WO2020128055A1 - Procédé de modification de formation d'hydrates de gaz - Google Patents

Procédé de modification de formation d'hydrates de gaz Download PDF

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Publication number
WO2020128055A1
WO2020128055A1 PCT/EP2019/086819 EP2019086819W WO2020128055A1 WO 2020128055 A1 WO2020128055 A1 WO 2020128055A1 EP 2019086819 W EP2019086819 W EP 2019086819W WO 2020128055 A1 WO2020128055 A1 WO 2020128055A1
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Prior art keywords
polypeptide
gas
source
hydrate
contacting
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Ceased
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PCT/EP2019/086819
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English (en)
Inventor
Prithwish Kumar Nandi NANDI
Mohammad Reza GHAANI
Niall Joseph ENGLISH
Christopher Curtis Royston Allen
Shamsudeen Umar DUNDARE
Jonathan Mark YOUNG
Timofey SKVORTSOV
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Queens University of Belfast
University College Dublin
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Queens University of Belfast
University College Dublin
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Publication of WO2020128055A1 publication Critical patent/WO2020128055A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G OR C10K; LIQUIFIED PETROLEUM GAS; USE OF ADDITIVES TO FUELS OR FIRES; FIRE-LIGHTERS
    • C10L3/00—Gaseous fuels; Natural gas; Synthetic natural gas obtained by processes not covered by subclass C10G, C10K; Liquefied petroleum gas
    • C10L3/06—Natural gas; Synthetic natural gas obtained by processes not covered by C10G, C10K3/02 or C10K3/04
    • C10L3/10—Working-up natural gas or synthetic natural gas
    • C10L3/107—Limiting or prohibiting hydrate formation
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G OR C10K; LIQUIFIED PETROLEUM GAS; USE OF ADDITIVES TO FUELS OR FIRES; FIRE-LIGHTERS
    • C10L2250/00—Structural features of fuel components or fuel compositions, either in solid, liquid or gaseous state
    • C10L2250/02—Microbial additives
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G OR C10K; LIQUIFIED PETROLEUM GAS; USE OF ADDITIVES TO FUELS OR FIRES; FIRE-LIGHTERS
    • C10L2250/00—Structural features of fuel components or fuel compositions, either in solid, liquid or gaseous state
    • C10L2250/04—Additive or component is a polymer
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G OR C10K; LIQUIFIED PETROLEUM GAS; USE OF ADDITIVES TO FUELS OR FIRES; FIRE-LIGHTERS
    • C10L2290/00—Fuel preparation or upgrading, processes or apparatus therefore, comprising specific process steps or apparatus units
    • C10L2290/14—Injection, e.g. in a reactor or a fuel stream during fuel production
    • C10L2290/141—Injection, e.g. in a reactor or a fuel stream during fuel production of additive or catalyst

Definitions

  • the present invention relates to a method of altering gas hydrate formation. Also disclosed is an isolated polypeptide, fragments and analogues thereof, for use in the method of altering gas hydrate formation. Further, there is disclosed a method of preparing an altered polypeptide, fragments and analogues thereof, for use in the method of altering gas hydrate formation.
  • Clathrate hydrates are non-stoichiometric crystalline inclusion compounds in which a water host lattice encages small guest atoms or molecules in cavities. Methane hydrates are the most widespread clathrate in nature - in the permafrost and relatively shallow continental-shelf ocean regions - and constitute a significant energy resource. The large quantity of marine methane hydrates has driven substantial interest in methane-gas-fuel potential.
  • Hydrate crystallisation for example, in pipeline flow assurance, is a major industrial processengineering problem in the gas sector, whilst suppression of hydrate crystallisation is also important for retrieving natural gas from marine sediments.
  • the promotion of clathrate formation is ideally an even more important industrial opportunity, for example for wastewater treatment, desalination (or gas storage, like carbon capture) on carbon dioxide streams (whether pure or in waste-flue gas form).
  • thermodynamic inhibitors Tl
  • classes of kinetic inhibitors are used to address the problem of hydrate crystallisation in pipeline flow assurance.
  • Tl thermodynamic inhibitors
  • a method of altering gas hydrate formation comprising the steps of:
  • polypeptide is isolated from a bacterium of the Genus Methylophaga.
  • the method is a method of altering the rate of gas hydrate formation.
  • the method is a method of increasing gas hydrate formation and comprises the steps of:
  • polypeptide is isolated from a bacterium of the Genus Methylophaga.
  • the method is a method of increasing the rate of gas hydrate formation.
  • the method is a method of decreasing gas hydrate formation and comprises the steps of:
  • polypeptide is isolated from a bacterium of the Genus Methylophaga.
  • the method is a method of decreasing the rate of gas hydrate formation.
  • an isolated polypeptide, or fragment or analogue thereof, for use in a method of altering gas hydrate formation comprising the steps of:
  • an isolated polypeptide, or fragment or analogue thereof for altering gas hydrate formation by: (a) providing a source of gas hydrates; and
  • a method of preparing an altered polypeptide, or fragment or analogue thereof, for use in a method of altering gas hydrate formation comprising the steps of:
  • polypeptide is isolated from a bacterium of the Genus Methylophaga.
  • the contacting step comprises contacting the source of gas hydrates with a bacterium of the Genus Methylophaga.
  • the contacting step comprises contacting the source of gas hydrates at a pressure of up to 120.0bar. Further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 19.5bar. Still further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 19.0bar. Still further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 18.5bar. Still further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 18.0bar. Still further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 17.9bar.
  • the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 17.8bar. Still further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 17.7bar. Still further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 17.6bar. Still further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 17.5bar. Still further optionally, the contacting step comprises contacting the source of gas hydrates at a pressure of up to 1 17.0bar.
  • the contacting step comprises contacting the source of gas hydrates at a temperature of at least 1 0°C. Further optionally or additionally, the contacting step comprises contacting the source of gas hydrates at a temperature of at least 1 .5°C. Still further optionally or additionally the contacting step comprises contacting the source of gas hydrates at a temperature of at least 2.0°C. Still further optionally or additionally, the contacting step comprises contacting the source of gas hydrates at a temperature of at least 2.5°C. Still further optionally or additionally, the contacting step comprises contacting the source of gas hydrates at a temperature of at least 3°C.
  • the contacting step comprises contacting the source of gas hydrates at a temperature of at least 3.5°C. Still further optionally or additionally, the contacting step comprises contacting the source of gas hydrates at a temperature of at least 4.0°C.
  • the contacting step is performed for at least one hour. Further optionally, the contacting step is performed for at least two hours. Still further optionally, the contacting step is performed for at least three hours. Still further optionally, the contacting step is performed for at least four hours. Still further optionally, the contacting step is performed for at least five hours. Still further optionally, the contacting step is performed for at least six hours. Still further optionally, the contacting step is performed for at least seven hours. Still further optionally, the contacting step is performed for at least eight hours. Still further optionally, the contacting step is performed for at least nine hours.
  • the bacterium is the species Methylophaga aminisulfidivorans.
  • the polypeptide has a molecular weight of about 40kDa.
  • polypeptide has the amino acid sequence:
  • LGGFISW LGGFISW; or a fragment thereof.
  • polypeptide has the amino acid sequence defined in SEQ ID NO:1.
  • polypeptide has the amino acid sequence: TAFDGGS.
  • polypeptide has the amino acid sequence defined in SEQ ID NO:2.
  • polypeptide has the amino acid sequence: AMPEINGLKVA.
  • polypeptide has the amino acid sequence defined in SEQ ID NO:3.
  • polypeptide has the amino acid sequence: LDRDSANGTPGGVADL.
  • polypeptide has the amino acid sequence defined in SEQ ID NO:4.
  • the step of providing a source of gas hydrates comprises contacting a gas and water.
  • the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 120.0bar. Further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 119.5bar. Still further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 19.0bar. Still further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 18.5bar. Still further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 18.0bar.
  • the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 17.9bar. Still further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 17.8bar. Still further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 17.7bar. Still further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 17.6bar. Still further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 17.5bar. Still further optionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a pressure of up to 1 17.0bar.
  • the step of providing a source of gas hydrates comprises contacting a gas and water at a temperature of at least 1 0°C. Further optionally or additionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a temperature of at least 1 5°C. Still further optionally or additionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a temperature of at least 2.0°C. Still further optionally or additionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a temperature of at least 2.5°C. Still further optionally or additionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a temperature of at least 3°C.
  • the step of providing a source of gas hydrates comprises contacting a gas and water at a temperature of at least 3.5°C. Still further optionally or additionally, the step of providing a source of gas hydrates comprises contacting a gas and water at a temperature of at least 4.0°C.
  • the gas is selected from methane, propane, carbon dioxide, and hydrogen. Further optionally, the gas is methane.
  • the gas hydrate is selected from methane hydrate, propane hydrate, carbon dioxide hydrate, and hydrogen hydrate. Further optionally, the gas hydrate is methane hydrate.
  • the water is selected from distilled water, wastewater, frack water, radioactive water, and seawater). Further optionally, the water is seawater.
  • the storing step comprises storing the bacterium of the Genus Methylophaga under conditions to prepare an altered polypeptide.
  • the storing step comprises storing the polypeptide or bacterium of the Genus
  • the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga under conditions for at least two weeks. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga under conditions for at least three weeks. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga under conditions for at least one month. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga under conditions for at least two months. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus
  • Methylophaga under conditions for at least three months.
  • the storing step comprises storing the polypeptide or bacterium of the Genus
  • the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 20°C. Further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 15°C. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 10°C. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 5°C. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 4°C.
  • the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 3°C. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 2°C. Still further optionally, the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 1 °C.
  • an isolated nucleic acid encoding the polypeptide isolated from the bacterium of the Genus Methylophaga.
  • a vector comprising the isolated nucleic acid encoding the polypeptide isolated from the bacterium of the Genus
  • host cell comprising the isolated nucleic acid encoding the polypeptide isolated from the bacterium of the Genus
  • the host cell comprises the vector comprising the isolated nucleic acid encoding the polypeptide isolated from the bacterium of the Genus Methylophaga.
  • the host cell is an Escherichia bacterial cell. Further optionally, the host cell is an Escherichia coli bacterial cell. Still further optionally, the host cell is an Escherichia coli BL21 bacterial cell. Still further optionally, the host cell is an Escherichia coli BL21 (DE3) bacterial cell.
  • Figure 1 illustrates an extended error-bar plot showing differences in taxonomic makeup between the treatment (methanol-enriched) and control flasks, wherein raw DNA sequence reads were taxonomically annotated as described herein, wherein the left panel shows a bar plot with the mean proportions of different species identified (where error bars are shown as standard error of the mean) and the right-hand panel shows the differences between these proportions with error bars shown as 95% confidence interval of the effect size, and wherein the statistical significance of the differences were inferred using Whites non-parametric t-test, and confidence intervals were calculated by Bootstrapping for 9999 replicates, P-values were corrected for multiple testing using Storeys false- discovery rate method, and only species with a corrected P-value ⁇ 0.05 are shown;
  • Figure 2 illustrates a schematic of a gas-hydrate rig, wherein the four main sections are: gas supplier, distribution terminal, reactor and refrigerator, wherein high-purity (N5-level) gases
  • Figure 3 illustrates metagenomic analysis of methanol-enriched seawater cultures and reveals significant enrichment of Methylophaga aminisulfidivorans from analysis of raw DNA sequence reads, wherein the figure shows the normalised proportion of reads which aligned to M.
  • Figure 4 illustrates a typical comparison of hydrate formation for cell-free supernatants, T1 and C1 , over a 6-hour timeframe, showing that - in a seawater milieu - there is an evident increase in hydrate formation due to the effect of a component in the T1 solution, wherein the T1 and C1 solution differ only in the fact that methanol was added to promote the growth of methanol-degrading bacteria in the seawater medium, wherein whole cells were removed by centrifugation prior to testing, and the supernatant was tested within one week of preparation (stored at 4°C);
  • Figure 6 illustrates a comparison of pressure behaviour through hydrate formation for TRIS-0.10% wt.
  • glycerol buffer solution 0.5mM TRIC HCI, 0.150 mM NaCI, pH 7.0
  • GHP1 protein solution 4.2 pg/ml
  • Figure 7 illustrates two-regime kinetic analysis of averaged GHP1 versus buffer-solution hydrate formation (framed as nucleation - on the right, and growth - on the left), wherein‘Alpha’, a, denotes the fractional conversion to hydrate, wherein up to about 1 hour, it can be seen that early-stage, incipient formation is dominated by a rapid drop in pressure and increase in conversion,
  • Figure 8 illustrates the behaviour of three selected polypeptide fragments in the formation of hydrate with deionized water; wherein, following a molecular dynamics simulation, we found that the interaction of a model for the polypeptide with methane and water can yield‘half cage’ precursors to full hydrate cages; which analysis also suggested that there were three separate domains that could have a catalytic role in facilitating the formation of energetically stabilised methane-hydrate precursors in the polypeptide of the invention.
  • the pH was measured as 6.9.
  • the experimental apparatus for hydrate-formation and dissociation kinetics (as well as estimation of dissociation temperature) employed a pressure vessel fabricated using 316 stainless steel with internal volume of approximately 340 cm 3 (see Fig. 2).
  • the vessel was agitated using a tilting shaker.
  • a pressure transducer with an uncertainty of 0.02 MPa, was used to measure pressure, whilst a thermocouple with an accuracy of ⁇ 0.1 K was inserted into the cell to measure the inner
  • The‘yield’ for conversion to hydrate during formation is calculated based on the number of absorbed moles of gas into the liquid/solid phase (i.e., by monitoring gas-phase pressure drop continuously on a mass-balance basis).
  • the first step in this number-of-gas-phase-moles-from-pressure determination lies in defining accurate the de-facto compressibility factor of the methane.
  • DNA was extracted from of each of the cell pellets using a Powersoil DNA extraction kit (Mobio) according to the manufacturers instructions, each extraction was eluted in 10Opl molecular grade water (Sigma-Aldrich). Final DNA concentrations were measured using a Quantus Fluorometer (Promega) in conjunction with the Quantiflour DsDNA dye system (Promega). Generally, treatment flasks showed a much higher DNA yield than control flasks indicating increased microbial biomass in the enriched cultures.
  • DNA-sequencing libraries were prepared using a Nextera library preparation kit (lllumina) and subsequent sequencing libraries were sequenced on a NextSeq500 DNA sequencer in mid-output mode for 300 cycles (2x150 bp) resulting in total of 340,959,304 paired-end sequence reads.
  • Adapter trimming was performed using bbduk from the bbtools package using the provided library of lllumina adapter sequences. Quality trimming was also performed using bbduk form the bbtools package; reads were trimmed to a minimum quality score of 20 over a sliding window of 10 bases.
  • Preliminary taxonomic analysis of the quality-trimmed reads was performed by using kaiju against a database of all proteins from bacteria, archaea, single-celled eukaryotes and viruses in the NCBI-nr protein database, allowing a maximum of 5 mismatches and a minimum bit score of 60. Significant differences in the taxonomic profiles between the treatment and control culture flasks were identified using STAMP (see Tables 2 & 3). Table 2. Total DNA yield per cell pellet from each culture flask and total paired-end sequencing reads returned from each sequencing library.
  • N50 is the contig size in base pairs over which 50% of the total assembly length is contained in contigs of this size.
  • L50 is the number of contigs of length N50 or greater.
  • N75 is the contig size in base pairs over which 75% of the total assembly length is contained in contigs of this size.
  • L75 is the number of contigs of length N75 or greater.
  • Open-reading frames were identified in all contigs > 1000bp using Prodigal30 with the -meta flag, resulting in a library of 385,489 ORFs.
  • the top-hit protein from the mass-spec analysis was then used as a query sequence in BLASTp31 search against the amino-acid translations of this library of ORFS with a percentage identity cutoff of 50% resulting in 10 hits with an amino acid sequence identity > 50% to the query sequence.
  • the ORF with highest sequence identity to the query sequence (ORF 64197) and highest relative abundance (i.e., coverage) in the T1 sample was selected for gene synthesis and downstream cloning.
  • top-hit ORFS were analysed for putative signal peptides using signalP (using the sensitive setting) and Phobius and subcellular localisation was predicted using Phobius and Psort 3.0 16. Both signalP and Phobius were in agreement that all top hit proteins contained putative signal peptides. Phobius also predicted all proteins were non-cytoplasmic in nature and Psort predicted that that all were gram negative outer-membrane proteins.
  • the total protein of the supernatant was recovered by acetone precipitation. This ensured concentration of the protein as well as purification from contaminants. Briefly, cold acetone four times volume of the protein sample was added to the sample, vortexed and incubated at -20°C for 60 min. After incubation, the mixture was centrifuges and the supernatant was decanted to obtain the protein precipitates. Protein precipitates were resuspended in HEPES (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) (pH 7.4).
  • HEPES 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid
  • the concentration of the recovered protein was estimated using the Coomassie (Bradford) protein assay kit (Thermo Scientific, IL) with Bovine serum albumin (BSA) used as the standard. A protein concentration of 0.4 mg/ml was determined. However, we estimate the concentration of the protein directly from the supernatant to be 40 pg/ml.
  • Recovered protein was mixed with equal volume of SDS loading dye, heated at 95°C for 5 min and ran on a 10% SDS-PAGE resolving gel (see Figure 5).
  • the gel band and gel chunk was cut into 1-mm cubes. These were then subjected to in-gel digestion, using a ProGest Investigator in-gel digestion robot (Genomic Solutions, Ann Arbor, Ml) using standard protocols. Briefly, the gel cubes were de-stained by washing with 50% ACN and subjected to reduction and alkylation before digestion with trypsin at 37°C. The peptides were extracted with 5% formic acid and concentrated down to 20 pL using a SpeedVac (ThermoSavant).
  • a portion of the resultant peptides were then injected on an Acclaim PepMap 100 C18 trap and an Acclaim PepMap RSLC C18 column (ThermoFisher Scientific), using a nanoLC Ultra 2D plus loading pump and nanoLC as-2 autosampler (Eksigent). Peptides were loaded onto the trap column for 10min at a flow rate of 5pl/min of loading buffer (98% H20/2% ACN/0.05% TFA). Trap column was then switched in line with the analytical column and peptides were eluted with a gradient of increasing acetonitrile, containing 0.1 % formic acid. Flow rate was 300 nl/min.
  • the eluate was sprayed into a TripleTOF 5600+ electrospray tandem mass spectrometer (ABSciex, Foster City, CA) and analysed in Information Dependent Acquisition (IDA) mode, performing 120 msec of MS followed by 80 msec MSMS analyses on the 20 most intense peaks seen by MS.
  • IDA Information Dependent Acquisition
  • the MS/MS data file generated via the‘Create mgf file’ script in PeakView (Sciex) was analysed using the Mascot search algorithm (Matrix
  • a protein was accepted as identified if it had two or more peptides with MASCOT Ion Scores above the Identity (p ⁇ 0.05) Threshold and, for those proteins identified by only two peptides, the MSMS spectral assignments match most of the peaks in the MSMS spectra.
  • GHP-1 An identified protein of the present invention was designated GHP-1.
  • Target DNA sequence of GHP-1 was codon-optimised, and the synthesised sequence was cloned into vector pET-30a(+) (Merck) with 6*His tag for protein expression in E. coli BL21 (DE3).
  • Bacterial cells transformed with the recombinant plasmid and stored in glycerol were used to inoculate TB medium containing kanamycin.
  • the bacterial culture was incubated at 37°C with sufficient agitation until OD600 reached 1.2, then induced with IPTG and further incubated at 15°C for 16 h.
  • Cells were harvested by centrifugation, resuspended in lysis buffer and disrupted by sonication on ice. The cell lysate was centrifuged and precipitate dissolved in urea. The supernatant containing denatured inclusion bodies of GHP-1 was collected and protein renaturation and refolding were carried out. The purity of the resulting protein preparation was assessed using SDS-PAGE and Western blot assay (Primary antibody: Mouse-anti-His mAb, GenScript, Cat. No. A00186), after which the purified protein was aliquoted and kept at -80°C in a storage buffer containing 50 mM Tris-HCI, 150 mM NaCI, 10% glycerol, pH 8.0.
  • mixed marine methylotroph cultures were grown on methanol as the sole carbon source, to ascertain if (and how) any extracellular elements affect methane-hydrate formation and stability at seafloor conditions.
  • a fresh seawater inoculum was used with a defined growth medium to establish six 300 ml cultures, three of which had no additional carbon source added (codified as‘C’ cultures) and three of which had 0.3% v/v methanol added (dubbed T cultures); the incubation time was 18 days at 22 °C, and further details of preparation are in‘Methodology’.
  • ORFs open-reading frames
  • the ORF with the highest identity to the major supernatant protein and the highest abundance (i.e., coverage) in the T1 metagenome was then selected for downstream synthesis, cloning and over-expression (E.coli pET-30a(+) vector); this is hereafter referred to as‘gas-hydrate protein’ 1 (GHP1).
  • GFP1 gas-hydrate protein
  • the E.coli-expressed GHP1 protein was purified into TRIS-glycerol buffer, to facilitate protein solubilisation and pH stability, using affinity chromatography (Genescript). The pure protein was then employed in further trials to assess the potential for acceleration of gas-hydrate formation.
  • clathrate-hydrate crystallisation kinetics as well as ultimate gas-hydrate-conversion yield
  • the present invention can be used to regulate and control hydrate crystallisation, whether to suppress (e.g., in pipeline flow-assurance applications) it, or to promote (e.g., in hydrate formation for wastewater- treatment applications).
  • Hydrates lead to highly purified water, acting as the ultimate molecular-level filter. Without being bound by theory, it is thought that the water lattice framework of the hydrates rejects anything other than the guest in the cages - with extreme prejudice. This rejection leads to subsequent melting of the hydrate to produce greatly- purified water.
  • the present invention uses biological accelerants or catalysts to alter this process of rapid clathrate growth and higher conversion yield.
  • the present invention demonstrates that controlling the temperature history of the protein allows for folding to the point where hydrate formation is actually inhibited, providing an approach to anti-hydrate action in flow-assurance applications, and may be of use in marine harvesting of gas from hydrates, to induce potential hydrate break-up and consequent latent-heat and gas release for natural-gas production from hydrates.

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  • Engineering & Computer Science (AREA)
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Abstract

La présente invention concerne un procédé de modification de la formation d'hydrates de gaz. L'invention concerne également un polypeptide isolé, des fragments et des analogues de ce dernier, destinés à être utilisés dans le procédé de modification de la formation d'hydrates de gaz. En outre, l'invention concerne un procédé de préparation d'un polypeptide modifié, de fragments et d'analogues de ce dernier, destinés à être utilisés dans le procédé de modification de la formation d'hydrates de gaz. Généralement, le procédé comprend la fourniture d'une source d'hydrates de gaz ; et la mise en contact de la source d'hydrates de gaz avec un polypeptide isolé, ou un fragment ou analogue de ce dernier, isolé à partir d'une bactérie du genre Methylophaga.
PCT/EP2019/086819 2018-12-21 2019-12-20 Procédé de modification de formation d'hydrates de gaz Ceased WO2020128055A1 (fr)

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Citations (2)

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US20050161631A1 (en) * 2002-04-12 2005-07-28 Walker Virginia K. Antifreeze proteins for inhibition of clathrate hydrate formation and reformation
WO2014202089A2 (fr) * 2013-06-18 2014-12-24 Roskilde Universitet Variants de polypeptides antigel

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050161631A1 (en) * 2002-04-12 2005-07-28 Walker Virginia K. Antifreeze proteins for inhibition of clathrate hydrate formation and reformation
WO2014202089A2 (fr) * 2013-06-18 2014-12-24 Roskilde Universitet Variants de polypeptides antigel

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HUANG ZENG ET AL: "Effect of antifreeze protein on nucleation, growth and memory of gas hydrates", AICHE JOURNAL, vol. 52, no. 9, 1 September 2006 (2006-09-01), US, pages 3304 - 3309, XP055676051, ISSN: 0001-1541, DOI: 10.1002/aic.10929 *

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