World Journal of Microbiology and Biotechnology, 8, 399-401 Steroid transformation glass air-lift fermenter in a laboratory-scale P.K. Roy,* A.W. Khan, J. Kumar, S.D.K. Chopra and S.K. Basu A glass airlift fermenter, 1550 ml working volume, was used for microbiological transformation of phytosterols. A gas hold-up of 1.6% was observed with the lowest superficial gas velocity (1.89 cm/s). The volumetric liquid circulation rate remained relatively constant (0.21 l/s to 0.23 l/s) for superficial gas velocity values up to 11.37 cm/s. A 72% conversion of sitosterol to 1,4-androstadiene-3,17-dione was obtained. Key words: Air-lift fermenter, biotransformation, Mycobacterium, phytosterols, steroids The transfer of oxygen from gas phase to aqueous medium is often critical in aerobic fermentation. Modifications of fermenter configurations have been made to improve the performance and efficiency of mechanically agitated reactors in various biological processes (Kennedy 1984). However, the overall requirement of energy input to attain adequate oxygen transfer is high (Malfait et al. 1981; Moresi 1981). An air-lift fermenter can, though, overcome many of the limitations of conventional stirred tank reactors (Mercer i981). In this report we describe the bio-conversion of sitosterol to androsta-l,4-diene-3,17-dione, which was efficiently performed by Mycobacterium sp. NRRL B 3683 in a glass air-lift fermenter designed in this laboratory. Materials and Methods Culture Mycobacterium sp, NRRL B 3683 was maintained on 1.4% (w/v) agar slants containing (g/l): NaCl, 1.25; KH2PO4, 0.75; Na2HPO 4, 1.8; (NH4)2SO4, 10; MgSO4.7H20, 0.05; dextrose, 10; sitosterol, I0. For steroid transformation it was grown at 28~ in maintenance medium without agar or sitosterol. For the induction of side-chain cleaving enzymes, Img sitosterol dissolved in 0.25 ml of N,N'-dimethylformamide (DMF) was added per 100 ml of 24-h grown culture and after about 24 h of induction the sitosterol (0.5 mg/ml) dissolved in DMF was added to the fermenter for side-chain cleavage reaction. P.K. Roy, A.W. Khan, J. Kumar, S.D.K. Chopra and S.K. Basu are with the Department of Fermentation Technology, Central Drug Research Institute, Lucknow-226 001, India. *Corresponding author. 9 1992 Rapid Communications of Oxford Ltd Steroid Analysis and Identification The steroid mixture obtained from the culture broth (culture broth extracted three times with an equal volume of ethyl acetate) was chromatographed on silica gel GF254 with chloroform:ether (lO:l,v/v) as solvent. The androsta-l,4- diene-3,17-dione (ADD) corresponding to an authentic sample (R~ value 0.46) was located under U.V. and eluted from the silica gel plates. The concentration of ADD was determined by high performance liquid chromatography by the method of Aihara et al. (1986). The residual sitosterol in the culture broth extract was estimated by the Libberman Burchard reaction (Katayama et al. 1974). Field desorption mass spectrum (FDMS) of the steroid mixture from the culture broth was recorded on a JEOL OIS 6-2 mass spectrometer using benzonitrile-activated tungsten wire emitters. A number of spectra were recorded at various emitter currents (8 to 18 mA) and summated. The summated FDMS spectra indicated molecular ions (M +) at m/z 414, 284 and 282. The compounds corresponding to these molecular weights are sitosterol, ADD and androst-4-ene- 3,17-dione (AD). The major component in the transformed products was ADD. This compound was further identified and the data obtained were as follows: R~ value (0.46), melting point (141~ mixed melting point (no depression). The identity was further confirmed by the superimposable spectra of the authentic sample. Air-lift Fermenter The glass fermenter of 2.11capacity was designed for laboratory-scale work (Figure 1). The fermenter consists of three basic sections as reported by Mercer (1981) and Malfait et al. (I981). In addition, a conical structure was added on the top of the downcomer section and served as a foam breaker. For continuous fermentation experiments the nutrients were fed through a pump from the T-joint on the top of the air sparger (A, Figure 1) and the reaction volume in the reactor was maintained by continuous removal of fermentation fluid through a pump from World Journal of Microbiology and Biotechnology. Vol 8, 1992 399