Resources, Conservation and Recycling, 5 ( 1991 ) 255-263 Elsevier Science Publishers B.V./Pergamon Press plc

255

Microbial oxidation of sulfur in dibenzothiophene

Judi th P. Kitchell a, Saraswathy V. Nochur a aDynaGen, Inc., Cambridge, MA 02139, USA

Judi th K. Marquis b'l bDepartment of Pharmacology, Boston University School of Medicine, Boston, MA, USA

Dennis A. Bazylinski c'2 and Holger Jannasch c c Woods Hole Oceanographic Institution, Woods Hole, MA, USA

( Received January 3, 1990; accepted after revision September 18, 1990)

ABSTRACT

Kitchell, J.P., Nochur, S.V., Marquis, J.K., Bazylinski, D.A. and Jannasch, H., 1991. Microbial oxi- dation of sulfur in dibenzothiophene. Resour. Conserv. Recycl., 5: 255-263.

An aerobic microorganism isolated from deep sea thermal vents in the Gulf of California and grown with the model coal compound dibenzothiophene (DBT) was found to produce DBT oxidation prod- ucts, including DBT-sulfoxide and DBT-sulfone. The organism can utilize DBT as sole carbon and sulfur source, but grows better when the medium is supplemented with yeast extract. After five to seven days of incubation with DBT, some DBT-sulfoxide and, to a lesser extent, DBT-sulfone, inter- mediates in the desulfurization of DBT, were detected. When the culture was grown in medium with DBT-sulfoxide as substrate, little oxidation occurred, but when DBT-sulfone was used as substrate, it was rapidly degraded. The extracellular extract is also active against DBT, producing sulfur oxidation products.

I N T R O D U C T I O N

The removal of sulfur from coal prior to combustion has been of great con- cern since the relationship of the sulfur combustion products to environmen- tal pollution was noted. Many physical and chemical approaches to this pro- cess are employed, but they all add substantially to the cost of coal utilization due to the energy and material input required. It has been shown that biolog- ical processing using microorganisms can also be employed for coal desulfur- ization [ 1-4 ]. However, it has not yet been shown that biological processing

~Present address: Life Sciences Section, Arthur D. Little, Inc., Cambridge, MA, U.S.A. 2present address: Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA, U.S.A.

0921-3449/91/$03.50 © 1991 - - Elsevier Science Publishers B.V./Pergamon Press plc

256 J.P. KITCHELL ET AL.

can be an economical method for reducing the pollution caused by coal com- bustion. The considerations in biological coal processing include: complete- ness of sulfur removal from the coal matrix; efficiency of separation of soluble sulfur from the product stream; maintenance of initial fuel value; and reduc- tion of energy and material costs.

Biological processing of coal could utilize whole organisms or only the bio- catalysts (i.e. enzymes ) in order to selectively remove the organic sulfur from the coal matrix. The potential problems with the use of whole cells are: (i) removed sulfur could be accumulated within the microorganisms, in which case the sulfur would have been moved from one bound form to another; (ii) whole cells could add significant bulk to the processing; (iii) cell viability may be a serious problem; (iv) whole organisms may not be as selective in the desulfurization process as specific enzymes might be; and (v) enzymes could function in water, buffer, or aqueous organic solvents, while cells would necessarily require a predominantly aqueous solution.

This paper describes the efforts to develop a clean coal bioprocessing tech- nology using crude enzyme preparations or isolated enzymes rather than me- tabolizing organisms. This approach has several theoretical strengths: only enzymes that perform the desired catalysis (e.g. sulfur oxidation or selected ring oxidations ) are used, cutting down on fuel loss; catalytic conditions (e.g. solvents) can be chosen with more flexibility because cell viability is not an issue; and the reaction product will be soluble sulfate, which is readily sepa- rated from the coal stream. The cost effectiveness of such an enzyme process would depend on the cost of enzyme/unit of clean coal, the physical enzyme reaction requirements (media and temperature), and the cost of separation processes.

As a preliminary means of evaluating enzymes for coal desulfurization, sources of enzymes that are functional in high sulfur environments were sought. An organism isolated from soil at a deep sea thermal vent has been shown to oxidize the sulfur in dibenzothiophene (DBT), a compound with a molecular structure similar to portions of the organic coal matrix [ 4 ]. Some activity is also obtained with extracellular fractions of the culture, indicating that the activity may be derived from one or more extracellular enzymes. The program attempts to demonstrate the efficacy of microbial enzymes in the oxidation of organic sulfur.

MATERIALS AND METHODS

Microorganisms

Two aerobic and mesophilic bacteria from oily, sulfur-rich sediments col- lected from deep-sea hydrothermal vents in the Guaymas basin have been used in this study. The organisms have not been identified and have been

MICROBIAL OXIDATION OF SULFUR IN DIBENZOTHIOPHENE 2 5 7

designated GB-1 and GB-2. GB-1 is a rod-shaped, motile bacterium, which stains variably with the Gram stain. GB-2 is vibrioid-to-helical in morphol- ogy, motile, and Gram-negative.

Growth-medium

GB-1 and GB-2 were routinely cultured in an artificial seawater medium containing (per liter): NH4C1, 0.5 g; yeast extract, 0.5 g; vitamin solution [ 5 ], 5 ml; and modified Wolfe's mineral elixir [6,7 ], 5 ml. The artificial sea- water formulation was that of Lyman and Fleming [8] except that MgC12" 6H20, CaC12" 2H20 and SrC12.2H20 replaced the corresponding an- hydrous salts. The pH of the medium was adjusted to 7.0 to 7.2 with 0.01N NaOH. Concentrations of DBT or other substrates used are as described in the text. DBT was added as a solid. Although insoluble, DBT was kept in suspension during the incubation period.

Growth conditions

Cultures were grown with shaking at 25 °C in a constant temperature room. A 1% inoculum was used. Cultures were harvested after several days of incu- bation as noted in the text.

Preparation and use of extracellular fractions (ECFs)

Cultures were harvested at various times and were centrifuged at 12,000g for 20 min. The supernatant fluids were carefully removed. The pH was ad- justed to 7.0, and the supernatants incubated with DBT at 25 °C with shaking. The supernatant fluids were essentially cell-free, for no growth occurred even after incubation at 25 °C for one week.

Extraction and characterization of DBT oxidation products

Extraction of culture medium After growth of the culture for several days, the culture medium was acidi-

fied with 1N HC1 to a pH of 2.1 to 2.3 and then extracted in an equal volume of methylene chloride. For controls, uninoculated medium was treated in the same manner. In some instances, the culture medium was centrifuged prior to acidification and extraction and the culture medium was also extracted without acidification. The methylene chloride extract was evaporated to dry- ness at room temperature and taken up in a small volume of acetonitrile. This was then analyzed by TLC or HPLC.

258 J.P. KITCHELL ET AL.

Characterization of DBT oxidation products Thin layer chromatography (TLC) was one of the methods used to identify

the components in the culture extract. Three different systems were used: (i) alumina plates (Eastman Kodak 13252 ) with fluorescence indicator were used with ethanol:water: ammonium hydroxide [ 2: 15 : 1 ] as eluent [ 4 ]; (ii) sil- ica gel plates (Merck No. 5628 ) were used with chloroform: acetone [ 80: 20 ] as eluent [ 3 ]; and (iii) silica gel plates (same as above ) were used with ben- zene: methanol [ 97: 3 ] as eluent [ 3 ]. The plates were examined under visible and ultraviolet light and after spraying with a 2% solution of Gibbs' reagent (Sigma) in ethanol.

HPLC was used for confirmation of the presence of DBT oxidation prod- ucts and for their quantitation. The HPLC conditions used were a modifica- tion of the method of Wyza [ 9 ]: column, Waters C 18 Resolve 5~tm spherical; solvent, tetrahydrofuran: acetonitrile: water [ 23 : 18 : 59 ]; flow rate, 1.5 ml/ min; injection volume, 2 ~tl; detection at 242 nm using a Waters Lambda Max 481 LC spectrophotometer.

Separation of components in the culture medium

To separate the products of microbial degradation of DBT, a silica gel col- umn ( 1 cmX20 cm) was prepared from a slurry of 6.65 g of silica gel G-60 (0-230 mesh ASTM) in 50 ml of methylene chloride. After the column had equilibrated, 1 ml of the concentrated culture extract was applied, the column was eluted with methylene chloride, and approximately 1 ml fractions were collected. After elution with 40 ml of methylene chloride, the column was eluted with 25 ml of ethanol. The fractions were analyzed by TLC using chlo- roform: acetone [ 80:20 ] as the eluent. Some of the fractions were analyzed using hexane: methylene chloride [ 50:50 ] as the eluent [ 9 ]. Fractions with the same material were pooled and further observed by HPLC.

EXPERIMENTAL RESULTS AND DISCUSSION

Studies of growth conditions of GB-1 and GB-2

GB- 1 and GB-2 were grown in artificial sea water medium containing DBT and supplemented with 0.05% yeast extract (YE). Within 24 hours of incu- bation, the GB-! cultures turned red, while the GB-2 cultures remained col- orless. GB-1 incubated in medium without DBT did not produce any red col- ored compound(s) . GB-1 grew without YE and with DBT as sole carbon source and sole sulfur source. GB-2 did not grow without YE supplementa- tion or without another carbon source. Both cultures are aerobic and do not grow above 28 ° C. Improved growth was obtained with shaking and with yeast

MICROBIAL OXIDATION OF SULFUR IN DIBENZOTHIOPHENE 259

extract supplementation. Since GB-2 did not show any indication of oxidiz- ing DBT, this culture was not studied any further.

Analysis of DBT degradation products in cell growth media

Cells were grown as described earlier with DBT and YE. Preliminary ex- periments involved extraction of the culture media with methylene chloride after different incubation times. There was no significant difference in the levels of oxidation products, whether or not acidification of the sample was done prior to extraction. Concentrated extracts were examined for products by UV spectroscopy; however, the spectra were not helpful because of the multiplicity of DBT, DBT-oxidation products and the media components.

The absorption max imum of the GB-1 extracts in the red range was at 528 nm. Kodama et al. [ 10] also reported the presence of a red colored substance in their cell extract which they identified as trans-4-[2-(3-hydroxy)-thiana- phthenyl]2-oxo-3-butenoic acid. The red colored compounds found in the GB-1 media seem to be either high molecular weight or cell bound/cel l asso- ciated, since the red color remained in the cell pellet when the culture was centrifuged prior to extraction. The red color was not pH dependent.

TLC with silica or alumina plates was used to identify DBT and DBT sulfur oxidation products in standard mixtures, and it was also used to observe and identify the components from the culture extracts. It was clear from the silica plates with benzene:methanol [97:3 ] that the GB-1 culture produced DBT- sulfoxide from DBT. No DBT-sulfoxide was observed in uninoculated and extracted culture media. TLC, however, is not a quantitative method and the samples were further analyzed by HPLC.

The HPLC system described in the Materials and Methods Section was used to separate DBT and its sulfur oxidation products and to analyze culture ex- tracts for these compounds. A HPLC chromatogram showing the separation of DBT, DBT-sulfoxide, DBT-sulfone, and o,o'-diphenol in a standard mix- ture is shown in Fig. 1. This method of analysis was used to identify DBT oxidation products in the media from cultures initially grown with 0.1% and 0.05% DBT. Subsequently, lower concentrations (0.01%) of DBT were used. The logical progression of sulfur oxidation in DBT is termed the "4S" path- way, resulting in the sequential formation of DBT-sulfoxide, DBT-sulfone, DBT-sulfonate, and the formation of o,o '-diphenol and simultaneous release of sulfate (Fig. 2 ). Estimations of released sulfate were not done since results would be complicated by the presence of inorganic sulfur in the medium components.

Cultures of GB-1 grown on 0.01% DBT were found to accumulate some DBT-sulfoxide and a small amount of DBT-sulfone at the end of 5 to 7 days. No o,o '-diphenol was produced. The results are shown in Fig. 3. Cultures of GB-I grown on 0.01% DBT-sulfoxide were found not to have accumulated

260 J.P. KITCHELL ET AL.

Fig. 1. HPLC separation of DBT, DBT-sulfoxide, DBT-sulfone and o,o'-diphenol from a stan- dard mixture using the conditions described in the test. (DBT-sulfoxide elutes at a retention t ime of 1.72 min, DBT-sulfone at 2.97 min, o,o'-diphenol at 4.5 min, and DBT at 22.41 min.)

D1BENZOTHIOPHENE [DBT]

DBT-SULFOXIDE O

O O DBT-SULFONE

DBT-SULFONATE

O,O'-DIPHENOL HH

Fig. 2. The sequential oxidation of DBT at sulfur.

any DBT-sulfone or o,o'-diphenol, and the amount of DBT-sulfoxide stayed unchanged after incubation. On the other hand, GB-1 incubated for 7 days with 0.01% DBT-sulfone revealed that most of the DBT-sulfone had been degraded, with no end-products identifiable by HPLC (no o,o'-diphenol was detected ).

When DBT was used as the sole carbon and sulfur source in the medium, the culture grew poorly with substantially lower degradation of DBT. Addi-

MICROBIAL OXIDATION OF SULFUR IN D1BENZOTHIOPHENE 261

I

Fig. 3. HPLC chromatogram of media extracted after 7 days of GB-1 growth on 0.01% DBT with shaking. (In the standard, Fig. 1, DBT-sulfoxide elutes at 1.72 min, DBT-sulfone at 2.97 min, o,o'-diphenol at 4.5 min, and DBT at 22.41 min. )

tion of 0.1% succinate to the medium substantially improved growth of the culture without a concomitant increase in DBT degradation (data not shown ).

Analysis of ECF activity versus DBT

The ECF of GB-1 cultures (prepared as described in the Materials and Methods Section) that had grown for 72, 114 and 168 h were adjusted to a pH of 7.0 and incubated with 0.01% DBT at 25°C with shaking for 7 days. The medium from each assay was then acidified and extracted with methyl- ene chloride and resuspended in a small volume of acetonitrile. The extracts were then examined by HPLC. Under the conditions tested, the 168 h and 114 h ECF had very little effect on DBT (i.e. only a small amount of DBT- sulfoxide and DBT-sulfone was produced). On the other hand, the 72 h ECF produced a significant amount of DBT-sulfoxide and a small amount of DBT- sulfone from DBT. The HPLC chromatogram of this sample is shown in Fig. 4. The 72 h ECF that had not been incubated with DBT served as the negative control. No o,o'-diphenol production was observed, but other as yet uniden- tified products of cell growth and DBT degradation were seen. The 72 h ECF was tested at pH 5.0, 6.0, 7.0, and 8.0. Activity against DBT was similar at

262 J .P. K I T C H E L L E T AL.

4

_ m _

Fig. 4. HPLC chromatogram of media extracted after 8 days incubation of GB-1 cell-free extract (collected at 72 hours) with 0.01% DBT. (In the standard, Fig. 1, DBT-sulfoxide elutes at 1.72 min, DBT-sulfone at 2.97 min, o,o'-diphenol at 4.5 min, and DBT at 22.41 min.)

pH 6.0 and 7.0, while it was almost negligible at pH 5.0 and pH 8.0. Further analysis of the ECF and the generation of other fractions from GB-1 to test for activity against DBT and coal will be the focus of future research.

CONCLUSIONS

The microbial culture GB-1 is capable of oxidizing the sulfur in dibenzo- thiophene (DBT), a model coal compound. Under the conditions tested, while the conversion to DBT-sulfoxide occurred readily, the subsequent oxidation to DBT-sulfone was slow. DBT sulfone, however, was well utilized; although the pathway of utilization of this compound has not been elucidated. No o,o'- diphenol, a likely oxidation product, was observed, and it is not clear whether o,o'-diphenol was produced but subsequently utilized, or whether another degradative pathway occurred. Similar activity (but at lower levels) was de- tected against DBT in extracellular fractions from GB-1 growth media. This activity was found to be pH-dependent and also varied depending on the age of the culture during harvest, with ECF harvest at 3 days being more active than that harvested at 5 or 7 days.

MICROBIAL OXIDATION OF SULFUR IN DIBENZOTHIOPHENE 263

ACKNOWLEDGEMENTS

We thank Ms. Y. Boyer and Mr. S. Crooker for their technical support. This research was supported by Grant DE-AC22-88PC-88855 from the U.S. De- partment of Energy. Contribution No. 7153 of the Woods Hole Oceano- graphic Institution.

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