibid., 198, 397 (1952). (370C) Sato, T., and Hayaski, il., Japan. Patent 1298 (RIarch 9, 1951). (371C) Saunders, A. P., Otto, R. H., and Sylrester, J. C., J . Bacterid.. 64. 725 (1952). . , (372C) Saunders,'R. R., Siu, R. G. H., and Genest, R. X., J . Bioi. Chem., 174, 697 (1948). (3736) Savard, J.. and Espil, L.. Centre tech. foiestier trop (Nogsntsur-Marne), Pub. No. 3, 7 (1951). (374C) Soatchard, G., Ann. A-. Y . Acad. Sei., 55,455 (1952).
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(375C) Schlussel, H., Maurer, W., Hock, A., and Hummel, O., Biochem. Z.,322, 226 (1951). (376C) Schwimmer, S., and Garibaldi, J. A., Cereal Chem., 29, 108 (1952). (377C) Scully, N. J., Stavely, H. E., Skok, J., Stanley, A. R., Dale, J. K., Craig, J. T., Hodge, E. B., Chorney, W., Watanabe, R., and Baldwin, R., Science, 116, 87 (1952). (3780) Seneca, H., Kane, J. H., and Rockenbach, J., Antibiotics & Chemotherapy, 2, 435 (1952). (379C) Serjak, W. C., Day, W. H., and Van Lanen, J. M., presented before Div Agr. Food Chem., 122nd Meeting, AMERICAN CHEMICAL SOCIETY, Atlantic City, N. J. (1952). (380C) Sher, I. H., and Mallette, M. F., J . Biol. Chem., 200, 257 (1953). (381C) Sherry, S., Trans. N . Y . Acad. Sci. (Ser. I I ) , 14, 138 (1952). (382C) Shiga, T., J . Antibiotics ( J a p a n ) ,5, 261 (1952). (3830) Shiga, T., and Arima, K., Ibid., 5,211 (1952). (3840) Shiga, T., Goto, S., and Arima, K., Ibid., 5, 255 (1952). (3850) Shu, P., Can. J . Bot., 30, 331 (1952). (3860) Shukla, J. P., and Seth, S. P., J. Sci. Ind. Research (India), 11B, 10 (1952). (387C) Simpson, F. J., and Stranks, D. W., Can. J . Tech., 29, 87 (1951). (3880) Singh, K., and Johnson, M. J., J . Bacterid., 56,339 (1948). (3890) Smith, E. L., Biochem. J. (London), 50, xxxvi (1952). (390C) Tbid., 52, 384 (1952). (391C) Smith, E. L., Ball, S., and Ireland, D. M., Ibid., 52, 395 (1952). (3920) Smith, E. L., Fantes, K. H., Ball, S., Waller, J. G., Emery, W. B., Anslow, W. K., and Walker, A. D., Ibid., 52, 389 (1952). (3930 Smith, E. L., Gurney, D. M., Howat, A . G., and Chalmers, J. N. M., Proc. Second Intern. Congr. Biochem., p. 19 (1952). (394C) Smith, E. L., Hockenhull, D. J. D., and Quilter, A. R. J., Biochem. J . (London), 52, 387 (1952). (395C) Smith, P. F., and Hendlin, D., J. Bacteriol., 65, 440 (1953). (3960) Smithies, W. R., Biochem. J . (London),51, 259 (1952). (397C) Smythe, C. V., and Neubeck, C. E. (to Rohm and Haas, Ino.), U. S. Patent 2,599,532 (June 10, 1952). (3980) Smythe, C. V., and Robb, L. A. (to Rohm and Haas, Inc.), U. S. Patent 2,606,899 (Aug. 12, 1952). (399C) Snell, E. E., Wittle, E. L., and Moore, J. A. (to Parke, Davis and Co.), U. S. Patent 2,625,565 (Jan. 13, 1953). (400C) Soltero, F. V., and Johnson, M. J., Appl. Microbial., 1, 52 (1953). (401‘2) Spencer, J. N., Payne, H. G., and Young, V., Federation Proc., 10 (1951). (402C) Spolek pro chemickou a hutni vyrobu, narodni podnik, (Austrian Patent 171,691) (June 25, 1952). (403C) Stacey, M., Chemistry & Industry, 1953, 318. (404C) Stacey, M., Endeavour, 12,38 (1953). (405C) Stacey, M., Research, 6, 159 (1953). (40GC) Stacey, M., and Paulard, F. G., Chemistry & Industry, 1952, 1058. (407C) Stavely, H. E., Baker, P. J., and Payne, H. C., presented before Div. Agr. and Food Chem., 122nd Meeting, AMERICAN CHEMICAL SOCIETY,Atlantic City, N. J. (1952). (408C) Stevens, G. M., Vohra, F., Inamine, E., and Roholt, D. A,, Federation Proc., 12, 275 (1953). (409C) Stokstad, E. L. R., Antibiotics & Chemotherapy, 3,434 (1953). (410C) Stoudt, T., and Brewer, B., presented before Div. Biol. Chem., 122nd Meeting, AMERICAN CHEMICAL SOCIETY, Atlantic City, N. J. (1952). (411C) Suzuki, T., J . Fermentation Assoc. ( J a p a n ) , 7, 59 (1949). (412C) Syrett, P. J., Ann. Botany, 7, 1 (1953). (413C) Ibid., 21 (1953). (414C) Tabenkin. B.. Lehr. H., Wasman. A. C.. and Goldberg. - M. W., Arch. Biochem. and Biophys., 38, 43 (1952). (415C) Taira, T., Yamatodani, S., and Fujii, S., J . Antibiotics ( J a p a n ) , 5, 313 (1952). (416C) Takaoka, K., Sci. of Foods (Japan), 3, 12 (1949). (4170) Takata, R., and Nagata, T., J . Fermentation Tech. (Japan), 27, 279 (1949). (418C) Ibid., 281 (1949). (419C) Ibid., 285 (1949). (420C) Ibid., 287 (1949). (421C) Talalay, P., Dobson, M. M., and Tapley, D. F., Nature, 170, 620 (1952). (4220) Tamboline, F. R., Can. J . Tech., 31, 70 (1953). (423C) Tanner, F. W., English, A. R., Lees, T. M., and Routien, J. B., Antibiotics & Chemotherapy, 2, 441 (1952). (424C) Tarr, H. L. A., Can. J . Tech., 30, 265 (1952). (425C) Tatum, E. L., and Peterson, W. H., IND.ENQ.CHEM.,27, 1493 (1935). (426C) Thaysen, A. C., Chemistry & Industry, 1953,446. (427C) Thierfelder, K., Ger. Pat,ent 805,347 (May 17, 1951).
1962
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INDUSTRIAL AND ENGINEERING CHEMISTRY
1968
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(LYJLI.
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(1951). SERMENTATION AS A UNIT PROCESS
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(44D) Hodge, H. AI., Hanson, C. T., and Allgeier, R. J., IND.EKG. CHEM.,44,132 (1952). (45D) Honig, P., “Principles of Sugar Technology,” Kern York, Elsevier Publishing Corp., 1953. (46D) Howe, E. E., and Putter, I. (to Merck and Co., Inc., and Rohm and Haas Co.), U. S. Patent 2,541,420 (Feb. 13, 1951). (47D) Humphrey, 8.E., and Gaden, E. D., presented before Division of Agricultural and Food Chem. at the 122nd Meeting of the AMERICAX CHEMICAL SOCIETY, Atlantic City, N. J., 1952. (48D) Hungate, F., and LIannell, T. J., Genetics, 37, 709 (1952). (49D) Inskeep, G. C., Bennett, R. E., Dudley, J. F., and Shepard, hl. W., IKD. EKG.CHEX., 43, 1488 (1951). (50D) Iso, C . , Yagishita, K., and Umezawa, H., J. Antibiotics ( J a p a n ) , 5, 274 (1952). (5lD) Jarvis, F. G., and Johnson, M, J., J . Am. Chem. ~ o c . ,69, 3010 (1947). (52D) Jinks, J. L., Proc. Roy. Soc., 140B,83 (1952). (53D) Karow, E. O., Sfat, 1%. R., and Bartholomew, W. H., Agr. Food Ckem., 1, 302 (1953). (54D) Karow, E. O., and Waksman, S. A , , IND.EKG.CHEM.,39, 821 (1947). (55D) Kiefer, 3. NI., Brewers Dig., 27, 4GT (April 1952). (5GD) Knoedler, E. L., and Babcock, S.H., IXD.ENG.CHEW,39, 578 11947). (57D) Kowkabany; G. N., Rinkley, W. m., and Wolfrom, M. L., Agr. Food Chem., 1, 84 (1953). (5SD) Legator, M., Ph.D. Thesis, University of Illinois (1952). (59D) Lindquist, W., J . Inst. Brewing, 59, 59 (1953). (GOD) Lindquist, W., Xature, 170,544 (1952). (61D) Lockwood, L. B., and Ward, G. E., IND.EXG.CHzar., 37, 405 (1945). (62D) Lodder, J., and Kreger-van Rij, K . W. T., “The Yeasts,” New York, Interscience Publishers, Inc., 1952. (G3D) Lott, W.A., Bernstein, J., and Heuser, L. J. (to E. R. Squibb & Sons), U. S. Patcnt 2,537,933 (Jan. 9, 1951). (G4D) Loutil. J. S., Australian J . Ezptl. B i d . M e d . S e i . , 30, 287 (1952). (65D) Lumb, M., and Fawcett, I
