Large- Scale Laboratory

Cultivation of Molds W. H. PETERSON, L. M. PRUESS, H. J . GOHCICA,

AND H. C GREENE

[ h i ) ersity of Wisconsin, Madison, Wis.

An upparutus (slerilizer-incuttor) arkd fer- mntution procedure for the growth of molds in pure culture on a large scale in the laboratory are described. Twenty-nine fermentations in which 120 kg. of mycelium were obtained have been made with the apparatus. Data on jfteen fer- mentations are reported in this paper.

The conversion of glucose into mycelium varies with different species of mold and for different fermentations with the same mold. The averages for three molds (A. fischeri, A . sydowi, and A . orzyae) are 23, 22, and 16 per cent, respectively.

HE growing of aerobic microiirganism on a hrge scak in the laboratory is attended with considerable dif- ficulty and has received only scant attention. The

principal difficulty in such work lies in excluding air-borne foreign microorganisms. In I930 an extended investigation of the biochemist.ry of molds was begun in these laboratories. Large quantities of mycelium were needed for certain phases of the project.&studies of the chemical nature and nutritive value of mold tissue. I t mas necessary to devise apparatus in which such quantities of mycelium could he grown under rigidly controlled bacteriological conditions. After consider- able experimentation the sterilizer-incubator described in this paper has been developed. It has been in use for ncarly two years, and during this time most of the difficulties which arr likely to appear in the operation of large-scale apparat,us have been encountered and, in R large measure, overcome.

At the time this work was begun, the only type of apparatus that seemed suitable for the purpose was that described by May and associates ( I ) . The aluminum pans which they employed had been fourid satisfactory, but a hctter means of housing these pans so as to avoid contamination was required. It \!-as also found desirable to start the mold growth a t or near pH 6.5 and, in certain cases, to use an organic medium. Such conditions of grou-th require not only a sterile inediurn a t the start, hut also the exclusion of air-borne bacteria, molds: and yeasts throughout the incubation period.

If the monograph of R,aistrick and co-workers (8’) had heen in print in the fall of 1030, it mould have saved much work in these laboratories, as the apparatus which has been developed here, though larger, is similar to that described by these in- vestigators. By the use of this apparatus, Raistrick and associates hare been able t.o @ow molds on a sufficiently large

ieale to permit the isolation of several new and interesting zompouiids ---citromycetin, citrinin, luteic acid, et.c..

APPARAKIti

Tlie sterilizer-incu~ator is n rectniiguiar cabiiiet 3 X 3 X 7 fcot (0.9 x 0.9 X 2.1 meters) made of KO. 16 galvanized iron, riveted together at all joints. The front is divided into halves which are removable and serve as doors. Each door is held in position by strap irons notched to rest on swing bolts, and can be drawn tight by means of v h g nuts. A re- cessed rubber insertion along the edge of the cabinet insures n close joint. Along the sides of the cabinet, spaced about 7 inches (17.8 rm.) apart, are narror shelves for the support of ten pans. These pans (32 X 28 X 3 inches or 81.3 X i l . 1 X 7.6 cm.) are made of highest g a d c aliiminum and furnish a total surface area of 82.2 stpiarc feet (5.78 square meters). Short lengths of pipe (1.5 X 2 inches or 3.8 X 5 em.), closed with screw caps or cotton plugs, nre located on both sides of the cabinet a t levels just ahove the top of each pan and serve as apertures for the introduction of the inoculum or the withdrawal of samples of medium, as well as for ob- servation puqmses. At the bottom of the cabinet is an inlet for high-pressure steam, and in the sloped bottom is an outlet for drawing off condensed steam. In later experiments the

213

214 I N D U S T R I A L A N D E N G I K E E R I N G C H E M I S T R Y Vol. 25, No. 2

steam inlet also served as an inlet for filtered air. The whole cabinet rests on an iron base that raises it about 6 inches (15.2 cm.) above the floor.

When another incubator was needed, experience gained from the one just described led to some modification in plan. The second incubator was built with essentially the same general design except that twenty-one pans (24 X 32 X 2 inches or 60.9 X 81.3 X 5 cm.) with a total surface area of 98 square feet (9.1 square meters) were used. Both the space between pans and the height of the pans were reduced, so that the incubator, although having a greater capacity, is some- what smaller than the first. Figure 1 shows the construction of this second sterilizer-incubator. The cloth fronts, which are held in place by means of studs and wing nuts, permit the doors to be opened slightly a t the bottom and still exclude appreciable contamination. The doors are opened only after a thick mat of mycelium is established and only if the rise in temperature and rate of fermentation become unusually rapid. The rubber joint packing, instead of being placed along the edge of the cabinet, is in a channel around the doors. This channel fits tightly about the projecting edge of the cabinet. The inoculation apertures are small tubes (0.75 X 1 inch or 1.9 X 2.5 cm.) soldered to the cabinet along one side only. One such tube on the opposite side permits observation of the mycelium. The tubes are closed by means of rubber stoppers, one of which holds a ther- mometer to record the temperature inside the cabinet. The pressure tubing attached to the top inoculation tube is a second steam inlet.

The two cabinets are kept in a constant-temperature room which is regulated by means of thermostatic control within 2-3' C.

FERMENTATION PROCEDURE

For each run of thirty-one pans the following quantities of material were used: commercial glucose ("cerelose") 31 to 46.5 kg. (68 to 102 pounds); ammonium nitrate, 3.1 kg. (7 pounds); potassium diphosphate, 2.1 kg. (4.6 pounds); MgS04.7Hz0, 1.55 kg. (3.4 pounds); FeCb.6Hz0, 0.043 kg. (0.095 pound); ZnS04.6H20, 0.015 kg. (0.033 pound); orthophosphoric acid (85 per cent, specific gravity 1.71), 0.3 liter (0.08 gallon); water to make 310 liters (82 gallons). This medium was made up in three batches in a 30-gallon (1 13.6-liter) earthenware crock. Ten liters were bailed out and placed in each pan. The cabinets were then closed and steamed for about 2 hours a t 96-100' C. Two days later the medium was again sterilized by steaming. As soon as the cabinets and medium were cool, 175 cc. of sterilized potas- sium hydroxide solution (25 per cent) were added to the medium in each pan, through the inoculation apertures, to bring the pH up to about 6.5. The inoculum, a suspension in sterile water of spores obtained from malt extract-agar slants, was then introduced through the same openings by means of sterile pipets.

Incubation for 10 to 12 days a t 28-32' C. resulted in the fermentation of about 80 to 95 per cent of the glucose and the formation of thick mycelial mats. A rise in temperature, sometimes as much as 5' C., was usually observed on the fourth or fifth day of incubation. At the end of the fermenta- tion period, determined by a previous analysis of unfermented glucose, samples of medium for bacteriological examination were withdrawn through the side openings by means of sterile pipets. The incubator was then opened, the mats examined, and samples of the medium taken for chemical analysis. The liquors contained only a small quantity of titratable acid (3 to 8 cc. of 0.1 N potassium hydroxide per 10 cc. of sample), and in terms of pH ranged between 2.5 and 4. Further evidence that the liquors contained no appreciable quantity

of fermentation products was obtained by determining their carbon content. Only about 0.4 per cent of carbon in the fermented liquors (runs 27 to 29) was not accounted for by unfermented glucose.

After the samples had been taken, the liquor was siphoned from the pans, and water was run in to remove the medium adhering to the mats. After standing for an hour, the water was siphoned off and the doors of the cabinets were replaced. Steam was passed into the cabinets for one hour to kill the spores, and the pads were then removed, dried a t 65" C., and ground. In some cases, when the pads were to be used for certain chemical analyses, they were harvested without pre- vious steaming and drying.

~IYCOLOGICAL TECHPI'IC

Bacteriological control was used to check the sterility of the incubator medium before and after inoculation and also to check the purity of the inoculum. Precautions were taken to perform all operations as aseptically as possible.

Before the incubators were to be inoculated, the room in which they were situated was thoroughly steamed. Samples of medium from alternate pans were removed by means of sterile pipets and placed in a neutral broth of beef peptone and glucose. This medium readily disclosed bacterial con- tamination if any existed. As a rule no contamination was found after the first steaming, and samples taken after the second sterilization were always free from visible growth. The high acidity of the medium (pH 2.5) also reduced the possibilities of bacterial growth in the incubators before the addition of the sterile potassium hydroxide solution. Another means used to check the efficiency of the intermittent steriliza- tion was to set small capped flasks, containing incubator medium, in the liquid in the pans before sterilization. The medium in the flasks was therefore exposed to the same heat treatment as the medium in the pans, and, if i t once became sterile, could not become contaminated by organisms that might enter the incubator subsequent to sterilization. At the end of the incubation period the flasks were examined and found free of growth, which demonstrated that the two steamings were sufficient to sterilize the medium.

The purity of the inoculum was checked by macroscopic and microscopic examinations, and also by inoculating pilot flasks containing autoclaved incubator medium. These flasks served a t the same time to check the manipulations incident to inoculation. N o contamination was detected in these flasks on transfer into beef peptone.

Sterile air was passed into the cabinets during cooling, between steamings, and throughout the period of incubation, in order to maintain a positive pressure outward and to pre- vent contaminating microorganisms from being drawn into the cabinet by changes in volume during cooling. Before this expedient was used, certain runs showed bacterial con- tamination although the inoculum was uncontaminated. Evidently contaminating microorganisms were drawn into the incubator through small leaks when the steam condensed. The introduction of sterile air immediately after the steam was turned off remedied the situation. The air was sterilized by passing it successively through two glycerol towers and a filter tube 24 inches (61 cm.) long, which was previously packed with cotton and sterilized. To check the purity of the air before it entered the incubators, it was bubbled through a broth of beef peptone and glucose after passing through sterile water (to prevent evaporation of the broth).

The microorganism capable of growing on the inorganic salts-glucose medium was isolated, and its general character- istics studied. The culture answered the description of B. subtilis.

A more rigid test of the possibility of growing molds in pure

Februar), 1933

culture in the above described apparatuz xvas made. The experiment was as follows:

I N D 7: S T 1t 1 A 1. h iX U E Ii G 1 N li E I< I N G C H E M I S T 11 Y 215

to tho medium and omitting orthopimpboric acid, (4) re- ducing the amount of salts in the medium, (5) increasing aeration, and (6) introducing wooden grids in the pans for support of the Inat of Inyceliurn. Altllough not enougll ex- periments have been conducted to warrant a hard and fast conclusion, in general the results haye bern less than the

Table I presents Some of the typical yields of A s w W g r o m in the two sterilizer-incubators. In all but three of the fifteen ru11s 80 per cent or more of tire glucose was fer- nsel,ted, and in eigi,t runs ver cent or more was destroyed,

Four pans of organic medium (5 per cent nrdt sprouts and 10 per cent glucose) and four pans of the regular inorganic salts- glucose medium plus an excess of calcium carhonrtt,e were p m pared. To test the efficiency of the sterilization, 20 cc. of me- dium were taken from each pan and put in small flasks which were capped and then returned to the pans. Sterilization was carried out in the regular manner. The pans were inoculated with A. jseheri and incubated for 10 days. At the end of the fermentation period all of the &&sku were stili free from growth, snd the heavily wrinkled pads showed no sigiis of cont,nnrination.

procedure.

TYPlCAL YIELDS OF M Y C E l X l M

Twenty-nine runs have been made with the steriliser-in- cubators, using three species of Aspergilli and two species of Pmicillia. About 120 kg. (264 pounds) of dry mycelia have been grorn.

12 14 15 16 17 26 27 28

Asperviiius fiacheri 21 ri 12.9 2 7 . 1 2 1 . 8 80.8 6.00 286 27.4 io 11 16.0 15.0 14 .5 96.6 3.80 380 2 6 . 2

27 12 16.0 40.5 35.6 87 .0 5.82 216 10.4 27 12 15.0 40 .5 36 .0 88.9 0 . ~ 3 249 18.7 14 11 12 .0 16.8 13.2 7 8 . ~ 3.87 240 26.5 23 11 1 2 . 0 a7.6 20 .0 72.4 4.77 207 23.8

16 12 1 5 ~ 0 2 4 . 0 2 2 . 4 93.4 4.20 288 19.1

31 12 12.0 37.2 3 2 . 6 87.6 9.12 294 28.0

228.7' 196.2* S5.8b 43.90' 2 6 8 b 23.16 Aap~igil lus svdawi

21 31 10 15.0 48.6 30.3 65.2 6.59 112 21.8 22 29 11 10.0 29.0 28.4 97.9 5.40 186 19.0 23 31 10 10.0 31.0 29.0 95.5 6.91 223 23.8 24 31 io 10.0 31 .0 80.6 88.4 8.17 263 20.8

. . 2 r 5 12 14.0 7 . 0 6.8 90.0 1.M) 2W 15.Y

4( 5 12 14.0 7 . 0 6 . 3 9 0 . 0 1.10 220 17 .5 3~ s 12 14.0 7.0 6 . 3 90.0 0.91 182 14.4

21.0- 18.9" 9o.oa 3.Vl ' f i ao1r IS 9 b Total.

6 Average. Ex~esa of oalcium oarboaate io a~ediun~.

In developing the procedure just described, various modi- fications have been tried for the purpose of increasing the yield of mycelium. Some of these are: (1) increasing the quantity of spore suspension used as inoculum, (2) using germinated spores as inoculum, (3) adding calcium carbonate

On the~average, about 1 per-cent of glucose was fermented per day of incubation.

The conversion of sugar into niyccliuin has averaged 23 per cent for A. sydowi, 22 per cent for A. jischa., and 16 per cent for A. oryzae. Because it tolerates a higher sugar con- centration and possesses almost the same conversion ef- ficiency, A. fisrhmi gives a larger yield of mycelium per unit of apparatus than does A . ngdowi. A. oryzae is the least productive of the three. The data show great variations, not only for different species, but also for different fermenta- tions with the same mold. In the case of A . fischeri the figures vary from 16.4 to 28.0 per cent. These values should be taken with some reservation because of uncontrolled cha.nges in the volume of the medium in different pans and because of errors incident to sampling such large quantities of material. The differences in yield could perhaps be elimi- nated by a more careful regulation of such factors as tem- perature, oxygen supply, and hydrogen-ion concentration. The extensive investigations necessary to bring such factors under control have not appeared worth while up to the present, as the first objective has been to secure a good rather than a uniform yield of mycelium.

ACRNOWLEDOMENT This work was supported in part by a grant from the Wis-

consin Alumni Research Foundation. The authors are in- debted to A. F. Lauglykke for assistance in the design of the apparatus and to J. U. Hnmes for assistance in ita operation.

LITERATURE CSTEU (1) May. Herrick. Moyer, and Hellbeah, IN". ENS. CHY~M., 21,

(2) ltaistrick and Collaborators, ?'ra,ia. Roll. SOC. (London), B220,

R s c s ~ v m Aupuat 6, 1932. Thia paper iucludes material prwented before the Diviaion of Bioloeioai Chsmiatry at the 83rd Meeting of the Ameriasn Chemioal Soaiety, New Orleaae. La.. Maroh 28 t o April 1, 1938. under the same title by W. H. Peteraon. L. M. Pruess. snd E. 8. Red.

1lYX (1Y20).

136 (1931).

. . ETHYLENE OxmE BEING WEIGBED INTO CANS OF CRUSHED SOLID CARBON DIUXIDE F-ROM

TANK@ ON PLATFORM SCALE