Purification, Stability, and Mineralization of 3-Hydroxy-2- Formylbenzothiophene, a Metabolite of Dibenzothiophene

Abstract

3-Hydroxy-2-formylbenzothiophene (HFBT) is a metabolite found in many bacterial cultures that degrade dibenzothiophene (DBT) via the Kodama pathway. The fate of HFBT in cultures and in the environment is unknown. In this study, HFBT was produced by a DBT-degrading bacterium and purified by sublimation. When stored in organic solvent or as a crystal, the HFBT slowly decomposed, yielding colored products. Two of these were identified as thioindigo and cis-thioindigo. The supernatant of the DBT-degrading culture contained thioindigo, which has not been reported previously as a product of DBT biodegradation. In mineral salts medium, HFBT was sufficiently stable to allow biodegradation studies with a mixed microbial culture over a 3-to 4-week period. High-performance liquid chromatography analyses showed that HFBT was removed from the medium. 2-Mercaptophenylglyoxalate, detected as benzothiophene-2,3-dione, was found in an HFBT-degrading mixed culture, and the former appears to be a metabolite of HFBT. This mixed culture also mineralized HFBT to CO 2 .For more than 4 decades, dibenzothiophene (DBT) has been the model compound for studying the biodegradation or biodesulfurization of sulfur heterocycles. 3-Hydroxy-2-formylbenzothiophene (HFBT) has often been found as a metabolite of bacterial degradation of DBT (1,9,11,12,19). Yamada et al. (27) reported that two Pseudomonas isolates produced a metabolite that upon purification yielded yellow crystal with a melting point of 107°C. Kodama et al. (11) conclusively identified this metabolite as HFBT (Fig. 1A). Figure 1B shows that two molecules of HFBT can undergo a spontaneous abiotic reaction forming a red dyestuff, 3-oxo-(3Ј-hydroxythianaphthenyl-2-methylene)-dihydrothianaphthene (compound III) (11, 12). Finkelstein et al. (8) described a different product of two molecules of HFBT spontaneously reacting to give compound IV (Fig. 1B). However, they based this tentative identification solely on the interpretation of a mass spectrum.No biodegradation products of HFBT have been conclusively identified, but compounds that may originate from HFBT have been detected in cultures biodegrading DBT. Examples of these are shown in Fig. 2. Bohonos et al. (1) found 3-hydroxybenzothiophene (compound V) and benzothiophene-2,3-dione (compound VIII). Eaton and Nitterauer (4) showed that when acidic conditions are used to extract culture supernatants containing 2-mercaptophenylglyoxalate (compound VII), an acid-catalyzed dehydration occurs to form compound VIII. Finkelstein et al. (8) detected 2,3-dihydroxybenzothiophene (compound VI) and 2-mercaptobenzoic acid (or thiosalicylic acid; compound IX). However, no one has conclusively shown that these metabolites result from further catabolism of HFBT and are not products of a concurrent metabolic pathway.To date, purified HFBT has not been used as a carbon and energy source for biodegradation studies. The only reported investigation into the biodegradation of HFBT is by Mormile and Atlas (21) who used filter-sterilized culture supernatant...

Identification of Disulfides from the Biodegradation of Dibenzothiophene

“…Previous studies in our laboratory (9,28) failed to detect these disulfides. The procedures used included adjusting the pH to approximately 2, extracting with dichloromethane, drying the extract by filtering it through anhydrous sodium sulfate, and analyzing the derivatized extract by GC-MS with an upper column temperature of 250°C.…”

“…This study was done with a culture of Pseudomonas sp. strain BT1d that was used previously to produce HFBT, via the Kodama pathway, while growing on dibenzothiophene as its sole carbon and energy source (9). This isolate was inoculated into a 2-liter Erlenmeyer flask that contained 1.5 liters of mineral salts medium (31) and 2 g of dibenzothiophene (most of which remained as crystals).…”

“…With the exception of the initial reactions which transform dibenzothiophene to HFBT, most of the other reactions are abiotic. Thioindigo, which was recently found in the culture extract of strain BT1d degrading dibenzothiophene (9), eluted from the GC column after the disulfides (Fig. 5) and is another example of a product which has a much higher molecular mass (296 Da) than the substrate (184 Da).…”

Aerobic Biodegradation of 2,2′-Dithiodibenzoic Acid Produced from Dibenzothiophene Metabolites

“…The GC-MS method was described previously (7). A spot test with cadmium acetate was used to detect H 2 S in some culture supernatants.…”

“…There have been no reports of the complete mineralization of DBT by bacteria using the Kodama pathway. However, HFBT can be mineralized (7), and benzothiophene-2,3-dione was detected in acidified HFBT-degrading cultures (7), and in DBT-degrading cultures (5,34). Bressler and Fedorak (8) proposed an abiotic mechanism for the formation of benzothiophene-2,3-dione from HFBT.…”