WO2020128055A1

WO2020128055A1 - A method of altering gas hydrate formation

Info

Publication number: WO2020128055A1
Application number: PCT/EP2019/086819
Authority: WO
Inventors: Prithwish Kumar Nandi NANDI; Mohammad Reza GHAANI; Niall Joseph ENGLISH; Christopher Curtis Royston Allen; Shamsudeen Umar DUNDARE; Jonathan Mark YOUNG; Timofey SKVORTSOV
Original assignee: Queens University of Belfast; University College Dublin
Current assignee: Queens University of Belfast; University College Dublin
Priority date: 2018-12-21
Filing date: 2019-12-20
Publication date: 2020-06-25
Anticipated expiration: 2021-06-21
Also published as: GB201820946D0

Abstract

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. Generally, the method comprises providing a source of gas hydrates; and contacting the source of gas hydrates with an isolated polypeptide, or fragment or analogue thereof, isolated from a bacterium of the Genus Methylophaga.

Description

Title of the Invention

A method of altering gas hydrate formation

Field of the Invention

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.

Background to the Invention

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. However, the promotion of clathrate formation (both more rapidly and in terms of ultimate conversion yield) is arguably 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).

Currently, methanol and other thermodynamic inhibitors (Tl) and, occasionally, classes of kinetic inhibitors are used to address the problem of hydrate crystallisation in pipeline flow assurance. However, the large economic expense and operationally challenging circumstances of having to remove the Tl downstream, for example to avoid poisoning people, is a disadvantage of these current solutions.

For wastewater treatment and desalination, the problem of hydrate crystallisation is addressed using settling tanks, reverse osmosis, ion-exchange processes, bubble columns, and membrane processes. However, these solutions pose problems such as high cost and slow water-treatment speed, in particular the very high cost associated with reverse osmosis in the process of desalination.

For gas storage, pressure-swing adsorption, vacuum-swing adsorption, chemical and physical storage (for example in ammonia boranes and zeolites/metal-organic frameworks (MOFs), respectively) are currently used solutions to the problem of hydrate crystallisation. However, there is an inability to deal with moisture (water vapour) in gas streams and n associated high cost with these solutions.

Summary of the Invention

According to a first aspect of the present invention, there is provided a method of altering gas hydrate formation, the method comprising the steps of:

(a) providing a source of gas hydrates; and

(b) contacting the source of gas hydrates with an isolated polypeptide, or fragment or analogue thereof,

wherein the polypeptide is isolated from a bacterium of the Genus Methylophaga.

Optionally, the method is a method of altering the rate of gas hydrate formation. Optionally, the method is a method of increasing gas hydrate formation and comprises the steps of:

(a) providing a source of gas hydrates; and

wherein the polypeptide is isolated from a bacterium of the Genus Methylophaga.

Optionally, the method is a method of increasing the rate of gas hydrate formation.

Optionally, the method is a method of decreasing gas hydrate formation and comprises the steps of:

(a) providing a polypeptide, or fragment or analogue thereof; and

(b) storing the polypeptide under conditions to prepare an altered polypeptide;

(c) providing a source of gas hydrates; and

(d) contacting the source of gas hydrates with the altered isolated polypeptide, or fragment or analogue thereof,

wherein the polypeptide is isolated from a bacterium of the Genus Methylophaga.

Optionally, the method is a method of decreasing the rate of gas hydrate formation.

According to a second aspect of the present invention, there is provided an isolated polypeptide, or fragment or analogue thereof, for use in a method of altering gas hydrate formation comprising the steps of:

(a) providing a source of gas hydrates; and

(b) contacting the source of gas hydrates with the polypeptide, or fragment or analogue thereof, wherein the polypeptide is isolated from a bacterium of the Genus Methylophaga .

According to a third aspect of the present invention, there is provided use of an isolated polypeptide, or fragment or analogue thereof for altering gas hydrate formation by: (a) providing a source of gas hydrates; and

(b) contacting the source of gas hydrates with the polypeptide, or fragment or analogue thereof, wherein the polypeptide is isolated from a bacterium of the Genus Methylophaga.

According to a further aspect of the present invention, there is provided a method of preparing an altered polypeptide, or fragment or analogue thereof, for use in a method of altering gas hydrate formation, the method comprising the steps of:

(a) providing a polypeptide, or fragment or analogue thereof; and

(b) storing the polypeptide under conditions to prepare an altered polypeptide;

wherein the polypeptide is isolated from a bacterium of the Genus Methylophaga.

Optionally, the contacting step comprises contacting the source of gas hydrates with a bacterium of the Genus Methylophaga.

Optionally, 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. Still further optionally, 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.

Optionally or additionally, 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.

Still further optionally or additionally, 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. Optionally, 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.

Optionally, the bacterium is the species Methylophaga aminisulfidivorans.

Optionally, the polypeptide has a molecular weight of about 40kDa.

Optionally, the polypeptide has the amino acid sequence:

MKLLKKSTLATLVGAAALTAAGAANATIVVGGENGYEFSVDGNINQFFIASDQDSASASANRDQDNQ

QVANGLLPTFFGFNVAMPEINGLKVAARVSISPSTNNGSYVNSDAAMEQREAFATVDGSFGQIMLG

KGLGLYSANNILLDQTLYGVGATGLTAAGDQNTGQTSLGRIGYGYEYANWRSQIRYTTPDMNGFKA

AVAVMDSDDFLTAKWNSSTTSNQFEKDARYEASLSYATAFDGGSMKLWLDGMTQDVRYSNATGS

EKSDAYTVGGQLVFGGIEAVAAYYDSKGQGVNGLGTGAFTADNKAREGDGYYAQLGYRFGGQTFV

AASYGKSTLDRDSANTVAGGVADLDTNSMTTIGIYHDVTANLKLVAEYSKIETEYHIQSSDDEVDVLS

LGGFISW; or a fragment thereof.

Optionally, the polypeptide has the amino acid sequence defined in SEQ ID NO:1.

Optionally, the polypeptide has the amino acid sequence: TAFDGGS.

Optionally, the polypeptide has the amino acid sequence defined in SEQ ID NO:2.

Optionally, the polypeptide has the amino acid sequence: AMPEINGLKVA.

Optionally, the polypeptide has the amino acid sequence defined in SEQ ID NO:3.

Optionally, the polypeptide has the amino acid sequence: LDRDSANGTPGGVADL.

Optionally, the polypeptide has the amino acid sequence defined in SEQ ID NO:4.

Optionally, the step of providing a source of gas hydrates comprises contacting a gas and water.

Optionally, 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. 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 17.9bar. 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.

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 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. 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 3.5°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.

Optionally, the gas is selected from methane, propane, carbon dioxide, and hydrogen. Further optionally, the gas is methane.

Optionally, the gas hydrate is selected from methane hydrate, propane hydrate, carbon dioxide hydrate, and hydrogen hydrate. Further optionally, the gas hydrate is methane hydrate.

Optionally, the water is selected from distilled water, wastewater, frack water, radioactive water, and seawater). Further optionally, the water is seawater.

Optionally, the storing step comprises storing the bacterium of the Genus Methylophaga under conditions to prepare an altered polypeptide.

Optionally, the storing step comprises storing the polypeptide or bacterium of the Genus

Methylophaga under conditions for at least one week. Further optionally, 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.

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. Still further optionally, 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.

According to a further aspect of the present invention, there is provided an isolated nucleic acid encoding the polypeptide isolated from the bacterium of the Genus Methylophaga.

According to a further aspect of the present invention, there is provided a vector comprising the isolated nucleic acid encoding the polypeptide isolated from the bacterium of the Genus

Methylophaga.

According to a further aspect of the present invention, there is provided host cell comprising the isolated nucleic acid encoding the polypeptide isolated from the bacterium of the Genus

Methylophaga.

Optionally, the host cell comprises the vector comprising the isolated nucleic acid encoding the polypeptide isolated from the bacterium of the Genus Methylophaga.

Optionally, 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.

Brief Description of the Drawings Embodiments of the present invention will now be described with reference to the following nonlimiting examples and the accompanying drawings in which:

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

(methane, propane, C0₂ and hydrogen) are supplied to the 0.34 litre, 200 bar-rated stainless-steel and rocker-mounted pressure vessel through the distribution terminal, with line-cleaning before purging the desired gas, by way of mass-flow controller and accurate measurement of gas loading into the (liquid-/sediment- loaded) reactor, and wherein the system operates under either isobaric or constant-volume modes, with a back-pressure cylinder for isobaric operation, where for the constant- volume case, the reactor’s inlet valve is closed upon reaching the desired pressure, and pressure logged digitally every second for the experiment’s duration (as described herein);

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.

aminisulfidivorans in the test cultures (T1-3) relative to the control (methanol-free) cultures (C1-3);

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 5 illustrates 10% SDS-PAGE resolving gel of total protein recovered from seawater- enrichment samples, wherein M: is pre-stained molecular-weight protein markers, where a relatively intense band was found around 40 kDa in the T1 acetone-concentrated supernatant (protein concentration post-acetone precipitation/resuspension = 0.4mg.ml-1); wherein the estimated protein concentration in the T1 supernatant before concentration (below the assay threshold) was 40pg.mf¹; 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) with GHP1 protein solution (4.2 pg/ml) in the same buffer; wherein each curve represents the average of 10 replicates for each test case; where a one-tailed Student’s t-test confirms the independence of the GHP1 and buffer solution result with a 99% confidence level (p-value = 2.21 *10-5), with GHP1 showing higher hydrate conversion, wherein at a pressure of 1 17.8 bar, the conversion is 15%, and preliminary rate analysis reveals that GHP1 -facilitated hydrate conversion is almost 5 times faster than that of the buffer solution in reaching this level (1.1 hours versus 5.2 hours);

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,

characteristic of nucleation towards critically-sized nuclei, wherein modelling this stage by the Avrami equation, a = 1-exp(-(kt)n), the reaction rate k for the GHP1 case (obtained by least-squares regression fitting) is an one order of magnitude higher; where during growth, after the steep pressure drop of nucleation in the first hour, the formation rate is almost linear (evidenced by straight-line regression fits) and the slopes are small, but that of GHP1 is still ~20% higher, although during the latter time at ~4.5-6 hours, this difference between any GHP1 and buffer-solution rate becomes essentially negligible; and

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.

Examples

Example 1

Culture preparation

Fresh seawater (82.5% v/v inoculum), obtained the evening before from Belfast Lough (Hollywood, Co Down, Northern Ireland) was used and stored overnight at 4°C added to a medium comprising (g/L): NaCI: 2.4, (NH₄)2S0₄: 1 .0, MgS0₄.7H₂: 1 .0, CaCI₂: 0.2, FeS0₄: 0.002, Na₂Mo0₄.2H₂0: 0.002, KH₂P0₄: 0.36, K₂HP0₄: 2.34, with 1 ml of Vishniacs’ trace-elements solution. This was added to Growth A medium to establish six 300 ml cultures, three of which had no additional carbon source (codified as‘C’ 1 , 2 & 3), and three of which had 0.3% v/v methanol (dubbed T 1 , 2 & 3); wherein incubation time was 18 days at 22°C. The final added phosphate concentration was approximately 2.34/174 = 13mM. There was noted to be considerable phosphate precipitation after the final medium was made up. Seawater contains circa 1 M salt, so this does not affect osmolarity significantly. The phosphate was autoclaved separately. Methanol has a density of 7.92 g/10ml and a molecular weight of 32 g/mol; thus, this corresponds to a molarity of (0.9 x 1000/300 x 0.792)/ 32 = 74 mM. The pH was measured as 6.9.

Example 2

Hydrate formation and mass-balance-based determination of hydrate-conversion yield

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 cm³ (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

temperature, with temperature/pressure readings every 1 s. Prior to each run, the vessel was sterilised by washing with ethanol solution (25 wt%). Each hydrate-kinetics experiment began with cooling down the main system to the desired temperature of 3.5°C (to mimic seafloor temperatures), via the temperature-control system. Once the system reached the desired temperature, the cell was charged with approximately 20 cm³ of deionised water with the selected amount of reagent (see Table 1). The reagents are selected in a way to keep the final solution in the same molar concentration. The cell was evacuated for 3 min by vacuum pump to remove any residual air, and then pressurised to the desired pressure of 120 bar (again, reflecting continental-shelf seafloor conditions) using pure methane. Due to inevitable Joule-Thomson thermal contraction, the cell pressure was decreased slightly by decreasing the temperature; however, within less than 10 minutes, the temperature stabilised and remains so until the end of the chose hydrate-formation period. Then, a continuous slow pressure decline was observed during the hydrate crystal-growth stage (always under the constant-volume conditions), after nucleation. In practice, however, there is some small temperature fluctuation during hydrate formation (i.e., the thermal trace of hydrate formation) due to its exothermic nature, but this is countered continually by the temperature-control system. Here, the average temperature of the production regime (i.e., the temperature plateau) is considered as the starting temperature of the reaction. The system was kept at (or very near) the desired temperature of 3.5°C for 6-9 hours to gauge the yield towards hydrate over this period. Table 1.

The‘yield’ for conversion to hydrate during formation (or, conversely, inferred when measuring dissociation) 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). Naturally, 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. The data reported by U.S. Dept of Commerce / National Institute of Standards and Technology was employed to calculate the compressibility factor of methane at various pressures and temperatures in the hydrate- formation runs from mass balances on thus-inferred gas-phase-number-of-moles data (from the gas-phase pressure), taking into account the temperature-variation of methane absorption in liquid with literature data for Henry’s Law constants for methane. Knowing the number of absorbed/released moles of methane, in addition to typical methane-hydrate cage-occupancy levels of 90% in the present P/T range 1 , allows for the percentage yield to methane hydrate to be calculated.

Example 3

DNA extraction, sequencing and bioinformatic analysis

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.

Sequencing read quality, length and adapter contamination were initially assessed using Fastqc. 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.

Table 3. Assembly statistics for the de-novo co-assembled metagenomes. Sequence reads were assembled using spades with the -meta flag and assembly quality was assessed using QUAST. 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.

The Statistical significance of the differences were inferred using Whites non-parametric t-test, whilst confidence intervals were calculated by Bootstrapping for 9999 replicates, P-values were corrected for multiple testing using Storeys false-discovery rate method.

For all three treatment samples, a de-novo co-assembly was performed using SPAdes (using the - meta flag) with the default settings. Prior to de-novo assembly, kmer-based normalisation was applied to reduce the impact a very low-abundance organisms on the assembly; normalisation was performed using bbnorm from the bbtools package, with the flags min = 10, max = 100, k = 32. To estimate the relative abundance (i.e. , coverage) of each contig in the original samples, quality- trimmed reads were mapped onto each contig using bbmap from the bbtools package with the flags k = 13 vslow = t, to ensure maximum mapping accuracy. Assembly quality was assessed using QUAST.

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.

In addition, the 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.

Example 4

Protein precipitation and identification from culture supernatants

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).

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).

Example 5

Mass Spectrometry

For individual protein band analysis, 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. For the gel band peptides were eluted using the following gradient: 2-40% acetonitrile in 16 min, 40-98% in a further 3 min, followed by 98% acetonitrile to clean the column before re-equilibration to 2% acetonitrile; for the gel chunk peptides were eluted using the following gradient: 2-20% acetonitrile in 60 min, then 20-40% in a 15 min and 40-98% in a further 5 min followed by 98% acetonitrile to clean the analytical column before re-equilibration to 2% acetonitrile. 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. The MS/MS data file generated via the‘Create mgf file’ script in PeakView (Sciex) was analysed using the Mascot search algorithm (Matrix

Science), against the NCBInr database (Aug 2016) all species (93482448 sequences), using trypsin as the cleavage enzyme and Carbamidomethylation as fixed modification of cysteine and methionine oxidation as a variable modifications. The peptide-mass tolerance was set to 20 ppm, with MSMS- mass tolerance to ± 0.1 Da.

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.

Example 6

Protein cloning and expression

An identified protein of the present invention was designated GHP-1. The synthesis of GHP-1 gene, followed by its cloning, heterologous expression and purification, was conducted at Genscript (USA) to produce enough protein for hydrate-formation experiments. 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.

In the present invention, 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’. The test (T) cultures alone harboured significant microbial growth after incubation when compared to the controls (‘C’). Metagenome analysis of recovered pelleted cells revealed a mixed culture dominated by the presence of known methylotrophic Methylophaga aminisulfidivorans in all three T1-3 cultures. Aged T1 supernatants, stored at 4°C for several weeks, did not produce this effect, suggesting a biological component was causing this kinetic enhancement. The post-centrifugation T1 -culture supernatant, with addition of further seawater, was then placed in a temperature-controlled hydrate- formation pressure vessel at 3.5 °C, and allowed to form methane hydrate (featuring chemically- and heat- treated marine sand, to neutralise any background effects), exposed to methane gas at 120 bar. Critically, we observed that the rate of accumulation of hydrate in the T1 test mixture, using freshly-obtained culture supernatant that was less than a week old, showed an enhanced rate of hydrate formation when compared to the C1 culture that was identical except for the addition of a methanol growth substrate. T1 stored at 3.5 °C for several weeks showed evidence of hydrate- formation inhibition vis-a-vis C1.

Naturally, TTs intriguing time-dependent promotion/inhibition‘dichotomy’ is suggestive of a potential microbe-derived activity underpinning this clear and substantial microbial effect, potentially based on a possible protein mechanism. To elucidate the precise biological origin, we next analysed a sample of the cell-free supernatant from the centrifuged T1 culture using mass spectrometry to reveal the presence of any secreted proteins/peptides that may have been present. Following concentration using acetone precipitation, a single 40 kD protein was revealed in the T1 supernatant after SDS page visualisation. Returning to the total acetone extract, the combined proteins were digested into peptides using a trypsin pre-treatment and then analysed using LC-MSMS. Proteins were identified and annotated based on the resultant peptide sequence information obtained. The spectra were searched against the MASCOT database revealing the highest probability score for the T1 c40Kd gel band as a hypothetical protein from Methylophaga aminisulfidivorans (probability score of 1023 and 62% sequence coverage). Other candidate proteins identified had markedly lower MASCOT probability scores. A subsequent BLAST search against the NCBI-nr protein database with the full 42.5.kD sequence of the hypothetical protein showed highest similarity (>95% amino acid sequence identity) to a porin-like protein from M. aminisulfidivorans [accession code: WP 007147016] Taxonomic analysis of the T1 -T3 metagenomes from the culture flasks revealed that the methanol- enriched cultures were overwhelmingly dominated by members of the genus Methylophaga. De-novo co assembly of the raw reads resulted in a library of 1 1721 contigs > 10OObp in length with an N50 of 1 1839. A library of 385,489 open-reading frames (ORFs) were identified in these contigs. The top-hit protein from the LC-MSMS analysis was used as a query sequence in a BLAST search against this library with a sequence identity cutoff of 50% and query-coverage cutoff of 90%, resulting in 10 hits. Further bioinformatic analysis of these ORFS using PSORT16 revealed that all appear to include putative signal peptides, suggesting that they are transported across the inner cell membrane.

Further, the PSORT analysis also predicted that the GHP1 was specifically an outer-membrane protein - although this observation would need further experimental confirmation. Taxonomic annotation of the top hit ORFS also revealed that all are most similar to proteins from the genus Methlyophaga and the most abundant hits in the T1 sample are all most similar to porins from M. aminisulfidivorans. The ORF with the highest identity to the major supernatant protein and the highest abundance (i.e., coverage) in the T1 metagenome (ORF 64197, 95.95% identity to the protein detected in the culture supernatant), 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).

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.

Comparisons were made between the protein solubilisation buffer and the GHP1 suspended in the same buffer.

Examples of the effect of purified GHP1 on hydrate formation over 10 replicates of each test case (i.e., GHP1 versus the buffer solution without GHP1 as the control) showed that the addition of the GHP1 protein led to an increase in hydrate-conversion yield by some 23 % under these experimental conditions against this control. The observed conversions over the 6-hour hydrate-formation times were 16.2 ± 1 .6 and 20.0 ± 1 .6 % in the case of absence and the presence of GHP1 , respectively. The statistical significance of the GHP1 -impact hypothesis (based on 10 replicates of test and control) was proved through a one-tailed Student’s t-test (reaching the 99 % confidence level). A comparison of two-stage hydrate-formation kinetics (nucleation and growth) shows that the presence of GHP1 leads to 15 % conversion almost five times faster (i.e., 1 .1 versus 5.2 hours. Using an Avrami analysis of hydrate nucleation points to a nucleation rate of an order of magnitude higher for GHP1 vis-a-vis the control. In addition, a boiled GHP1 showed no apparent hydrate-nucleation- promoting activity relative to the control; nor did use of pure deionised water (with no GHP1) instead.

By using microbially-grown progenitor enzymes, and their biomimetic, protein-engineered analogues (and even peptide sub-sequences); the inventors have demonstrated that clathrate-hydrate crystallisation kinetics, as well as ultimate gas-hydrate-conversion yield, may be altered. Indeed, if desired, by controlling the age and storage history of proteins, this hydrate-formation kinetics can be suppressed, with proteins and peptide sequences acting as hydrate-formation inhibitors. 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.

Claims

1 . A method of altering gas hydrate formation, the method comprising the steps of:

(a) providing a source of gas hydrates; and

wherein the polypeptide is isolated from a bacterium of the Genus Methylophaga.

2. A method according to Claim 1 , wherein the method decreases gas hydrate formation and comprises the steps of:

(i) providing the polypeptide, or fragment or analogue thereof; and

(ii) storing the polypeptide, or fragment or analogue thereof, under conditions to prepare an altered polypeptide;

prior to the providing a source of gas hydrates step (a).

3. A method according to Claim 1 or 2, wherein the contacting step comprises contacting the source of gas hydrates at a pressure of up to 120.0bar.

4. A method according to any one of Claims 1 -3, wherein the contacting step comprises

contacting the source of gas hydrates at a temperature of at least 1 0°C.

5. A method according to any one of Claims 1 -4, wherein the contacting step is performed for at least one hour.

6. A method according to any one of Claims 1 -5, wherein the bacterium is the species

Methylophaga aminisulfidivorans.

7. A method according to any one of Claims 1 -6, wherein the polypeptide has the amino acid sequence defined in SEQ ID NO:1 , or a fragment or analogue thereof.

8. A method according to any one of Claims 1 -7, wherein the polypeptide has the amino acid sequence defined in SEQ ID NO:2, or a fragment or analogue thereof.

9. A method according to any one of Claims 1 -8, wherein the polypeptide has the amino acid sequence defined in SEQ ID NO:3, or a fragment or analogue thereof.

10. A method according to any one of Claims 1 -9, wherein the polypeptide has the amino acid sequence defined in SEQ ID NO:4 or a fragment or analogue thereof.

11. A method according to any one of Claims 1-10, wherein the step of providing a source of gas hydrates comprises contacting a gas and water.

12. A method according to Claim 11 , wherein the gas is selected from methane, propane,

carbon dioxide, and hydrogen.

13. A method according to Claim 11 or 12, wherein the water is selected from distilled water, wastewater, frack water, radioactive water, and seawater.

14. A method according to any one of Claims 2-13, wherein the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga under conditions for at least one week.

15. A method according to any one of Claims 2-14, wherein the storing step comprises storing the polypeptide or bacterium of the Genus Methylophaga at a temperature of less than 20°C.