ENZYMES FOR REMOVING SULFUROUS COMPOUNDS IN DOWNHOLE FLUIDS

Abstract

Cysteine synthase enzymes (e.g. O-Acetyl-L-Serine Sulfhydrylase enzymes) may be used in combination with sulfide quinone reductase enzymes in additive compositions, fluid compositions, and methods for decreasing or removing hydrogen sulfide from recovered downhole fluids and/or the subterranean reservoir wellbore from which the downhole fluid was recovered. The fluid composition may include at least one cysteine synthase enzyme with at least one sulfide quinone reductase, and a base fluid, such as a water-based fluid, an organic-based fluid, and combinations thereof.

Claims

1. An additive composition for a base fluid selected from the group consisting of a drilling fluid, a completion fluid, a production fluid, a servicing fluid, an injection fluid, a refinery fluid, and combinations thereof; wherein the additive composition comprises at least one cysteine synthase enzyme and at least one sulfide quinone reductase enzyme; wherein the at least one cysteine synthase enzyme is at least 75% homologous to the cDNA sequence of SEQ ID NO:1; and wherein the at least one sulfide quinone reductase is at least 75% homologous to the cDNA sequence of SEQ ID NO:2.

2. The additive composition of claim 1, wherein the concentration of the at least one cysteine synthase enzyme within the additive composition ranges from about 1 nanomolar (nM) independently to about 5 millimolar (mm).

3. The additive composition of claim 1, wherein the concentration of the at least one sulfide quinone reductase enzyme within the additive composition ranges from about 1 nanomolar (nM) independently to about 5 millimolar (mm).

4. The additive composition of claim 1, further comprising at least one additional component selected from the group consisting of a pyridoxal phosphate, O-acetyl-serine, dithiothreitol (DTT), coenzyme Q10, plastoquinone, vitamin K2, and combinations thereof.

5. The additive composition of claim 1, wherein the at least one cysteine synthase enzyme forms cysteine in the absence of pyridoxal phosphate.

6. The additive composition of claim 1, further comprising the at least one additional component in a concentration ranging from about 0.25 mM to about 20 mM.

7. A fluid composition comprising: a base fluid selected from the group consisting of drilling fluids, servicing fluids, production fluids, completion fluids, injection fluids, refinery fluids, and combinations thereof; at least one cysteine synthase enzyme that is at least 75% homologous to the cDNA sequence of SEQ ID NO:1; and at least one sulfide quinone reductase enzyme is at least 75% homologous to the cDNA sequence of SEQ ID NO:2.

8. The fluid composition of claim 7, wherein the concentration of the at least one cysteine synthase enzyme within the additive composition ranges from about 1 nanomolar (nM) independently to about 10 millimolar (mm) as compared to the total base fluid.

9. The additive composition of claim 1, wherein the concentration of the at least one sulfide quinone reductase enzyme within the additive composition ranges from about 1 nanomolar (nM) independently to about 5 millimolar (mm).

10. The fluid composition of claim 7, wherein the fluid composition further comprises at least one additional component selected from the group consisting of a pyridoxal phosphate, O-acetyl-serine, dithiothreitol (DTT), coenzyme Q10, plastoquinone, vitamin K2, and combinations thereof.

11. The fluid composition of claim 7, wherein the fluid composition further comprises at least one sulfur-containing compound; and wherein the fluid composition comprises the sulfur-containing compound in a decreased amount as compared to an identical fluid composition absent the at least one cysteine synthase enzyme and the at least one sulfide quinone reductase enzyme.

12. The fluid composition of claim 7, further comprising the at least one additional component in a concentration ranging from about 0.25 mM to about 20 mM.

13. The fluid composition of claim 7, wherein the at least one cysteine synthase enzyme and the at least one sulfide quinone reductase enzyme are added at the same time or different times.

14. A method comprising: contacting at least one fluid with at least one cysteine synthase enzyme that is at least 75% homologous to the cDNA sequence of SEQ ID NO:1 in an effective concentration to decrease an amount of sulfur-containing compounds within the at least one fluid; wherein the at least one fluid is selected from the group consisting of drilling fluids, servicing fluids, production fluids, completion fluids, injection fluids, refinery fluids, and combinations thereof; contacting the at least one fluid with at least one sulfide quinone reductase enzyme that is at least 75% homologous to the cDNA sequence of SEQ ID NO:2 in an effective concentration to decrease an amount of sulfur-containing compounds within the at least one fluid; decreasing an amount of sulfur-containing compounds in the at least one fluid.

15. The method of claim 14, wherein the at least one cysteine synthase enzyme and the at least one sulfide quinone reductase enzyme contact the at least one fluid at the same time or different times.

16. The method of claim 14, wherein the at least one cysteine synthase enzyme and/or the at least one sulfide quinone reductase enzyme is disposed within a downhole fluid selected from the group consisting of a drilling fluid, a completion fluid, a production fluid, a servicing fluid, an injection fluid, a refinery fluid, and combinations thereof.

17. The method of claim 14, wherein the at least one cysteine synthase enzyme and/or the at least one sulfide quinone reductase enzyme is disposed within a water-based fluid, an organic-based fluid, and combinations thereof.

18. The method of claim 14, wherein the effective concentration of the at least one cysteine synthase enzyme ranges from about 1 nanomolar (nM) independently to about 5 millimolar (mm) as compared to a total base fluid.

19. The method of claim 14, wherein the effective concentration of the at least one sulfide quinone reductase enzyme ranges from about 1 nanomolar (nM) independently to about 5 millimolar (mm) as compared to a total base fluid.

20. The method of claim 14, further comprising circulating at least one additional component selected from the group consisting of a pyridoxal phosphate, O-acetyl-serine, dithiothreitol (DTT), coenzyme Q10, plastoquinone, vitamin K2, and combinations thereof.

21. The method of claim 7, further comprising the at least one additional component in a concentration ranging from about 0.25 mM to about 20 mM.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more fully understand the drawings referred to in the detailed description, a brief description of each drawing is presented here:

[0024] FIG. 1 (SEQ ID NO:1) represents the nucleotide sequence that codes for the cysteine synthase enzyme;

[0025] FIG. 2 represents the plasmid pBEn-SET3a used for expressing the cysteine synthase enzyme;

[0026] FIG. 3 is a graph depicting the effect of reaction components on cysteine synthase activity where the cysteine formed was treated with acid ninhydrin, and the absorbance was measured with a spectrometer at 560 nm;

[0027] FIG. 4 is a graph depicting the hydrogen sulfide levels measured by colorimetry using an HACH sulfide assay;

[0028] FIG. 5 is a graph depicting an amount of H.sub.2S measured in head space after treating a bottle or condition containing an equilibrated sulfide and hydrogen sulfide where the measurements were acquired by using gas chromatography;

[0029] FIG. 6 (SEQ ID NO:2) represents the nucleotide sequence that codes for the sulfide quinone reductase enzyme;

[0030] FIG. 7 is a graph depicting use of a cysteine synthase enzyme and a sulfide quinone reductase enzyme in sour water;

[0031] FIG. 8 is another graph depicting use of a cysteine synthase enzyme and a sulfide quinone reductase enzyme in sour water;

[0032] FIG. 9 is a graph depicting a combination of cysteine synthase enzyme and sulfide quinone reductase enzyme use to decrease a percentage of sodium sulfide in a solution; and

[0033] FIG. 10 is another graph depicting a combination of cysteine synthase enzyme and sulfide quinone reductase enzyme use to decrease a percentage of sulfide in a sour refinery water.

DETAILED DESCRIPTION

[0034] It has been discovered that an additive composition having at least one cysteine synthase enzyme and at least one sulfide quinone reductase enzyme may be added to a base fluid to decrease an amount of sulfur-containing compounds in the base fluid. Alternatively, a fluid composition comprising a cysteine synthase enzyme and a sulfide quinone reductase enzyme may be circulated in a subterranean reservoir wellbore to decrease an amount of sulfur-species compounds present in the subterranean reservoir wellbore and/or any downhole fluids recovered therefrom. In addition to being biodegradable, the cysteine synthase enzyme(s) and the sulfide quinone reductase enzyme(s) may be less toxic to the environment and may be made from renewable resources. The use of cysteine synthase enzyme(s) and sulfide quinone reductase enzyme(s) in a base fluid may provide a renewable alternative to conventional additives (non-biodegradable) that are used in downhole fluids to decrease sulfur-containing compounds.

[0035] In a non-limiting embodiment, the cysteine synthase may be or include, but is not limited to, O-Acetyl-Serine Sulfhydrylase (OASS). The cysteine synthase enzyme(s) and sulfide quinone reductase enzymes may be derived from various organisms. In a non-limiting embodiment, the cysteine synthase enzyme(s) may be derived from Aeropyrum pernix.

[0036] The additive and/or fluid composition may further include an additional component, such as but not limited to, a pyridoxal phosphate, O-acetyl-serine, DTT, coenzyme Q10, plastoquinone, vitamin K2, and combinations thereof; the additional component(s) may be added to the base fluid at the same time or a different time from the cysteine synthase enzyme(s) and at the same time or different time from the sulfide quinone reductase enzyme(s). In a non-limiting embodiment, the additional component(s) may be added to the base fluid in a concentration ranging from about 0.25 mM independently to about 20 mM, alternatively from about 0.1 mM independently to about 15 mM, or from about 1 mM independent to about 5 mM in another non-limiting embodiment.

[0037] In a non-limiting embodiment, pyridoxal phosphate may be added to the base fluid in a concentration ranging from about 0.1 mM independently to about 5 mM, alternatively from about 1 mM independently to about 4 mM. In a non-limiting embodiment, the additive and/or fluid composition does not include pyridoxal phosphate. In another non-limiting embodiment, the DTT may be added to the base fluid in a concentration ranging from about 0.25 mM independently to about 5 mM, alternatively from about 1 mM independently to about 3 mM.

[0038] In a non-limiting embodiment, the cysteine synthase enzyme(s) and/or the sulfide quinone reductase enzyme(s) may be added to the base fluid in a concentration substantially equal to or more than the amount of suspected sulfur-containing compounds present in the base fluid and/or the subterranean reservoir wellbore. In an alternative non-limiting embodiment, the cysteine synthase enzyme(s) may be added to the base fluid in a concentration ranging from about 1 mM independently to about 20 mM, alternatively from about 5 mM independently to about 15 mM. Similarly, the sulfide quinone reductase enzyme(s) may be added to the base fluid in a concentration ranging from about 1 mM independently to about 20 mM, alternatively from about 5 mM independently to about 15 mM.

[0039] Cysteine synthase enzymes and sulfide quinone reductase enzyme(s) may remove or decrease sulfur-containing compounds, e.g. hydrogen sulfide (H.sub.2S) or other types of mercaptans in a non-limiting embodiment, within a recovered downhole fluid from a subterranean reservoir wellbore and/or decrease the amount of hydrogen sulfide or other sulfur-containing compounds in the wellbore from which the downhole fluid was recovered. In a non-limiting embodiment, sulfur-containing compounds may be or include, but is not limited to any compound having the formula R—SH where R is an unsubstituted or substituted alkyl, thiol, carboxylic acid, dithio acid, and combinations thereof. In yet another non-limiting embodiment, R may have or include from 1 independently to 40 carbons, alternatively, from 6 independently to about 20 carbons.

[0040] The cysteine synthase enzyme may catalyze the reaction of an O-acetyl-L-serine and hydrogen sulfide to form L-cysteine and acetate. In a non-limiting embodiment, hydrogen sulfide is converted into L-cysteine in a 1:1 ratio, i.e. one mole of H.sub.2S forms one mole of L-cysteine. Likewise, O-acetyl-L-serine and hydrogen bind to the enzyme in a 1:1 ratio.

[0041] The sulfide quinone reductase enzyme may catalyze the reaction of a quinone (e.g. coenzyme Q10, plastoquinone, vitamin K2, and combinations thereof in a non-limiting embodiment) and hydrogen sulfide to form quinol and an oxidation product, such as but not limited to elemental sulfur, a sulfite, polysulfide and combinations thereof.

[0042] ‘Enzyme’ as used within ‘cysteine synthase enzyme’ and/or ‘sulfide quinone reductase enzyme’ is defined herein to be the active site of each respective enzyme to convert sulfur-containing products into less hazardous materials, such as but not limited to L-cysteine, elemental sulfur, a sulfite, polysulfide, acetate, and combinations thereof. The active site of the enzyme may be or include the whole protein, an active fragment of the protein, a mimetic of the protein, and combinations thereof. ‘Fragment’ as used herein is meant to include any amino acid sequence shorter than the full-length enzyme, but where the fragment maintains similar activity to the full-length enzyme. Fragments may include a single contiguous sequence identical to a portion of the enzyme sequence. Alternatively, the fragment may have or include several different shorter segments where each segment is identical in amino acid sequence to a different portion of the amino acid sequence of the enzyme, but linked via amino acids differing in sequence from the enzyme. ‘Mimetic’ as used herein may include polypeptides, which may be recombinant, and peptidomimetics, as well as small organic molecules, which exhibit similar or enhanced catalytic activity as compared to the enzymes described herein.

[0043] The gene for the cysteine synthase enzyme may be codon optimized to increase the efficiency of its expression in E. coli. The nucleotide sequence of one embodiment of the cysteine synthase enzyme is set forth in FIG. 1 (SEQ ID NO:1). The gene coding for the cysteine synthase enzyme may have a nucleotide sequence that is substantially homologous to the nucleotide sequence of FIG. 1 (SEQ ID NO:1). The term “substantially homologous” is used herein to denote nucleotides having at least 75% sequence identity to the sequence shown in FIG. 1 (SEQ ID NO:1), alternatively from about 80% independently to about 99.5%, or from about 85% independently to about 95%. As used herein with respect to a range, “independently” means that any threshold may be used together with another threshold to give a suitable alternative range, e.g. about 75% independently to about 85% is also considered a suitable alternative range.

[0044] The gene for the sulfide quinone reductase enzyme may be codon optimized to increase the efficiency of its expression in E. coli. The nucleotide sequence of one embodiment of the sulfide quinone reductase is set forth in FIG. 6 (SEQ ID NO:2). The gene coding for the sulfide quinone reductase enzyme may have a nucleotide sequence that is substantially homologous to the nucleotide sequence of FIG. 6 (SEQ ID NO:2). The term “substantially homologous” is used herein to denote nucleotides having at least 75% sequence identity to the sequence shown in FIG. 6 (SEQ ID NO:2), alternatively from about 80% independently to about 99.5%, or from about 85% independently to about 95%.

[0045] The cysteine synthase enzyme may be a homodimer, i.e. two subunits that are the same, where each subunit may have a pyridoxal phosphate as a cofactor. However, as previously mentioned, the cysteine synthase enzyme may function in the absence of a pyridoxal phosphate cofactor.

[0046] The primary structural sequence of an enzyme is the linear sequence of amino acids that are linked together by peptide bonds to form the primary structure of the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme. The secondary structure of the protein refers to the base pairing interactions within a single molecule or set of interacting molecules, such as a beta-helix in the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme. Tertiary structure refers to the three-dimensional structure of the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme formed from the nucleotide sequence. Quaternary structure refers to the interaction between at least two tertiary structures.

[0047] To obtain the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme, a particular bacterium known for producing the respective enzyme may be plated on a growth medium, such as an agar, which is conducive to the growth of the particular bacterium in a non-limiting embodiment. As a non-limiting example, Aeropyrum pernix may be plated on a growth medium for growing the Aeropyrum pernix bacterium for subsequently producing a cysteine synthase enzyme. The enzyme may be directly isolated from its bacteria to be added to or used within a fluid composition for decreasing a sulfur-containing compound in a fluid composition and/or a subterranean reservoir wellbore. ‘Isolated’ is defined herein to denote that the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme has been removed from the intact cells or cellular debris, and is in a condition other than its native environment, is free of other extraneous or unwanted nucleic acids, proteases, and lipids, in a form suitable for use as a cysteine synthase enzyme and/or the sulfide quinone reductase enzyme as described herein.

[0048] In a non-limiting embodiment, a particular gene of a cysteine synthase gene from the Aeropyrum pernix bacterium may be inserted into a plasmid vector. Similarly, a gene of the sulfide quinone reductase gene may be inserted into a plasmid vector. A vector is a DNA molecule that may be used as a vehicle to artificially carry genetic material from a foreign cell and/or organism. A plasmid is defined as a circular extrachromosomal element found naturally in bacteria and some other organisms, which may be genetically engineered to clone DNA fragments. The plasmid may then be inserted into a host bacterium cell, such as Escherichia coli, where the host cell may replicate and/or express the foreign DNA. The E. coli cells may be plated on a growth medium, such as an agar, which is conducive to the growth of E. coli. The growth of E. coli propagates the enzyme as clones within each E. coli cell. The enzyme(s) may be isolated from the E. coli cells and added to or used within a fluid composition.

[0049] FIG. 2 is a depiction of the plasmid pBEn-SET3a before cloning the cysteine synthase enzyme thereinto. P T7/lacO is a promoter site with a lac operator, which is a site for starting transcription. The extended RBS is a ribosomal binding site that is a sequence on mRNA that is bound by a ribosome when initiating protein translation.

[0050] The SET3tag may increase the solubility of any problem proteins within E. coli. Although the mechanism by which the SET tag may improve solubility has not been confirmed, the SET tag may enhance solubility of the fusion protein by providing a net negative charge, which is thought to prevent aggregation and provide more time for correct protein folding in vivo.

[0051] The multiple cloning site (MCS) is a short segment of DNA that has several restriction sites, such as Nde1 and BamH1 in a non-limiting embodiment. Nde1 is a type II restriction enzyme that may cut open specific sequence targets; specifically, Nde1 may be used to cut open the reading frames in the plasmid to insert the cysteine synthase enzyme genes. BamHI is also a type II restriction endonuclease that recognizes the sequence 5′-GGATCC-3′, and cleaves these sequences just after the 5′-guanine on each strand to leave sticky ends that are 4 base pairs long.

[0052] Ampicillin (Amp.sup.R) regulates the expression of β-lactamase, pBR322 ori is the DNA sequence that signals for the origin of replication (also known as ‘origin’), and lacI codes for the lactose repressor.

[0053] To isolate or obtain the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme from E. coli, the E. coli cells may be harvested via centrifugation to produce a cell pellet. The cell pellet may be lysed either by physical means or by chemical means, such as detergents and/or enzymes (e.g. lysozyme) to produce a lysate. The raw lysate may contain the recombinant protein, as well as other proteins originating from the bacterial host.

[0054] The cysteine synthase enzyme and/or the sulfide quinone reductase enzyme may be in a powder form and/or a liquid form (e.g. in solution) when added to or included in the base fluid. The cysteine synthase enzyme and/or the sulfide quinone reductase enzyme may be part of an additive where the additive includes the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme, as well as other components to aid the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme in decreasing the amount of sulfur-containing compounds in a fluid composition and/or a subterranean reservoir wellbore.

[0055] The additive may include a cysteine synthase enzyme and/or the sulfide quinone reductase enzyme where the cysteine enzyme is at least 75% homologous to the cDNA sequence of SEQ ID NO:1 and where the sulfide quinone reductase enzyme is at least 75% homologous to the cDNA sequence of SEQ ID NO:2. cDNA is defined herein as DNA synthesized from a messenger RNA (mRNA) template in an enzymatic catalyzed reaction using reverse transcriptase. The additive may include the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme within the additive composition in a concentration ranging from about 1 nanomolar (nM) independently to about 5 millimolar (mm), alternatively from about 10 nM independently to about 1 mm, or from about 1 micromolar (μM) independently to about 5 (μM) as compared to the total base fluid.

[0056] In a non-limiting embodiment, the reaction where sulfur-containing components are catalyzed into less hazardous materials via at least one of the cysteine synthase and/or the sulfide quinone reductase enzyme(s) may occur for at least 30 minutes, alternatively from about 30 minutes independently to about 4 hours, or from about 1 hour independently to about 4 hours. In a non-limiting embodiment, the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme may maintain optimal function at a temperature ranging from about 75° F. independently to about 200° F., alternatively from about 100° F. independently to about 175° F. The cysteine synthase enzyme and/or the sulfide quinone reductase enzyme may maintain optimal function at a pressure less than about 15,000 pounds per square inch (psi). The cysteine synthase enzyme and/or the sulfide quinone reductase enzyme may maintain optimal function at a pH ranging from about 4 independently to about 11, alternatively from about 5 independently to about 8, or from about 6 to about 7.5 in another non-limiting embodiment. The cysteine synthase enzyme and/or the sulfide quinone reductase enzyme may still function at a decreased reaction rate, if at all, outside of the ranges mentioned for temperature, pressure, and/or pH.

[0057] The additive may be added to a base fluid to form a fluid composition. The base fluid may be or include, but is not limited to, a drilling fluid, a completion fluid, a production fluid, a servicing fluid, an injection fluid, a refinery fluid, and combinations thereof. In a non-limiting embodiment, the base fluid may be an aqueous fluid, a non-aqueous fluid, an organic fluid, and combinations thereof. In another non-limiting embodiment, the base fluid or fluid composition may be contained in an oil pipeline, gas pipeline, a storage tank, a transportation vehicle, a refinery (e.g. separation vessels, dehydration units, gas lines, and pipelines), and combinations thereof.

[0058] The cysteine synthase enzyme(s) having at least 75% homology to the cDNA sequence of SEQ ID NO:1 may be circulated into a subterranean reservoir wellbore to decrease an amount of sulfur-containing compounds within the subterranean reservoir wellbore and/or any downhole fluids recovered therefrom. The sulfide quinone reductase enzyme(s) having at least 75% homology to the cDNA sequence of SEQ ID NO:2 may be circulated into a subterranean reservoir wellbore to decrease an amount of sulfur-containing compounds within the subterranean reservoir wellbore and/or any downhole fluids recovered therefrom. In yet another non-limiting embodiment, the cysteine synthase enzyme(s) and/or the sulfide quinone reductase enzyme(s) may be circulated into an oil pipeline, gas pipeline, a storage tank, a transportation vehicle, a refinery (e.g. separation vessels, dehydration units, gas lines, and pipelines), and combinations thereof.

[0059] The cysteine synthase enzyme(s) and the sulfide quinone reductase enzyme(s) may be circulated at the same time or different times. ‘Same time’ is defined herein to be substantially the same time, i.e. both enzymes are added to a base fluid or fluid composition prior to circulating the fluid composition through a subterranean reservoir wellbore and/or refinery equipment and/or storage/transportation equipment. ‘Different times’ is defined herein to mean the sulfide quinone reductase enzymes may be added separately to a base fluid or fluid composition before or after the cysteine synthase enzyme is added to the same base fluid or fluid composition. However, even when added at different times, the sulfide quinone reductase enzymes and the cysteine synthase enzymes are considered to be added/used ‘in combination’ to decrease the sulfur-containing components when added to the same base fluid or fluid composition.

[0060] The cysteine synthase enzyme(s) and the sulfide quinone reductase enzyme(s) may be used together but in different concentrations. For example, sulfide quinone reductase enzymes is currently less expensive than cysteine synthase enzymes; therefore, the sulfide quinone reductase enzyme(s) may be added to either a base fluid(s) and/or the fluid composition(s) first to decrease the sulfur-containing compounds as much as possible. Then, the cysteine synthase enzyme(s) may be added to the same base fluid or fluid composition to further decrease the sulfur-containing compounds below a pre-determined threshold. However, a greater concentration of the sulfide quinone reductase enzyme may be used because it is currently less expensive than the cysteine synthase enzymes. Alternatively, the cysteine synthase enzymes may be used in a greater concentration as compared to the sulfide quinone reductase enzymes in another non-limiting embodiment. And in yet another non-limiting embodiment, the cysteine synthase enzymes and the sulfide quinone reductase enzymes may be used in substantially the same concentrations.

[0061] In yet another non-limiting embodiment, the fluid composition may include a salt, such as but not limited to, a brine, sea salt, and combinations thereof. The brine may be or include, but is not limited to potassium chloride, sodium chloride, calcium chloride, zinc chloride, potassium bromide, sodium bromide, calcium bromide, zinc bromide, sodium formate, potassium formate, ammonium formate, cesium formate, and combinations thereof.

[0062] ‘Derived from’ with respect to the enzymes is meant to include whole enzymes or enzyme fragments, where the enzyme originated from its respective bacterium and was isolated from its particular species; ‘derived from’ also encompasses polypeptides identical in DNA and/or amino acid sequence to the active site (e.g. the cleft between C and N terminal domains and Lysine 47 site of the cysteine synthase enzyme in a non-limiting example) of the bacterium that are recombinantly expressed in a host cell expression system or chemically synthesized. ‘Recombinant DNA’ is DNA that has been formed artificially by combining constituents from different organisms, such as inserting the enzyme into an E. coli host cell for a cloned reproduction of the cysteine synthase enzyme in a non-limiting example.

[0063] ‘Derived from’ also includes derivatives of the enzymes, such as a polypeptide or fragment that may be substantially similar in primary structural sequence to the enzymes described herein, but which may include chemical and/or biochemical modifications that are not found in the native polypeptide. Such modifications may be or include, but are not limited to a label, such as a radioactive isotope, a fluorophore, or an enzymatic label useful in tracing the enzymes. The label or other modification may be useful in isolating the enzymes from the bacterium and/or other expression system (E. coli as described below). The label or other modification may be used to identify the enzymes within a fluid composition and/or recovering the enzyme from the fluid composition. Other non-limiting modifications may be or include a nucleotide mutagenesis to impart additional thermostability and pH tolerance to the enzymes.

[0064] The method may include decreasing the amount of at least one sulfur-containing compound within the fluid composition and/or within a subterranean reservoir wellbore, storage facility, transportation vehicle, a refinery, and combinations thereof. Parameters that may be used to assess the effectiveness of the enzymes may include measurements of formation kinetics where the sulfur-containing compounds are catalyzed into their respective oxidized components, amount of sulfur-containing compounds present in the recovered fluids, fluid compositions, and/or subterranean reservoir wellbore before and after treatment with the additive and/or fluid composition, and the like. Methods for measuring these parameters may be used to assess the ability of the enzymes to reduce, decrease, or inactivate sulfur-containing compounds. ‘Effective concentration’ is defined herein to mean any concentration of the enzymes that may decrease or reduce the amount of sulfur-containing compounds within the fluid composition, a subterranean reservoir wellbore and a downhole fluid recovered therefrom; alternatively, ‘effective concentration’ is defined herein to mean any amount of the cysteine synthase enzyme and/or the sulfide quinone reductase enzyme that may decrease the amount of sulfur-containing compounds.

[0065] The invention will be further described with respect to the following Examples, which are not meant to limit the invention, but rather to further illustrate the various embodiments.

EXAMPLES

Example 1

[0066] Two separate experiments were conducted with two sets of samples where cysteine and H.sub.2S concentrations were measured after completion of each reaction within each sample.

[0067] Samples 1 and 5 were the blanks for each set that do not contain cysteine synthase. Samples 2 and 6 included an O-Acetyl-Serine (OAS) in a concentration of 5 mM, pyridoxal-5′-phosphate (PLP) in an amount of 0.25 mM, and a cysteine synthase enzyme, i.e. an O-Acetyl-Serine Sulfhydrylase (OASS) enzyme, in an amount of 50 μL (about 200 nanograms of enzyme). Samples 3 and 7 included OAS in a concentration of 5 mM, PLP in a concentration of 0.25 mM, and the OASS enzyme in an amount of 50 μL. Samples 4 and 8 included OAS in a concentration of 5 mM, no PLP, and the OASS enzyme in an amount of 50 μL.

[0068] An increased absorbance measurement at 560 nm represents an increased amount of cysteine formed from each sample. The reactions treated with the cysteine synthase enzyme showed higher concentrations of cysteine compared to the blanks (samples 1 and 5), indicating that the cysteine was synthesized from the enzyme. The cysteine synthase enzyme used H.sub.2S or sulfide during the enzymatic reaction to synthesize cysteine. No significant differences in H.sub.2S reduction (decrease) occurred regardless of whether PLP was present during the enzymatic reaction.

[0069] As shown in TABLE 1, each set of samples produced about the same amount of cysteine where the second set produced more cysteine than the first set. Samples 4 and 8, which did not have PLP, produced similar amounts of cysteine indicating that the addition of PLP is optional. The lack of PLP in samples 4 and 8 also indicates that the PLP is optional from the concentrations of H.sub.2S measured, which was the lowest amount of H2S as compared to the other samples 1-3 and 5-7.

[0070] Samples 1 and 5, the experiment blanks for each set that did not include the cysteine synthase enzyme, had high amounts (14.5 and 15 mg/L) of H.sub.2S as compared to at least a 50-60% decrease in H.sub.2S present in samples 2-4 and 6-8 treated with the cysteine synthase enzyme.

TABLE-US-00001 TABLE 1 Measured Amounts of Cysteine and Sulfide produced form Cysteine Synthase H.sub.2S conc Cysteine H.sub.2S conc Cysteine Condition Set 1 Synthesis Set 2 Synthesis Enzyme Blank 14.5 mg/L  0 15 mg/L  0 OAS + Pyridoxal 8.5 mg/L 0.223 6.5 mg/L.sup.  0.409 phosphate + enzyme OAS + Pyridoxal .sup. 7 mg/L 0.218 6 mg/L 0.387 phosphate + enzyme OAS + no pyridoxal 4.5 mg/L 0.228 5 mg/L 0.458 phosphate + enzyme

Example 2

[0071] FIG. 3 is a graph depicting the effect of reaction components on cysteine synthase activity by detecting the amount of cysteine produced by reacting the completed reaction products with acid ninhydrin and measuring the absorbance with a spectrometer at 560 nm (amino acids reacts with ninhydrin to produce purple color). Sample 1 included 5 mM O-Acetyl serine, 1 mM sulfide, 1 mM DTT, 0.25 mM PLP, and 100 mM Tris HCl buffer, pH 7.5; sample 1 did not include the OASS enzyme. Sample 2 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl (about 200 nanograms of the enzyme), 5 mM O-Acetyl serine, 1 mM sulfide, 1 mM DTT, 0.25 mM PLP, and 100 mM Tris HCl buffer, pH 7.5. Sample 3 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, and 100 mM Tris HCl buffer, pH 7.5; sample 3 did not include dithiothreitol (DTT), nor did sample 3 include PLP. Sample 4 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, 0.25 mM PLP, and 100 mM Tris HCl buffer, pH 7.5; sample 4 did not include DTT. Sample 5 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, and 100 mM Tris HCl buffer, pH 7.5; sample 5 did not include dithiothreitol (DTT), nor did sample 3 include PLP. Sample 6 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, and 100 mM Tris HCl buffer, pH 7.5; sample 6 did not include DTT, nor did sample 6 include PLP; however, sample 6 included 50% O-acetylserine OAS. Sample 7 was substantially similar to sample 6, except that sample 7 only included 25% OAS.

[0072] As noted from FIG. 3, sample 2, which included all of the reaction components, formed the largest amount of cysteine. Worth noting here, an absence of DTT in the sample may reduce detection of cysteine as the cysteine is oxidized after formation; however, the absence of DTT may not affect the reaction efficiency of the cysteine synthase enzyme. In addition, an absence of PLP in the sample may not affect the reaction efficiency of the cysteine synthase enzyme, as the PLP molecule is added to the cysteine synthase enzyme during protein expression.

Example 3

[0073] FIG. 4 is a graph depicting the amounts of H.sub.2S measured in each sample after 4 h.

[0074] Sample 1 included 5 mM O-Acetyl serine, 1 mM sulfide, 1 mM DTT, 0.25 mM PLP, and 100 mM Tris HCl buffer, pH 7.5; sample 1 did not include the cysteine synthase (OASS) enzyme. Sample 2 included the OASS cysteine synthase enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, 1 mM DTT, 0.25 mM PLP, and 100 mM Tris HCl buffer, pH 7.5. Sample 3 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, and 100 mM Tris HCl buffer, pH 7.5; sample 3 did not include dithiothreitol (DTT), nor did sample 3 include PLP. Sample 4 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, 0.25 mM PLP, and 100 mM Tris HCl buffer, pH 7.5; sample 4 did not include DTT. Sample 5 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, and 100 mM Tris HCl buffer, pH 7.5; sample 5 did not include dithiothreitol (DTT), nor did sample 3 include PLP. Sample 6 included the cysteine synthase (OASS) enzyme in an amount of 50 microliters μl, 5 mM O-Acetyl serine, 1 mM sulfide, and 100 mM Tris HCl buffer, pH 7.5; sample 6 did not include DTT, nor did sample 6 include PLP; however, sample 6 included 50% O-acetylserine OAS. Sample 7 was substantially similar to sample 6, except that sample 7 only included 25% OAS.

[0075] As noted from FIG. 4, sample 7 had the lowest amount of H.sub.2S remaining after treatment. The H.sub.2S concentration was decreased the most in samples 6 and 7, which may be due to more efficient use of OAS by the enzyme in the absence of DTT and PLP.

Example 4

[0076] FIG. 5 is a graph depicting the amount of H.sub.2S present in the head space in four samples measured by gas chromatography (GC). ‘Head space’ refers to the gaseous volume within each sample. Sample 1 was the blank where no cysteine synthase (OASS) enzyme was added to the sample. Samples 2-4 included 400 mL mM Tris-HCL, pH 7.0, 5 mM cysteine synthase, 1 mM DTT, and 0.025 mM PLP. Samples 2-4 were sparged with 10% H.sub.2S for two hours to reach an H.sub.2S concentration of 1500 ppm in the vapor phase. One ml of an cysteine synthase (OASS) enzyme lysate was added to 90 mL of the solution for sample 2, and 2 ml of an OASS cysteine synthase enzyme lysate was added to samples 3 and 4, and then each sample was incubated for 4 hours at 37° C. The cysteine synthase (OASS) enzyme lysate was obtained by the process mentioned above. The H.sub.2S concentration in each sample was then measured by GC.

[0077] Two bars are noted for sample 1 and sample 2; the bar on the left for each sample represents the amount of H.sub.2S as detected with a lower sensitivity probe, and the bar on the right for each sample represents the amount of H.sub.2S as detected with a higher sensitivity probe. As noted in FIG. 5, 2 mL of the enzyme lysate of cysteine synthase completely eliminated the H.sub.2S in samples 3-4.

Example 5

[0078] FIG. 7 is a graph depicting use of a cysteine synthase enzyme and a sulfide quinone reductase enzyme in sour water. The first set of samples to the far left is sour water without any treatment, which has a sulfide concentration of about 35 ppm. Each sample had a total volume of 100 mL of sour water, and enzyme where noted. The second set of samples includes a sour water sample having a sulfide concentration of about 35 ppm, and the second sample of the second set of samples included the same sour water sample with 2 mL of sulfide quinone reductase enzyme. The third set of samples includes a sour water sample having a sulfide concentration of about 40 ppm, and the second sample of the third set of samples included the same sour water sample treated with 2 mL of cysteine synthase enzyme. The 2 mL of each enzyme was obtained from a crude lysate, which is about 500 ppm of each enzyme.

[0079] As can be seen by FIG. 7, the sulfide quinone reductase and the cysteine synthase enzymes both decrease the amount of sulfide concentration present in the sour water.

Example 6

[0080] FIG. 8 is another graph depicting use of a cysteine synthase enzyme and a sulfide quinone reductase enzyme in sour water. The first set of samples to the far left is sour water without any treatment, which has a sulfide concentration of about 1800 ppm. Each sample had a total volume of 100 mL of sour water and enzyme where noted. The second set of samples includes a sour water sample having a sulfide concentration of about 1900 ppm, and the second sample of the second set of samples included the same sour water sample with 2 mL of sulfide quinone reductase enzyme. The third set of samples includes a sour water sample having a sulfide concentration of about 1700 ppm, and the second sample of the third set of samples included the same sour water sample treated with 2 mL of cysteine synthase enzyme. The 2 mL of each enzyme was obtained from a crude lysate, which is about 500 ppm of each enzyme

[0081] As can be seen in FIG. 8, the sulfide quinone reductase and the cysteine synthase enzymes both decrease the amount of sulfide concentration present in the sour water.

Example 7

[0082] FIG. 9 is a graph depicting a combination of cysteine synthase enzyme and sulfide quinone reductase enzyme use to decrease a percentage of sodium sulfide in a solution. Eight samples having the same solution and same percentage of sodium sulfide were subjected to various treatment amounts of cysteine synthase enzyme and sulfide quinone reductase enzyme combinations. The solution (in the absence of enzyme) comprised sodium sulfide from a stock solution having 1000 ppm, de-ionized water, 0.05 M Bis Tris buffer having a pH of 7.4, and 0.1 M Tris buffer having a pH of 7.5.

[0083] The first sample did not include any treatment and had a 100 percent sodium sulfide therein. The second sample included 300 ppm of sulfide quinone reductase enzyme in the absence of cysteine synthase enzyme. The third sample included 60 ppm cysteine synthase and 240 ppm sulfide quinone reductase enzyme. The fourth sample included 120 ppm cysteine synthase and 180 ppm sulfide quinone reductase enzyme. The fifth sample included 180 ppm cysteine synthase and 120 ppm sulfide quinone reductase enzyme. The sixth sample included 240 ppm cysteine synthase and 60 ppm sulfide quinone reductase enzyme. The seventh sample included 270 ppm cysteine synthase and 30 ppm sulfide quinone reductase enzyme. The eighth sample included 300 ppm cysteine synthase in the absence of sulfide quinone reductase enzyme. When both the cysteine synthase enzyme and the sulfide quinone reductase enzyme were used, the enzymes were added at the same time.

[0084] As can be seen from FIG. 9, varying combinations of cysteine synthase enzyme and sulfide quinone reductase enzyme were effective at decreasing the percentage of sodium sulfide present in the solution.

Example 8

[0085] FIG. 10 is another graph depicting a combination of cysteine synthase enzyme and sulfide quinone reductase enzyme use to decrease a percentage of sulfide in sour refinery water. Six samples having the same solution and same concentration of sulfide were subjected to various treatment amounts of cysteine synthase enzyme and sulfide quinone reductase enzyme combinations. The solution (in the absence of enzyme) comprised sulfide from a sour refinery water having 300 ppm of sulfide.

[0086] The first sample did not include any treatment and had 100 parts per million (ppm) of sulfide therein. The second sample included 150 ppm of sulfide quinone reductase enzyme and 750 ppm cysteine synthase enzyme. The third sample included 300 ppm of sulfide quinone reductase enzyme and 600 ppm cysteine synthase enzyme. The fourth sample included 600 ppm of sulfide quinone reductase enzyme and 300 ppm cysteine synthase enzyme. The fifth sample included 900 ppm of sulfide quinone reductase enzyme in the absence of any cysteine synthase enzyme. The sixth sample included 900 ppm cysteine synthase enzyme in the absence of any sulfide quinone reductase enzyme. When both the cysteine synthase enzyme and the sulfide quinone reductase enzyme were used, the enzymes were added at the same time.

[0087] As can be seen from FIG. 10, varying combinations of cysteine synthase enzyme and sulfide quinone reductase enzyme were effective at decreasing the percentage of sodium sulfide present in the solution. In addition, the sulfide concentration is decreased more when using a combination of the sulfide quinone reductase enzyme and the cysteine synthase enzyme than when either of the enzymes is used alone.

[0088] In the foregoing specification, the invention has been described with reference to specific embodiments thereof, and has been described as effective in providing methods, additive compositions, and fluid compositions for decreasing and/or removing hydrogen sulfide in downhole fluids circulated in a subterranean reservoir wellbore. However, it will be evident that various modifications and changes can be made thereto without departing from the broader spirit or scope of the invention as set forth in the appended claims. Accordingly, the specification is to be regarded in an illustrative rather than a restrictive sense. For example, specific base fluids, additional components, pyridoxal phosphates, and the like falling within the claimed parameters, but not specifically identified or tried in a particular composition or method, are expected to be within the scope of this invention.

[0089] The present invention may suitably comprise, consist or consist essentially of the elements disclosed and may be practiced in the absence of an element not disclosed. For instance, the additive composition for a base fluid, such as a drilling fluid, a completion fluid, a production fluid, a servicing fluid, an injection fluid, a refinery fluid, and combinations thereof may consist of or consist essentially of at least one cysteine synthase enzyme that is at least 75% homologous to the cDNA sequence of SEQ ID NO:1 and at least one sulfide quinone reductase enzyme that is at least 75% homologous to the cDNA sequence of SEQ ID NO:2.

[0090] The fluid composition may consist of or consist essentially of a base fluid, at least one cysteine synthase enzyme that is at least 75% homologous to the cDNA sequence of SEQ ID NO:1, and at least one sulfide quinone reductase enzyme that is at least 75% homologous to the cDNA sequence of SEQ ID NO:2; the base fluid may be or include, but is not limited to a drilling fluid, a completion fluid, a production fluid, a servicing fluid, an injection fluid, a refinery fluid, and combinations thereof.

[0091] The method may consist of or consist essentially of contacting at least one fluid with at least one cysteine synthase that is at least 75% homologous to the cDNA sequence of SEQ ID NO:1 and at least one sulfide quinone reductase that is at least 75% homologous to the cDNA sequence of SEQ ID NO:2 in an effective concentration to decrease and/or remove hydrogen sulfide in the fluid(s).

[0092] The words “comprising” and “comprises” as used throughout the claims, are to be interpreted to mean “including but not limited to” and “includes but not limited to”, respectively.