11 Feasibility Studies of a Biochemical Desulfurization Method A. J. DAVIS, III and T. F . Y E N Departments of Biological Sciences and Chemical Engineering, University of Southern California, University Park, Los Angeles, Calif. 90007
The microorganism, Thiobacillus thiooxidans, is a possible desulfurizing agent of organically bound sulfur with an efficiency greater than 20%. It oxidizes inorganic sulfur to sulfate, producing sulfuric acid as a by-product. In this study, T. thiooxidans was grown on tertiary butylsulfides (mono-, di-, and poly-) for 24 days. During this time, growth of the organism was followed by a decrease in pH and an increase in sulfate production, gravimetrically assayed for in the barium salt form. There was significant growth on the tertiary butyldisulfide and the tertiary butylpolysulfide. Significant growth was not reported on the tertiary butylmonosulfide.
' T p he removal of sulfur from high sulfur containing petroleum has long presented a problem. W i t h the depletion of so-called "sweet" oil (i.e., oil low i n sulfur content), the problem has escalated into major proportions to contend with a clean and livable environment. The development of practical methods of decreasing or eliminating the sulfur content of petroleum has proved repeatedly to be an economical obstacle to industry. The amount of dollars spent on corrosion technology alone is phenomenal ( I ) . Rudimentary investigations of microbial desulfurization have received little attention in the literature at this time (2). One successful example of desulfurization is the removal of pyrite from coal by Tfttobacillus sp. and Ferrobaccus sp. (3). W h i l e studies of the complex hydrocarbon-sulfur systems are of great value, being closer to in situ reality, investigation of a defined system should form the foundation of these more detailed studies. 137
138
SHALE
OIL, T A R SANDS, A N D R E L A T E D
FUEL
SOURCES
Our present study was intended to explore the ability of a micro organism as a possible desulfurizing agent. To this end, a pure, defined system was used that would give some idea as to the ease with which such an agent could abstract organically bound sulfur. W h i l e this pro gram is preliminary, future investigations may lead to a feasible industrial application. Materials and Methods Media and Culture. The strain of Thiobacillus thiooxidans used i n these experiments was originally obtained from the National Types C u l ture Collection. Inoculate substrain was obtained by further culture in this laboratory (4). Fernbach culture flasks (2.8-1. borosilicate glass) containing 1.0 1. of Waksmans medium were prepared i n duplicate. The medium contained the following concentrations of salt i n g/1. (all Mallinckrodt " A R " grade): ( N H ) S 0 , 0.20; K H P 0 , 3.00; M g S 0 • 7 H 0 , 0.50; C a C l · 2 H 0 , 0.25. The medium was prepared with distilled water. In place of the elemental sulfur substrate of Waksman s medium, different symmetric organic sulfides of the tertiary butyl class w i t h a normalized sulfur equivalent of 10.0 g/1. were used. To provide a greater surface area, the normally viscous sulfides were emulsified with Triton X-100 surfactant (J. T. Baker Chemical C o . ) . The first series of flasks received 49.0 m l terf-butyl sulfide and one drop Triton X-100 each. The second series of flasks received 28.0 m l terf-butyl disulfide and one drop Triton X-100 each. The third series of flasks received 17.0 m l ferf-butyl polysulfide and two drops Triton X-100 each. In addition, flasks of ele mental sulfur and flasks of organic sulfur substrates were prepared to act as controls. The p H of the media was adjusted to 3.5 with 1.02V H P 0 (phosphoric acid) (Mallinckrodt " A R " grade), and sterilization was accomplished by autoclaving or by membrane filtration. After steriliza tion all flasks containing sulfur or organic sulfur compounds, except the organic sulfur control flasks, were inoculated with 10.0 cc Γ. thioxidans suspensions. A l l flasks were incubated at room temperature. 4
2
2
2
4
2
4
4
2
3
4
Sulfide Samples. The teri-butyl sulfide and the terf-butyl disulfide used i n these experiments were obtained from the Aldrich Chemical Co. The purity of the monosulfide ( M W 146.30) was i n excess of 9 7 % . The technical grade disulfide ( M W 178.36) was 3 8 % pure and contained 6 2 % tertiary butyl bisulfide. The terf-butyl polysulfide used was kindly supplied by Phillips Petroleum Co. The polysulfide ( M W 190.00) was greater than 9 0 % pure. The polysulfide contained an average of four and five sulfur atoms per molecule. N o pretreatment of the sulfides was performed.
11.
DAVIS AND Y E N
Biochemical Desulfurization Method
139
A n a l y t i c a l M e t h o d s . Insoluble barium sulfate precipitate, one of the two criteria by which growth of the bacteria was established was determined by standard gravimetric methods. The amount of barium sulfate is directly proportional to the amount of sulfide that has been oxidized by the sulfur bacteria. Hydrogen ion concentration ( p H ) was the second criterion by which growth of the bacilli was established. Sulfuric acid is a natural metabolic by-product of sulfur oxidation by the acidophillic Thiobacillus thiooxidans (5). As sulfur is used, acid is built-up in the medium thus lowering the p H . Studies in this laboratory have shown that the bacteria grow well in a p H as low as 0.5. Samples for the barium sulfate determinations were prepared by removing a 5.0 m l aliquot portion from each flask and then centrifuging them on an Adams centrifuge for 15 min at 3000 rpm. To the supernatant of each was added 2 drops concentrated hydrochloric acid, an acid buffer, and 11 drops of a saturated solution of barium chloride (Mallinckrodt " A R " grade). The precipitate was filtered on a Millipore apparatus (borosilicate glass), washed with distilled water, and then allowed to dry three days i n an evaculated dessicator. The samples were then ignited, and the weight of the barium sulfate established. The controls were handled i n the same manner. The amount of barium sulfate produced from the organic sulfides less the controls was averaged and then plotted. The p H was read directly with a Beckman Zeromatic, SS-3 p H meter. Procedure Modified Waksman's medium was prepared in a Fernbach culture flask. A n amount of organic sulfide normalized to an equivalent sulfur content of the standard medium (10 g/1.) was added followed by an emulsifier. The medium was then autoclaved for 30 min at 15 psi (121 ° C ) , or, as with the polysulfide and elemental sulfur, sterilization was achieved by membrane filtration. U p o n cooling, the medium was inoculated with 10 cc of the pure strain of Thiobacillus thiooxidans. The cultures initial p H value was read, and an initial gravimetric sulfate assay was performed. Thereafter, p H and sulfate values were determined at two-day intervals for 25 days. Results The model was designed to study the ability of sulfur-oxidizing bacteria to use the organically bound sulfur as substrate. The symmetry of the sulfides provided an insight into the sulfur abstracting prowess of T. thiooxidans. Figure 1 shows that the oxidizing potential of the organism is enhanced by the availability of unshielded sulfur i n the molecular structure. The sulfur-sulfur bonds of the d i - and polysulfides are easily disrupted; but access to the carbon-sulfur-carbon bond of the monosulfide seems
140
SHALE
'
è
OIL,
TAR
SANDS,
AND
Î0
RELATED
F U E L
20
SOURCES
$5—
Days
Figure
1.
Conversion
of organic sulfide 25-day period
into sulfate
in
The amount of sulfate ion represents the difference of that produced in the inoculated flasks containing different organic sulfide and that in the controls containing the same organic sulfide without inocuhtion. —.—, di-tert-butul volusulfide; , di-tertbutyl disulfide; , di-tert-butyl monosulfide; , control on elemental sulfur.
questionable. W h i l e some sulfate ion is produced by the organism on the monosulfide, it is not known how much of the sulfate arises from residual sulfur previously incorporated by the bacteria. The values of p H seem to bear this out (Figure 2). Based on sulfate ion and p H values, however, it appears that the organism is growing on the d i - and polysulfides. Discussion The polysulfide and elemental sulfur were unstable to autoclaving. This fact necessitated the use of a membrane to achieve sterility i n those cases. The surfactant not only provided greater surface area but also minimized the amount of sulfide i n contact with atmospheric oxygen since the sulfide was generally dispersed throughout the medium. The fate of the non-sulfur-containing hydrocarbons, while of real interest, is beyond the scope of this present work. This study indicates that the sulfur of our sulfide samples was susceptible to bacterial attack i n two of the three cases. The aliphatic sulfides could be ranked in the order of their ease of oxidation as: di-tertbutyl polysulfide > di-ferf-butyl disulfide > ài-tert-butyl sulfide. Seemingly, T. thiooxidans is able to attack the sulfur-sulfur bond quite readily and the sulfur-carbon bond w i t h some difficulty, if at all. Since a large portion of the sulfur compounds i n petroleum are of the monosulfide-aromatic type, more attention is paid to this facet.
11.
DAVIS AND Y E N
Biochemical Desulfurization Method
141
The simplest scheme of the bacterial oxidation would be: s-
2
T.
T. thiooxidans
S0 4
2
A few mechanisms were postulated for this reaction (6). N o matter what mechanism is considered, the oxidation of elemental sulfur or thiosulfate is accompanied by reductive cleavage of the sulfur-sulfur bridges. In the case of sulfur, the intermediate involved is a cyclic form of sulfur, probably S , although there is little difference observed for different allotrophic forms of sulfur such as rhombic, precipitated, and amphorous. These cyclic sulfides form the basis of polysulfanes and polythionates which could be metabolized readily by Thiobacilli (as shown i n Figure 3 ) . Actually, sulfur oxidation begins with its reduction, i n which the gluathione-sulfhydryl groups located near the cell surface take part: 8
NAD-H + S* +
G.-S-S-G.
-> N A D +
2 G - S H -> H S + 2
2G-SH
G.-S-S-G.
^control 40
y^/^-"' " polysulfides m
i
35
disulfide
monosulfide 25 Days
Figure 2. Variation of pH after inoculation of sulfur bacteria. , di-tert-hutyl monosulfide; , ditert-butyl disulfide; , di-tert-butyl polysulfide; , control of elemental sulfur.
142
SHALE
Figure 3.
O I L , T A R SANDS,
AND RELATED FUEL
SOURCES
Reductive cleavage of S-S bridges of polysulfanes and polythionates
Figure 4.
Possible mechanism of disulfide cleavage
11.
DAVIS AND YEN
Biochemical Desulfurization Method
143
It is thought that the S H " of the glutathione-sulfhydryl group acts as the functional agent in the reductive cleavage of the sulfur. For thiosulfate oxidation it is hypothesized that an enzyme system on the cell surface initiates the formation of polythiosulfonic acid and consequently splits the terminal S 0 " as sulfate. This process may evolve the intermediate of mixed anhydride -S-O-PO4" from phosphorylation (Figure 4). The overall reaction is one of electron transfer in the enzyme system: 3
The critical step in the use of multiple sulfur linkages is the availability of H S " from the reduced enzyme system. The H S " could possibly attack the cyclic or the acyclic multiple sulfur linkages through a nucleophilic mechanism. The cleaved sulfide linkages undergo oxidation to sulfite or thiosulfate. This seems a plausible pathway for the oxidation of the sulfur compounds i n the present study. Finally, removal of the sulfate ion creates a new contamination prob lem. In connection with this, sulfate reduction bacteria for the complete removal of sulfur are being examined. The conversion of the organic sulfide into inorganic sulfide w i l l be one future objective of this study. Acknowledgment The authors thank Judith H i g a for technical help. Literature
Cited
1. Bertness, Τ. Α., Mobil Oil Corp., personal communication, 1973. 2. Isenberg, D. L., "Microbial Desulfurization of Petroleum," Ph.D. Disserta tion, Louisiana State University, 1961. 3. Silverman, M. P., Rogoff, M. H., Wender, I., "Removal of Pyritic Sulfur from Coal by Bacterial Action," Fuel (1963) 42, 113. 4. Findley, J. E., Appleman, M. D., Yen, T. F., "Abstracts of Papers," 166th National Meeting, ACS, Div. of Microbial Chemistry and Technology, Chicago, 1973. 5. Roy, A. S., Trudinger, P. Α., "The Biochemistry of Inorganic Compounds of Sulfur," p. 207, Cambridge University, 1970. 6. Sokolova, G. Α., Karavaiko, G. I., "Physiology and Geochemical Activity of Thiobacilli," Israel Program for Scientific Translations, p. 48, 1968. RECEIVED December 26, GI-35683, AER-74-23797,
1974. This work is supported by NSF and ACS-PRF Grant No. 6272-AC2.
Grant
No.
