CURRENT MICROBIOLOGY Vol. 21 (1990), pp. 229-231 Current Microbiology © Springer-Verlag New York Inc. 1990 Oxidation of Dibenzothiophene by Cunninghamella elegans Donald L. Crawford and Rajinder K. Gupta Department of Bacteriology and Biochemistry, Institute for Molecular and Agricultural Genetic Engineering, University of Idaho, Moscow, Idaho, USA Abstract. The metabolism of dibenzothiophene (DBT) by the catabolically versatile fungus Cunninghamella elegans (ATCC 36112) was investigated. This fungus has been previously shown to metabolize polycyclic aromatic hydrocarbons including nitropolycyclic aromatics, but has not been shown to metabolize DBT. The results show that C. elegans oxidizes DBT to its corresponding DBT-5 oxide and then to DBT-sulfone, without the formation of any biphenyl intermediates. Dibenzothiophene (i)BT) and its derivatives have been characterized as a representative group of the organic sulfur compounds in fossil fuels. A growing interest in the pre-combustion desulfurization of fos- sil fuels has attracted considerable interest in micro- bial processes [5, 7, 16]. A number of research groups have reported the isolation of microorgan- isms capable of metabolizing DBT including Acinet- obacter [12], Arthrobacter [9], Be(jerinckia [4, 11], Bacillus [8], Pseudomonas [10, 13-15], Rhizobium [12], Rhodococcus [8], and Sulfolobus [6] species. Most of the aerobic organisms have been shown to degrade DBT to 3-hydroxy-2-formyl benzothio- phene [10, 11, 13, 14]. An alternate pathway utilized by some is the oxidation of DBT to DBT-5-oxide [11, 15] and then to DBT-sulfone [14, 15]. Recently, Kilbane and Beilaga have reported the degradation of DBT to 2-hydroxybiphenyl and biphenyl deriva- tives by Bacillus sphaericus and Rhodococcus rho- dochrous [8]. As a part of our studies of microbial transforma- tion of coals, we have investigated the metabolism of dibenzothiophene by the fungus Cunninghamella elegans (ATCC #36112). This fungus has been re- ported to metabolize a number of polycyclic aro- matic hydrocarbons (PAHs) including nitropolycy- clic aromatic hydrocarbons [1-3]. The results of the current investigation show that this strain also oxi- dizes DBT, first to its corresponding DBT-5-oxide and then to DBT-sulfone, without the formation of any biphenyl derivatives. Paper from the Idaho Agricultural Experiment Station. Address reprint requests to: Dr. Donald L. Crawford, Department Agricultural Genetic Engineering, University of Idaho, Moscow, ID Materials and Methods Chemicals. Dibenzothiophene, dibenzothiophene-5-oxide and di- benzothiophene-sulfone were purchased from Aldrich Chemical Co. (Milwaukee, Wisconsin). Microorganism and culture conditions. Stock culture of the fungus Cunninghamella elegans (ATCC-36112) was obtained from Dr. Carl E. Cerniglia (U.S. FDA National Center for Toxicological Research, Jefferson, Arkansas). Stock cultures were maintained at 4°C on Sabouraud dextrose agar slants. An Erlenmeyer flask (1 liter) containing 200 ml of Sabouraud Dextrose Broth (SDB; Difco Laboratories, Detroit, Michigan) and 20 mg of dibenzothio- phene (DBT) dissolved in 1 ml of dimethylformamide was inocu- lated with a 10% (vol/vol) inoculum with the mycelial mass from a 20-ml seed culture. The seed culture was obtained by growing cells from a slant inoculum for 48 h in SDB at 28°-30°C with shaking on a rotary shaker (150 rpm). DBT and DBT-metabolites extraction. To estimate the extract- able amount of DBT and its metaholites present in these cultures, 5 ml of the broth was withdrawn at 0, 24, 36, 48, 72, and 96 h and again after 7 days' incubation. The sample was acidified with dilute HC1 to pH 2 and extracted with 4 ml of ethyl acetate. The ethyl acetate extract (2 ml) was evaporated under vacuum, and the residue was dissolved in 1 ml of methanol for analysis by reverse-phase high performance liquid chromatography (HPLC). Two additional sets of experiments were conducted with DBT-5- oxide and DBT-sulfone in place of DBT as substrate for the fungus. After 7 days, the fungal mycelium (30 g wet weight, or 1.2 g dry weight) was collected, extracted, and the extract ana- lyzed for DBT, DBT-5-oxide, and DBT-sulfone. HPLC analysis of DBT and DBT-oxidation products. DBT and DBT-oxidation products were analyzed on a Hewlett-Packard 1090A, high performance liquid chromatograph equipped with an HP-1040A diode-array detector with a 5-/~m ODS column (Alltech Assoc., Deerfield, Illinois; 25 cm × 4.6 mm, i.d.). A linear gradi- ent of methanol-water (50 : 50 to 95 : 5 in 18 rain, then back to of Bacteriology and Biochemistry, Institute for Molecular and 83843, USA.