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INDUSTRIAL AND ENGINEERING CHEMISTRY
Power: Mill, 420 h. p.; classifier fan, 120 h. p. Remarks: One of the unusual features about this installation is the extremely low maintenance in face of the heavy duty imposed on the mill. For example, the cost of ball replacement is only one cent per ton of cement ground. Grinding Iron Pyrites. Purpose: Flash-roasting for manufacture of sulfuric acid. Unusual requirements: To obtain a product through 35 mesh with a minimum on 200 mesh to enable close control of the furnace conditions by virtue of obtaining a comparatively uniform grain product. Mill used: One 7 foot x 48 inch Hardinge Ball Mill with rotary air classifier. Feed size: Minus 0.75 inch. Capacity: 9 tons per hour. Product: 95 per cent through 35 mesh with 60 per cent plus 200 mesh. Power: Mill, 140 h. p.; classifier fan, 45 h. p. Remarks: This unit contains recent improvements to the air classifier which increases the capacity nearly one-third over the expected results, with a corresponding decrease in operating costs.
Vol. 26, No. 11
Grinding Silica.
Purpose: For use in high-grade pottery manufacture and special processes. Unusual requirements: Mill produces two products simultaneously, one very fine and the other granular but with sharp, angular grain particles. Mill used: One 8 foot X 60 inch Hardinge Conical Pebble Mill and 4.5-foot superfine air classifier with by-pass. Feed size: Minus 20-mesh beach sand. Product: From collector, 99.9 per cent minus 200 mesh; granular product from by-pass, 11 per cent on 60 mesh, 6 per cent passing 325 mesh. Capacity: Superfine product, 1.66 tons per hour; granular product, 1 ton per hour. Power: Mill, 65 h. p.; classifier fan, 20 h. p. Remarks: The product has t o be absolutely free from metal contamination; hence the grinding is done with flint pebbles and the mill lining is also flint. As the classifier fan handles only return air, there is no contamination here. RBCBIYED September 4, 1934.
Citric Acid Fermentation W. P. DOELGER AND S. C. PRESCOTT, Massachusetts I n s t i t u t e of Technology, Cambridge, Mass.
A
MONG the classic studies
t h o u g h m a x i m u m y i e l d s of The important factors governing the rate of incitric acid are obtained from suof Pasteur were interestcrease in citric acid fermentation by organisms of . ing investigations on the the Aspergillus niger type are indicated. When crose; this finding has been confirmed by B e r n h a u e r (3) and chemical changes produced by the technic governing these factors has been so Molliard (15). fungi other than yeasts, and imregulated that the rate of increase is uniform in When sucrose i s e m p l o y e d portant fundamental facts refor the fermentation, theoretis u l t e d therefrom. It is now successive fermentations in a "standard mecally 1 molecule of the sugar known that certain fungi, pardium," the actual eficiency of the fermentation produces 2 molecules of citric ticularly black or brown organwill depend on the ratio of volume to surface area acid or 1 gram of sucrose proisms b e l o n g i n g t o the group of medium employed. The depth of medium in duces 1.12 grams of citric acid : A s p e r g i l l u s n i g e r , have t h e the container must be such that a maximum ability to ferment sugar solutions CJL20ii Hz0 60 = to citric acid. amount of acid will be produced, in the shortest 2CsHsOi 4Hz0 Wehmer (20), about 1892, was time, with a minimum amount of sugar remainHowever, not all of the suthe first to report that fungi, ing unconverted to citric acid at the conclusion of crose in the medium is available which he named C i t r o m y c e s , the fermentation period. for direct conversion to citric produced citric acid from sugar acid by the organism, as much s o l u t i o n s . The fungi which Wehmer observed were green Penicillia, and it was not until of it is utilized in the formation of the mat or mycelium and sometime later that strainr of Aspergillus niger were recog- some is lost in the form of carbon dioxide as a result of the nized as citric acid producers. Currie (8),in 1917, as the organism's respiratory activity. The large majority of the fungi belonging to the group result of his work with Thom (18), showed that citric acid could be produced in considerable amounts by certain strains Aspergillus niger, and particularly the black species, accordof Aspergillus niger when grown in acidified solutions contain- ing to Angeletti (2) can be made to produce citric acid by reing sucrose and relatively small amounts of inorganic salts. peated transfers of the spores in media containing sucrose or Currie also showed that the formation of oxalic acid, which had dextrose with the addition of the proper type and amount of been previously recognized by Wehmer ( H ) ,Emmerling ( l l ) , inorganic salts (8). The major difficulty encountered in Heinze ( I S ) , and others as an important product of the fer- dealing with the fermentation is concerned with the discovery mentation of Aspergillus niger could be considerably reduced of a method or methods for the production of uniform yields by adjusting the nitrogen supply and the initial hydrogen-ion of citric acid in successive fermentations. The difficulty concentration of the medium. The maximum yield of citric mentioned is due, in a large measure, to the fact that these acid is usually obtained within a 9- to 12-day period of incuba- organisms are particularly sensitive to many conditions which govern the fermentation; slight differences in these contion at any temperature within the limits of 24" to 28" C. The general mechanism of the citric acid fermentation is ditions in apparently similar fermentations will result in still obscure in spite of many attempts to isolate the reaction wide variations in the yield of citric acid. It is the purpose of this paper to suggest certain convenient products, May and Herrick (14) have compiled a bibliography of the work accomplished. Changes occur rapidly methods for maintaining uniformity of conditions governing during the fermentation, and for this reason investigators the citric acid fermentation, based on some of the results have found it difficult to make satisfactory analyses. Ame- that have been obtained in connection with a comprehensive lung (1) has shown that citric acid can be produced by A s - investigation dealing with the normal characteristics of the pergillus niger from 3-, 4-, 5-, 6-, and 12-carbon sugars, al- fermentation.
+
+
+
I N U U S T I I I A L A N D E N GI I N E E I+ 1 N G
xr>\en,1>er,1934
I'KBCAUTIONS 'ro Be O n s ~ n v m Tlie ~ a l u eof expcriniental results dcpeiids to a groat cxtent mi tlie adoption of a uuiforin tecluiic in setting UJJ the citric acid fermentations. At tlie outset, tlirrefore, it has beeti found useful to observe the following precautbn.~:
c H E f r i I S T 1%Y
I143
siiinll quantities of inorganic salts coutaiiiirig the cleinents nitro#!ri, potassium, pliosphorus, magnesium, and cliloririe. 'I'lie presence of all of tlicse eleinerits in the inodium has been foiind to play a particiilarly important role in the nature and regulation of rate US tlie metabolic cimiges brought about by the organism.
Tho stock cultures of Aspergillus r ~ i g e rmust be tmnafenad at, definite intervals on II medium simiiar in chcniicd cornpositinn mid ?;"gar conoentmtion t o that, which is tu l x employcd for the fermentrrtions. The Aspergillus niger orgmisms, likc the majority of related fungi, grow on thr surfm? of the mcdium, and characteristic changes are brought about by nieaiis of enaynm contained inside of the fungus miit or myeeliurn. The urgrrnisms begin their growth from round, dark colored spores formed in groat numhcrs R t the end of branches protruding from the white surface of the mnt (Fikwre 1). It is diliiciilt to obttiin uniform yieids of citric scid ualess a t least four YU spores arc mitile in tho culture medium at In order i,o iireveiit unsuspected contnminntian of the stock eukure, n mcthnd of purifimtion must be employed at definite intervals. The stock cultures which are to he used to inoculate the test fermentations must be of the same a m at 1,he time of each irtoculetion. Tho shapes of the containers u-hich are to be employed for the fermentation should he as nearly alike as possible so that the relation between the volume (cubio oentimeters) and the surhce area (saume centimeters) of the medium udl be substsnthlly constint for e a h contain&. When making inoculations, it is sdvisabie t,o perform them in un inoculating chamber since it requires niueh time and care to seed each Rnsk with il uniform amount of uncontaminated spores. The metlit should ha made up with dist,illed tmter. In fermnmtntions ruu over a period of 6 months in substrates of "uniform" cornpsition, results have shown that yields of citric ncid ~ w i m :1 , n F I C A L ~I,~~nt.:-~,r:nrrrNr. ~irmosOF ~ s p e r q i i i u s were more stable in the modin made up with di;rt,illed water than nr,qcr in t,lie ease of similar media y p n r , ~ dwith Cambridge tap WatCT. C'i,*b-ai,ilpadhead boars t w o I O W B 01 o"I.mn&r d / B W i L l i "".,rea lorming It would appear t.hat the dig t vurirrtiun in cheniical composition nt t h e ends of the seound row 01 < : e i / ~ , of bhe tap water over n period of t i m e mi~yalter the composition of the pure sucrose and inorganic salt solution sufficiently to Experirnents werc designed with the priiiiary purpose of affect the t.itratsb1e acidity produced. Fininally, t,he sugar and inorganic d t s used in the ~~rspamtioii developing a simple rnediurn supplied with the proper type of the medium must, bo of a uniformly good c. P. grade. aiid aim~uiitof inorganic salt8 so that the sugar could be fermented to a high titratable acidity by the organism in a Mzritoo OF C i i o o s i ~A X~ 01tc~l;islr IO-day jmiod OS iiicubatioii. Fermentations were run in subFor the niir~osesof this iavestimition eight species of black strates made up with inorganic salts containing nitrogeri, pctassium, ~~liosphorus, sulfur, magnesium, and chlorine, added in various combinatiuus and amounts t o a 14 pcr ceiit sucrose tion at 28' C. '"For this numoie the s e v c ~ dorganisins were solution. Each batch of medium was adjusted t o pH 3.00 subcultured in test tubes Eontaining 10 cc. oi Ciapek's broth witti hydrochloric acid and then autoclaved for 30 minutes (IO),with a11 initial reaction of pH 3.00, rind uviiig n 13 per cent nt 8 to 9 pounds per square iricli (0.56 to 0.63 kg. per sq. cm.) q,,,Tnse IIF a Imsc. Five tmnsfrrs were m d c a t inter.I ..... aoll,ti",, ~.~~~ titt!mi pressurc. The 250-cc. flasks eiiiployed for the fervds of 8 days; after-the completion of the %day incubation period of thr fifth t.r;tnsier, spores were seeded lightly over the nierrtations were "filled" with 75-cc. portions of medium and serl:tce of 75-cc. portions of the Caapek broth medium in 2 5 k c . seeded with spores taken from test tube cultures 10 days old. Erlenmeyer flasks. I( the purposes of inoculation, a quarterThe hydrogen-ion concentratioir of the fermented and uninch (0.635-cm.) por n of a straight %%gage platinum needlr ferniented niedium was determined by use of the quinliydrone was moistened, nnd the spores adhering to the moistened end electrode method; in determining tlie pEI by this means there \rere distributed on to the surface of the medium. Determinntions were made for titclbuble acidity on tho ninth were never any instances of a poisoning action of tlie electrode and tonth days of the fermentation eriod. Qualitative testa by the solution teyted, arid results checked the hydrogen elecwere mnde for citric acid b y mems of t i e Iknige method (6). trode method with a madiriurri error of 0.05 pII. TESTS ON FEKMENTED SUIISTTHATRS. A sufficient number A review of the results obtained stiowed that a type of organism isolated by the authors from rotted oak bark pro- of flasks was employed for the fermentation so that several duced a titratable anidity varying from 0.40 to 0.50 normal ut ferniented solutions could be drained, filtered, and analyzed tlie conclusion of the l 0 d a y fementatiou period, which wus a on the ninth and tenth days of the fermentation period. Afhigher average range of titratable acidity than that produced ter measuring the vnluine of the fermented medium. the folhy the other seven organisms. In morphological characteris- lowing tests were made: tics the organism isolated from rotted oak bark resembled Titratable acidity WBY determined with sodium hydroxide Aspergillus niger Van Tieghem, described by Tliom and wing phenolphthalein as the indicator, and titrations were carChurch (17), and listed as (TC 167) in Catalog 1004 in the ried to B stable oink color since both citric and oxalic acids are Arnericau Type Culture Collection, McCorrnick Institute, neutraliaed by chis procedure. Normality in t,lie tables i s extm per cent normal; a I N solution of pure citric wiclChicago, If{. This organisin was empk~yedfor all of the tests i.pressed e., 70 gri~msper liter--is considered as 100 per cent. As at dealing with citric acid fermentation. le& 90 per cent of the titratable acidity on the ninth and tonth dnvs of the fermentation was found tu be due to the ~resenceof DEYYELOPMENT OF A STANDAXD MEDIUM ~
~
~
INORGANIC SALT8 FOB PREPARING SUBSTRATES. While sugar is the principal component of the medium used for the production of citric acid, there is a physiological necessity for
CeHBOO,-H,O, bf molecular weight 210)
1144
INDUSTRIAL AND ENGINEERING
The large number of fermentations run demanded the use
CHEMISTRY
Vol. 26, No. 11
formation of oxalic acid became quite marked while the yield of citric acid was greatly reduced. In general, substrates prepared with larger quantities of the three salts than those mentioned in the preceding paragraph resulted in the production of a heavy mat and heavy spore Oxalic a c i d was produetion, accompanied by a low yield of citric acid. In precipitated in t h e particular the addition of greater amounts than 2.50 grams cold from a neutral per liter of ammonium nitrate increases the weight of mat 20-cc. sample with calcium chloride, formed, while the addition of more than 0.30 gram of magand, when quantita- nesium sulfate per liter tends to increase spore formation. tive determinations This is probably because the changed buffering effect of the f o r t h e acid were medium for any given amount of acid is decreased thereby, ING made, the precipitate WASHEP. was dissolved with thus permitting a more alkaline reaction. The production PACKING sulfuric acid a n d of a high yield of citric acid was always associated with a STOPPER very light sporulation to a complete absence of spores on the TS (PLUGG.ED titrated with potassium permanganate. surface of a thin mat, and this combination of factors, unCOTTON) The filtrate remaining after the oxalic natural in the normal growth of the organism, occurred only acid p r e c i p i t a t i o n in media prepared with a minimum amount of inorganic salts, CALlBK A T ED was reduced t o a Taking these facts into consideration, i t would appear that definite volume in the production of citric acid is associated with a type of each instance by boilmetabolism induced by a limitation rather than by an ample i n g a n d was t h e n autoclaved f o r 30 supply of certain elements which are normally required for PINCH COCK minutes at 20 pounds maximum vegetative growth. This is analogous to the UST GUARD per square inch (1.4 effect on diphtheria toxin production reported by Bunker ( 5 ) . kg. per sq. cm.) steam The composition of the medium shown below was found to pressure. T h e calcium citrate precipi- be most satisfactory because the organism was able to fertate was washed with ment such a medium to a high average yield of citric acid 50 cc. of hot water, with the production of less than 2 per cent oxalic acid. The then dried t o a constant weight a t 60" medium is subsequently referred to as the standard medium : C . , a n d weighed. Grams T h e method was FIGURE2. APPARATUS FOR ADDING Sucrose 140 found t o be 90 to 96 Ammonium nitrate 2.23 MEDIUM TO TEST TUBES UNDER per cent of the true Potassium monohydrogen phosphate 1.00 STERILE CONDITIONS value as compared t o Magnesium sulfate heptahydrate 0.23 Insert shows set-up for sterilization of Salts and sugare are dissolved and made u p t o 1 liter with distilled water quantitative determedium. to p H 2.20 t o 1.60 with 1 N hydrochloric acid, a n d sterrliaed at 6 minations run by a adjusted pounds per square inch (0.56 to 0.7 kg per sq. cm.) steam pressure for method based on that t30o 10 minutes. of Shaffer and Marriott (16), and discussed by Camp (6). This latter method involved the conversion of citric acid to acetone by I~IPORTAXCE OF INITIAL HYDROGEZT-ION COKCENTRATION potassium permanganate, with the subsequent determination of ON TITRATABLE ACIDITYPRODUCED. The initial hydrogenthe acetone with thiosulfate. ion concentration of the standard medium was varied with INORGAXIC SALTSIN THE STANDARD MEDIUM. The sub- 1 N hydrochloric acid in order to determine the importance strates containing different combinations of the inorganic of such variation on the appearance of the resulting growth salts potassium nitrate, sodium nitrate, ammonium nitrate, and on the titratable acidity produced by the organism a t the ammonium sulfate, ammonium carbonate monohydrate, conclusion of a 10-day period of fermentation. The results potassium monohydrogen phosphate (K2HP04), potassium of the tests served to show that the initial hydrogen-ion condihydrogen phosphate (KH2P04),potassium chloride, magne- centration of the medium was one of the important factors sium hydrogen phosphate heptahydrate (MgHP04.7H20), affecting the yield of citric acid. As the initial hydrogen-ion concentration of the medium magnesium sulfate heptahydrate, and calcium nitrate were fermented and tested in the manner just described. The was increased from p H 2.80 to 5.00, a tendency to form spores experimental data obtained served to show that the important was increased, accompanied by a downward trend in yield of elements necessary for growth, other than carbon, hydrogen, citric acid produced. Maximum yields of citric acid in the and oxygen, were made readily available to the organism in standard medium were obtained a t pH 1.60 using Cambridge salts of ammonium nitrate, potassium monohydrogen phos- tap water, and a t approximately p H 2.00 with distilled phate, potassium dihydrogen phosphate, and magnesium water as the solvent. Adjusting the initial hydrogen-ion sulfate. These findings are in agreement with those of Currie concentration of the standard medium at any point from pH (8) who was able to produce good yields of citric acid from 2.20 to 1.60 was found to be advantageous as a means both sucrose by eliminating certain salts (particularly salts of of obtaining good yields of citric acid and of minimizing the heavy metals) previously considered necessary to establish danger of contamination of the fermentation by other organisms, particularly during the early stages of the fermenthe best growth of the organism. All the data indicated that spore production, weight of mat tation. While sterilized media have generally been employed, formed, and the nature of the changes brought about by the a number of fermentations have been successfully carried organism could be governed to a great extent by adjusting through in pans and flasks with this procedure without the weights and proportion of the three salts ammonium sterilization of the medium. The addition of hydrochloric acid in adjusting the pH of the nitrate, potassium monohydrogen phosphate, and magnesium sulfate contained in the medium. The organism produced medium is of distinct benefit as it adds the element chlorine the most citric acid in a medium containing less than 2.50 to the constituents of the medium. Experimental evidence grams of ammonium nitrate, 1.50 grams of potassium mono- tends to indicate that the acid radical is an important factor hydrogen phosphate, and 0.30 gram of magnesium sulfate governing the production of citric acid. When sulfuric, per liter; if greater amounts of the three salts were used, the nitric, and acetic acids, respectively, were employed in adjust-
of a rapid method for the quantitative determination of citric acid. Tests were based on the varying solubilities of the calcium salts of oxalic and citric acids:
November, 1934
I N D U S T R I A L h N D E N G I N E E R I N G C H E M I ST R Y
ing the initial pH of the various batches of standard medium through a pH range of 2.60 to 2.80, lower yields of citric acid mere obtained in the resulting fermentations as compared with yields produced by the organism in media adjusted to similar hydrogen-ion concentrations with hydrochloric acid. When formic acid was employed in amounts sufficient to adjust the medium to p H 3.00, the spores seeded on the surface would not sprout a t all, probably because of the toxic effect of the acid radical at the concentration of acid used. SUGARIN THE STANDARD MEDIUM.Analyses served to show that all of the sugar that the organism could utilize in a straight-run 9- to 12-day fermentation was made available when a liter of medium contained 140 grams of sucrose. Under optimum conditions approximately 60 per cent (83 grams) of the sugar is converted largely to citric acid in 9 days; the remaining 40 per cent (54 grams) is both utilized in the building u p of the mycelium or mat and lost in the liberation of carbon dioxide as a result of the process of respiration. Employing more than 15 per cent sucrose in the preparation of the standard medium will result in a greater amount of sugar remaining unconverted to citric acid, as there are limits to the amount of sugar that the organism can convert to citric acid in a given time period. When a good yield of citric acid is obtained in a 9- to 10-day fermentation in the standard medium, less than 3 per cent of sugar, calculated as glucose, will remain in the medium. It was thought by the authors that the addition of either glucose or fructose to the sucrose might increase the yield of citric acid, assuming that the sucrose is hydrolyzed as a preliminary step in the production of the acid. Fructose in particular has been considered as an important precursor to the formation of citric acid by Bernhauer (3). Working on the above assumption, varying amounts of glucose (1 to 5 per cent) were added to the standard medium to make a total sugar content of 14 per cent in each instance, but all of the fermentations resulted in a decrease in the amount of citric acid produced as compared to the results obtained in the standard medium containing 14 per cent sucrose. Similarly, the yields of citric acid oLitained by fermenting the standard medium with the addition of varying amounts of fructose (1 to 5 per cent) were lower in all instances than the yields normally obtained with the standard medium made u p with sucrose. It would appear, therefore, that the normal sequence of changes leading to the production of citric acid depends either on the proper proportion of fructose and glucose as they are formed by the organism itself from sucrose during the course of the fermentation, or that sucrose is split to other products such as saccharic acid, as suggested b y Franzen and Schmidt (12) and also b y Challenger and his associates (7',19), but refuted by Bernhauer (4). PREPARATION O F S T l N D A R D hfED1TJM
STOCKSOLUTION.Owing to the large amount of medium used and to the necessity for adopting a uniform technic for the preparation of each batch of medium, it was found most convenient to prepare a stock solution of the inorganic salts contained in the formula for the standard medium. Every batch of stock solution was made up to a 2-liter volume, and each 50 cc. of the stock solution contained the amount of salts required to make u p 1 liter of the standard medium. The following procedure was employed in the preparation of the stock solution: A 9.2-gram (40 X 0.23) portion of magnesium sulfate heptahydrate was weighed and washed into a 1-liter beaker with 150 cc. of distilled water. Then 40 grams (40 X 1.00) of potassium monohydrogen phosphate were accurately weighed and washed into the magnesium sulfate solution with 300 cc. of distilled water. On the addition of the potassium monohydrogen phosphate, a heavy white precipitate of magnesium phosphate
1145
formed. Then 280 cc. of 1 N hydrochloric acid were slowly added; this is more than ample to dissolve the precipitate of magnesium phosphate. Finally 89.2 grams (40 X 2.23) of ammonium nitrate were added to the salt and acid mixture in the 1-liter beaker. The whole mixture was then made up to 2 liters in a volumetric flask. Each 50 cc. of the stock solution thus prepared contained 7 cc. of 1 A' hydrochloric acid.
METHOD FOR ~IIXIIIIIZHYDROLYSIS. Cons i d e r a b l e hydrolysis of the sucrose occurs when the standard medium is autoclaved in the presOTTON PLUG ence of acid employed to adjust its r e a c t i o n within a range of p H 2.60 t o 1.60. When 3 oer c e n t o r m o r e of t h e sucrose in the standard medium has been hydrolyzed following sterilization, the total acidity p r o d u c e d in a 10-day f e r m e n t a t i o n in such media will be considerably less than that produced by f e r m e n t i n g a medium containing unhydrolyzed sucrose. The reason for this can possibly be explained by assuming that best en- FIGURE 3. APPARATUS FOR ADDzmeProduction leading ING MEDIUMTO FLASKSUNDER to the p r o d u c t i o n of STERILECONDITIONS citric acid is stimulated by a splitting of the sucrose, while the monosaccharide present interferes with the normal sequence of changes from sucrose to citric acid. In order to limit the amount of hydrolysis during sterilization, the following method was employed in the preparation and autoclaving of the standard medium : ING
I! -
The standard medium was prepared in individual batches of 3 liters, the salts and acid solution being prepared separately from the sugar solution. In the preparation of 3 liters of medium, 150 cc. of the stock inorganic salt solution were added to a 500-cc filter flask. Approximately 17 cc. of 1 N hydrochloric acid per li-. ter of standard medium were required to adjust its reaction to pH 2.00. Therefore, since each 50 cc. of the stock solution contained 7 cc. of 1 N hydrochloric acid, 30 cc. of the acid remained to be added to the 150-cc. stock salt solution in the 500-cc. filter flask. After the addition of the acid, 420 grams of sucrose were added to a Pliter Erlenmeyer flask; this was equivalent to 140 grams of' sucrose per liter of standard medium. The sugar was dissolved and made up t o such a volume with distilled water that, on the addition of the salt and acid solution in the filter flask, the sum of the two volumes would be exactly 3 liters. After the salt and acid mixture and sugar solution had been prepared, the 500-cc. filter flask was connected to the 4-liter Erlenmeyer flask by means of a siphon arrangement shown in the insert on Figure 2. Rubber tubing was employed to connect the glass tube to the side arm of the filter flask, I, and a screw clamp was tightened on this rubber connection in order that the siphon would form to that point following sterilization. When ready for sterilization, the two flasks, connected as shown, were placed in the autoclave for 30 minutes at 8 pounds per squaw inch (0.56 kg. per sq. em.) steam pressure. Following sterilization, the solution of salts and acid in filter flask I was mixed with the sugar solution in the 4-liter flask 11, in the following manner: First, the screw clamp was removed, and the sugar solution was allowed to run into the filter flask until the liquid reached the level of the side arm. At this point the flow of sugar solution was arrested by pinching the rubber tubing. The bottom of the filter flask was then raised above the level of the liquid in the 4-liter flask so that the flow of liquid would proceed in the opposite direction. In order to retain the siphon, the flow of liquid from the filter flask was stopped just
1146
I N D U ST R I A L A N D E N G I N E E R I N G C H E M IST R Y
before it was completely drained. The 4-liter flask was then shaken vigorously to mix the salts, acid, and sugar. For rinsing purposes the filter flask was refilled through the siphon in the manner just described and again resiphoned into the 4-liter flask. When the standard medium was prepared and autoclaved as described, any considerable amount of hydrolysis was avoided, and, all other conditions being equal, fermentations in such media have resulted in the production of 10 t o 20 per cent more citric acid than in the case of fermentations run in media with the sugar autoclaved in the presence of the acid. The method of sterilizing and mixing the medium presented no difficulties and insured absolute sterility during the mixing operation.
Vol. 26. No. 11
in titratable acidity during a 20-day period of incubation at 24" C. is illustrated by the solid line in the lower part of Figure 4. The increase in titratable acidity is most rapid from the sixth to the twelfth day of the fermentation period. The concentration of citric acid in the fermented solution mill be a t a maximum just prior to the time when the rate of increase in titratable acidity diminishes; thereafter the orgacism produces increasing amounts of oxalic acid and begins to split citric acid to compounds of lower molecular weight. In comparison to the increase i n t i t r a t a b l e FILLING OF APPARATUS.Sterile flasks and test tubes can acidity, the curve reprebe conveniently filled with standard medium whenever they senting the acid increase are needed by use of the apparatus shown in Figures 2 and 3 . in hydrogen-ion concenAny number of flasks and test tubes can be filled Kith the tration rises gradually for apparatus illustrated with a minimum chance of infection a time and then falls as during the filling operation. the rate of increase in tiAfter the lower and upper exposed ends of tubing have been t r a t a b l e acidity dimincovered with suitable paper dust guards, the apparatus is ishes. The d o t t e d line autoclaved a t 15 pounds per square inch (1.05 kg. per sq. cm.) shown on Figure 4 illussteam pressure for one hour. The sterile medium is then cont r a t e s the gradual rate nected to the filling apparatus in the following manner: of increase in weight of mat (calculated as dry Filter flask I (Figure 2) is disconnected at point A , and the end of the glass tubing is immediately inserted into the rubber conweight) up to the tenth nection leading into the main body of the filling apparatus. day of the fermentation The bulb of the filling apparatus shown in Figure 3 is calip e r i o d , followed by a brated t o deliver exactly 75 cc. of medium to the 250-cc. Erlengreatly diminished rate meyer flasks used for the fermentation. In filling a sterile 250-cc. flask, the cotton plug is removed and the mouth of the flask is of i n c r e a s e f r o m that flamed. The flask is then placed under the dust guard so that time on. the delivery tube will run well down into the neck of the flask. DAYS The rate of increase The mouth of the flask is reflamed, and the cotton plug replaced FIGURE4. RATE OF INCREASE in titratable acidity diwhen the 75 cc. of medium have been delivered. Sterile cotton I N TITA.4TARLE ACIDITY,WEIGHT or sterile paper is kept over the dust guard of the filling appaminishes noticeably OF MAT, AXD PH RANGEIN ratus when it is not in use. FLASK FERMENTATIONS when approximately 90 In order to fill the culture test tubes, the apparatus shown in Der cent of the sucrose Figure 2 is used in essentially the same manner as described for the filling of the 250-cc. Erlenmeyer flasks. The main body of originally present in the medium has been converted to other the apparatus is a calibrated buret, permitting the addition of products. When the concentration of citric acid has risen any desired volume of medium from 1 t o 50 cc., although 10-cc. beyond approximately 7 per cent, it tends to restrain or posportions of medium are usually employed in the culture tubes. sibly become toxic to the organism, and citric acid is broken down; in its effect it becomes a second factor gradually tendCHARACTERISTICS OF THE CITRICACID FERMEA-TATIOX ing to diminish the rate of increase in titratable acidity. Normally, in the case of a good fermentation, titratable RATE OF ACID RISE IN HYDROGEX-ION CONCENTRATION, AND TITRATABLE ACIDITY. When the standard medium acidity will rise rapidly up to the ninth or tenth day of the containing 14 per cent sucrose has been prepared in the man- fermentation period or until the concentration of citric acid ner indicated in the preceding sections, the resulting fermen- is about 7 to 8 per cent in the solution a t the end of the fertation in such a medium will display certain normal charac- mentation, and the amount of oxalic acid formed is less than teristics as regards the rate of growth of the organism and the 1 per cent. When the fermented medium contains 8 per cent rate of increase in titratable acidity and hydrogen-ion con- citric acid, the titratable acidity will be equivalent to a t least 1.2 normal. centration. EFFECTOF TEMPERATURE OF INCUBATION ON TITRATABLE Microscopic observations have revealed that the spores The nature of the begin to sprout approximately 5 hours after they have been ACIDITYAND CITRICACID PRODUCED, seeded on the surface of the medium, provided, however, that changes brought about by the organism will depend to a conthe temperature of incubation has been maintained within siderable extent on the temperature of incubation employed. the limits of 24" to 28" C. Photomicrographs of the sprout- Generally speaking, if the fermentation is run a t or above ing spores are shown in the paper by Doelger and McLaugh- temperatures of 30" C., citric acid production will decrease, and a greater proportion of the titratable acidity will be due lin (9). It is found as an empirical fact that best results are obtained to the formation of oxalic acid. On the other hand, the when one-fourth to one-half of the surface area of the medium amount of citric acid produced will be on a rising scale as the is seeded with spores. If the entire surface area of the me- temperatures of incubation are increased from 8" to 28" C . Table I shows the titratable acidity and citric acid prodium is seeded, the first actively growing ones will prevent the growth of others and uniform sprouting will be delayed. duced, as well as the percentage evaporation in fermentations The unsprouted spores which become embedded in the mat run at five different temperatures of incubation. Portions apparently have a toxic effect as far as citric acid production (75 cc.) of standard medium in 250-cc. flasks were fermented and analyzed after a 10-day incubation period. The flasks is concerned. A t optimum temperatures of incubation an interval of 3 were plugged firmly with nonabsorbent cotton. Sets of t o 5 days will elapse before the entire surface area of the ten flasks were run a t each temperature of 20" to 22", 24", medium is covered with a firm, well-developed mat. Once 26O, 28", and 30" to 33" C. All of the fifty flasks used in the the mat has formed, the citric acid concentration of the experiment were inoculated from the same 10-day-old spore medium begins to rise rapidly. The actual rate of increase culture. I
INDUSTRIAL I N D ENGINEERING CHEMISTRY
November, 1934
TABLEI. EFFECTOF TEMPERATURE OF INCUBATION ON TITRATABLE ACIDITYAND CITRICACID PRODUCED TEMP. OF
TITRATABLE
INCUBATION ACIDITY
c.
Normality
CITRICACID PRODUCED Per flask Per 100 EVAPN. fermengrama OF sugar MEDIUM tation Grams Grams %
The data of Table I show that there is little actual difference in average titratable acidity produced at any of the five temperatures of incubation employed in the tests. It is evident, however, that a t the incubation temperature of 30" to 33" C. there is a considerable reduction in yield of sugar to citric acid and in the amount of citric acid produced per container, as compared with the results obtained at the other temperatures of incubation. Although the titratable acidity produced a t the conclusion of the 10-day incubation period a t 30" to 33" C. is about the same as the averages produced a t the other temperatures, the total amount of citric acid produced is low enough to indicate the formation of other acids than citric-e. g., oxalic. The data obtained also show that the best citric acid production occurs at temperatures of incubation varying from 26" to 28" C. RELATION OF TITRATABLE ACIDITYTO MOISTURELoss. Too rapid evaporation retards the formation of citric acid by the organism; therefore it is best to employ covered containers for the fermentation, allowing only a minimum of air circulation, or in open containers a t a relative humidity of a t least 80 per cent.
The volume of the fermented solutions was always measured and recorded after the completion of a 10-day incubation period, the procedure simply involving the draining of the liquid and the squeezing of the remaining traces from the mat. By grouping the fermentations according to the titratable acidity produced, the average amount of medium remaining in fermentations falling within each of the groups could be readily compared. The percentage moisture loss due to respiration alone was determined by subtracting the total percentage moisture lost in the fermented samples from the average percentage evaporation in the control flasks incubated for the same time period. The results shown in Table I1 illustrate the fact that the volume of medium remaining after the completion of a given period of fermentation a t uniform temperatures of incubation and relative humidity depends largely upon the titratable acidity produced by the organism. When the titratable acidity falls within a range equivalent to 0.1 to 0.2 normal, the original volume of the medium is reduced by 18 per cent, and from this point on there is a gradual reduction in the amount of medium remaining in the fermentations grouped according to increasing amounts of titratable acidity produced. In fermentations having a titratable acidity equivalent to greater than 0.9 normal, the original volume of the medium is reduced by 32 per cent, or a 14 per cent greater reduction compared to fermentations falling within a titratable acidity range corresponding to 0.1 to 0.2 normal. It is evident that, in any series of fermentations by Aspergillus niger in the same type of containers and a t uniform temperatures of incubation and relative humidity, the rate of respiration increases in direct proportion to the titratable
TABLE11. VARIATION OF MOISTURE Loss RANQE OF TITRATABLE ACIDITY No. of flsske Vol. remaining, cc. Vol. loss. % Vol. loss due t o respiration,
Less than 0.2 N 25 61.4 18.1 11.1
1147
WITH
TITRATABLE ACIDITYPRODUCED
TITRATABLE ACIDITY 0.2-0.3 N 84 57.7 23.1 16.1
0.3-0.4 N 52 56.7 24.4 17.4
When fermentations are run in Erlenmeyer flasks with nonabsorbent cotton stoppers, evaporation is most rapid during the early stages of the fermentation while the mat has not completely covered the surface of the medium. Most of the reduction in volume takes place after the mat has formed as a direct consequence of the process of respiration, and the extent of the loss in volume is indicated by the following data:
0.4-0.5 N 83 55.9 25.5 18.5
0.5-0.6 N 75 55.1 26.5 19.5
0.6-0.7 N 112 54.3 27.6 20.6
0.7-0.8N 122 53.1 29.2 22.2
0.8-0.9 N 118 52.6 30.0 23.0
.More than 0.9 N 210 51.0 32.0 25.0
the process of respiration carried on by the organism. In the process of respiration much carbon dioxide is liberated, and, as the sugar content is reduced thereby, the volume of the medium diminishes. The evolution of carbon dioxide is always greatest during the interval when titratable acidity is rising rapidly, and the amount of carbon dioxide produced will vary directly with the amount of citric acid produced.
acidity produced. The percentage volume lost increases with respiratory activity and is an index to the approximate titratable acidity of the medium. EFFECTOF THE NUMBEROF SPORETRANSFERS ON TITRATABLE ACIDITYPRODUCED. Experimental results have shown that organisms of the Aspergillus niger type display a type of adaptation. The spores tend to receive a definite stimulation from the constituents of the medium, particularly inorganic salts, in which they are being transferred, and this important characteristic must be utilized in the culturing operation previous to the transfer of the spores into a large batch of medium if maxirnum acid production is desired. The process of adaptation is best illustrated by the fact that increasing amounts of citric acid are produced with repeated spore transfers of the same organism on media of uniform composition. Table I11 illustrates the adaptive process as i t applies to spore transfers. Results in Table I11 were obtained by adopting the following experimental procedure:
Table I1 illustrates the effect of respiration on moisture loss in fermentations run a t uniform temperatures of incubation and relative humidity. Results were compiled from several hundred fermentations run in the course of experiments on the citric acid fermentation. All of the fermentations employed in obtaining the data for Table I1 were run in 250-cc. flasks containing 75 cc. of medium and incubated at 28" C. for 10 days, with the relative humidity in the incubator varying, on the average, from 40 to 50 per cent.
Transfers were made at intervals of 10 days and were continued for a period of approximately 8 months. Twelve 250-cc. flasks containing 75 cc. of standard medium (14 per cent sucrose, pH 2.00) were inoculated from a single spore culture which had previously been cultured in a medium containing 10 per cent sucrose. The twelve 250-cc. flasks were placed in an incubator at 26' C., and at the conclusion of the tenth day of the incubation period the spores produced were reinoculated into another set of twelve flasks also containing standard medium. The average titratable acidity produced and the spore production were measured at the completion of each 10-day interval. Spore production is re-
When 250-cc. Erlenmeyer flasks are filled with 75-cc. portions
of standard medium and plugged with nonabsorbent cotton, 4 to 5 cc. of the liquid will evaporate in a 10- t o 12-day period of incubation at 28" C., with the relative humidity i n the incubator varying from 40 to 50 per cent. When fermentations are run in the 250-cc. flasks, the volume lost increases greatly because of
1148
INDUSTRIAL AND ENGINEEKING CHEMISTRY
corded as the percentage of the surface area covered with spores. Column 3, Table 111, lists the increases in average titratable acidity from one transfer t o the next as compared with the lowest 0.2844 normal. The titratable acidity produced-namely, figures for average spores on the mat are actual averages of twelve cultures for each step in the series; obviously no single observation could be made with an accuracy as small as one per cent. TABLE
111. AVERAGETITRATABLE ACIDITYPRODUCED EIGHTEEN SUCCESSIVE SPORE TRANSFERS
NO. O F Av. INCREASE I N TITRATABLE Av. ACIDITY FROM SPORES INOCULATION TITRATABLE Low POINT ON hfAT SERIES ACIDITY Normality Normality % 10.1 .... 53.2 32.7 0.0000 0,0454 14.5 0.0586 9.5 0.0936 8.0 0.0406 6.7 0,1859 5.0 0.1464 5.5 0.1956 7.1 0.0751 32.2 0.2663 3.3 0.2369 3.1 0.2774 3.3 0.1873 1.4 0.4115 1.6 0,3388 3.3 0.5272 1.3
WITH
Vol. 26, No. 11
about by intracellular enzymes; therefore the fermentation cannot proceed normally unless the mat is in constant contact with the medium. As the enzymes are contained in the mat, it can be assumed that the sucrose solution directly beneath the mat i? first converted to citric acid, and this statement is borne out by the fact that samples taken directly from the undersurface of the mat have a higher acid content than bottom samples. As the fermentation is carried out a t rest, the acid layer diffuses into the bottom sugar layer a t a rather slow rate. Provided the layer of medium is not too deep, all of the sugar will finally come into contact with the mat, and the formation of citric acid will continue until the concentration of the whole solution becomes so great (concentration of 7 to 8 per cent) that it is split to other products by the organism. These experiments have shown that hastening the diffusion of the acid layer into the unconverted sugar layer by gentle to moderate shaking will retard the rate of citric acid production as compared with a resting fermentation. The authors believe that resting fermentations are best, as the concentration of citric acid a t the undersurface of the mat must always be high enough to stimulate the production of more acid by the enzymes in the mat. Since enzymic activity can take place only when the mat is in contact with the medium, it follows that there will be a greater increase in rate of conversion of sucrose to citric acid as the surface area of the mat is increased. If fermentations are run in shallow containers allowing the formation of a broad mat, a rapid rate of conversion of sucrose to citric acid will result. On the other hand, in deep containers, allowing a large volume of medium in relation to its surface area, the rate of acid formation will be retarded by dilution and by the fact that there is only a small surface area of mat in contact with the medium. Therefore, for the commercial production of citric acid by fermentation, shallow pans are used in order to allow the formation of a large surface area of mat in relation to the volume of substrate. Aluminum pans are used for the fermentation of pure sucrose, as aluminum is the only practicable metal which does not have anpdirect effect in stunting growth o r in hindering the enzymic activity of the organism.
It is shown that there is a drop in titratable acidity after the first three transfers owing to the fact that the spores, previously transferred on a medium containing only 10 per cent sucrose, are not accustomed to the sugar concentration of the standard medium. Thereafter, with the continuation of the number of transfers, the organism becomes accustomed to the new medium by reason of certain changes occurring in the spores. From microscopic observations it appears that with increasing numbers of transfers the spores tend to increase in size, and the threads or hyphae composing the mat show a thickening of the cell walls as well as an increase in the number of vacuoles and fatty bodies inside of the cell wall. Although there is not a uniform increase in acid production from one transfer to the next, the trend is always to a higher level. Under the conditions of the experiment it has been found that the organism will continue to form increasing amounts of acid in each new fermentation until a limit is reached when it is producing a t i t r a t a b l e a c i d i t y equivalent to 1.2 normal in a 9 d a y fermentation. A sharp drop in acid production in any flask in the series will be due to the entrance of conI, t a m i n a t i n g or g a n i s m s, chiefly n n wild yeasts or s o m e t i m e s o t h e r fungi growing on the surface of the mat. The tabulation of data on the relation of spore production to acid produced indicates that spore production progressively diminishes as the organism gains in its ability to L, ' ALUMLNUM P A N produce increasing a m o u n t s of citric acid. Scanty spore formaCAIEINLLTb) FRONT VIEW (BAFFLE) tion is usually a good criterion of the efficiency of the fermentation. Mats producing high yields of citric acid showed either a few spores or complete absence of spores on their C S L O T bfXg(1N COPPER COVER) surface.
If
I I
i y
EFFECT OF RATIOOF VOLUME TO SURFACE AREA O F M E D I U MO N Y I E L D SO F C I T R I CACID. The
FIGURE 5. TYPEOF PANAND COVER USEDFOR FERMENTATIONS
formation of citric acid is brought
(Scale 1 em. = 1 inch)
Fermentation in Aluminum Pans. Several hundred c i t r i c a c i d fermentations were run in shallow pans made of aluminum. Ready-made b a k i n g pans (Figure 5 ) were first employed, constructed of an ordinary commercial grade of aluminum with a purity of 99 per cent plus and a thickness of 0.16 cm. inch gage). It was found that the hydrochloric acid used to adjust the reaction of the medium, as well as the citric acid produced by the organism d u r i n g the fermentation period, reacted with the impurities in the commercial-grade metal. E t c h e d a r e a s frequently became so deep that leakage occurred during the third fermentation in a pan, with the result that the pan had to be discarded. It became necessary to construct specially prepared pans made with a l u m i n u m of v a r i o u s grades of purity in order to test the resistance of the metals to the acid of the fermentation. It was found that etching could be almost entirely
INDUSTRIAL AND ENGINEERING CHEMISTRY
November, 1934
eliminated by the use of aluminum rated as 99.80 or a 99.98 per cent pure by the Aluminum Company of America. When the pans were constructed with this metal of 0.12-em. thickness (3/’M-in~h gage), they were firm and durable, and showed little wear after more than twenty fermentations. Square angled rather than flared pans were made. Copper pan covers were made to fit snugly over the sides of the aluminum pans. The pans and covers were similar to those shown in Figure 5 , but the covers were constructed without the air baffle. The circulation of air obtained by forcing a current of air through the baffle was found t o affect yields of citric acid adversely. It is of benefit to allow only a mininium amount of air circulation over the surface of the mat, as a slight carbon dioxide pressure will promote better yields of citric acid. The six 1.9-cm. (3/&ch) vents in the cover shown in Figure 5 give a satisfactory amount of ventilation, preventing the rapid escape of carbon dioxide and also limiting the amount of evaporation during a 10- to 12-day fermentation period. TABLEI v . EFFECT OF VARYING RATIO OF VOLUME TO SURFACE AREAOF MEDICMON YIELDSOF CITRICACID 1
2
3 TOTAL
4
6
14
PERCENT VOL.: SCGAR SURFACE SOLN. /LREA FINAL ORIGIIV.4L PER RATIO VOL. PAN VOL.
cc./
cm. 2.45 2.20 2.08 1.83 1.22
sq.
Cc. 2000 1800 1700 1500 1000
Grams 280 25% 238 210 140
Cc. 1810 1620 1490 1310 780
CITRIC ACID PER
7
6
TOTAL CITRIC ACID PER
100 cc.
PAN
Grams 6.35 7.05 7.40 7.75 8.80
Grams 114.9 114.2 110.3 110.2 68.6
TIELD OF
SUGAR TO
CITRIC ACID
When the pans and covers were ready to be sterilized, the air vents on the tops of the covers were plugged with a thin layer of cotton. A small U-shaped glass tube was inserted into the 0.95-em. (3/s-inch) hole shown on the corner of the pan cover, and the sterile medium was allowed to flow into the pan through this tube. A siphon was formed in the tube so that samples could be withdrawn from time t o time during the fermentation period. The dimensions of the specially made pans were 25 X 33 em. (approx. g3/,, X 13 inches), making a total surface area of 825 sq. em. (approx. 126 square inches), and allowing a depth of 2.5 em. (1 inch) of medium for each 2 liters of medium added to the pan. Table IV shows the effect on acid formation when varying amounts of medium are fermented in the 25 X 33 cm. pans. The fermentations were run at an incubation temperature of 26’ C., employing the standard medium adjusted to p H 2.40 in all cases. All of the data listed are based on analyses made on the ninth day of the fermentation; a t that time the pans were drained and the volume was measured in order to obtain the percentage evaporation during the fermentation period. In the inoculation of the pans approximately one-fourth of the surface area of the medium was seeded with a 10-day-old spore culture.
EXPERIMENTAL FARMOF
THE:
Analyses for citric acid were made by the calcium chloride method previously described. A maximum yield of 73 to 74 per cent citric acid (molecular weight 210) can be recovered from calcium citrate (molecular weight 570) whereas a yield of 70 to 71 per cent was actually obtained, corresponding to the purity of the U. S. P. grade of calcium citrate on the market. The percentage sugar of the medium (grains per liter) must be held at the point to n-hich the organism hai been accustomed; therefore the sugar content per pan can be increased only by increasing the volume of medium in the pan. Table IV, column 6, indicates that the total amount of citric acid formed per pan tends to increase as the volume of medium fermented is increased up to 2 liters. As the type of medium and technic in setting up the fermentation is uniform throughout, the rate of increase in citric acid does not vary appreciably with an increase in the volume of medium or amount of sugar fermented per pan; therefore, as indicated in column 7, the percentage yield of sugar to citric acid tends to decrease. The downward trend in yield of sugar to citric acid will be maintained if the volume of medium fermented is increased above 2 liters in the 25 X 33 cm. pans. An increase in volume of medium will result in a corresponding increase in depth of mediurn in the pan, and, as previously explained, in deeper layers of medium the percentage of citric acid will be reduced by dilution. The actual reduction in percentage of citric acid in this series of fermentations is shown in column 5.
yo 41.0 45.3 46.3 48.3 49.0
1149
LITERATURE CITED (1) Amelung, H . , Z.p h ~ s i o l Chem., . 166,161-209 (1927). (2) Angeletti, A , , Ann. SchiappareZli, 7,72-5 (1933). (3) Bernhauer, K., Biochem. Z., 197,309-26, 327-42 (1928). I
Bernhauer, K., Siebeniiuger, H., and Tschinkel, H., Biochem.
Z.,230,46&74
(1931).
Bunker, G. W.B., J . Bad., 4, 379-407 (1916). Camp, F. A , , Ann. X i s s o u r i Botan. Garden, 10,No.3 (1923). Challenger, F., Klein, L., and Walker, T. K., J Chem. Soc., 1927,200-8; Nature, 119,674 (1927). Currie. J. N.. J . Biol. Chem.. 31. 15-37 (1917). Dodger, W.’P., and MMcLaughiin, G. D., J.’Am. Leather Chem. Assoc., 35,S o . 6 (1930). Dox, A. W., U. S. Dept. Anr., Anima2 Ind. Bull. 120 (1910). Emmerling, O., Centr. Bak;., Parasitenk., 11, 10,273 (1903). Franaen. H., and Schmidt, F., Ber., 58,222-8 (1925). Heinae, B., Ann. Myc., 1, 334-53 (1903). May, 0. E . , and Herrick, H . T., U. S. Dept. Sgr., Circ. 216 (1932).
Molliard, M., Compt. rend. SOC. bid., 90, 1395-7 (1924). Shaffer, P. A., and Marriott, W. McK., J . BioZ. Chem., 16,26580 (1913). Thom, C., and Church, M. B., “The Aspergilli,” Williams 8: Wilkins Co., Baltimore, 1926. Thom, C., and Currie, J. N., J.Agr. Research, 7,1-15 (1916). Walker, T. K., Subramaniam, V., and Challenger, F., J . Chem. SOC.,1927,3044-54. Wehmer. Carl, Bull. soc. chim., [31 9, 728-30 (1893); Compt. rend., 117,332-3 (1893). Wehmer, Carl, Centr. Bakt., Parasitenk., 11, 3, 102 (1897). RECEIVED August 17, 1934. Contribution 38 from the Department of Biology and Publio Health, Massachusette Institute of Technology.
U . S. DEPARTMENT OF AGRICULTURE AT ARLINGTON, VA.
