BACKGROUND

Sulfur is an objectionable element which is nearly ubiquitous in fossil fuels, where it occurs both as inorganic (e.g., pyritic) sulfur and as organic sulfur (e.g., a sulfur atom or moiety present in a wide variety of hydrocarbon molecules, including for example, mercaptans, disulfides, sulfones, thiols, thioethers, thiophenes, and other more complex forms). Organic sulfur can account for close to 100% of the total sulfur content of petroleum liquids, such as crude oil and many petroleum distillate fractions. Crude oils can typically range from close to about 5 wt % down to about 0.1 wt % organic sulfur. Those obtained from the Persian Gulf area and from Venezuela (Cerro Negro) can be particularly high in organic sulfur content. Monticello, D. J. and J. J. Kilbane, "Practical Considerations in Biodesulfurization of Petroleum" IGT's 3rd Intl. Symp. on Gas, Oil, Coal, and Env. Biotech., (Dec. 3-5, 1990) New Orleans, La., and Monticello, D. J. and W. R. Finnerty, (1985) Ann. Rev. Microbiol. 39:371-389.

The presence of sulfur has been correlated with the corrosion of pipeline, pumping, and refining equipment, and with premature breakdown of combustion engines. Sulfur also contaminates or poisons many catalysts which are used in the refining and combustion of fossil fuels. Moreover, the atmospheric emission of sulfur combustion products such as sulfur dioxide leads to the form of acid deposition known as acid rain. Acid rain has lasting deleterious effects on aquatic and forest ecosystems, as well as on agricultural areas located downwind of combustion facilities. Monticello, D. J. and W. R. Finnerty, (1985) Ann. Rev. Microbiol. 39:371-389. To combat these problems, several methods for desulfurizing fossil fuels, either prior to or immediately after combustion, have been developed.

One technique which is employed for pre-combustion sulfur removal is hydrodesulfurization (HDS). This approach involves reacting the sulfur-containing fossil fuel with hydrogen gas in the presence of a catalyst, commonly a cobalt- or molybdenum-aluminum oxide or a combination thereof, under conditions of elevated temperature and pressure. HDS is more particularly described in Shih, S. S. et al., "Deep Desulfurization of Distillate Components", Abstract No. 264B AIChE Chicago Annual Meeting, presented Nov. 12, 1990, (complete text available upon request from the American Institute of Chemical Engineers; hereinafter Shih et al.), Gary, J. H. and G. E. Handwerk, (1975) Petroleum Refining: Technology and Economics, Marcel Dekker, Inc., N.Y., pp. 114-120, and Speight, J. G., (1981) The Desulfurization of Heavy Oils and Residue, Marcel Dekker, Inc., N.Y., pp. 119-127. HDS is based on the reductive conversion of organic sulfur into hydrogen sulfide (H.sub.2 S), a corrosive gaseous product which is removed from the fossil fuel by stripping. Elevated or persistent levels of hydrogen sulfide are known to inactivate or poison the chemical HDS catalyst, complicating the desulfurization of high-sulfur fossil fuels.

Moreover, the efficacy of HDS treatment for particular types of fossil fuels varies due to the wide chemical diversity of hydrocarbon molecules which can contain sulfur atoms or moieties. Some classes of organic sulfur molecules are labile and can be readily desulfurized by HDS; other classes are refractory and resist desulfurization by HDS treatment. The classes of organic molecules which are often labile to HDS treatment include mercaptans, thioethers, and disulfides. Conversely, the aromatic sulfur-bearing heterocycles (i.e., aromatic molecules bearing one or more sulfur atoms in the aromatic ring itself) are the major class of HDS-refractory organic sulfur-containing molecules. Typically, the HDS-mediated desulfurization of these refractory molecules proceeds only at temperatures and pressures so extreme that valuable hydrocarbons in the fossil fuel can be destroyed in the process. Shih et al.

Recognizing these and other shortcomings of HDS, many investigators have pursued the development of commercially viable techniques of microbial desulfurization (MDS). MDS is generally described as the harnessing of metabolic processes of suitable bacteria to the desulfurization of fossil fuels. Thus, MDS typically involves mild (e.g., physiological) conditions, and does not involve the extremes of temperature and pressure required for HDS. Additionally, the ability of a biological desulfurizing agent to renew or replenish itself is viewed as a potentially significant advantage over physicochemical catalysis.

The discovery that certain species of chemolithotrophic bacteria, most notably Thiobacillus ferrooxidans, obtain the energy required for their metabolic processes from the oxidation of pyritic (inorganic) sulfur into water-soluble sulfate has stimulated the search for an MDS technique for the desulfurization of coal, in which pyritic sulfur can account for more than half of the total sulfur present. Recently, Madgavkar, A. M. (1989) U.S. Pat. No. 4,861,723, has proposed a continuous T. ferrooxidans--based MDS method for desulfurizing coal. However, a commercially viable MDS process for the desulfurization of coal has not yet emerged.

Because of the inherent specificity of biological systems, T. ferooxidans MDS is limited to the desulfurization of fossil fuels in which inorganic sulfur, rather than organic sulfur, predominates. Progress in the development of an MDS technique appropriate for the desulfurization of fossil fuels in which organic sulfur predominates has not been as encouraging. Several species of bacteria have been reported to be capable of catabolizing the breakdown of sulfur-containing hydrocarbon molecules into water-soluble sulfur products. One early report describes a cyclic desulfurization process employing Thiobacillus thiooxidans, Thiophyso volutans, or Thiobacillus thioparus as the microbial agent. Kirshenbaum, I., (1961) U.S. Pat. No. 2,975,103. More recently, Monticello, D. J. and W. R. Finnerty, (1985) Ann. Rev. Microbiol. 39:371-389, and Hartdegan, F. J. et al., (May 1984) Chem. Eng. Progress 63-67, have reported that such catabolic desulfurization of organic molecules is, for the most part, merely incident to the utilization of the hydrocarbon portion of these molecules as a carbon source, rather than a sulfur-selective or -specific phenomenon. Moreover, catabolic MDS proceeds most readily on the classes of organic sulfur molecules described above as labile to HDS.

Although Monticello and Finnerty report that several species of bacteria have been described as capable of desulfurizing the HDS-refractory aromatic sulfur-bearing heterocycles, in particular Pseudomonas putida and P. alcaligenes, this catabolic pathway is also merely incident to the utilization of the molecules as a carbon source. Consequently, valuable combustible hydrocarbons are lost, and frequently the water-soluble sulfur products generated from the catabolism of sulfur-bearing heterocycles are small organic molecules rather than inorganic sulfur ions. As a result, the authors conclude that the commercial viability of these MDS processes is limited. Monticello, D. J. and W. R. Finnerty, (1985) Ann. Rev. Microbiol. 39:371-389.

None of the above-described desulfurization technologies provides a viable means for liberating sulfur from refractory organic molecules, such as the sulfur-bearing heterocycles. The interests of those actively engaged in the refining and manufacturing of petroleum fuel products have accordingly become focused on the need to identify such a desulfurization method, in view of the prevalence of these refractory molecules in crude oils derived from such diverse locations as the Middle East (about 40% of the total organic sulfur content present in aromatic sulfur-bearing heterocycles) and West Texas (up to about 70% of the total).

SUMMARY OF THE INVENTION

This invention relates to a continuous process for desulfurizing a petroleum liquid which contains organic sulfur molecules, a significant portion of which are comprised of sulfur-bearing heterocycles, comprising the steps of: (a) contacting the petroleum liquid with a source of oxygen under conditions sufficient to increase the oxygen tension in the petroleum liquid to a level at which the biocatalytic oxidative cleavage of carbon-sulfur bonds in sulfur-bearing heterocycles proceeds; (b) introducing the oxygenated petroleum liquid to a reaction vessel while simultaneously introducing an aqueous, sulfur-depleted biocatalytic agent to the reaction vessel, the agent being capable of inducing the selective oxidative cleavage of carbon-sulfur bonds in sulfur-bearing heterocycles; (c) incubating the oxygenated petroleum liquid with the biocatalytic agent in the reaction vessel under conditions sufficient for biocatalytic oxidative cleavage of said carbon-sulfur bonds, for a period of time sufficient for a significant number of cleavage reactions to occur, whereby the organic sulfur content of the treated petroleum liquid is significantly reduced and a significant amount of water-soluble inorganic sulfate is generated; (d) removing the desulfurized petroleum liquid from the reaction vessel; (e) retrieving the spent aqueous biocatalytic agent from the reaction vessel, the spent agent being significantly enriched in inorganic sulfate; (f) treating the sulfate-enriched spent aqueous biocatalytic agent in a manner sufficient for the removal of a substantial amount of inorganic sulfate from the agent, whereby the biocatalytic activity of the agent is regenerated; and (g) reintroducing the regenerated aqueous biocatalytic agent to the reaction vessel while simultaneously introducing a petroleum liquid in need of biocatalytic desulfurization.

This invention further relates to a continuous process for desulfurizing a liquid fossil fuel which contains organic sulfur molecules, a significant portion of which are sulfur-bearing heterocycles, comprising the steps of: (a) contacting the liquid fossil fuel with a source of oxygen under conditions sufficient to increase the oxygen tension in the liquid fossil fuel; (b) introducing the oxygenated liquid fossil fuel to a vertically elongated reaction vessel having means to collect and decant organic liquid from an upper region and means to remove an aqueous liquid from a lower region, while simultaneously introducing an aqueous, sulfur-deprived biocatalytic agent to the reaction vessel at a site spatially distinct from the site of introduction of the liquid fossil fuel, in such a fashion as to create a countercurrent flow system within the vessel, the agent comprising one or more microbial organisms expressing an enzyme that catalyzes the sulfur-specific oxidative cleavage of heterocyclic aromatic rings in sulfur-bearing heterocycles to produce desulfurized organic molecules and inorganic sulfur ions, enzymes derived from such microbial organisms, or mixtures of such microbial organisms and enzymes, the establishment of countercurrent flow providing sufficient mixing between the liquid fossil fuel and the aqueous biocatalyst for a significant number of carbon-sulfur bonds to be biocatalytically cleaved within a reasonable period of time; (c) incubating the oxygenated liquid fossil fuel with the biocatalytic agent in the reaction vessel under conditions sufficient for biocatalytic oxidative cleavage of said carbon-sulfur bonds, whereby the organic sulfur content of the treated liquid fossil fuel is significantly reduced and a significant amount of water-soluble inorganic sulfur is generated; (d) removing the liquid fossil fuel from the reaction vessel by decanting it from the upper region of the vessel; (e) removing the spent aqueous biocatalytic agent from the reaction vessel by recovering it from the lower region of the vessel, the spent agent being significantly enriched in inorganic sulfur; (f) treating the inorganic sulfur-enriched aqueous biocatalytic agent in a manner sufficient for the removal of a substantial amount of inorganic sulfur from the agent, whereby the biocatalytic activity of the agent is regenerated; and (g) introducing regenerated aqueous biocatalytic agent to the reaction vessel while simultaneously introducing thereto a liquid fossil fuel in need of biocatalytic desulfurization, in such a fashion as to maintain countercurrent flow.

In a preferred embodiment of the invention, the biocatalytic agent comprises a culture of mutant Rhodococcus rhodocrous bacteria, ATCC No. 53968. This microbial biocatalyst is particularly advantageous in that it is capable of catalyzing the selective liberation of sulfur from HDS-refractory sulfur-bearing aromatic heterocycles, under mild conditions of temperature and pressure. Therefore, even crude oils or petroleum distillate fractions containing a high relative abundance of refractory organic sulfur-bearing molecules can be desulfurized without exposure to conditions harsh enough to degrade valuable hydrocarbons. Additionally, the biocatalyst is regenerated and reused in the continuous method described herein; it can be used for many cycles of biocatalytic desulfurization. Moreover, the method and process of the instant invention can be readily integrated into existing petroleum refining or processing facilities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the structural formula of dibenzothiophene, a model HDS-refractory sulfur-bearing heterocycle.

FIG. 2 is a schematic illustration of the cleavage of dibenzothiophene by oxidative and reductive pathways, and the end products thereof.

FIG. 3 is a schematic illustration of the stepwise oxidation of dibenzothiophene along the proposed "4S" pathway of microbial catabolism.

FIG. 4 is a schematic flow diagram of a preferred embodiment of the instant continuous process for biocatalytic desulfurization (BDS) of this invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention employs a biocatalytic agent which is capable of selectively liberating sulfur from the classes of organic sulfur molecules which are most refractory to current techniques of desulfurization, such as HDS. The instant biocatalytic agent is used in a continuous process for desulfurizing a petroleum liquid containing organic sulfur molecules, a significant proportion of which are comprised of sulfur-bearing heterocycles. These HDS-refractory molecules occur in simple one-ring forms (e.g., thiophene), or more complex multiple condensed-ring forms. The difficulty of desulfurization through conventional techniques increases with the complexity of the molecule.

The tripartite condensed-ring sulfur-bearing heterocycle dibenzothiophene (DBT), shown in FIG. 1, is particularly refractory to HDS treatment, and therefore can constitute a major fraction of the residual post-HDS sulfur in fuel products. Alkyl-substituted DBT derivatives are even more refractory to HDS treatment, and cannot be removed even by repeated HDS processing under increasingly severe conditions. Shih et al. Moreover, as noted above, DBTs can account for a significant percentage of the total organic sulfur in certain crude oils. Therefore, DBT is viewed as a model refractory sulfur-bearing molecule in the development of new desulfurization methods. Monticello, D. J. and W. R. Finnerty, (1985) Ann. Rev. Microbiol. 39:371-389. No naturally occurring bacteria or other microbial organisms have yet been identified which are capable of effectively degrading or desulfurizing DBT. Thus, when released into the environment, DBT and related complex heterocycles tend to persist for long periods of time and are not significantly biodegraded. Gundlach, E. R. et al., (1983) Science 221:122-129.

However, several investigators have reported the genetic modification of naturally-occurring bacteria into mutant strains capable of catabolizing DBT. Kilbane, J. J., (1990) Resour. Cons. Recycl. 3:69-79, Isbister, J. D., and R. C. Doyle, (1985) U.S. Pat. No. 4,562,156, and Hartdegan, F. J. et al., (May 1984) Chem. Eng. Progress 63-67. For the most part, these mutants desulfurize DBT nonspecifically, and release sulfur in the form of small organic sulfur breakdown products. Thus, a portion of the fuel value of DBT is lost through this microbial action. Isbister and Doyle reported the derivation of a mutant strain of Pseudomonas which appeared to be capable of selectively liberating sulfur from DBT, but did not elucidate the mechanism responsible for this reactivity. As shown in FIG. 2, there are at least two possible pathways which result in the specific release of sulfur from DBT: oxidative and reductive.

Kilbane recently reported the mutagenesis of a mixed bacterial culture, producing one which appeared capable of selectively liberating sulfur from DBT by the oxidative pathway. This culture was composed of bacteria obtained from natural sources such as sewage sludge, petroleum refinery wastewater, garden soil, coal tar-contaminated soil, etc., and maintained in culture under conditions of continuous sulfur deprivation in the presence of DBT. The culture was then exposed to the chemical mutagen 1-methyl-3-nitro-1-nitrosoguanidine. The major catabolic product of DBT metabolism by this mutant culture was hydroxybiphenyl; sulfur was released as inorganic water-soluble sulfate, and the hydrocarbon portion of the molecule remained essentially intact. Based upon these results, Kilbane proposed that the "4S" catabolic pathway summarized in FIG. 3 was the mechanism by which these products were generated. The designation "4S" refers to the reactive sulfur intermediates of the proposed pathway: DBT-sulfoxide, DBT-sulfone, DBT-sulfonate, and the liberated product, inorganic sulfate. The hydrocarbon portion of the DBT molecule remains essentially intact; in FIG. 3, the theoretical hydrocarbon product, dihydroxybiphenyl is shown. In practice, monohydroxybiphenyl is also observed. Kilbane, J. J., (1990) Resour. Cons. Recycl. 3:69-79, the teachings of which are incorporated herein by reference.

Subsequently, Kilbane has isolated a mutant strain of Rhodococcus rhodocrous from this mixed bacterial culture. This mutant, ATCC No. 53968, is a particularly preferred biocatalytic agent for use with the instant method of continuous biocatalytic desulfurization. The isolation and characteristics of this mutant are described in detail in J. J. Kilbane, U.S. Pat. No. 5,104,801, the teachings of which are incorporated herein by reference. This microorganism has been deposited on Nov. 28, 1989 at the American type Culture Collection (ATCC), 12301 Park Lawn Drive, Rockville, Md., U.S.A. 20852 under the terms of the Budapest Treaty, and has been designated as ATCC Deposit No. 53968. One suitable ATCC No. 53968 biocatalyst preparation is a culture of the living microorganisms, prepared generally as described in U.S. Pat. No. 5,104,801 and in prior U.S. patent application, Ser. No. 07/631,642 now abandoned. Intact heat-killed ATCC No. 53968 microorganisms can also be used, as can cell-free enzyme preparations obtained from ATCC No. 53968 generally as described in U.S. Pat. No. 5,132,219 and in pending U.S. patent application, Ser. No. 07/897,314 now U.S. Pat. No. 5,358,870. In the instant method for biocatalytic desulfurization (BDS), the ATCC No. 53968 biocatalytic agent is employed in a continuous desulfurization process for the treatment of a petroleum liquid in which HDS-refractory organic sulfur molecules, such as the aromatic sulfur-bearing heterocycles, constitute a significant portion of the total organic sulfur content.

Biocatalytic conversion of sulfur-bearing heterocycles into molecules that are not substituted by sulfur can proceed via a reductive (anaerobic) pathway, such that molecules similar to biphenyl (FIG. 2 are produced. Thus, preparations of the microorganism disclosed by Kim et al. (1990), Degradation of organic sulfur compounds and the reduction of dibenzothiophene to biphenyl and hydrogen sulfide by Desulfovibrio desulfuricans M6, 12 BIOTECH. LETT. (No. 10) 761-764, incorporated herein by reference can be used as a desulfurization biocatalyst in the present invention.

Preferably, an oxidative (aerobic) pathway can be followed. Examples of microorganisms that act by this oxidative pathway, preparations of which are suitable for use as the biocatalyst in the present invention include the microbial consortium (a mixture of several microorganisms) disclosed in Kilbane (1990), 3 RESOUR. CONSERV. RECYCL. 69-79, the microorganisms disclosed by Kilbane in U.S. Pat. Nos. 5,002,888 (issued Mar. 26, 1991), 5,104,801 (issued Apr. 14, 1992) [also described in Kilbane (1990), Biodesulfurization: future prospects in coal cleaning, in PROC, 7TH ANN. INT'L. PITTSBURGH COAL CONF. 373-382], and 5,198,341 (issued Mar. 30, 1993); and by Omori et al. (1992), Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1, 58 APPL. ENV. MICROBIOL. (No. 3) 911-915, all incorporated herein by reference.

Each of the foregoing microorganisms can function as a biocatalyst in the present invention because each produces one or more enzymes (protein biocatalysts) that carry out the specific chemical reaction(s) by which sulfur is excised from refractory organosulfur compounds. Lehninger, PRINCIPLES OF BIOCHEMISTRY (Worth Publishers, Inc., 1982), p. 8-9; cf. Zobell in U.S. Pat. No. 2,641,564 (issued Jun. 9, 1953) and Kern et al. in U.S. Pat. No. 5,094,668 (issued Mar. 10, 1992). Mutational or genetically engineered derivatives of any of the foregoing microorganisms can also be used as the biocatalyst herein, provided that appropriate biocatalytic function is retained.

Additional microorganisms suitable for use as the biocatalyst or biocatalyst source in the desulfurization process now described can be derived from naturally occurring microorganisms by known techniques. As set forth above, these methods involve culturing preparations of microorganisms obtained from natural sources such as sewage sludge, petroleum refinery wastewater, garden soil, or coal tar-contaminated soil under selective culture conditions in which the microorganisms are grown in the presence of refractory organosulfur compounds such as sulfur-bearing heterocycles as the sole sulfur source; exposing the microbial preparation to chemical or physical mutagens; or a combination of these methods. Such techniques are recounted by Isbister and Doyle in U.S. Pat. No. 4,562,156 (issued Dec. 31, 1985); by Kilbane in 3 RESOUR. CONSERV. RECYCL. 69-79 (1990), U.S. Pat. Nos. 5,002,888, 5,104,801 and 5,198,341; and by Omori and coworkers in 58 APPL. ENV. MICROBIOL. (No. 3) 911-915 (1992), all incorporated by reference.

As explained above, enzymes are protein biocatalysts made by living cells. Enzymes promote, direct or facilitate the occurrence of a specific chemical reaction or series of reactions (referred to as a pathway) without themselves becoming consumed or altered as a result thereof. Enzymes can include one or more unmodified or post-translationally or synthetically modified polypeptide chains or fragments or portions thereof, coenzymes, cofactors, or coreactants which collectively carry out the desired reaction or series of reactions. The reaction or series of reactions relevant to the present invention culminates in the excision of sulfur from the hydrocarbon framework of a refractory organosulfur compound, such as a sulfur-bearing heterocycle. The hydrocarbon framework of the former refractory organosulfur compound remains substantially intact. Microorganisms or enzymes employed as biocatalysts in the present invention advantageously do not consume the hydrocarbon framework of the former refractory organosulfur compound as a carbon source for growth. As a result, the fuel value of substrate fossil fuels exposed to BDS treatment does not deteriorate.

Although living microorganisms (e.g., a culture) can be used as the biocatalyst herein, this is not required. In certain suitable microorganisms, including Rhodococcus rhodocrous ATCC No. 53968, the enzyme responsible for biocatalytic cleavage of carbon-sulfur bonds is present on the exterior surface (the cell envelope) of the intact microorganism. Thus, non-viable (e.g., heat-killed) microorganisms can be used as a carrier for an enzyme biocatalyst. Other biocatalytic enzyme preparations that are useful in the present invention include microbial lysates, extracts, fractions, subfractions, or purified products obtained by conventional means and capable of carrying out the desired biocatalytic function. Generally, such enzyme preparations are substantially free of intact microbial cells. Kilbane and Monticello disclose enzyme preparations that are suitable for use herein in U.S. Pat. No. 5,132,219 (issued Jul. 21, 1992), and in pending U.S. patent application Ser. No. 07/897,314 (filed Jun. 11, 1992), now U.S. Pat. No. 5,358,870. Rambosek et al. disclose additional enzyme preparations, engineered from Rhodococcus rhodocrous ATCC No. 53968 and suitable for use herein, in U.S. patent application Ser. No. 07/911,845 (filed Jul. 10, 1992), now abandoned. Enzyme biocatalyst preparations suitable for use herein can optionally be affixed to a solid support, e.g., a membrane, filter, polymeric resin, glass particles or beads, or ceramic particles or beads. The use of immobilized enzyme preparations facilitates the separation of the biocatalyst from the treated fossil fuel which has been depleted of refractory organosulfur compounds.

It is preferable to prepare a BDS-active suspension of lysed microorganisms, substantially free of intact cells. Any lysis process, whether conventional or adapted from conventional techniques, can be used, provided that the enzyme responsible for BDS reactivity remains functional. For example, the ATCC No. 53968 bacteria can be subjected to one or more freeze-thaw cycles, treated with a suitable detergent and/or chaotropic agent, processed using a French press, or, more preferably, can be sonicated by conventional means comprising the use of a bath or immersion probe sonicator and incubation on melting ice.

It is particularly preferred to prepare a substantially cell-free aqueous extract of the microbial source of BDS reactivity, wherein the extract contains a substantial proportion of the total BDS activity functionally expressed by the microorganism. In certain suitable microorganisms, the BDS reactive enzyme may be functionally expressed as a cell envelope-associated enzyme. In the case of the ATCC No. 53968 microorganism and its functional derivatives, it was previously disclosed in U.S. Ser. No. 07/486,597 now U.S. Pat. No. 5,132,219 that BDS activity appears to arise from an enzyme associated with the exterior cell membrane and/or cell wall of the intact bacterium.

A cell free extract suitable for use as biocatalyst in the present BDS method can be prepared according to standard techniques, such as centrifugal fractionation, ammonium sulfate fractionation, filtration, bioaffinity or immunoaffinity precipitation, gel permeation chromatography, liquid chromatography, high pressure liquid chromatography, reverse-phase liquid chromato-graphy, preparative electrophoresis, isoelectric focussing, and the like. For example, a centrifugal fractionation procedure, wherein it is shown that a substantial proportion of ATCC No. 53968 expressed BDS reactivity is associated with the "cell debris" fraction of sonicated, lysed bacterial cells. This fraction, which comprises fragments of cell walls and/or outer cell membranes, was obtained as a pellet following centrifugation of lysed ATCC No. 53968 cells for 5 minutes at 6,000 xg.

In another embodiment, recombinant enzymes can be employed. These enzymes can be prepared by methods known in the art, such as by complementation, as exemplified below.

Mutant strains of a R. rhodochrous, which are incapable of cleaving carbon-sulfur bonds, are produced by exposing a strain of R. rhodochrous to a mutagen to produce R. rhodochrous mutants. Suitable strains of R. rhodochrous include any strain of R. rhodochrous containing DNA which encodes a biocatalyst capable of selective cleavage of carbon-sulfur bonds, such as ATCC No. 53968 as reported in U.S. Pat. No. 5,104,801, the teachings of which are incorporated herein by reference. In one embodiment, the IGTS8 strain of R. rhodochrous, from Institute of Gas Technology (Chicago, Ill.) is used.

Suitable mutagens include radiation, such as ultraviolet radiation or chemical mutagens, such as N-methyl-N'-nitrosoguanidine (NTG), hydroxylamine, ethylmethane-sulphonate (EMS) and nitrous acid.

R. rhodochrous mutants are allowed to grow in an appropriate medium and screened for carbon-sulfur bond cleavage activity. Mutants without carbon-sulfur bond cleavage activity are labelled CS.sup.-. Any method of screening which allows for an accurate detection of carbon-sulfur bond cleavage activity is suitable in the method of the present invention. Suitable methods of screening for this activity include exposing the different mutants to carbon-sulfur bond containing molecules and measure carbon-sulfur bond cleavage. In a preferred embodiment, the mutants are exposed to DBT, whose breakdown product, 2-hydroxybiphenyl (2-HBP), fluoresces under short wave ultraviolet light. Other methods include gas and liquid chromatography, infrared and nuclear magnetic resonance spectra. See Kodama, et al., Applied and Environmental Microbiology, pages 911-915 (1992) and Kilbane and Bielaga, Final Report D.O.E. Contract No. DE-AC22-88PC8891 (1991). Once CS.sup.- mutants are identified and isolated, clones are propagated for further analysis.

Concurrent with the mutagenesis of one culture of R. rhodochrous, a second culture is maintained, R. rhodochrous, that expresses a substance with carbon-sulfur bond cleavage activity (CS.sup.+). DNA is extracted from this organism. Various methods of DNA extraction are suitable for isolating the DNA of this organism. Suitable methods include phenol and chloroform extraction. See Maniatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, page 16.54 (1989), herein referred to as Maniatis et al..

Once the DNA is extracted from R. rhodochrous, the DNA is cut into fragments of various kilobase lengths, collection of which makes up the DNA library. Various methods of fragmenting the DNA of R. rhodochrous to free the DNA of the present invention, may be used including enzymatic and mechanical methods. Any four-base recognition restriction endonuclease such as TaqI or Sau 3A is suitable for fragmenting the DNA. Suitable methods of fragmenting DNA can be found in Maniatis et al..

The various DNA fragments are inserted into several mutant clones of R. rhodochrous, with the purpose of isolating the fragment of DNA, which encodes a biocatalyst. The transformation of a previously CS.sup.- mutant cell to a CS.sup.+ transformed cell is evidence that the inserted DNA fragment encodes a biocatalyst. Any method of inserting DNA into R. rhodochrous which allows for the uptake and expression of said fragment is suitable. In a preferred embodiment, electroporation is used to introduce the DNA fragment into R. rhodochrous. See Maniatis et al..

Once transformed mutant R. rhodochrous has been produced and identified, DNA fragment encoding the CS.sup.+ biocatalyst can be identified and isolated. The encoded biocatalyst can then be produced using the isolated DNA in various methods well-known and readily available to those skilled in the art. In addition the isolated DNA can be sequenced and replicated by methods known by those skilled in the art (See Maniatis et al.).

DNA isolated by the above described method can be isolated from any organism which expresses a biocatalyst capable of selectively cleaving carbon-sulfur bonds in a sulfur-bearing hydrocarbon. They include Bacillus sphaericus ATCC No. 53969 as reported in U.S. Pat. No. 5,002,888, the teachings of which are incorporated herein by reference.

Other methods of isolating the DNA of the present invention, include variations on the rational used above. For example, it would be possible to randomly insert a CS.sup.- DNA plasmid into clones of a CS.sup.+ strain of R. rhodochrous. DNA encoding a CS.sup.+ biocatalyst could then be identified by screening for clones that have been transformed from CS.sup.+ to CS.sup.-.

The recombinant DNA molecule of the present invention is intended to encompass any DNA resulting from the insertion into its chain, by chemical or biological means, a gene encoding a biocatalyst capable of selectively cleaving carbon-sulfur bonds, said gene not originally present in that chain. Recombinant DNA includes any DNA created by procedures using restriction nucleases, nucleic acid hybridization, DNA cloning, DNA sequencing or any combination of the preceding. Methods of construction can be found in Maniatis et al., and in other methods known by those skilled in the art. The term "recombinant DNA", as used herein, is intended to encompass any DNA resulting from the insertion into the chain, by chemical or biological means, of a DNA not originally present in that chain.

Procedures for the construction of DNA plasmid vectors of the present invention include those described in Maniatis et al. and other methods known by those skilled in the art. Suitable plasmid vectors include pRF-29 and pRR-6. The term "DNA plasmid vector" is intended any replication competent vector which has the capability of having DNA inserted into it and, subsequently, the expression of that DNA insert by an appropriate host cell. In addition, the plasmid vector must be receptive to the insertion of a DNA plasmid containing the genes of the present invention where the gene encodes a biocatalyst which has the capability to selective cleave carbon-sulfur bonds. Procedures for the construction of DNA plasmid vectors include those described in Maniatis et al. and others known by those skilled in the art.

The plasmids of the present invention include any DNA fragment containing the genes of a DNA which encode a biocatalyst which has the capability to selective cleave carbon-sulfur bonds. The term "plasmid" is intended to encompass any DNA fragment. The DNA fragment should be transmittable to a host microorganism by transformation or conjugation. Procedures for the construction or extraction of DNA plasmids include those described in Maniatis et al. and others known by those skilled in the art.

The transformed microorganisms of the present invention can be created by various methods by those skilled in the art. For example, transfection electroporation as explained by Maniatis et al. can be used. By the term "microorganisms" or "organism" is intended any organism capable of the uptake and expression of foreign DNA, i.e., DNA not originally a part of the organism nuclear material. Suitable organisms may include Corynebacterium or Escherichia.

In the biocatalytic desulfurization stage of multistage deep desulfurization, the liquid fossil fuel containing sulfur-bearing heterocycles is combined with the biocatalyst preparation. The relative amounts of biocatalyst preparation and liquid fossil fuel can be adjusted to suit particular conditions, or to produce a particular level of residual sulfur in the treated, deeply desulfurized fossil fuel. The amount of biocatalyst preparation to be combined with a given quantity of liquid fossil fuel will reflect the nature, concentration and specific activity of the particular biocatalyst used, as well as the nature and relative abundance of inorganic and organic sulfur compounds present in the substrate fossil fuel and the degree of deep desulfurization sought or considered acceptable.

The specific activity of a given biocatalyst is a measure of its biocatalytic activity per unit mass. Thus, the specific activity of a particular biocatalyst depends on the nature or identity of the microorganism used or used as a source of biocatalytic enzymes, as well as the procedures used for preparing and/or storing the biocatalyst preparation. The concentration of a particular biocatalyst can be adjusted as desired for use in particular circumstances. For example, where a culture of living microorganisms (e.g., ATCC No. 53968) is used as the biocatalyst preparation, a suitable culture medium lacking a sulfur source other than sulfur-bearing heterocycles can be inoculated with suitable microorganisms and fermented until a desired culture density is reached. The resulting culture can be diluted with additional medium or another suitable buffer, or microbial cells present in the culture can be retrieved e.g., by centrifugation, and resuspended at a greater concentration than that of the original culture. The concentrations of non-viable microorganism and of enzyme biocatalyst preparations can be adjusted similarly. In this manner, appropriate volumes of biocatalyst preparations having predetermined specific activities and/or concentrations can be obtained.

BDS Treatment of a Typical Middle Distillate with a culture of living ATCC No. 53968 microorganisms

A petroleum distillate fraction, similar in specific gravity and other properties to a typical middle distillate or a heavy atmospheric gas oil or a vacuum gas oil or the material from a delayed coker, having an initial sulfur content of 0.51 wt %, was treated with a preparation of Rhodococcus rhodochrous ATCC No. 53968. The biocatalyst preparation consisted of an inoculum of the bacteria in a basal salts medium, comprising:

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    Component           Concentration
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    Na.sub.2 HPO.sub.4  0.557%
    KH.sub.2 PO.sub.4   0.244%
    NH.sub.4 Cl         0.2%
    MgCl.sub.2.6H.sub.2 O
                        0.02%
    MnCl.sub.2.4H.sub.2 O
                        0.0004%
    FeCl.sub.3.6H.sub.2 O
                        0.0001%
    CaCl.sub.2          0.0001%
    glycerol            10 .mu.M
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                  TABLE 1
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    Biocatalytic Desulfurization by intact, lysed, and a cell-free
    fraction obtained from ATCC No. 53968
                Change in Absorbance
                (610 nm) per Hour per
                                Number of
    Biocatalyst Cell Material   Determinations
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    Washed intact
                0.085 .+-. 0.007
                                n = 4
    cells
    Freeze-Thaw 0.060 .+-. 0.001
                                n = 2
    lysed cells
    (unfractionated)
    Sonicated lysed
                0.035 .+-. 0.002
                                n = 2
    cells (cell debris
    fraction)
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