)METER
REMOVABLE FRONT
THERMOMETER FIGURE1. CABINETINCUBATOR
SECTION
Production of Fat from Glucose by Molds Cultivation of PeaiciZZium javaaicum van Beijma in Large-Scale Laboratory Apparatus GEORGE E. WARD,LEWISB. LOCKWOOD, ORVILLE E. MAY,AND HORACE T. HERRICK B u r e a u of Chemistry and Soils, Washington, D. C.
M
ORE than three years ago the CoIor and Farm Waste Division of this bureau completed a survey of the
action upon glucose and xylose of a large number of molds of the genera Aspergillus and Penicillium. This work led to the accumulation of many samples of mycelium and, as it was thought that a t least a preliminary examination of some of these might be of interest] the heavier mats were subjected to a crude fat determination, made by treating one-gram portions of the dried, ground material with ethyl ether about 16 hours in Soxhlet extractors. Of the sixty-one different organisms studied, thirty-nine were Penicillia and twenty-two Aspergilli. Ten organisms contained more than 15 per cent, and only six contained
more than 20 per cent of ether-soluble material (Table I). The fact that nine of the ten organisms yielding more than 15 per cent fetty material were Penicillia and only one an AspergiIlus suggests that within the genus Penicillium there would perhaps be a great,er probability of finding organisms the mycelia of which would contain large quantities of fat. However, it must be borne in mind that culture conditions have a marked influence on the fat content of the mold mycelia. Such an effect is reported later in this paper and is also shown by the recent studies of Pruess, Eichinger, and Peterson (IO) who made a survey of the lipid content of twenty-four molds that were grown on two different media.
318
I N D U S T R I A L -4 N D E N G I N E E R I N G C H E M I S T R Y
March, 1935
STUDY OF Penicillium jovanicttm
A surzqey of sixty-one organisms of the genera Aspergillus and Penicillium showed nine Penicillia and one Aspergillus whose mycelia conBecause of the high yields of tained more than if5 per cent ether-soluble mateether-soluble substances f r o m a n u m b e r of these molds, it rial. A n intensice study of Penicillium javaniwas thought advisable to incum van Beijma showed that its mycelium may v e s t i g a t e further the nature contain as much as 41.5 per cent fat, depending of this f r a c t i o n . Penicillium on culture conditions. I n flask cultures, media javanicum v a n Beijma was containing 40 per cent glucose gazte mycelia of chosen for thebe studies because i t developed c o m p a r a t i v e l y highest f a t confent. vigorously in glucose nutrient A cabinet f o r experimental study of shallow-pan s o l u t i o n s and produced large is described, and representative fermentations quantities of fat in its mycelium. results of culture experiments conducted therein Nevertheleqs, a s t h e work progreised, considerable diffiare presented. I n such cultures, increase of the culty was encountered in obglucose content of the medium apparently does not taining consistently good yields increase the f a t content of the mycelium of P. of mycelium and fat, and it javanicum, as it does in flask cultures. The free was only after e x t e n s i v e exfatty acid content of fhefat obtained from the myperimentation and s t a n c l a r d i zation of the culture technic celium grown on 30 and 40 per cent glucose soluthat fat production was stabitions is much higher than that of fat similarly delized. ricedfrom 20per cent glucose solutions. I n addiMATERIALSAND M E T H O D S . tion to fat, the mycelium of P. jaeanicum yielded The culture of P. javanicum a complex carbohydrate and a chitinous material. was r e c e i v e d d i r e c t l y from VAN
BEIJM.4
Charles Thom, w h o h a d obtained it from van Beiirna ( 1 ) . Van Beijma isolated the mold from a culture deiived from rotten tea roots in Java. A description of the culture is presented in another communication ( 6 ) . The standardized culture procedures which proved successful with P. javnnicuin comprised the following steps: (1) Stock cultures of the organism were grown on a corn meal-agar medium, one liter of which contained 2.0 grams agar and the hot water extract of 60 grams corn meal. (2) One generation of the organism was grown on a liquid sporulation medium. (3) Nutrient solutions for the study of fat production were inoculated with the mycelium and spores developed on the sporulation medium.
The sporulation medium had the following composition : Commercial glucose "&NO2 MgSO4.7HzO HsPOi KC1
Grams/Ziter 275 0.225 0.040 0.0043 0.008
When grown on this medium for 3 to 15 days, P. javanicum developed a thin mat covered with many conidia; this material fragmented readily and hence was suitable for inoculation of other culture flasks. The glucose used in all media discussed in this paper was of a commercial hydrated grade, and its composition was about 91.5 per cent d-glucose, 8.0 per cent moisture, and 0.4 per cent dextrins and other nonreducing carbohydrates. The inorganic nutrients were all of c. P. quality. All solutions were made with distilled water. Two nutrient media well adapted to the needs of the organism when fat production is desired are as follows: G SOLUTION Commercial glucose NHiNOa
M SoLcrrox Grams/lzter 220 4.50 2.00
0.216 0.40
Grams/liter Commercial glucose 220 NH4NOa 2.25 MgSO4.7HzO 0.25 KHlPOl 0.30
319
More consistent results, however, have been obtained with the h1 solution. These nutrient solutions, or slight variations of them, have also given good r e s u l t s in other fermentation studies conducted by this division. Flask cultures fur the study of fat p r o d u c t i o n were prepared in the following manner:
Seventy-five cubic centimeters of the desired nutrient solution were p l a c e d in 200-cc. Pyrex Erlenmeyer flasks, which were stoppered with cotton plugs and s t e r i l i z e d by a u t o c l a v i n g a t 15 to 20 pounds per square inch (1.0 t o 1.5 k g . p e r s q . cm). pressure for 15 minutes. After being cooled, t h e s o l u t i o n s were i n o c u l a t e d and the flasks were placed in a constant-temperature room m a i n t a i n e d a t 30" C. At the end of the desired incubation period, which was usually 12 to 14 d a y s , t h e c u l t u r e s were heated almost to boiling and filtered t h r o u e h c h e e s e c l o t h , The mycelia \&e pressed, again heated with 50 cc. of distilled mater, filtered through cheesecloth, pressed, dried at about 100" C. for 24 to 48 hours, weighed, and stored in small bottles awaiting extraction. The combined filtrates and wash liquors were cooled and aliquots taken for the determination of sugar and of acidity. All acidity determinations were made by titrating 10 cc. of the culture solution with 0.1 S sodium hydroxide, using phenolphthalein as an indicator. Qualitative tests for the acids which commonly arise as products of mold metabolism (i. e., gluconic acid, oxalic acid, and citric acid) showed the presence of citric acid only, and on several occasions the acidity as determined by titration was checked by a determination of the acid as calcium citrate, wit,h very good agreement. The yields of citric acid were never very high, however, the maximum yield being about 10 per cent. based on the added glucose. Sugar content mas determined by the ShafferHartmann method (12) using the cuprous titration. The crude fat determinations in all studies of P . javanicun were made by extracting the dried, ground mycelium in Soxhlet extractors for about 16 hours with petroleum ether (boiling point, %'to 85'C.), which dissolved almost as much material as ethyl ether but gave a much less colored product'.
A detailed study of the physiological responses of P. javanicum to changes in the composition of the nutrient solution has been presented in another paper ( 6 ) . It may be stated that the organism was found not to be sensitive to slight variations in the concentration of the nutrient salts. Variation in the concentration of the glucose, however, had a pronounced effect on the mat weights and their fat content, as is illustrated by the following experiment, conducted in 200-cc. flasks containing a medium corresponding to the M solution except that variable amounts of glucose were used. Triplicate cultures were grown, and each value reported is the average for three flasks. Table I1 shows the results of this study. The experiment shows that under the conditions described the mycelium contains the maximum quantity of fat when the glucose concentration approximates 40 per cent, and that the maximum yield of fat per flask (or per unit surface area) is obtained when the glucose concentration is between 30 and 40 per cent. Higher concentrations of sugar cause a decrease both in mat weights and in their fat content.
INDUSTRIAL AND ENGINEERING CHEMISTRY
320
Earlier workers have also reported that fat production by microorganisms is favored by a high sugar concentration in the medium. Smedley-MacLean (IS) found that an abundance of carbohydrate in the medium augmented greatly the fat content of yeast; and Belin (2) reported that the fat content of Stwigmatocystis nigra increased from 4 to 18.5per cent when the glucose concentration was increased from 2 to 12 per cent. Analogous results were obtained with the Fl6ole bacillus. Terroine and Bonnet (15) showed that Sterigmatocystis nigra, cultured on media differing only in glucose content, yielded mycelia containing more f a t as the medium became richer in glucose.
TABLE I. MOLDSYIELDINQ MOSTFATIN
THE
SURVEY
CRUDEFATI N MYCELICM ORGANISM . Penicillium bialowierense Zal. P . cilrinum Thorn P . hirsutum Dierckx P . soppi Zal. P. javanicum van Beijma P. roqueqorti Thorn P . oza&m Currie and Thorn P . piscarum Westling P. pat ocinerium Biourge Aspergillus Aavus Thom and Church
cy, ,"
~
17.0 18.1 18.4 20.2 22.2 22.9 24.4 26-28 28.5 16.0
I n order to obtain sufficient SHALLOW PAN CULTURES. fatty material for a complete analysis of its constituents, i t was necessary to have a large quantity of mycelium. This was secured by growing the organism in shallow pans constructed of relatively pure aluminum, the composition of which has been described in a previous communication (7). The present work was conducted in pans of two sizes; the earlier studies were conducted in vessels of dimensions 43 X 43 x 2 inches (109 X 109 X 5 cm.) and the later experiments in pans 22 X 36 X 2 inches (56 X 91.5 X 5 cm.). Pan fermentations were conducted on a table top in a constant-temperature room maintained at 30" C., and also in a metal cabinet (described at the end of this article) which served as both sterilizer and incubator. I n the table-top experiments, the pans were covered with two thicknesses of cheesecloth supported by a wooden frame which rested on the edges of the vessel. The covered pan was sterilized with steam before the inoculum and the previously sterilized and cooled nutrient solution were added. The principal disadvantages of this procedure were the necessity for a large table-top area and the danger of contamination with other organisms. TABLE11. EFFECTOF GLUCOSE CONCENTRATION ON FATPRODUCTION BY P. javanicum WHENCULTURED IN FLASKS GLUCOSE GLUCOBIC DRY WEIQHT CONGLUCOSE ADDED CONCN. PER FLASK BUMED OF MATS Grams Grams Grams 9.%21.3 31.6 41.6 53.6 60.3
16.0 23.7 31.2 40.2 45.2
10.3 11.3 9.9 5.2 2.0
2.52 2.40 1.96 1.02 0.26
CRUDE W E I Q E T OF FAT F A T P E R IN
MATS
Yo 29:O 34.6 41.5 35.2 17.5
FLASK Grams 0.73 0.83 0.81 0.36 0.05
A typical fermentation in the cabinet (Figure 1) was conducted in the following manner: The re uired quantity of glucose, nutrient salts, and water was place] in each pan, the rakes and door were screwed in place, and steam was blown through the apparatus (valve E remaining open) until a temperature of slightly over 100" C. was reached (2 ,to 4 inches of water pressure), and this temperature was maintained for an hour. During the f i s t 20 minutes of heating, steam was by-passed through the air filter to sterilize it, after which the cotton was dried by the heating coil. After an hour the steam was cut off and sterile air was blown through the system, cooling being hastened by running cold water through the copper tubing. Care was exercised that the apparatus was always under a positive pressure, thereby avoiding danger of collapse or of entrance of nonsterile air. In 3 to 4 hours the temperature was reduced to about 35" C., and the solutions in the pans were inoculated through the aluminum tubes from
Vol. 27, No. 3
sporulation cultures. These had been grown in 3-liter flasks fitted with rubber stoppers containing two glass tubes, one covered with cotton for the admission of sterile air and the other attached to a length of sterile rubber tubing used for the conveyance of the inoculum to the aluminum tube. The inoculated solutions were stirred by moving the rakes back and forth, the aluminum tubes being wiped with cotton saturated with alcohol to prevent entrance of contaminating organisms by this route. After the air flow was adjusted to the desired rate, the apparatus required practically no further attention during the incubation period, except adjustments necessitated by the heat developed by the fermentation. An increase in temperature occurred on the third day and again about the ninth day after inoculation. To maintain the environment at about 30' C., cold water was at such times run through the cop er tubing. At the termination of the incubation period gsually 12 to 14 days) the front of the cabinet was removed and the cultures were harvested. Tables 111, N, and V show representative results obtained when P. javanicum was grown on glucose solutions in aluminum pans, inside or outside the cabinet. The smaller type of pan was used in all experiments except in runs 2 and 3 (Table 111) and the volume of culture solution in the smaller pans was 12 liters in all cases except the two noted in run 12 (Table V). CULTURES (ON TABLETOP) TABLE111. SINGLE-PAN AGE
YGLUCOBEPAN OF HARConsurned TYPE SOLN.VEST Added VOL.
AT
Liters Day8 Grams Grams
%
CRCDB
CITRIC
FAT ACID M A TWEIGHT I N PRO- Per Per MYDUCED D a n sa. m. CELIUM Grams Grams Grama %
RUX 2, MEDIUX Q BOLUTION
Large
20
12
4000
3440
86.0
88.5
832
700
20.4
741
622
13.9
RUN S, MEDIUM Q SOLUTION
Large
20
13
4000
2980
74.5
..
RUN 4, MEDIUM 15% QLUCOBE, OTHER SALTS AB I N M BOLUTION
Small
12
12
1800
1310
72.8
118
352
688
14.1
The data accumulated in these pan fermentations lead to several conclusions and also raise several questions. One is impressed by the large variation in mat weights, acid contents of the solutions, and fat contents of the mycelia from various pans. Even when all the pans in one cabinet run were filled with the same medium and inoculated with the same preparation, unexplainable differences occurred. Although special means were taken to have atmospheric conditions within the cabinet simulate closely those conditions prevailing around the pans on the table top, the mycelia developed in the two cases differed widely in appearance, and in some cases (runs 8 and 11, Table N) the fat content of mats from outside control pans greatly exceeded that of the corresponding pans grown in the cabinet. All cabinet cultures yielded mats which were completely covered with a white, velvety, aerial growth, and the lower surfaces of which were light brown in color. I n contrast, the mats which developed in control cultures displayed very little aerial growth and were dark brown on the lower surfaces. The original rate of aeration of the cabinet, 1.6 liters per minute, was sufficient to give a complete change of air every 24 hours. As previous studies in this division had shown that organisms taking part in oxidative fermentations require large quantities of air, it was thought that perhaps an increase in the rate of aeration would yield better results. T h a t i t did so is borne out in runs 11 and 12 (Tables N and V) where increase of the aeration rate to 4 to 5 liters per minute caused a n increase in sugar consumption, mat weights, acid production, and fat content of the mats. The data obtained from run 12 indicate that in cabinet fermentations, increase in the sugar concentration of the nutrient solution does not result in increased fat content of the mats, such as occurs in small flask cultures. Although the 40 per cent glucose concentration gave t h e mats of highest fat content in flasks, it apparently gave the mat of lowest f a t content in the pan fermentations.
I: N D U S T R I A L .4N D E N G I N E E R I N G C H E M I S T R Y
March, 1935
That the high fat yields encountered in run 12 were probably due more to the high rate of aeration than to the .lightly greater age of these cultures is indicated by results obtained in a study of the time factor in flask cultures. This study, which is described in detail in another communication (6), shorn-ed no great differences in fat content of mats of different ages. Each of the duplicate pan cultures in this run gave almost identical amounts of fat, as calculated from the mat weights and the percentage of material soluble in petroleum ether. TABLEIV.
REPRESENTATIVE SHALLOW-PAN CULTURES GROWNIN CABINET
RUN No,
MEDIUM
No.
Days 13 17 14
G
8 9 11
PAN
AGE AT HARVEST
G M
GLUCOSE CONSUMED Grams
%
CITRIC ACID PRODUCED
Grams
RATEOF
A~~RATION Liters/min. 1.6 1.6 4-5
CRUDE FAT
MATWEIQEIT
IN
MY-
Per pan Grams
Per sq. rn. Grams
CELIUM
413 382 406 395 449 414 422 483
808 746 793 772 879 810 824 945
11.1 10.8 13.9 12.0 17.3 11.2 14.9 21.8
414 334 317 333 317 392 430
810 653 620 6.50
620 766 840
11.4 6.6 7 ,o 8.6 7.0 10.2 13.1
376 469 435 498 492 489 417 435
735 918 850 975 962 957 815 850
15.5 13.8 18.9 13.4 14.9 11.2 15.9 24.4
%
RUN 0
1 2 3
1240 1110 1280 4 1120 5 1420 6 1200 7 1080 Control" 1450
50.4 45.1 52.0 45.6 57.8 50.8 43.9 59,O
48.4 42.5 50.2 47.3 68.9 44.9 49.3 54.7 RUN 9
1 2 3 4
5 6 7
1320 1150 1080 1020 1130 1100 1350
53.7 46.7 43.9 41.5 46.0 44.7 54.9
1 2 3 4
1210 1900 1540 1830
50.5 79.2 64.2 76.2 ~8 0. .8 75.0 58.0 57.5
49.2 40.1 34.8 33.8 35.4 41.0 57.4 R U N 11
101.0 85.0 101.0 146.0 195.0 153.0 124.0 101.0
321
stearic, tetracosanic, oleic, and a- and /3-linoleic acids, and a small amount of unsaponifiable matter. Strong and Peterson (14) have also recently reported finding tetracosanic acid in the fat produced by Aspergillus sydowi, and these two reports have been the only ones identifying the 24-carbon acid as a constituent of the fats elaborated by molds. Since the publication of Ward and Jamieson's paper describing the composition of the oil obtained from mycelia developed on 20 per cent glucose solutions, an examination has been made of the fat from the mycelia of run 12, in which the glucose content of the medium was varied. The fatty fractions obtained from these mycelia were of similar physical appearance, except that the portions from pans 3 and 7 (30 per cent glucose medium) and pan 4 (40 per cent glucose medium) were solid a t room temperature instead of liquid, as had been observed for all fat produced from 20 per cent glucose solutions. To ascertain the reason for this condition, several chemical constants were determined, and these results, together with comparative data for the oil previously described (16)are presented in Table VI. TABLEVI. CHEMICAL CONSTANTS OF FATPRODUCED BY P. javanicum WHENGROWN ON SOLUTIONS OF DIFFERENTGLUCOSE CONCENTRATIONS (Pans from run 12)
GLU-
SOURCE FAT
OP
MEAN
COSE MOL. C O N C N . SAUNWT. OF PONIFI12 UN- B A P O N I - OF ME- CATION No. SATD. SATD. FIABLE SATD. ACID DIUM N O . (HANUS)4 C I D S a A C I D SMATTBR ~ ACIDSVALUE
% Previously described (16) 20 Pans 1 and 5 20 Pane2and6 30 Pans 3 and 7 30 Pan 4 40 Uncorrected.
191 193 192 193 192
84 78 84 88 88
%
%
%
32.5 33.0 33.3 33.5 34.6
61.1 61.3 62.0 61.4 60.1
2.00 1.02 1.13 1.90 1.67
272 272 271 272 271
10.6 6.4 15.2 55.5 50.7
These figures show that the samples are chemically similar, except for the fact that those obtained from 30 and 40 per cent glucose solutions contain about one-fourth of their fatty acids in the free state, rather than combined as glycerides. The control cultures were grown outside the cabinet on the table top. This condition must be responsible for the higher solidification temperature of such samples. The low acid value of TABLEV. EFFECTOF VARYINGGLUCOSE CONCENTRATIONthe fat from pans 2 and 6 (30 per cent glucose medium) can I N SHALLOW-PAN CULTURES GROWN I N CABIhTET be attributed t o the fact that, because of the smaller volume (Run 12: medium, glucose concentrat:on varied, each pan contained 27.0 of solution in these pans (8 liters instead of 12) the glucose grama NHINOJ, 3.0 grams MgSOc7HzO,3.6 grama KH1P04; age a t harvest, 19 days; rate of aeration, 4 t o 6 liters per minute) available per unit area of mycelium was the same as that in GLUthe usual 20 per cent glucose solutions. cos1 5
1940 ~
6 1810 7 1390 Controla 1380
.
Q
IN
NUTRI-VOL. GLUCOSE PAN ENT OF ConNo. SOLN. SOLN.Added surned
%
1 2 3 4 5 6
7
20 30 30 40 20 30
30
CIT- MATW E I Q H T
RIC Per Per ACID pan n q . m. Liters Grams Grams % Grams Grams Grams 12 2420 1720 7 1 . 1 198 427 835 8 2420 1680 6 9 . 5 223 407 795 12 3600 1850 51.3 249 445 870 12 4840 2060 42.5 162 427 835 12 2420 1810 74.7 233 430 840 8 2420 1990 82.2 201 467 912 12 3600 2100 58.3 219 468 915
CRUDEFAT % Grams 22.3 21.8 23.9 18 1 22:4 19.3 22.3
95.2 88.8 106.5 77.4 96.3 90.6 104.5
NATUREOF FATTY MATERIAL
A 6240-gram portion of the dried, ground mycelium of P. iavanicum obtained from the best pan cultures, exclusive of runs 11 and 12, was placed in a n extractor of the type recommended by Sando (11) for the extraction of large quantities of plant materials, and was extracted with redistilled petroleum ether (boiling point, 35" to 65" C.) for 7 days. After clarification the extract yielded 691 grams of a clear, orangecolored oil, corresponding t o 11per cent of the dried mycelium taken for extraction. The physical characteristics and chemical composition of this oil have been described in a previous communication (16). It has been shown to be a typical vegetable f a t composed of glycerides of palmitic,
INVESTIQAT~ON OF A CARBOHYDRATE CONTAINED IN THE
MYCELIUM
When the dried mycelium of P. javanicum was treated with hot water, there was obtained an orange-colored extract which, when boiled, displayed a strong tendency to froth and foam. Upon cooling this extract, there was deposited a voluminous, flocculent, white precipitate which was filtered off and washed several times with cold water. I t gave a positive Molisch test, gave no color with iodine, and reduced Benedict's solution only after boiling for one-half hour with 6 N hydrochloric acid. These reactions indicated that the substance was a carbohydrate. The specific rotation a t 90" C. was determined in a jacketed tube [(a)900 = +280°]. This material is apparently the same complex carbohydrate as that obtained by Birkinshaw, Charles, and Raistrick (4) from the mycelia of three strains of P . digitaturn by a similar hot water treatment. Their product showed similar solubility behavior, displayed no color with iodine, and various samples gave specific rotation values from +261° to +299" at 90" C. These workers further showed that the substance yielded only glucose on hydrolysis. The
322
I N D U S T 11 I A L A N D E N G I N E li, 13 I N G C H E M I S T R Y
Vol. 27, No. 3
"mycodextran" prepared by I)ox and Xeidig (5) from P. ezpansum also had the same properties as tile carbohydrate
isolated from P. jmanicum. Apparently this complex carbohvdrate is rather widely distributed throughout the various species of molds. Alkaline treatment of the mvcelium of P. iavanicum has yielded variable amounts of a chitinous complex which is the subject of another coinniunieiition (8) from tliis division. L)ESCRIPTIOX OF CABINET INCUBATOR
During the course of t i m e investigations, a stcriliaerincubator cabinet became available for continuing tire largepan cuitnres. T h e cabinet was designed and const,ructed in this division for general mold culture investigations. It follows the basic design of the sterilizer-incubator deacrihed by Birkinshaw, Charles, Lilly, and Raistrick (3) and is also somewltat similar t o the one described by Peterson, I'ruess, Gorcica, and Greene (9). Figure 1 SIIOWS the apparatus in section: The walls were constructed of No. 18 gdvaniaed-imn shooting, riveted and soldered 80 as to he air-tight. Grossb~rshalted to reGnforcing angle irons a t seven different levels served us supports for the seven aluminum fermentation pans. I3afIles oonstructed of thin aluminum were suspended by means of hea,vy copper wires from the crossbars and also from the top of the cabinet, Theso baliles served to carry the condensate formd during sterilization of the apparatus toward the wails of ihe cabinet, thus preventing dilution of the nutrient solutions in t.he pans. For the purpose of circulating cold water to cool the entire apparatus after sterilization and to regulate the temperature when excessive heat was generated by the fermontatinn process, 175 feet of O.25-inch copper tubing was led throuzh the auuaratus. about 25 feet of it beine woven across each of the baffl& Steam for sterilization of the apparatus was admitted through valve A . Valve B served to by- 599 steam through air filter F to sterilize it a t the beginning o?each run. Thiq air filter was merely B piece of &inch pipe, capped a t each end, filled with cotton, and equipped with steam and air inlet tubes (air inlet not shown) and one outlet tube leading to the cabinet through valve C . Drying of the cotton after sterilization was facilitated by an electric heating coil surroundin the cylinder. Upon mtering the chamber through valve C, t f e sterile air was humidified by bubbling through water in the reservoir a t the bottom of the cabinet. More than enough water to fill this reservoir was alwa s supplied by the condensate formed during sterilization of t i e appzriitus. the excess water being run out through outlet valve D. Tho n i p left the cabinet a t vaive E or a t needle valve G, to which could be attached a flowmeter. The pressure inside the cabinet was indicated by the manometer, while provision for automatic:4y relieving any pressuie in excess of about 4 inches of water was afforded by.~~ pipe P , wnnected directly to the water reservoir. The removable front was likewise fabricated of galvanized-iron sheeting, re8nforoed by an& irons as shown. It contained eight sight glasses, as did also one side of the cabinet. The glasses served for illuminationand observationofeach panand the water reservoir during the incubation period. The front also contained glands for the insertion of 0.5.inch aluminum tubes (one j w t above each pan) through which the solutions were inoculated after sterilization and cooling. These tuhes were 48 inches in length, and to the inner end of each was fastened a perforated aluminum strip which was slightly narrower than the mns. This rakelike device was useful in distributing the I
each t;be was Grovided with a screw cap. The front was attached to the main body of the cabinet, through a felt gasket by means of thirty-four brass screws. The entire cahinet was mounted on four eastnrs. and to the base were attached four jacks, which facilitated leveling of the apparatus
ACKNO WLEDGMZNT T h e authors are greatly indebted to R. Hellbach, of this division, for the construction of the cabinet and for assistance in its design; t o R. M. Baker and I. Bauseman, of the Chemical Engineering Division of this bureau, for the drawing.
F. H. van, Verhandel. Akad. Wdens c h a p p a Amslcrdam, Sco. If. 26. IB-19 (1929).
(1) Beijrua thoe Kiiigma,
(2) Belii1, P., Bull.soc.chirn. bid.. 8, 1081-1102 (1926) I . H. V., Lilis, C . 11.. and Rniatriok. (3) Birkinahaw, J. Si.. (:hark. . IT. . . ., Tvnnr. .. . Rog. Soc. (London), 13220, 136-8.366-7 (1931). (4) Birkinahaw,. .I. H.., Chnrlor. J. H. V.. and Raistriok, H., Ibid.. nnnn Y el__: rlnl,/ Y Y ,'""'i. Ye*".
( 5 ) Dox. A. W., and Neidie. R. E., J . Bid. Chem.. 18, 167 (1914). (G) Lockwood. L. B.. Ward, G. E., May, 0. E., Herriok H. T., snd O'Neill. H. T., Zed?. Bahl. Parouilak., IS. 90, 411-25 (1034). (7) Mas. 0. E.. Herrick, 11. T.,M a w , A. J., and Hallhach. R.,
1x0. E ~ o . < ; m ~ . . 211!18 1 , (1920).
(8)
Mus, 0. E., nnd Ward, G.E.. I . Am. C l ~ e mSoc., . 56, 1307-9
. . .
.
(11) Baodo. C. E., SNO. E m . C e ~ x .16, , 1125 (19'24). (12) Shsffcr. P. A,. and liartmann, A. P., J . Bid. Cliem., 45, 36fr90 (1021).
(13) Sniodloy-MaoLean, I., Biodrern. J.. 16,370-9 (1922). (14) Strong, F. M., andPeterson. W. H.. J . d m . Cham. Soc., 56,052-5 (1934). (15) Torroine. E. F., erird Bonnet, R., Bull. WC. chim. hid., 9. 588 (1027). (16) W a d . G. E., and Jamiemn, G.S., J . Am. Chern. Soc., 56, 073-5 (1034). Il~c~~v November eo 2, 1984. Contribution 245 from the Culoi siid Farm Waate Diviaiaii, Ruiesu of Clrerniatiy and Sella.
