Energy & Fuels 1993, 7 , 159-164
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Characterization of Intermediates in the Microbial Desulfurization of Dibenzothiophene Edwin S. Olson,* Daniel C. Stanley, and John R. Gallagher Energy and Environmental Research Center, University of North Dakota, Box 8213, University Station, Grand Forks, North Dakota 58202 Received September 22, 1992. Revised Manuscript Received October 29, 1992
The desulfurization of dibenzothiophene by Rhodococcus rhodochrous IGTSS gave several microbial products which were isolated by extraction from the culture media. Two key compounds that were proposed as intermediates in the 4 s pathway, for dibenzothiophene desulfurization, are 2'hydroxybiphenyl-2-sulfinicacid and 2'-hydroxybiphenyl-2-sulfonic acid. By utilizing a gas chromatographic/Fourier transform infrared/mass spectrometry (GC/FTIR/MS)system, these compounds were identified in the sultine form, dibenz[c,el[1,2loxathiin 6-oxide, and sultone form, dibenz[c,e][1,2loxathiin 6,6-dioxide, respectively. High-confidence identifications of these metabolites were provided by matching spectral data obtained from the components in extracts with that obtained for synthesized standards. Introduction
The microbial removal of sulfur from petroleum and coal has interested scientists for a number of years. Dibenzothiophene (DBT) was often used as a model compound to represent the organic sulfur in the fossil fuel matrix. The objectives of many of these model compound studies were to screen organisms for their effectiveness in converting the heterocyclic sulfur compound to inorganic sulfur (sulfate or sulfite) and to elucidate the metabolic pathways by which the sulfur is excised. This research has defined two distinct oxidative routes for the microbial degradation of DBT. The Kodama pathway' involves oxidation and cleavage of one of the benzo rings to benzothiophene derivatives and further oxidation to sulfate. Several of the intermediates of the pathway were isolated,' and a large number of other metabolites resulting from benzo cleavage were recently characterized.2 The alternative 4s pathway involves initial oxidation of the sulfur and cleavage of the carbon sulfur bonds resulting in the formation of 2-hydroxybiphenyl,3s42,2'-dihydroxybiphenyl,4q5 or benzoate.6 The intermediates in the pathway were suggested to be DBT 5-oxide (sulfoxide) (I), DBT 5,5-dioxide (sulfone) (2), and 2'-hydroxybiphenyl2-sulfonic acid (3),3but no evidence for the existence of the sulfonic acid in the culture media could be ~ b t a i n e d . ~ The 4s pathway is more interesting for utilization in desulfurization of fossil fuels because carbon is not oxidized and lost as carbon dioxide. (1)Kodama, K.; Nakatani, S.; Umehara, K.; Shimizu, K.; Minoda, Y.; Yamada, K. Agro. B i d . Chem. 1970,34, 1320-1324. (2) Gallagher, J. R. Third International Symposium on the Biological Processes of Coal, May 4-7, 1992, Clearwater Beach, FL. Abstract No. 2-4. (3) Kilbane, J. J. Bioprocess Coals Workshop-111,Proc., Aug. 25-17 1988, Tysons Corners, V A 1988, 156-166. (4) ARCTECH Enhancementof Microbial Coal Desulfurization. Final Report, DOE/PC/79876-T1, Nov. 1990. ( 5 ) Krawiec, S. Bioprocess Coals Workshop-III, Proc., Aug. 15-1 7, 1988, Tysons Corners, V A 1988, 189-198. (6) van Afferden, M.; Schacht, S.; Klein, J.; Truper, H. G. Arch. Microbiol. 1990, 153, 324-328. (7) Bielaga, B. A.; Kilbane, J. J. Molecular Biological Enhancement of Coal Biodesulfurization. Sixth Quarter Report for Feb. to April 1990, DOE/PC/88891-T6, April 1990.
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In work intended to verify the results of cloning of 4s pathway genes, several metabolites derived from DBT were rountinely isolated from culture media to which DBT was added. Identification and quantitative analysis of DBT &oxide, DBT 5,5-dioxide, and 2-hydroxybiphenyl by GC retention times and GC/MS were routinely carried out. In addition, we carefully examined the products from DBT degradation by Rhodococcus rhodochrous (IGTS8) and derivative organisms by using a serial GC/FTIR/MS system, and in this paper we present the chromatographic and spectral evidence for the presence of the 2'-hydroxybiphenyl-2-sulfonic acid as well as very small amounts of the related sulfinic acid. Experimental Section Cultures. Rhodococcusrhodochrous IGTS8 (AmericanType Culture Collection No. 53968) was maintained on Luria-Bertani media.8 Media for growth with dibenzothiophene consisted of K2HP04, 4 g; NaHZPOd, 4 g; NH4C1, 2 g; MgC12.6Hz0, 0.2 g; CaCl~~2H20,O.OOl g; FeC&.6H20,0.001g/L distilled and deionized water. The final pH of this media was 6.5. Glycerol was added as a source of carbon and energy at 0.2 5% (w/v). For testing with DBT, cells were pregrown at 30 "C with shaking into late stationary phase (ca. 48 h), pelleted at 4000g for 15 min, and suspended in the same media without glycerol but with DBT crystals added at 0.1 % (w/v). The final cell concentration was approximately 1.5 au at 550 nm. The cells plus DBT were incubated at 30 "C with shaking for 48 h and then stored at -20 "C until analyzed. Sample Workup. The samples were centrifuged to remove suspended insoluble solid materials. The decantates were (8) Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: New York, 1982.
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acidified to pH 1 with hydrochloric acid and extracted with 50 mL of ethyl acetate (three times). The solids that were removed by centrifugation were also extracted with ethyl acetate. The ethyl acetate extracts were dried with anhydrous calcium chloride and filtered, and the ethyl acetate was removed by rotary evaporation. A known amount of octadecane in chloroform solution was added to each sample as an internal standard and the samples analyzed by gas chromatography/flame ionization detection (GC/FID) and GC/FTIR/MS. Methylations of acidic components in the ethyl acetate extracts were conducted by adding ether solutions of diazomethane to portions of the extracted material. The aqueous layers left over from the extraction were rotary evaporated to dryness and were also treated with ether solutions of diazomethane. Instrumentation. The GC/FTIR/MS analyseswere obtained with a serial interfaced GC/FTIR/MS system as previously de~cribed.~JO The Finnigan ion trap (ITD 800) was operated with the automatic gain control (AGC) on, but mass spectra of the polar oxysulfur species exhibited small ( m+ 1)+ions due to self-chemical ionization and protonation of the basic oxygens. The gas chromatography for these analyses was conducted with a 30 m X 0.32 mm DB5 column (l.O-rm phase thickness) with a 2.0 mL/min helium carrier flow rate measured at 330 OC. Oncolumn injections were utilized for sample introduction, since the sulfoxidesand sulfonesare thermally unstable and decompose in split or splitless injectors." The oven temperature program was as follows: 40 "C injection, 20 deg/min to 80 "C, 5 deg/min to 200 OC, 10 deg/min to 330 OC, hold 5 min. GC/FID analyses were performed with a HP 5880A with similar column, flow rate, and oven temperature program. Materials. Dibenzothiophene was purchased from Kodak or Fluka; the material from both sources contained a small amount of biphenyl impurity. Dibenzothiophene 5-oxide (sulfoxide)was synthesized by oxidation of DBT with tert-butyl hypochlorite according to the procedure of Johnson and McCants.12 Dibenzothiophene 5,bdioxide (sulfone) was purchased from Pfalz and Bauer and used without further purification. The dibenz[c,e][1,210xathiin &oxide (sultine) (6) and dibenz[c,e] [ 1,210xathiin 6,6-dioxide (sultone) (5) were synthesized from P-hydroxybiphenyl (Aldrich) by the methods of Hanson and Kemp.13 Diazomethane was prepared from diazald (Aldrich) by the procedure of V~gel,~' except that methanol was used instead of ethanol.
Olson et al.
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Figure 1. Reconstructed Gram-Schmidt (bottom) and totalion (top) chromatograms of ethyl acetate-extracted material. Numbered peaks are identified as follows: (1)biphenyl, (2) hydroxybiphenyl, (3) DBT, (4) phthalate plasticizer, ( 5 ) sultine, (6) sultone, (7) DBT 5-oxide. Scheme I
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Rssults In order to understand the pathway(@ that result in cleaving the carbon-sulfur bonds and lead to the formation of 2-hydroxybiphenyl, the identification and analysis of several compoundsproduced by Rhodococcus rhodocrow IGTSS and its modifications were desired. Reconstructed Gram-Schmidt (infrared) and total ion (mass spectrometry) chromatograms of the products extracted by ethyl acetate from an IGTS8 culture are shown in Figure 1. In addition to large amounts of the DBT substrate and the major product 2-hydroxybiphenyl, several other components appeared in the chromatograms. Some of these minor Components in the extract could be intermediates (9) Diehl, J. W.; Kleinjan, S. B.; Oleon, E. S. A Gas Chromatographic/ Fourier Infrared Spectroscopy/Mass Spectrometry/Atomic Emission Detection/Flame Ionization Detection System, Spectrosc. Int. J . 1990, 8, 43-72. (10) Olson, E. S.; Diehl, J. W. Serially Interfaced Gas Chrometograph/ Fourier Transform Infrared/IonTrap Mam Spectrometer System. Anal. Chem. 1987,59, 443-8. (11) Vignier, V.; Berthou, F.; Picart, D. J.High Resolut. Chromatogr. Chromatogr. Commun. 198S, 6, 661-6f35. (12) Johnson, C. R.; McCanta, D., Jr. J. Am. Chem. Soc. 1965, 87, 1109-1114. (13) Hanson, C.; Kemp, D. S. J . Org. Chem. 1981,46,5441-5443. (14) Vogel, A. I. Textbook ofPractical Chemistry, 3rd ed.; Longmans, Green, and Co: London, 1956; p 971.
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in the pathway for desulfurization of DBT to 2-hydroxybiphenyl, and other components could be compounds that are not further metabolized. The suspected metabolites or intermediates were DBT 5-oxide,DBT 6,5-dioxide, 2'-hydroxybiphenyl-2-sulfonicacid (31, and 2'-hydroxybiphenyl-2-sulfiiicacid (4). These strong acids are present as the sulfonate and sulfiinate ions, respectively, in the aqueous culture medium, but the 2'-hydroxybiphenyl-2sulfonic acid and the sulfiinicacid will cyclize to the sultone, dibenz[c,e][1,210xathiin 6,fMioxide (51, and sultine, dibenz[c,el[1,210xathiin6-oxide (6), respectively, under the acidic conditions used in the workup (Schemes I and 11). The possible biological degradation of the DBTto the sulfinate is of interest, because it is a product of the thermal degradation of DBT sulfone in the presence of KOH/crown ether.15 (15) Squires, T. C.; Venier, C. G.;Hodgson, B. A.; Chang, L. W. J. Org. Chem. 1981,46, 2372-2376.
Energy t Fuels, Vol. 7, No. 1, 1993 161
Microbial Desulfurization of Dibenzothiophene
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Figure 3. Infrared spectrum of sultine (peak 5 ) from GC/FTIR/ MS of Rhodococcus extract. Figure 2. ITD mass spectrum of sultine (peak 5 ) from GC/ FTIR/MS of Rhodococcus product extract. Scheme I11 -so
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Figure 4. Infrared spectrum of sultine from GC/FTIR of standard.
The sultine and sultone were identified in the acidified extracts of culture media by spectral matching in the GC/ FTIRlMS analysis. High-confidencedeterminations can be obtained by the integrated GC and spectral analyses provided by this instrumentati~n.~J~ Mass spectra, FTIR spectra, and retention times for the components extracted from the media matched those of pure synthetic standards prepared by the procedure described by Hanson and Kemp. The amounts of the sultine were very small in all of the products examined, sometimes below the detection limit for the FTIR. Detailed descriptions of the mass spectra and FTIR spectra are presented here so that the reader will be able to utilize the information to not only unequivocally recognize thesc DBT metabolites but also to identify other similar oxidation products that might be produced in various other organisms or from variants of oxidative enzymes used in aromatic sulfur metabolism. The ion trap mass spectrum (Figure 2) of the sultine from the gas chromatogram of the Rhodococcus product extract matched the standard and was essentially the same as the spectrum reported by Hanson and Kemp." The mass spectrum of the sultine may be easily confused with that of the DBT dioxide (Figure 9),since the two spectra exhibit the same fragment ions at mlz 188,187,168,160, and 115,as well as the molecular ion at mlz 216. These fragmellt ions observed in the spectra of either compound are consistent with the loss of CO, CO + H, SO, and 2CO from the molecular ions to give ions corresponding to the peaks at mlz 188,187,168, and 160,respectively (Scheme 111). The reason for the similarity is the rearrangement of the DBT dioxide molecular ion to the sultine molecular ion, which was postulated in previous reports to explain the losses of CO observed in the DBT dioxide fragmentation.16J7Although the molecular ion of the DBT dioxide
appears to be more intense relative to the other peaks in the spectrum shown in the figure, this is not always the case and is not a reliable distinction between the sultine and the DBT dioxide. The best way to distinguish the two components on the basis of the mass spectra is the ratio of mlz 188 to 187 ions, which is close to or greater than 1 for the sultine and less than 0.4 for the DBT dioxide. Comparison of the infrared spectrum (Figure 3)of the very small peak (peak 5) in the reconstructed chromatogram shows an exact match with that of the sultine standard (Figure 4) obtained on the same GClFTIR system. The vapor-phase infrared spectra of the sultine and other DBT metabolites selectively identify the types of sulfur functionalities in each of these analytes. The spectrum of the sultine (Figure 3) features a strong absorption at 1170 cm-l corresponding to the S-O stretching vibration. The other major absorption in the vaporphase spectrum is the C-H out-of-planering deformation band that occurs as a strong peak at 754 cm-l. This absorption occurs at a little higher wavenumber in the sultine and in other DBT metabolites than it does in DBT (739 cm-9. Other peaks in the vapor-phase infrared spectrum of the sultine are of medium or weak intensity. Infrared spectral bands were reported earlier for the sultine,13Js but it is difficult to directly compare the spectral data obtained in chloroform solution with the vapor-phase spectrum. The mass spectrum of the sultone obtained with the ion trap detector (ITD) (Figure 5) was similar to the spectrum described by Hanson and Kemp13with a base peak at m / z 139 and another large fragment ion at mlz 163. The molecular ion at mlz 232 distinguishes the sultone from other known DBT metabolites, but obviously not from other hypothetical DBT oxidation products. Smaller characteristic fragment ions are present at mlz 204 and 168 that correspond to loss of CO and S02,respectively, from the molecular ion (Scheme IV). The mlz 139 fragment is probably the cyclic ion resulting from stepwise loss of all heteroatom functionality as S02,CO, and H,as is common for oxygen and sulfur heterocyclicsof this type. ~
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(16)Bowie,J. H.; Willinms,D. J.;Lawesson,S.-O.;Madeen,J.O.;Nolde, (17)Fields, E. K.;Meyereon, S. J. Am. Chem. SOC.lS66,88,2836C.; Schroll, G . Tetrahedron 1966,22,3515-3525. 2837.
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Figure 6. Infraredspectmm of sultone (peak6)from GC/FTIR/ M8 of Rhodococcus extract.
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Figure 5. ITD mass spectrum of sultone (peak 6) from GC/ FTIR/MS of Rhodococcus product extract.
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