APPLIED MICROBIOLOGY, Feb. 1968, p. 393-400 Vol. 16, No. 2Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Large-Scale Transformation of Steroidsby Fungal Spores

KARTAR SINGH, S. N. SEHGAL, AND CLAUDE VEZINADepartment of Microbiology, Ayerst Laboratories, Montreal, Quebec, Canada

Received for publication 24 November 1967

Spores of Aspergillus ochraceus and Septomyxa affinis were produced on a largescale by surface sporulation on moist wheat bran and barley. 1lca-Hydroxylationof progesterone and Reichstein's compound S by spores of A. ochraceus and 1-dehy-drogenation of compound S by spores of S. affinis were carried out in 5-liter fer-mentors. It was shown that, above a certain minimum, increase in aeration andagitation did not significantly affect steroid conversion. The industrial feasibility ofthe spore process was further demonstrated by 1la-hydroxylation of 6a-fluoro-16a ,17a-dihydroxypregn-4-ene-3,20-dione in a modified 200-gal stainless-steelvessel with spores of A. ochraceus. Strict aseptic conditions are not necessary, eitherduring harvesting of spores or during steroid transformation.

Steroid transformations by fungal spores wasdiscovered by Schleg and Knight (5), who re-ported the 1la-hydroxylation of progesterone byconidia of Aspergillus ochraceus. Subsequentreports from our laboratory (6, 7, 9) establishedthat steroid interconversions by spores is wide-spread in streptomycetes and in fungi of variousclasses, and applies to many steroids for a varietyof reactions. Factors involved in the hydroxyla-tion of progesterone by spores of A. ochraceus (9)and the 1-dehydrogenation of compound S byspores of Septomyxa affinis in shake flasks havebeen reported (8). During conversion, strictaseptic conditions are not necessary: acidic pH(4 to 6) and absence of nitrogenous nutrientsprevent spore germination and bacterial growth.Germination is also inhibited by the high sporeconcentration (>108) used (our unpublishedresults; B. T. Lingappa and Y. Lingappa, Bac-teriol. Proc., p. 35, 1964). Bacterial contaminationcan be further minimized by the addition of anti-biotics. These observations prompted us toinvestigate the feasibility of using the sporeprocess for large-scale transformation of steroids.

Translation of these observations into an in-dustrial process involved two distinct steps: theproduction of spores and the interconversionproper. Optimal conditions for submerged sporu-lation of several microorganisms have alreadybeen described (10). Surface sporulation couldalso be scaled up, and methods for mass produc-tions of A. ochraceus and S. affinis spores bysurface culture are described in the present paper.The feasibility of large-scale transformation of

steroids by spores is exemplified by the resultspresented on the lla-hydroxylation of progester-one and compound S by spores of A. ochraceusand the 1-dehydrogenation of compound S bythose of S. affinis in 5-liter fermentors. 1 lcl-Hydroxylation of 6a-fluoro-16a, 17a-dihydroxy-pregn-4-ene-3, 20-dione in a nonsterile 200-galvessel is also described.

MATERIALS AND METHODSSpore inocula. A. ochraceus NRRL 405 and S.

affinis 6737 were maintained as lyophilized pelletsand as soil stocks (9). A. ochraceus was grown onSabouraud Dextrose Agar at 28 C for 4 to 5 days,and S. affinis was grown at 25 C for 7 to 10 days ona medium consisting of 5% malt extract (Difco),0.5% Edamine (Sheffield Chemical, Norwich, N.Y.),and 2% agar. Conidia from each Roux bottle wereharvested and suspended in 100 ml of a 0.01% solu-tion of sodium monolaurylsulfonate; the suspensionswere usually filtered through sterile glass wool. Theseinocula contained about 5 X 108 spores ofA. ochraceusand 3 X 108 spores of S. affinis per ml, as determinedby direct counting in the hemacytometer.

Sporulation on barley. A 200-g amount of "pot"barley (Ogilvie Flour Mills, Montreal, Canada) in a2.8-liter Fernbach flask was moistened with tap water(80 ml for A. ochraceus, 120 ml for S. affinis), sterilizedat 121 C for 1 hr, and cooled to a suitable incubationtemperature while being shaken vigorously to breakclumps. Each flask was inoculated with 5 ml of thespore inoculum. A. ochraceus spores were incubatedat 28 C, 98% relative humidity, for 5 to 7 days, andS. affinis spores were incubated at 25 C, 98% relativehumidity, for 10 to 12 days.

Spores were harvested by adding 500 ml of watercontaining 0.01% Tween 80 to each flask; the cotton

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plug was replaced by a rubber stopper, and the flaskwas shaken vigorously to dislodge spores from barleygrains. The spore suspension was decanted through acopper screen which fitted the flask neck and kept thegrains in the flask; a second harvest was effected byuse of 300 ml of water. Combined spore suspensionswere filtered through glass wool to remove mycelialfragments and to break chains and clumps of conidia.Spores were collected in a Sharples centrifuge operat-ing at 50,000 rev/min, and washed twice by resus-pending the paste in water and centrifuging. Each flaskyielded 5 to 6 g of spore paste of A. ochraceus or S.affinis: dry weight was about 30%c, total yield wasabout 2.5 X 1011 spores.

Sporulation on bran. When barley in Fembachflasks was replaced by 100 g of hard wheat bran(Ogilvie Flour Mills, Montreal, Canada) moistenedwith 500 ml of tap water, A. ochraceus producedsignificantly more spores (3.5 X 1011 to 4 X 10"1 perflask).On a larger scale, the following procedure was used:

hard wheat bran (24 kg) and tap water (9 liters) weremixed and sterilized with live steam in a jacketedmixer (Charles Ross and Sons, Inc., New York,N.Y.), cooled to 28 C, and inoculated with 1.2 litersof threefold diluted spore inoculum of A. ochraceus.Inoculated bran was distributed into 24 aluminumtrays (30 X 18 X 1.75 inches; 66.2 X 45.7 X 4.5cm), and the trays were incubated at 28C, >98%relative humidity, for 7 days. Spores were harvestedby suspending and mixing the moldy bran in 120liters of tap water containing 0.01% Tween 80. Branwas removed by centrifugation at low speed in abasket centrifuge (or a Sharples Super-D-Cantor,model P-600). The spore suspension was filteredthrough glass wool, and the spores were collected ina Sharples supercentrifuge (15,000 rev/min). Thespore paste was washed twice with about 100 litersof tap water each time. Average yields of 7 X 1012conidia per kg of bran were obtained. During theseoperations, aseptic conditions were not maintained.

Storage of spores. Spores of A. ochraceus couldbe stored as a paste at -20 C for more than 1 yearor at 4 C for more than 3 months without any detecta-ble loss in hydroxylating activity. Spores of S. affiniswere unstable and showed gradual loss in dehydro-genating activity on storage at 4 C; stored at -20 C,they were stable for at least 8 weeks.

Glucose determination. Glucose was determinedby the Somogyi method, as modified by Nelson (4).

Transformation in 5-liter fermentors. Most of thesteroid conversions were carried out in New Brunswick5-liter fermentors (model FO-5). Frozen spores werethawed, counted in the hemacytometer, diluted withwater or buffer and adjusted to the required concentra-tion with 0.07 M phosphate (pH 5 to 6). For 1 lC-hydroxylation with spores of A. ochraceus, glucosewas added to yield a concentration of 5 g/liter.Steroid, dissolved in a water-miscible solvent or inthe form of a micronized powder (average particlesize, about 5 A) suspended in 0.%l, Tween 80, wasadded slowly below the surface of the liquid, whichwas kept under vigorous agitation. To insure goodtransformation, it was important to obtain a uniformsuspension of the steroid in the aqueous medium.

Each 5-liter fermentor contained 2.4 liters of steroid-spore suspension (2 X 108/ml spores), and, unlessotherwise indicated, the agitator was operated at 300rev/min; 0.6 liter of air per min was introduced intothe fermentor, and the contents were held at a tem-perature of 25 to 28 C. Antifoam agents were usuallynot necessary, although some of the mixtures foamedheavily in the early stages of transformation. A me-chanical foam breaker installed 10 cm above thesurface of the liquid was generally effective in breakingthe foam. With A. ochraceus spores, a substantialamount of spores and steroid were thrown out of thesystem by foam, and it was necessary periodically toreincorporate them into the system. This was achievedby lifting the fermentor and vigorously swirling thecontents. In larger vessels, the problem of foam wasless serious and was easily controlled by occasionaladdition of a solution containing 3% Alkaterge C inmineral oil.

Spore suspensions for 5-liter fermentors wereprepared in sterile water or buffer, and the fermentorswere sterilized before use. However, when transforma-tions were carried out in larger vessels, nonsterilewater was used for preparing buffer and spore suspen-sions. Usually, no contamination was observed forthe first 48 hr of incubation. Bacterial contamination,if any, did not significantly affect steroid conversion,but, as an extra precaution, tetracycline hydrochlorideand neomycin sulfate (4.0 ,ug/ml of each) were added.At the concentrations employed, these antibioticsdid not influence steroid conversions.

Steroid determination. At the end of incubation,the reaction mixture was extracted twice with an equalvolume of ethylene dichloride; the combined extractswere filtered, dried over sodium sulfate, and evapo-rated to dryness under vacuum. The Zaffaroni systemof chromatography (1) was used for determininghydroxylated steroids, as previously reported (9).Compound S and 1-dehydro S in samples were deter-mined quantitatively by a modified spectrophotomet-ric procedure, based on the difference in the rates ofthiosemicarbazone formation by compound S and1-dehydro S (3).More recently, steroid transformation was followed

by thin-layer chromatography (TLC) on silica-gelplates developed in a 1:7 mixture of isopropanol andbenzene, and viewed under a Black-Ray ultraviolet,short-wave lamp, sprayed with 50% p-toluenesulfonicacid in ethyl alcohol or 5% H2SO4 in methanol, andheated at 120 C for 10 min. For quantitative estima-tion, a sample of the dry residue was spotted on aTLC plate; the plate was developed and air-dried;the ultraviolet absorbing bands were scraped off andeluted in methanol-chloroform (1:1); and the eluateswere evaporated to dryness. Results were expressedin percentage of total steroid, or in grams per liter.A gas-liquid chromatography method was also

used to follow steroid transformation (S. N. Sehgal,G. Schilling, K. Singh, and C. Vezina, Intern. Congr.Microbiol., 9th, Moscow, 1966).

RESULTS

Time-course experiments. Figure 1 illustratesthe course of progesterone hydroxylation by

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.

,

20 / N

10

20 30 4050

6070

Incubation (Hr.)

FIG. 1. Time course of progesterone hydroxylationin 5-liter fermentors by spores ofAspergillus ochraceus.Spores 2 X 108/ml; progesterone, 5 glliter; glucose,5 glliter; pH 5.2; volume, 2.4 liters; temperature,28 C; agitation, 300 rev/min; air flow, 0.6 liter/min.*, progesterone; 0, lJa-hydroxyprogesterone; 0,63,JJla-dihydroxyprogesterone; L\, glucose utilized.

spores of A. ochraceus. After an initial lag period,progesterone was transformed to the monohy-droxy derivative and glucose was metabolizedrapidly. On longer incubation, small amounts of6,3,1 a-dihydroxyprogesterone were formed. Theamount of the dihydroxylated compound in thereaction mixture was higher when low concentra-tions (0.5 g/liter) of progesterone were used. Atthe relatively high progesterone concentrationsused in the present studies (2.5 g or more perliter), the percentage of the secondary dihydroxyderivative formed was usually low.Transformation of compound S by A. ochraceus

spores yielded one product, namely 1 la, 17a,21-trihydroxypregn-4-ene-3, 20-dione (epi-cortisol).The rate of transformation (Fig. 2) was slowerthan that of progesterone hydroxylation. Thedifference in the rates of transformation of thesetwo steroids (and other steroids) could be at-tributed to a variety of factors, such as substratespecificity, solubility of steroid, and other physico-chemical factors. Visual observation under amicroscope indicated that, whereas spores ofA. ochraceus adhered to progesterone, they didnot adhere to compound S.As described earlier (8), transformation of

compound S by S. affinis spores yielded mainly17a, 21 - dihydroxypregna - 1, 4 - diene - 3,20 - dione(1-dehydro S), with small amounts of side-chaincleavage products (mainly androsta-1 ,4-diene-3,17-dione). The time course of this transforma-tion in 5-liter fermentors is shown in Fig. 3.

Carrier solvents. For lla-hydroxylation byspores of A. ochraceus, progesterone could beadded as a solution in ethyl alcohol, acetone,propylene glycol, methanol, dimethyl formamide,or dimethyl sulfoxide (DMSO). Acetone was

routinely used because of the higher solubility ofprogesterone in this solvent. Acetone also sup-pressed foam formation in the early stage oftransformation. At acetone concentrations of 2to 4%, an initial lag period of 4 to 6 hr was ob-served in the transformation of progesterone andin glucose utilization; this extended with increas-ing concentrations of acetone (up to 10%).

100

c0U

* 80I.

EL. 600

* 40

o 20a.E0U

0 20 40 60Incubation (Hr.)

80 100

FIG. 2. Time course of compound S hydroxylationby spores ofAspergillus ochraceus. Spores, 2 X 108/ml;compound S, 2.5 glliter; glucose, 5.0 glliter; pH, 5.2;volume, 2.4 liters; temperature, 28 C; agitation, 300rev/min; airflow, 0.6 liter/mim.

100

C

v

;A.

S

E

0

o

C

U

I-

C0

c

E0v

80

60

40

20

20 40 60Incubation (Hr.)

80 100

FIG. 3. I-Dehydrogenation of compound S byspores of Septomyxa affinis. Spores, 2 X 108/ml;compound S, 2.0 glliter; pH 6.0; volume, 2.4 liters;temperature, 28 C; agitation, 300 rev/min; air flow,0.6 liter/nin.

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However, higher concentrations of acetone (10to 14%) did not irreversibly inactivate the system.After an initial lag period, during which most ofthe acetone probably evaporated, the rate ofprogesterone transformation was restored tonormal. Any lag in steroid transformation couldbe almost completely eliminated by the additionof steroid in the form of a micronized powderslurried in 0.1% Tween 80 (final concentration,0.005%). With a progesterone charge of 5 g perliter or less, this mode of addition did not offerany significant advantage over the addition as asolution in acetone (final acetone concentrationin fermentor, 2%), as measured by the overallrate of conversion. At higher steroid concentra-tions, when more acetone was needed for dissolu-tion, the addition of steroid in micronized formoffers the advantage of a reduced lag period.However, the disadvantages of using micronizedpowder were excessive foaming encouraged byTween 80 and some germination of spores after24 to 48 hr of incubation.Compound S showed greater solubility in

methanol than in acetone; therefore, for hydrox-ylation, the former was used as a carrier solvent.However, for 1-dehydrogenation with spores ofS. affinis, dimethylformamide (final concentration1 %) was found to be the solvent of choice.

Incubation temperature. The effect of incubation

TABLE 2. Effect of steroid concentration andincubation temperature on 1-dehydrogeniation ofcompound S by spores of Septomyxa affinis-

Com-pound Scharged

g/liter1.0

1.0

1.0

1.0

2.0

2.0

2.5

Incuba-tiontemp

C

24

28

32.5

38

28

32.5

28

Incuba- Compoundtion S trans-peid formedpeid (A)

hr

4367

4367

4367

4367

4367

4367

4367

7183

8690

8088

<10

6884

5878

6377

Androsta-1, 4-diene-3, 17-dione

(B)b

12

20

traces

15

traces

13

l-De-hydro- S(A - B)

71

70

88

<10

69

78

64

a Spores, 2 X 108/ml; pH, 6.0; volume, 2.4 liters;agitation, 300 rev/min; air flow, 0.6 liter/min.

b The main 17-side-chain cleavage product.

TABLE 1. Effects of incubation temperature onprogesterone hydroxylation by spores of

Aspergillus ochraceusa

Steroids in conversion mixture (%)Incuba-

Incubation tiontemp (C) period IaHdoy6,l-iy(hr) Progesterone pres-Hydrone droiyprgseoeprogesterone

22 24 65 34 148 24 72 4

25 24 40 59 148 8 87 5

28 24 29 70 148 8 88 4

34 24 56 4448 9 90 1

38 24 76 2448 42 55 3

42 24 100 _ -48 100 _

a Progesterone, 5 g/liter; glucose, 5 g/liter;spores, 2 X 108/ml; pH, 5.2; volume, 2.4 liters;agitation, 300 rev/min, air flow, 0.6 liter/min.

temperature on lla-hydroxylation of progester-one by A. ochraceus spores is shown in Table 1.The optimal rate of transformation was obtainedat 28 C. Similar results were obtained for 1 la-hydroxylation of compound S by A. ochraceusspores and 1-dehydrogenation of compound Sby S. affinis spores. At 32 to 34 C, the rate oftransformation of compound S was somewhatslower (Table 2), but the formation of side-chaindegradation products was considerably reduced.Unless otherwise indicated, all transformationswere carried out at 28 C.

Glucose utilization. As reported previously, (9),glucose or any other suitable source of carbonwas essential for 1 a-hydroxylation of steroids inshake flasks by spores of A. ochraceus. The effectof glucose concentration on steroid hydroxylationin 5-liter fermentors is shown in Fig. 4. A minimalconcentration of glucose was necessary to main-tain optimal transformation rates. At a steroidcharge of 5 g/liter, 2.5 g/liter of glucose wassufficient to give complete transformation ofprogesterone at the optimal rate under the stand-ard conditions described in Materials and Meth-ods. The rate of glucose utilization was dependentupon the concentration of spores (Fig. 5) andvirtually independent of the initial glucose con-

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3.2

1 1x

c

24

0

IL

o1.6

20 40 60 80

Incubation (Hr.)

FIG. 4. Effect of glucose concentration on hy-droxylation of progesterone by spores of Aspergillusochraceus. Spores 2 X 108/ml; progesterone, 5.0g/liter; pH, 5.2; volume, 2.4 liters; temperature, 28 C;agitation, 300 rev/min; aeration, 0.6 liter/min. Sym-bols: 0, no glucose added at zero time; 1, at 43 hr2.5 g/liter of glucose added; *, glucose, 1.2 g/liter;0, glucose, 2.5 glliter; A (dotted line), glucose, 5glliter; (solid line), glucose, 10 glliter.

centration. At 2 X 108 spores/ml, about 6 g ofglucose was utilized per fermentor in 24 hr. Whenglucose was completely utilized prior to completetransformation of steroid, the rate of steroid trans-formation was retarded. Therefore, to maintainthe optimal rate of transformation, an excess ofglucose was usually maintained in the reactionmixture. When higher concentrations of steroid(10 to 20 g/liter) were transformed and the trans-formation cycle was longer than 48 to 72 hr,glucose was added periodically.

Spores of S. affinis also metabolize glucoserapidly (8). However, 1-dehydrogenation reac-tion with S. affinis spores was not glucose-requir-ing and glucose was therefore omitted from thereaction mixture.

Effect ofspore concentration. The rate of steroidtransformation increased with increasing con-centration of spores. Effect of spore concentrationon lla-hydroxylation of compound S by A.ochraceus spores is shown in Fig. 5. Similar resultswere obtained for the 1-dehydrogenation reactionwith S. affinis spores (Table 3). In general, aspore concentration of 2 X 108 per ml was foundto be the most convenient in terms of period oftransformation and amount of steroid transformedper unit concentration of spores. In the trans-formation of certain steroids for which the con-

t 1.6la

E

1.2

0.8I?

0.4

2 3 4 5

Spore Concentration per Ml x1X

6

1-0

-

up

la0m

FIG. 5. Effect of spore concentration on compoundS hydroxylation and glucose utilization by spore. ofAspergillus ochraceus. Compound S, 2.5 glliter; pH,5.2; volume, 2.4 liters; temperature, 28 C; agitation,300 rev/min; air flow, 0.6 liter/min; initial glucoseconcentration per liter was 5 g at 108 to 2 X 108/mIspore concentration, 10 g at 4 X 108/ml spore concen-tration, and 15 g at 6 X 108/ml spore concentration.Epi-cortisol formed in A, 21 hr; B, 33 hr; C, 45 hr.Glucose utilized in A1, 21 hr; B1, 33 hr; C1, 45 hr.

TABLE 3. Effect of spore concentration on1-dehydrogenation of compound S by

spores of Septomyxa affinis

Spores/ml Incubation period Compound Stransformed

hr %I X 108 41 31

65 43

2 X 108 41 5065 66

4 X 108 41 6565 88

Compound S, 1.0 g/liter; volume, 2.4 liters;incubation temperature, 28 C; pH 6.0; agitation,300 rev/min; air flow, 0.6 liter/min.

version rates were low, a spore concentration of4 X 108 to 5 x 108 per ml was used.

Steroid charge. In the presence of adequateglucose concentration, spores of A. ochraceus(2 x 108 per ml) completely transformed 5.0 g ofprogesterone per liter in 40 to 50 hr (Table 4)and 2.0 to 2.5 g of compound S per liter in 50 to70 hr. Similarly, with spores of S. affinis 1.5 to2.0 g of compound S per liter was dehydrogenatedin 48 to 72 hr. The amount of steroid transformedcould be increased by increasing the concentra-tion of spores.

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TABLE 4. Transformation ofprogesterone by spores of Aspergillus ochraceus"

Initial pH

5.2

6.0

6.0

6.0

6.0

Glucose added

g/liter5.0

7.5

7.5

7.5

10(+10 g after70 hr)

Progesteronecharge

glliter5.0

10.0

10

10

20b

Incubationperiod

hr

2347

237194

4871

4871

4367139

Steroids in conversion mixture

Progesterone

30

7925

23

10

7358

lia-Hydroxyprogesterone

6997

217090

7694

8285

274295

60, 1la-Dihydroxyprogesterone

1

3

5

10

1

6

815

5

a Incubation temperature, 28 C; progesterone added as a solution in acetone; acetone concentration,4%; volume, 2.4 liters; agitation, 300 rev/min; air flow, 0.6 liter/min.

bAcetone concentration, 7%.

Effect ofpH. As reported earlier (9), in shakeflask experiments, the 1la-hydroxylation ofprogesterone by spores of A. ochraceus was unaf-fected by pH in the range from 4 to 8.5. The1-dehydrogenation by spores of S. affinis showeda pH optimum between 6.7 and 7.5 (8). However,at this pH, excessive foaming was observed in5-liter fermentor experiments. Therefore, 0.07 M

phosphate buffers atpH 5.2 for ll1a-hydroxylationand pH 6.0 for 1-dehydrogenation were routinelyused.

Aeration and agitation. Above a certain mini-mal level, any further increase in agitation andair flow did not enhance the rate of steroid trans-formation by spores of A. ochraceus or S. affinis.With A. ochraceus spores, it was difficult to ascer-tain the optimal levels of aeration and agitationbecause excessive foaming occurred at higherlevels of aeration and agitation; spores andsteroids were quickly thrown out of the system.However, limited experiments indicated that,below an agitation rate of 150 rev/min (air flow,0.6 liter/min), the rate of transformation was low.Agitation only (300 rev/min without aeration) oraeration only (1 liter/min without agitation)resulted in low steroid transformation. Routinely,an agitation rate of 300 rev/min and an air flowof 0.6 liter/min were used in 5-liter fermentors.Variation in agitation rate from 300 to 500 rev/min and increase in air flow from 0.6 to 1.2liter/min did not significantly influence steroid

hydroxylation. Transformation was also unaf-fected by increasing the oxygen tension in the air.With S. affinis spores, foaming was not a serious

problem, and a range of agitation (150 to 750rev/min) and aeration (0.6 to 3.0 liter/min) rateswere tried. As with S. ochraceus, increases inaeration-agitation above 300 rev/min and airflow of 0.6 liter/min did not significantly enhancethe rate of transformation. Unless otherwisementioned, the agitation rate was 300 rev/minand aeration was 0.6 liter/min.

Transformation of steroids in large vessels. Thepossibility of transforming steroids under non-sterile conditions was investigated in stainless-steel vessels ranging in size from 30 liters to 200gal and equipped with facilities for agitation andaeration. Progesterone, compound S, and 6a-fluoro-16a, 17a-dihydroxypregn-4-ene-3, 20-dionewere transformed in quantities up to 1 kg at atime. The transformation of 6a-fluoro-16a,17a-dihydroxypregn-4-ene-3, 20-dione to the cor-responding lla-hydroxylated derivative (2) in a200-gal stainless-steel vessel is described below.A stainless-steel extraction tank (total capacity,

200 gal) was suitably modified as shown in Fig. 6.The air inlet, which served to sparge air in thetank during fermentation, was also used forpassing the steam required for cleaning the tankat the end of each run. A turbine impeller fixedto the lower end of the drive shaft provided goodmixing and incorporation of air. The upper

Spores/ml

2 X 108

2 X 108

4 X 10I

6 X 108

2 X 108

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FIG. 6. Diagram of 200-gal stainlessusedfor transformation of steroids.

1001

c

0

aE0

c

3-

0

0

IL

801

20

0 20 40 60

Incubation (Hr.)

FIG. 7. Transformation of 6a-fluoro-droxypregn-4-ene-3,20-dione by spores oJochraceus. Spores 5 X 108/ml in 0.07 AipH 5.9; steroid, I glliter; glucose, 5 g/li24-hr intervals; temperature, 26 to 2810, 50-gal fermentor; volume of reaction.liters; agitation, 1,750 rev/min; airflow, 1

0, 5-liter fermentor; volume, 2.4 liters; a

rev/min; air flow, 0.6 liter/mim; *, 2Avolume of reaction mixture, 400 liters; a

to 300 rev/min; airflow, 52 liters/min.

impeller fixed slightly below the sur

liquid helped to reincorporate the Itaining spores-steroid) into the systema slight vortex. The blade above the

Drve Shaft the liquid was used as a precautionary measure tohelp break the foam. A 3-hp Lightnin mixer was

Temperature used, and a speed of 260 to 300 rev/min was obContrd Coil tained at a liquid volume of 400 liters.

Buffer and spore suspensions were prepared inBaffles nonsterile tap water and the conditions for trans--Level of Liqaid formation were as follows: spores, 5 X 108/ml;

steroid, 400 g (in 8 liters of acetone); 0.07 Mphosphate buffer, pH 5.9; agitation, 260 to 300rev/min; air, 52 liters/min (dissolved oxygen asmeasured with a Beckman model 777 oxygenanalyzer was more than 2%); temperature, 26 to28 C; and volume, 400 liters. Tetracycline hydro-chloride and neomycin sulfate (4 Ag of each per

Turbine impellef ml) were added to prevent bacterial contamina-Spef Ring tion. Foaming was controlled by the occasional

addition of 3% Alkaterge C in mineral oil. Thetime course of transformation is shown in Fig. 7.It will be noted that the rate of transformationwas better than in a 50-gal vessel and in 5-literfermentors. In 15 runs, the transformation prod-

s-steel tank uct was consistently obtained in 85 to 98% yield,and 5 kg of crystalline product was obtained.

DISCUSSION

Results presented above clearly indicate thecommercial feasibility of steroid transformationwith spores of fungi which sporulate abundantly.Preparation of spores such as those of A. ochra-ceus could be easily scaled up by increasing thenumber of trays or other containers used to con-tain barley or bran. For harvesting the spores,equipment such as the Sharples Super D-Cantorcould be used for separating spores from bran ona large scale. The transformation process itselfcould easily be scaled up by using modificationsof relatively simple pilot-plant equipment, asaseptic conditions are not essential for trans-formation. Moreover, the transformation medium

80 100 used is simple (buffer with or without glucose)and the spores do not germinate. Consequently,the extraction of the transformation products

16,17a-dihy- offers fewer problems than those existing in thef Aspergillus conventional process where more complex mediaIf phosphate;iter added at are used for fermentation.C. Symbols: Spores can be stored in a freezer for a con-mixture, 150 siderable length of time without significant lossS5 liters/min; in activity. Therefore, a bank of spores could bergitation, 300 maintained for carrying out steroidal transforma-gitation, 260 tion on a short notice.

Industrial transformation of steroids by sporesof fungi appears to be a feasible process and

face of the offers some attractive features worthy of consider-foam (con- ation, especially if one does not have, or does notby creating want to invest in, large-scale fermentation equip-surface of ment.

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400

ACKNOWLEDGMENTSWe are grateful to W. F. Bourchier for conducting

many 5-liter fermentor experiments. Technical assist-ance of Rene Saucier and Paul Duchesne is gratefullyacknowledged.

LITERATURE CITED

1. BURTON, R. B., A. ZAFFARONI, AND E. H. KEUT-MAN. 1951. Paper chromatography of steroids.II. Corticosteroids and related compounds.J. Biol. Chem. 188:763-771.

2. DEGHENGHI, R., M. BOULERICE, J. G. ROCHEFORT,S. N. SEHGAL, AND D. J. MARSHALL. 1966.Antiinflammatory A4-pregnenolone derivatives.J. Med. Pharm. Chem. 9:513-516.

3. FoRus, A. A. 1959. Spectrophotometric deter-mination of residual A4-3-ketosteroids in bulkA1,4-3-ketosteroids. Differential rates of thio-semicarbazone formation. Anal. Chem. 31 :913-915.

4. NELSON, N. 1944. A photometric adaptation ofthe Somogyi method for the determination ofglucose. J. Biol. Chem. 153:375-380.

APPL. MIcRoBIoL.

5. ScHLEG, M. C., AND S. G. KN1GHT. 1962. Hy-droxylation of progesterone by conidia fromAspergillus ochraceus. Mycologia 54:317-319.

6. SEHGAL, S. N., K. SINGH, AND C. VEZINA. 1963.Transformation of Reichstein's compound Swith Didymella lycopersici. Steroids 2:93-97.

7. SINGH, K., S. N. SEHGAL, AND C. VEZINA. 1963.C-1-Dehydrogenation of steroids by sporesof Septomyxa affinis. Steroids 2:513-520.

8. SINGH, K., S. N. SEHGAL, AND C. VEZINA. 1965.Transformation of Reichstein's compound Sand oxidation of carbohydrates by spores ofSeptomyxa affinis. Can. J. Microbiol. 11:351-364.

9. VEZINA, C., S. N. SEHGAL, AND K. SINGH. 1963.Transformation of steroids by spores of micro-organisms. I. Hydroxylation of progesteroneby conidia of Aspergillus ochraceus. Appl.Microbiol. 11:50-57.

10. VEZINA, C., S. N. SEHGAL, AND K. SINGH. 1965.Sporulation of filamentous fungi in submergedculture. Mycologia 57:722-736.

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