FERMEN TA TI0N -
DAVID PERLMAN and CHARLES L. KROLL T H E SQUIBB INSTITUTE FOR M E D I C A L RESEARCH, N E W BRUNSWICK,
N. J.
Quring the past year no significant expansion of operating facilities occurred in the fermentation industries. Production of antibiotics increased slightly, but the quantities of ethyl alcohol, butyl alcohol, and acetone produced by fermentation processes did not change markedly from 1952. Microbiological processes were introduced for the production of the antibiotics, erythromycin, and tetrecvcline, and microbial oxidations were utilized as essential steps in new processes for the manufacture of cortisone and hydrocortisone.
R
EVIEWS of the literature which have appeared in this series ( 6 A , BA, Q A )have surveyed available information describing industrial fermentations as well as the products of microbial origin which in the near future may be produced on an industrial scale. This review is divided into three major sections: economics of the fermentation industries; industrial fermentation processes; and fermentation as a unit process. Several publications have appeared during the past year which have reviewed various aspects of the fermentation industries. Ledingham ( 5 A ) has surveyed the recent (1947-52) literature concerned with the microbial production of antibiotics, alcohols, organic acids, vitamins, enzymes, and food and feed products. Similar reviews covering a shorter period have been prepared by Kawamura ( 4 A ) and Rao (10A). Allen ( I A ) has emphasized new techniques and diverse aspects of fermentation operations. General surveys of biosyntheses and degradations carried out by microorganisms have been prepared by Sainclivier (IIA), de Ley ( ? A ) , and Dooren de Jong (JA). Birkinshaw (WA) has reviewed the recent literature, discussing aspects of the chemistry of the fungi, and Waksman (12A) has surveyed the biology of the actinomycetes and their economic importance.
ECONOMICS
OF
THE FERMENTATION INDUSTRIES
The previous reviews of this series (849 A ) include a brief glance a t the economic status of the fermentation industries. This has been done again this year, covering trends in the industrial application of fermentation processes. Bohmfalk (IB-4B) and Larsen (40B) have studied the organization of the modern pharmaceutical industry and have shown the importance of fermentation products, including antibiotics, vitamins, hormones, and polysaccharides, in current activities. I n general it can be stated that the fermentation industries and the related pharmaceutical industries in the United States encountered a period of slightly increased earnings in 1953 as compared with the earnings in 1952. Only a slight expansion of fermentation plant facilities was reported in the United States during 1953. In gcneral, the antibiotic industry had difficulties in utilizing the excess capacity resulting from stable markets and improved processes, and the only marked addition was the completion of the streptomycin production unit a t the Bristol Laboratories plant (WQB). There is some possibility that this excess capacity will be absorbed eventually as other products appear and the market for antibiotics expands (JOB). In other countries, construction of new fermentation facilities for antibiotic production has been speeded, and a number of production units have started operations. These include two plants in Brazil (14B, 36B) and single plants in 4WB), Norway ( Q B ) ,Chile ( 5 J B ) ,and Yugoslavia Hungary (IYB, (63B) which should produce sufficient quantities of penicillin to satisfy domestic needs in these countries and provide for export. Other units for the production of penicillin and streptomycin are under construction or in the planning stage in India
(53B), Indonesia ( I S B ) , and Pakistan and ilrgentina (JYB). Chlortetracycline will be produced in Germany ( 4 I B ) . The use of fermentation processes in the disposal of sulfite waste liquor has been mentioned in the past reviews. The Lake States Yeast Co. has operated a plant in Rhinelander, Wie., for nearly 3'/2 years and has produced over 10,000,000 pounds of torula yeast from this substrate (5B). This material has found a ready market as a n animal feed supplement and has aided the Rhinelander Paper Co. in disposal of this waste material to such an extent that a similar plant is being built by the Charmin Paper Mills in cooperation with the Red Star Yeast and Products Co. The new unit will have a capacity for production of 10,000,000 pounds of yeast annually (BB). The expansion and use of fermentation facilities for the production of vitamin concentrates for animal feeds has been announced by Hiram Walker and Sons (45B) and the Eli Lilly & Co. (1QB). Construction of a plant for the recovery of vitamin Bl2 from sen-age sludge and related fermented materials has been considered feasible on the basis of pilot plant trials and may be carried out in the near future (d2B).
INDUSTRIAL FERMENTATION PROCESSES In the present review discussion is limited to a critical survey of the literature concerned with the fermentations that appear to be of industrial interest in this or other countries. A brief summary has also been included of recent developments in fermentation processes where the process itself, the process requirements, or the products contain some elements of novelty or scientific significance that yields greater understanding of basic factors in the field. Since the literature has grown so voluminous i t is not feasible to attempt to mention all the publications that have appeared during the past year, and only those contributions yhich appear to be especially noteworthy are included in the folloaing discussion. SOLVENTS
Acetone and Butyl Alcohol. bIcCutchan and Hickey (SOSC) have provided a detailed summary of the literature as well as a description of techniques used in producing these solvents on an industrial scale. Research on fermentation processes for production of acetone and butyl alcohol has continued in Japan where synthetic processes are not used extensively on a commercial scalo. Fermentation of grain mashes containing 10% solids has been successfully carried out on a pilot plant scale (S96C). Rice and grain mixtures were satisfactorily fermented in laboratory experiments with solvent yields equivalent to those obtained with corn (297'C). Pilot plant scale experiments in Germany have shown that the pentoses remaining in sulfite waste liquor after alcoholic fermentation can be profitably converted to acetone and butyl alcohol (308C). Ethyl Acetate. Production of ethyl acetate by selected strains of Hansenula anomala has been studied rather extensively. Yields of the order of 30% of the glucose or ethyl alcohol fcr-
1809
1810
INDUSTRIAL AND ENGINEERING CHEMISTRY
mented have been obtained (5C, 6C,l%C, 3SOC, S37C, S38C). D a t a collected in experiments using diisopropyl fluorophosphate, as well as studies of fermentation dynamics, showed that ethyl acetate formation is probably an energy coupled reaction and not the result of a reversal of a simple hydrolysis mediated by an esterase. Ethyl Alcohol. The competitive situation in the production of industrial ethyl alcohol in the United States, mentioned in the last revieTv, has continued during the past 12 months. Johnson and Nelson (162C) reviewed aspects of the fermentation and synthetic processes. Stark (328C) prepared a very complete description of the processes used in producing ethyl alcohol on an industrial scale from grain; Hodge and Hildebrandt (140C) described the production from molasses; and McCarthy (Z&C), the production from sulfite waste liquor. The fermentability of maltose was found to depend in part on the presence of traces of glucose. The still residues from incompletely fermented maltose solutions were further fermented t o produce the expected quantity of alcohol based on sugar determination following hydrolysis; the reducing power of these residues was lower than t h a t calculated for unfermented maltose. The results were consistent with the interpretation that a polymerization reaction is one of a series of competing reactions involved in the fermentation of maltose (19C). The amylo process for production of ethyl alcohol from grain mashes continued to receive study in laboratories in Japan. Selected strains of Aspergillus species including A. niger, A . usamii, A. oryzae, and A . awamori were found in laboratory and pilot plant studies to be useful in hydrolyzing the mashes in a two-stage process. Pields equivalent to SS% of theory were obtained (%94C, 286C). The addition of nitrogen containing supplements to media used in preparing the diastatic preparation was advantageous in the two stage process (22.i.C). I n a one stage process best results were obtained when a strain of Rhizopus javanzcus was used to inoculate the grain mash (2282). Pretreatment of the concentrated mashes with dilute acid reduced viscosity and permitted more extensive hydrolysis of the polysaccharide by the fungal enzymes (289C). il number of oligosaccharides, including sakabiose, kojibiose, isomaltose, dextrantriose, and Panose, were prepared by the action on nialtoee of diastatic preparations from fungi (3132, 314C). Glycerol. While all of the glycerol produced commercially in the United States has been made either as a by-product of the manufacture of soap or from propylene, there has been a continuing interest in fermentation processes, and plans have been made to use the latter in emergencies (46C). Underkofler (367C) summarized many of the aspects of the processes that have been suggested for the production of glycerol by yeast or bacterial fermentation of molasses or grain mashes. An alternative to the processes where alkali or sulfite salts are added to yeast fermentations to induce production of glycerol is the growth of the yeast on media containing an antivitamin BI. Addition of sulfathianole to media resulted in glycerol yields equivalent to 347, of the glucose present in the medium (100C). ORGANIC ACIDS
Acetic Acid. While the production of acetic acid from solutions containing ethyl alcohol is one of the oldest recognized fermentations, there are still many problems to be solved if the maximum process efficiency is to be realized. Vaughn (360C) has described in detail the industrial production of vinegar as carried out in the United States where annual production by the Frings generator process (or similar units) has been of the order of 24,000,000 gallons. The facets of the submerged culture process include a relatively small equipment investment, a continuous fermentation operation, and attainment of theoretical yields (149C). An evaluation has been made of the conventional generator process on a pilot plant scale. The nitrogenous compo-
Vol. 46, No. 9
nents of the nutrient solution markedly affected rate of alcohol oxidation by Acetobacter xylinum (W). Ascorbic Acid. In the Reichstein process for the synthesis of ascorbic acid, sorbitol is oxidized t o sorbose by Acetoba , suborydans, the sorbose is converted by chemical synthesis to diacetone-2-keto-Z-gulonic acid and ascorbic acid. An alternative process to this predoniinant'ly chemical synthesis has been suggested in which glucose is converted by A . suboxydans t,o 5-keto-d-gluconic and the latter is reduced with hydrogen t o 1-idonic acid. This product is then converted by Psertdomo??as jfuorescens to 2-keto-1-gulonic acid and subsequently by acid to ascorbic acid (144C, 145C). The chemical preparation of Z-idonic acid from 5-keto-d-gluconic acid has received attention and methods proposed for reducing the quantity of gluconic acid formed as a by-product (S86C). Selection of culture, presence of other nutrients in the fermentation medium, and ferment'ation conditions all affected the conversion of l-idonic acid to 2-keto-1-gulonic acid (SSSC-SS6C). Under optimal conditions nearly theoretical yields were obtained in a 3-day fermentation. Fact'ors affecting the conversion of sorbitol to I-sorbose have also been studied, and the instability of sorbose in neutral solution has been shorn ( S C ) . Mother liquors from the productioii of sorbose also contained significant quantities of galactose. glucuronic acid, acetic acid, and galact'uronic acid which may have arisen in part from microbial action (182C). Citric Acid. Shu (317C) has s h o m the import'ance of adequatc: aeration in the production of citric acid in submerged culturc. When a selected strain of Aspergillus niger was gronm for 6 days on a sucrose medium and aerated with air, a 42% yield (based 011 sucrose available) was obtained. A i O % yield was obt'ained when oxygen was passed over the agitated culture. The presence of carbon dioxide in the gas above the liquid m-as essential to high yields of acid in surface culture ( 1 Z l C ) and submerged culturo (318C). Other investmigationsshon-ed that citric acid yield JTas inversely proportional to the iron content of the 47 samples of Indian conimercial sugars used in the experiment's (189C). Similar results were obtained by Shiga (316C)in studies on the effect of medium composition, purity of sucrose or molasses, and strains on cit,ric acid production. Untreated molasses w a s not suitable for cit'ric acid fermentations b u t maximum yields (about 527,) were obtained when molasses was purified by a coprecipit'ation met,hod using H,POa and Ca(OH)*. Ion exchange treatment was not so effective as this coprecipitation procedure. Procedures useful with one strain were not always satisfactory for the others studied. Addition of cyanide to fermentations reduced the utilization of citric acid by A. niger during the fermentation of glucose to citric acid (57C). Aspergillus fumaricus was also used to convert sugars to citric acid. Yields of 527, were obtained when sucrose LTas fermented. Lon-er yields were obtained when media containing raffinose maltose, lactose, fructose, galactose, glucose, arabinose, or xylosc were fermented (311C). Johnson ( 1 6 S C ) has reviewed the literature concerned with production of citric acid by the surface culture and submerged culture processes, and included a discussion of medium composition, fermentation conditions, and commercial scale operations. Gluconic Acid. Underkofler (367C)reviewed t'he literaturc concerned with the production of gluconic acid by the bacterial and mycological processes and included a discussion of commercial operations. Other investigations showed that 2,5-dilretogluconic acid was formed from glucose, gluconic acid, or 2-ketogluconic acid by Acetobacter ?~zelanogenum(171C). Enzymatic processes for the conversion of glucose t o gluconic acid (SC)and 2-ketogluconic acid (5bC) have been described. Lactic Acid. Schopmeyer (509C) has prepared a n authoritative discussion of problems encountered in the production of lactic: acid on a commercial scale in the United States; substitution OS cheaper carbohydrate sources for the glucose used as the sub-
INDUSTRIAL AND ENGINEERING CHEMISTRY
September 1954
strate has nearly always resulted in increased costs of recovery of the lactic acid and no economic saving. Process development in Japan has included a study of lactic acid production by aerobic spore-forming bacteria. Acid-hydrolyzed sweet potato starch mash (15% sugar) was fermented to 1-(dextro)-lactic acid in 4 to 5 days on a semi-industrial scale with nearly theoretical yields (122C, I W C ) . Operation of this process in open wooden tanks often resulted in racemization of the lactic acid due to the activities of contaminating bacteria ( I a d c , I73C). Water-soluble growth factors required for the growth of the lactic-acid producing aerobic sporeforming bacteria may be supplied by rice bran (12SC). The use of an extract from mycelium obtained from penicillin processes has been suggested as a source of these factors for the growth of Lactobacillus delbruckii (279C) as an alternative to corn steep liquor or malt sprouts. VITAMINS
Riboflavin. Hickey ( I 3 4 C ) has reported that yields of the order of 1 mg. per ml. were obtained when Eremothecium ashbyii was grown on a medium containing glucose, maltose, sodium glutamate, inorganic salts, inositol, thiamine, biotin, oleic acid, and a polypeptide fraction from casein or gliadin. This requirement for a polypeptide fraction, biotin, inositol, and thiamine was also related to riboflavin production by others (7'2C, %lSC-d15C, 317C-219C). Some stimulation of riboflavin production was observed when hexachlorobenzene or plant hormones were added to media (d13C, S S I C ) . Yields of 1.5 mg. per ml. were obtained when E. ashbyii was grown in submerged culture on a medium containing peanut meal, corn steep liquor, and glucose (132). A process for the production of riboflavin by the growth of E. ashbyii on a mixture of copra meal and rice bran has also been described ( I 5 6 C ) . A t the end of the incubation period the mash was dried, pulverized, and used as an animal feed supplement. So (324C) has reported a number of compounds produced by E. ashbyii which may be responsible for the odor of growing culture. Processes for the production of riboflavin by the related microorganism, Ashbya gossypii, have been described by Van Lanen (359C) and by Hickey (135C). I n the former the yeast was grown on a medium containing carbohydrate, salts, biotin, thiamine, inositol, and a protein hydrolyzate or amino acids. I n the latter it was found that addition of 5 p,p.m. of cobalt, as Co(NO&, to a medium containing glucose, corn steep liquor, beef stick liquor, casein hydrolyzate, and yeast extract increased riboflavin production (in shaken flask experiments) from 0.58 to 0.75 mg. per ml. Presumably this supplement also enhances vitamin BIZ production by this microorganism (32SC). Cobalamins. Studies of the chemistry and production of the vitamin BIZor cobalamin complex ( 1 8 C ) have continued in a number of laboratories. Reviews of the information on the chemistry of this group of compounds have been prepared by Wolf and Folkers ( S 7 5 C ) and Wijmenga (37fC),while Wuest (S80C) has discussed their industrial preparation. The relationships of the various factors may be summarized as follows (d78C): Cobalamin
2
Pseudocobalamin Adenylic acid
Factor A The isolation of 2-methyladenine from pseudovitamin BUD distinguishes this growth factor from the pseudovitamin BIZA
1811
produced by anaerobic bacteria and which contains an adenine residue (7SC, 142C, 276c). A rather detailed description of the process used in production of cobalamin on a commercial scale a t the Dawe's Laboratories has been prepared by Hester and Ward ( I S S C ) . A vitamin BIZ producing strain of Streptomyces olivaceus (118C) as grown for 3 to 5 days on a medium containing 4% distiller's solubles, 0 5 to 1.0% glucose, 0.5% CaC08, and 0.00015 to 0.001% CoCh 6Hn0. The reported yields of 1 to 2 mg. per liter are somewhat lower than those reported elsewhere (I2UC). The fermented medium was concentrated by evaporation, dried on steamheated drum dryers, and used for animal feeds. Significant quantities of niacin, biotin, folic acid, riboflavin, pyridoxine, and pantothenic acid were synthesized by this microorganism
-
(laoc).
Several processes employing Streptomvces griseus have been described, I n one the organism was grown on a fish mealglucose-yeast-salts medium for 44 hours to yield 0.28 mg. of vitamin BIZactivity per liter ( 7 4 C ) , while in another a yield of 0.6 mg. per liter was obtained using a sucrose-salts-glycerolprotein hydrolyzate medium ( 8 5 C ) . Substantially all the vitamin activity produced in this latter process was found in the microbial cells. To produce cobalamins or related factors it is generally necessary to add a cobalt salt to the media. S. griseus was reported to be capable of utilizing the cobalt present in a large number of inorganic and organic complexes as a precursor for cobalamin synthesis (268C). A cobalt complex found in yeast grown on cobalt-containing medium was found to be more efficiently converted to cobalamin than was the cobalt of C o ( N 0 3 ) ~(27UC). McDaniel and Woodruff (207C) stated that when S. griseus is grown on a medium containing casein hydrolyzate, beef extract, and salts and supplemented with X&Fe(CN)G,Ca(CN)2or KCN, cyanocobalamin is formed by transfer of the cyanide from these complexes to the cobalamin formed. The cyanides were added to the media, before autoclaving, or R ere sterilized separately. No study of the disappearance of these cyanides during preparation of the medium or during the fermentation was reported. Other sources of microbial origin that have received attention recently have been sewage ( S I l C ) , herring meal ( 3 4 4 3 , Ustilago zeae fermentations ( I 2 8 c ) ,and fermentations employing various streptomyces species ($5C, 119C, SUSC). Sufficient data are not available to determine what forms of the vitamin Bls-like materials can be obtained from these sources. DEXTRAN
The literature concerned with the use of and requirements to be met by substances designed as blood plasma expanders has been evaluated by Bulloff and Novak (38C). Polyvinyl pyrrolidone, oxygelatine, and dextran have all been approved by the National Research Council for use as blood plasma expanders (55C).
A number of reports have dealt with production of dextran by Leuconostoc mesenteroides. When the nutrient content of the media was reduced in order to slow the growth rate, the sucrose content was increased to 30%, and dextran and maltose were added to the medium as primers, the average molecular weight of the dextran formed was 75,000 i.25,000 (243C). This process may be more economical than that described by Lockwood (19SC) where the fractionation of the depolymerized dextran was found to be the most costly operation in the process. James (169C) has described some of the steps in the production of dextran on a commercial scale including depolymerization of the dextran by exposure to ultrasonic energy and fractionation of the hydrolyzed product by acetone. Holmlund (143C) summarized a search for native bacterial polysaccharides as potential plasma extenders. Screening procedures were developed for native polysaccharides based on dialysis through gelatin-agar membranes of known porosity, viscosity determinations, and osmotic pressure measurements of t h e polysaccharide solutions. In all the studies clinical grade dextran
1812
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
was used as the standard of comparison. Study of the polyEaccharide produced by a spore-forming organism of the Bacillus subtzlzs type showed that a levan with a molecular weight equivalent t o that of the clinical material could be obtained in a 24-hour fermentation in yields equivalent to 40 to 60% of theory. Other investigations showed that in the hexosan mixture produced by Baczllus subtihs ( 7 l C ) and by Bacillus polymyxa (dZQC),the ratio of fructose to glucose units varies from 1:2 to 6: 1. Koepsell studied the use of a cell-free preparation of dextransucrase t o produce a dextran having a molecular weight of 75,000 from sucrose on a pilot plant scale. Best results were obtained when dextran with a molecular weight of 25,000 was used as a primer for the reaction. Yields of clinical dextran (mol. wt. 75,000) equivalent to 25 t o 33% of the sucrose added were obtained ( I S I C , f8OC, S62C). The rate of addition of sucrose t o the dextran-sucrase solution had little effect on the size of the dextran formed. High molecular weight dextrans were formed when incubation was a t 30" C., Trhile low molecular weight dextrans predominated when the mixture was held a t 15" C. Best riclds of clinical dextran were obtained when the reaction mixture 6 a s incubated a t 15" C. and contained (per ml.): 40 dextransucrase units; 100 mg. sucrose; 2 to 20 mg. of primer (mol. wt. 25,000); pH 5.0 buffer ( S 6 S C ) . The chemistry of bacterial dextrans has been reviewed by Bourne IR6C) and Stacev (SW7C). The relationship of the structule t o the method used in fractionation has been emphasized in a number of studies concerned with depolymerization of native dextrans and recovery of particles Jvith molecular weights of the order of 75,000. Acid hydrolysis (14 f C. 209C, 376C), treatment with ultrasonic energy ( 7 9 C ) , and degradation in an alternating electrical field (Z66C) have been used. Mellies (209C) concluded, on the basis of a study of hydrolysis of dextrans from different sources (and presumably of different composition), that special hydrolysis conditions were required to depolymerize each type of native dextran to materials mith a molecular weight of 75,000. Dextrans have been suggested as ingredients in lacquers, adhesives, candy fillings, beverages, and emulsifiers, as well as suspending and stabilizing agents in pharmaceuticals and fillers in textiles (48C).Use as a soil conditioner apparently will depend on the economics of production ( I Q S C ) . Sulfated dextran has been compared in clinical tests with heparin and found t o have similar action without the side effects often noted when heparin is used t o delay blood clotting (Z90C-292C). MICROBIOLOGICAL TRANSFORMATION OF STEROIDS
Vol. 46, No. 9
and Peterson ( Z S I C ) . The use of cell-frec enzymes has been considered (272C, SdOC, S42C) and may lead t'o better control of the reactions. I n addit'ion to making use of microorganisms to osidize or reduce st'eroide, i t has been possible to hydrolyze steroidal sapogenins b y microbial action to release the steroid nucleus. Bacterial ( S O I C ) and fungal (186C) fermentations for hydrolyzing the saponins obtained from Dioscorea compositu and other roots have been described, as has a method for hydrolyzing digitonin by Bacilius i?aacerana (SO6C). Another report ( 2 6 I C ) suggested that mild acid hydrolysis of cells of Bacillm licheniJormis, Bacil2us subtilis. Sermtia marcescans, Lactobacillus leichmanii,a n d EscheTichia coli j-ielded substances xhich had adrenocorticotropic activity as measured by the Savers bioassay for adrenocorticotropic hormones. ANTiBlOTlCS
Several general art'icles and books have appeared during thc past year covering the broad field of ant'ibiotic production, properties, and uses. Two reviews ( I C , lR6C) vere concerned primarily n-ith penicillin, while another mentioned the naturally occurring peptide antibiotics (JIG'). Actinomycetes and their antibiotics were reviewed by Benedict ( 1 6 C ) and Waksman and Lechevalier (366C), while t'he role of antibiotics in the physiology of the organisms was covered by Gottlieb ( I O S C ) ,Villemin(S6'1C), and Nickerson and Ram Xohan (26%'). Penicillin. As noted above, penicillin has maintained its posit,ioii as t,he largest, volume antibiotic and extensive research on its production has continued. This has been concentrated on finding better precursors for benzylpenicillin biosynthesis (6ZC) and new types of penicillins. A penicillin recently int,rnducetl in Europe in clinical practice is penicillin V (SSC, I Z 9 C ) or phenoxymethylpenicillin. It is claimed to be more acid stable t,han benzylpenicillin, and of value when administered orally. A study of the metabolism of labeled phenylacetic acid added to fermentations has shown that all the carboxyl carbon ol the precursor was concentrated in the C=O group in the side chain ( S I O C ) . I n a similar study, 93% of t,lie total radioactivity found in the molecule was located in the C=O adjacent to the benzyl
Earlier review in this series have summarized the background of researches on microbiological transformation of steroids, and considerable effort along- these lines has been expended during the past 12 months. Peterson (B72C) reviewed the literature concerned ivith the microbioTable 1. Microbial Transformation of Steroids logical approaches to the preparations of cortisone and hyEfficiency of Transformation, drocort,isone ( 1ia-hydroxl-corProduct Formed Microorganisms % Substrate ticosterone). These a p p r o a c h 17a-hydroxycorticosterone Cuiininghamella blakesleeana 35 ll-desoxy17y h y d r o x y - 17a-hydroxycorticosterone Strepfomycesjradiae 1.; have been considered economiC u r d a r i a lunata 35 corticosterone 17 a-hydroxycorticosterone cally feasible by Djerassi ( 7 5 C ) 8 , 17a, 21 trihydroxy-4-pregHelicostylium p i r i f o r i n e 40 nene-3-20-dione \Tho reviewed synthetic chemiAI-dehydrotestololactone Cylindrocarpon radicicola 50 Ad-androstenedione Penicillia and Aspergilli ... cal and microbiological p1.0~Testololactone Penicillia and Aspergilli essep. -1 combination of miBp-hydroxy-1 1-desoxycortiStreptomyces fmdiae Pi'(appr0x.) ll-desoayC G I ticosterone costerone crobiological a n d c h e m i c a l Corticosterone Streptomyces endus 2.0 Aspergillus fumigafus 1 4 Corticosterone treatment,s has led to steroids 1Ga-Hydroxy-11-desoxycortiStreptomycetes 20 (approx.) with more biological activit'y costerone Aspergillus nidulans 10 11a-Hydroxycorticosterone than the naturally occurring A ) ,%ndrostadiene-3,17-dione Streptomyces lauendulae 7 Progesterone Cylindrocarpon radicicola 50 A 1-dehydrotestololactone substances (SjC, 96C). .. A l~~-androstadiene-3,17-dione Fusarium solani T h e yarious t,ransfornlatioils Gliocladium calenulatum , . A4-androstene-3,17-dione Penicillium lilacium ,. of steroids carried out by miAspergillus s a w s .. Gliocladium catenulatum .. Gp-hydroxyandrostenedione croorganisms have been reAspergillus f l a w s ; Penicil.. Testololactone vien-ed by Dorfinan and Unlium adametzi R h i z o p u s nig-ricans 25 11 a-hydroxyprogesterone gar ( 7 6 C ) . A number of trans36 17a-hydroxy11a-17a-dihydroxyprogesterone Rhizopus nigricans Rhizopus n i g w a n s .. progesterone Gp-17a-dihydroxyprogesterone formations reported since their A s p e ~ g i l l u spauus .. Testololactone 28 tabulat,ion are summarized in 11a-hydroxy-17a-progesterone Rhizopus nigricans 1G-dehydroprogesterone Table I. A general discussion Rhizopus arrhizus 23 11a-hydroxyandrostenedione 4-androstene3 17-dione of techniques used in anaRhizopus nigricans 10 11a-hydroxy-17n-methyltes17;-methyltestosterone tosterone lyzing steroid mixtures has been presented by Murray
Lit.
Cited (196C)
(62C) (319C) (OS7C) (97C) (273C) (273Cj (IlSCj
(1IGC) (IlGC) (,?62C)
September 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
group, 1.5% in the p-lactam C=O, and 5.5% in the remainder of the molecule. Furthermore, 82% of the benzyl chain must have come from the phenylacetic acid (1OSC). Phenylacetic acid, when used as a precursor for biosynthesis of benzylpenicillin, can be oxidized to o-hydroxyphenylacetic acid. This latter compound can either be metabolized to carbon dioxide and water or serve as a precursor for o-hydroxybenzylpenicillin biosynthesis (261C). On the other hand, N-methylphenacetamide will not assist in the formation of o-hydroxybenzylpenicillin. The acetic, propionic, or butyiic acid esters of phenethanol were used more efficiently than phenylacetic acid as precursors of benzylpenicillin (271C). Up t o 40% of the compounds were converted to benzylpenicillin with a single addition to the medium while only 5 to 10% of the phenylacetic acid added under the same conditions was converted to benzylpenicillin. Similar results were reported when N-(2-hydroxyethyl)phenacetamide TT as used as a replacement for phenylacetic acid (86C). When glycerol or ethylene glycol esters of phenylacetic acid or castor oil esterified with phenylacetic acid were used as antifoam agents, penicillin yields were increased above those attainable with a mixture of soybean oil and phenylacetic acid (222C). A total of 0.08% of the esters was added in four aliquots during the fermentation period. Penicillin W, an apparently new biosynthetic penicillin, was found in filter-paper chromatograms of samples taken from cultures of Penicillzum chrysogenum &176 growing in a corn steeplactose medium without added precursor (117C). Benzylpenicillin, p-hydroxybenzylpenicillin, and cephalosporin N were also present in these samples. When p-aminophenylacetic acid was added to fermentation media, p-aminobenzylpenicillin was produced (3OC). Best results were obtained when 0.01% of the precursor was added t n ice daily during the fermentation period. This penicillin has been shoivn to maintain higher blood levels in animals than those obtainable with benzylpenicillin. Allylmercaptoacetic acid, the ethanolamide of allylmercaptoacetic acid, and allylmercaptoethanol were found to be satisfactory precursors for biosynthesis of allylmercaptomethylpenicillin (penicillin 0) ( 9 4 3 . Limited work has been reported on compounds proposed to be precursors for the other portions of the penicillin molecule (dC, 6%'). Three-carbon (aminomalonsemialdehyde) and five-carbon (a-amino-p-thio isovaleric acid) fragments have been used as possible precursors for the thiazolidine portion of the molecule. A study of the metabolism of P. chrysogenum strains Q 1 7 6 and KN2326 revealed that the oxidation-reduction potential, glucose content, and total nitrogen declined during the first 7 days of incubation (S87C), while the values rose with increased incubation. hfycelial yield reached a maximum 2 days before the peak yield of penicillin was attained. Penicillin was produced by nearly half the Penicillium strains studied (2442, 246C) in a survey of antibiotic production by cultures isolated from soil samples. hfetabolism of formate, acetate, and lactate labeled with C14 has been studied and incorporation of formate, acetate (both carbons), and carboxyl carbon of lactate in the thiazolidine portion of the penicillin was found ( I & X ) . When cultures are grown on media containing glucose or ~ u crose as the sole carbohydrate source only minimal amounts of penicillin are produced unless the sugars are added continuously throughout the fermentation period (SSC). I n a corn steep medium with phenylacetic acid as the precursor (added continuously), optimum feed rates for glucose were determined as 0.042% per hour (3.0% total) 0.07% per hour (total 5.0%) for sucrose, and 0.034% per hour (2.5% total) for lactose. Feed was initiated 24 hours after inoculation of the fermentation, and continued for 72 hours. At the optimum feed rates maximum potencies obtained were 1520 units/ml. for glucose, 1470 units/ml. for lactose, and 1410 units/ml. for sucrose, compared to 1610 units/ml. for the
1813
control fermentation in which the medium a t batching contained
3% lactose. Thus glucose and sucrose, which are cheaper than lactose, might well be used on a commercial scale and offer economic advantages. Other studies have shown that arabinose and xylose can be used in media designed for the production of penicillin (16OC). When a synthetic medium was used, yields of penicillin higher than those obtained when the cultures were grown on lactose media were obtained by feeding 0.03% glucose per hour (326C). Other potential energy sources in the fermentation are oils. It has been shown that P. chrysogenum mycelium, particularly older cells, will oxidize lard oil (298C). I n other Etudies, when oil was fed continuously to a fermentation, starting 20 hours after inoculation, yields were increased 50% above those obtained when oil was used only as a defoamer (S49C). One gram of oil, 80% of that added, was utilized per 100 ml. of medium over the complete fermentation period. The pH control was simplified by the use of oil. Unsaturated fats were utilized a t a more rapid rate than saturated fats, and any not used were almost completely hydrolyzed to free fatty acids. The utilization of various sulfur containing amino acids and peptides by the organism have been investigated. The sulfur from I-cystine, I-cysteine, dl-methionine, and glutathione was used in preference to that in inorganic sulfates (331C). In other studies, using labeled substrates, glycine and dl-cystine were found to be incorporated into the p-lactam C=O, while dlvaline was incorporated into the penicillamine moiety (2C). Of these amino acids, dl-cystine was found to be the best precursor for the biosynthesis of the thiazolidine moiety of the three substances reported. Another report states that d-cystine was found to be the only one of the four forms tested (1, d l , m, and d ) effective in penicillin synthesis (335C). Streptomycin. Waksman (365C) in his Nobel prize address reported on the history, properties, and uses of streptomycin. When Streptomyces griseus was grown on a medium containing organic sources of nitrogen and carbon, mineral salts, and certain growth-promoting substances 100 to 200 pg. of streptomycin per mI. were produced after 3 to 5 days' incubation. Other reports have shown t h a t yields of 300 pg. of streptomycin were obtained when a selected strain of S. griseus was grown for 110 hours on a peptone-meat extract-glucose-sodium chloride medium (16OC) and somewhat higher yields were obtained by adding a supplement of iron salts to this medium (287C). Considerably higher yields are probably obtained in commercial operations. Hydroxystreptomycin, produced by Streptomyces griseocarneus ( I Y C ) has been shown to be identical Xyith reticulin produced by 8. reticuli (146C-148C). Certain strains of Streptomyces olivaceus which are primarily important in the production of vitamin Biz are also capable of synthesizing streptomycin according to one report (17 8 2 ) . Tetracycline Group. The newest addition t o the group of broad spectrum antibiotics is tetracycline, produced by chemical synthesis (SSC, Z4C, 64C) and directly by fermentation (EX, 63C, 155C, 21dC). The compound has the same basic structure as chlortetracycline (Aureomycin) and oxytetracycline (Terramycin), differing only in the absence of the C1 and OH groups, respectively. The three compounds, with similar antibacterial spectra, were shown to behave differently with regard to diffusion rates in agar, stability in aqueous solution, and behavior in filter-paper chromatography (2bC). The steps taken to determine the structure of oxytetracycline and the structure itself were reported by Hochstein (139C). Although no details have been made available concerning the production of tetracycline by the fermentation process, some improvement in production was gained by culture selection studies (212C). Reports have been made of laboratory and clinical tests (28SC, S84C, S Y S C ) and the antibiotic has been approved for medical use. Chloramphenicol. The production of chloramphenicol has
1814
INDUSTRIAL AND ENGINEERING CHEMISTRY
continued primarily by chemical synthesis. Hox-ever, fermentation studies showed that glucose, starch, glycerol, and maltose were suitable carbohydrate sources, while sucrose was unsatisfactory (199C). Pyruvic, citric, ketoglutaric. succinic, malic, and acetic acids Rere detected in the broths. A general description of the fermentation changes occurring during groath of Streptomyces venezuelae with no antibiotic formation was reported (11OC). Erythromycin. -4product patent was issued to Bunch and McGuirecoveringerythromycin, its salts, and methods of preparation ( S 9 C ) . When S . erythreus was grown in a synthetic medium containing glucose, glycine, alanine, and salts, a second crystalline antibiotic, designated as erythromycin B, was found (274C). This was separated from erythromycin by paper and column chromatography. Although the spectra of the tivo substances are similar, erythromycin B is only 75 to 85% as active as erythromycin in inhibiting the growth of microorganisms. Chemical properties, derivatives, and degradation studies of these antibiotics have also been reported (59C, 91C, SSOC). Carbomycin. Production of carbomycin by Streptomyces halstedii was mentioned in the last report. More recent studies have sho\\n that this cultuie produces a second antibiotic chemically related to carbomycin (1SSC). Production of carbomycin by other cultures has also been reported (78C, WSOC), and there is evidence that griseomycin is related chemically to the carbomycins (268C). Neomycin. A patent has been issued covering the production of neomycin by Streptomyces fradzae when grown on a medium containing soy peptone, meat extract, glucose, and sodium chloride (SOtC). Other studies have shoan that the inorganic content of the meat extract was responsible for directing the fermentation to the production of neomycin A (neamine) rather than the other neomycins. Lipids. maltose. or starch could be utilized as energy sources for production of neomycin by this actinomycete (269C). Other Antibiotics. I n addition to the antibiotics of commercial importance a t the present time there are certain others t h a t are of interest because of the organisms which they are reported to inhibit. Viomycin, produced by Actinomyces vinaceus ( I 9 7 C ) , Streptomyces puniceus (88C), and Streptomyces JEoridae (SZC) appears t o be another substance which can be used in the treatment of tuberculosis, particularly in combined therapy with other antimicrobial agents (64C, 137C). Synnematin (111C)has shown in vivo activity against Salmonella infections and may be of value (256%'). I t s relationship t o cephY has not been clarified ( I I 7 C , ISOC, 252C). Procealosporin i dures for the production, recovery, and purification of synnematins A and B have been reported (257C, 277C). Several antibiotics that are active against fungi have received intensive study. Ascosin, produced by S t r e p t m y c e s canescus when grown on a dextrin-soybean meal medium ( I S S C ) , may be related to candicidin (49C, 15gC, 187C). The latter has been found to have in vivo activity as n d l as in vitro activity (177C, 187C), but some undesirable toxicity was noted, limiting the potential of this material as a chemotherapeutic agent. The preparation and properties of nystatin (formerly called fungicidin) ( 3 6 C ) were reported ( 7 7 C ) . Another microorganism, Streptomyces aureus, n as discovered which produces an antifungal substance similar t o nystatin (288C),while a variant of Streptomyces noursei, the original nystatin producing species, was found to produce an antibacterial agent, phalamycin ( S S C ) . Of possible interest in the field of tumor chemotherapy are a number of antibiotics including actinomycins (SSC-S5C, 19OC, S64C),azaserine (IOC, 60C, 8OC, 81C, 102C, ZOSC, 254C, SSWC, S S S C ) , puromycin (99C, ISZC, S51C), and sarkomycin (258C, S Z C , 356C), all metabolites of streptomycetes. Encouraging results were obtained Tith all of these substances in laboratory tests. Helenine ( S 1 6 C ) isolated from Penicillium funiculosum was shown t o possess antiviral activity in in vivo studies in mice. Substances from Nocardia formica (127C, 205C) and Penicillium stoloniferum (281C, 282C) were also reported t o be active against viruses. Fumagillin ( 1 2 5 C ) is of interest since it has specific activity against intestinal amoebiases while other intestinal flora are unaffected (5OC, 162C).
Vol. 46, No. 9
MICROBIOLOGICAL SULFUR PRODUCTION
The microbiological production of sulfur has been shown to be quite feasible t'echnically. I t s industrial application is a matter of economics. Butlin (42C-44C) has reviewed the literature and evaluated the progress in making the process economically attractive. While the technical efficiency of the process has been demonstrated in laboratory and pilot plant studies (43C, 47C, $64C! 2?9C, 88OC), the primary economic problem is the supply of sufficient cheap raw maberials. The chief raw materials required are sulfate salts, a reducing agent which may be either an organic material or hydrogen gas, and nutrients for the growth of the bacterium, Desulfovibrio desulfuricans. The nutrients for bacterial growth would probably be present in any suitable organic reducing material b u t would have t o be supplied if hydrogen were employed as reducing agent. The cost of sodium or magnesium sulfat'es recently has been about $40 per ton, with unconditioned sulfur costing approximately the same price. Sewage sludge is apparently the only suitable organic reducing agent available in large quantities. I t s use would depend on whether sulfide product'ion can be combined with the use of sludge as a source of methane. Hydrogen gas possesses many advantages as a reducing agent but t'here are no cheap supplies available at present
(47C). An alternative to fermentations in factories is the exploitat,ion of sulfide-containing nat,ural mater. Sulfur deposits on lakes in Cyrenaica, Australia. and elsexhere have been observed to be formed by microbial action. The only requirements for the process would be sunlight for the photosynthetic, sulfide-oxidizing bacteria, Chromatiurn and Chlorobium, and a source of nutrient. The latter requirement might be supplied by sewage and the natural process of conversion of sulfide to sulfur hastened, according to Butlin (42C, 2 g I C ) . MICROORGANISMS FOR FOOD AND FEED
Within the past year considerable attention has been focused on methodj of production of algae with the objectives of providing food in areas where population densities and/or limited natural resources result in a shortage of food and food materials. Whether this will provide a solut,ion for the shortage still remains to be determined. Algae. The summary by Gaffron (104C)and the more extensive discussion edited by Burlew (4OC) have included consideration of many of the economic factors concerned in the production of Chlorella on a large scale. Yields were estimated to be as high as 35 tons per acre per year, as compared with 0.75 ton per acre per year of soybeans. These estimates have been questioned by responsible investigators (I14C, 167C, 276C). Furthermore, the cost of machinery needed to produce 50 tons of cells per day may be as high as $25,000,000 or about $140 per ton of cells (based on a 10-year amortization). Such an investment appears excessive in light of the present cost of soybeans ($75 per ton) or menhaden meal ($35 per ton) (114C). When the cost of operat,ing the machinery is added to this, the cost of algae becomes even more prohibitive. Studies on the efficiency of light conversion by Chlorella cultures, using both continuous and intermittent light, have been carried out on a laboratory and pilot plant scale (179C, S@C, 3432, SSSC), and progress has been made in obtaining efficient processes. Yields of the order 0.3 gram dry cell material per liter have been obtained in pilot plant units operated on a. COIIt,inuous basis (6'?C, 191C). Use of strains grorTing a t high temperatures may help t,o reduce the refrigerat,ion needed to maint'ain the ponds a t uniform temperatures (326C). Information on the nut,rition of algae has been summarized in several reviews (ZOC, 92C, SSC, 183C, 184C, 242C). The fermentation conditions which viere used affected the composition of the cells markedly, with t'he nitrogen content of the medium indirectly determining t,he lipid content (93%'). .4study of
September 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
the composition of the Chlorella produced in pilot plant and other operations gave no indications that the cells contain materials of apparent industrial interest (89C);the sterols present in Xcenedesmus obliyuus might be of interest as a starting material for cortisone synthesis if made available in quantity (183C). The nutritional value of Chlorella appeared to be about the same as other plant materials according to preliminary analyses, but large scale feeding trials have not been completed (QOC,5102). The pilot plant studies which have been summarized in a number of papers (6QC, 84C, 106C, 116C, ’164C, 191C, SdOC, 374C) have shown that conditions used in one area are not entirely satisfactory in other places where sunligiit periods, temperature controls, and available machinery are not equivalent. Burlew ( 4 f C )has called for the construction of a demonstration plant with a total growth area of one a( re where data concerning the labor required and cost of operation of maintenance of large installations could be collected. Combined algal growth and sewage disposal has been suggested as a useful process (168C, 19@, S69C). The drawbacks appear to include the large space required (15 acres per million gallons of sewage per day), the cost of recovery of the algae from such dilute suspensions, variations in the composition of the sewage, and fluctuations in operating conditions arising from variations in temperature, light intensity, and light periodicity. An alternative to the use of algae directly as food might be that of feeding the material to fish and using the latter for food (166C), or using the material as a substrate for a methane fermentation (16SC). Yeast. Irvin (157C) discusses pure-culture maintenance, contamination problems, yeast propagation, handling of fermentation operations, recovery procedures, and properties of the yeast. An extensive survey of a more technical nature has been prepared by Walter (367C). Laboratory studies conducted by Maxon and Johnson (203C) using continuous fermentation techniques with three synthetic media showed that in aeration the gas film was the limiting factor in oxygen transfer. Production rates as high as 13.2 grams of yeast per liter per hour were attained with airflow rates of 6.1 liters per minute per 1.5 liters of medium with a holdup time of 3.14 hours in the fermentor. The protein content and methionine value of 8.cerevisiae N-ere increased to 10% by adding more nitrogen salts to the cane molasses-cornsteep liquor medium (66C). Wiley (372C) has collected available information concerned with the preparation and properties of food and feed yeast including raw materials, economics, production methods, and a description of the process used in the production of food yeast from sulfite waste liquor. The primary use of this material has been in the preparation of animal feed supplements. The use of potato starch wastes has also been suggested as an economic process for profitable disposal of this waste material, and yields of the order of 45% of the 1.2% solids from potato waste were obtained in batch and continuous fermentations in pilot plant apparatus (28SC). The potato waste contained sufficient nitrogen and carbohydrate for growth of the yeast. I n another study, i t was found to be a superior substrate to sulfite waste liquor (37C). Molasses ( 1 1 C ) spent amyl acetate liquors from penicillin recovery operations (Sf SC),and pentose-containing liquors (255C) have also been used as substrates for the growth of T. ulilis. Thaysen (16C, 346C) has pointed out that T. ufilis converts 80% of the ammonia added to molasses media to protein a t extremely high speed. Certain strains of Candida were found to be useful in the production of yeast protein from mannitol-containing sea-weed hydrolyxates (347C), orange juice (1Q2C), and sulfite waste liquor ( l 7 5 C ) . Synthesis of radioactive sulfur-containing amino acids by the growth of 8.cerevisiae or T . ufilis on media containing S3504 salts has been found to be practical since 95% of the sulfur added was converted to cystine and methionine (307C, d77C, 378C). Recent studies have also shown the presence of hemoglobin in S. cereuisiae, T . utilis, and fungi ( l 7 2 C ) .
1815
Bacteria. Processw for producing animal growth factors and feed supplements by the growth of certain bacilli in nutrient media have been described. D r y cell yields from 20 to 60 grams per 100 grams of sucrose were obtained in 6 to 12 hours when Bacillus megaterium was grown on a molasses-salts medium (188C). Symbiotic growth of S. cerevisiae and Aerobacter cloacae or A. aerogenes on distillers slops resulted in a vitamincontaining product with value as a feed supplement ( 7 0 C ) . Fungi. The value of mycelium of Penicillium and Streptomyces as feed supplements has been studied and found equal to such materials as fish solubles as a source of “unknown” growth factors (306C). An analysis of S. griseus rells showed the presence of 55% protein and 2.7% lipid making this material comparable to materials of animal and vegetable origin as a feed ingredient
(304C). The application of the submerged culture process to the growth of Psalliota campestris was mentioned in the last review. I n more recent publications (51C, 336C) growth of this organism on media containing peanut meal, peat, wheat, and sulfite waste liquor has been described. The resulting mycelium had a flavor not unlike the mushrooms grown on flats but morphologically was markedly different. Block ( 2 1 C ) has grown a mutant strain of Agaricus blazei on synthetic media, as well as a medium based on orange juice and one based on citrus press water. Approximately 35% of the glucose (initial concentration, 50 grams per liter) of the salts-glucose medium was converted to mycelium in 5 to 8 days. The B vitamin content of the mycelium, including thiamine, riboflavin, niacin, and pantothenic acid, compared favorably with that of torula yeast but vias lower than that of brewer’s yeast. The mycelium grown on synthetic media had little or no flavor when fried or toasted, while that grown on other media had some of the taste of the substrate (Sf C). Antibiotic Feed Supplements. Stokstad (334C) has reviewed the recent literature concerned with the role of antibiotics in animal nutrition, while Braude (19C)has critically evaluated the use of antibiotics as growth-promoting agents for swine. Coates (61C) concluded that the presence of antibiotics in the swine and poultry rations counteracts the “normal” growth depression caused by intestinal bacteria and increases vitamin absorption by the animal. This hypothesis was strengthened by the observations that antibiotics have little effect on the growth of germfree animals (334C) and that subcutaneous implantation of pellets containing bacitracin, the procaine salt of benzylpenicillin, or chlortetracycline had no effects on the weaning weights or survival rates of suckling pigs (345C). However, administration of penicillin-resistant microorganisms, alone or with penicillin, resulted in increased chick growth (296C, 299C). The administration of chlortetracycline to swine has been shown t o result in a shift of intestinal flora to one consisting predominantly of Aspergillus Jlavus. This fungus u-as found to produce a new growth factor (286C) and when present in the intestinal tract resulted in lowered blood-glucose levels in the host animal (286C). Synthesis of Fats. Studies on the synthesis of fats by microorganisms have continued, although in the past production on a commercial scale has been attempted only during periods of extreme economic stress. While the mycelium of Penicillium javanicum contained 16% fat and that of Aspergillus niger 25%, the conversion rate of glucose in the culture medium to fat was only 6 and 8%, respectively (11SC). The iodine number of the fat produced increased with the length of the incubation period. I n other studies, A . nidulans when grown in surface culture (surface:volume ratio of 2.3 square em. per cc.) produced 0.0218 gram of fat per square cm. of mycelium a t a conversion rate of glucose to fat of approximately 15% (105C). Both Aspergilli and Penicillia have been grown for fat production successfully on media containing wood hydrolyzates as an energy source (168C). Experiments on fat formation by a microorganism related to the Endomyces showed that glucose, fructose, galactose, mannose, lactate, and glycerol were converted to fat. When the fungus was
1816
INDUSTRIAL AND ENGINEERING CHEMISTRY
grown on media containing peptone together with starch, dextrin, lactose, sucrose, malose, or arabinose as energy sources, little fat was formed (24SC). Cells containing 45Yo fat were obtained when t,he fungus was grown in submerged culture for 3 days on starchfree sweet potato juice-sucrose medium (247C). The fat content of the cells did not change when the medium was diluted fivefold although the cell yield decreased to one fifth. An examination of factors affecting f a t synthesis by Rhodot o ~ ~ gracilis la shon-ed that decreasing the temperature of incubation from 28" to 22' C. reduced the synthesis rate by a factor of two. Increasing the carbohydrate content of the medium up to 16% did not affect the synt,hetic rate (SWSC). For fat synthesis from acetatmeor glucose the optimum pH was betwcen 6.3 and 6.5 (SSZC). When Torula utilis lvas gron-n on molasses or other fermentable carbohydrates, 40 kg. of yeast was produced per 100 kg. of reducing substances fermented. The fat cont'ent was approximat'ely IS%, n-hich x a s a slightly higher conversion of carbohydrate to fat than obtained v i t h F u s n ~ i i o nspecies (37SC'). E N Z Y M E S OF I N D U S T R I A L INTEREST
During the past year a number of report8shave emphasized the multiplicity of enq-mes produced simultaneously by microorganisms. The sequence of appearance of enq-mes in the cellfree medium when Aspergillus oryzae was grown in surface culture on a sucrose-t'artrate protein-free medium n amylase, phosphatase, prot,eases, esterases, and cat,alase (65C, S S C ) . Paper e1ectropheret)ic separation of the carbohydrases of another strain of A . oryzae showed the presence of arnj vertase, maltase, transfruct,osiclaee, and transglycosidase ($66C). Filter-paper chromatography of a number of enzymes derived from A . oryzae including proteinase, amylase, catalase, esterase, acid phosphatase, sucrase, salicinase, and p-nitrophenyl-pglucosidase showed the presence of two or more entities in each enn>-maticpreparation ( 1 6 l C j . Amylases. High aeration was required for maximum ainylnse production of Bacillus mesenfeiicus. I n anot'her study (2OUC) it was found t,hat, growth paralleled amylase production (ZOIC). The optimum incubation temperature for growth was 30" C., while incubation a t 36" C. appeared opt,imal for amylase production. Starch and ethyl alcohol \\-ere satisfactory energy sources for grov-th and enzyme formation by this strain, while peptone was found to be the best nitrogen source (202C). In another study ( Z 2 1 C ) a strain produced large amounts of amylase when gron.n at 60" C. in submerged culture on a medium containing the mycelium and spent fermentation liquor obtained from the production of penicillin. A study of the cell-free liquid obtained from fermentations of strains of Clostridium acetobiitylicunz showed the presence of a-am>-lase, @-amylase,limit destririase, and maltase (SOOC). A study of the amylases produced by five streptomyces ehoii-ed that only a-amylase was produced (SBOC) 17-hen these species were grown on a starch-containing medium. Comparison of crystalline preparations of bacterial snccharogeiiic amylase and liquefying amylase showed the former to have a loner optimal p H as well as a stability in a narrower pH range than the lat,ter ( I O I C ) . Amylase production by fungi grown in submerged culture has continued to receive attention. The @-amylase and maltase found in the filtrate during Koji fermentat'ion did not change when the incubation period vas increased from 7 to 11 days, while the a-amylase content decreased significantly (173C. 174C). Certain enzymes from bacteria were found to destroy t,he amylase formed by the fungi (SSSC). Examination of the amylases produced by Aspergillus niger sholyed high maltase and loiv amylase production when the organism was grown on either synthet'ic media or media containing natural products (87C). Media containing 6% glucose favored wamylase production and resultedin lowered maltase production by Aspergillus kawachii
Vol. 46, No. 9
(I7OC). Stillage from Clostridium acetobutylicum butyl alcohol-acetone fermentations supplemented with 1% ground corn meal. 0.5% calcium carbonate, 0.02% zinc sulfate, and 0.014% aluminum powder was a suitable medium for growth and ami-lase production by ,4. niger in submerged culture ( I S C ) . a-Amylase from A. oryzne hydrolyzes cornstarch, potato starch, and dextrins but not dextran and maltose which do not cont,ain 1,4-a-glucosidic chains (184C). Taltoaka (S39C') has described crystallization of an a-amylase devoid of maltase activity from culture. filtrates of Aspergillus candidus var. am!/lolyticus. Best results lvere obtained with fungal amylases in the baking industry when t'he dough contained 20 to 80 S.K.B. (Sandstedt. Kneen, and Rlish) units per 100 grams of flour (118C). I n nuother study more amylase from A. oryzae than t,hat from cereal wap needed to give the same viscosity change in starch suspclnsions. Bact,erial amylases were quite inferior to either the fungal or cereal amylases in this study (27C). Lipases. ?;ashif (2@C-S6OC) found that the lipase producwl by Pseudomonas fragi hydrolyzes tricaprylin more rapid1 other glycerides. Production of extracellular lipase o when the bacterium Tyas grown at 75' C. under vigorous tieration on nutrient broth containing glycerides. No enzyme was found when incubation 'Ivas a t 30' C., although the culture grew well. Examination of the lipases of 12 cultures drawn from the Paeitdom o m s and Achrotnobncter genera showed the presence of lipases Tvith different substrate specificit,ies. Proteases. Mortierella renispora (36YC, 370C), Bacterizm linens (98C),and Sspergillus orgsae (MC, 66C)each produced several proteolytic enzymes. A4single proteolyt,ic enzyme appeared to be produced by two streptomycetes (107C, 354C'). A prot,einase free of collagenase activity arid a po\yerful colhgcnase, which, although it still contained other proteinases, was considerably enriched and concentrated by precipit,at,ion methotis, mere isolated from Clostridium histolyticutn ( I S 6 C j . I n another study collagenase activity in bact'erial preparat,ions was found to be accompanied by gelatinase ( 8 J C ) . Transferases. The presence of transglycosidases and ot,her transferases (223Cj in microbial preparations has received considerable attention, Synthesis of oligosaccharides by amylase preparations from fungi and Bacillus subtilis has been studied by P a n (26RC, 26SC) who obtained trisaccharides from maltose, sucrose, and isomaltose. Similar results were obtained in other laboratories (4C,IRC, 186C, SfSC). Enzymatic conversion of lactose to 4- to 10-galactosyl oligosaccharides was observed when lactase from Saccharonzyces fragilis hydrolyzed lactose (Z67C, bSSC,
Zsgc).
Other Enzymes. A combination of glucose oxidase and catalase obtained from A . niger has found wide use in the desugaring of egg whites on a commercial scale (9C). Maltose, galactose, marinose. xylose, and ylucosamjne were not oxidized by this preparation. Thc use of glucose oxidase for product,ion of gluconic acid from glucose has been described in a recent patent, (SC) but apparently i t has not been exploited commercially. A lactose ouidase from Pseudomonas graceolens has been used to convwt lactose t o lactobionic acid ( 6 S C ) . Siu and Reese (.%?IC) and Tracey (S'5UC) have reviewed the literature concerned with decomposition of cellulose b y microorganisms Tvith special emphasis on the role of cellulase in this decomposition and t h e product,ioii and properties of thrse enzymes.
FERMENTATION AS A UNIT PROCESS Consideration has been given to fermentation a8 a unit process in this series of reviews ( 6 A , 8-4, SA). Since many of the principles involved have been summarized in the previous reports, only those which have been emphasized in recent publications will be covered in this review. Since methods of recovery of fermentation products have often been a deciding factor in deter-
September 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
mining the economic success of fermentation processes, surveys of this aspect of fermentation operations have been included. THE MICROORGANISM
Cultures or strains of a given species vary considerably in their ability to produce a given nietmabolicproduct. This “natural” variation has led to the large scale screening of cultures isolated from diverse natural habitats from ability to perform the desired reactions most efficiently. A number of reports appearing during the past year have shown that’ such screening programs have been successfully operated for the selection of actinomycetes for the production of vitamin Blz (25C, lWOC), of fungi for amylase pruduction ( 4 8 0 , 5 8 0 , 74D), of fungi for the production of proteolytic enzymes (70D-73D), ol fungi for the production of penicillin (245C), and of bacteria for the digestion of collagen ( 6 7 1 ~ ) . ‘(Rough” variants of Serratia marcescans were found to be superior for the produetion of prodigiosin. Fermentations inoculated with rough strains foamed less and filtered easier than fermentations inoculated with “smooth’’ strains ( 3 S D ) . A natural variant of Streptomyces noursei produced an antibacterial agent rather than the antifungal agent produced by the parent culture (SBC). The role of the microbial geneticist in increasing yields of antibiotics and other metabolites by microorganisms has been appreciated during the past year. Increased yields of benzylpenicillin (as compared with yields produced by the parent cultures) were produced by survivors of ultraviolet light and nitrogen mustard treatment of P e n i c i h m chrysogenum spores ( M C , 10911, 1 1 8 0 ) . I n one study yields were increased stepwise over a sequence of six experiments, in which irradiation and selection of natural variants were employed, from 1285 to 2575 units pcr ml. (86C). In another study (&C) mutants found to produce high yields of allylmercaptomet~hylpenicillin.ivhen grown on appropriate media produced low yields of benzylpenicillin when grown on media used for production of the latter. Pontecorvo and Sermonti ( l O I D ) , in the course of their studies on Penicillium chrysogenum Q1rs, developed heterozygous diploid strains which had some of the properties of the starting strains. Increases in production of streptomycin have resulted from culture selection programs using ultraviolet light, nitrogen mustard compounds, and x-rays as mutational agents (2BD, 7 5 D , 8 g 0 , 8.Ql, lWlD, 1220). I n one series of experiments yields were increased from 250 to 2000 mg./liter ( 2 2 D ) . Increased production of amylases ( 9 2 D , 9 S D ) and citric acid ( 4 4 0 )was obtained with cultures of Aspergilli selected aft,er exposure to ultraviolet light and single-cell isolation procedures. Morphological mutants resulting from ultraviolet light treatment of spores of Aspergillus niger ( 1 3 9 D ) and other organisms including Aspergillus oryzae ( 9 1 0 ) and Penicillium ?,oquejorti (BOD) were found to have different pigmentation than the parent cultures but carried out the same desired chemical changes. Maintenance of Strain. The desired strain of a given microorganism must be maintained in a manner conducive to the procurement of uniform results. Lyophilization or desiccation using soil or proteinaceous materials as supporting menstra are favored in certain laboratories ( 4 0 , 1 4 0 ) . Record and Taylor ( 1 0 6 D ) reported t h a t survival of Escherichia coli during lgophilization depended 011 the presence in the suspension of a soluble material derived from the organisms; this protective effect was enhanced by the addition of glucose to the suspension. Haskiris and Anastasiou ( 4 l D ) reported that the viability of Aspergillus niger spores following a freeze-drying method using a vacuum centrifuge was significantly higher than that following two other methods involving snap-freezing a t low temperatures. Over SO% of the fungous spores survived desiccation when 20% sucrose was used as a suspending solution. Preservation of certain strains of Saccharomyces cerevisiae by suspension of the cells in a solution of lactose (which is not fermented by the yeast) under oil was recommended a t a suitable method ( 9 7 D ) . When cultures of certain organisms are repeatedly t,ransferred
1817
withoutmadequate opportunity for cellular maturation, variants often arise which have undesirable characteristics. This has been observed in streptomycin-producing cultures of Streptomyces griseus (99D, 137D), patulin-producing cultures of Aspergillus clavatus ( 3 8 0 ) , citric acid-producing strains of A . niger ( 9 5 D ) , and penicillin-producing strains of P. chrysogenum (W44C). Various methods have been proposed for recovering strains producing high yields of the desired substances from these “degenerated” cultures. These include selection of natural variants ( 1 3 7 0 ) ,passage through nonsterile soil ( 3 8 0 ) )and repeated transfer on “minimal” agar media ( 9 5 0 ) . It has been found undesirable to transfer certain solvent-forming clostridia more thai? six times in the vegetative state in preparing inocula for fermentations ( 1 1 2 0 ) . Bacteriophage. Among other problems concerned with the selection of cultures is that of susceptibility to phages. A phagr infection affecting a culture used in a fermentation process may cause complete disruption of operations. McCutchan and Hickey (2OBC) reported that t’he manufacture of butyl alcohol and acetone in certain plants has been suspended because of bacteriophage infection. Repeated subculture of the susceptible cultures in the presence of the lytic agent map result in the eventual growth of strains resistant to the phage. Phage infections of streptomycin-producing fermentations have been mentioned in earlier reviews in this series, and more recent reports of infections in 850). Polyvalent phages-Le., other plants are available (QD, phages attacking more than one species-as well as monovalent phages have been found (WD, Q D , 860, 8 9 0 ) . Shirling ( l l 4 D ) and Jones ( 5 5 0 ) reported that the asporogenous character of certain streptomycetes was due to the presence of a lytic principle LThich could be transferred from culture to culture. Studies on the origin of bacteriophage have shown that many normal-appearing bacterial cells carry phage which map be released by exposure of the cells to ultraviolet light or certain other mut’ational agents (64D, 115D, l b 7 D , 1SOD). SUBSTRATE
Strain specificity is important in formulating media for use in fermentation operations, and in some industries all raw materials are evaluated on a pilot plant scale before being used in commercial operations ( 3 2 0 ) . Carbohydrates and Other Energy Sources. The major product of the fermentation determines the choice of carbohydrates or other energy source to be used, especially if the product results from the direct dissimilation of such a compound. For example, in the production of ethyl alcohol the cost of the product is largely determined by the cost of the carbohydrate (ldOC, 16BC, 328C). During the past year there has been a surplus of blackst,rap molasses and sucrose in the Carribean area. As a result of this situation much of the cane juice produced during the past, year has been converted into high test molasses rather than sugar and blackstrap molasses. This is apparently an unusual circumstance because high-test molasses is not normally available (14OC). The use of cereal grains for fermentation media has not been profitable in recent years because of their comparatively high cost as compared with molasses. Thus, even with a record corn drop of 3.2 billion bushels in 1953 in the United States, little of this material has been used in the production of ethyl alcohol by fermentation for industrial chemical use. Use of other source$ of carbohydrates for ferment’ation purposes has not been profitahlo in the United Statw although potatoes have been used in Europe (lOSD, lO4D) and ryce in the Orient. Anew process has been described for hydrolyzing wood wastes t’oproduce “ E U ~ M ” for fermentation purposes (1OD). The composition of molasses used in the fermentation industries has been discussed by Hodge and Hildebrandt (140C) and by Binkley and Wolfrom ( 6 D ) . The nonfermentable substances found in molasses (which may account for as much as 5% of the total reducing solids present) have been studied (goOD). Raffinose has been found as a contaminant of sucrose obtained from sugar beets ( 3 7 D ) . The composition of grain used in the production of ethyl alcohol has been discussed in detail by Stark (328C). McCarthy (2OO4C) has considered the composition of sulfite waste liquor wed for the same purpose.
1818
INDUSTRIAL AND ENGINEERING CHEMISTRY
The reactions brought about by heating carbohydrates with prot'einaceous material have been studied in some detail. Significant quantities of glucose and fructose were lost when these sugars were heated wit'h tryptophane, arginine, histidine, lysine, or t'hreonine (25D). Hodge has reviewd the literature available on t.his aspect of the browning reaction (490). Reductones formed by heating glucose in alkaline media increased tlie lagphase portion of t'he growth curve of bacteria ( 8 7 0 ) . A recent report has shown that a considerable amount of inversion of sucrose took place when slightly acid media were autoclaved ( 5 0 ) . Other energy sources include animal and vegetable oils used in processes for the production of penicillin (298C,S49C),neomycin (26'9C), and various anbibiotics produced by streptomyces ( I 2 7 D ) . Occasionally the carbohydrate has been replaced b y oils on a caloric basis. Animal and vegetable oils have also been added to fermentations as antifoaming agents either alone or x i t h surface active substances. The presence of t,hese surface active agents may influence the fermentation process and their compoaition should be known if possible ( 7 7 0 , 2280). Alcohols have been suggested as energy sources for the formation of fat by Endoinyces ( 8 2 0 ) . The yeast, grew well in media containing 4.1yo ethyl alcohol. Sorbitol, mannitol, and dulcitol were satisfactory energy sources in media used in a process for the production of prodigiosin by Serratia mamscans (400). Nitrogen Sources. The use of proteinaceous materials such as extracted seed meals, yeast', distiller's solubles, corn steep liquor, fish stick liquor, and meat stick liquor as ingredients of fermentat'ion media has been mentioned in past reviews in this series and in the reviem by Schopmeyer (309C), McCutchan and Hickey (WOSC), Irvin ( I L V C ) , and Hodge and Hildebrandt (Z4OC). More recent reports have, in general, confirmed previous observat'ions. The use of stillage from acetone-butyl alcohol fermentations as an ingredient in media for the production of fungal amylase v a s found advantageous ( 2 3 C ) , while a protein hydrolyzate was reported of value as an ingredient of media used for the production of vitamin BIZby Streptmyces yriseus (85C). A study of azaserine synthesis by a streptomycete showed that the proteins added to the media served as the chief source of energy and that antibiotic production was not markedly affected by t'he carbohydrate content of the medium ( S I C ) . -4mmonia gas and ammonium salts have been used in media for the production of ethyl alcohol ( l 4 O C ) , torula yeast (EVWC), and baker's yeast (257C). Other reports have shown t h a t addition of nitrogen salts to the molasses medium causes an increase in t,he protein and methionine values of yeast, although the effect is dependent in part on the type of molasses used (56C). The type of nit.rogenous material used iu t'he preparation of media for the groivt,h of mushrooms aficct,ed the taste of the resulting mycelium ( S I C , 6 2 C ) . Blthough growth in media containing citrus press water was satisfactory the mycelium had a bitter taste and mas considered to be unsuitable for food use. Inorganic Nutrients. The mineral requirement,s of Phgcoinyces blakesleeanus, a mold useful in the synthesis of carotene and in the oxidation of steroids (9@), and Chlorella pyrenoidosa (2842) have been discussed in recent reviews. Other studies have shown that Chlorella uulgaris requires magnesium for cell multiplication and catalase formation, but that the total cell weight is independent of the magnesium content of the medium ( 2 6 0 , 2 7 0 ) . h recent report associates the effect of metallic ions with the incubation temperature of the fermentation. Highest yields of citric acid were obtained at 25' C. At this temperature the variation of the quantities of iron, manganese, and copper in the medium had the least effect on the yield (59D).This relat'ionship between the incubation temperature of the fermentation and the effect of mineral coniposition of the media has been thought to be related to the destruction of citric acid by the mold (980). This destruction may be inhibited by the addition of cyanide to the growing culture (57C) or by the addition of low molecular weight alcohols or methyl acetate a t levels of 1 t'o 5 % of the medium volume ( 7 8 0 ) . The latter method has also been used to reduce the undesirable effect of met'allic ions on the biosynthesis of it,aconic acid and ethylene oxide dicarboxylic acid by fungi ( 7 8 0 ) .
Vol. 46, No. 9
The yield of antibiotic substances produced by streptomycetes was increased when sea water (or the salts present in sea water) was substituted for t'ap water in the media ( 6 1 0 ) . I n other invest'igations the addition of iron to niedia resulted in increased yields of streptomycin (R87C) and decreased yields of chloraniphenicol ( i R 3 0 ) . The latter effect was reduced when sufficient inorganic phosphate was added to the niediuin to complex the soluble iron salts present. Precursors. Some chemical substances, when added to certain fermentations, are directly incorporated into the fermentation products. The use of cobalt compounds in the formation of vit,amin I312 and phenylacetic acid in the formation of benzylpenicillin are examples of fermentations where precursors are commercially important. However, in the oxidation of steroidal compounds the st'eroid is the substrate, which is undergoing change, and not a precursor, XThich is being incorporated into a larger unit. Recent studies have shown that under certain conditions pht:11ethanol and phenacetyl derivatives are more efficiently converted to benzylpenicillin than is phenylacetic w i d (86C, SRBC, %IC', 2?1C). I n studies on the forinatiou of allylmercaptomethylpenicillin, certain strains of P. chrysogen~inzthat efficiently converted the derivatives of allylmercaptoacetic acid to the penicillin XTere much less efficient in the use of phenylacetic acid or S-(2-hydroxyethyl)phenacetamide in benzylpenicillin formation
(Q4C). As mentioned earlier, the search for precursors of tlie thiazolidine portion of the penicillin molecule has not been successful, Studies of the chemical changes occurring during the fermentation have failed to identify the intermediates important in the biosynthesis of this portion of the penicillin molecule (80, 6 6 0 , 6 7 D ) . Fermentations using nonpenicillin producing strains oi' P . chrysogenum have been suggested as a possible source of these intermediates ( 2 2 6 D ) . Cystine isomers are more efficiently converted to penicillin (the thiazolidine port,ion) than are inorganic sulfates, but the efficiency of the conversionsreportedmas low, and there are conflicting reports on the value of the d and I isomers (2C, SSiC, 335C). Precursors (other than cobalt derivatives) for the biosynthesis of vitamin BIZhave not been reported. A number of degradation product's of the molecule were not useful (WWO), although a few, when added in high concentrations to the media, stimulated the production of the vitamin slight,ly. Organic complexes of cobalt found in microorganisms grown on cobalt-containing media were more efficiently converted to vitamin BIZthan were inorganic cobalt salts (Z70C). Glucose or metabolites formed from glucose were the major source of the carbon of the streptomycin formed when Streptomyces griseus was grown on a synthetic medium ( 9 0 0 ) . I n the formation of dextran by dext,ran-sucrase from sucrose the addition of a polysaccharide primer to the enzyme-substrate mixture directed the reaction to the formatmionof a dextran with a molccular weight of 80,000. The primer may not be a direct precursor as it \vas not shown to be present in all the dextran molecules formed (SO'dC). Growth Factors. The importance of vit'amins and growth factors in t,he nutrition of microorganisms has been stressed in this series of reviews. Certain of the known vitamins, including biotin and p-aminobenzoic acid, were necessary for the growth and solvent production by a number of species of Clostridia (470,1360), although under some circumstances the biotin could be replaced by a mixture of thiamine and pyridoxine (860). Thiamine, biotin, inositol, and unknown growth factors were necessary for the production of maximum yields of riboflavin by Eremothecium ashbyii (234C, IIGC, 217C). On the other hand, addition of large amount,s of inositol to a synthetic medium resulted in reduced riboflavin biosynt.hesie (Z28C). S-Glucosylglycine, formed when media containing glucose nnd glycine were autoclaved, was found to be a growth factor
September 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
for Lactobacillus gayonii ( I l O D ) , and may be important in other fermentation processes. The reaction products formed when glycine was heated with other sugars also stimulated growth and acid-formation by this and other species of Lactobacilli ( 1 l l D ) . The inhibition of growth of Lactobacillus bifidus, observed when an autoclaved cystine solution was added to the medium, was not found when the cystine was autoclaved with glucose (113Dj. White and Munns (1320) studied the effect of the vitamin content of the medium on the growth rate of yeast. The bioassay methods based on growth of bacteria did not give satisfactory results when used for the determination of vitamin content of raw materials used in yeast fermentations (1020, 1330, 134D), and they recommended the use of a yeast bioassay. Another author reports that the growth of Sacchardmyces cerevisiae was stimulated when ergosterol (suspended in Tween SO) was added to a medium containing water soluble vitamins (10).
#
.j
FERMENTATION OPERATIONS
Dawson and Pirt (160) outlined the relationship between engineering and the microbiological processes pointing out that sterility control is no longer a major problem. They stated that the major developments presently required are (1) suitable equipment for controlling the nutrient concentration, pH, and available oxygen; (2) continuous fermentation processes; and (3) an aeration system where shearing forces do not injure the organism. Warner ( 1 2 9 0 ) in a related analysis explained the role of engineering in connection with economic considerations and a number of fermentation operations including air and media sterilization, oxygen transfer, p H and temperature control, and equipment design. Fermentation Equipment. A number of reports include descriptions of equipment, covering all scales of operation from shaken flasks through full size manufacturing plants. A description and pictures of equipment used a t Glaxo Laboratories antibiotics plant a t Ulverston in which penicillin, streptomycin, and vitamin BlS are manufactured has been presented (110, 490). The plant operated by Dawe's Laboratories and designed for producing a vitamin Blz supplement was described in considerable detail ( I S S C ) . Seed tanks with a capacity of 400 gallons, batched to 220 to 260 gallons, are used for preparing inoculum of Streptomyces olivaceus. The fermentation is carried out in 5200-gallon fermentors equipped with coils for steam or cooling water, agitators, and air spargers. A pilot plant scale unit for the production of 2,3-butanediol was described, with equipment including a continuous sterilizer, flash cooler, and 1500-gallon stainless steel fermentor (1510). The fermentor was equipped with a vaned disk agitator, porous stainless steel sparger, and mechanical foam breaker. Temperature and p H were controlled automatically. Other pilot plant scale equipment which was described included: 100-gallon stainless steel fermentors and 30-liter stirred jars used for the production of synnematin (M7'C); wooden and iron tanks used for an acetone-butanol fermentation (29%'); and a Waldhof type fermentor used in the production of feed yeast from citrus wastes (192C). The mass culture of the pathogen Shigella sonnei, carried out in a 7-gallon cylindrical glass jar, has been reported ( 5 4 0 ) . This fermentor was equipped with an internal p H electrode, a mercury manometer for indicating tank pressure, and agitator, and a ring sparger. A mercury seal on the agitator shaft and a slight negative pressure on the jar prevented the escape of the organism to the surroundings. A 30-liter glass fermentor was also used for studies on the continuous production of torula yeast from potato starch wastes (289C). Based on the results of this study and on conditions reported for a sulfite-liquor yeast plant, a preliminary cost of 5 cents per pound of yeast was estimated for a 4.5-ton-per-day plant.
1819
A 4-liter glass fermentor equipped for automatic control of pH, air flow, and addition of inoculum and nutrients was used for small scale fermentation studies of the preparation of dextransucrase ( 6 8 0 ) . A flask designed for continuous culture propagation (5%'0)was used in the study of ribonucleic acids and protein synthesis by Polytomella caeca. Nutrients were fed dropwise through a capillary, and excess broth was removed through an overflow tube connected to a pump. Several pilot plants designed for specific processes have been reported. These include: ( I ) a-vinegar generator, packed with beechaood shavings, for the oxidation of ethanol by Acetobacter z ~ l i n u m( 7 C ) ; ( 2 ) a plant unit for the study of algal culture in which broth m-as circulated through polyethylene tubing (191C); and (3) a prepilot plant, also for the study of the growth of Chorella, which used a concrete trough covered with polyethylene plastic sheet as the culture vessel (2,t3OC). Glass has been used instead of polyethylene for containing the broth while allowing light to penetrate to the algae ( 7 6 0 ) . Feed units were designed and used for maintaining a constant rate of carbohydrate addition to shaken flasks. One consisted of a miniature rotary displacement pump. Solutions were forced through a rubber tube by rollers attached to the shaft of a 1-r.p.m. motor (68C). I n another unit, gas generated by electrolysis forced the liquid from a reservoir into the fermentation unit (386C). Sterility. The aspects of sterility as applied to fermentations can be subdivided into measures taken to obtain initial sterility and those employed to prevent contaminations subsequent to the start of the fermentation. Steam a t 10 to 15 pounds per square inch gage was maintained for 15 to 18 hours on all equipment used in a pilot plant designed for the production of 2J-butanediol. A continuous cooker with jet injection of steam was used for sterilization of medium ( I S I D ) . I n the acetone-butanol process sterilization of the corn mash by maintaining it for 3 hours a t 135" C. was satisfactory while oversterilization resulted in lower yields (296C). I n a plant scale experimental unit designed for continuous sterilization of 3000 gallons of penicillin medium per hour, all heat transfer was carried out in a three-section plate-type heat exchanger. Under the conditions employed, 9 minutes a t 121 O C., penicillin yields were increased by 10% above those obtained with batch sterilization (1560). Sykes ( l Z 0 0 ) reviewed several methods of air sterilization and reached the following conclusions: chemicals are impractical because of carry-over into the fermentation broth; heat is too expensive; ultraviolet light and electrostatic precipitation are inefficient; and slag wool is the best of the filter materials. A study of the mechanism of air filtration has shown that the design of the filter should be based on the air stream velocity; type of filter medium; microbial load in the entering air; and an economic balance on filter size, pressure drop, and life expectancy of the filtering material (46D). Other methods for air sterilization have included ozone (only 1 p.p.m. being necessary) ( 5 0 D ) , heat ( 1 7 0 ) , and a membrane filter ( 6 1 0 ) . Air filter efficiency was tested in an experimental unit, using a buffer to catch bacteria in the treated air stream (35D). Other laboratory equipment used in testing filtering materials has been described ( 6 6 0 ) . Antibiotics and other chemical compounds have been used for controlling contaminations in fermentations. Polymyxin, at a level of 0.005 pg. per ml., almost completely eliminated bacterial infection in a beer fermentation and resulted in a stimulation of the yeast fermentation (1190). When polymyxin and thiolutin were used together, secondary yeast growth was controlled. Tyrothricin, oxytetracycline, chlortetracycline, chloramphenicol, and benzylpenicillin were effective as contamination-control agents in the grain alcohol fermentation (160). Of these, penicillin, a t 0.75 to 2.0 units per ml., has been tested on a commercial scale. Chlortetracycline was effective in minimizing bacterial contaminants in the growth of ure yeast strains (480). I n the penicillin fermentation bacteriaf contaminants were inhibited by compounds having side chains at the 2-position in 5nitrofuran (1840). Fermentation Conditions. The physical conditions during fermentation have, in many cases, a decided effect on the production of the desired material. The gas film was found to be the
1820
INDUSTRIAL AND ENGINEERING CHEMISTRY
limiting resistance to oxygcan transfer in the propagation of baker's yeast in :t continuous fermentation (203C). Consequently the maxirnuni production rate wns at,taiiied a t the highest air-flon. rate teated. Shu (317C) studied, in shaken flasks, the effect, oi oxygen uptake on three fermentations lvit,h dissimilar aeratioii characteristics. With an Aspergillus nigei fermentation for the production of a-amylase tjhe optimum oxygen uptake ratc (for maximum euzyme production) was lower than bhe maxiinuni ratv of oxygen tleillzncl by the organism. With a Ustilago ~ e a e ferment:ition oxygen demaricl was low mid the rate of ust,ilagic acid production pti,ralleled the osygcn uptake rate. Finally> u-ith an A. nigei. fermentation for the product,ion of csitric acid, oxygen !VAS the controlling fa.rtor, the demand Leiiig high a n d t,he rat(' of' :+&I production ag8in running parallel t o the upt:ilw 1xte. I n surface culture studies 011 the metabolism of acot:lte I)>. -1.nigw (f21C),the rate of utilization of acetic acid incrcawd t o :i maxinium with incrensing carbon dioxide concentration or c k creasing air flow rate. Whereas the production of oxalate wiii iiot greatly affected by the aeration rate or carbon dioxide cmirentrat,ion, citric acid yields could bc markedly increased ljy i,rduced aeration and regulated carbon dioxide conceritratioii. I n the production of ruticin, the antibiotic was irreversibly inact,ivat,ed when aeration of the culture was halted ( 2 9 D ) . The addition of hyclrogen peroxide t o bhe broth TWS effective in S to preventiug this inactivation. Since the inactivation L V V ~due iyducing enzymes, i t was concluded that. ruticiri 1%-ouldbe un,sstisfact,oryas a chemotherapeutic, agent'. Severe foaming is often a problein in fermentation processes when high aeration rates are used. Several subPtances have beon reported to be effective in controlling foam. These include : corn, soy, or lard oil addecl tit batching and during Che S.olivaceirs felmentation process for vitamin Bl*( / 3 3 C ) , a commercial product (Swift 1000) used in the torula yeast process (28861,11butyl phosphate used in the mass-culture of bacteria (c5SL)), a,nd hlkaterges (products of Commercial Solvents Corp.), mixed n-ith mineral oil, used in penicillin fermentatiolis (Ian). Mechanical foam breakers have been suggested for use in place of oils or chemical compounds for the control of foam in ferinentations. In one arrangement the Eoani was pumped out of the fermentor int,oa second tank ( 3 6 D ) ,while in another arrangement. the foani w i s dispersed by a truncated cone rvith vanes inside, mounted on the agitator shaft 3 feet, above the liquid level ( I S 1 ). In the pilot plant, production of xanthomycin, an in temperature from 25' t o 30" C. reduced the fernientatio by 24 hours and increased t,he antibiotic yield from 3L to 45 ,ug. per inl. I-Iomver, the streptomycete c u h r e inactivated 60% of t h e antibiotic in 2 t,o 5 how it 30" C., a more rapid rate of inactivation than t,hat observed a t 25' C. This inactivation could be retarded by adjusting the p H of the medium t o 2.0 (1050). The rate of fat, production by Rhodotorula gracilis also varied with temperature; a t 22" C. i t was only one half the rate a t 28" C. (SPQC). Similarly, in ail ethyl alcohol fermentation the cycle varied ~ v i t htemperat,ure from 3 days at 33" C. to 15 days a t 15' C. ( 9 6 D ) . I n the pubmerged oxidat,ion of ethyl alcohol to acetic acid, the optimum fermentation temperature was inversely proportional to the total concentration of medium ingredients (460). Further temperature fluctuations between 24.6" and 32" C. are harmless if t,hey occur infrequent,ly during the fermentation period, but' repeated variations R-ithin this range tend to cause a decrease in the fermentation rate. The sensitivity of algal culture to ternperature fluctuations has been noted (@C, 155C). One of the major costs in the mass culture of algae in open ponds or controlled manufacturing equipment would be the control of the temperature throughout the year. It may be possible to reduce the cost of cooling the growing culture if a strain of Chlorella, capable of growing satisfactorily a t temperatures higher than
n
Vol. 46, No. 9
26" C. is used. iZ c~ultiii.c'-~l'o\.;ii~g woll :it 39" C. has been tic,icribed (S26GCj. The effects of the ui rirc of trticee elcmcnts in the niediuni oii 1itr.ic acid production -4.niyri. are greatly influenced k)y ilii: incukiation teinpcrature (5$~/1),For thi, highest yields at 25" C., twice the concentrations o l ziiir: iron, nitiiiganese, a n d coplicr ~ r v r erequired than h:td i)c~ii wt:d)lislied as optimal a t 30" C. .\t 2.5" C. thc pi.oduotiori oi' ci tr:itc, was not so sensitive to vxc'c-ws of iron, inangaiicso, :i11(1 w p p i ~ i ' t, the higher teniperatlll'e,
'l'lie opi,iniuin p1-I for. thtx piwluc+ion ol tonilti yeast from pot:ato *(:irc.li wastes was found l o I](> 5 in :1 rtutly of levels betwc i :mtl 8 (PRDC). In ac!tiition, t l i r ici.nic,iit:ition resists h:u rwtitaniination a t a p l i of 5 01'lo\vc~i~. In the production of 2,:i-lj~,t,~iiedi01, imirx!r.sion electrodes JIWiiiittcd closer caontrol of t l i ( ~plT t,h:tn electrodes installed i i i HII c,sterniil leg. I n addition, 1 i i ~ i h l corit:iniinat,ion e from the, pump i n the leg was avoided (1310!. Inirnwsioii electrodcs iii a +liter fermcrjtor pernii-tttd c.oiitro1 of the pf-1 a t 6.75 d= 0.1 in the ration of dest,raiisucmw 1)y Leuconostoc mesenteroides (69D). Fermentation Cycles. Fornicntation processes can be clmfiified iiito tu-o groups depcwding o u whcthcr a batch or continuous process is employed. .Qlt'hough t'he former type of operation is ;till more widely used, in sing rolisidcwtion is being given to c~ont,jnuousoperation-: anti Inany new dwclopments havo bwn 1.i~portPd. Iri :i production plant! 1 t o 2 ~ g of. vitamin I312 per ml. wwe pi.oduccd in 3 t o 5 day^ of f'wmentation (139C). Of tho (j% .iiigar in citrus press liquor, 57.1% nvw converted to Candida o h o w n cells in 10 hours iri a Waldhof fermentor (19PC). By (*hangingthe phy,sical conclitionr during the riboflavin fcrmcliitation. 1740 p g . per ml. iverc produced in a %day fermentation (8811). The broth, inoculated with E. ashbyii, x-as aerated ivith 2 volumes of air/volume of medium/minutr a t 30" C. for 100 3ioiirs and Lhen Jvitli 1 voluiiie!volume/tiiiriutc at 28" ( > ,lor, an :itlditional 110 hours:. During the fermentation pi,oduction of i)ut:inol and 2-prop:iiiol i'roin sugar with Clostridiim toanurn, t evolution of rarkioii f'ter inocuhtion :rnd dioxide and hydrogen began 6 t,o 7 hou rcnched a maximum after 24 houfs ( 3 0 ) . l'he total gas evolved o w 26 times the mash volume x-hilc the maximum rate of evolution was two times t,he inash volumc pcr hour. Although liydrogen predominated during the early nl-t~ges,after 12 t,o 13 hours of fermentation the concentration of citrbon dioxide equaled that of hydrogen, and afterwird carbon dioside evolut,ion exwcded that of hydrogeii. Since experiments showed t,hat>growth l,atcs of bacilli are higher than those of yeasts, an ecoriomical p r o c c s ~for the production o€ I d supplements was developed ( I S S C ) . Under opt'imum rontlitions, 50 to 60 grams of B. ?neguteriuin cells were produced in 7 to 9 hours from 100 grams of sugar. lssuming that equivalent over-all productiTTity rates can be obtained in batch and continuous fermentation processes, the latter have an obvious advantage, particularly ait'h short cycles wh.en t,he time required to clran cquipmrnt, batch the fermentor, ~ i i dstwilize the medium is a major portion of the total time. Equations have been developed (203C, 280, 9 4 D ) that. \how the relationships hct\wen thc flon rate of medium, geiierntion tinic of the organism, of equipment, and product' conccntration. Golle ( 3 d D ) notcd th:rt a distinct advantagc mists in using a second culture vessel in series, and possibly a third, in order t,o gain a higher eoncentration of product a t the same rate of feed of the nutrient solution. It was further indicated t'hat a Pyetem in equilibrium could be disrupted by select'ive grov3h of mutants or contaminants. In a plant for continuous yeast, production from sulfit'c waste liquor, 145 kg. of Candida utilis was produced per hour in a 54,000liter bat,ch with a cycle time of 4.5 hours (17bC). Cycle times of 3.14 hours (ZOSC) and 12 hoiirq ( 3 7 C ) were found to he optimum
September 1954
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
in sniail scale yeast fermentations. I n the former (SOSC), a production rate of 13.2 grams of yeast/liter-hour was attained. In the continuous culture of yeast, incomplete buffering of mcdium van result in steady cycling of the population, 90" out of phase with the pI-1 fluctuations (280). As growth proceeds, ~ u g a ris converted to acid. When the pH decreases the growth rate is slowed down also so that less acid is produced, the pH rises, and the cycle begins again. In one operating unit for continuous alcohol fermentation three fermentors are used in series. A 2.5% yeast inoculum is used in the first, while 25% inoculum for the second tank is obtained from the first, and 25% for the third from the second (680). I n another process, a modification of the Amatos process for alcohol production, two fermentors are used to reduce idle time while cleaniiig a fermentor after a contamination (180). The procedures used in the continuous fermentation of beet juice in five French distilleries were described in detail (690). Waste Disposal. It is often necessary to dispose of fermentation wastes in order to eliminate or reduce stream pollution. Techniques employed for disposal of other industrial wastes arc normally employed, I n one plant antibiotic, vitamin, and sulfa drug wastes were combined and treated by a trickling filter (1080). I n mother, the B.O.D. of penicillin wastes was reduced from 5000 to 30 p,p.m. by continuous filtration through percohting filters ($40). Recovery of Fermentation Products. The problem of recovery of acetic acid froin dilute aqueous solutions was reviewed and i t was reported that, with concentrations below IS%, extraction with ethyl acetate W ~ Ha satisfactory niethod ( 2 3 0 ) . Riboflavin was extracted from E. ashbyii broths, with and without mycelium, wit,h isobutanol (125D). A solvent extraction procedure was reported for the recovery of vitamin Biz also. In the study, diethylacetic acid was found to be a specific extractant for Bp2from fermented media and the vitamin was precipitated from the solvent layer with hexane ( S I D ) . -4liquid-solid extraction process made use of small spheres of 2,4,5-t,richlorophenol. Broth from a S. griseus fermentation was passed through a column conthining the spherel;. After being washed with water, the solids were dissolved in acetone and carbon tetrachloride and the vitamin Biz was extracted with benzyl alcohol (190). The sensitivity of the vitamin to recovery methods was demonstrated when an artificial product with antipernicious anemia activity was isolated from vitamin B12-containing fermentation broths in an extraction process using phenol, acetic acid, sodium nitrite, chloroform, a,nd butanol (116D). Since unused precursor usually accompanies penicillin through the recovery steps, it is important to be able to separate them when residual precursor concentrations are high. Phenylacetic acid was removed from benzylpenicillin solutions by extraction a t pH 4 to 5 with toluene, carbon tetrachloride, or petroleum ether ( 7 0 ) . Penicillin 0 was separated from unused precursor by forming the insoluble 2-chloroprocaine salt of the penicillin
(son).
Phenol mas used to extract st'reptomycin from broth a t pH 8
to 9 and acid extraction of the phenol solution removed the antibiot,ic product (1580). Another process would eliminate the need for acid-resistant equipment since streptomycin could be freed from the fermentation solids of the broth by addition of a mixture of inorganic salts (650). I n another study, recovery of the antibiotic was improved by adding a sequestering agent to the broth before the isolation procedure (330). An electrolytic process for the reduction of strept)omycin to dihydrostreptomycin was patented ( l S D , 69D). New recovery procedures for other antibiotics were also proposed. Neomycin was absorbed on ion exchange resin from broth a t acid pH and eluted with a nitrogen base such as ammonium hydroxide or solutions of quaternary ammonium salts (79D). Oxytetracycline (1070) and viomycin (1000)were precipitated
1821
iroiii fermentation broths by the addition of dyes. For final i.ecovery of the former, the precipitate was dried, ground, suspcnded in acetone, and treated with trimethylamine sulfate at pIl 2. The dye was filtered off and the solution was adjusted to p€I 6 to precipitate the oxytetracycline (1070). Prodigiosin was extracted from Serratiu niarcescens cells with solvents eont,ainirig six or more carbon atoms and the compound was crystallixcd tliiwily from the solvent, (son).
BIBLIOGRAPHY GENERAL
Allen, L. A., 12epts. Prop. A p p l . Chem., 37, 190 (1952). Birkmshaw, J. H., Ann. Rev. Bioehem., 22, 371 (1953). Jong, L. E. den Doolen de, "AZiorobenalsVriendenEnVljanden Van de mens," H. Nellssen, Bilthoven, 1952. Kawamura, S.,HulckB Ky6kai Shi, 11, 13, 69 (1953). Ledmgham, G. A., Ann. Rev. Il.licrobiologg, 7, 433 (1953). Lee, S. B., IND. ENG.CHEM.,43, 1948 (1953). Ley, J. de, P h a r m . Tijdschr Belgae, 30, 184 (1953). Pcrlman, D., Brown. ITr E , and Lee, S. B., Zbid , 44, 1996 (1952).
Perlman, D., Tempel, A. E., Jr , and Brown. W.E., Ibid., 45, 1944 (1953).
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Saniclivier, &I., Inds. agr. et aliment. ( P a r i s ) , 70, 995 (1953). Waksman, S. A., Biology of Actinomycetes and Their Economic Importance in "Symposium: Actinomgcctttles," Fondazionc Emanude Pttterno, Roma, 1953. ECONOMICS
(1B) Bohmfalk, J. P., Chem fino. Mews, 31, 4182 (1953). (2B) Ibid., p. 5006. (3B) Ibid., p. 5186. (4B) Ibid., p. 5356. (513) Chem. Eng., 60, 150 (October 1953). (6B) Chem. Eng. AVeuis,31,192(i (1R53). (7B) Ibid., D. 2298. (8Bj Ibid., p. 3553. (9B) Ibid., p. 3773. (IOB) Ibid., p. 4254. (11B) Ibid., p. 4694. (12B) Ibid., p. 5242. (13B) Ibid., 32, 1170 (1954). (14B) Chem. Week, 72, 32 (Mar. 28, 1963). (15B) Ibid., p. 48 (May 9, 1953). (16B) Ibid., p. 76 (May 16, 1953). (17B) I6id., 73, 26 (Aug. R, 1953). (t 8B) Ibid., p. 30 (Sov. 28, 1963). (I9B) Ibid.,74, 8 (Jan. 9, 1954). (20B) Ibid., p. 28 (Jan. 16, 1954). (21B) Ibid., p. 86 (Jan. 23, 1954). (2213) Ibid., p. 12 (Feb. 6, 1954). (23R) Ibid.. p. 87 (Feb. 20, 1954). (24B) Ibid., p. 16 (April 3, 1954). (Z5B) Ibid., p. 14 (April 10, 1954). (2613) Ibid., p. 22 (April 10, 1954): (2713) Chemistry & Industrv, 1953,p. 691. (28B) Ibid., 1954,p. 246. (29B) FDC Reports, 15,W4 (June 27, 1953). (30B) Ibid., 16, W6 (Jan. 23, 1954). (31H) Ibid., W14 (Feb. 27, 1954). (3213) Ibid., W8 (March 20, 1954). (3313) Ibid., W9 (March 20, 1954). (34B) Ibid., W7 (April 3, 1954). (35R) Ibid.. W 8 (April 3, 1954). ENG.CHEM.,45,9A (August 1953). (36B) IND. (37l3) Ibid., p. 7 8 (October 1953). (3813) J . Agr. Food Chem., 1, 203 (1953). (39B) Ibid., p. 653. (40B) Larsen, R. F., Abel, F. K.. and E'oden, A. E., C h m . Week, 74,24 (Jan. 9 , 1954). (41B) M j g . Chem., 23, 483 (1952). (42B) Nature, 171, 1010 (1953). (4313) Zbid., 172,329 (1953). (44B) Ibid., 173,22 (1954). (45B) New Yolk Times (Jan. 10, 1964). (46B) Sato, N., personal communication. (47B) U. S . Dept. Commerce, special report (Nov. 10, 1950). (48B) U. S. Dept. of Commerce, Bureau of Census, Rept. FT410,
1953.
(49B) U. S. Tariff Commission, Synthetic Organic Chemicals, 1962.
INDUSTRIAL AND ENGINEERING CHEMISTRY
1822
(50B) U. S. Treasury Dept., Annual Rept. of Commissioner of Internal Revenue, June 30, 1953. (SIR) Weirich, L., personal communication. (52B) Welch. H., Antibiotics & Chemotherapy, 4, 87 (1954). (53B) World Health Organization, Chronicle, 7, 327 (1953).
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INDUSTRIAL FERMENTATION PROCESSES
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“Antibiotics-Survey of Properties and Uses,” Pharmaceutical Press, London, 1952. Irnstein, H. R. V., and Grant, P. T., Biochem. J., 55, v (1953). Asahi, Y . , A n n . Repts., T a k e d a Research Lab., 11, 24 (1952). ilso, K., and associates, J . Ferm. Technol. (Japan),31, SI (1953). Baba, T., Bull. Fac. E n g . , Himshima Univ., No. 1, 111 (1952). Baba, T., and associates, Ibid.. S o . 2, 33 (1963). Baetsle, R., Fermentation, 1953, p. 190. Baker, D. L. (to B.L. Sarett), U. S.Patent 2,651,592 (Sept, 8, 1953). Baldwin, R. R., and associates, Food Technol.. 7, 275 (1953). Bartz, Q. R., Haskell, T. H., and associates, S a t u r e , 173, 7 2 (1954). Basaca, WI. G., Philipp.lne J . Sci., 81, 75 (1952). Bealing, F. J., Biochein. J., 55, 93 (1953). Beesch, S.C. (to Publicker Industries), U. S.Patent 2,641,568 (June 9, 1953). Beesch, S. C., and Fraser, B. W,, Ibid., 2,647,074 (July 28, 1963). , Bender, 8.E., Nature, 171, 917 (1953). Benedict, R. G., Botan. Reo., 19, 229 (1953). Benedict, R. G., and Stodola, F. H. (to U. S. A,, Sew. of Am.). U. S.Patent 2.617.755 (SOT-. 11. 1952). Biochem. J., 52, 1 (1952). Blair, 11. G., and Pigman, W.W ,Arch. Biochem. and RLOphys., 48, 17 (1954). Blinks, L. R., Physiology of the algae, in “Mauual of Phycoiogy,” (G. 11. Smith, editor), Chronica Botanica, Waltham, Mass., 1951. Block, S.S.,and associates, J . B g r . Food Chem., 1, 599 (1953). Bohonos, N., and associates, Antibiotics A n n u a l , 1953-4, p. 49. Boothe, J. H., and associates, J . Am. Chenz. Sue., 75, 4621 (1953). Boothe, J. €I., and associates, Antibiotics Anneial, 1953-4, p. 46.
Borenztajn, D., and Kurylowicz, K,, M e d . Doswiadczak~sai Mikrobiol., 5, 283 (1953). Bourne, E. J., Ann. Repts. Progr. C‘hem., 49, 235 (1952). Brabender, C. W., and Pagenst,edt, R., Bakers Big., 27, 17 (1953). Brandl, E., and associates, W i e n e r Medizinsche Wochenschr., 103, 602 (1953). Braude. R.. and associates. Antibiotics and Ciiemotiiempy, 3 , 271 (1953). Brewer, G. il., and Johnson, hl. J , , A p p l . i ~ f i c r o b i o l . ,1, 163 (1953). Bricase, E., and Fromageot, C., Advances in Pmfein Chem., 8 4 (1953). Brockmann, H., Angew. Chem., 66, 1 (1954). Brockmann, H., Bohnsack, G , , aud GrSne, H., .Vat!~rwiss.. 40, 223 (1953). Brockmann, H., and Grone, €I., Ibid., 40, 222 (1953). Brockmann, H., Linge, H.. and Grone, H., Ibid., 40, 224 (1953). Brown, R., and Hazen. E. L.. Antibiotics & Chemot%er.apy,3 , 818 (1953). Bujack, S.,A c t a Microbiol. Polon., 1, 65 (1962). Bulloff, J. J., and Novak, D. J., Presented at 125th Meeting, ACS. Kansas City, 1954. Bunch, R. L., and McGuire. J . &I. (to Eli Lilly & Co., Inc.). U. S.Patent 2,653,899 (Sept. 29, 1953). Burlew, J. E., Carnegie Inst. of Washington, Pub. 600, 1953. Ibid., p. 3. Butlin, K. R., Abstracts S’th Congr. Intern. Microbiol., Rio de Janieiro, 1950, p. 183. Butlin, K. R., Research ( L o n d o n ) , 6, 184 (1953). Butlin, K. R., and Postgate, J. R., in “Symposium on l i c r o bial Metabolism,” Fondazione Emanuele Paterno, Roma, 1953. Chem. Eng., 61, 195 (January 19d4).
(5OC) (6lC)
(54C) (55C) (56C)
Vol. 46, No. 9
Chem. E n g . N e w s , 31, 2786 (1953). Ibid., p. 3393. Ibid., p. 4033. Cliem. W e e k , 72, 54 (March 7, 1953). Ibid., p. 59 (March 14, 1953). Ihid., p. 50 (March 28, 1953). Ibid., p. 42 (April 18, 1953). Ihid., 73, 11 (Oct. 10, 1953). Ibid., p. 43 (Kov. 21, 1953). Ibid., p. 10 (Dec. 12, 1953). Chiao, J. S.,and Peterson, W. H., J . Am. Food Chem., 1, 1005 (1953). Chugtai, I. D., and Walker, T. K.. Biociieni. J., 56, 484
(1954). Claridge, C. A , and Kerkman, C. T i . , Arch. Biocheni. and Biophys.. 47, 99 (1953). Clark, R. K., Jr., Antibiotics & Chemotherapg, 3, 663 (1953). Clarke, D. A . , and associates, Presented a t 125th Meeting, dCS, Kansas City, 1954. Coates, M. E., Chemistry 6: Ilid?LSh’~,1953, p. 1333. Colingsworth, D. R., aiid associates, J . Biol. Chenz., 203, 807 (1953). Comers, W. AI.. and Cort, W.31., Presented at 124th Meeting ACS,Chicago, 1963. Conorer, L. H., and associates, J . Anz. C h m . Sac., 75, 4622 (1953). Crewthcr, W.G.. and Lennox, F. G., Rust. J . Biol. Sei., 6, 410 (1953). Ibid.. p. 428. Current Sci. ( I n d i a ) , 22, 382 (1953).
Davey, F;. F.,and Johnson, M. J., 4 p p l . M i ~ r ~ b i o Z .1,, 208 (1953). Davis, E. A., and associates. Carnegie Inst. of Washington, Pub. 600, p. 105 (1953). De Becze. G. I. (to Schenley Industries), U. 8. Patent 2,636,823 (April 28, 1953). Dedonder, R., and Soblesse, C., A?in. Inst. Pastew, 85, 356 (1953).
Dikanskaya, E. A$., MikrobioZogiya, 22, 256 (1953). Dion, H. W.,and associates, J . Am. Cham. Soc., 76, 948 (1954). Distillers Co. Ltd., and AIcCornbie, J. J.. Brit. Patent 685,531 (Jan 7 , 1953). Djerassi, C., V i t a m i n s and Hormones, 11, 205 (1953). Dorfman, R. I., and Ungar, F., “Xetaholism of Steroid Hormones,” Burgess, Minneapolis, 1953. Dutcher, J., and associates, Antibiotics An?lual, 1953-4, p. 191. , Dutcher, J., and associates, Antibiotics and C h e m o t h e r a ~3, 910 (1953). East Anglia Chemical Co., Ltd., and IIaworth, V. C., Brit. Pat. 681,548 (Oct. 29, 1952). Ehrlich, J., Anderson, L. E., and associates, Nature, 173, 72 (1964). 13, Ehrlich, J., Coffey, G. L., and associates, Federation PTOC., 351 (1954). and associates, Am. Rea. TubercuEhrlich, J., Smith, R. M., l o s i s , 63, 7 (1951). Evans, D. G., and Wardlaw, A. C., b. Gen. Xicrobiol., 9, 481 (1953). Evenari, M . , and associates, Carnegie Inst. of Washington, Pub. 600, p. 197 (1953). 5, 1952). Farbenfabriken Bayer, Brit. Patent 682,329 (SOT. Farrell, L., C a n . J . M e d . Sei., 31, 512 (1953). a , 28 (1953). Fenilrsova, R. V., i ~ f i k ~ o b i o l o g i y 22, Finlay, A. C., and associates, Am. Res. Tuberculosis, 63, 1 (1951). Fisher. A. W..Carnezie Inst. of Fashineton. I . Pub. 600. D. 311 ‘(1953). Fisher, A. W., and Burlev J. S., Ibid., Pub. 600, p , 303 (1953). Flynn. E. H., and associates, Presented a t 124th meeting, ACS, Chicago, 1953. Eogg. G. E., “The Metabolism of Algae,” John Kiley, Kew York, 1953. Fogg, G. E., and Collyer, D . AI., Carnegie Inst. of Washington. Pub. 600. 0 . 177 (1963). Ford. J. H., and associate3,‘Anltbiotics arkd Chemotherapy. 3, 1149 (1953). Fried, J., and Sabo, E. F., J . Am. Chem. Soc.. 75, 2273 (1953). Ibid., 76, 1455 (1954). Fried, J., and associates. Ihzd., 75, 5764 (1953). ~
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I
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September 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
(98C) Friedman, M. E., and associates, J . D a i r y Sci., 36, 1124 (1953). (99C) Fryth, P. W., Chem. Eng. N e w s , 32, 813 (1954). (1OOC) Fukui, S., and Mohara, K., J . Perm. Technol. ( J a p a n ) , 29, 198 (1951). (101C) Fukumoto, J., and associates, Symposium on Enzyme Chemistry, 8, 40 (1953). (102C) Fusari, 9. A., Frohardt, R. P., and associates, Presented a t 125th Meeting, ACS, Kansas City, 1954. (103C) Fusari, S. A., Haskell, T. H., and associates, Presented at 125th Meeting, ACS, Kansas City, 1953. (104C) Gaffron, H., Research ( L o n d o n ) , 6, 222 (1953). (105C) Garrido. J. M.. and Walker, T. K.. A n a l e s real SOC. esaafi. fh y quim., 49B, 81 (1953). (106C) . . Geoghegan, 1LI. J., Carnegie Inst. of Washington, Pub. 600, p.-182 (1953). (107C) Goldsmith, M. T., Fertile Research J., 20, 613 (1950). (108C) Gordon, M., Pan, S. C., and associates, Science, 118, 43 (1953). (109C) Gottlieb, D., in “Symposium on Actinomycetales,” Fondazione Emanuele Paterno, Roma, 1953. Mycologia, 45, 507 (1953). (llOC) Gottlieb, D., and Legator, M., (111C) Gottshall, R. Y., and associates (to State of Michigan), U. S. Patent 2,658,018 (Xov. 3, 1953). (112C) Gray, P. P. (to Wallerstein Co.), Ibid., 2,665,215 (Jan. 5, 1954). (113C) Gregory, M. E., and Woodbine, M., J . Ezptl. B o t a n y , 4, 314 (1953). (114C) Groggins, P. H., J . Agr. Food Chem., 1, 1193 (1953). (115’2) Gummert, F., and associates, Carnegie Inst. of Washington, Pub. 600, p. 166 (1953). (1lGC) Haines, W.J., and Colingsworth, D. R. (to the Upjohn Co.), U. S. Patent 2,649,401 (Bug. 18, 1953). (117C) Hale, C. W., and associates, Nature, 172, 545 (1953). (118C) Hall, H. H. (to U. S.A., Sew. Agr.), U. S.Patent 2,643,213 (June 23, 1953). (119C) Hall, H. H., Renedict, R. G., and associates, Bact. Proc., 1954, p. 18. (120C) Hall, H. H., Benedict, R. G., Wiesen, C. F., and associates, AppZ. MicrobioZ., 1, 124 (1953). (121C) Halliwell, G., J . E r p t l . B o t a n y , 4 , 369 (1953). (122C) Hanoaka, A., and associates, Mem. I n s t . Sei. and I n d . Research, Osaka Univ., 9, 202 (1952). (123C) Hanoaka. A., and Kikuni, J., J . A g r . Chem. SOC.J a p a n , 26, 14 (1952). , Caldwell, bl. L., J . Am. Chem. SOC., (124C) Hanrahan, V. M ~ and 75, 2191 (1953). (125C) Hanson, F. R., and Eble, T. E. (to the Upjohn Co.), U. 5. Patent 2,652,356 (Sept. 15, 1953). (126C) Hanson, F. R., Mann, K. M., and associates, J . Am. Chem. Soc., 75, 5369 (1953). (127C) Harris, D. A , , and Woodruff, H. B., Antibiotics A n n u a l , 1953-4, p. 609. (128C) Haskins, R. H., and associates, Abstracts Vth Intern. Congr. Microbiology, 1950, p. 189. (129C) Hausmann, E., and Zischinsky, H., W i e n e r Medizinische Wochenschr., 103, 725 (1953). (130C) Heathy, N. G., and Florey, H. W., Brit. J . Pharmacol., 8,252 (1953). (131C) Hellman, K.K.,and associates, Presented at 125th meeting, ACS, Kansas City, 1954. (132C) Hesseltine, C. W., and associates, Mycologia, 46, 16 (1954). (133C) Hester, -4.S., and Ward, G. E., IND.ENG. CHEM.,46, 238 (1954). (134C) Hickey, R. J., J . Bact., 66, 27 (1953). (135C) Hickey, R. J. (to Commercial Solvents Corp.), U. S.Patent 2,667,445 (Jan. 26, 1954). (136C) Hickey, R. J., Corum, C. J., and associates,.Antibiotics & Chemotherapy, 2, 472 (1952). (137C) Hobby, G. L., Trans. 12th Conf. Chemotherapy of Tuberculosis, 1953, p. 300. (138C) Hochstein, F. A,, and associates, Presented at 125th Meeting, ACS, Kansas City, 1954. (139C) Hochstein, F. A., Stephens, C. R., and associates, J . Am. C h e m . Soc., 75, 5455 (1953). (140C) Hodge, H. M.,and Hildebrandt, F. M., in “Industrial Fermentations,” Chemical Pub., New York, 1954. (141C) Hofreiter, B. T., and associates, Presented at 124th meeting, ACS, Chicago, 1953. (142C) Holdsworth, E., and associates, .Vatwe, 171, 148 (1953). (143C) Holmlund, C. E., and associates, Presented a t 124th Meeting, ACS, Chicago, 1953. ~I
1823
(144C) Hori, I., and Nakatani, T., J . Ferm. Technol. ( J a p a n ) , 31, 72 (1953). (145C) Ibid., 32, 33 (1954). (146C) Hosoya, S. (to Inst. for Infectious Diseases), Japan. Patent 2249 (’52) (June 17, 1952). (147C) Hosoya, S., Komatsu, N., and associates. J a a a n . J . E z d . M e d . , 22, 303 (1952). (148‘2) Hosoya, S., and associates, J . Antibiotics ( J a p a n ) , 6, Ser. B, 61 (1953). (149C) Hromatka, O., and associates, Enzymologia, 15, 337 (1953). (150C) Ichino, K., J . Ferm. Technol. ( J a p a n ) , 28, 459 (1950). (151C) IND. ENG.CHEM.,45, 8-4 (Xarch 1953). (152C) Ibid., p. 9A. (153C) I b i d . , p. 7.4 (November 1953). (154C) I b i d . , p. 8.4 (December 1953). (155C) I b i d . , 46, 158 (February 1954). (156C) Inoue, N., Japan. Patent 4995 (’52) (Nov. 26, 1952). (157C) Irvin, R., in “Industrial Fermentations,” Chemical Pub., New York, 1954. (l58C) Iwata, Y., and Ikeda, T., J . Ferm. Technol. ( J a p a n ) , 29, 14 (1951). (159C) James, A. E., Chemistry & I n d u s t r y , 1954, p. 38. (160C) Janicki, J., and associates, X e d . Doswiadczawa i MikrobioZ., 5 , 283 (1953). (161C) Jermyn, M. A,, A u s t r a l i a n J . Biol. Sci., 6, 77 (1953). (162‘2) Johnson, -4.J., and Selson, C. R., Chemistry & I n d u s t r y , 1953, p. 28. (163C) Johnson, M. J., in “Industrial Fermentations,” Chemical Pub., New York, 1954. (164C) Jorgenson, J., and Convit, J., Carnegie Inst. of Washington, Pub. 600, p. 190 (1953). (165C) Joslyn, &I.A., J . A g r . Food Chem., 1, 36 (1953). (166C) J . A g r . Food C h e m . , 1, 398 (1953). (167C) Ibid., p. 596. (168C) Ibid., p. 863. (169C) I b i d . , p. 869. (170C) Kanie, M., Mem. Fac. Agr. Kagoshima Univ., 1, 87 (1952). (171C) Katznelson, H., and associates, J . B i d . Chem., 204, 43 (1953). (172C) Keilin, D., Nature, 172, 390 (1953). (173C) Kitahara, K., and Fukui, S., Symposium on Enzyme Chem., 8, 108 (1953). (174C) Kitahara, K., and Murata, U., J . FeTm. Technol. ( J a p a n ) , 31, 349 (1953). (175C) Klaushofer, H., Mitt. Versuch. Garwngsgew. W e i n , 6, 124 (1952). (176C) Kleinseller, A., and Beran, K., Chem. L i s t y , 47, 447 (1953). (177C) Kligman, A. M., and Solotorovsky, bl., Trans. 12th Conf. Chemotherapy of Tuberculosis, 1953, p. 323. (li8C) Kobayashi, S., and associates, TBhoku J . EzptZ. ‘Wed., 55, 273 (1952). (179C) Kodama, R., and X’agai, T. (to Takeda Pharmaceutical Industries, Co.), Japan. Patent 4993 (‘52) (Kov. 26, 1952). (ISOC) Koepsell, H. J., and associates (to U. S. A,, Secy. -4gr.), U. S. Patent 2,660,551 (Nov. 11, 1953). (18lC) Kok, B., Carnegie Inst. of Washington, Pub. 600, p. 63 (1953). (182C) Kondo, K., and Wada, S., J . Ferm. TeehnoZ. ( J a p a n ) , 27, 331 (1949). (183C) Krauss, R. W., Carnegie Inst. of Washington, Pub. 600, p. 85 (1953). (184C) Krauss, R. W., and hIcAleer, W.J., Ibid., p. 316. (185C) Krider, M. &I.,and associates, Bact. Proc., 1954, p. 24. (186C) Kurasaw, F., and associates, Symposium on Enzyme Chemistry, 8, 122 (1953). (187C) Lechevalier, H., and Waksman. S. A,, Trans. 12th Conf. Chemotherapy of Tuberculosis, 1953, p. 321. (188C) Lewis, J. C., Ijichi, K., and associates, J. A g r . Food Chem., 1, 897 (1953). (189C) Lewis, Y. S.,and Johar, D. S., Current Sei. (Indie),21, 311 (1953). (19OC) Lima, 0. G. de, and Sanchez-Marroquin, A., Microbial. E s p a n . 6, 127 (1953). (191C) Little, A. D., Inc., Carnegie Inst. of Washington, Pub. 600, p. 235 (1953). (192C) Lobo, J. M. V., and associates, A n a l e s real SOC. espafi. P s y guim., 49B, 245 (1953). (193C) Lockwood, A. R., Chemistry & I n d u s t r y , 1954, p. 40. (194C) Ludwig, H. F., and associates, Sewage a n d I n d . Wastes. 23, 1337 (1951). (195C) Lumb, M., Repts. Prog. A p p l . Chem., 37, 677 (1952).
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1824
(196C) Mandl, I., and associates, J . Clin. Invest., 32, 1323 (1953). (197C) Narsh, W. S., and associates (to CIBA, Inc.), IJ. S. Patent 2,633,445 (;\Tar. 31, 1953). (198C) Martin, E., Berky, J., and associates, J . B i d . Chern., 203, 239 (1953). (199CI hIatsuoka, M., and associatea, Japa.7r.J. ;Med. Sci. Hiol., 6 , 161 (1953). (200C) Matsushima, K., J . Fertn. ‘Z’echnol. (Japan),28, 90 (1950), (ZOIC) Ibid., p. 173. (202C) Ihid., 30, 111 (1952). (203C) Maxon, W. D., and Johnson, 31. J., IND. EN^. CHEM.,45, 2554 (1953). (204C) McCarthy, J. L., in “Industrial Fermentations,” Cheinical Pub., New York, 1954. (205’2) McClelland, L., Antibiotics Annual, 19534, p. 615. (206C) McCutchan, W. K., and Hickey, R. J., in ”Industrial Fermentations,” Chemical Pub., New York, 1954. (207C;) MeDaniel, L. E., and Woodruff. H. B. (to Merck & Co., Iiic.), U. S. Patent 2,650,896 (Sept. I, 1953). (208C) &fellies, R. L., Mehltretter, C. L., and associates, Presented at 124th meeting, .4CS, Chicago. 1953. (209C) Mellies, R. L., Rogovin, S. P., arid associates, Ibid., 115th meeting, Kansas City, 1954. (21OC) Rlilner, H. W., Carnegie Inst. of Washington, Puh. 600, y. 286 (1953). (211C) bliner, C. S., and Wolnak, B. (to Sewerage Corniiiission of City of Milwaukee), CJ. S. Patent 2,646,386 (Sept. 21, 1953). (2lZC) Minieri, P. P., and associates, Antibiotics Annual, 1953-4, p. 81.
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i270C) (271C) (87%) (273C) (2742)
(275C)
406 (1953). Ihid., p . 421.
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*
(1954).
(276C) Pirie, N , W., Ciirinistry & I n d i i a t i y , 1953, p , 442. (277C) Pisano. :\I,,and Olson, W. T I . , Prezented at 124th ACs, Chicago, 1953. (278C) Porter, ,J. W. Q.. personal corniiiiiiiic.iit,ioii. (279C) Posteate. J. R.. .I. &?a. M?‘ciobioL.,5. 714 (19513.
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1004 (1953). (293C) Roberts, H. R., and I1
yen,
KC. Y.,Arch. 1 ~ i o ~ : h c iwr t.i d
Biophijs., 43, 233 (195, . (294C) Roberts, H. R,,, and McFarrcii, 1:. I,’,,.1.Dai,y/ ,4ci., 36, (1953).
1?%1
1
(1853).
(243C) Sadel, K.,and associates, B y p l . itficrobiol., 1, 217 (192.3) (2446) Kagao, M., A&. Antibiotics ( J a p a n ) , 4, 61 (1951). (2432) I b i d . , p. 65. (246C) Nakahama, T., J . F E T V ~Tecimol. . ( J a p a n ) , 29, 331 (1951’1. (247C) Nakahama, T., and Ishida, H., Ibid., 30, 100 (1952). (2486) Kashif, S. A., and ?;elson, F.R., 6.D a i r y Sei., 36, 459 ( I 953 I . (2490) I b i d . . D. 471. 698. (250C) Ibid.; (261C) Nelson, J. W., and asmciates, Science, 119, 379 (1954). (252C) Newton, G. G. F., and Abraham. E. P., N a t u r e , 172, 3 Y i (1953). (253C) Nickerson, R.J., and Rain Mohan, R,, in “Syinpoiiurri on Actinomycetales,” Fondazione Emanuele Faterno, Iionia, 1953. (2540) Nicolaides, E. D., and assoriates, Presented at 125th Meeting, ACS, Kansas City, 1954
i.
(1953).
83, 17 (1953). (300Ci Roy. D.
I
