Novel microorganism and use thereof

Abstract

A microorganism according to the present disclosure is a microorganism (accession number: NITE BP-04014) belonging to a species (Geobacillus sp.) closely related to Geobacillus icigianus.

Description

Novel microorganisms and their uses

[0001] The present invention relates to a novel microorganism and the use of said novel microorganism.

[0002] Degradable resins such as biodegradable resins are used as materials that can be removed by decomposition after use. Microbial decomposition has been considered as one method for promoting the decomposition of biodegradable plastics such as polylactic acid. Methods for decomposing biodegradable plastics such as polylactic acid using microorganisms belonging to the genus Geobacillus have been reported (Patent Documents 1 and 2, Non-Patent Document 1).

[0003] EP 3944906 A JP 2017-086040 A

[0004] Polymer Degradation and Stability (2018), 154, 46-54

[0005] Polyglycolic acid is also used in well drilling applications, agricultural applications, and other applications as a material that can be removed by decomposition after its intended use has been achieved. For example, polyglycolic acid is used as a component for degradable downhole tools in the hydraulic fracturing process of well drilling applications. After the downhole tool temporarily seals a portion of the well and changes the fluid pattern, the polyglycolic acid is hydrolyzed by the water in the well. The hydrolyzed polyglycolic acid loses its strength, releasing the seal in the well.

[0006] However, since the hydrolysis of polyglycolic acid slows in a well environment with a relatively low temperature (for example, 66°C or lower), there is a problem that it is difficult to remove the seal in the well within the suitable time range required for the hydraulic fracturing process. Also, a method of using an acidic fluid to promote the decomposition of polyglycolic acid is known, but there is a problem that the corrosion of the metal casing is promoted, which poses a risk of damaging the integrity of the well.

[0007] Therefore, no microorganisms are known that promote the decomposition of polyglycolic acid in a relatively low-temperature borehole environment (for example, 66°C or lower and pH 6 to 10), and there is a strong demand for microorganisms that promote the decomposition of polyglycolic acid in the above-mentioned environment.

[0008] One aspect of the present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a novel microorganism that promotes the degradation of polyglycolic acid.

[0009] As a result of screening, the present inventors have discovered a novel microorganism that promotes the decomposition of polyglycolic acid even in a relatively low-temperature composition environment, for example, at 66° C. or lower. They have also found that this microorganism can promote the decomposition of polyglycolic acid in an environment of pH 6 or higher and pH 10 or lower. In other words, they have found that this microorganism can promote the decomposition of a downhole tool component for well drilling, which is made of polyglycolic acid, in a well environment, and have completed the present invention.

[0010] A microorganism according to one embodiment of the present invention is a microorganism belonging to a species closely related to Geobacillus isigianus (Geobacillus sp.) (Accession number: NITE BP-04014).

[0011] According to one aspect of the present invention, a microorganism that promotes the degradation of polyglycolic acid can be provided.

[0012] FIG. 1 is a schematic diagram of an agar medium used for screening microorganisms. FIG. 2 is a diagram showing the colony shape of DB14. FIG. 3 is a diagram showing a Gram stained image of DB14. FIG. 4 is a diagram showing a simplified molecular phylogenetic tree based on the 16S rDNA base sequence of DB14. FIG. 5 is a diagram showing the results of a second stage bacterial test. FIG. 6 is a diagram showing changes in weight retention of PGA sheets due to DB14 culture. FIG. 7 is a diagram showing changes in pH of DB14 culture. FIG. 8 is a diagram showing a calibration curve for glycolic acid. FIG. 9 is a diagram showing changes in glycolic acid concentration due to DB14 culture. FIG. 10 is a diagram showing the results of decomposition of polyglycolic acid by crude enzyme treatment derived from DB14.

[0013] Unless otherwise specified in this specification, the expression "A to B" representing a numerical range means "A or more (including and greater than A) and B or less (including and less than B)."

[0014] As used herein, "promoting the decomposition of polyglycolic acid" refers to promoting the hydrolysis reaction of polyglycolic acid. Furthermore, as used herein, "microorganisms having the activity of promoting the decomposition of polyglycolic acid" refers to microorganisms that promote the hydrolysis reaction of polyglycolic acid.

[0015] For example, whether or not a microorganism has the activity of promoting the degradation of polyglycolic acid can be evaluated by the following procedure: contacting a liquid containing the microorganism or a product derived from a culture of the microorganism with a molded article formed from polyglycolic acid; measuring the weight of the molded article 18 hours after the contact or measuring its weight average molecular weight in accordance with ISO 6014-1:2019; calculating the weight retention rate or weight average molecular weight retention rate as the ratio of the weight or weight average molecular weight of the molded article 18 hours after the contact to the weight or weight average molecular weight of the molded article before the contact; setting the weight retention rate or weight average molecular weight retention rate of the control (polyglycolic acid contacted with a liquid not containing a product derived from a culture of the microorganism) to 1, if the calculated weight retention rate or weight average molecular weight retention rate is 0.9 or less, the microorganism is evaluated to have the activity of promoting the degradation of polyglycolic acid. Microorganism culture-derived products will be described later.

[0016] In this specification, an oil well or a gas well, which is a well having a porous and permeable subterranean formation for extracting hydrocarbon resources such as oil or natural gas, is collectively referred to as a well.

[0017] [Novel Microorganism] A microorganism according to one embodiment of the present invention is a microorganism belonging to a species closely related to Geobacillus icigianus (Geobacillus sp.). Hereinafter, this microorganism may be referred to as DB14.

[0018] DB14 is a microorganism that has the activity of promoting the degradation of polyglycolic acid, and can promote the degradation of polyglycolic acid in low-temperature environments such as well borehole environments.

[0019] DB14 can promote the decomposition of polyglycolic acid at least in an environment of pH 6 to pH 10.

[0020] A microorganism that is a progeny lineage of DB14 and has the activity of promoting the degradation of polyglycolic acid also falls within the category of the microorganism according to one embodiment of the present invention. A microorganism that is a natural or artificial mutation of DB14 or a progeny lineage of DB14 and has the activity of promoting the degradation of polyglycolic acid also falls within the category of the microorganism according to one embodiment of the present invention.

[0021] The progeny strains of DB14 and naturally or artificially mutated strains of DB14 have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the following characteristics (1) to (10): (1) are motile Gram-positive bacilli and do not form spores; (2) do not grow at 37°C, but grow at 45°C and 70°C; (3) do not grow at pH 5.0, but grow at pH 9.0; (4) are catalase-positive; (5) are oxidase-positive; (6) produce acid from glucose, but do not produce gas; (7) hydrolyze casein, but do not hydrolyze starch; (8) do not grow under anaerobic conditions; (9) oxidizes glycerol, ribose, D-xylose, glucose, fructose, mannose, esculin, maltose, esculin, maltose, melibiose, sucrose, trehalose, melezitose, raffinose and starch; (10) has gelatinase activity.

[0022] The results of 16S rDNA base sequence analysis, morphological observation, and physiological and biochemical property tests showed that DB14 is closely related to Geobacillus isigianus.

[0023] DB14 was deposited at the National Institute of Technology and Evaluation (NPMD), Room 122, Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture, Japan (original deposit date: November 21, 2023, accession number: NITE BP-04014).

[0024] DB14 may be cultured according to a general culture method used for Geobacillus microorganisms. The culture method is batch culture using a liquid medium or fed-batch culture in which a carbon source and / or an organic nitrogen source is continuously added to the culture system, and aeration and agitation are preferred. The culture medium may contain a carbon source, a nitrogen source, or a necessary nutrient source such as inorganic salts that can be assimilated by Geobacillus microorganisms. The culture pH is preferably 6 to 10, the culture temperature is preferably 45°C to 65°C, and the culture time is preferably 1 to 7 days.

[0025] [DB14 Culture-Derived Product] A culture-derived product according to one embodiment of the present invention is a DB14 culture-derived product. A culture-derived product according to one embodiment of the present invention is a culture, cultured bacterial cells, culture supernatant, or extract. The culture-derived product may also be a mixture of two or more of the culture, cultured bacterial cells, culture supernatant, and extract. The culture-derived product may also be a processed product, such as a disrupted product, concentrate, purified product, or dilution of a mixture of one or more of the culture, cultured bacterial cells, culture supernatant, or extract.

[0026] As used herein, the term "culture" refers to a culture obtained by culturing under any conditions and containing DB14 cells and a medium.

[0027] As used herein, the term "cultured bacterial cells" refers to bacterial cells obtained by separating them from the culture by centrifugation, membrane separation, or the like.

[0028] As used herein, the term "culture supernatant" refers to a substance obtained by removing bacterial cells from the culture by centrifugation, membrane separation, or the like.

[0029] In this specification, the term "extract" refers to a residue obtained by adding a solvent (water, surfactant, buffer solution, etc.) to the cultured bacterial cells, stirring the mixture, and then centrifuging or filtering the resulting mixture; a solution separated by removing the residue; a precipitate formed by adding salts to the solution; or a product obtained by separating the solution using HPLC, etc. An example of an extract is a crude enzyme solution derived from DB14.

[0030] The DB14 contained in the culture-derived product according to one embodiment of the present invention may be viable or dead. Whether or not the culture-derived product is a culture-derived product according to one embodiment of the present invention can be determined by confirming whether or not the culture-derived product contains DB14 or a portion thereof. The presence of DB14 or a portion thereof can be confirmed by a method known in the art, such as polymerase chain reaction (PCR), for a gene extracted from the culture-derived product or the degradation accelerator. Furthermore, if the culture-derived product or the degradation accelerator contains viable cells, the presence of DB14 can be confirmed by separately culturing and growing the viable cells. For example, NB agar medium sprayed with the culture-derived product or the degradation accelerator can be aerobically cultured at 55°C for one day, and the resulting colonies can be isolated. Genes extracted from the cells present in each colony can then be confirmed by a method known in the art, such as PCR.

[0031] [Decomposition promoter for decomposition of polyglycolic acid] A degradation promoter for decomposition of polyglycolic acid according to one aspect of the present invention (hereinafter, may be referred to as "the degradation promoter according to this embodiment") contains DB14 or a product derived from DB14 culture as an active ingredient.

[0032] The degradation accelerator according to this embodiment may be DB14 or a culture-derived product of DB14 as it is. Furthermore, the degradation accelerator according to this embodiment may further contain other components in addition to DB14 or a culture-derived product of DB14. For example, DB14 or a culture-derived product of DB14 may be mixed with a carrier (diluent) such as a solid carrier or a liquid carrier, a surfactant, and other formulation adjuvants, and formulated into various forms such as dusts, granules, powders, wettable powders, water-soluble powders, emulsions, liquids, oils, aerosols, microcapsules, pastes, liniments, fumigants, fumigants, and microsprays. Carriers (diluents), surfactants, and other formulation adjuvants used in known microbial formulations may be used.

[0033] The concentration of DB14 contained in the decomposition accelerator according to this embodiment is, for example, 1.0 × 10 ４ ~1.0 x 10 １０cfu / ml, and 3.0 x 10 ４ ~1.0 x 10 １０ cfu / ml is preferred, 7.0 x 10 ５ ~1.0 x 10 １０ cfu / ml is more preferred.

[0034] The decomposition accelerator according to this embodiment can also decompose polyglycolic acid under the same pH conditions as DB14.

[0035] The decomposition accelerator according to this embodiment may be used in combination with other decomposition accelerators that are known as decomposition accelerators for the decomposition of polyglycolic acid. Such combination may involve the application of the decomposition accelerator according to this embodiment in combination with the other decomposition accelerator, or the two may be applied separately.

[0036] The decomposition accelerator according to this embodiment can be used as a decomposition accelerator for decomposing molded articles formed from polyglycolic acid. There are no particular limitations on the molding method or shape of the molded article that the decomposition accelerator is intended to treat, and the decomposition accelerator according to this embodiment can be used as a decomposition accelerator for decomposing molded articles of any shape obtained by any molding method.

[0037] The polyglycolic acid may be a homopolymer of glycolic acid, a copolymer containing repeating units derived from glycolic acid and repeating units derived from other monomers, or a mixture thereof. When polyglycolic acid is a copolymer, examples of other monomer components include hydroxycarboxylic acids such as L-lactic acid, D-lactic acid, 3-hydroxybutanoic acid, and 1-hydroxyhexanoic acid; ester compounds composed of diols and dicarboxylic acids, such as condensates of 1,4-butanediol and succinic acid and condensates of 1,4-butanediol and adipic acid; cyclic esters and lactones produced by intramolecular condensation of the other monomer components described above; and cyclic carbonates such as trimethylene carbonate. When polyglycolic acid is a copolymer, the content of glycolic acid-derived structural units is 70 mol% or more, preferably 80 mol% or more, and more preferably 90 mol% or more, from the viewpoint of promoting decomposition in a low-temperature environment.

[0038] For example, the decomposition accelerator according to the present embodiment can be used as a decomposition accelerator for decomposing a molded product for well drilling formed from polyglycolic acid, such as a downhole tool, a downhole tool component, and a temporary sealant.

[0039] (Downhole tool) In this specification, a tool that is used for various well treatments such as well drilling, well plugging, and fracturing (hydraulic fracturing) and that is installed in a well is referred to as a downhole tool. The shape of the downhole tool is not particularly limited, and it can be, for example, a conventionally known shape. Examples of downhole tools include a frac plug, a bridge plug, a cement retainer, a perforation gun, a ball sealer, a plugging plug, and a packer.

[0040] The downhole tool is preferably a well drilling plug, and more preferably a frac plug or a bridge plug. The well drilling plug includes, for example, a mandrel and various downhole tool components placed on the outer peripheral surface of the mandrel perpendicular to the axial direction. The mandrel may have a hollow portion, or may have a shape whose diameter changes along the axial direction, or may have a fixing portion, a step portion, a recessed portion, a protruding portion, or the like on its outer surface. In addition to the downhole tool components, the well drilling plug may further include known components such as a sensor.

[0041] (Downhole tool components) Examples of downhole tool components include a mandrel, load ring, cone, socket, bottom, ball or seal member in the above-mentioned frac plug or bridge plug; a sleeve, ball, ball seat or seal member in a sleeve system (frac sleeve); a seal member such as a ball valve or flapper valve in a downhole tool; a plug or seal member that can temporarily block fluid by being placed in an opening between the downhole tool and the casing; and a seal member that covers a metallic downhole tool component, sensor, flow path, etc. to provide protection or a seal, and that seals the wellbore by expanding the diameter of these metal parts, etc.

[0042] The downhole tool component may be a molded body obtained by molding polyglycolic acid, or may be a secondary processed molded body obtained by subjecting the molded body to conventionally known machining (e.g., cutting).

[0043] The shape of the downhole tool component may be determined depending on the intended use, and may be a sheet (thin film, thick plate, etc.), a rod (round bar, square column, etc.), a rectangular parallelepiped (including a cube), a ball, an annular, cylindrical, or other block (regular, irregular, etc.).

[0044] (Temporary Plug) In this embodiment, the term "temporary plug" refers to a material that is blended into a well treatment fluid for the purpose of temporarily plugging the well during various well drilling processes. Fluid leakage due to naturally occurring fractures or pores can result in a loss of circulating fluid and a drop in fluid pressure. This can result in the risk of wellbore collapse and a decrease in the efficiency of drilling and treatment operations. Therefore, to prevent fluid leakage and the resulting drop in fluid pressure, a method of temporarily plugging the well wall using a temporary plug is used. Another method is to create multi-stage fractures in different locations by repeatedly forming artificial fractures by fracturing and temporarily plugging the open holes with a temporary plug.

[0045] Calcium carbonate and the like are commonly used as temporary sealants, but after sealing, the temporary sealant must be decomposed by acid treatment. Therefore, if the acid treatment is insufficient, there is a risk that the temporary sealant will remain, reducing the amount of resource recovery. Furthermore, there is a risk that the acid treatment will corrode the metal casing. Therefore, in order to reduce the cost of acid treatment, shorten the excavation time, and prevent a reduction in the amount of resource recovery due to the remaining temporary sealant, it is preferable to use a temporary sealant made of a degradable material that decomposes and loses its sealing function after a certain period of time.

[0046] The form of the temporary sealing material may be in accordance with its application, and may be in the form of a sheet (a thin film, a thick plate, etc.), a spherical (ball-like), granular, powdery, thread-like, or other lumps (shaped regularly, shaped irregularly, etc.), etc.

[0047] The decomposition accelerator according to this embodiment can accelerate the decomposition of polyglycolic acid in a wellbore environment. Therefore, for example, it can accelerate the decomposition of a molded article for wellbore drilling that temporarily seals the wellbore to prevent the inflow of liquids such as water, thereby removing the seal. Furthermore, the decomposition accelerator according to this embodiment can accelerate the decomposition of polyglycolic acid at a pH of 6 or higher and 10 or lower. Therefore, it can accelerate the decomposition of polyglycolic acid forming the molded article for wellbore drilling while suppressing corrosion of the metal casing placed inside the wellbore.

[0048] In addition to use in well environments, the decomposition accelerator according to this embodiment can also be used in other environments where the resin needs to be removed by decomposition after temporarily exhibiting the function of polyglycolic acid. Examples of other environments include agricultural environments (e.g., promoting the decomposition of agricultural films made from polyglycolic acid). The decomposition accelerator according to this embodiment can also be used as a medical material (e.g., a material for regenerative medicine of bone or teeth, a porous substrate serving as a scaffold for cells).

[0049] [Method for Decomposing Polyglycolic Acid] A method for decomposing polyglycolic acid according to one aspect of the present invention includes a contacting step of contacting the decomposition accelerator according to this embodiment with polyglycolic acid. This can promote the decomposition of polyglycolic acid. For example, the decomposition accelerator according to this embodiment can be contacted with polyglycolic acid by adding, applying, spraying, sprinkling, or the like, the decomposition accelerator according to this embodiment to polyglycolic acid. The decomposition accelerator according to this embodiment can also be contacted with polyglycolic acid by, for example, immersing polyglycolic acid in a liquid containing the decomposition accelerator according to this embodiment. Furthermore, the decomposition accelerator according to this embodiment can also be contacted with polyglycolic acid by mixing the decomposition accelerator according to this embodiment with polyglycolic acid by, for example, stirring.

[0050] In the contacting step, the decomposition accelerator according to this embodiment may be diluted 1 to 10,000 times with water or the like before contacting with polyglycolic acid, or may be contacted with polyglycolic acid without dilution.

[0051] The pH in the contact step is preferably 6 or higher, more preferably 6.5 or higher, and even more preferably 7 or higher, from the viewpoint of suppressing corrosion of metals present in the surrounding environment and stimulating microbial activity and facilitating growth. Furthermore, the pH is preferably 10 or lower, more preferably 9.5 or lower, and even more preferably 9 or lower, from the viewpoint of further accelerating the decomposition of polyglycolic acid by the enzyme derived from the microorganism according to one embodiment of the present invention. Therefore, the pH range in the contact step is preferably pH 6 or higher and pH 10 or lower, and more preferably pH 7 or higher and pH 9 or lower. To adjust the pH in the contact step, a pH adjuster may be used together with the decomposition accelerator according to one embodiment of the present invention, and a step of adjusting the pH of the surrounding environment may also be included.

[0052] The temperature in the contact step may be 25° C. or higher, 30° C. or higher, or 35° C. or higher. The temperature may be 75° C. or lower, 65° C. or lower, or 55° C. or lower. A temperature of 35° C. or higher and 65° C. or lower is preferred, as this promotes the decomposition of polyglycolic acid by the microbial enzyme according to one aspect of the present invention.

[0053] The concentration and amount of the decomposition accelerator according to this embodiment used in the contact step can be appropriately selected depending on the form of the active ingredient of the decomposition accelerator, the dosage form, the time of use, the method and place of use, etc.

[0054] [Well Drilling Method] A well drilling method according to one aspect of the present invention includes a step of contacting a decomposition accelerator according to this embodiment with a molded article for well drilling formed of polyglycolic acid in a well environment. The molded article for well drilling may be a downhole tool, a downhole tool component, or a temporary sealant. Specific examples of the downhole tool, the downhole tool component, or the temporary sealant are as described above in the section [Decomposition Accelerator for Decomposition of Polyglycolic Acid].

[0055] An example of a method for contacting the decomposition accelerator according to this embodiment with the molded article for well drilling is to introduce the decomposition accelerator into a well and bring it into contact with the molded article for well drilling after the molded article for well drilling has been used as a sealing material. The decomposition accelerator may be brought into contact with the molded article for well drilling together with a fluid originally present in the well, such as groundwater, or a fluid supplied into the well after downhole insertion.

[0056] Alternatively, the well drilling molded product and the decomposition accelerator according to this embodiment may be introduced into the well so as not to come into contact with the well drilling molded product, and the decomposition accelerator and the well drilling molded product may be brought into contact for a desired period of time after the well is sealed with the well drilling molded product. One example is to encapsulate the decomposition accelerator in a container, then support the container on the well drilling molded product and introduce it into the well, controlling the decomposition accelerator to elute from the container for a desired period of time. If the container is made of a decomposable resin such as polyglycolic acid, the decomposable resin is hydrolyzed by water in the fluid. Hydrolysis reduces the strength of the container encapsulating the decomposition accelerator, and the decomposition accelerator elutes, allowing the decomposition accelerator to come into contact with the well drilling molded product.

[0057] Alternatively, the molded article for well drilling may be introduced into a well while the decomposition-accelerating agent according to the present embodiment and the molded article for well drilling are in contact with each other in advance. For example, a molded article for well drilling containing the decomposition-accelerating agent according to the present embodiment may be molded and introduced into a well so that the molded article for well drilling decomposes within a desired period after sealing.

[0058] The well drilling method according to one aspect of the present invention may include other steps in addition to the step of contacting the decomposition-accelerating agent according to this embodiment with the molded article for well drilling. Examples of the other steps include a sealing step.

[0059] The plugging process is a process of temporarily plugging the inside of a wellbore using a downhole tool. An example of the plugging process is shown below. The downhole tool applies a force in the axial direction of the mandrel to compress the downhole tool member, which is a sealing member, in the axial direction and expand the diameter in a direction perpendicular to the axial direction of the mandrel. Then, the sealing member abuts against the inner wall of the downhole or casing, and the inner part in the direction perpendicular to the axial direction abuts against the outer peripheral surface of the mandrel. In this way, the inside of the wellbore is temporarily plugged using the downhole tool.

[0060] Furthermore, the well drilling method according to one aspect of the present invention may further include a step of supplying a temperature-adjusting medium (e.g., water) to the well after plugging to adjust the temperature of the downhole tool or downhole tool component that is plugging the well. This step makes it possible to control the onset of decomposition of the downhole tool or downhole tool component in the well by adjusting the ambient temperature of the downhole tool or downhole tool component. Furthermore, this temperature adjustment can improve the polyglycolic acid decomposition-promoting activity of the decomposition promoter of this embodiment. Therefore, this step is preferable from the viewpoint of performing the unplugging step at a desired timing.

[0061] The well drilling method according to one aspect of the present invention may further include a step of recovering decomposition products resulting from the decomposition of the molded article for well drilling by recovering fluid in the well after contacting the decomposition-promoting agent of the present embodiment with the molded article for well drilling. Such a step makes it possible to easily recover the decomposition products in the well, and is suitable from the viewpoint of suppressing the occurrence of well clogging in the production of hydrocarbon resources.

[0062] The well drilling method according to one aspect of the present invention uses the decomposition accelerator of the present embodiment to decompose well drilling moldings within a desired period of time. Furthermore, the well drilling moldings can be rapidly decomposed in a low-temperature well environment. Furthermore, the well drilling moldings can be rapidly decomposed in an environment with a pH of 6 or higher and 10 or lower. Therefore, the well drilling method according to one aspect of the present invention can promote the decomposition of well drilling moldings while suppressing corrosion of metal casings or setting tools placed inside the wellbore and maintaining the integrity of the well. As a result, the steps and costs required for the decomposition of well drilling moldings can be reduced, and the mining efficiency of hydrocarbon resources such as oil and natural gas can be improved.

[0063] [Summary] The microorganism according to aspect 1 of the present invention is a microorganism (Accession No.: NITE BP-04014) belonging to a species closely related to Geobacillus isigianus (Geobacillus sp.).

[0064] The culture-derived product according to the second aspect of the present invention is a culture, cultured bacterial cells, culture supernatant, or extract of the microorganism according to the first aspect of the present invention.

[0065] A degradation promoter according to Aspect 3 of the present invention is a degradation promoter for decomposing polyglycolic acid, which contains, as an active ingredient, the microorganism according to Aspect 1 of the present invention or the culture-derived product according to Aspect 2 of the present invention.

[0066] The decomposition accelerator according to Aspect 4 of the present invention is a decomposition accelerator for decomposing a molded product for well drilling, and is the decomposition accelerator according to Aspect 3 of the present invention, wherein the molded product for well drilling may be formed from polyglycolic acid.

[0067] The decomposition accelerator according to Aspect 5 of the present invention may be in Aspect 4 of the present invention, wherein the molded article for well drilling may be a downhole tool, a downhole tool component, or a temporary sealing material.

[0068] A method for decomposing polyglycolic acid according to Aspect 6 of the present invention includes a contacting step of bringing the decomposition accelerator according to any one of Aspects 3 to 5 of the present invention into contact with polyglycolic acid.

[0069] A method for decomposing polyglycolic acid according to Aspect 7 of the present invention may be such that in the contacting step of Aspect 6 of the present invention, the decomposition accelerator is brought into contact with the polyglycolic acid under conditions of pH 6 or higher and pH 10 or lower.

[0070] A well drilling method according to an eighth aspect of the present invention includes a step of contacting the decomposition accelerator according to any one of the third to fifth aspects of the present invention with a molded article for well drilling formed of polyglycolic acid in a well environment.

[0071] A well drilling method according to a ninth aspect of the present invention is the method according to the eighth aspect of the present invention, wherein the well drilling molded product is a downhole tool, a downhole tool component, or a temporary sealing material.

[0072] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents cited in this specification are incorporated by reference.

[0073] In the following examples, polyglycolic acid may be abbreviated as "PGA." The polyglycolic acid shown in the following examples is a homopolymer of glycolic acid.

[0074] Evaluation Example 1 Screening of Polyglycolic Acid-Decomposing Bacteria Steam condensate from a steam distribution pipe at a factory in Fukushima Prefecture was used as a source for isolating microorganisms.

[0075] The screening medium (agar plate) used was a two-layered medium consisting of a lower layer of NB agar medium shown in Table 1 and an upper layer of PGA agar medium containing dispersed polyglycolic acid particles shown in Table 2. A schematic diagram of the screening medium is shown in Figure 1. To match the growth of the degrading bacteria with the decomposition rate of PGA, the weight-average molecular weight (Mw) of PGA in the PGA agar medium was set to approximately 40,000. Furthermore, to significantly lower the pH of glycolic acid eluted after PGA decomposition, KPi was used in the PGA agar medium, resulting in a PGA concentration of 0.5%. KPi is a buffer solution prepared by adding equimolar amounts of dipotassium hydrogen phosphate and potassium dihydrogen phosphate to distilled water to prepare a 0.1 M mixed solution, and then adding KOH aqueous solution to adjust the pH to 7. In Table 2, 50% D indicates the 50% average particle size.

[0076]

[0077]

[0078] After mixing the components, the mixture was sterilized in an autoclave at 121°C for 20 minutes, and the mixture was poured into a petri dish and cooled to room temperature to solidify, thereby preparing an NB agar medium. For the PGA agar medium, a lower layer of NB agar medium was prepared in the same manner, and then an upper layer of agar medium containing dispersed polyglycolic acid microparticles was prepared, forming a two-layer structure.

[0079] Evaluation Example 2 Isolation of Polyglycolic Acid-Degrading Bacteria The steam condensate was diluted 1000-fold with sterile distilled water and applied to the screening medium, followed by incubation at 55°C for 7 days. The periphery of one of the resulting colonies became transparent, confirming the degradation of polyglycolic acid. The microorganism isolated from the colony was inoculated into NB medium and incubated at 55°C for 1 day, after which the purity of the colony was confirmed. The microorganism collected from the colony was inoculated again into PGA agar medium and incubated at 55°C for 7 days. The periphery of the newly grown colony became transparent, confirming the degradation of polyglycolic acid. The microorganism collected from the colony was designated DB14 and used in the subsequent studies.

[0080] [Evaluation Example 3] Identification of DB14 Identification tests were outsourced to Techno Suruga Co., Ltd. A comprehensive assessment was made based on morphological observation, physiological tests, and the sequence of 16S rDNA. The colony shape of this strain is shown in Figure 2, and a Gram stained image is shown in Figure 3.

[0081] The method used to identify DB14 is described below.

[0082] (1. Culture conditions) Culture medium: Oxoid Nutrient Agar (Oxford, ENG) Culture temperature: 65°C Culture confirmation time: 24 hours Other conditions: Aerobic culture

[0083] (2. 16S rDNA base sequence analysis) DNA extraction: Cica Genesis DNA Extraction Reagent ST (Kanto Chemical, Japan) PCR amplification: Tks Gflex DNA Polymerase (Takara Bio, Japan) Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) Primers used: PCR amplification: 9F, 1510R; sequence (approximately 1,500 bp): 9F, 515F, 1099F, 536R, 926R, 1510R Sequencing: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) Base sequencing: ChromasPro 2.1 (Technelysium, Australia) BLAST homology search: Analysis software: ENKI v3.2 (TechnoSuruga Laboratory, Japan), International Nucleotide Sequence Database (DDBJ / ENA / GenBank), Search date: November 10, 2022. Simple molecular phylogenetic analysis. Phylogenetic tree estimation: Neighbor-joining method, Base substitution model: Kimura-2-parameter, Tree shape reliability evaluation: Bootstrap method (1,000 round trips).

[0084] (3. First stage bacterial test) Colony observation was performed using a stereoscopic microscope, morphological observation using an optical microscope, and tests were performed based on the Barrow & Feltham method for catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F). ・Gram staining: Faber G "Nissui" (Nissui Pharmaceutical, Japan) ・Microscope: Optical microscope BX50F4 (Olympus, Japan) ・Stereoscopic microscope: SMZ800N (Nikon, Japan)

[0085] (4. Bacterial Tier 2 Test) A bacterial tier 2 test was performed using API 50 CHB (b90 Merieux, FRA).

[0086] The 16S rDNA sequence of DB14 analyzed by 16S rDNA sequence analysis is shown in SEQ ID NO: 1. The results of a BLAST search of DB14 are shown in Table 3, and a simplified molecular phylogenetic tree based on the 16S rDNA sequence of DB14 is shown in Figure 4. The "a" in Table 3 is not a valid scientific name based on the International Code of Prokaryotic Nomenclature and was therefore excluded from this analysis. The line in the upper left of Figure 4 indicates the scale bar, and the numbers at the branches of the phylogenetic branches indicate bootstrap values. The T at the end of the strain name indicates the type strain of that species.

[0087]

[0088] The results of the first stage bacterial test are shown in Table 4. The first stage bacterial test was carried out by using cultures that had been cultivated for 24 hours to about two weeks, and observing them multiple times.

[0089]

[0090] The bacterial second stage test results are shown in FIG.

[0091]

[0092]

[0093] Simple phylogenetic analysis revealed that DB14 was included in the cluster consisting of the genus Geobacillus. Furthermore, 16S rDNA analysis revealed that Geobacillus icigianus G1w1 T The DB14 strain was supported by a high bootstrap value of 99.7% with the strain (KF631430), indicating a close relationship, but some distance was observed between the two strains. Results of the first-stage bacterial test indicated that DB14 grew well at 65°C, was a motile Gram-positive bacillus, had positive catalase and oxidase reactions, produced acid from glucose, and did not produce gas. These properties were nearly identical to those of the Geobacillus genus, to which 16S rDNA base sequence analysis assigned it, except that no spores were detected. Results of the second-stage bacterial test using a biochemical and physiological property test kit (API kit; bioMérieux Japan) indicated that DB14 oxidized D-xylose, melibiose, sucrose, raffinose, etc., but did not oxidize rhamnose, inositol, mannitol, or lactose, and exhibited gelatinase activity but did not exhibit β-galactosidase, arginine dihydrolase, or urease activity. Furthermore, DB14 did not grow under anaerobic conditions, in the presence of 5% NaCl, or at pH 5.0, but grew in the presence of 1% NaCl and at pH 9.0, hydrolyzing casein but not starch. These properties are similar to those of Geobacillus icigianus, which 16S rDNA sequence analysis suggested was its closest relative. However, there were differences, such as a positive oxidase reaction, no urease activity, and no nitrate reduction. Therefore, although DB14 belongs to the genus Geobacillus, it was difficult to estimate its taxonomic group at the species level. For this reason, it was tentatively identified as Geobacillus sp.

[0094] [Evaluation Example 4] Decomposition of Polyglycolic Acid 3 g of polyglycolic acid pellets were sandwiched between two aluminum sheets and heated and compressed at 5 MPa for 2 minutes in a heat press heated to 260 °C. The aluminum sheets were then sandwiched between cooling plates at 5 °C to produce a 170 μm PGA sheet. 20 mg of the PGA sheet was cut out, immersed in a 70% aqueous ethanol solution, and allowed to stand for 2 hours. One side of the sheet was then sterilized by drying for 30 minutes under UV irradiation in a safety cabinet. The sheet was then inverted and dried for another 30 minutes under UV irradiation to sterilize the other side. A sterilized PGA sheet sample was obtained by the above procedure.

[0095] 10 ml of NB medium (8 g / L) was poured into the test tube, sealed with a silicone stopper, and then sterilized by autoclaving at 121 ° C for 20 minutes. A pre-weighed sterilized PGA sheet was then added to the test tube, and one platinum loop of DB14 that had been cultured was inoculated and then sealed with a silicone stopper. Control A, in which only the PGA sheet was added but no DB14 was inoculated, Control B, in which no PGA sheet was added but no DB14 was inoculated, and Control C, in which no PGA sheet was added but no DB14 was inoculated, were also prepared. Two samples were prepared for each condition.

[0096] The test tubes were stored in a shaking incubator at 55 ° C and 180 rpm, and were removed at predetermined times. 1 ml of the solution was recovered from the test tube and used for subsequent pH measurement and glycolic acid quantification. The decomposition solution in the test tube was subjected to suction filtration using a filter to separate the solid and liquid. The filter and the residue were then dried in a desiccator for 12 hours, after which the weight of the residue was measured. The weight of the PGA decomposition product was determined by subtracting the weight of the filter measured before use.

[0097] As shown in Figure 6, it was found that the weight retention rate of the PGA sheet was significantly reduced by inoculating DB14. This suggests that the decomposition of polyglycolic acid was promoted by inoculating DB14. Furthermore, as shown in Figure 7, it was found that the pH temporarily increased to approximately 8-9 due to the growth of DB14. However, the pH tended to decrease as the culture time continued. This is presumably due to the production of glycolic acid, an acid component, as the PGA sheet decomposes.

[0098] Evaluation Example 5: Quantification of Glycolic Acid After the incubation period, the digested solution was diluted 5-fold with the eluent and then quantified for glycolic acid using HPLC. Shimadzu Corporation's HPLC systems (LC-20AD, SIL-20A, SPD-20A, and CTO-20A) were used as the measurement device. An Ultra AQ C18 5 μm column (Restek Corporation) was used, and the temperature was 40°C. Furthermore, 53.2 g of phosphoric acid and 23 g of ammonium dihydrogen phosphate (both manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in ultrapure water and made up to 1 L. This solution was used as the eluent for HPLC, and measurements were performed at a flow rate of 0.5 ml / min. UV at a wavelength of 210 nm was used for peak detection. First, weighed glycolic acid was dissolved in distilled water to prepare aqueous glycolic acid solutions of 0.1 mg / ml, 0.5 mg / ml, and 1.5 mg / ml. The aqueous glycolic acid solutions were measured by HPLC, and the peak area on the HPLC chart between RT (retention times) of 4.10 and 4.50 minutes was determined, and the calibration curve for glycolic acid shown in Figure 8 was created.

[0099] When the NB medium was measured by HPLC, peaks of components other than glycolic acid were also present in the RT region of 4.10 to 4.50 minutes. Therefore, the released concentration of glycolic acid was calculated as the difference from the control group prepared in Evaluation Example 4, using the following formula.

[0100] - Glycolic acid release concentration of Control A = {0.00281 × (area value of Control A − area value of Control C) + 10.23813} × 5 (dilution concentration) / 1000 (unit conversion) [mg / ml] - Glycolic acid release concentration of DB14 + PGA sheet = {0.00281 × (area value of DB14 − area value of Control B) + 10.23813} × 5 (dilution concentration) / 1000 (unit conversion) [mg / ml] The area values ​​in the formula are the peak area values ​​for R.T. 4.10 to 4.50 minutes on the HPLC chart.

[0101] The HPLC measurement results are shown in Figure 9 and Table 7. The error bars in Figure 9 indicate the range obtained by adding or subtracting the value calculated from the standard deviation of the measurement results for each sample solution to or from the average value of the results. The results in Table 7 show the average values ​​obtained when each sample solution was measured. These results demonstrate that the inoculation of DB14 significantly increases the concentration of glycolic acid released into the degradation solution. This demonstrates that the inoculation of DB14 promotes the degradation of polyglycolic acid.

[0102]

[0103] [Evaluation Example 6] Fractionation of crude enzyme derived from DB14 Fractionation of crude enzyme derived from DB14 was performed by reprecipitation using ammonium sulfate. First, 300 ml of NB medium was placed in a 500 ml flask, sealed with a silicone stopper, and sterilized by autoclaving at 121 ° C for 20 minutes. Next, one platinum loop of DB14 was inoculated and cultured with shaking at 55 ° C and 180 rpm for 1 day. The bacterial cells and solution were then separated using a centrifuge, and ammonium sulfate was added to the solution side to achieve a 40% saturation concentration, followed by stirring. Further, the 40% saturated precipitate and solution were separated using a centrifuge, and additional ammonium sulfate was added and stirred to achieve an 80% saturation concentration in the solution side. The 80% saturated precipitate and solution were separated using a centrifuge, and 2 ml of 0.1 M KPi was added to the 80% saturated precipitate to dissolve it, thereby preparing a crude enzyme solution.

[0104] Evaluation Example 7: Esterase Activity of Crude Enzyme Derived from DB14 The esterase activity of the crude enzyme derived from DB14 was measured according to the method of Kay et al. (MJ Kay, RW McCabe, and LHG Morton. Chemical and physical changes occurring in polyester polyurethane during biodegradation, International Biodeterioration & Biodegradation 31 (1993) 209-225). 0.268 ml of 1 mM p-nitrophenyl acetate and 0.132 ml of crude enzyme solution were added to 1.6 ml of 0.1 M KPi, and the increase in p-nitrophenol liberated by decomposition of the ester bond was determined by measuring the absorbance at 405 nm using a spectrophotometer. The amount of enzyme required to produce 1 μmol of p-nitrophenol per minute was defined as 1 unit (U). To examine the effect of the pH of the liquid medium, esterase activity was also measured using 0.4 M sodium phosphate buffer adjusted to pH 8 instead of 0.1 M KPi. The results of the esterase activity measurements are shown in Table 8.

[0105]

[0106] As shown in Table 8, the crude enzyme derived from DB14 exhibited esterase activity, and was found to exhibit stronger activity at pH 8 than at pH 7. Because of its strong esterase activity, it is suggested that it may also exhibit stronger activity in the hydrolysis of polyglycolic acid at a pH higher than pH 7, for example, at pH 8.

[0107] [Evaluation Example 8] Decomposition of PGA using DB14-derived crude enzyme To investigate the effect of pH on the decomposition of PGA sheets by fractionated DB14-derived crude enzymes, a decomposition test was conducted using 0.1 M KPi adjusted to pH 7, 0.4 M sodium phosphate buffer adjusted to pH 8, and 0.5 M Tris-HCl buffer adjusted to pH 9. A 2 mg piece of sterilized PGA sheet, 900 μl of the above-mentioned buffer, and 100 μl of DB14-derived crude enzyme solution were added to a 2 ml vial, sealed with a lid, and then decomposed for 18 hours at 60 °C and 80 rpm using a shaking incubator. The decomposition solution in the test tube was subjected to solid-liquid separation by suction filtration using a filter. The filter and the remaining solution were then dried in a desiccator for 12 hours, and the weight of the residue was measured and used as the weight of the PGA decomposition product. The molecular weight of the resulting PGA decomposition product was also measured using HPLC. For comparison, a degradation test was also conducted in which 100 μl of sterile distilled water (control) and a solution obtained by autoclaving DB14-derived crude enzyme solution at 121°C for 20 minutes (autoclaved enzyme) were added instead of the DB14-derived crude enzyme solution. Two samples were prepared for each condition. The weight and weight-average molecular weight (Mw) of the PGA sheet before and after the degradation test were compared, and the weight retention rate (%) and weight-average molecular weight retention rate (%) were calculated. The results are shown in Figure 10 and Table 9. The error bars in Figure 10 were set by adding and dividing the standard deviation calculated from the measurement results by the average value of the measurement results, and the values ​​in Table 9 indicate the average value of the measurement results.

[0108]

[0109] As shown in Figure 10, the addition of the DB14-derived crude enzyme solution promoted the weight loss and Mw decrease of PGA in buffer solutions of all pH levels, demonstrating the PGA degradation activity of the DB14-derived crude enzyme solution. The fact that the weight loss and Mw decrease of PGA were slower when the crude enzyme was denatured by autoclaving compared to the control also supports the PGA degradation activity of the crude enzyme. Furthermore, it was found that PGA degradation activity was highest at pH 8.

[0110] Furthermore, as shown in Evaluation Example 4 and Figure 7, DB14 has the property of increasing the pH of the surrounding environment to about 8 to 9. Therefore, it was found that the culture of DB14 is efficient for maximizing the hydrolysis ability of an enzyme having PGA degrading activity.

[0111] The microorganisms of the present invention can be used in drilling wells for extracting hydrocarbon resources such as petroleum or natural gas.

[0112] NITE BP-04014

Claims

1. A microorganism (accession number: NITE BP-04014) belonging to a species closely related to Geobacillus ishigianus (Geobacillus sp.).

2. A culture-derived product, which is a culture, cultured cells, culture supernatant or extract of the microorganism according to claim 1.

3. A decomposition accelerator for polyglycolic acid, containing as an active ingredient the microorganism according to claim 1 or the culture-derived product according to claim 2.

4. The decomposition accelerator according to claim 3, which is for decomposing a molded article for well drilling, and the molded article for well drilling is formed of polyglycolic acid.

5. The decomposition accelerator according to claim 4, wherein the molded article for well drilling is a downhole tool, a downhole tool member or a temporary plugging material.

6. A method for decomposing polyglycolic acid, comprising a contacting step of bringing the decomposition accelerator according to claim 3 into contact with polyglycolic acid.

7. The method for decomposing polyglycolic acid according to claim 6, wherein in the contacting step, the decomposition accelerator is brought into contact with the polyglycolic acid under the condition of pH 6 or more and pH 10 or less.

8. A well drilling method, comprising a step of bringing the decomposition accelerator according to claim 3 into contact with a molded article for well drilling formed of polyglycolic acid in a well.

9. The well drilling method according to claim 8, wherein the molded article for well drilling is a downhole tool, a downhole tool member or a temporary plugging material.

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