Clean biocide

Abstract

The present invention relates to methods of producing halophyte extract. In particular the present invention relates to the use of halophyte extract for reducing MIC-bacteria, especially sulphate-reducing bacteria and / or biofilm.

Description

[0001] Clean biocide

[0002] Technical field of the invention

[0003] The present invention relates to methods of producing halophyte extract. In particular the present invention relates to the use of halophyte extract for reducing MIC-bacteria, especially sulphate-reducing bacteria and / or biofilm.

[0004] Background of the invention

[0005] Offshore oil production facilities are subjectable to internal corrosion, potentially leading to human and environmental risk and significant economic losses. Microbiologically influenced corrosion (MIC) and reservoir souring - sulfide production by sulfate-reducing microorganisms - is believed to be an essential factor in this significant problem for the petroleum industry. Souring occurs mainly due to water flooding during secondary oil recovery, where seawater can contain large amounts of sulfate (up to 25-30 mM). MIC is often seen as localized pitting attacks that are generally associated with the presence of microbial communities embedded in a matrix (often with bioinorganic matrixes) referred to as biofilms. Currently, toxic and non-eco-friendly biocides are being used to reduce biofilm formation and subsequent MIC.

[0006] Halophytes are plant species that grow in seawater. These plants have been shown to produce a wide variety of very active phytochemicals, including potent antimicrobials. Halophytes specialize in thriving under competitive saline aquatic environments, protecting themselves from attacks by seawater bacteria and archaea.

[0007] US2021 / 0324526 discloses e.g. a method for mitigating or eliminating microbially influenced corrosion (MIC) at a site of interest i.e., a metal surface of an equipment for refining, storing, transporting of oil and natural gas is provided.

[0008] WO2018 / 125694, which is within the field of skin formulations, discloses dried extracts of e.g. Salicornia herbacae collected after Soxhlet extraction and drying, milling milled to a fine powder. The dried plant powder were extracted for eight hours at 100°C with ethanol (v / v) using a Soxhlet extractor followed by drying.

[0009] W02019 / 014061 discloses compositions and methods for controlling souring and corrosion causing prokaryotes, such as SRP, by treating oil and gas field environments or treatment fluids with a bacterial strain as a self-propagating whole cell that produces an anti-SRP bacteriocin in-situ. In another aspect, the methods use one or more toxic peptides or proteins isolated therefrom in methods to control unwanted prokaryotic growth in these environments.

[0010] Chaturvedi T. et al., 2018 ("Investigation of microbial souring mechanisms and test of natural antibiotics for MIC prevention") discloses e.g. that active phytochemicals including strong antimicrobials from halophytes can be used for combating MIC (microbiologically influenced corrosion) by inhibiting MIC microorganisms such as methanogens and SRPs (sulfate-reducing prokaryotes).

[0011] AMPP paper no. 18089, Annual conference & expo 2022, ("Effect of Antimicrobial Halophilic Plant Extracts on Microbiologically Influenced Corrosion (MIC)") discloses i.e. halophyte extracts diluted to different concentrations of 5, 10, 15, and 20% and their H2S reducing effects .

[0012] Chaturvedi T. et al., 2019 (" Investigation of natural antimicrobial compounds for prevention of microbiologically influenced corrosion (MIC)") discloses that in steel reactors inoculated with sediments from Wadden Sea, 5-15 % (v / v) halophytic plant extract mixture significantly inhibited MIC bacteria, reduced biofilm formation, reduced the microbiological abundance for carbon steel and for stainless steel.

[0013] At an industrial scale, several different techniques have been tried and tested for eliminating MIC. Eliminating sulfates through seawater filtration is a possible way to control sulfides; however, it is expensive. Adding nitrate to injection water could abate H2S production but requires repeated treatments and is associated with high chemical costs. Adding nitrate might also increase the biomass of microorganisms in the water, causing plugging and even increased corrosion rates in some fields. The current industrial methods of controlling MIC are maintenance pigging and chemical treatment (biocides and corrosion inhibitors). A combination of mechanical cleaning and chemical treatment is the most efficient technique currently, and chemical controls are generally considered to be the most effective in performance and cost. While biocides readily destroy planktonic cells, biofilm cells on the pipeline surfaces (sessile cells) are protected by a polysaccharide covering, which wards off the effects of toxic biocides.

[0014] Conventional biocides used today in the oil and gas sector, most commonly THPS and / or glutaraldehyde in e.g. Denmark, have been reported to be ineffective against biofilms. This necessitates pipeline pigging (physical removal by scraping) which is risky for the operation since the pig can get stuck, blocking the pipeline. Hence, breaking down established biofilms is a beneficial property of the biocide on top of the potential environmental concerns.

[0015] Hence, an improved methods of producing clean, natural and non-toxic halophyte extract would be advantageous, and in particular a more environmentally friendly and efficient use of halophyte extract for reducing MIC-bacteria and inhibiting biofilm formation would be advantageous.

[0016] Summary of the invention

[0017] Thus, an object of the present invention relates to improved methods of producing clean, natural and non-toxic halophyte extracts, and in particular a more environmentally friendly and efficient use of halophyte extract for reducing MIC- bacteria and inhibiting biofilm formation, e.g. on steel surfaces.

[0018] Hence, one aspect of the invention relates to a method of producing extract from halophytes, the method comprising the following consecutive steps: (i) performing Soxhlet or subcritical extraction on dried ground halophyte biomass using a solvent resulting in a fraction of left over halophyte fibres; followed by

[0019] (ii) drying of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by (iii) subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment in a subcritical hydrothermal reactor under agitation; followed by

[0020] (iv) cooling of the resulting liquid hydrolysate; followed by

[0021] (v) a step of filtrating the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.

[0022] • The biocide can be a mixutre of the extract obtained through the less potent Soxhlet extract (i) and the filtrate (v); or

[0023] • From the filtrate (v) alone

[0024] Another aspect of the present invention relates to halophyte extracts that can be produced by a method according to the present invention.

[0025] Yet another aspect of the present invention is to provide a use of the halophyte extracts that can be produced by a method according to the present invention as a biocide.

[0026] Brief description of the figures

[0027] Figure 1 shows H2S concentrations measured every week for 28 days. Each column of graphs represents the addition of extract from a specific halophyte species. Each row represents the extraction method. The result for the untreated control samples is shown as a black line. The lowest addition of any specific extract is shown as a dashed line, and the highest addition is a solid grey line.

[0028] Figure 2 shows ATP Measurements of the samples treated with Type B (see details in table 2). Each column of graphs represents the addition of extract from a specific halophyte species. The result for the untreated control samples is shown as a black line. The lowest addition of any specific extract is shown as a dashed line, and the highest addition is a solid grey line. 3-B and 4-B does not have an ATP measurement on day 28 due to instrument failure.

[0029] Figure 3 shows (Left) Coupons from control samples. Control samples were inoculated, but were not treated with extracts. (Right) Coupons from test samples 3-B and 4-B. These coupons were from flasks treated with 20% of halophyte extract 3-B and 4-B, respectively.

[0030] Figure 4 shows a heatmap of the sulfate-reducing bacteria and the methanogens which were present in the control sample. On the left the order and genera of the bacteria are shown. Each number should be interpreted as the relative abundance of a specific genera of microorganism. A value of 100 in a cell would mean that the sample contains only a single organism. The bottom row is the Shannon Diversity Index, which is a logarithmic term for the amount of different species present. An index of 1 is a pure strain, while >3 is very diverse.

[0031] Figure 5 shows weekly H2S concentration measurement of serum flasks. The different grey scales (graph line indicators) correspond to extracts produced at various temperatures between 140°C and 240°C as per Table 3. Dashed lines indicate the lowest tested concentration (10%), and solid lines indicate the highest concentration (20%).

[0032] Figure 6 shows corrosion rates measured by converting coupon weight loss into mm / year. Black / grey / white scale-coded to reflect the corresponding black / grey / white scale-codes of Figure 5.

[0033] Figure 7 shows 3D scanning of coupons. Left: A coupon from a control sample (no extract) is shown. Right: A coupon from a flask treated with 20% of the 190°C is shown. The top row is the optical image of the coupon, and the bottom row is the aligned height map of the same coupon. The real scale of the image width is 8.5 mm (10: 1 magnification).

[0034] Figure 8 schematically shows the experimental setup and image of the biofilm reactor.

[0035] Figure 9 shows an overview of the 40 most abundant genera is provided across samples 01-12. The most abundant genus is Marinobacterium, followed by Desulfovibrio, and the third-most abundant genus is Dethiosulfovibrio. The most abundant genera across samples 01-12. The lowest assigned taxonomic classification is given if no genus-level classification can be obtained. In addition, the phylum level classification is given (Proteobacteria at class level).

[0036] Figure 10 shows incubations of steel coupons after 25 days. All the incubations were shaken before the pictures were taken. From left to right: Sterile control, Incubated control, 630 ppm Halophyte biocide (HB), 1260 ppm HB, 2520 ppm HB, 250 ppm THPS, 250 ppm Glutaraldehyde.

[0037] Figure 11 shows (light gray) H2S measurements. The error bars represent 95% confidence. Due to the large difference between the control and the other incubations, a log scale is used. All the H2S results are statistically significant— (n = 5); (dark grey) Corrosion rate, calculated from weight loss. Error bars represent a 95% confidence interval. (n = 5).

[0038] Figure 12 shows KEYENCE VR-3200 scanned images of one coupon from each set of quintuplicate incubations are shown above. All samples were scanned for numerical analysis. Each coupon is represented by an optical image of the surface on top and a height map at the bottom. The top rows are samples from the least corroded coupon face, and the bottom rows are the most corroded coupon face.

[0039] Figure 13 shows the pitting rate in mm / year. The pitting rate was calculated by measuring the depth of the deepest pit and extrapolating the result from 25 days to one year. Error bars represent the sample standard deviation (n = 5)

[0040] The present invention will now be described in more detail in the following.

[0041] Detailed description of the invention

[0042] The inventors surprisingly observed that MIC bacteria were significantly inhibited in liquid samples from the reactors treated with halophyte extracts produced according to the method of the present invention.

[0043] Specifically, biofilm formation was reduced by three times on carbon steel and four times on stainless steel coupons in reactors treated with halophyte extracts produced according the method of the present invention. Established biofilm was dissolved after the addition of the halophyte extracts produced according to the method of the present invention. A visible and measurable reduction in pitting and corrosion damages on carbon steel coupons was observed with the help of 3D surface scanning.

[0044] The addition of the halophyte extracts produced according to the method of the present invention reduced the microbiological abundance (number of microorganisms measured by qPCR) by 20 times for Carbon steel and at least five times for Stainless steel.

[0045] Even further, the halophyte extracts produced according to the method of the present invention reduced the microbiological diversity, measured for the top 40 genera (see Figure 9). The halophyte extracts inhibited sulfate-reducing species especially well (see heatmap in Figure 4).

[0046] Embodiments

[0047] In one embodiment of the present invention there is provided a method of producing extract from halophytes, the method comprising the following consecutive steps:

[0048] (i) performing Soxhlet or subcritical extraction on dried ground halophyte biomass using a solvent resulting in a fraction of left over halophyte fibres; followed by

[0049] (ii) drying of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by

[0050] (iii) subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment in a subcritical hydrothermal reactor under agitation; followed by

[0051] (iv) cooling of the resulting liquid hydrolysate; followed by

[0052] (v) a step of filtrating the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.

[0053] In another embodiment of the present invention there is provided a method of producing extract from halophytes selected from the group consisting of Saiicornia ramossisima, Suada Maritima, Tripolium Vulgare and Atriplex portulacoides, the method comprising the following consecutive steps: (i) performing Soxhlet or subcritical extraction on dried ground halophyte biomass having average particle sizes <6 cm and a dry matter content of 70-100% (w / w) using a solvent, preferably water, resulting in a fraction of left over halophyte fibres; followed by

[0054] (ii) drying of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by

[0055] (iii) subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment in a subcritical hydrothermal reactor under agitation; followed by

[0056] (iv) cooling of the resulting liquid hydrolysate; followed by

[0057] (v) a step of filtrating the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.

[0058] In still another embodiment of the present invention there is provided a method of producing extract from halophytes selected from the group consisting of Salicornia ramossisima, Suada Maritima, Tripolium Vulgare and Atriplex portulacoides, the method comprising the following consecutive steps:

[0059] (i) performing Soxhlet or subcritical extraction on dried ground halophyte biomass having average particle sizes <6 cm and a dry matter content of 70-100% (w / w), preferably 80-100% (w / w), more preferably >95% (w / w), using a solvent, preferably water, resulting in a fraction of left over halophyte fibres; followed by

[0060] (ii) drying of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by

[0061] (iii) subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment in a subcritical hydrothermal reactor under agitation; followed by

[0062] (iv) cooling of the resulting liquid hydrolysate; followed by

[0063] (v) a step of filtrating the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.

[0064] In still another embodiment of the present invention there is provided a method of producing extract from halophytes selected from the group consisting o Salicornia ramossisima, Suada Maritima, Tripolium Vulgare and Atriplex portulacoides, the method comprising the following consecutive steps:

[0065] (i) performing Soxhlet or subcritical extraction for 1-16 hours on dried ground halophyte biomass having average particle sizes <6 cm and a dry matter content of 70-100% (w / w), preferably 80-100% (w / w), more preferably >95% (w / w), using a solvent, preferably water, resulting in a fraction of left over halophyte fibres; followed by

[0066] (ii) drying of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by

[0067] (iii) subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment in a subcritical hydrothermal reactor under agitation; followed by

[0068] (iv) cooling of the resulting liquid hydrolysate; followed by

[0069] (v) a step of filtrating the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.

[0070] In still another embodiment of the present invention there is provided a method of producing extract from halophytes selected from the group consisting of Salicornia ramossisima, Suada Maritima, Tripolium Vulgare and Atriplex portulacoides, the method comprising the following consecutive steps:

[0071] (i) performing Soxhlet or subcritical extraction for 1-16 hours on dried ground halophyte biomass having average particle sizes <6 cm and a dry matter content of 70-100% (w / w), preferably 80-100% (w / w), more preferably >95% (w / w), using a solvent, preferably water, resulting in a fraction of left over halophyte fibres; followed by

[0072] (ii) drying at between 50-70°C, preferably at 55-65, most preferably at 60°C, of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by

[0073] (iii) initial loading of dried biomass from step (ii) into the subcritical hydrothermal reactor from step (iii), said loading being 2-20% (w / w), preferably 5-10% (w / w), most preferably 7.5% (w / w) of the water loading, thereby subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment in a subcritical hydrothermal reactor under agitation; followed by

[0074] (iv) cooling of the resulting liquid hydrolysate; followed by

[0075] (v) a step of filtrating the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.

[0076] In still another embodiment of the present invention there is provided a method of producing extract from halophytes selected from the group consisting o Salicornia ramossisima, Suada Maritima, Tripolium Vulgare and Atriplex portulacoides, the method comprising the following consecutive steps:

[0077] (i) performing Soxhlet or subcritical extraction for 1-16 hours on dried ground halophyte biomass having average particle sizes <6 cm and a dry matter content of 70-100% (w / w), preferably 80-100% (w / w), more preferably >95% (w / w), using a solvent, preferably water, resulting in a fraction of left over halophyte fibres; followed by

[0078] (ii) drying at between 50-70°C, preferably at 55-65, most preferably at 60°C, of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by

[0079] (iii) initial loading of dried biomass from step (ii) into the subcritical hydrothermal reactor from step (iii), said loading being 2-20% (w / w), preferably 5-10% (w / w), most preferably 7.5% (w / w) of the water loading, thereby subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment carried out at 3.6- 33.5 bar pressure and 140-240°C, preferably 170-220°C, most preferably 190°C for 5-60 minutes, preferably 7-15 minutes, most preferably 10 minutes, in a subcritical hydrothermal reactor under agitation; followed by

[0080] (iv) cooling to below 50°C of the resulting liquid hydrolysate; followed by

[0081] (v) a step of filtrating the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.

[0082] In still another embodiment of the present invention there is provided a method of producing extract from halophytes selected from the group consisting of Salicornia ramossisima, Suada Maritima, Tripolium Vulgare and Atriplex portulacoides, the method comprising the following consecutive steps:

[0083] (i) performing Soxhlet or subcritical extraction for 1-16 hours on dried ground halophyte biomass having average particle sizes <6 cm and a dry matter content of 70-100% (w / w), preferably 80-100% (w / w), more preferably >95% (w / w), using a solvent, preferably water, resulting in a fraction of left over halophyte fibres; followed by

[0084] (ii) drying at between 50-70°C, preferably at 55-65, most preferably at 60°C, of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by

[0085] (iii) initial loading of dried biomass from step (ii) into the subcritical hydrothermal reactor from step (iii), said loading being 2-20% (w / w), preferably 5-10% (w / w), most preferably 7.5% (w / w) of the water loading, thereby subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment carried out at 3.6- 33.5 bar pressure and 140-240°C, preferably 170-220°C, most preferably 190°C for 5-60 minutes, preferably 7-15 minutes, most preferably 10 minutes, in a subcritical hydrothermal reactor under agitation; followed by

[0086] (iv) cooling to below 50°C of the resulting liquid hydrolysate; followed by

[0087] (v) a step of filtrating with a filter mesh of 5-20 pm, preferably 7-15 pm, most preferably 11 pm, the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.1

[0088] In still another embodiment of the present invention there is provided halophyte extracts that can be produced by any of the methods of the above embodiments.

[0089] In still another embodiment of the present invention there is provided halophyte extracts that can be produced by any of the methods of the above embodiments for use as a biocide.

[0090] In still another embodiment of the present invention there is provided halophyte extracts that can be produced by any of the methods of the above embodiments for:

[0091] (i) reducing MIC-bacteria, such as sulphate-reducing bacteria and / or

[0092] (ii) inhibiting biofilm formation and / or

[0093] (iii) reducing microbiological diversity as determined by the Shannon Diversity Index.

[0094] In still another embodiment of the present invention there is provided halophyte extracts that can be produced by any of the methods of the above embodiments for use as a biocide wherein the halophyte extracts is in solution with a halophyte extract concentration between 5-20% (v / v) or an equivalent amount of dehydrated extract.

[0095] In still another embodiment of the present invention there is provided halophyte extracts that can be produced by any of the methods of the above embodiments for:

[0096] (i) reducing MIC-bacteria, such as sulphate-reducing bacteria and / or (ii) inhibiting biofilm formation and / or

[0097] (iii) reducing microbiological diversity as determined by the Shannon Diversity Index, wherein the halophyte extracts is in solution with a halophyte extract concentration between 5-20% (v / v) or an equivalent amount of dehydrated extract.

[0098] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0099] In addition to the above-indicated embodiments, the halophytes antimicrobials and bio-chemicals could have applications in a wide variety of industries besides the petrochemical industry, e.g. food, feed, and pharmaceutical industry as natural preservatives, microbial contamination control, and MIC-mitigation in other water systems such as industrial cooling and fire suppression.

[0100] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0101] The invention will now be described in further details in the following non-limiting examples.

[0102] Examples

[0103] Example 1 - obtaining dried around halophyte biomass

[0104] Rinsing

[0105] The halophyte biomass is rinsed from sand, mud and otherimpurities. The washing process is designed to remove sand, small sea creatures e.g. snails, and surface salt from the biomass. The biomass is placed in a filter basket, placed in a washing drum. Tap water or demineralized water is spayed on the biomass as step one. Once the excess dirt is rinsed off the biomass is submerged in a bath of tap water. The drum reciprocator is turned on and the biomass is washed for a given time to ensure all contaminants are removed. The washing water is drained of by opening the bottom valves. Drying

[0106] The rinsed and fractionated biomass is then subjected to a drying step, preferably at around 60 °C, by sundrying, oven-drying or other industrial drying processes such as fluid bed, tray, or rotary drying. The drying step may alternatively be carried out after the fractionating / grinding step.

[0107] Fra ctionatin g / grin din g

[0108] The rinsed halophyte biomass is then subjected to a fractionating / grinding step. A simple screw press is suitable for efficiently fractionating / grinding the halophyte biomass to smaller pieces (preferably less than 1 cm). Screw pressing also gives a preliminary mechanical distortion to the lignocellulosic fibres, and shredding reduces the size further. Screw pressing also allows for a good separation of solid and liquid fractions, leading up to relatively high fibre dry matter content.

[0109] Example la - preparation of inoculum mixture

[0110] A mixed bacterial culture was collected from the Wadden Sea using handheld sediment samplers. The sediment is added to one litre of Postgate media and stored at 25° C. This mixture serves as inoculum for the experiments. Postgate media is prepared using DSMZ's procedure for making Postgate media for Desulfovibrio. Three solutions, A, B, and C, are prepared using the below- mentioned chemicals (Table 1).

[0111] Table 1

[0112] The ingredients of solution A are boiled, then cooled to room temperature while sparging with 100% N2 gas. Solutions B and C are added to solution A, and pH is adjusted to 7.8 with NaOH and distributed under 100% N2 gas. The mixture is autoclaved for 60 min at 121°C. If the media volume is more than one litre, autoclave for 20 mins for every additional litre.

[0113] Example 2 - Serum flasks experiments

[0114] The mixed microbial culture from the Wadden Sea as produced in Example 1 was used to inoculate two sequential series of aerobic, 100 ml_ serum flasks containing Postgate medium (described in Example 1 above) and various halophyte extracts produced according to the method of the present invention.

[0115] The first series investigated the choice of halophyte and extraction method (Table 2), and the second investigated the temperature at which the extraction was performed (Table 3).

[0116] Halophyte and extraction method screening

[0117] A steel coupon was put into each flask. In the first set of serum flask experiments SS316 stainless steel was used. In the second experiment, a low-alloy carbon steel was used.

[0118] Once per week, samples were taken from the flasks. H2S and ATP concentrations were measured from these samples. After four weeks the experiment concluded, and samples of the liquid were sequenced using 16S rRNA amplicon sequencing.

[0119] Table 2: Halophyte species, extraction methods, and concentrations tested in the first series of serum flask experiments: halophyte screening.

[0120] Out of the three tested extraction methods, "Type B" (subcritical hydrothermal treatment of halophyte fibers) consistently performed better than the other two. Three of the four tested halophytes (Figure 1), resulting from the "Type B" extraction method, performed better than the untreated controls at the 5% (v / v) concentration. Every extract resulting from the "Type B" extraction method completely prevented H2S production at 20% (v / v)

[0121] ATP was also measured from the same samples every week. Halophyte extracts from the "Type B" extraction method were the best at preventing H2S production, so only the results from "Type B" extracts are shown in Figure 2.

[0122] A sharp decrease in ATP is observed in the samples. However, the decrease is often less pronounced than for the H2S, indicating a selective inhibition of sulfatereducing bacteria. The sequencing results in Figure 4 further backed this up.

[0123] Steel coupons from 3-B and 4-B (Figure 1) were compared against coupons from the controls. The controls were inoculated with the same microbial culture, but no halophyte extracts were added to the flasks. A clear reduction in corrosion products is observed on coupons from flasks treated with extracts.

[0124] 16S rRNA amplicon sequencing was performed on samples of the ferment. The sequencing returns the relative abundance of all the bacterial species in a sample. Because halophyte extracts produced according to Extraction method B ("Type B") as shown in Table 2, performed much better in terms of reducing H2S production in the flasks, these samples were prioritized in the sequencing. More than 300 genera of bacteria were identified. Figure 4 shows the relative abundance of the species which have been linked to microbial corrosion.

[0125] Halophyte 1, 3, and 4 (see Figure 2) are the best performing in terms of reducing the abundance of sulfate-reducing genera. A common trend is that only Shewanella can survive being exposed to the extract. An important thing to note is that the sequencing only shows the relative abundance which means that it does not say anything about the total amount of bacteria. This means that Shewanella was very like inhibited, but not to the same extent as the other species, because a sharp decrease in ATP (overall activity) was also seen in these samples (Figure 2).

[0126] Example 3 - Extraction temperature optimization

[0127] SaHcornia ramosissima ("Halophyte 1" in Example 2 and Figure 2) performed well in the first serum flask experiment and was chosen as the model biomass for this experiment. It was concluded that "Type B extracts", subcritical hydrothermal treatment, was the most effective. The next step was determining the optimal temperature for the best biocidal effect. The temperatures chosen were: 190°C, 210°C, and 240°C and extracts were made with 7.5% dry matter. All extractions lasted 10 minutes.

[0128] Serum flasks containing Postgate medium and a low-alloy carbon steel coupon were inoculated with Wadden Sea sediment like in the previous experiment (Example 2). One of the three extracts was added to each flask at either 10% or 20%.

[0129] Table 3 below shows the parameters used in the second series of serum flask experiments and their black / gray / white coding in the graphs of Figure 5. H2S was measured weekly for the samples. A general trend was that increasing temperature increased the performance at low concentrations. At temperatures above 190°C, no H2S was produced in any of the flasks. The concentrations in the inoculated samples were the same as those in the un-inoculated sample.

[0130] For these serum flasks, low-alloy carbon steel was used, which meant that corrosion was measurable over the 28-day course of the experiment. Weight loss was measured and converted into mm / year using the density and dimensions of the coupons.

[0131] Any value beneath the horizontal black line of Figure 6 had a lower corrosion rate than the control, which was inoculated with bacteria without any extract present. The blind was just the sterilized growth medium without any extract, being the analog for non-microbial corrosion.

[0132] The extracts all have a corrosion-preventing effect against MIC, but increasing the temperature above 210°C had little biocidal effect and only a marginal effect in corrosion prevention at the highest concentration. Heating water from 210°C to 240°C increases the saturation pressure from 19 to 33 bars, so the added hazard and exponentially increasing energy requirements are likely not worth the increase in biocidal performance.

[0133] 3D scannings were performed, too, which show that the pitting corrosion, which is characteristic of MIC, is entirely absent on coupons from flasks treated with halophyte extract, whereas pitting corrosion is visible to the naked eye on untreated coupons. Figure 7 shows a picture of the coupon surface along with the heightmap measured using 3D scanning.

[0134] Example 4 - Biofilm reactor experiments

[0135] Experimental plan:

[0136] Reactors were inoculated with sediments obtained from the Wadden Sea and continuously circulated with Postgate media while maintained at 25°C. The reactor has four inlets. The first inlet is connected to the inlet pump, and the second is to the outlet pump. The third inlet is connected to a nitrogen source, and the fourth inlet is to a filter that acts as the exit for excess nitrogen in the reactor. The holding container (in this case, a 5L glass bottle) has four openings, two for connecting the inlet and outlet pumps to the reactor and the remaining two holes to allow for nitrogen circulation (inlet and outlet) in the bottle headspace. The schematic of the experimental design is shown below. Nitrogen was flushed through the reactor and substrate bottle to maintain anaerobic conditions.

[0137] Results:

[0138] ATP (a measure of cellular activity) was reduced to half in the continuous reactor and remained steady in the pulse reactor compared to the initial growth levels in the respective reactors. Biofilm formation on coupons was reduced by three times on carbon steel and four times on stainless steel coupons in reactors treated with the extractive mix. A visible and measurable reduction in pitting and corrosion damages on carbon steel coupons was observed with the help of 3D scanning, where the depth of corroded pits was three times more in the control reactor when compared to the continuous reactor. Additionally, twice the surface area was corroded in the reactor where the extractives were not used.

[0139] The extract mix from halophytes does reduce the microbiological abundance (number of microorganisms measured by qPCR) by several orders of magnitude in biofilm samples. Additionally, the extracts significantly reduce the microbiological diversity (diversity of microorganisms measured by Amplicon Sequencing). The microbial species in the biofilm matrix react differently to the presence of extract mix - the majority are inhibited, and a minority are stimulated.

[0140] Conventional biocides used today in the oil and gas sector, most commonly THPS and / or glutaraldehyde in e.g. Denmark, have been reported to be ineffective against biofilms. This necessitates pipeline pigging (physical removal by scraping) which is risky for the operation since the pig can get stuck, blocking the pipeline. Hence, breaking down established biofilms is a beneficial property of the biocide on top of the potential environmental concerns.

[0141] Example 5 - Comparative study of biocidal effectiveness

[0142] This example compares the biocidal effectiveness of the SaHcornia ramosissima extract obtainable according to the method of the invention against the effectiveness of two biocides used in the oil and gas industry: 50% Tetrakis hydroxymethyl phosphonium sulfate (THPS) in water and 50% glutaraldehyde in water.

[0143] In this example, the halophyte extracts from Saiicornia Ramosissima, produced according to the methods of the invention were added to flasks containing Postgate medium and carbon steel coupons. These flasks were inoculated with a mixed microbial culture derived from sea sediment. The inhibitory effects of the halophyte extracts on the bacterial community and the corrosion rate of the coupons were compared to those of said two conventional biocides, THPS, and glutaraldehyde, as well as a control without biocides.

[0144] Specifically, rinsed / washed, salt-reduced halophyte biomass was dried at 60 °C and ground into smaller pieces (less than 1 cm). Following this, the grinded biomass was subjected to the extraction process of the invention.

[0145] Serum flasks was used with postgate medium inoculated with a mixed microbial culture and added halophyte extracts to one set of incubations and the two conventional biocides to other sets of incubations.

[0146] Growth medium preparation - Postgate medium

[0147] Two media were prepared simultaneously with the medium preparation protocol found for the Desulfovibrio medium by DSMZ GmbH (for details see "Desulfovibrio (Postgate) Medium," 2017. https: / / www.dsmz.de / microorganisms / medium / pdf / DSMZ_Medium63.pdf, incorporated herin by reference). The difference between the two was that yeast extract was omitted from one medium (highlighted in 4 below).

[0148] Table 4: Chemicals used for Postgate medium (for details see DSMZ, "HALODESULFOVIBRIO (POSTGATE) MEDIUM." pp. 1-1, 2022, Available: https: / / www.dsmz.de / microorganisms / medium / pdf / DSMZ_Mediuml63.pdf, incorporated herin by reference) with salt. Solution A is heated to the boiling point; solutions B and C are added once the solution is cool.

[0149] Solution A (4) was brought to a boil and cooled to room temperature in an ice bath while sparging with 100% N2 gas. Solutions B and C (Table 4) were added, and pH was adjusted to 7.8 with NaOH. The solution was transferred to blue-cap bottles and autoclave for 15 min at 121°C. After autoclaving, the bottles were stored in a dark cupboard at room temperature. Serum flasks of 100 ml capacity were filled with 75 ml of the sterilized Postgate Medium containing 32 g of NaCI per liter. A coupon of corrosion-resistant low-alloy carbon steel (AISI 1080 Carbon Steel) with a diameter of 9 mm and height of 4 mm was polished using 500P SiC wet-sanding sandpaper. This coupon was then rinsed with demineralized water, dried using 99% ethanol, allowed to air dry in a sterilized laminar air flow (LAF) bench, weighed on an analytical scale with 0.1 mg precision, sterilized again in ethanol, and then placed into a serum flask. Each incubation received a dose of biocide. The halophyte-based biocide was added at 5%, 10%, and 20% of the medium volume, corresponding to 630, 1260, and 2520 ppm dry weight concentrations, respectively. Two commercial biocides, 50% THPS, and 50% glutaraldehyde, were added at a rate of 500 ml / L resulting in an active compound concentration of 250 ppm. The incubations were sparged with Nitrogen gas, sealed with a butyl rubber cap, and inoculated with a 72-hour- old preculture derived from 2 g of anaerobic sea sediment in 100 ml of Postgate Medium containing 32 g / L NaCI.

[0150] The incubations were placed in a fume hood at 20 °C and left for 25 days.

[0151] Dissolved H2S

[0152] Following the 25-day incubation, the dissolved H2S was examined using a UniSense H2S (SULF) microsensor. The flasks were agitated, and an 8 mL sample of the liquid was extracted from an incubation through the rubber cap using a syringe equipped with a needle. This sample was then introduced into the microsensor chamber, and the H2S concentration was recorded once the signal had stabilized (typically took 5 to 10 seconds). The sensor was calibrated using a UniSense H2S calibration kit. Steel coupon cleaning and weight loss

[0153] Before and after the experiment, the coupons had their corrosion products removed. This was done by adhering to a protocol based on NACE SP0775-2018 (for details, see "NACE Standard: SP0775-2018. AMPP, [Online]. Available: https: / / store.ampp.org / sp0775-2013-formerly-rp0775-2", incorporated herin by reference).

[0154] The coupons were cleaned using water that contained a small quantity of dish soap. Thereafter, the cleaned coupons were immersed in 99% ethanol and left to dry to remove any remaining water.

[0155] Following this, inhibited HCI was prepared. This was done by adding 1 w / v% N,N- Dibutylthiourea to 37% HCI and subjecting the mixture to sonication. Once the N,N-Dibutylthiourea was fully dissolved, the HCI solution was diluted with an equal volume of water.

[0156] The coupons were then cleaned by immersing them in a sequence of solutions. The first solution was acetone, followed by the inhibited HCI, which was sonicated for 10-20 seconds. The coupons were then immersed in a solution of saturated NaHCOs in water, followed by demineralized water, and finally in 99% ethanol. Each immersion was done sequentially, ensuring the coupons were thoroughly exposed to each solution. After being submerged in ethanol, the coupons were air-dried on a paper towel. Once dry, the coupons were weighed on a 0.1 mg precision scale. The weights before and after were used to calculate weight losses, which were then converted to corrosion rates using the corrosion rate formula.

[0157] For the cleaning before the experiment, The coupons were polished with P500 SiC wet-sanding sandpaper after the acid cleaning protocol, which was followed by another submersion in ethanol, after which the weighing was done.

[0158] Steel coupon topography scanning Once cleaned, the coupons were preserved in 99% ethanol for one week. After this storage phase, they were scanned using a KEYENCE VR-3200 Wide-Area 3D measurement system, with no additional sample preparation needed for this scanning procedure. Post- processing was carried out using the KEYENCE VR-3000 Series software. This process involved excluding the outer 0.5 mm of each coupon due to the edge curvature and correcting the plane tilt. In addition, individual reference heights were established, set to the average height of each coupon.

[0159] The pitting rates were calculated once the reference heights and plane tilts were established. This was done by identifying the deepest pit on each coupon and comparing it to the reference height.

[0160] After 25 days, the experiment ended. Figure 10 shows pictures of the every fifth incubation.

[0161] H2S and Coupon weight loss

[0162] H2S was measured at the end of the 25-day experiment.

[0163] All incubations treated with biocides had their H2S concentration reduced 300 times more (see Figure 11). Glutaraldehyde incubations had a smaller concentration of H2S than the abiotic sample. The reason for this is unknown, but the glutaraldehyde-based biocide may contain other chemicals with F S-scavening properties.

[0164] The corrosion rate for the halophyte biocides was reduced significantly for all the biocides compared to the control sample. The lowest weight loss was observed with Glutaraldehyde and THPS, which both performed statistically insignificantly different from the sterile control. The halophyte extracts also reduced corrosion rate significantly, resulting on average in 93%, 83%, and 83% decreases in MIC rate for the 630, 1260, and 2520 ppm incubations, respectively.

[0165] As seen in Figure 10, the scans visually represent the coupons as photography and in the same area as a height map. The reference (0 pm) height is set on a coupon-to-coupon basis since the exact height of the coupons varies due to the sanding prior to the experiment. Therefore, the reference is set as the average height of the coupons, meaning that some parts of the coupons may appear to be raised. However, because pitting is a process that happens more rapidly than general corrosion, any pitting will still appear as blue spots.

[0166] The differences in pitting rates are lower than the general corrosion rates or the H2S concentrations (Figure 11). The average pitting rates, see Figure 13, of coupons in halophyte biocide-treated incubations tend to decrease with increasing biocide concentrations. They are all lower than the untreated control incubation. The average pitting rate for coupons in incubations treated with the two conventional biocides is slightly higher, with THPS above the untreated control incubations. Regarding pitting, glutaraldehyde performs on par with coupons from the 630 ppm DM halophyte biocide incubations.

[0167] There seems to be a good correlation between the pitting results in Figure 4 and the most corroded face rows in Figure 12, with the corrosion density decreasing with increasing halophyte concentration and THPS performing worse than the other treated samples and the control sample.

[0168] Conclusions

[0169] The halophyte extracts obtained according to the method of the invention resulted in a similar reduction in H2S production, and weight loss was reduced by up to 93% with these extracts. Halophyte extracts demonstrated a higher inhibitory effect on bacteria than conventional biocides and showed selectivity for SRB between THPS (lower selectivity) and glutaraldehyde (higher selectivity).

[0170] Dissolved H2S concentration was reduced to at most 1 / 300 of the control incubation's concentration. Differences between the samples and the sterile control were negligible for all treated samples. Microbial corrosion rate reduction ranked from most effective biocide to least effective: Glutaraldehyde (99%), THPS (99%), 630 ppm extract (93%), 1260 ppm extract (83%), 2520 ppm extract (83%). Pitting rates tended towards less pitting with increasing concentration of halophyte extract.

Claims

Claims1. A method of producing extract from halophytes, the method comprising the following consecutive steps:(i) performing Soxhlet or subcritical extraction on dried ground halophyte biomass using a solvent resulting in a fraction of left over halophyte fibres; followed by(ii) drying of the left over halophyte fibres from step (i) or without drying the left over halophyte fibres from step (i); followed by(iii) subjecting the dried biomass from step (ii) to subcritical hydrothermal treatment in a subcritical hydrothermal reactor under agitation; followed by(iv) cooling of the resulting liquid hydrolysate; followed by(v) a step of filtrating the cooled hydrolysate from step (iv) thereby resulting in the halophyte extract.

2. The method of producing extract from halophytes according to claim 1, wherein the halophytes are selected from the group consisting of Salicornia ramossisima, Suada Maritima, Tripolium Vulgare and Atriplex portulacoides.

3. The method of producing extract from halophytes according to any of claims 1-2, wherein the dried ground halophyte biomass of step (i) has average particle sizes <6 cm and a dry matter content of 70-100% (w / w).

4. The method of producing extract from halophytes according to any of claims 1-3, wherein the solvent is water.

5. The method of producing extract from halophytes according to any of claims 1-4, wherein the Soxhlet or subcritical extraction in step (i) is running for 1-16 hours.

6. The method of producing extract from halophytes according to any of claims 1-5, wherein the drying in step (ii) is carried out between 50-70°C.

7. The method of producing extract from halophytes according to any of claims 1-6, wherein the initial loading of dried biomass from step (ii) into the subcritical hydrothermal reactor from step (iii) is 2-20% (w / w) of the water loading (preferably 7.5% (w / w))8. The method of producing extract from halophytes according to any of claims 1-7, wherein the subcritical hydrothermal treatment of step (iii) is carried out at 3.6-33.5 bar and 140-240°C (preferably 190°C) for 5-60 minutes (preferably 10 minutes).

9. The method of producing extract from halophytes according to any of claims 1-8, wherein the hydrolysate in step (iv) is cooled to below 50°C.

10. The method of producing extract from halophytes according to any of claims 1-9, wherein the filter mesh in filtration step (v) is 5-20 pm (preferably 11 pm)11. Halophyte extracts that can be produced by the method according to any of claims 1-10.

12. Use of the halophyte extracts according to claim 11 as a biocide.

13. Use of the halophyte extracts according to claim 11 for:(i) reducing MIC-bacteria, such as sulphate-reducing bacteria and / or(ii) inhibiting biofilm formation and / or(iii) reducing microbiological diversity as determined by the Shannon Diversity Index.

14. The use of the halophyte extracts according to claims 12-13, wherein the halophyte extracts is in solution with a halophyte extract concentration between 5-20% (v / v) or an equivalent amount of dehydrated extract.

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