ms
FERMENTATION SYLVAN B. LEE,
COMMERCIAL.
SOLVENTS CORPORATION, TERRE HAUTE, IND.
Antibiotics were chiefly responsible for the rapid progress in the fermentation industry during the period under review. During the past year improved processes and increased plant capacities resulted in greatly increased production of the major antibiotics-namely, penicillin, streptomycin, bacitracin, Chloromycetin, aureomycin, and tenamycin. During 1950 the discovery of tenamycin was announced and it was produced in commercial quantities. The ability of antibiotics to stimulate the growth of poultry and swine was announced and large quantities of several antibiotics are being produced and sold as feed supplements. A series of studies b y several investigators, during recent years, has been concerned with the production of citric acid in submerged fermentation? a large production plant is now being constructed for the purpose of producing citric acid b y this technique. Outstanding studies are reported concerning the fundamentals of aeration and mechanical agitation and their effect on dissolved oxygen concentrations and oxygen transfer in aerobic fermentations. Other reports deal with the effects on various fermentation processes, of such factors as mutation, trace materials, precursors, sterilizetion, inoculum development, and general fermentation techniques and equipment.
I
INDUSTRIAL FERMENTATIONPROCESSES Except where otherwise stated, progress in the different procemes up to 1950 has been reviewed (2f7, 618,328) and consideration only of more recent developments is given here. With the exception of newer fermentations, little attention i s given t o the extraction, purification, and uses of the fermentation products.
The technology of alcohol production has been reviewed in detail by Jacobs (190). As raw materials for its production he emphasized the use of agricultural wastes rather than those valuable as food or feed. Arnold and Kremer (6), in a review and economic study of various processes for ethyl alcohol production, placed particular emphasis on the future of corn as a raw material.
Recent Alcohol Production (392)
N T H I S series of fermentation reviews (217,618, 588) the
chief emphasis has been on present or potential commercial fermentations and particularly on factors concerned with fermentation as a unit process. This review is divided into two sections. The first summarizes recent literature on various industrial fermentation processes and the second considers factors of importance in the fermentation unit process. An attempt also is made t o cover as completely as possible the foreign literature on industrial fermentation despite lack of access, in some instances, t o the original publications. Several textbooks and reviews on industrial microbiological processes have recently appeared (86, 39, 78, 87, 137, 146, 612, 288, 294). Various phases of the fermentation industry have been reviewed by de Becze (as), Bishop ( 3 9 ) , Corran (87), and Petty (a88). Govindarajan (146) has reviewed recent progress in industrial mycology in India. Vogel (384) has prepared a comprehensive summary of the raw materials used in the fermentation industry. Brownlee (61)described a plant scale experiment on t h e production of penicillin t h a t was planned, controlled, and evaluated statistically. Attention is drawn t o this work because i t is believed that this approach should be widely applied in the fermentation industry. From a n economic viewpoint, the fermentation industry made excellent progress during 1950 and has every prospect of continuing t o d o so. The war emergency has been partially responsible for this situation, particularly with regard to the production of ethyl alcohol, butyl alcohol, acetone, and antibiotics. Expanded production facilities in this country for many fermentation products were completed during the past year or are being constructed. Examples are those for industrial alcohol (67), citric acid ( 7 3 ) ,antibiotics (66, 64,S38),antibiotic feed supplements (66), and dextran (68). The foreign fermentation industry is also expanding, particularly for the production of antibiotics (76-77').
ETHYL ALCOHOL
1948 1949 1950 Total production= 171,800,000 187,500,000 203,000,000 Produced by fermentation. % 64 52 47 a Production expressed as gallons (95% alcohol) to the nearest 100,000 gallons.
The total production of industrial alcohol continues to rise, while the per cent produced by fermentation continues t o decrease. The production increase during 1950 occurred principally during t h r last quarter. Then, due t o the war emergency, considerable fermentation capacity was again placed in operation. Government estimates of the country's 1951 needs are for 350,000,000 gallons. I n view of the shortage of molasses and the greater demand for alcohol, grains are again a feasible raw material. The normal trend, however, is R lessening in the use of grain. Interesting developments greatly affecting the ethyl alcohol fermentation industry occurred in the economic-s of molasses. Whereas United States buyem expected to purchase. molasses for as little as 3 cents per gallon in 1950, shortagw. due to compotition from foreign buyers and the use of molasses in cattle feeds, resulted in a price of 18 cents or more per gallon. Additional facilities for producing alcohol hy fermentation are under construction (.57). T h e long-term trend is, however, toward a relatively greater expansion of synthetic production plants (67. 76). Koshland and Westheimer (110)have reinvestigated the mechanism of alcoholic fermentation by fermenting gluco=e-l-C1*. About 95y0 of the total radioactivity fermented appeared in the methyl group of the alcohol obtained, giving quantitative support t o the currently accepted mechanism for the alcoholic fermentation. Nord (266) presented some facts and interpretations of the mechanism of :~lcoholicfermentation b y yeasts snd molds. Genevois (140, 141) has developcd equations which summarize his quantitative studies of the balance between the principal products and secondary products formed in alcoholic fermentation b y yeast. Berand and Millet (3f)have investigated the alcohol-producing properties of yeasts cultivated at low temperatures. Their d a t a show that when the fermentation of glucose is conducted at 7" C. the alcohol formation per cell is two to six times greater than at 25" C. The ethyl alcohol yield per unit of sugar fwmented is also greater at the lower temperature. The superiority of the low tem-
1948
September 1951
INDUSTRIAL AND ENGINEERING CHEMISTRY
perature fermentation is retained through a series of fermentations a t low temperatures. Miner and Wolnak (248) have recently been granted a patent covering the addition of m a l l quantities of activated sludge or hydrolyzed sludge to media used for ethyl alcohol production. They claim that the addition of this sludge reduced the total fermentation time and increased the yields. Laqomasino (214) described the Melle-Boinot alcoholic fermentation method in which the yeast cells are used repeatedly. This method makes it possible t o avoid almost completely the formation of new cells a t the expense of sugar. Alcohol yields as high as 97% of theoretical are thus obtained. The yeast was separated from the fermented beers before distillation by a special centrifuge and immediately re-used. The following advantages were claimed for this process: Beers containing 9 to 10% alcohol are obtained. The use of sugar for yeast production is largely eliminated. Fermentation time is reduced to 12 to 18 hours. Rapid alcohol formation inhibits t h e growth of foreign organism?. A higher degree of cleanliness is maintained in t h e still and “vat lees” are noticeably reduced. About 607, of the still residue can be used for diluting the molasses for fermentation while the remainder, low in organic matter, offers less of a disposal problem.
A number of publications deal with the utilization of inexpensive raw materials for alcohol production. Ekelund (111) received a patent on a process for “sorping” the sugars from an industrial liquor containing low concentrations. Example. A mass of yeast cells was added to sulfite waste liquor to “sorp” the sugar from the solution. The yeast was separated from the sulfite liquor before any substantial fermentation could occur and was added t o another solution of much smaller volume. As the result, higher concentrations of carbohydrates were fermented. i\ccording to Sundman (563) when acetaldehyde is formed as a by-product of the alcoholic fermentation of sulfite liquor, two moles of alcohol are lost for every mole of acetaldehyde produced. Variation in the pH, the amount of yeast, and the temperature had little effect on the amount of aldehyde formed. However, the amount of aldehyde seemed t o be proportional t o the amount of sulfur dioxide in the liquor Therefore, it was suggested t h a t acetaldehyde, obtained during distillation of fermented liquor, be added to the subsequent fermentation in order to bind free sulfur dioxide and thereby inhibit the formation of further acetaldehyde. Flygare (18%) found that milo meal, which resulted from the conversion of milo t o pearl barley. was a suitable raw material for alcoholic fermentation, and particularly for beverage production. Mickelson and coworkers (246) have shown t h a t theoretical yields of ethyl alcohol could be obtained when sugar beet crowns were used as a substrate for ethyl alcohol production. Clarified extracts of beet crowns, however, gave inferior results. Continuous Fermentation. Little material has been published during the past year on continuous methods for the alcoholic fermentation. According t o Rao et al. (299) pumice impregnated with distillery yeast in packed columns of a fermentor for continuous operation accelerated the rate of fermentation of molasses and increased the efficiency of the yeast by preventing supersaturation of the medium with carbon dioxide and the caking of yeast cells. Microbial Amylase. The submerged fungal amylase process developed by the Northern Regional Research Laboratory (317, 218) has been tested on a large scale (69, 74, 576). Commercial scale experiments with this process a t the Grain Processing Corp., Muscatine, Iowa, showed that a plant using 12,000 bushels of grain per day could save more than $1000 a day by shifting t o
a949
the new process. Conversion to the fungal process nas relatively simple. The process had no adverse effect on the qualitv of the alcohol and the by-product feeds were practically identical with those from the usual malt process. However, this process cannot be used for the production of beverage alcohol because of government regulations. Cheol (79) came to the conclusion that the amylo process waR superior to the malt process. Deckenbrock (96, 95) found that the main advantages of the fungal amylase process as compared with the malt process were greater alcohol yields, decreased fermentation time, and decreased foaming. Teixeira, Andreasen, and Kolachov (363) reported the production of alcohol from cassava root using submerged fungal amylase as the starch-converting agent. The authors claimed that it is possible t o obtain plant efficiencies of 90% from cassava mashes converted with submerged fungal cultures. This is compared to alcohol yields of 43 to 74% \+ith acid hydrolysis and 70 t o 74% with barley malt. Tsuehiya et al. (875) reported further studies on the submerged production of fungal amylase. Fungal a-amylase and maltasr could be controlled to some degree by adjusting the concentration of the thin stillage solids and corn meal in the medium. The u w of calcium carbonate in the medium resulted in a lowered yield of maltase. Rao and Sreenivasaya (898) described a process for growing Aspergillus oryzae on fibrous material for 3 to 6 days, then drying the entire mass t o procure material having a high amylase activity. Pan et al. (280) have shown that submerged fungal amylase cultures converted an appreciable portion of the starch into dextrins difficult to hydrolyze. Secondary alcoholic fermentation depends on the rate of hydrolysis of these dextrins. They have also demonstrated that submerged fungal amylase will convert maltose into an unfermentable carbohydrate. Bacterial amylase is also being tested as a replacement for barley malt (74). Since the bacterial amylase can be reduced to a dry powder or a stable concentrated solution, it does not have to be produced a t the point where it is used. A further advantage of bacterial over fungal amylase is its greater heat stability. AIthough at the present time not enough data are available, it is estimated that the cost of these two amylase preparations will be about the same. Saiuno and Ano (319)have obtained a patent on the produrtion of bacterial amylase from Bacillus mesentericus and on its application in the production of alcohol. Smythe et al. (535) have been granted a patent on a process for producing amylase concentrates using submerged cultures of Bacillus subtilis and Bacillus mesentericus. The broader field of enzymes and enzyme technology has been reviewed by Reed (504). ACETONE-BUTYL ALCOHOL
The acetone-butyl alcohol fermentation industry has shown a very marked upward trend beginning with the latter half of 1950. Total production in 1950 of n-butyl alcohol was 147,300,000 pounds, and of acetone 481,600,000 pounds (592). Information indicating the proportion produced by fermentation and by synthetic processes is not available. The production of butyl alcohol by fermentation was reviewed by Abai (10, 11) and by WUiams (400) who also described the synthetic production of butyl alcohol from acetaldehyde although he did not compare the merits of the two methods. Aschner and Kutzenok (13) described a Cbstridium butylicu~n which produced a high yield of butyl alcohol, some isopropyl alcohol, and minute amounts of acetone and butyric acid In fermentations where clostridial formation was diminished or absent, butyric acid replaced butyl alcohol in the end products. Nakahama and Harada ($54) isolated a bacterium classified as
1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
Clostridium h n e b o i which fermented staxch as well as cane sugar and gave high yields. In a single semi-industrial scale experiment using Clostridium kaneboi symbiotically with Bacillua m e s a t e r i CUS, the quantities of acetone, butyl alcohol, and ethyl alcohol were claimed to be in the range of 36%, based on the weight of the original cane sugar used (266). Vergnaud (381) patented a process for obtaining increased yields of butyl alcohol and acetone from low-cost carbohydrate materials such as molasses and Jerusalem artichoke juice, through the use of mineral acids, such as hydrochloric, sulfuric, or phosphoric acid, in the fermentation medium after the peak acidity has occurred, thereby maintaining a more acid pH. It waa claimed that this resulted in a more complete conversion of the carbohydrate into the desired solvents. Stark and McGhee (34W) described an improved procedure for the pretreatment of waste sulfite liquor, which resulted in considerable improvement in yield over earlier methods, when it was used as the raw material for the acetone-butyl alcohol fermentation. The process consisted of pretreating the liquor by stripping; addition of lime; precipitation; filtration; and neutralisation of the filtrate with carbon dioxide. These improvements could be readily adapted to a commercial operation. The authors reported that 20 to 23% of the total sugars, or 33 to 42% of the yeast-fermentable sugars, were converted to solvents. To date the commercial process is not completely worked out and a number of steps, outlined by the authors, require further development. Mickelson et al. (245) found that sugar beet crowns could he readily fermented by Clostridium acetobutylicum. The rate of fermentation and the solvent yields were equivalent to those obtained in corn mashes. However, a saccharolytic Clostridium culture did not ferment sugar beet crowns as readily as blackstrap molasses, and solvent yields were only ahout one third those obtained from molasses. Artigas and Jane (9) described a combined acetone-butyl alcohol and ethyl alcohol fermentation of grapes and Jerusalem artichoke musts. Primary fermentation was carried out a t 37" C. with Bacillus butylicus followed by the yeast alcohol fermentation. Butyl alcohol, acetone, and ethyl alcohol were formed in the ratio of 6 to 3 to 1. P,3-BUTANEDIOL
The lack of commercial interest in the 2,Sbutanediol fermentation mentioned in previous reviews (917, 218) haa continued throughout the current period. Canadian Research Council investigations discussed in the ,previous review (218) have continued (963, 269, 329, 389). The effect of the pH on the fermentation was investigated. An electronic pH monitor, which gave automatic control, was employed. T h e organism most often used was Bacillus polymyxa (269, 329, 389). The pH range was 5 to 8; the yield of 2,3butanediol decreased above pH 6.4, and a t pH 7.6 little or no diol was formed. The yield of acetylmethylcarbinol and acetic and lactic acids increased in the alkaline PH range, reaching a niaximum a t 7.6 under anaerobic conditions. T h e most efficient conversion t o butanediol under anaerobic conditions occurred a t p H 6.0 t o 6.4. At pH 6.8 there was a sharp increase in the production of organic esters and a corresponding decrease in 2,3-butanediol. Under anaerobic conditions the rate of fermentation was greater than under aerobic conditions; the yields of butanediol and ethyl alcohol were increased, and the yield of organic esters decreased. The most efficient dissimilation of sucrose to butanediol occurred anaerobically at a concentration of 8% after 30 hours at pH 6.2; 65 millimoles of butanediol were produced per 100 millimoles of sucrose fermented. Under aerobic conditions the fermentation was slower, and a t sugar concentrations of 8 and 1 0 7 was incomplete even after 48 hours. The total yields of butaneliol and acetylmethylcarbinol remained fairly constant a t about 80 millimoles per 100 millimoles of sucrose fermented.( 2669, 389). By acclimatization, strains of Bacillus p o l y m y x a , selected for glucose fermentation , fermented beet molasses satisfactorily.
Vol. 43, No. 9
The molasses process required pH adjustment of the medium to 5.6 before sterilization. 6.2 after sterilization. and addition of organic substances such as yeast extract and wheat bran. Sufficient phosphorus, articularly orthophosphate, was essential for satisfactory yields 629). Natural organic substances such aa wheat bran, yeast extract, and cornsteep liquor also contained some minor unidentified factors which produced a small but noticeable stimulation of the fermentation in molasses media (263).
As a result of these and previous investigations by the same group, a Canadian government corporation obtained a patent (61), available for licensing, on an improved and effertive method for producing 2,Sbutanediol by bacterial fermentation of mashes of whole cereal grain. Similar observations on the effect of aeration and p H were made by Orlova in Russia (266, 967). The organism used was Aerobacter aerogenes which had also been used in some of the experiments of the Canadian investigators (663,269). Orlova found that in alkaline media the organism fermented glucose to form acetic acid, lactic acid, ethyl alcohol, and formic acid, with very little formation of acetylmethylcarbinol or 2,3-butanediol. In peptone medium containing no sugar, the organism fermented butanediol t o form acetylmethylcarbinol under aerobic conditions, or ethyl alcohol and volatile acids under anaerobic conditions. The p H for maximal yields of acetylmethylcarbinol and butanediol was 6.0. In general, conversions up t o 30% were obtaincd, but in some cases converPions were as high as 50% (d66, 167) An improved 2,Sbutanediol process was the subject of United States and British patents by Vergnaud (377, 380). One of the main features of the patent was the use of certain strictly aerobic bacteria of the Bacillus mesentericus group, instead of strictly o r optionally anaerobic organisms such as Bacillus polymuxa. It was claimed that ethyl alcohol or organic acids were formed in amounts no greater than 2%. The total yield of butanediol and acetylmethylcarbinol exceeded 40% of the weight of the sugar used. Extreme aeration resulted chiefly in the formation of acetylmethylcarbinol. The addition of small amounts of acetylmethylcarbinol at the beginning of the fermentation wm claimed to increase the economy of the process. When the products introduced before fermentation were subtracted, the yields, based on sugar utilized, were 40.2% butanediol and 0.9% acetylmethylcarbinol ($77, 380). Stark et al. (Sdl).described the production of butanediol from grain with Aerobacter aerogenes. The yields per bushel of grain used were 15.7 pounds of the diol, 1 pound of ethyl alcohol, and 1 to 1.5 pounds of acetylmethylcarbinol. According to Mickelson et al. (245)sugar beet crowns could be used as a substrate for the formation of 2,Sbutanediol with Aerobacter aerogenes. Yields in 96 hours ranged from 40 to 48% of the weight of the sugar fermented in beet diffusion juice and molasses media. I n beet crown media more rapid fermentation resulted, but yields were only 30 to 34% of the sugar fermented. CITRIC ACID
During 1950 a very great expansion of the production facilities for citric acid occurred. While production was approximately 35,000,000 pounds last year, it is estimated that by the end of 1951 the citric acid capacity will be over 50,000,000 pounds. Even this production may not be enough for current demand (73). Up to the present Chaa. Pfizer & Go., Inc., has produced over 90% of the acid in this country. Miles Laboratories, a large user of citric acid, is building a production plant, which will employ the submerged technique. This plant is estimated to have a capacity of 5,000,000 pounds per year and should be in production b y July 1951 (73). Several patente issued to Miles Laboratories concrrriing the production of citric acid by submerged fermentation techniques have been reviewed prrviously (618).
September 1951
INDUSTRIAL AND ENGINEERING CHEMISTRY
The economics of citric acid production in South Africa were discussed by Cillie and Rapson (81). Conventional production nwtliods as well as the economics, properties, and uses of this acid are reviewed by Stone (360). Perlman (283) has reviewed the factors affecting the mycological production of citric acid by the submerged method. He also discussod the chemical changes which occur during the conversion of sucrose into citric acid. Various theories concerning the formation of citric and other acids by Aspergillus niger and similar molds were reviewed and discussed in detai? by Walker (388). A French patent (2.44) has been granted o n a method for the production of citric acid by Aspergillus wentii. This method has been reviewed previously (388). The effect of trace metals on citric acid production has been further investigated by Tomlinson et al. (371). Using reagentgrade cheniirals, it was demonstrated that wit,h a strain of ilspergil(us niger zinc, iron, manganese, and copper were required for maximal citric acid production. Omitting zinc caused a 96% decrrme of acid formation; iron, 91%; manganese, 44%; and coppei,. 76%. However, excessive amounts of these metals intert c w d with the accumulation of citric acid When sucrose was pwified by cation exchange treatment, the yield with optimal concentration of the above metals was 17% lower than with unpurified sucrose. This indicated t h a t some additional factor was essential for high acid production (371). Oxford et al. (277) found that chlorides of sodium, potassium, and magnesium, and t o a lesser extent, sulfates and nitrates, suppressed the formation of oxalic acid in the citric acid fermentation of molasses. Terui (365) was granted a Japanese patent in which he claimed that the production of citric acid from sucrose by Aspergillus niger could be increased by adding a small amount of ferrocyanide or ferricyanide to the sporulation medium. Oxford et al. (274-277) studied the production of citric acid from a number of samples of cane nil,iass-es. Their data confirmed the findings of many previous investigators. O T H E R ORGANIC ACIDS
Lactic Acid. The potentialities of the lactic acid fermentation industry appeared excellent a year ago due to the availability of low cost molasses (218, 256). The preeent high cost of molasses and other carbohydrate materials has changed this picture consider ab I y. A very thorough study of the effect of continuously controlling the pH in lactic acid fermentations has been made by Finn el al. (199, 1850) and Kempe et ai. (604). The former studied the rate of fermentation of glucose t,o lactic acid by Lactobacillus delbruckii a t rigidly controlled p1-I levels from 3.5 to 6.0. The p H was kept constant b y automatic control throughout each fermentation and the equipment was devised so as to record continuously the rate at which acid was produced. The maximal rate of acid production was increased a8 much as fourfold by raising the p H only 0.5 unit. At all pII levels a high concentration of accessory nutrients -for (.sample, peast extract-was necessary for rapid production of acid; however, there was an optimal concentration of yeast extract beyond which no further rate increase resulted. The rate of lactic acid fermentation was maximal a t a p H of 5.7 and w ~ t s apparently independent of the glucose concentration, at least in the range of A to 1Oyo. The maximal rate of acid roduced was about 0.13 g r a m g r hour per 100 ml. of broth. T k s confirmed earlier nork by empe and coworkers (904), who investigated the lactic acid production in wheat grit mashes using contiriuously eoiitrolled pH. I n both invest'igations the acid production d e creased when the p H was outside the range of 5.4 to 5.7.
Suzuki et al. (354) found that rice bran contained a lactic acid fermentation accelerator. More accelerator %'as found in fat-free than in untreated bran. It was most efficiently extracted with distilled wattlr a t 50" C., less with aqueous hydrochloric acid, and not a t all with et,her. The factor W E heat $table and could not be precipitated.
1951
Considerable work has been done on the utilization of raw materials for lactic acid production (86, 111, 180, 136, %@). Friea (136) ohtained a patent o n the utilization of sulfite wa&e liquor in which he recommended treatment of the liquor with sufficient lime to raise the p H t o 12.5, thereby precipitating both calcium sulfite and lignin compounds. H e recommended alternatively the addition of enough lime to adjust the pH to the range of 7.5 to 9.5, thereby precipitating only the sulfite. After removal of the precipitate, t h e temperature was adjusted to 30" t o 40' C., the p H to 6.3 to 6.7, nutrients such as malt sprouts or cornsteep liquor were added, and the liquor was inoculated with various lactabacilli. B strain of Lactobacillus pentosus was most, acceptable. I n a more recent patent of Ekelund (111) the carbohydrate was extracted from sulfite waste liquor by "sorping" on lactic acid bacteria prior to fermentation. Two Spanish investigators (120) produced lactic acid by ferment,ing the juices of Jerusalem art,ichokes which had been fortified with nitrogenous materials such as peptone, ammonium sulfate, and an excess of calcium carbonate. Grape juices were similarly fermented. Yields approaching 100% of the theoretical were claimed. Cordon and coworkers (86) used potatoes as the raw material for lactic acid production. The starch was first converted t o sugar with a n amylase preparation from Aspergillus niger. Various species of lactobacilli were used to produce lactic acid and the yields were 80 to 90%, based on the carbohydrate consumed. Highest yields were obtained with Lactobacil[us pmtosus. Bernhauer et al. (36) made an extensive investigation of the production of lactic acid by Rhizopus species. A strain of Rhizopus oyrzae gave best results; 80% of the glucose could be transformed into lactic acid in the presence of calcium carbonate. With starch as the starting material, the yield was only 45 t o 48%. I n a preliminary investigation of the effect of trace metals it waa found t,hat, within the concentration limits used, iron and manganese ions did not seem to have any effect; however, zinc had a favorable effect. Copper had some inhibiting action which could, however, be corrected by the addition of iron OF zinc ions. i t d o - and bromoacetic acids, as well as quinones, favored lactic acid production and limited the formation of ethyl alcohol. Selenium derivatives had the opposite effect,. The best nitrogen sources were urea and ammonium sulfate. The use of submerged mycelium or partially germinated spores, instead of a spore suspension, markedly accelerated the fermentation of glucose t o lactic acid. Yields of 70 to 80% were attained in about 7 days. Itaconic Acid. The production of itaconic acid from sucrose hy Aspergillus terreus was invefitigated by Vicenty et al. both in surface (382) and submerged fermentations (3885). I n surface culture the optimal conditions were a fermentation period of 12 days on a medium 1.7 cxn. in depth, a p H of 1.8 to 2.3, and a sucrose content of 14 t o 17%. Roiled or decanted sugar cane juice was also a satisfactory carbohydrate source. Yields on this substrate were 35% it,aconic acid. With glucose as a substrate, p H adjustment wit,h nitric acid gave a yield of 20%, while the addition of hydrochloric or sulfuric acid yielded 36 to 38% itaconic acid, based on the weight of the glucose used. Technical dextrosc gave 36 t o 38% yield with all three acids. Using the same organism in submerged culture on a semipilot plant scale, it was found that the yields of itaconic acid from sucrose decreased with increasing concentration of the carbohydrate. A mechanically agitated and aerated 20-liter fermentation yielded 38% itaconic acid in 7 to 8 days (583). Gluconic, Ketogluconic, and Related Acids. The mechanism of the formation of gluconic and other organic acids produced by Aspergillus niger and related molds was reviewed b y Walker (S88).
Bose ( 4 1 ) used molasses as the medium for gluconic acid fermentation with Aspergillus niger and obtained yields of 80 to Moj, based on glucose used. Three Indian investigators (91) obtained an Indian patent on the oxidation of glucose-containing
1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
solutions. They used the mycelial mat of Aspergillus niger resulting from the citric acid fermentation. Ground mycelium plus calcium carbonate was added to 10 t o 15% glucose solutiou and the mixture was aerated. After the fermentation was completed, the protein matter was coagulated, and the solution of calcium gluconate filtered off and evaporated. Ikeda el al. (186)tested various types of microorganisms and found, as had previous investigators, that Aspergillus niger was the most efficient converter of glucose t o gluconic acid. Yields in a 10% glucose solution were 70 to SO%, based on glucose used, a t a temperature of 30 O C. and a fermentation period of 42 hours. The use of Acetobacter in the oxidation of glucose to gluconic acid, and of the latter to ketogluconic acids, was studied in Germany (37, 808) and Japan (852). Bernhauer and Riedl-Tumova ( 3 7 ) studied methods for maintaining the activity of the cultures and for growing the inoculum. They recommended the frequent transfer of the original cultures and their further cultivation on media rich in nutrients. For active inocula it was especially important that these cultures were not older than 48 hours in order to ensure the most rapid oxidation rate. Acetobacter suboxydans gave yields of 95% gluconic acid in a 42-hour fermentation. Investigating the effect of Acetobacter on gluconic acid, Bernhauer and Riedl-Tumova (87, 308)found that one strain of Acetobacter suboxydans produced mainly 5-ketogluconate, while another strain yielded mainly 2-ketogluconate. Acetobacter melanogenum produced another aa yet unidentified oxogluconic acid ~ a l tbesides 5-ketogluconate (808). Sumiki and Hatsuda (362) obtained a 54% yield of 5-ketogluconic acid from glucose with a strain of Acetobacter. Miscellaneous Acids. Turski and Iwanowski (374)described the methods for the production of concentrated acetic acid. Hromatka and Ebner (180)reviewed the development of acetic acid production by fermentation. They studied the fermentative oxidation of alcohol to acetic acid by submerged fermentation in which it waa found that acid formation increased with time as an exponential function, This was in contrast to the usual industrial method, using the vinegar generator, in which the fermentation was determined t o be a linear function of time. The velocity of the submerged fermentation method was reported t o be approximately thirty times as great as the vinegar generator technique. Fumaric acid formation by Rhizopus was investigated by Rauch et al. (846,302,303). As the result of a comparison, in surface culture, of 16 fumaric acid-forming species of Rhizopus, i t was shown that these fungi primarily form fumaric acid. However, in order t o obtain the highest yields, it was necessary t o neutralize the acid with calcium carbonate as it was formed. Highest yields were obtained with a strain of Rhizopus delemar which, in 17 days, yielded 58.8% fumaric acid and 19.4% lactic acid, based on the glucose used, This'was the only culture which formed fumaric acid in a medium containing urea and zinc and no calcium carbonate (802). Using the organism which proved t o be the highest fumaric acid producer in surface culture, the same group of investigators also studied the submerged production of fumaric acid (303). Moderate quantities could be produced by submerged cultivation only if the acid were neutralized by maintaining a p H of 5.0 to 6.0 with sodium carbonate. Even under these conditions a yield of only 30 to 40%, based on glucose, was obtained. The rest of the glucose was used for the formation of the mycelium and fermentation products such as ethyl alcohol, lactic and other acids. An investigation of the effect of heavy metal ions on the Rubmerged fumaric acid fermentation (246)showed that zinc, iron, and manganese had very little influence in a medium with technical glucose and t a p water. Copper in low concentrations had a stimulating effect, but in high concentrations it inhibited strongly. However, inhibition could be reversed by zinc or aluminum. Fermentation inhibitors such as selenium derivatives, sodium fluoride and iodoacetic acid inhibited fumaric acid accumulation i i i high concentrations but had little effect in low concentrations.
Vol. 43, No. 9
Another compound, p-benzoquinone, increased the yield of fumaric acid (246). MlCROBlOLOGlCAL PRODUCTION OF VITAMINS
Riboflavin. T h e use of riboflavin in the pure form and M riboflavin-rich concentrates for food and feeds (818)continued to increase during the past year. At least one producer has released a new feed supplement of high riboflavin potency (63). Reviews on the synthesis of riboflavin by microorganisms were prepared by Dikanskaya (97)and Hickey (174). Pridham and Raper (,996) have presented an extensive review of the literature on Ashbya gossypii, one of the more promising riboflavin-producing organisms. This review was written from the viewpoint of the mycologist. Dulaney and Grutter (107)studied the nutritional requirements of Eremothecaum ashbyii, the chief microorganism used for producing riboflavin. They found that with their basal medium which contained hydrolyzed casein and inorganic salts very little growth and riboflavin production occurred. The addition to this medium of biotin and vitamin B,, alone or in combination, had no stimulatory effect but I-inositol alone or in combination with these vitamins stimulated both growth and riboflavin production. The organism also required EL fairly complex nitrogen source. Glycine alone was found t o be insufficient; however, when other amino acids, such as proline, arginine, or glutamic acid were suhstituted for glycine, satisfactory growth and riboflavin production occurred, provided 2-inositol was added. Under all these conditions, much lower yields were obtained than in complex organic media. The authors concluded that factors, as yet unidentified, stimulate riboflavin production by Eremothecium ashbyii. Riboflavin production was not increased by glutamine, pantothenic acid, vitamin B12, nicotinic acid, pyridoxin, choline, p-aminobenzoic acid, and folic acid. Chin (80), also using Eremothecium ashbyii, found that peptone was the best nitrogen source for riboflavin production. Other suitable nitrogen sources were glutamine, asparagine, alanine, and ammonium sulfate. Tryptophan, tyrosine, and urea were not suitable. Rice germ extract was an excellent source of growth factors. James (191) obtained a patent in which he claimed that much higher yields of riboflavin could be obtained when malted wheat mashes were used in the medium. I n preparing the malted wheat, the amount of barley malt used should be a t least 8% of the wheat solids. The final mash contained between 9 and 17% solids and the pH was between 5.8 and 6.2. Using a fermentation temperature between 28" and 30" C., yields of 1175 micrograms of riboflavin per ml. were claimed. A recent patent by de Becze et al. ( 2 7 ) , on the production of riboflavin-containing materials from distiller's stillage, claimed an increased riboflavin concentration in the final broth by adding inorganic nitrogen and phosphorus nutrients as well as a carbohydrate source. Takata (860) described an improved culture method and large scale production using Eremothecium ashbyii grown on solid media such as germ rice and germ wheat. Maximal yields were 20,000 microgram per gram. Sekido and Kichina (866) claimed in a Japanese patent that, if the riboflavin in a medium fermented by Eremothecium ashbyii and which contained 5000 microgram of riboflavin per gram, were dissolved in water, filtered, and left standing open to the air, riboflavin was precipitated. It was claimed that, after being centrifuged and dried, the product contained 50,000 micrograms of riboflavin p6r gram. Yamasaki (411) discussed the effect of calcium carbonate and ferrous sulfite on the production of riboflavin by certain anaerobic bacteria. The production of riboflavin by Ashbya gossypii received much
September 1951
INDUSTRIAL AND ENGINEERING CHEMISTRY
attention (2@,289,339). Cornsteep liquor, peptone, and tankage itre claimed to be suitable nitrogen sources (243). Smiley and coworkers (332)have shown t h a t by careful control of the sterilizing conditions and by the use of the proper concentration of various ingredients, high yields of riboflavin could be obtained, under submerged aerobic conditions, from a medium containing grain ethyl alcohol stillage, animal stick liquor, and ca i,bohy drate I'feifer el al. (289)conducted extensive experiments on the pilot plant scale to supply engineering data on the process for the production of riboflavin by submerged culture of Ashbya gossypii. Thc effect on the yield of the sterilizing methods, composition of the medium, aeration, agitation, temperature, and strain were investigated. Satisfactory yields could be obtained if the variables were closely controlled. The medium contained 2y0 glucose, 1.8 to 2.1% cornsteep liquor, 1.0% animal stick liquor, :ind a small amount of an appropriate antifoam agent. Most satisfactory sterilization resulted from a retention time of 5 minutes a t 275" F. a t pH 4.5. Seed cultures were developed on the produrtion medium and used a t 0.5 t o 1% by volume to inoculate the fermentor. Mechanical agitation should be sufficient to keep the medium well nlixcd but not to interfere with the growth of the organism. No definite expression of the degree of agitation was given The most satisfactory air flow was 0.25 volume of air per minute per volume of fermentation medium and t h t ~most suit,a'ule fermentation temperature was 28" to 30 O C. 17nder these conditions yields of 500 to 848 micrograms per ml. \yore obtained in a 9 6 to 120-hour fermentation. The fermented liquor was evaporated to a sirupy consistency and drum dried. Tliv firial product contained 2.5y0riboflavin. Under the conditions prevailing at t,he time of t,he study-that is, in the summer of 1949-preliminary cost calculations, based on an annual production of 9000 kg. of riboflavin, indicated a production cost of 3.75 cents per gram (289). Schopfer and Guilloud (394) reported comprehensive nutrit ional studies with Candida guilliermondii. The maximum riboflavin production wm 280 micrograms per mi. hIitra (250) in India, described a mutant strain of top yeast, obtained by treatment of a brewery yeast strain with acenaphthrne, which showed greater growth and riboflavin excretion than its parent strain, Two other Indian investigators (997)described artificially produced top yeasts which produced considerable miounts of riboflavin. Zalesskaya (412) described the riboflavin formation in Asperqillus flatus mycelium which had been grown in filt'ered cereal mashes. The riboflavin concentrates contained 30 to 92 micrograms per gram. In his st,udies of metabolic antagonists, Uroolley (410) demonstrated t h a t 1,2-dichloro-4,5-diaminobenzene inhibited the synthesis of riboflavin and vitamin Blz in bacterial cultures. Vitamin BI2and Animal Protein Factor. Research on vitamin B,, and the animal protein factor, now referred to commercially as vitamin B12feed supplement, has been extremely active during the period under review, and it is impossible to discuss here all the published material. From a n economic viewpoint, vitamin BIZ for human use, and especially as a n animal feed supplement, has hccome a t,hriving business and several firms are engaged in its fermentative production. Several review articles on the preparation, chemistry, and physical properties, uses, etc., of vitamin Blz have been publishcd (12, l S 3 , SS3, $79). The actinomycetes :ire the most prolific producers of the vit'aniin BIZgroup. Hall et al. (166) screened approximately 5000 molds, yeasts, aciinomycetes, and bacteria for production of vitamin Blzand found t h a t several of the actinomycetes and bactt~ria,but none of the yeasts and molds, produced B12-like subYtances. In another paper Ha11 et al. (156) described a survey of several
.
1953
hundred isolates of actinomyeeks, principally species of Streptomyces, which were found t o produce vitamin Bl2 activity. I n a study of neomycin production wit,h Streptomyces jradiae, Kelson et al. (260) reported t h a t little vitamin B I Z activity was produced in the ordinary neomycin media, but t h a t the addition of a small amount of cobalt chloride improved the yield. Maximal yields of neomycin and B,, did not occu'r under the Fame ferment;).tion conditions. Increased BIZproduction due to the addition of cobalt t o the basal medium was first reported by Hendlin and Ruger (169) in experiments with Streptomyces yriseus. The same organism was used by Chaiet et al. (55) for the biosynthesis of radioactive vitamin B12 containing cobalt 60. Petty (286) received a patent relating to a chick growth factor produced in aerobic fermentation by Flai'obackrium soiare. The medium contained glucose, yt:ast extract, peptone, malt cxtract, urca, and inorganic salts. This factor was believed to be similar to vitamin BIZ. Halbrook et al. (153) tested the vitamin Blz product,ion by microorganisms which were isolated from poultry house litter. A strain of Aerobacter aerogenes gave more vitamin B1. than either Bacillus megatherium or severai species of Streptomyces. I n another paper Halbrook et al. (154) described how poultry house litter can be used as a growth-promoting supplement for chicks being fed a n all-vegetable vitamin Bl?-deficient diet. An anaerobic process for the production of vitamin B12-rich concentrates was described by Hodgc et al. ( 17 9 ) . Smiley et al. ($32)reported that in their process for the production of riboflavin with Ashbya gossypii, vitamin B12 or vitamin Blz-like substances were also produced. Brink and Folkers (44, 45) isolated 5 , 6 - d i m e t h y l ~ ~ i m i d a z o l e from a n acid hydrolyzate of vitamin BIZ. This compound was further degraded t o 1,2-diamino4,5-dimethylbensene, a moiety of riboflavin. Both of these compounds showed some Rlz activity when fed to rats on a diet lacking B12 (112). 1,2-Dichloro-4,5diaminobenzene was found t o act as a metabolic antagonist by inhibiting t,he synthesis of vitamin Bin and of riboflavin in bacterial cultures (410). There has been considerable discussion in the literature concerning the naturc of B12,and Bl.'b. BI,, was obtained by catalytic hydrogenation of vitamin B,, (133). Vitamin &2b, originally isolated from Streptomyces aureofaciens Iiroth, had an absorpt,ion spectrum differing from BIZ but had similar biological activity (221). It has recently been demonstrated t h a t B12, and B1zb are identical and are hydroxocobalamins (197, 198) while BIZis cyanocobalamin ( 4 6 ) . Further work has been done on the microbiological assay method for vitamin B12using Lactobacillus leichmanii (82, 170, 313). There has been much discussion and speculation regarding the relationship of the vitamin BIZ group to animal protein factor (305, 347). This speculation v a s supported in part by the greater growth stimulation from some animal protein factor suppleixenta (305). The superior qualities of these products were found to be due t o residual traces of aureomycin in the crude supplc,nient pippared from Streptomyces aureofaciens broths (349,397). Thc growth effect noted with aureomycin led 'to the testing of many other antibiotics and it has been observed that, penicillin, bacitracin, streptomycin, and tcrraniycin cause growth responses similar to aureomycin in nonruminant animals. This subject will be considered in a separate section of the review. At the present time, it appears t h a t t'he vitamin B,z group is largely responsible for the growt,h effect ascribed t o animal protein factor (414). Miscellaneous Vitamins. THECITROVORUM FACTOR.I n 1948, Sauberlieh and Baumann ($81 ) found t h a t Leuconostoc citrovorum failed t o grow on a synthetic medium satisfactory for Leuconostoc ?uesenteroides. The nutritional defect could be remedied by ad-
1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
dition to the medium of liver extract, yeast extract, etc. Further work on this factor (17,196,320,322,403) established t h a t i t was not identical with the Lactobacillus bulgaricus factor (LBF), vitamin B,z, folic acid or thymidine. More recently it was found thttt the factor is a derivative of folinic acid. Urockman and coworkerp (47) have reported the synthesis and isolation of a crystalline substance with the biological properties of the citrovorum factor. This compound was obtained by catalytic hydrogenation of pteroyl glutamic acid or its N-formyl derivativc. The new compound also competitively reverses the tosicity of 4-aminopteroylglutamic acid for the mouse. Snell (336) reviewed the recent developments in the nutrition of microorganisms with greatest emphasis on growth factors and vit nmins. LACTOBACILLUS BULGARICUS FACTOR (LBF). Williams, HoffJorgenson, and Snell(40l) described in 1949 the conditions for the cstraction and purification and also some of the properties of a. growth factor essential for a strain of Lactobacillus bulgaricus. T h e richest source of the factor was yeast extract although tho substance is widely distributed in natural materials. Rasmussen et al. (300) found t h a t this factor was synthesizctl by numerous species of yeasts, bacteria, and fungi. A preliminary vspcriment indicated t h a t it was required for the growth of chicks (300). In another paper Smiley et al. (338)demonstrated t.hat the factor was formed, in unusually large amounts, simultanc,ously with the formation of riboflavin by Ashbya gossypii. They also found that the factor was related to pantothenic acid :ind that its production was increased when pantothenic acid or i ~ r:iin t precursors of pantothenic acid were supplied in the metliunt (332). DEXTRAN
Ikxtran is a fermentation product which, because of its possit)l(x use as a plasma extender, has gained both national and inter-
rintional prominence within the last few years, and especially during the ycar covered by this report. I t is a polysaccharide which ro.;ults from thc fcrmentation of sucrose by Leuconostoc meaentwoides and other microorganisms. The dextran fermentation has been a scourge t o the sugar industry for many centuries. It i*Iogsfilters, retards crystallization of the sugar and is, in general, :I Iiiiiclrance in the refining process. I t was first studied in the early part of th9 nineteenth century by 1;rench sugar chemists, who a t first thought it t o be a constitw i l t of sugar beets. It was not until the latter part of the ninetivnth century t h a t dextran was proved to be of bacterial origin. Until recent years the chief interest in dextran was limited t o the ~ w a n of s avoiding its formation in the sugar refining process. The early history of dextran was reviewed by Evans and Ililhert (119) and by Tarr and Hibbert (368). I n 1931, Hucker arid Peterson (181) published a classical study on the physiology arid classification of the chief organisms producing dextran-Le., buctcbria of the genus Leuconostoc. During the same year, Tarr : i n d Ilibbert (862) published the first detailed study of the optininl conditions for the preparation of dextran. Recent interest in dextran, as a blood plasma extender, dates from 1944 when two Swedish investigators, Gronwall and Ingelman, demonstrated t h a t if the molecular size of dextran was somewhat decreased by partial acid hydrolysis, the resulting material could be used a s a substitute for blood plasma (148,1-@). Sative dextran was obtained by fermenting sucrose media with a wlected strain of Leuconostoc mesateroides (119, 164, 19S, 223, 273, 339, 362). The media usually contained sucrose, inorganic s d t s , and yeast extract or a source of growth factors and organic nitrogen. During the fermentation, sucrose was transformed largely into dextran and fructose. The most important recent study of the production of dextran was that of Jcanes et al. (193). Their medium contained 10%
Vol. 43, No. 9
sucrose, together with yeast extract, dipotassium hydrogen phosphate, magnesium and ammonium sulfates, and sodium chloride. Increasing the age of the inoculum affected the type of dextran produced and the final viscosities attained wem lower. Higher initial p H values in the medium resulted in greater viscosities. Aeration of the medium or buffering with calcium carbonate, either singly or in combination, gave no increase in the dextran yield. The type of dextran formed was greatly influenced by the cultural conditions. However, dextran of high or low viscosity could be obtained by suitable control of the fermentation conditions. Hehre (166) obtained dextran and fructose by the use of a n enzyme complex isolated from a strain of Leuconostoc mesenteroides. It is of interest t h a t glucose-phosphate, which is necepsary for the formation of glycogen and starch, was not a n intermediary in the formation of dextran (281). Dextran was scparatcd by precipitation with methanol or ethyl alcohol or acetone (164,193, 873,339). Before being used as a plasma extender, the native dextran must be degraded from a molecular weight of several million to about 40,000 t o 300,000. This has been done by partial acid hydrolysis (2, 160, 187). Other methods which have been mentioned for this partial degradation include alkaline hydrolysis (883), hydrolysis by bacterial enzymes (182, 883), and ultrasonic vibration (68, 228). From a n industrial point of view, the main problems in the production of dextran for use as a plasma extender are the hydrolysis, the purification of the partially hydrolyzed dextran, and the sterile packaging. A simplified flow sheet of a dextran production process has been published (68). The most important producer of this plasma extender in this country is Commercial Solvents Carp.; however, Refined Syrups and Sugars, Inc., and others are also entering the field (68). Abroad, dextran for use as a plasma extender is produced in Sweden where the product originated (68, 187), England (68, 231 ), and the Union of South Africa (69). Dextran is administered a t a concentration of 6% in isotonic salt solution. The pharmacological and clinical results have been excellent (68, 148, 14.9, 187, 231). Dextran containing CI4 is being used t o determine what happens to this material in the human body. Dextran’s chief competitor as a plasma extender is polyvinylpyrrolidone (PVP), which is obtained synthetically from acetylene, formaldehyde, and ammonia by a process developed in Germany (68). Other actual and potent,ial uses for dextran are: t o prevent water loss in oil-well drilling muds (271, 272); as a stabilizer for ice cream (119); in viscosity and moisture control (68); as a stabilizer in other foods and beverages (269); as a substitute or as a n extender for vegetable gums (270); and in the form of ethers and esters for use as lacquer bases (226-227). M I C R O O R G A N I S M S F O R FOOD AND FEED
Interest throughout the world in microorganisms for food and feed has continued during the past year. Bergner ( 3 4 ) has reviewed the synthesis of foods by microorganisms. Yeasts. During the past year a report appeared of the symposium held in late 1948 entitled “Yeasts in Feeding” (296). This is a n excellent review relating t o the composition and production of yeast and its value in foods and feeds. Bunker (62) has reviewed the production of protein food by yeast. Brahmer (42) has developed a formula for the maximal growth of yeast from glucose with a consideration of the requirements for nitrogen and inorganic substances. According to Benesch (30) the maximal yield of yeast from molasses depends on the number of cells which bud. A large concentration of unfermentable organic substance also favors multiplication.
September 1951
INDUSTRIAL AND ENGINEERING CHEMISTRY
Atkin (i4)reviewed the literature on the yeast growth factors. Atkin et al. (16) conducted a n experimental investigation on the growth and fermentation factors for different brewery yeasts. Emphasis was placed on growth under one set of conditions, followed by alcoholic fermentation under another set of conditions. From the results, it was possible to evaluate, in a relatively short time, the vitality and charactelistics of “pitching” yeast. The yeasts were grouped into five categories according to their vitamin requirements. The required factors for lager, ale, and baker’s yeast were tabulated (16). Scalf and Stier (323) found t h a t the production of anaerobic crops of distillery yeast could be increased almost 100% by the addition of commercial malt sprouts to a basal medium n Ilich &is apparently complete with respect t o water-soluble growth factorb. The active agent responsible for this increase was not contained in a n aqueous extract of the malt sprouts, but was extracted by alcohol and ether. All of the active lipide substance, or substances, was shown t o be present in the unsaponifiable fraction. White has continued his series of papers reviewing the principles and practices of yeast production. His most recent papers dealt with modern practice in the production of baker’s yeast (994); the preparation of molasses wort for fermentation (595); and the harvesting of the yeast crop (396). Hatch and coworkers (162, 163) have patented two vessels for growing aerobic organisms, particularly baker’s and brcn-er‘a yeasts. Several papers have been published on the use of inexpensive ram materials for the production of yeasts. A number of reviews on fat synthesis, especially by Rhodotorula gracilis, have been published (36, 173, 228-230). Bernhauer ( 3 5 )concluded t h a t although a great deal of work has been done on submerged culture processes for fat production, a t the present time, fat synthesis by yeast is not economical. Lundin (228250) revierred the last 10 years of European work. An excellent review of the industrial biosynthesis of fats by yeasts and other organisms was given by Hesse (17S),who concluded t h a t present processes are uneconomical and should be used only in a n emergency. Pan et al. (279) investigated the effect of the composition of the nutrient medium on the fat synthesis by Rhodotomla gracilis. Nielseen and Nilsson (264), also working with Rhodotorula gracilis, dernonstrated t h a t this yeast was able t o utilize the saccharides prcsent in wood hydrolyzates. The organism after adaptation was also capable of utilizing xylose. The production of food yeast during the occupation of Belgium \vas reviewed by van Laer ($13). Harris et ai. ( 1 6 1 ) have investigated protein production by atrains of six species of yeast in a Waldhof-type propagator. S o n e of the samples produced was the equivalent of casein as n source of protein. Thompson et ai.( S 8 ) developed a number of equations which were claimed t o provide a rational basis (1) for analyzing the propagation process of Torulopsis utilis in a constant-volume, continuous-flow propagator; ( 2j for evaluating controlled characteristics of the system; and (3) for research on environmental factors affecting yeast growth. One of the decisive factors which would help t o make the production of yeasts for food and feeds economical is the use of inespensive waste-type raw materials.- Inexpensive materials which have been studied include sulfite liquor (398, 399), wood processing by-products (160, 211, 264), straw (38, d l l ) , dairy wastes (94, 692), tvaste pear juice (367), and miscellaneous plant materials ( 7 ) .
Other Microorganisms. Hesse ( 173) presented a n excellent review of the literature on the industrial production of fat with such microorganisms as O O S ~ O Tlactis, U PeniciElium, Aspergillus, Mucor, and Fusarium. The main emphasis was on submerged culture processes.
1955
Kaibara (200-202) in Japan has investigated the effect of the cultural conditions such as t8he pfI, nitrogen nutrition, carbon sources, and other factors on fat, production by Penicillium gavanzcunt. Grosser and Bernhauer (151) st,udied the enrichment of cellulose with protein using penicillia in submerged culture. An especially intense decomposition of the cellulose medium occurred in the presence of technical zinc, which was believed to have a stimulating effect on c-ellulose. Fink (186) described esperiments on the formation of fodder protein with Aspergiilus OT@XZe grown on potato slices. I t was claimed that a very Jigestible material of high nutritional value was produced and t h a t the method described would make it possible for the individual farmer to produce his own fodder protein. Oospora lactis is another microorganism suitable for .feed production. Eberlein ( 110) studied fat production in submerged fermentation with this organism, while Drews and Specht (101) itudictl protein formation by this organism under similar conditio~~~;. Diemair and Boresch (96) used Mucor as a fat producer. T h y found t h a t technical glucose was a better source of carbon than pure glucose. The fat production was independent of the nitrogen source and a high-fat mycelium was obtained with the use of low-nitrogen media. The data collected in the study were used for the design of a semicornniercial process. >LIyceliuin yields of 8 to 12 grams and fat of 3 t o 5 grams per liter of medium were obtained. The production and economic importance of soy sauce pi’epared with Aspergillus oryeae and other organisms has been reviewed ( 186). Szuecs (369) was granted a patent on the preparation of a11 essence of mushrooms which comprised distilling the essence from a mushroom mycelium which had been grown on a n aerated substrate of seed-oil residues inoculated with small mycelial particles. Algae such as Chlorella continued to receive attention as sourres of food and feed (70,159,206). Harder and JJ-itsch (15’9) have reported on experiments on fzt production by a diatom. The physical and chemicai characteristics of various algae grown in mass culture have been described by Ketchum and Redfield (205). ANTIBIOTICS
The number of new antibiotics described in the literature is increasing rapidly each year. Baron ( 1 9 ) in his handbook listcd over 130 antibiotics. Florey et a,l. (1S1) have assembled in two large volumes the essence of what was known by 1949 on antibiotics. Now, less than 2 years after its publication, this sumin:ii~y in many respects is out of date. Besides treatisesexclusivelydevoted to the discussionof the :Intibiotics, a large number of more general review articles have bccn published during the period covered by this review. A fen. of these which are concerned, a t least in part, with fermentation :ire enumerated here (SS, 114-1 27, 172, 247, 288, 301, S1 4 , 330, 385, S86). Robinson (311, 312) and Vonderbank (386, 386) havc presented progress reports on antibiotics and Erdman (114) hiis prepared a bibliography of the German literature on antibiotjc,q. Of major interest are the reviews by Herrel (172) and Kiser :md Woodruff (208). During the early months of 1950, competition in antibiotic production, particularly penicillin, remained very keen. However, the JT a r emergency created greater demand for antibiotics and the price of penicillin has risen slightly and remained firm. Skccn (33oj has prescnted a. survey of commercial antibiotic procluct,ion. The volume of antibiotics produced continued to increase rapidly as evidenced by the comparative production figures for penicillin and streptomycin in the last 3 years. Production figures for other antibiotics are not available due to the limited number of producers (Table I). It has recently been estimated that antibiotics account for more than 50% of the dollar sales of all ethical drugs sold in this country. Further details on the economics of the conimc~rcial antibiotics havc been rcported (307).
INDUSTRIAL AND ENGINEERING CHEMISTRY
1956 Table
1.
Production of Penicillin and Streptomycin (392) 1948
Penicillin Billion unitsa Grams Pounds Streptomycin Billion unita Gramsa Pounds
95 855
67,513:OOO
126,700
37 700
37,709:160 83,000
1949
1950
133,464 80,078 000 176: 400
133,382,000
83,700
83,699 137 184: 200
222,305
293,822
92,447
92,446,342 203,800
a Figures supplied by U. 5. Department of Commerce: conversion figures were derived by assuming 0.6 microgram per unit for penicillin and 1.0 miorogram per unit for streptomycin.
Recent developments indicate t h a t only a portion of the potential antibiotic market has been tapped. Foreign demands are being met by new and enlarged antibiotic production facilities in South America, Great Britain, France, Germany, Denmark, Belgium, Italy, Sweden, India, South Africa, and Japan. T h e use of antibiotics i n feed supplements for poultry and swine has also provided impetus for greatly expanded antibiotic production facilities. Penicillin. The total production of penicillin in this country in 1950 was approximately 65% greater than in 1949. This increase, which was prompted largely by the war emergency and a more favorable price picture, resulted from improved fermentation yields, greater efficiency of extraction and purification processes, and enlarged plant facilities. Several reviews and publications concerned with penicillin technology have appeared during the past year (19, 131, 311, 312). Perlman (282) reviewed the mycological aspects of penicillin production, and discussed selection of cultures, formulation of media, addition of precursors, aeration, agitation, and other fermentation conditions. He also discussed the chemical changes which occur during the growth of the fungus and the production of penicillin, and the available information concerning the mechanism of penicillin formation. The technology of penicillin production was reviewed by Ganapathi (158)and Terjesen (364). A flow sheet has been published on penicillin production giving the materials of construction used in the equipment (64). New strains from mutation programs have continued t o increase penicillin yields. Otani and Okads (268) desoribed the variation of Penicallium chrysogenum Q-176 induced by radium irradiation. They obtained a pigmentless fitrain which did not give high yields of penicillin but in yhich, due t o the lack of pigment production, the penicillin was more easily purified t o a white product. Woodru? and Larsen (409) obtained a patent in which it was claimed t h a t , by successive irradiation and selection of high penicillin-producing isolates of Penicillium chrysogenum Q-176, a mutant was obtained which not only was incapable of secreting the undesirable yellow pigment, but also produced enhanced penicillin yields. They claimed 1700 units per ml. in large-scale production. Penicillin G accounted for more than 80% of the total penicillin formed. It was claimed t h a t the use of this nonpigment-secreting mutant made it possible t o increase the efficiency of the purification process. Anderson (6) gave a description of pilot plant experiments, in 30-liter fermentors, with a pigmentle-ss Q-176 mutant which had been obtained by ultraviolet or nitrogen-mustard treatment. Yields of over 2000 units per ml. were obtained in some experiments. Duckworth and Harris (104) gave a description of the morphology of the Penicillium chrysogenum Q-176 in submerged fermentation. The authors reported several observations of morphology in penicillin-producing cultures and intend, with further work, t o correlate microscopic appearance with biochemical behavior. Negroni and Fischer (267) linked penicillin production with two genetic factors called M and C. Both factors
Vol. 43, No. 9
were stated t o be necessary for high production of penicillin. A pure strain of type M did not produce penicillin and pure type C was an irregular producer. Interesting experiments on a plant scale were reported by Brownlee (61)in which he evaluated six process variables. These studies were prompted, at least i n part, by the discrepancies which had occurred in attempts t o translate pilot plant results into full-scale production. Using factorially designed experiments and statistical analyses of the data, Brownlee (61) has developed, by mathematical means, a n experimental plan which permits a reduction in the number of plant batches needed for proper evaluation of fermentation variables. Peterson (285) has reviewed his earlier experience with the factors affecting the kinds and quantities of penicillin produced by strains of Peniczllium chrysogenum. A British patent (242) described penicillin production under submerged conditions in a medium which contained cottonseed meal as the chief ingredient. Kato (203)summarized a series of experiments on the production of penicillin in synthetic media in which he studied factors such as pII, sterilization, inoculum development, and age and quantity of inoculum. Wolf (408)grew Penicillium chrysogenum Q-176 in a basal synthetic medium t o which he added a single amino acid as the sole nitrogen source. Small amounts of penicillin were produced with glycine, alanine, &alanine, serine, and glutamic acid. Penicillin was not produced when leucine, valine, and tryptophan were present in the basal medium. Weigner and Weigner (391) confirmed the results of other investigators t h a t a n appreciable iron content in the medium decreased penicillin yields. I n a n investigation of the carbon sources suitable for penicillin production, Bautz et al. (25) conducted experiments in which glucose was added a t varying rates. T h e penicillin yields obtained were lower t h a n those with lactose regardless of the rate of glucose addition. The authors concluded t h a t the superiority of lactose i n penicillin fermentation was due t o causes other than its slow fermentability. Bautz et al. (26)studied the effect of p H on the growth rate of Penicilliumchrysogenum Q-176. They found t h a t a t p H 4.5 t o 4.7 growth was the most rapid and the highest total weight of mycelium was obtained. After completion of the growth, the p H was maintained at 7.0 for penicillin production. Brown and Peterson (48), controlle,d the p H of penicillin fermentations using both manual and automatic techniques ,Steady utilization of lactose and penicillin production was associated with w p H range 6.8 t o 7.4. Yields of 1800 units per ml. were obtained through the use of p H control, incremental addition of precursor, and proper agitation-aeration conditions. Ninety-seven per cent of the total penicillin was G. When using submerged processes for penicillin production, proper aeration and agitation are of great importance. Brown and Peterson (48, 49) confirmed earlier reports t h a t aeration is a function of both the rate of aeration and degree of agitation, and t h a t proper control of these factors is vital t o highest penicillin yields. Bartholomew et al. (22, 24) reported the results of a thorough investigation on the effect of aeration and agitation o n various characteristics of antibiotic fermentations. Specially designed 5-liter laboratory fermentors were used (21 ), and the prime variables studied were mechanical agitation, rate of airflow, and sparger design. Dependent variables were residual sugar, antibiotic activity, mycelial weight, pH, dissolved oxygen, and oxygen diffusion rates. The experimental results were presented a s correlation of the effect of the horsepower input and air flow upon antibiotic production.
An apparatus has been described (2326) for the production of penicillin by submerged fermentation which ermitted the recycling of air fortified with oxygen and freeaof carbon dioxide. Bergel et al. (5%')described an apparatus, for submerged penicillin production, constructed so t h a t t h e organisms may be grown with the use of relatively small volumes of air which was
September 1951
INDUSTRIAL AND ENGINEERING CHEMISTRY
more readily sterilized. The apparatus contained a high speed stirrer which provided agitation and at the same time drew air from the space above the liquid and distributed it in fine particles throughout the medium. The excess air escaped continuously through a passage around the agitator shaft at its point of entry into the vessel. Haller (167) described an apparatus, for the.semicontinuous production of penicillin by surface fermentation, which was claimed t o be effective in reducing the fermentation period. The main features of the invention were a tank having a valved outlet in the side to permit decantation of the finished solution from the area immediately beneath t h e surface mold pad. Liebmann and de Becze (2%2)patented a continuous process for the roduction of penicillin and other antibiotics. The fermented [quid was continuously withdrawn and fresh nutrient added to the propagation tank at t h e stage when maximal propagation had occurred and the quantity of the by-pro9ucts had not yet accumulated to a toxic level. The fermentation was completed a t a slow rate in a second tank under conditions of limited aeration. This was claimed to be a more economical production method and had other advantages such as less danger of contamination, less undesirable mutations, and need for a lesser quantity of inoculum. Gilbert (243)obtained a new chlorine-containing penicillin by adding o- or p-chlorophenylacetan~idet o the medium. Thorn and Johnson (369)obtained aliphatic penicillins when appropriate acids were added t o the medium. Caproic and a,y-hexanoic acids were identified as precursors, respectively, for dihydro-F penicillin and penicillin F. The study suggested t h a t a configuration capable of blocking the beta oxidation of the acid is important in precursor compounds. Goldschmidt, and Koffler ( 1 4 4 ) investigated the ability of sur. face active ag+nts t o increase penicillin yields. They found t h a t the stimulatory effect of commercial defoaming agents could be reproduced by the unsaturated fatty acids contained therein. The authors were of the opinion that the action of oleic acid, which increaEd t,he yield of both mycelium and penicillin, could not be completely explained by its foam-cutting properties nor by its role as precursor for penicillins with long aliphatic side chains. No real explanation of this phenomenon has been presented t o date. Colingsworth (84) received a patent on the use of longchain fatty acid esters in penicillin production in which he stipulated that thew “growth stimulants” have to be added t o the nutrient medium before the end of the first 24 hours of the fermentation. He likewise failed to offer any satisfactory explanation of the mechanism leading t o the increased yield. Sakaguchi and Murao (318) reported preliminary work on a new enzyme, called penicillin-amidase which is said t o catalyze the decomposition of penicillin G into phenylacetic acid and a residual amine called penicin. The enzyme was found in the mycelium of Penicillium chrysogenum Q-176 and in the mycelium of Aspergillus wyzae. The authors attributed t o this enzyme the drop in penicillin activity which often occurs in fermenting tanks after the penicillin has been formed. Berhens and Garrison (28) discovered a series of compounds which exhibited a marked antipenicillinase activity. Among these compounds were 2-benzylimidazole, 2-methylimidazole, benzylpenicillic acid, histamine, and others. Streptomycin. The total streptomycin production in the United States in 1950 was approximately 92,000,000 grams. This represents an increase of only about 10% over 1949. This small increase may be a result, at least in part, of the increased production of this antibiotic in foreign countries. Streptomycin plants have been or are being built in practically all of the countrieR where penicillin plants are located. The literature on streptomycin has been reviewed by Florey et al. (131)and by =vera1 other authors (2,43, 272, 322,318). An interesting development during 1950 was the isolation of a new streptomycin-type antibiotic, called hydroxy-streptomycin, differing from streptomycin only in having a n additional oxygen atom in the molecule (2.9,156, 346). Hydroxy-streptomycin was produced by Streptomyces griseocarneus. I t s formation and
1957
structure were announced independently by the Northern Regional Research Laboratory (29, 346) and Abbott Laborat,ories (15%). A recent patent granted to Dulaney (106) further illustratcs the profitable results which may be obtained from mutation studies. Dulaney developed a strain of Streptomyces griseus which produced ztreptomycin yields of a t least 800 micrograms per ml. hlcDanie1 and Hodges (237) have developed strains of Streptomyces yriseus which are resistant t o streptomycin concentrations as high as 500 micrograms per ml. Several patents were granted during 1950 on nutrient media for the production of streptomycin with Streptomuces grisew (18, 83, 86, 99, 335-337, 372). Baron ( 1 8 ) received a patent for a synthetic medium cont,aining technical glucose, sodium citrate! and inorganic salts. Colingsworth (83) replaced the peptone and beef extract with “fermentat.ion solubles,” a by-product of the molasses-thy1 alcohol fermentation. Donovick et al. (99) proposed to replace the peptone and meat extract in the original Waksiiian medium with whole vegetable, meals, and infusions. McDaniel ($34) claimed that a medium containing yeast, sucrose, andinorganicsalts resulted in more than50% increase over previous results. Another medium patented by McDaniel (236)contained soybean meal, distiller’s solubles, glucose, and sodium chloride. McDaniel and Hendlin (236)claimed that a medium containing distiller’s solubles, glucose, and inorganic salts was acceptable for streptomycin production. Trussell (376) claimed t h a t a calture medium which contained both cornsteep liquor and soybean flour produced higher yields than media containing either of these ingredients alone. Perlman and Langlykke (284) have shown that the stmptomycin-producing organisms will utilize a number of vegetable and animal oils as energy sources when these are present in the media. When these oils completely replace the carbohydrate component of a soybean meal medium, the antibiotic yield was not affected. However, when only part of the carbohydrate is replaced, significant decreases in yield occurred. A study of the metabolic changes associated with high yields in a streptomycin fermentation has been made by Garner et al. (139). These investigators demonstrated that, in a high yielding fermentation, the respiratory activity was lower in t h initial stage and cell dry weight was maintained a t a higher level than in runs giving low yields. Fermentations giving highest yields also showed very active production of volatile nitrogen compounds on the first day followed by a decrease in the courve of the nest 10 days when the greatest portion of the streptomycin waa formed. Phosphate was shown to reduce yields while added calcium increased them. During a n investigation of the role and the measurement of aeration in biological culture media, Wise (405, 406) found that the maximal yield reached in streptomycin fermentation depcnded on the rate of solution of oxygen. No specific effect of vcssel size or agitation was noted. The yield increased cr,til the rate of solution of the oxygen was equal t o the maximal oxygen denisnd of the culture, a t which time the yield became independent of the two factors. Wise expressed the aeration of culturc media i n terms of +L,which is dependent on the rate of agitation, air flow, etc. Bartholomew et al. (22, 24) reported that for t.heir strain of Streptomyces griseus oxygen uptake was independent of oxygen concentration in the broth except a t very low concentrations. Streptomycin is extremely resistant t o destruction by microorganisms; however, it has been demonstrated recently by Pramer and Starkey (2995) that streptomycin can be decomposed by bacteria which, although incompletely identified, appeared to belong t o the genua Pseudomonas. Koerber et al. (209) studied the multiplication of phages a f fecting Streptomyces griseus and, having distinguished two tyEn.8 of phages, they found strains of the mold which were resistuiit t o one or both types.
1958
INDUSTRIAL AND ENGINEERING CHEMISTRY
Chloromycetin. Chloromycetin (chloramphenicol) continued to gain the favor of the medical profession during the past year, and it is believed, although the production figures are not available, that it is now being produced a t the rate of several hundred kilograms per month (43, 131, 172). This antibiotic is produced chiefly in this country and here only by Parke, Davis & Co., which produces both fermentation and synthetic Chloromycetin, and by the Monsanto Chemical Co., which uses only the synthetic process (330). It is believed that only 2001,or less was produced by fermentation in 1950 (830). Various aspects of chloramphenicol chemistry, production, and uses have been reviewed (49,131,172,247). Oyaas, Ehrlich, and Smith (278) studied the metabolic changes in Chloromycetin fermentation by Streptomyces venezuelae. The formation of this antibiotic was most rapid during the period of the organism's maximal growth rate, indicating that the antibiotic was a product of relatively young cells. The same period was characterized by a rapid decrease in assayable glycerol and ammonium nitrogen. Autolysis of the mycelium during the last 2 days of incubation accounted for much of the observed increase in ammonium nitrogen and the rise in pH. Crooks et al. have received a patent on a process for the chemical synthesis of chloramphenicol ( 8 3 ) . Aureomycin. Aureomycin, an antibiotic produced in this country only by the Lederle Laboratories Division of the American Cyanamid Co., has increased rapidly in production volume and sales during the past year (67, 330). It is the leading broad spectrum antibiotic, followed in order by Chloromycetin and terramycin. Plans are being made to produce it in Great Britain (68). The production of aureomycin has every prospect of further increases since, in addition to being a leading therapeutic agent, it also has proved to be an excellent growth stimulant when used in the rations of poultry and swine (196, 233, 348). A series of pertinent review articles have appeared during the past year which are concerned with the nature, production, and utilization of this antibiotic (43, 67, 30, 131, 142, 171, 172, 83.4, 847). Petty and Matrishin (887)found that Streptomyces aureofaciens effectively utilized chlorine to produce aureomycin in fermentation media containing minimal quantities of this element. This efficient utilization was found to hold for several types of media and a number of forms of chlorine were found to be used. Aureomycin production waR proportional to the amount of chlorine present. Bacitracin. Bacitracin, a peptide antibiotic produced by Bacillus subtilis Tracy is steadily growing in favor with the medical profession. I t is particularly valuable for topical application, especially in patients allergic to penicillin. Some lots of the drug, having lower toxicity, have been used parenterally. Bacitracin is produced principally by Commercial Solvents Corp., which recently completed a new bacitracin production plant ( 1 88). Various aspects of this antibiotic are covered in several review articles (8, 30,131, 172,241, 386). During 1950 investigators a t Oxford University (8, 868, 864, 887) showed that the principal components of the antibiotic ayfivin,produced by Bacillus licheniformis, were identical with those components in commercial bacitracin. Johnson and Meleney (134) obtained a patent for the production, recovery, and isolation of bacitracin. They used several complex media such as tryptone broth and meat infusion broth, and also synthetic media containing glucose, glutamic acid, and inorganic salts. The active material was extracted from t h e medium with butyl alcohol. Commercial production employs a submerged fermentation process based on investigations which have been published (217 , 218). Hendlin (168) investigated the nutritional requirements of a bacitracin-producing strain of Bacillus subtilis. A rate of aeration
Vol. 43, No. 9
and a temperature higher than reported previously improved the rate of growth and the rate of bacitracin production but not the total yield. Addition of a number of growth factors such :1s pantothenic acid and riboflavin decreased the lag phase but did not affect the total cell or antibiotic yield. When the initial pH was increased from 4.5 to 8.3, bacitracin productio? increased but not cell growth. The requirements of the organism for carbon sources, nitrogen sources, and trace materials wcre also detcrmined. Hills et al. (176), who made a comparison between the various media for the growth and antibiotic production by Bactllus licheniformis, demonstrated that, depending on the composition of the medium, this organism produced either ayfivin (bacitracin) or another antibiotic which resembled licheniformin. The identity of ayfivin and bacitracin was determined ~ J \ F countercurrent distribution methods which resolved the crude products into seven components. Three of these had antibiotic activity and were called bacitracin A, B, and C (262, 268). Sharp and coworkers (327) have determined the amino acids obtained from an acid hydrolysis of this antibiotic. Earlier pharmacological and clinical results with bacitracin have been reviewed in previous papers of this series (217, 618), also by Cutting (30) and by Meleney and Johnson (841). More recently Graupner and coworkers (147) were able t o show that bacitracin appears t o be effective against intestinal amebiasis and that, in combination with streptomycin and glucuronolactone, it may be effective against nonspecif-: ulcerative colitis. Thomas and Prier (866) obtained promising results in the treatment of hemorrhagic septicemia of cattle. Bacitracin shows promise of being one of the leading antibiotics for feed supplements (230)and is being produced and sold in large quantities for this purpose (66). Terramycin. The discovery of terramycin, by a group from Chas. Pfizer and Co. ( l a @ , and its rapid development into large scale production is an excellent example of close teamwork in an industrial laboratory. The culture was isolated in the summer of 1949, and the antibiotic was on sale in March 1950 (66, 68, 60). Over 100,OOO cultures were screened prior to the discovery of Streptomyces rimosus, the microorganism which produces terramycin. Weyer (333) reviewed the development, chemistry, pharniacological and clinical evaluation, administration, and dosage of this new antibiotic. These aspects were also the subject of a symposium (8.4249). Sobin, Finlay, and Kane (337) were granted a patent on the production, recovery, and isolation of terramycin by Streptomyces rimosus This antibiotic was produced in an aerated and agitated submerged fermentation process. The medium contained organic nitrogen sources such as soybean meal, wheat gluten, cottonseed meal, or casein digest; carbohydrates such as corn sugar or corn starch; and other ingredients such as calcium rarbonate, sodium nitrate, and Rodium chloride. The p H of the medium was adjusted t o 7.0 and calcium carbonate was added prior to sterilization. The incubation period was 4 days on a laboratory shaker a t 28' C. The antibiotic was isolated by extraction with butyl alcohol or by adsorption on charcoal. This antibiotic has bcen prepared in crystalline form. Preliminary results have becn reported on the chemical and physical properties of terramycin (1.@). The antibiotic and its salts have characteristic spectra. Terramycin is an amphoteric substance having an empirical formula estimated to he Ct2HL2--24NZOg.2HZ0. It contains a phenyl and a carbohydixte group and its molecular weight is about 440. Terramycin is an effective a F n t against pneumococcus pnrumonia, staphylococcus infections, streptococcus infectioiis brucellosis, rickettsial diseases such as scrub typhus and Rochy Mountain s ~ o t t e dfever, vencrcal diseases such as gonorrhea, intestinal amebiasis, bacillary dysentery, and otherp. When usrd for diseascs such as cpidcmic influenza virus infections, prieu-
September 1951
INDUSTRIAL AND ENGINEERING CHEMISTRY
mococcus meningitis, Friedlander’s pneumonia, actinomycosis, peritonitis, pertussis, and syphilis, the results of treatment are encouraging, but not positive. Terramycin ie not effective against infections caused by Bacillus proteus, Pseudomona.s aeruginosa, and infections due t o the typhoid bacterium and to Salmonella (249). The use of terramycin ae a feed supplement for animals IS described in another section of this review Neomycin. Interest in neomycin has decreased considerably during the past year, particularly with regard t o its possible use in the treatment of tuberculosis. This is due chiefly to its toxicity. However, because of the potent antibacterial action of neomycin, especially against gram-negative bacteria, it may be usable for certain types of therapy (122). Several phases of the literature on neomycin have been reviewed (43, 131, 218). Swart et al. (365, 356) demonstrated by countercurrent distribution that neomycins A and B are not single substances. Differences in potency and in clinical results with neomycin preparations may be due to differing proportions of these substances. Dutcher and coworkers (109) described a neomycin fraction which they called C, which was rather different from previously described neomycins A and B. Of economic importance from the point of view of neomycin production was the fact t h a t vitamin BIZwas produced concurrently with neomycin in the fermentation broth (189, 260); however, the conditions for maximal yields of the two products were not the same (2660). Polymyxins. Little has been published during the past year o n the polymyxins, a group of antibiotics produced by Bacillus polymyza. The previous literature has been adequately covered in several reviews (60, 90,172, 217, 218, 24’7, 386). The polymyxins are being produced in comparatively small quantities in this country. Regna et al. (506) investigating the recovery, purification, and properties of polymyxin B found t h a t the composition of the hydrolyzates of polymyxin B and the minor active component were related, since they contain the same amino acids-namely, threonine, leucine, phenylalanine, a,r-diaminobutyric acid, and a 9-carbonoptically active fatty acid. Kagan and coworkers (199) found that polymyxin B could be utilized with satisfactory results in serious infantile infections which do not respond to safer therapy. It waa demonstrated in laboratory and clinical tests t h a t both polymyxin B and E were useful against infections due to Escherichia coli, Aerobacter aerogenes, Pseudomonas aeruginosa, Shigella, and Hemophilus inJEuenzae. The nomenclature of the polymyxin antibiotics is based on a proposal by Stansly and Brownlee (340). Xelson et al. (261) described laboratory and pilot plant procedures for the fermentation and extraction of circulin, an antibiotic produced by Bacillus circulans, which has a n antibacterial spectrum and other characteristics similar to t h a t of the polymyxins. Subtilin. The literature on subtilin, an antibiotic produced by Bacillus subtilzs, has been previously reviewed (217, 218, 291, 38s). Lewis (2.20) of the Western Regional Research Laboratory, where most of the work on subtilin has been done, has prepared a n annotated bibliography of subtilin which reviews the literature up to October 1950 It is believed t h a t subtilin has been produced only in experimental quantities in this country. Production on a limited scale in Italy has been reported (66). Two patents (121, 351) were granted during the past year to investigators a t the Western Regional Research Laboratory. According to Feeney and Garibaldi (121) previous methods have not given uniform yields of subtilin because it was not realized that small amounts of certain mineral elements caused great differences in the yield of this antibiotic. They determined the amount of potassium, magnesium, manganese, iron, zinc, sulfur, and phosphorus required for optimal subtilin yields. It was
.
1959
produced in a continuous manner in a medium containing the mineral elements mentioned, a source of energy such a~ glucose, smrose, or glycerol and nitrogen sources such aa ammonium nitrate o r ammonia. Yields of subtilin as high as 900 milligrams per liter were obtained in 72 hours. I n a later patent, Stubbs et al. (351) claimed t h a t waste products such as asparagus juice and beet molasses, or sucrose supplemented with amino acids, yeast extract, and mineral salts produced high subtilin yields in submerged fermentation. Andersen and Michener (3,4) obtained very promising results in preserving food through the complementary action of subtilin and a mild heat treatment. It appears a t the present time, however, that the use of subtilinor other antibiotics for food preservation is rather indefinite pending further laboratory and commercial scale experimentation and approval by the Food and Drug Administration, Miscellaneous Antibiotics. I n general, these reviews have covered only those new antibiotics that have some commercial significance, This review, however, mentions briefly some of the newer antibiotics even though, in most instances, they have not reached the stage of commercial production. ANTIMYCIN.I n 1949, Leben and Keitt (216) announced t h a t various crude antibiotic preparations, derived from cultures of a n unidentified species of Streptom ces, were effective in controlling apple scab and tomato bligEt. During the same year, Dunshee et al. (108) described the isolation and properties of a highly active fungicidal fraction which they called antimycin A. Kid0 and Spyhalski (206)found that antimycin A was a powerful insecticide and miticide, being active against the housefly, carpet beetle, and the red spider mite. FRADICIN. I n 1949, Swart, Hutchison, and Waksman (355)reported in a paper concerned with the recovery and purification of neomycin t h a t they had isolated from the culture filtrate of Streptomyces fradiae another antibiotic which had antifungal properties and which they called “factor X.” I n 1950, Swart et al. (567, 568) announced the isolation of one of the constituents of the factor X which was called fradicin. Recently Hickey and Hidy (1’75)reported the isolation of crystalline fradicin. The pure conipound, which showed marked activity against yeast and filamentous fungi, was believed i o be too toxic for parenteral uses and may also cause skin irri,ntion when used topically. FUMAOILLIN. I n 1949 Hanson and Eble (168) isolated, from a n AspmgilEus culture designated H-3, active concentrates which were capable of inhibiting the bacteriophage of Staphylococcus aureus 209. McCowen et al. (233) recently reported t h a t this antibiotic, fumagillin, was a n extremely potent amebicide. Its effectiveness was not only shown in vitro but also in vivo with rats, mice, and rabbits. I n t h e opinion of these investigators, it was possibly one of the best direct-acting amebicides. F U N G r C I D I N . Early in 1951, Hazen and Brown (165) announced the isolation of two antibiotics of differing chemical and biological properties from a Streptomyces species. One of these was extracellular and has been called fungicidin. Fungicidin is both fungistatic and fungicidal, b u t apparently lacks antibacterial action. This new antibiotic appears t o be of value in the treatment of histoplasmosis and cryptococcosis in mice. KETRUPSIN. Finlay et al. (127, 128) announced the isolation of a new antibiotic, netropsin, from the culture broths of a previously undescribed actinomycete, Streptomyces netropsis. Xetropsin has been prepared as the crystalline salt of various acids. The free base was unstable and has not been isolated t o date. It was suggested t h a t netropsin is a tetracidic base corresponding to the formula CJ2HdI804; A portion of the molecule seemed to be a substituted guanidine, which, however, did not account for all the nitrogen present. It had, in vitro, a wide antibacterial spectrum. Netropsin has also shown considerable insecticidal activity, particularly against clothes moths and carpet beetles ( 1 2 7 ) . I t s parenteral toxicity was fairly high but its oral toxicity was low. THIOLUTIN. Another recently discovered antibiotic of potential industrial importance is thiolutin. This new antibiotic was isolated from several strains of Streptomyces albus by Tanner el aE. (361). It inhibits both gram-negative and gram-positive bacteria and many fungi. It is a stable neutral compound readily extracted by solvents. I t s toxicity for animals is fairly high; however, it shows great promise as a practical fungicide.
INDUSTRIAL AND ENGINEERING CHEMISTRY
1960 ANTIBIOTIC HED SUPPLEMENTS
One of the outstanding developments in the fermentation industry during &hepast year waa the large scale production of feed Supplements containing antibiotics either alone or in conjunctioo with vitamin Biz. Such products are produced by a number of industrial fwms in this country. The chief antibiotics used on a large scale for this purpose are aureomycin (196, 348), bacitracin (66), penicilh (103),and terramycin (66, 102). A t present, these feed supplements are used mainly for swine and poultry, The growth-promoting effect of antibiotics bn animals was first noted by Stokstad and Jukes (349)who pointed out that small amounbs of aureomycin in a crude BIS feed supplement gave a markedly superior growth of chicks. Other investigators quickly demonstrated a similar effect with a number of antibiotics. Increased growth responses in the range of 10 t o 25% and greater feed efficiencies are claimed for poultry and swine. Matterson et ul. (239) compared aureomycin, streptomycin, penicillin, terramycin, and bacitracin as growth stimulants in practical chick-starting rations. Streptomycin was found t o be least effective in promoting growth response. Penicillin and bacitracin were effective in maintaining an increased growth response, over the basal rations, for a longer period of time than the other antibiotics. In general, the data suggested that the higher the protein and vitamin quality of the basal rations, the less was the growth response from antibiotic supplements.
FERMENTATIONAS A UNIT PROCESS In the previous review of this series (218) consideration was given t o fermentation aa a unit process. The discussion was based on the Eterature cited previously (217, 818, 328). This section of the review is presented in a manner similar t o that of the previous review ($18),and is intended t o extend the discussion t o include pehinent, literature which has become available during this review period. The reviewer particularly wishes t o call attention to the presentation of fermentation as a unit process in the previous survey (218) and to stress that this section of this review is an extension of the previous unit process discussion. During the past year Corran (87), de Becze (26), Petty (288) and others b v e presented discussions pertinent to this subject. Also, during the past year, several publications have appeared which deal more directly with considerations of the quantitative principles of fermentation processes (22-24, 31, 124, 177, 178, 367, 368). THE MICROORGANISM
Corran ( W )briefly reviewed the role of the microorganism in has made a survey of industrial fermentations, McClung (,%?32) the recent developments in microbiological technique. He stressed the techniques used with bacteria and discussed such problems aa enrichment techniques, single cell isolation, preservation of cultures, cytological and genetic studies, continuous culture methods, and techniques for production of large quantities of cells. Monod (262)reviewed the more recent literature on the theoretical aspects of the growth of bacterial cultures and thereby provided a baa$ for a sounder approach to industrial fermentations. In another paper (261)he investigated the theory and application of the continuous culture technique and discussed the rate of growth in relation to limiting concentration of the nutrient, the growth yield, the speed of biosynthesis, and the mutation rate. The morphology, cytology, and taxonomy of the Actinomycetes, the most prolific group of antibiotic-producing microorganisms, were reviewed by Erikson (117). The morphological and physiological characteristics of Ashbya gossypzi, which is used industrially for the production of riboflavin, have been discussed by Pridham and Raper (2996). Duckworth and Harris (104)
Vol. 43, No. 9
have described the morphology, in submerged fermentations, of Penicillium chrysogenum. Although the work reported was mainly descriptive, the authors expressed the hope that it may be possible to define, by morphological examination, the conditione under dhich cultures have been grown and to correlate microscopic appearance with biochemical behavior. I t is imperative that the parent or stock-culture be handled with utmost care. The preferred approach is to preserve the culture in an inactive state; this can be done by several techniques. Mejlbo (240) claimed that Lactobacillus acidophilus can be preserved, through a seemingly unlimited period without any perceptible reduction in vitality, by freezing it, a t temyeratures as low as -25' C., in a sodium chloride solution. Lyophylization methods are frequently used with great success. While investigating the preservation of mold cultures, Fennel1 et al. (123) concluded that lyophylization waa the most reliable means of maintaining a mold culture in unaltered form over long periods of time. However, if the culture cannot be 80 preserved, alternative methods must be used. Preservation in soil offers certain advantages, but this method is not applicable to the maintenance of mycelial forms The authors suggested that, if the structure is strictly mycelial or if very few spores are produced, preservation under oil affords the best means of conservation. According t o Dopter (100) two strains of yeast, lyophylized and reconstituted after storage in vacuo for one year, possessed morphological and biochemical characteristics identical with those of the untreated parent cultures which were maintained by periodic transfer. Barrett and Tatum (20)have described a simplified method, which required a minimum of special e q u i p and materials, for lyophylizing microorganisms. I t is usually possible, in a new fermentation process, markedly to improve the yields obtained through the application of culture selection or induced mutation. A classic example of such a pursuit is the selection of appropriate high yielding organisms for penicillin production. The literature on this subject has recently been reviewed by Perlman (282). Woodruff and Larsen (409), in a recent patent, described a high penicillin-~roducingmutant of Penicillium chrysogenum Q-1 76 which has the further advantage of being incapable of secreting objectionable pigments into the fermentation medium. Anderson et al. (5) described pilot plant results obtained with pigmentless mutants of Penicillium chrysogenum Q-176. These mutants, which had been produced by the action of ultraviolet and nitrogen-mustard, gave lower penicillin yields than the parent culture. %ani and Okada (268) produced, by radium irradiation, a ponpigment-secreting mutant which also failed to produce higher penicillin yields than the parent Penicillium chrysogenum Q-176. Mutants of yeasts which produce riboflavin have been described (260, 289, 297). Pfeifer el al. (289) demonstrated the effect of strain variations on riboflavin yields. Mitra (260)obtained a high-yielding riboflavin-excreting yeast by treating the parent culture with acenaphthene. Skovsted (831)used camphor to induce mutations in Saccharomyces cereuisiae. Latarjet and coworkers (216) used water-soluble carcinogenic agents, such as alkyl carbamates, to induce mutations in E. coli. Dickey and coworkers (96) used organic peroxides for the induction of mutation. The general problem of the occurrence and inductionof mutations in fungi has been reviewed by Backus (16). The establishment of a correlation between genetic factors and high yields should in the future make the selection of desirable strains easier. Negroni and Fischer (267) have linked penicillin production by Penicillium notaturn with two genetic factors, h1 and C, both of which must be present for high penicillin production. The Inoculum. The economic significance of this problem has been discussed by Corran (87), while Petty (288)reviewed the role of the inoculum in antibiotic productim.
INDUSTRIAL AND ENGINEERING CHEMISTRY
September 1951
Several examples of the effect o f inoculuni on fernient:ttion yitxltls are cited below Although more could be mcntion~d, thcse should he sufficient to strcss the i m p r t a n c e of t,his problei11 and to point out the necessity for drscrihing as accuratcly as pcmiblr, i n any account of a fernicntatiori prowss, the state, quantity, and othcxr 1)imprrtic.s of thc inoculum used.
1961
One of the explanations for difforcnt results with various carbon compounds may be the adaptive production of enzymesfor example, Goodman (145) found that Aspergillus ~ Z U ~ I L and S Asperyillits fcrreus produced more aniylase with starch in the mcdiuni than wit.h sucrose, and that greater amounts of lipase are forincd whcn corn oil wrn in t,he mcdiuni. This type of adaptation heen kno\vri for several ycars and it appears tu be a normal haL. Studying streptomycin production, Bartholornew el al. ( 2 1 ) characteristic of microorganisms. were able t o sho\v that a uniform inoculum wa:, necessary for good Various proteinaccwus materi:ils commonly used for large scale replication among different fermentors. These workers demonstrated that the standard deviation hctwcen fermrnt,ots was production oper&tionshave been listed (218). il:Syowhen t h e inocula jverc ident ic*:tl,Lvhereas when individual Sclson et al. (261).in their study of the production of c:ii~culin, s l i ~ k e rflasks \yere used for inoculating carh fernicntor, the dcviinvestigated the effect of different arotein sources in a daxti ination was 1 2 . 1 5 . Jarvis antl Johnson (Ic92)have sho\vn that protein-salt8 medium; the yiclds progressively increased with i nig. per nil. of caliloride in thc inoculum medium was detrimental t o penic illin pi,oiluc t ion. t,he folloning protein sources: yeast, soybean meal, peanut 13errihauei~an(l coworkers ( 3 6 ) , in thcir study of lartic acid granules, cottonseed meal, oatmenl, crirn meal, and rolled o;tts. formation in sut)nierged fei,nicntation ivith Ilhizopics spcvics, obIIaller (267) proposed as protein sourccs for penicillin product.ion, served thxt prcgcrminat,ed spores or 2-da~.-old niyccliuni gave hominy nical, whole ground ryt,, wheat bran, and untreated whole twt tor results tli:tii spore suspensions. Tlic iniporta1ic.e of the irioculum for dextran production was corn mcal. Colingsworth (83) suggested niolasms-ethyl fernic,ntl(~iiionati~ated b ~ Jcanes . el al. (193) who pointed out t.hat the tation solubles for streptomycin production, while McDanic~l ni:isiiiial viscosit!. of thc fcrnicntation mcdia decreased progres(635, 256) claimed that distiller’s solubles, soybean meal, sotliuni sively with incre:ised incu1)ation time of the inoculum. nitrate. and other nitrogenous substances were cspccially suitNelson et a l . (261 ), in their study of the production of circulin, clemonst~~ateii that the inoculum medium and t,lie phase of dealile for streptomycin production. vt:lopnimt of the inoculum cultures have a marked effect on ferAlany nat,ural carbohydratr-coiit,ainirig materials are used in n i ( ~ i t i i t i ~>.ields. n Optinium yields were obtained only \\it11 an industrial fermentations. A few additional exaniplcs will tie iiii~r~uluni that appeared t o be at the rnd of the logarithmic phase, cited to supplement the previous review (218). BLJE(41) used \ + I i ( a i i most of the cells werc in the spore form. Younger seed rultui~es,B-itliout spo~’(’s,gcnerally resulted in slower fermentaniolmws as a medium for gluconic acid fermentation, and Cillie tions tvitli lo\v(>t,find yields. and Rapson (81) reported satisfactory results with niolassos i n the citric acid fermentation. Grape and Jerusalem art,ichokc SUBSTRATE musts have been proposed as raw materials for the combincd aeetone-b;ltyl alcohol and ethyl alcohol fermentations ( 9 ) . Tlic~i i i ~ l i i ias ne11 HY the grnerul inulation and ~ii~~t~al)olisni of iiiirr(J(i~g~~~ii~1iis 11:ive lieen discussed by several invwtipators Sugar txet crowns have been found suitable as a raw material for the ethyl alcohol, 2,Sbutylene glycol, lactic acid, and butyl ( Z I ,282, 288, 336, 344, 345, 376). Snell (336) reviewed the iiualcohol-acetone fermentations (2445). Sulfite waste liquor has ti.it i o i i o f niic.i.ocJrganisnis, placing special emphasis o n iticntified aiitl uiiitl(intified groivth factors. Umbreit (376), reviewing the been studied as a carbohydrate source for ethyl alcohol (111, litc,r:itiii.e o n the metatiolism of microorganisms, emphasized 378), yeast (378, 399), butyl alcohol-acetone (349), and lactic acid (111) ferment,ations. Dufrenoy (10.5) proposed the use of ctii~l)oli~drate and nitrogrn nietabolism. He also discussed the niride of action of penicillin, streptomycin, and other drugs. by-products of the manufacture of preserved asparagus for the production of tyrothricin. Milo meal, which results from thc Steinlicrp (3.44) i n 1939 published a detailed review of the literaconvcrsion of milo to war1 barley, was found suitable for ethyl ture o n t h e g r o ~ r t i iof fungi in synthetic nutrient solutions and alcohol production (152). Teixeira e t al. (363) demonstral cd (luring tlie pwt \par he again reviewed this subject (546). Petty that cassava is an inexpensive source of starch for the production (288)t1iwusi;eti tlii> influence o f the substrate composition on o f alcohol. :intibiotics production. Pcrlman (286) discussed the media used Synthetic Media. Synthetic mcdia have been developed in for t,hr production of pcniciilin. the laboratory for practically all industrial fermentation procI t has been demonstrated repc:itc~clly that a change in t,he esses. Howcvcr, due to genci,aIly higher cost and lower yields, composition of tlic niedia c : ~ ntiec,isively influence the fernientathey are very rarely used on a n industrial scale, although they t ion results. S t i ~ ~ p t o m y c ejsm d i a c has 1)ccn found to produce do have ccrtain advantages. Baron (18)claimed that difficultii3s but the yields of hoth thr x n t i l r i ~ i t i cneomycin :inti vit.:tiiiin Ih, t l i ( w t i v o l ) ~ ’ o d ~ i c t , < not funrti(i~isof the sanie net of conditions in the extraction and purification o f streptomycin could I)e avoided by using a synthetic medium. ). 1liIl.- ct a i . (f76)found that RS the rcault of medium variPretreatment of Raw Materials. Quite often industrially :it ion+, N ( i l c . i l i , t r / ; ~ / ~ l c , t ? ~ , ~p~r~c/i,dtu~c~~ ~d s an antibiotic differing I J f J t i i i n ~ . i c ~ :l i ~~ il c I i l l genc,r:il c1i:~r:cctc’i.istic.s t.hrough changes in the availahlc raw materials are only suitable for fermentation af‘ter having undergone some type of pretreatment procedure. Stark Iiic~iliuin. A ( , x i , t ~ ( i r it o iiitrogcin r;itio of 15 a n d a neutral pH g i v e t l i c I,wt l,:ic,iti.win yic.lti, n ~ l i i l ca ratio o f 6 antl an :ilkalinr and 3IcGhee (3.62)developed an improved procedure for the prct,reatment of waste sulfite liquor. The pretreatment included p I I f:ivoi,c,tl t i i t , p r ( j ( ! i i ( > t i ( i r i of : i n ;intiliiotic ~~cwmbling liclienistcam stripping, addition of lime, precipitation, filtration, and 1’1 t I i i i i i Types of Raw Materials. Several investigators have reported neutralization of the filtrate with carbon dioxide. The authom stated that this treatment. made the utilization of this raw niateo t i the effect of the cartion source (25, 145, 284). I’crlnian and rial economically attractive for the butyl alcohol-acetone fcr1,:iriglykke (284)demoriatrated that Streplonzyccs qriseits utilized nicntation. :i nurnher of vegetable and animal oils as enc’rgy sourws for Ekelund (111) described a proccss for recovering carbohystreptomycin production Ileplacement, on a caloric bayis, of drates from solutions having low concentrations. He adclcd t l i c cartmhydrate componcnt of a soybean meal nicdium by thcm large quantities of active microorganisms, such as yeast, for the c l i k did not affect antibiotic yield. On the other hand. when only purpose of “sorping“ the sugar. The microorganisms iwre ptt1.t of the carbohydrate was replaced by these glycrrides, antiquickly separated from the liquor by centrifugation, or otlier iiiotic production was significantlv reduced. J h u t z anti comeans, and transferred imniediately to nutrient salt solutions workers (65) compared glucose and lactose rn carbon sourct’s i n after which the fermentation could take place under optiinal periicillin production with Peniciliiurn chrysogcnuni (2-1;6 and conditioris. The method was stated to be suitahlc for the profound the latter far superior They concluded that this cupcriorduction of alcohol from sulfite waste liquor and for the preparation ity is due tQ causes other t h a n the slow fermcntability o f the of lactic acid from whey. I aptose. nr(3
1962
INDUSTRIAL AND ENGINEERING CHEMISTRY
a n d e r (413)claimed a molasses purification procedure which involved the use of a composite laminar membrane which comprised three dialyzing semipermeable membranes of films of regenerated cellulose. Two of the membranes were separated by a continuous layer of a cation exchange resin, while the third membrane was separated from the center membrane by a layer of anion exchange resin. Molasses purified by Zender's process should be usable in the citric acid and other fermentations where raw molasses is unsatisfactory. Precursors. Precursors may be defined as substances, added prior to or simultaneously with the fermentation, which are incorporated without any major change into the molecule of the fermentation product and which generally serve to increase the yield or improve the quality of that product. A classical example of the use of such precursors is in the production of penicillins. This problem has been reviewed in this series (f217, 218, S28), by Perlman (282), and by Petty (288). Several publications have appeared recently in foreign journals which demonstrated increased yield of penicillin G through the use in the media of phenylacetic acid, of esters or amides of phenylacetic acid or of phenylacetylethanolamine. These results confirm those previously obtained. Gilbert (148)patented the preparation of new chlorine-containing penicillins by adding 0- or pohlorophenylacetamide t o the nutrient medium. Wintersteiner (404)prepared penicilloates which were said to be useful as precursors for penicillin production. Thorn and Johnson (869) identified caproic acid as a precursor of dihydro-F penicillin and a,y-hexanoic acid as a precursor of F-penicillin. The same authors (S69) also prepared new biosynthetic penicillins such as propyl-penicillin from tributyrin and butyl-penicillin from tri-n-valerin. A configuration capable of blocking the beta oxidation of the acid was suggested as a n important characteristic of precursor compounds. Another example of the use of precursors in fermentation is the incorporation of cobalt into vitamin B I ~ . Nelson et al. (260) found that the addition of cobalt chloride improved markedly the vitamin Blr yields in media suitable for the formation of neomycin by Streptomycesfradiae. Hendlin and Ruger (169), using Streptomyces griseus, first demonstrated the necessity of cobalt for vitamin BI2 production. But the most striking proof that cobalt is precursorlike in action for vitamin BIZwas obtained by Chaiet and coworkers (65) when they prepared radioactive vitamin B12 by the addition of cobalt 60 to a broth inoculated with Streptomycesgriseus. Woolley (410) obtained some evidence that 1,2-dimethyl-4,5diaminobenzene would act as a precursor for the synthesis of vitamin BIZ and of riboflavin. H e also demonstrated that 1,ZJichloro-4,5-diaminobenzenewas a metabolic antagonist of this compound and inhibited the synthesis of these two vitamins in and other bacterial cultures. 1,2-Dimethyl-4,5-diaminobenzene degradation products of vitamin Biz were shown to act as precursors for vitamin B12&henfed to rats (112,118). According to Petty and Matrishin (E??'), Xtreptomycps aureofaciens could efficiently utilize the chlorine in fermentation media for the synthesis of aureomycin.
TRACE MATERIALS
A discussion of the effect of trace materials on industrial fermentations is difficult because there is no clear-cut definition of what should be comidered as a trace material. For future use the following definition is suggested: An essential trace material is an organic o r inorganic compound which, when added to the fermentation medium in minute quantities, increases the yield and/or the rate of production of a fermentation product; it is not a precursor for the product but may be a n essential micronutrient which functions chiefly as a catalyst. Admittedly,
Vol. 43, No. 9
such a definition cannot be strictly adhered to since it will be impossible to define the role of many metals, vitamins, and growth factors, present in fermentation media in very small quantities, until the details of the fermentation mechanisms are known. As pointed out previously (118),the use of natural materials in industrial fermentation media largely eliminates the necessity for the discussion of trace materials in connection with present industrial fermentation processes. The following discussion will be divided into inorganic and organic trace materials instead of, as previously, into stimulatory and inhibitory materials. Inorganic Materials. The role of inorganic trace materials in the nutrition of microorganisms has been reviewed by Hutner et al. (184). Steinberg (846)has brought u p to date a previously published paper (344) on the nutrition of fungi in which he gave special emphasis t o the role of metals. Tomlinson et al. (370,37i)re orted t h a t zinc, iron, manganese, and copper were essential for figh yields of citric acid with the Aspergillus niger strain they used. Omission of any of these elements caused a marked reduction in acid formation. Miksch et al. (246)demonstrated that zinc, iron, and manganese have very little influence on fumaric acid production by submerged cultures of Rhizopus in a medium containin glucose and t a p water. Lower concentrations of copper showefstimulation, while larger concentrations were strongly inhibitory, but this effect could be reversed by zinc or aluminum. Selenium derivatives and sodium fluoride inhibited fumaric acid formation in high concentrations b u t had practically no effect in low concentrations. Hendlin (168)studied the nutritional requirements of a bacitracin-producing strain of Bacillus subtilis. Magnesium, iron, potassium, and phosphate were essential for growth and bacitracin production, while manganese was essential only for bacitracin production. Calcium, copper, and nickel reduced the yield of bacitracin but had no effect on growth. Zinc, molybdenum, and chromium affected neither bacitracin production nor growth. Webb (390)investigated the effect of magnesium on the growth of gram-positive and gram-negative bacteria. The former required ten t:mes as much magnesium in the media as the latter. Webb attributed this difference to the incorporation of the magnesium into the structure of the gram-positive bacteria. If the organisms were killed, passed through a bed of ion exchange resins, and dialyzed, the gram-positive cells retained a portion of the magnesium, whereas the gram-negative organisms, with one exception, did not. Two Italian inveatigators (98) examining the effect of molybdenum on microorganisms and plants found t h a t molybdenum had a noticeable influence on the biological fixation of nitrogen and seemed to be indispensable t o the life of nitrogen-fixin bacteria. The action of molybdenum on fungi varied. It gad a positive effect on Aspergillus niqm and a negative one on Penicillium notatum. Molybdenum did not appear to influence the alcohol fermentation. Organic Materials. I n his review of the nutrition of microorganisms, Snell (SS6)placed special emphasis on the literature pertainin t o organic growth factors. Atkin f14) has reviewed the literature on growth factors for yeast. H e has found (16)that brewery yeasts could be grouped into five categories depending on their requirements for B vita-
nuns.
According to Miksch et al. (246) the addition of p-benaoquinone increased the yield of fumaric acid in submerged fermentation, with Rhizopus, from 52 t o 67% of theoretical, while decreasing at the same time the formation of by-products. Finn et al. (129) demonstrated that increasing the concentration of yeast extract stimulated the rate of lactic acid fermentation with Lactobacillus delbruckii. Dulaney and Grutter (IO?') reported studies on the nutritional requirements of Eremothecium ashbyii. They found t h a t 1-inositol was indispensable for growth as well as for riboflavin production. Other B vitamins were not essential. However, there was some evidence t h a t unidentified factors existed which stimulated the production of riboflavin by this organism. Hendlin (168)studied the effect of several vitamins of the B group, and other growth factors, on a bacitracin-producing strain of Bacillus subtilis. H e observed that these factors decreased the lag phase of growth but did not affect the total cell or bacitracin yields. Behrens and Garrison (28) found a number of substances such as imidazob derivatives, benzyl penicillic acid, penicillamine, histamine, and others which exhibited a marked antip~nicillinase activity.
September 1951
INDUSTRIAL AND ENGINEERING CHEMISTRY
1963
STERILIZATION
Sterilization of Media. Fortune et al. (194) have called attention to the fact that when a completely liquid medium is sterilized, only the insulation characteristics of the fermentors are effective as variables. However, with a medium composed of both liquids and solids, a second insulating quality, t h a t of the solids, must be considercd. By measuring the temperature inside the pilot plant fermentors with thermocouples, Fortunc et al. (134) found that, although the temperature of the autoclave reached 115' t o 120' C. within a few minutes after autoclaving began, the temperature of t,he solids-containing medium in the bottom of the jar did not exceed 100' C. until 30 minutcs later and usually did not reach a temperahre greater than 110" C. during sterilization periods of 30to 60 minutes. No practic:il stirring device mas developed by these authors to minimize thy insulating effect of the solids in the medium during the steril tion. Although this difficulty was encountered only in the ste zation of laboratory fermentors in autoclaves, the results are of general significance to emphasize the necessity of having all portions of media and all areas of plant equipment a t the desircd sterilization temperature for the desired time. During the investigation of the production of riboflavin witli Ashbya gossypii on a pilot plant scale, Pfeifer et al. (289) studied the following methods for sterilizing the medium: batch cooking at high and low. pH; continuous sterilizat.ion a t high and low p I I ; and continuous sterilization a t low pH with separate sterilization of a portion of the medium. The medium used contained glucose, cornsteep liquor, and animal stick liquor. Consistent and satisfactory results were obtained Then the p H during sterilization was h w , and the sterilization was conducted continuously at 275' F., with aretention time of 5 minutes. Wilson and Bruno (40,g) have proposed to sterilize bacteriological media and other fluids with ethylene oxide. They test,ed solutions containing gram-positive and gram-negative microorganisms, aerobic and anaerobic spore-forming bacilli, fungi, and vaccinia virus, and claimed to have encountered no microorganism which withstood t,he action of this gas. The method was especially recommended by the authors when heat and prcssure could not be used. Undoubtedly this sterilization technique will he examined further by other investigators. Rentschler and Nagy (SOT)have obtained a patent for an apparatus used in sterilizing fluids by exposure to ultraviolet radiation of 2537 A. wave length so that the fluid receives an average intensity of about 0.038 microwatt per square centimeter for a period of eight hours. I t was claimed that the exposure had no effect on the properties of the solution other than to kill contaminating bacteria. Sterilization of Air. The sterilization of air in the fermentation industry has been discussed by Corran (87) and in the previous review (218). The problem mas thoroughly considered a t a symposium o n air disinfection and sterilization conducted by the Society of Chemical Industry in London (88). Problems discussed were (I) methods of sampling air for bacteria; ( 2 ) air disinfection by ultraviolet irradiation and chemical vapors; (3) removal of bacteria from air by filtration and its application to industrial fermentations; and (4)industrial large-scale purification of air rrith electrostatic precipitators. Stark and Pohler (343) reviewed air sterilization by heat of compression, using a reciprocating compressor, and presented experiment,al data from actual installations. F E R M E N T A T I O N EQUIPMENT
I n the last review (218) a gmeral discussion was presented of the equipment used in the fermentation industry. Pilot plants for studies of aerobic fermrntations have been deacribcd in a number of recent articlrs (194, 261, $90). Petty (288) reviewed the techniques and equipment used for the production of anti-
Laboratory Fermentation Apparatus Containing Several Five-Gallon Capacity Fermentors
biotics and h:Le included a flow sheet of a pure culture fermcntation plant (287). Iiiskeep et nl. (188) described a nen-ly constructed fermentation plant. VersaCilit,y was the keynote in the design of this plant which may he usable for a variety of aerobic fermentation processes. One of the special design features was the use of a central control board from which nearly all operations can be activated and controlled. A flow sheet of a penicillin plant (64)emphasized the materials used for construction of the equipnient. Corran (87) in his review of the technical development of fermentation processes also discussed the materials of construction. Waldo and Shaw (387) have described a n e r pilot plant designed for organic chemical development. RIany of the features of this plant, which stressed safety arid flesibilit,y, are applicable t,n the design of fermentation pilot plants. Fermentor. No significant developments have occurrcd since the problem of tlie fermentor was previously discussed (218). A standard fermentor, which is allegedly used by over SOY0 of the industry, was pictured by Petty (288). The fermentor equipment consisted of the mechanical agitator, baffles, inoculation nozzle at the top, and an air inlet at the lower side and a jacket for heating and cooling the fermentation substrate. Similar ferment,orshave been descrihed by others (48, 689,391). According to a penicillin plant flow sheet the preferred material for fermentor construction is carbon steel (54). Llost reports in the literature support the content,ion that the iron concentration in the medium, which is att,ained in a carbon steel vessel, is not harmful t o penicillin yields. I t is believed, however, that most of the nevi' fermentors nom being const.ructed by several industrial firnis for penicillin and other antibiotic production, are of the noncorrosive t,ype-principally stainless steel, Such fermentors are much easier t o keep eleiin and free from contamination. I n Germany it has been reported (391) that aluminum fermentors were used for pilot scale production of penicillin. The authors had found a deleterious effect of iron and, in order to avoid this, they sprayed the iron portions of their fermentors with aluminum. The design and application of special fermentors have been reported. The Waldhof-type fermentor., as described by Brown and Peterson (@), was construc.ted so that the agitator-aerator forced the liquid a t the bottom of the trtnk outward and iip~vard and simultaneously aerated it. The liquid, toget>hern-ith foam
1964
INDUSTRIAL AND ENGINEERING CHEMISTRY
generated by aeration and agitation, returned to the bottom of the fermentor throu h a central draft tube whioh revented the accumulation of nongbreaking foam on the top of t i e liquid. As the foam passed through the draft tube and out throu h the agitator i t was subjected to a beating action which reduce8 its quantity. Brown and Peterson (49) found that this fermentor produced higher penicillin yields, more uniform sugar utilization, more constant p H values, and better aeration than t h r standard type fermentor. Other devices have been suggested (32, f18) which have been designed mainly for providing better aeration. A number of laboratory fermentors have been described (21, 48, 68, 104, 309). In all these fermentors an attempt lias been made t o duplicate as closely as possible all t h e features found in fermentors used in the large scale fermentation plants. Most closely related t o the usual type of laborator fermentor 'is the culture vessel which Duckworth and Harris f i 0 4 ) used for laboratory scale penicillin formation. Their flask contained three openings of different sizes, the largest for the stirrer, anothrr for inoculation and for air exhaust, and a third for introducing the aerating tube and the sampling tube. The 30-liter fermentor described by Ilivett et al. of the University of Wisconsin (%9) resembled more closely the standard-type fermentors used in industrial fermentation plants. The apparatus was complete with paddle-t).pe ropeller, ring sparger, baffle, air exhaust, sampling tube, a n 8 other fittings. This fermentor was usell, among others, by Brown and Peterson ( 4 8 ) in their study of penicillin production. A similar type laboratory fermentor was designed by Bartholomew et a f .( 2 1 )and has been used for a series of stwiiies of the effect of aeration and agitation on antibiotk fermentations (22, 24). A scientific apparatus company has recently arinounced a new laboratory fermentor ( 6 9 ) the design of which is nearly identical to t h a t described by IIumfeld (183). Fraset (136) designed a special l h o r a t o r y apparatus, having no moving parts, which perniittzd a large air flow with excellent agit,ation and which was es ecially adapted for the prqaration of bacte1,iophage and for t i e growth of aerobic bacteria on a rather large laboratory scale. Equipment for Agitation and Aeration. In the previous review (218) there were separate discussions of equipment for agitating and aerating fermentations. This year it seemed desirable to discuss them togetlter because a number of devices have been developed which carry out these two functions simultaneously. T h e conventional type of equipment having a separate agitator and air distributor was used by Fortune and coworkers (f34), Nelson el al. (261 ), and Rivett et al. (300). During the period under review, there have been sev.era1 reports dealing with t,he theoretical as well as the practical understanding of the mechanical mixing of liquids. The recent literature on this subject was reviewed by Rushton (316)who discussed such problems as power characteristics of mixers, theoretical aspects of mixing, and the application of mixing to fermentation processes. Rushton and coworkers (316, 317) ublished a theoretical and experimental study on the power ciaracteristics of mixing impellers. T h e common forms of impellers used for agitation and mixing of liquid systems, such as marinetype three-blade propellers; paddles; flat, curved, and arrowhead-bladed turbines; and fan turbines, were studied under conditions existin in the laboratory and in large scale operation. Correlations o f the most important variables were presented in the form of dimensionless groups, characteristic plots, and genera1 equations. Helmbold (157), in France, published a paper on agitators which uses a less theoretical amroach. Various t m e s of aeitators were discussed with regaid t o their practical iiplicatiotk Instead of using mathematical equations containing empirical factors which changed with viscosity and type of operation, Helmbold used a nomograph to obtain the horsepower required, using such factors aa the volume, densit and viscosit of the liquid, and the type of agitators. Mack $38) developedYequations and a plot which made it possible to estimate, for simple turbinetype agitators, the power requirements in baffled tanks from the number and dimensions of the blades, when speed and fluid rop erties are known. A summary description has been assembgd of new mixing equipment recently announced by manufacturing firms in this country (71 f . Many investigators have attempted to develop equipment which combines agitation and aeration. Bergel and associates (3.2) have patented such a n apparatus. T h e fermentations were conducted in an apparatus provided with a high-speed stirrer which agitated the liquid efficiently and also drew surface air into the liquid and distributed it effectively. The apparatus was also provided with a conventional sparger system. -4 restricted outlet for sir was provided around the point of entry of the agitator shaft into the top of the vessel.
Vol. 43, No. 9
Hatch et al. (16'2,163) patented an apparatus in which aeration without excessive foaming was accomplished. This apparatus had many features similar to those of the Waldhof fermentor. Boresch et al. (40)used a special agitator which, instead of havin the usual blades carried at the end of the shaft, had a pear-staped attachment with four small lateral openings and one arger opening a t the bottom. The centrifugal force of the agitator caused the liquid to flow through the lateral openings, the vacuum formed thereby being compensated by the larger opening. It was claimed that with this arrangement the efficiency of aeration was increased tenfold as compared with the usual type of agitator. It was also claimed that this type of agitator could be used for all sizes of equipment, that it facilitated sterilization and decreased the dangers of mechanical breakage. In a French patent (118) an apparatus was described which comprised an internal vertical tube in which the foam formed ran up through the tube and was deflected by a plate at the top of the tube. This assured intimate mixing and aeration of the fermentation liquid. THE FERMENTATION
General fermentation factors were discussed in the previous review (618). Corran (87) and Petty (288) have reviewed the technical development of fermentation processes. I n addition many of the variables which must be considered in fermentation processes were discussed by Lee and McDaniel (219) and by Bartholomew et al. (21) in connection with their report on the design and operation of a laboratory fermentor. pH. The necessity for p H control is well known. The type and quantity of microbial growth and the enzymes produced in any metabolic process are influenced by the pH of the medium; the optimum being a function of the individual process. The effect on lactic acid fermentation of continuously controlling p H has been reported (129, 130, 904). The yield and rate of lactic acid production were found to be functions of pH. The maximal rate of arid production during a fermentation cycle was increased as mucah as fourfold by raising the p H only 0.5 unit. I n a dextrose medium, the rate of fermentation waa greatest a t p H 5.70 (129) and in wheat grit mashes the optimal value was found t o be 5.40 (204). I n fermentations such as those involving Baci22us polymyxa and Aerobacter aerogenes, the ratio of the products was reported to be dependent on the p H (269,266,387) Further reports have appeared (96,48, 49) concerning the importance of p H control in the production of penicillin. Bautz et al. (96)found that the growth of the organism wm most rapid at pH 4.5 to 4.7 while for penicillin production, optimal results were obtained by maintaining the p H at 7.0. Brown and Peterson (48) emphasized the necessity for automatic p H control and reported t h a t t h e optimal p H for penicillin formation was 7.2. In a later paper (49) they demonstrated t h a t in a Waldhof-type fermentor p H values were more constant than in the standard type fermentors, thrreby eliminating the necmsity for further p H control after initial adjustment to p H 7.0. Nelson et al. (261j found, in their study of t h e production of circulin, that the fluctuation of pH during fermentation could be limited by the addition of potassium dihydrogen phosphate Jeanes and coworkers (193) were able to show t h e importance of the p H for the production of dextran; in general, the higher the p H of the culture medium, the higher Kas the viscosity of the resultant ferments. Temperature. Fortune and coworkers (134) have pointed out that determination and control of fermentation temperature has always presented a problem. It is necessary t o control not only the heat produced b y the fermentation but also the energy put into the fermentation process through vigorous mechanical agitation. Cooling coils or jackets are required in all fermentors to make possible a close control of temperature. For all fermentation processes there is generally a n optimal temperature for maximal yield and fermentation rate. The insulating quality of solids suspended in the medium is an important tactor in temperature control.
September 1951
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
The recent, literature has scv(9rd ( ~ ~ a n i p lof r s the striking effect of temperature on fernicntation yields. 1i:trtholoniew r t a / . (21) have presented data on the effrct of teiiipc:r:it,ure on the production of streptomycin 1 ) ~ . Slreplomyces yriaeus in a soybean meal medium. They found t h a t 27" and 25' C. gavc cxscntially the sBme tntal yield but that the r:tt,e of production ivas nmre ra1)id a t 27" C. Temperaturcs of 29" and 31" C. gave fast initial rates follo>ved by plateaus considerably below the yield obtained a t 25" and 27" C. IIentllin (168) has shown that liiq strain of Nrcciiiits sttbfilis produced larger quantitirs of Ixwiirwiti by incrcasiiig tlie tem11on.evcr. a t the higher pcrature in the range of 24" to 27" (': t,eniperatures the ilacitracin content of tho fwnients also deerttased more rapidly aftor re:iching the niasiniiini. Aeration-Agitation. It has been pointed out (218) that reports in the literature, n-ith regard to tlie i i n g l r or coiiil)inrtl effects of aeration and mechanical agitation, cont,:iili inforn~ationon product yields which is largely empiricnl. O m (If tho leading dcvelopments in the fermentation field during tlie past year was the series of reports on the fundamentals of agitat,ion and aeration and their effect on fermentation yields (29, 24, 178, 406, 407) I n all of these studies the polarograph was used to determine the dissolved oxygen concentration in the medium, They measured the concentration of dissolved oxygen in a culture medium which was initially saturated and thereby determined the rat,e of oxygen consumption by the organism. The rate of solution of oxygen into the medium was estimated by aerating an oxygen-free medium and measuring t,hr concentration of dissolved oxygen at suitable intervals. This r,ate o f solution was expressed by an equation (405, 407) which contained a constant and which was found to be dependent on the size antl shape of the vessel; was directly proportional to the rate of air flo~z-;and TVQS greatly increased by mechanical stirring. Hixson and Gaden (178) and ptrticularly Bart,holomew el al. (82, 24) reported comprehensive studies of the relation of oxygen transfer to agitation in submerged fermentation. IIixson and Gaden (178) treated the transfer of oxygen supplied by aeration rn a series of rate proeesscs; this perniittcd the development of a quantitative oxygen transfer equation for oach step. They demonstrated that the physical absorption of oxygen was a function of the design and operating chziractcristics of the equipment and the physical properties of tho medium. They ascribed variations in the calculated absorption coefficient over the course of a single fermentation to changes in the absorbing phase. The absorption cnefficient \vas correlated Kith acration rates for different systems of agitation and air dispersal, giving relationship which aided in predicting the most suitable aeration sq-tems for different processes. The experiments w r e conducted in a small specially designed laboratory fermentor. A voltammetric method, bLqed on the use of the dropping mercury electrode, was described for the instantaneous determination of the concentration of dissolved oxygen. A somewhat different approach was used by B a r t h o l o ~ n eand ~ his coworkers ($2, 24) who proposed a theory for oxygen absorption by suspended mycelium in aerated broths. This theory involved a pattern of diffusional rate steps, between air and organism, coupled with the rate of oxygen uptake by the cells The results were discussed in terms of diffusion mechanism steps which included a n oxygen transfer resistance at all air-liquid interfaces; a resistance through cell clulnps and liquid films around individual cells; and a mechanism which involved direct contact between cells and air bubbles. Air supply and agitation rate affected the oxygen transfer resistances in many ways and determined whether the local oxygen concentrations at the cells led to a state of oxygen saturation or deficiency. These theoretical considerations were applied to an experimental study of the effect of air fiow and agitation variables on antibiotic formation try Penicillium chrysogenum and Streptomyces griseus. Mechanical agitation, air flow rate, and type of sparger were the
1965
I'cpmfent vat~i;it!lt~s\ v w r rwid1i:LI sug;tr. CI 11t e n t , imtihiotic activity, mycelial weight, pH, dissolvctl ox>.p'rt, aiicl oxygen saturation level. The mechanical power i n p u t to 1 l i t ' ])t.iii~.(, v:iri:iIiles.
fermentors and the specific oxygen absorption coefficient Ii( ' I I . measured. The power contributed by air flowiiig through t11r fermentors was also computed. These workprs p i valuable data pertaining to the effect of the qw agitation intensity on air tl: Idup and oxygcri di'fusiori r:irc,Q; effect of the concentration of mycclium and inctliuin conit i t i i r t i i 9 on rate of oxygen uptake; dissdved oxygen level> ;Lnd o x ~ . g ( ' n uptake rate during the entire feriiichntation periocl : coi,rrIatio11 o f the quantity of air flowand intensity of agitation lv'tli tlic >.icslriy of penicillin and streptomycin; thc horsepoiz-c:r input t x i 1 fermenting b r o t h which resulted botli f w m mrr:llanir:ll ;igil antl nc,ration. Itivett et a(. (309) ancl 13artholotiir1z- :rntl vo\z-orkc>rs( $ 1 ) 1 1 , ) Eigned latioratory fermviitiirs for :ic'i,ol)ic: fcrni(,nt:ttions \\ liibll permitted a inore accur:itc dritcriiijii:itioli o f the cfTcs(,tof : I ~ ~ r : i i i o t i . agitation o n experimental fcrnirnt:itioii~th:in i q pllhSiiJlV \ \ i t 1 1 i l l , * usual laboratory equipment such as s1iaLr flasks Pfeifer et a/.(28n), in t,licir rtutly of t h e ri1)ufl:ivin fvrtiLciili:,tion with d s h b y a gossypi'i, wportctl o n the c,ffc.ct o n yil:l[l+ I , ; aeration and agitation. Thi: rate of :acr:ition was varied ~ I I J I I I 0.215 t o 1.0 volume of air per minute por voluriie of fernient:tiion medium with an agitator speed of 100 revolutions per iiiinut(3. At higher rates of aeration, fermentation time ant1 yields i v c t ~ ~ ~ both reduced. Increased agitator speed decremcti the ferment i t tion time but lowered the yield. The authors conjecturcd t h : i t the "agitation interfered wit.h niycelii growth to an cxirsnt i,h:tt the normal productionof riboflavin w m disturbed." It would indeed be dif?icult to translate the dnt:t r e ~ x i i ~ t i ~i)?.c l Pfeifer et a!. (289) into various size fermentation vrserl,~. .i f:iotor which should be considered, a.ccording t o 1 3 : i r t l d o r n ~r i~I ~ : / .~ (22, 94),is the air flow which is nreded. This woultl v:irJ. t , a t l i ! ~ ~ . widely with ( I ) the tyrp of air spargel', (2) tho type :inti i t i t r t i , q i t , y of agitation-i.e., the horscpoww input to the fc,rnic~ntiiri)y the> mechanical agitator, and (3) the air throughput. Tht. o ~ i t i i i i ; i I agitator sDred mould vary with the six'. shape, antl iiiini1)(>1. i ~ tlic> f agitator blades and would also lie tiepriidcnt o n tho rate of : i i r flow. Brown and Peterson (48)studied t,ho effects of :ier:itiott :I 1 1 1 1 agitation on penicillin production and found that, a t nriy o m ' ; i ~ ' t ' ation levc.1, H linear relationship existed betwren ~x~ni(~illiit 111'11duction and the rate of agitation Of the thrcc rirtc,s of :icr:itioIi eniployed4.21, 0.64, and 1.64 lit.ers o f air per liter of niediuni I ) ( I. minute-the middle value gave c~ptinidyields. .Juct as i n t h case of ritioflavin, there is an optimal aeration r:rtc'. l,o\v : I L , I ' ation rates could be employed if adequate disper*ii~ir, i f t l i ( x :lit, throughout the medium was obtaincd 1)y mecIi:iiiii.:~l:igit:{t iotl, Selson arid coxorkers (261), in t>heirstudy of thr 01)titii:~II ' I I I I ditions for the production of rirculin, slso found tli:tt it i v : i ~ till. dcgwe of air dispersion which affected the antibiotic pr(idu~i~i!~tt and that the yield improved to the degree that thc dispi~sioii was improved. l t ( h
ACKNOWLEDGMENT The author wishes to acknowledge thc :wsistancc of F. C,. ( ' ( ~ l i ~ y during the literature search, in t h c tr:tnslat,ion of sonic' o f tl11. foreign literature, and in the propnration o f tlie ni:inuuc*ril)t, The author wishes also to a c ~ k n o ~ ~ lthe ~ ~ lv:ilu:tliIit pr :Lssist:iti(.,s of G. C. M.Harris in the prrp:Lr:itiori n f this mnnusci,ipt. BIBLIOGRAPHY (1) Addinall, C. R.. Can. Chrm. Proccss I n d s . , 34, 108 ( 1 : ~ ) ) . ( 2 ) ilktiebolaget Phormaria. Brit. Patent 588,378 ( I l c r . 1 7 , i:xilj). . ( 3 ) Andersen, A. A . a n d Milichcncr. H. L)., i h c t . Pro----
