Novel microorganisms and uses thereof

Abstract

The microorganism of the present disclosure is a microorganism belonging to the genus Paenibacillus (deposited under Accession No. NITE BP-04014) that is closely related to Bacillus glacialis.

Description

Technical Field

[0001] This invention relates to novel microorganisms and their applications. Background Technology

[0002] Biodegradable resins and other biodegradable resins are utilized as raw materials that can be removed through degradation after use. Furthermore, the use of microbial degradation is considered as a method to promote the degradation of biodegradable plastics such as polylactic acid. To date, methods for degrading biodegradable plastics such as polylactic acid using microorganisms belonging to the genus *Geotrichum* have been reported (Patent Documents 1 and 2, Non-Patent Document 1).

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: EP 3944906 A

[0006] Patent Document 2: Japanese Patent Application Publication No. 2017-086040

[0007] Non-patent literature

[0008] Non-patent literature 1: Polymer Degradation and Stability (2018), 154, 46-54 Summary of the Invention

[0009] The problem that the invention aims to solve

[0010] Polyglycolic acid (PGA) is also used in applications such as well drilling and agriculture as a raw material that can be degraded after its intended use. For example, in hydraulic fracturing processes for well drilling, PGA is used as a component of degradable downhole tools. After temporarily sealing off a portion of the well using downhole tools to change the fluid pattern, PGA hydrolyzes due to the water within the well. The hydrolyzed PGA loses its strength, and the seal within the well is broken.

[0011] However, in the relatively low-temperature (e.g., below 66°C) pit environment, the hydrolysis of polyglycolic acid (PGA) is slowed down, thus posing a problem that it is difficult to remove the seal within the pit within the appropriate time range required in the aforementioned hydraulic fracturing process. Furthermore, while methods using acidic fluids to promote PGA degradation are known, there is a risk that corrosion of the metal casing is accelerated, potentially compromising the integrity of the pit.

[0012] Therefore, microorganisms that promote the degradation of polyglycolic acid in relatively low-temperature pit environments (e.g., below 66°C and pH 6–10) are unknown, and a microorganism that promotes the degradation of polyglycolic acid under such conditions is highly anticipated.

[0013] One aspect of the present invention was made in view of the above-mentioned problems, and its object is to provide a novel microorganism that promotes the degradation of polyglycolic acid.

[0014] Solution for solving the problem

[0015] Through screening, the inventors discovered a novel microorganism that promotes the degradation of polyglycolic acid (PGA) in relatively low-temperature environments, such as below 66°C. Furthermore, they found that this microorganism can promote PGA degradation in environments with a pH above 6 and a pH below 10. In other words, they discovered that this microorganism can promote the degradation of downhole tool components for well excavation that are made of PGA in a pit environment, thus completing this invention.

[0016] One aspect of the present invention is a microorganism belonging to a species closely related to Bacillus glaciformis (Bacillus genus) (accession number: NITE BP-04014).

[0017] Invention Effects

[0018] According to one aspect of the present invention, a microorganism that promotes the degradation of polyglycolic acid can be provided. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the agar medium used in the screening of microorganisms.

[0020] Figure 2 This is a diagram showing the colony shape of DB14.

[0021] Figure 3 This is a diagram showing a Gram staining image of DB14.

[0022] Figure 4 This is a diagram showing a simplified molecular phylogenetic tree of DB14 based on its 16S rDNA base sequence.

[0023] Figure 5 This is a graph showing the results of the second phase of the bacterial experiment.

[0024] Figure 6 This is a graph showing the change in weight retention of PGA slices cultured in DB14.

[0025] Figure 7 This is a graph showing the pH changes in the DB14 culture.

[0026] Figure 8 This is a graph showing the standard curve of glycolic acid.

[0027] Figure 9 This is a graph showing the changes in glycolic acid concentration during DB14 culture.

[0028] Figure 10 This is a graph showing the degradation results of polyglycolic acid after crude enzyme treatment with DB14. Detailed Implementation

[0029] In this specification, unless otherwise stated, “A~B”, which indicates a numerical range, means “A or above (greater than or equal to A) and below B (less than or equal to B)”.

[0030] In this specification, promoting the degradation of polyglycolic acid (PGA) means promoting the hydrolysis reaction of PGA. Furthermore, in this specification, microorganisms with PGA degradation-promoting activity refer to microorganisms that promote the hydrolysis reaction of PGA.

[0031] For example, whether a microorganism possesses polyglycolic acid (PGA) degradation-promoting activity can be evaluated through the following process: contacting a liquid containing the microorganism or a culture source of the microorganism with a molded article formed from PGA; measuring the weight of the molded article 18 hours after the contact or determining the weight-average molecular weight according to ISO 6014-1:2019; calculating 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 as the weight retention rate or weight-average molecular weight retention rate; if the calculated weight retention rate or weight-average molecular weight retention rate is 0.9 or less when the weight retention rate or weight-average molecular weight retention rate of the control (where a liquid without a culture source of the microorganism is contacted with PGA) is set to 1, the microorganism is evaluated as having PGA degradation-promoting activity. The culture source of the microorganism will be described later.

[0032] In this specification, oil wells or gas wells with porous and permeable underground layers used for the extraction of oil or natural gas resources are collectively referred to as pit wells.

[0033] [New Microorganisms]

[0034] One aspect of the present invention involves a microorganism belonging to a species closely related to *Geobacillus icigianus* (genus *Geobacillus* sp.). Hereinafter, this microorganism will sometimes be referred to as DB14.

[0035] DB14 is a microorganism that promotes the degradation of polyglycolic acid (PGA). It can promote the degradation of PGA in low-temperature environments such as pits and wells.

[0036] DB14 can promote the degradation of polyglycolic acid at least in the pH range of 6 to 10.

[0037] Progeny strains of DB14, i.e., microorganisms with polyglycolic acid degradation-promoting activity, also fall within the scope of the microorganisms of one aspect of this invention. Naturally mutated or artificially mutated microorganisms of DB14 or its progeny strains, i.e., microorganisms with polyglycolic acid degradation-promoting activity, also fall within the scope of the microorganisms of one aspect of this invention.

[0038] The progeny strains of DB14 and the naturally mutated or artificially mutated strains of DB14 possess one, two, three, four, five, six, seven, eight, nine, or ten of the characteristics (1) to (10) described below:

[0039] (1) It is a motile Gram-bacterium that does not form spores;

[0040] (2) It does not grow at 37℃, but grows at 45℃ and 70℃;

[0041] (3) It does not grow at pH 5.0, but grows at pH 9.0;

[0042] (4) The catalase reaction is positive;

[0043] (5) The oxidase reaction is positive;

[0044] (6) Glucose produces acid, but no gas;

[0045] (7) Casein is hydrolyzed, but starch is not;

[0046] (8) It does not grow under anaerobic conditions;

[0047] (9) Glycerol oxide, ribose, D-xylose, glucose, fructose, mannose, aescin, maltose, aescin, maltose, melibiose, sucrose, trehalose, melibiose, melibiose and starch;

[0048] (10) It has gelatinase activity.

[0049] Results of 16S rDNA base sequence analysis, morphological observation, and physiological / biochemical trait tests showed that DB14 is closely related to Bacillus glaciers.

[0050] DB14 has been deposited at the Patent Microbial Collection Center (NPMD) of the National Institute of Technology and Evaluation (hereinafter referred to as "NITE"), Room 122, 2-5-8 Kazusa-Kamazutsu, Kisarazu City, Chiba Prefecture, Japan (Original deposit date: November 21, 2023, deposit number: NITE BP-04014).

[0051] The cultivation method for DB14 can be performed according to the usual cultivation methods for microorganisms of the genus *Geotrichum*. The cultivation method is either batch culture using liquid medium or fed-batch culture with continuous addition of carbon and / or organic nitrogen sources, preferably with aeration and stirring. The culture medium can contain essential nutrients such as carbon sources, nitrogen sources, or inorganic salts that can be assimilated by microorganisms belonging to the genus *Geotrichum*. The preferred cultivation pH is 6–10, the preferred cultivation temperature is 45°C–65°C, and the preferred cultivation time is 1–7 days.

[0052] [Culture source of DB14]

[0053] One embodiment of the present invention uses a culture source of DB14. Another embodiment of the present invention uses a culture, cultured cells, culture supernatant, or extract as the culture source. Furthermore, the culture source may be a mixture of two or more of the following: culture, cultured cells, culture supernatant, and extract. Additionally, the culture source may be a processed product such as a fragment, concentrate, purified product, or diluted product of a mixture of one or more of the following: culture, cultured cells, culture supernatant, or extract.

[0054] In this specification, "culture" refers to a culture containing DB14 cells and culture medium obtained under any conditions.

[0055] In this specification, "cultured cells" refers to bacterial cells isolated from the above-mentioned cultures by means of centrifugation or membrane separation.

[0056] In this specification, "culture supernatant" refers to the culture supernatant obtained by removing bacterial cells from the above-mentioned culture through centrifugation or membrane separation.

[0057] In this specification, "extract" refers to the residue obtained by adding a solvent (water, surfactant, buffer, etc.) to the cultured bacterial cells and stirring, followed by centrifugation or filtration; the solution obtained by removing and separating the residue; the precipitate produced by adding salts to the solution; or the substance obtained by separating the solution using HPLC or similar methods. As an example of an extract, a crude enzyme solution derived from DB14 can be cited.

[0058] In one embodiment of the present invention, the DB14 contained in the culture source can be either live or dead bacterial cells. Whether a culture source contains DB14 or a portion thereof can be determined. Confirmation of the presence of DB14 or a portion thereof can be made by methods known in the art, such as polymerase chain reaction (PCR), to confirm the presence of genes extracted from the culture source or the degradation promoter. Furthermore, if the culture source or the degradation promoter contains live bacterial cells, the presence of DB14 can be confirmed by further culturing the live bacterial cells to proliferate. For example, after aerobically culturing the culture source or NB agar medium sprinkled with the degradation promoter at 55°C for one day, the resulting colonies are isolated, and genes extracted from the bacterial cells present in each colony are confirmed by methods known in the art, such as PCR.

[0059] [Degradation accelerator for polyglycolic acid degradation]

[0060] One embodiment of the present invention provides a degradation accelerator for the degradation of polyglycolic acid (hereinafter, sometimes referred to as "the degradation accelerator of this embodiment") containing DB14 or a culture source of DB14 as an active ingredient.

[0061] The degradation promoter of this embodiment can be used directly as a degradation promoter, either DB14 or a culture source of DB14. Furthermore, the degradation promoter of this embodiment may also contain other components besides DB14 or a culture source of DB14. For example, DB14 or a culture source of DB14 can be mixed with a carrier (diluent) such as a solid or liquid carrier, a surfactant, and other formulation adjuvants to formulate various forms such as powders, granules, wettable powders, soluble powders, emulsions, liquids, oils, aerosols, microcapsules, pastes, coatings, fumigants, fumigants, and micro-sprays. The carrier (diluent), surfactant, and other formulation adjuvants can be substances used in known microbial preparations.

[0062] The concentration of DB14 in the degradation promoter of this embodiment is, for example, 1.0 × 10⁻⁶. 4 ~1.0×10 10 cfu / ml, preferably 3.0 × 10⁻⁶ 4 ~1.0×10 10 cfu / ml, more preferably 7.0×10 5 ~1.0×10 10 cfu / ml.

[0063] In the degradation accelerator of this embodiment, polyglycolic acid can also be degraded under the same pH conditions as DB14.

[0064] The degradation accelerator of this embodiment can be used in combination with other degradation accelerators known as degradation accelerators for polyglycolic acid degradation. This combination can be a mixed application of the degradation accelerator of this embodiment with other degradation accelerators, or it can be applied separately.

[0065] The degradation accelerator of this embodiment can be used as a degradation accelerator for degrading molded articles formed from polyglycolic acid. There are no particular limitations on the molding method and shape of the molded article to which the degradation accelerator is applied; the degradation accelerator of this embodiment can be used as a degradation accelerator for degrading molded articles of any shape obtained by any molding method.

[0066] Polyglycolic acid (PGA) can 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. Other monomeric components in the case of PGA as a copolymer include, for example, hydroxycarboxylic acids such as L-lactic acid, D-lactic acid, 3-hydroxybutyric 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 formed by intramolecular condensation of the aforementioned monomeric components; and cyclic carbonates such as trimethylene carbonate. In the case of PGA as a copolymer, from the viewpoint of promoting degradation at low temperatures, the structural units derived from glycolic acid are 70 mol% or more, preferably 80 mol% or more, and more preferably 90 mol% or more.

[0067] As an example, the degradation accelerator of this embodiment can be used as a degradation accelerator for degrading molded articles for pit excavation formed from polyglycolic acid. Examples of molded articles for pit excavation include downhole tools, downhole tool components, and temporary plugging materials.

[0068] (Downhole tools)

[0069] In this specification, the tools used for various well treatments, such as well excavation, well plugging, and fracturing (hydraulic fracturing), and placed within wells, are referred to as downhole tools. The shape of downhole tools is not particularly limited; for example, they can be made into previously known shapes. Examples of downhole tools include: fracturing plugs, bridge plugs, cement retainers, perforating guns, sealing balls, sealing plugs, and packers.

[0070] The downhole tool is preferably a pit excavation plug, more preferably a fracturing plug or a bridge plug. The pit excavation plug includes, for example, a mandrel and various downhole tool components disposed on an outer circumferential surface orthogonal to the axial direction of the mandrel. The mandrel may have a hollow portion, a shape in which the diameter changes along the axial direction, and may have a fixing portion, a stepped portion, a recess, or a protrusion on its outer surface. In addition to the downhole tool components, the pit excavation plug may further include known components such as sensors.

[0071] (Downhole tool components)

[0072] Examples of downhole tool components include: mandrels, bearing rings, cones, bearing seats, bottom joints, balls, or sealing components in the aforementioned fracturing plugs or bridge plugs; sleeves, balls, ball seats, or sealing components in sleeve systems (fracturing sleeves); sealing components such as ball valves and flap valves within downhole tools; plugs or sealing components located between the downhole tool and the casing, thereby temporarily cutting off fluid flow; and sealing components that exist in the form of protecting or sealing downhole tool components, sensors, or flow paths made of metal, etc., and that seal the wellbore by expanding the diameter of these metal parts, etc.

[0073] Downhole tool components can be molded bodies obtained by molding polyglycolic acid, or they can be secondary processed bodies obtained by performing conventionally known machining (such as cutting) on ​​the molded body.

[0074] The shape of downhole tool components can be determined according to their purpose, and can be sheet-like (thin film-like, thick plate-like, etc.), rod-like (round rod-like, prismatic, etc.), cuboid-like (including cubic), spherical, ring-like, cylindrical, or other block-like (regular shape, irregular shape, etc.).

[0075] (Temporary filling material)

[0076] In this embodiment, temporary plug refers to a material used in various processes of well excavation for the purpose of temporarily sealing the fluid within the well. When fluid leakage occurs due to naturally occurring cracks or pores, circulating fluid may be lost, causing a decrease in fluid pressure. This results in the risk of well collapse or reduced efficiency in excavation and treatment operations. Therefore, to suppress fluid leakage and the accompanying decrease in fluid pressure, a method of temporarily sealing the well wall using temporary plug is employed. Furthermore, a method is also used to create multi-stage cracks in different locations by fracturing to create artificial cracks and repeatedly using temporary plug to temporarily seal the opening.

[0077] Calcium carbonate and similar materials are commonly used as temporary plugging materials. However, after sealing, these materials require acid treatment to degrade. Therefore, insufficient acid treatment poses a risk of residual temporary plugging material and reduced resource recovery. Furthermore, there is a risk of corrosion of metal casings due to acid treatment. Therefore, to reduce acid treatment costs, shorten excavation time, and prevent reduced resource recovery due to residual temporary plugging material, it is preferable to use a degradable temporary plugging material that loses its plugging function after a specified period.

[0078] The form of temporary filling material can be determined according to its purpose, and can be sheet-like (thin film-like, thick plate-like, etc.), spherical, granular, powdery, linear, or other block-like (regular shape, irregular shape, etc.).

[0079] The degradation promoter of this embodiment can promote the degradation of polyglycolic acid in a pit environment. Therefore, for example, it can promote the degradation of molded pit excavation products used to temporarily seal pits and suppress the inflow of liquids such as water, thereby removing the seal. Furthermore, the degradation promoter of this embodiment can promote the degradation of polyglycolic acid at pH 6 or higher and pH 10 or lower. Therefore, it can promote the degradation of polyglycolic acid in the molded pit excavation product while suppressing corrosion of the metal casing disposed inside the pit borehole.

[0080] In addition to its use in pit and well environments, the degradation promoter of this embodiment can also be used in other environments where the resin needs to be removed through degradation after temporarily exhibiting the function of polyglycolic acid. Examples of other environments include agricultural environments (e.g., promoting the degradation of agricultural films formed from polyglycolic acid). Furthermore, the degradation promoter of this embodiment can also be used as a medical material (e.g., a regenerative medical material for bones or teeth, or a porous substrate that serves as a scaffold for cells).

[0081] [Degradation methods for polyglycolic acid]

[0082] One aspect of the present invention provides a method for degrading polyglycolic acid (PGA) including a contact step in which the degradation promoter of this embodiment comes into contact with PGA. This promotes the degradation of PGA. For example, by adding, coating, spraying, or sprinkling the degradation promoter of this embodiment into PGA, the degradation promoter of this embodiment can come into contact with PGA. Furthermore, by impregnating PGA with a liquid containing the degradation promoter of this embodiment, the degradation promoter of this embodiment can also come into contact with PGA. Moreover, by mixing the degradation promoter of this embodiment with PGA through stirring or the like, the degradation promoter of this embodiment can also come into contact with PGA.

[0083] In the above contact process, the degradation promoter of this embodiment can be diluted with water or the like to 1 to 10,000 times and then contacted with polyglycolic acid, or it can be contacted with polyglycolic acid without dilution.

[0084] In terms of inhibiting corrosion of metals present in the surrounding environment and promoting the active growth of microorganisms, the pH in the above-mentioned contact process is preferably 6 or higher, more preferably 6.5 or higher, and even more preferably 7 or higher. Furthermore, in order to further promote the degradation of polyglycolic acid by enzymes derived from microorganisms according to one aspect of the present invention, the pH is preferably 10 or lower, more preferably 9.5 or lower, and even more preferably 9 or lower. Therefore, the pH range in the contact process is preferably pH 6 or higher and pH 10 or lower, more preferably pH 7 or higher and pH 9 or lower. In addition, to adjust the pH in the above-mentioned contact process, a pH adjusting agent can be used together with the degradation promoter of one aspect of the present invention, and a step of adjusting the pH of the surrounding environment may also be included.

[0085] The temperature in the above-described contact process can be 25°C or higher, 30°C or higher, or 35°C or higher. Alternatively, the temperature can be 75°C or lower, 65°C or lower, or 55°C or lower. In promoting the degradation of polyglycolic acid by enzymes derived from microorganisms according to one aspect of the invention, the temperature is preferably 35°C or higher and 65°C or lower.

[0086] The concentration and amount of degradation accelerator used in the above-mentioned contact process in this embodiment can be appropriately selected according to the form, dosage form, usage period, usage method and usage location of the effective component of the degradation accelerator.

[0087] [Pit and well excavation methods]

[0088] One aspect of the present invention provides a well excavation method comprising a step of contacting a degradation accelerator of this embodiment with a molded article for well excavation formed of polyglycolic acid in a well environment. The molded article for well excavation may be a downhole tool, a downhole tool component, or a temporary plugging material. Specific examples of downhole tools, downhole tool components, or temporary plugging materials are as described in the above-described item regarding the degradation accelerator for polyglycolic acid degradation.

[0089] As an example of the contact method between the degradation accelerator and the molded article for well excavation in this embodiment, a method can be listed such as introducing the degradation accelerator into the well after it has been used as a sealing material for the molded article for well excavation, so that it comes into contact with the molded article for well excavation. This degradation accelerator can come into contact with the molded article for well excavation along with fluids already present in the well, such as groundwater, or fluids supplied to the well after drilling.

[0090] Alternatively, the molded article for pit excavation and the degradation accelerator can be introduced into the pit in a manner that prevents the degradation accelerator of this embodiment from contacting the pit excavation molded article. After sealing the pit with the molded article, the contact between the degradation accelerator and the molded article is controlled for a desired period. As an example, a method can be described where a degradation accelerator is sealed in a container, which is then loaded with the molded article for pit excavation and introduced into the pit. The degradation accelerator is controlled to dissolve from the container for a desired period. In the case where the container is formed of a degradable resin such as polyglycolic acid, the degradable resin is hydrolyzed by water in the fluid. Through hydrolysis, the strength of the container containing the degradation accelerator decreases, the degradation accelerator dissolves, and thus the degradation accelerator can contact the molded article for pit excavation.

[0091] Furthermore, the molded article for pit excavation can be introduced into a pit after the degradation accelerator of this embodiment has been brought into contact with it beforehand. For example, in order to degrade the molded article for pit excavation during a desired period after sealing, a molded article for pit excavation containing the degradation accelerator of this embodiment can be molded and introduced into the pit.

[0092] In addition to the step of bringing the degradation accelerator of this embodiment into contact with the molded article for pit excavation, one embodiment of the present invention may include other steps in its pit excavation method. Examples of other steps include a sealing step.

[0093] A plugging procedure is a process of temporarily sealing a pit or well using downhole tools. An example of a plugging procedure is shown below. The downhole tool applies an axial force to the mandrel, causing the downhole tool component, which serves as the sealing member, to compress axially and expand in diameter in a direction orthogonal to the mandrel's axial direction. Furthermore, the sealing member abuts against the inner wall of the downhole wall or casing, and its inner portion, in the direction orthogonal to the axial direction, abuts against the outer circumferential surface of the mandrel. In this way, the pit or well is temporarily plugged using the downhole tool.

[0094] Furthermore, one embodiment of the well excavation method of the present invention may further include the following steps: after plugging, supplying a temperature-regulating medium (e.g., water) to the well to adjust the temperature of the downhole tool or downhole tool component used to plug the well. This step can control the initiation of degradation of the downhole tool or downhole tool component within the well by adjusting the ambient temperature of the downhole tool or downhole tool component. Furthermore, this temperature adjustment can enhance the polyglycolic acid degradation-promoting activity of the degradation promoter of this embodiment. Therefore, from the viewpoint of performing the unplugging step at the desired time, this step is preferred.

[0095] Furthermore, one embodiment of the well excavation method of the present invention may further include the following step: after contacting the degradation promoter of this embodiment with the molded article for well excavation, the degradation products generated by the degradation of the molded article for well excavation are recovered by recovering the fluid in the well. From the viewpoint of easily recovering the degradation products in the well and suppressing the occurrence of well blockage in oil and gas resource production, such a step is preferred.

[0096] One aspect of the well excavation method of the present invention utilizes the degradation promoter of this embodiment to degrade well excavation molded articles within a desired period. Furthermore, it enables rapid degradation of well excavation molded articles in a low-temperature environment, i.e., a well environment. Moreover, it enables rapid degradation of well excavation molded articles in an environment with pH above 6 and pH below 10. Therefore, the well excavation method of the present invention can suppress corrosion of metal casings or setting tools disposed inside the well borehole, maintain the integrity of the well, and promote the degradation of well excavation molded articles. As a result, it can reduce the steps and costs required for the degradation of well excavation molded articles, and improve the extraction efficiency of oil and gas resources such as petroleum or natural gas.

[0097] 〔Summarize〕

[0098] The microorganism in Scheme 1 of the present invention is a microorganism belonging to a species (Bacillus genus) closely related to Bacillus glacis (accession number: NITE BP-04014).

[0099] The culture source of Scheme 2 of the present invention is the culture, culture cells, culture supernatant or extract of the microorganism of Scheme 1 of the present invention.

[0100] The degradation promoter of Scheme 3 of the present invention is a degradation promoter for the degradation of polyglycolic acid containing microorganisms of Scheme 1 of the present invention or culture sources of Scheme 2 of the present invention as active ingredients.

[0101] The degradation accelerator of Scheme 4 of the present invention is the degradation accelerator of Scheme 3 of the present invention. Alternatively, the degradation accelerator may be a degradation accelerator for degrading molded articles for pit excavation, which are formed of polyglycolic acid.

[0102] In Scheme 5 of the present invention, the degradation promoter can also be, in Scheme 4 of the present invention, the molded article for pit excavation is a downhole tool, a downhole tool component or a temporary plugging material.

[0103] The degradation method of polyglycolic acid according to Scheme 6 of the present invention includes a contact step in which the degradation promoter of any one of Schemes 3 to 5 of the present invention is brought into contact with polyglycolic acid.

[0104] In the contact step described in Scheme 6 of the present invention, the degradation method of polyglycolic acid in Scheme 7 of the present invention may also involve contacting the degradation promoter with the polyglycolic acid under conditions of pH 6 or higher and pH 10 or lower.

[0105] The pit excavation method of Scheme 8 of the present invention includes a step of contacting the degradation promoter of any one of Schemes 3 to 5 of the present invention with a molded article for pit excavation formed of polyglycolic acid in a pit environment.

[0106] In Scheme 9 of the present invention, the pit excavation method can also be in Scheme 8 of the present invention, wherein the molded product for pit excavation is a downhole tool, a downhole tool component, or a temporary plugging material.

[0107] The following embodiments illustrate the implementation of the present invention in further detail. It is understood that the present invention is not limited to the following embodiments, and various details may be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims. Embodiments obtained by appropriately combining the disclosed technical means are also included within the technical scope of the present invention. In addition, all documents described in this specification are incorporated herein by reference.

[0108] Example

[0109] In the following examples, polyglycolic acid is sometimes abbreviated as "PGA". Furthermore, the polyglycolic acid shown in the following examples is a homopolymer of glycolic acid.

[0110] [Evaluation Example 1] Screening of polyglycolic acid degrading bacteria

[0111] Steam condensate from a steam delivery pipe at a factory in Fukushima Prefecture was used as the source for microbial isolation.

[0112] As the screening medium (agar plate), a two-layer medium was used, with NB agar medium as shown in Table 1 as the bottom layer and PGA agar medium with dispersed polyglycolic acid particles as shown in Table 2 layered on top. A schematic diagram of the screening medium is shown below. Figure 1 To ensure the growth of degrading bacteria matches the degradation rate of PGA, the weight-average molecular weight (Mw) of PGA in the PGA agar medium is set to approximately 40,000. Furthermore, the glycolic acid released after PGA degradation significantly lowers the pH; therefore, KPi is used in the PGA agar medium, with the PGA concentration set at 0.5%. KPi is prepared by adding equimolar amounts of dipotassium hydrogen phosphate and potassium dihydrogen phosphate to distilled water to prepare a 0.1M mixed solution, and then further adding KOH aqueous solution to prepare a buffer solution with a pH of 7. In Table 2, 50%D represents the 50% average particle size.

[0113] [Table 1]

[0114]

[0115] [Table 2]

[0116]

[0117] After mixing all the components, sterilize the mixture in an autoclave at 121°C for 20 minutes. Pour the mixture into petri dishes and allow it to cool and solidify at room temperature to prepare NB agar medium. For PGA agar medium, similarly prepare the lower layer of NB agar medium, and then prepare the upper layer of agar medium with dispersed polyglycolic acid microparticles, thus creating two layers.

[0118] [Evaluation Example 2] Isolation of polyglycolic acid degrading bacteria

[0119] The steam condensate was diluted 1000 times with sterile distilled water and spread onto the screening medium, then incubated at 55°C for 7 days. The periphery of one of the resulting colonies became clear, confirming the degradation of polyglycolic acid. The microorganisms isolated from this colony were inoculated into NB medium and incubated at 55°C for 1 day to confirm colony purity. The microorganisms collected from this colony were re-inoculated into PGA agar medium and incubated at 55°C for 7 days. The newly grown colonies became clear, confirming the degradation of polyglycolic acid. The microorganisms collected from this colony were designated DB14 for subsequent studies.

[0120] [Evaluation Example 3] Identification of DB14

[0121] Identification tests were commissioned to TECHNO SURUGA Co., Ltd. The determination was based on a comprehensive assessment of morphological observation, physiological tests, and 16S rDNA sequence. The colony morphology of this strain is shown below. Figure 2 The Gram staining image is shown in Figure 3 .

[0122] The following describes the method used for the identification of DB14.

[0123] (1. Cultivation conditions)

[0124] • Culture medium: Oxoid Nutrient Agar (Oxford, ENG)

[0125] • Culture temperature: 65℃.

[0126] • Culture confirmation time: 24 hours.

[0127] • Other conditions: Aerobic culture.

[0128] (2.16S rDNA base sequence analysis)

[0129] • DNA extraction Shikagenius DNA extraction reagent ST (Kanto Chemical, Japan).

[0130] ・PCR amplification of Tks Gflex DNA Polymerase (Takara Bio, Japan).

[0131] • BigDye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, USA).

[0132] • PCR amplification using primers: 9F, 1510R, sequencing (approximately 1500bp): 9F, 515F, 1099F, 536R, 926R, 1510R.

[0133] • Sequencing instrument: ABI PRISM 3500xL gene analyzer system (Applied Biosystems).

[0134] • Base sequence determination ChromasPro 2.1 (Technelysium, AUS).

[0135] • BLAST homology retrieval analysis software: ENKI v3.2 (TechnoSuruga Laboratory, Japan), international base sequence database (DDBJ / ENA / GenBank), retrieval date: November 10, 2022.

[0136] • Inference of phylogenetic tree for simplified molecular systematics analysis: neighbor-joining method, base substitution model: Kimura two-parameter model, tree reliability evaluation: bootstrap method (1000 repetitions).

[0137] (3. Phase I bacterial assay)

[0138] Based on colony observation using stereomicroscopy, morphological observation using optical microscopy, and the Barrow & Feltham method, experiments were conducted on catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F).

[0139] • Gram staining Favor G "Nissui" (Nissui pharmaceutical, Japan).

[0140] • Microscope, optical microscope BX50F4 (Olympus, Japan).

[0141] • Stereo microscope SMZ800N (Nikon, Japan).

[0142] (4. Phase II bacterial assay)

[0143] Phase II bacterial assays were performed using API 50 CHB (b90Merieux, FRA).

[0144] The 16S rDNA sequence of DB14, analyzed by 16S rDNA base sequence analysis, is shown in Sequence No. 1. Furthermore, the BLAST search results for DB14 are shown in Table 3, and a simplified molecular phylogenetic tree of DB14 based on its 16S rDNA base sequence is presented. Figure 4 The term “a)” in Table 3 is not a valid scientific name based on the International Rules for Nomenclature of Prokaryotes, and therefore is excluded from this analysis. Figure 4 The line at the top left represents the scale bar, and the number on the branch in the system branch represents the expansion value. The T at the end of the strain name indicates the type strain.

[0145] [Table 3]

[0146]

[0147] The results of the first-stage bacterial assay are shown in Table 4. The first-stage bacterial assay used cultures from approximately 24 hours to 2 weeks post-incubation, and involved multiple observations.

[0148] [Table 4]

[0149]

[0150] The results of the second phase of bacterial testing are shown in Figure 5 Tables 5 and 6.

[0151] [Table 5]

[0152]

[0153] [Table 6]

[0154]

[0155] Preliminary systematic analysis showed that DB14 was contained within a cluster of *Geotrichum* species. Furthermore, 16S rDNA analysis showed a high degree of confidence (up to 99.7%) in its close relationship to *Geotrichum glaciers* G1w1T (KF631430), but a discrepancy was noted. Phase I bacterial assays showed that DB14 exhibited good growth at 65°C, was a motile Gram-positive bacterium, and showed positive catalase and oxidase reactions, producing acid from glucose but not gas. These characteristics are largely consistent with those of *Geotrichum* species identified in the 16S rDNA sequence analysis, but the difference lies in the inability to confirm spore formation. The results of the second-stage bacterial assay using biochemical / physiological trait tests (API kit; bioMérieux, Japan) showed that DB14 oxidizes D-xylose, melibiose, sucrose, and melitriose, but not rhamnose, inositol, mannitol, or lactose. It exhibited gelatinase activity but not β-galactosidase, arginine dihydrolase, or urease activity. Furthermore, DB14 did not grow under anaerobic conditions, in the presence of 5% NaCl, and at pH 5.0. However, it grew under 1% NaCl and at pH 9.0, hydrolyzing casein but not starch. These traits are similar to those of *Bacillus glacialis*, which is the closest relative indicated by 16S rDNA sequence analysis. However, DB14 differs in that it showed a positive oxidase reaction, no urease activity, and no nitrate reduction. Therefore, although DB14 belongs to the genus *Bacillus*, it is difficult to presume its group classification at the species level. Thus, it is tentatively identified as belonging to the genus *Bacillus*.

[0156] [Evaluation Example 4] Degradation of Polyglycolic Acid

[0157] 3g of polyglycolic acid (PGA) granules were sandwiched between two aluminum sheets and heated and compressed using a hot press at 260°C under 5MPa for 2 minutes. The PGA sheets, along with the aluminum sheets, were then cooled on a 5°C cooling plate to produce 170μm PGA sheets. 20mg of the PGA sheet was cut and immersed in a 70% ethanol aqueous solution, allowing it to stand for 2 hours. Then, it was sterilized on one side by irradiating with UV light for 30 minutes in a biosafety cabinet. The sheet was then flipped over and dried again for 30 minutes by irradiating with UV light to sterilize the remaining half. These steps yielded a sample of sterilized PGA sheets.

[0158] 10 ml of NB medium (8 g / L) was injected into the test tube, sealed with a silicone stopper, and autoclaved at 121°C for 20 minutes to sterilize. Then, pre-weighed sterilized PGA tablets were added to the test tube, and DB14 cultured in a platinum ring was inoculated. The tubes were then sealed with silicone stoppers. Additionally, control A (with only PGA tablets added but no DB14 inoculation), control B (without PGA tablets added but with DB14 inoculation), and control C (without PGA tablets added but no DB14 inoculation) were prepared. Two samples were prepared for each condition.

[0159] The test tubes were stored in a shaking incubator at 55°C and 180 rpm for the specified time. 1 ml of solution was recovered from the test tubes for subsequent pH determination and glycolic acid quantification. The degradation solution in the test tubes was filtered to separate the solid and liquid phases. The residue, along with the filter, was dried in a desiccator for 12 hours. The weight of the residue was then measured, and the value obtained by subtracting the weight of the filter before use was taken as the weight of the PGA degradation product.

[0160] like Figure 6 As shown, DB14 inoculation significantly reduced the weight retention of the PGA tablets. This suggests that DB14 inoculation promoted the degradation of polyglycolic acid. Furthermore, as... Figure 7 As shown, the pH temporarily rises to around 8-9 during DB14 growth. However, with further cultivation, the pH tends to decrease. This is presumably because the degradation of the PGA tablets generates glycolic acid, an acidic component.

[0161] [Evaluation Example 5] Quantitative analysis of glycolic acid

[0162] For the degradation solution after the incubation period, the glycolic acid was quantified by HPLC after being diluted 5 times with eluent. As the measuring apparatus, HPLC systems (LC-20AD, SIL-20A, SPD-20A, and CTO-20A) manufactured by Shimadzu Corporation were used. An Ultra AQ C18 5μm column (manufactured by Restek Corporation) was used, and the temperature was set to 40°C. Furthermore, 53.2 g of phosphoric acid and 23 g of ammonium dihydrogen phosphate (both manufactured by Fujifilm and Kojun Pharmaceutical Co., Ltd.) were dissolved in ultrapure water, and the resulting aqueous solution was brought to a final volume of 1 L as the eluent for HPLC. The flow rate was 0.5 ml / min. Peak detection was performed using UV light at a wavelength of 210 nm. First, the 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 peak area of ​​the above-mentioned glycolic acid aqueous solution was determined by HPLC, with a retention time (RT) of 4.10–4.50 minutes, and the solution was prepared. Figure 8 The standard curve of glycolic acid.

[0163] When the NB medium was measured by HPLC, peaks of components other than glycolic acid were observed in the region of RT 4.10–4.50 min. Therefore, the concentration of glycolic acid released was calculated as the difference from the control group prepared in Evaluation Example 4, as shown in the following formula.

[0164] • Glycolic acid release concentration of control A

[0165] ={0.00281 × (area value of control A - area value of control C) + 10.23813} × 5 (dilution concentration) / 1000 (unit conversion) [mg / ml]

[0166] Glycolic acid release concentration of DB14+PGA tablets

[0167] ={0.00281 × (area value of DB14 - area value of control B) + 10.23813} × 5 (dilution concentration) / 1000 (unit conversion) [mg / ml]

[0168] The area values ​​in the formula are the peak area values ​​at RT 4.10–4.50 minutes on the HPLC chromatogram.

[0169] The HPLC determination results are shown in Figure 9 See Table 7. Figure 9The error bars in the table show the range of values ​​calculated from the standard deviation of the results obtained from measuring each sample solution when performing addition and subtraction operations relative to the average of the results. Table 7 shows the average values ​​for measuring each sample solution. Based on these results, the concentration of glycolic acid released in the degradation solution is significantly increased after DB14 inoculation. That is, it shows that DB14 inoculation promotes the degradation of polyglycolic acid.

[0170] [Table 7]

[0171]

[0172] [Evaluation Example 6] Grading of crude enzymes derived from DB14

[0173] The fractionation of crude enzyme derived from DB14 was performed using an ammonium sulfate reprecipitation method. 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, a platinum ring of DB14 was inoculated and cultured with shaking at 55°C and 180 rpm for one day. Then, the bacterial cells and solution were separated by centrifugation. Ammonium sulfate was added to the solution side and stirred to achieve a 40% saturation concentration. Subsequently, the 40% saturated precipitate and solution were separated by centrifugation, and additional ammonium sulfate was added to the solution side and stirred to achieve an 80% saturation concentration. The 80% saturated precipitate and solution were separated by centrifugation, and 2 ml of 0.1 M KPi was added to the 80% saturated precipitate and dissolved to prepare the crude enzyme solution.

[0174] [Evaluation Example 7] Esterase activity of crude enzyme derived from DB14

[0175] The esterase activity of the crude enzyme derived from DB14 was determined according to the method of Kay et al. (MJKay, RWMcCabe, and L.H.G.Morton. Chemical and physical changes occurring in polyesterpolyurethane during biodegradation, International Biodeterioration & Biodegradation 31 (1993) 209-225). 0.268 ml of 1 mM p-nitrophenylacetate and 0.132 ml of the crude enzyme solution were added to 1.6 ml of 0.1 M KPi, and the absorbance at 405 nm was measured using a spectrophotometer to determine the increase in free p-nitrophenol due to ester bond degradation. The amount of enzyme producing 1 μmol of p-nitrophenol within 1 minute was defined as 1 unit (U). Furthermore, to investigate the effect of pH on the liquid medium, the esterase activity was also measured using a 0.4 M sodium phosphate buffer prepared to pH 8 instead of 0.1 M KPi. The results of the esterase activity measurements are shown in Table 8.

[0176] [Table 8]

[0177]

[0178] As shown in Table 8, the crude enzyme derived from DB14 exhibited esterase activity, with stronger activity at pH 8 than at pH 7. This strong esterase activity suggests the possibility that higher pH levels, such as pH 8, would exhibit even stronger activity in the hydrolysis of polyglycolic acid than pH 7.

[0179] [Evaluation Example 8] Degradation of PGA using crude enzyme derived from DB14

[0180] To investigate the effect of pH on the degradation of PGA tablets by graded crude enzyme derived from DB14, degradation experiments were conducted using 0.1M KPi (pH 7), 0.4M sodium phosphate buffer (pH 8), and 0.5M Tris-HCl buffer (pH 9). 2 mg of sterile PGA tablets, 900 μl of the aforementioned buffer solutions, and 100 μl of crude enzyme solution derived from DB14 were added to 2 ml vials. The vials were sealed and incubated at 60°C and 80 rpm for 18 hours using a shaking incubator. The degradation solution in the vials was filtered to separate the solid and liquid phases. The residue, along with the filter, was dried in a desiccator for 12 hours, and the weight of the residue was determined as the weight of the PGA degradation product. Furthermore, the molecular weight of the obtained PGA degradation product was determined using HPLC. For comparison, the following degradation tests were also performed: instead of the crude enzyme solution derived from DB14, 100 μl of sterile distilled water was added (control), and an autoclaved enzyme solution was obtained by autoclaving the crude enzyme solution derived from DB14 at 121°C for 20 minutes. Two samples were prepared for each condition. The weight and weight-average molecular weight (Mw) of the PGA tablets 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 presented below. Figure 10 And Table 9. It should be noted that... Figure 10 The error bars recorded in the table are set by adding and dividing the standard deviation calculated from the measurement results relative to the average value of the measurement results. The values ​​in Table 9 represent the average value of the measurement results.

[0181] [Table 9]

[0182]

[0183] like Figure 10 As shown, the addition of a crude enzyme solution derived from DB14 promoted the reduction of PGA weight and Mw in buffer solutions at any pH, demonstrating the PGA degradation activity of the crude enzyme solution derived from DB14. When the crude enzyme was denatured by autoclaving, the changes in PGA weight and Mw reduction were slower compared to the control, further supporting the PGA degradation activity of the crude enzyme. Furthermore, the highest PGA degradation activity was observed at pH 8.

[0184] In addition, as in evaluation example 4 and Figure 7 As shown, DB14 has the property of raising the pH of the surrounding environment to around 8-9. Therefore, it can be concluded that DB14 cultures are effective in maximizing the hydrolytic capacity of enzymes with PGA-degrading activity.

[0185] Industrial availability

[0186] The microorganisms of this invention can be applied to well excavation and other operations for the extraction of oil and gas resources such as petroleum or natural gas.

[0187] Collection Number

[0188] NITE BP-04014. PCT / RO / 134 form

Claims

1. A microorganism belonging to the genus *Geobacillus*, which is closely related to *Geobacillus glacians*, and the preservation number of said microorganism is NITE BP-04014.

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

3. A degradation promoter for the degradation of polyglycolic acid, comprising the microorganism according to claim 1 or the culture source according to claim 2 as an active ingredient.

4. The degradation promoter according to claim 3, wherein, The degradation accelerator is a degradation accelerator used to degrade molded articles for pit excavation, which are formed from polyglycolic acid.

5. The degradation promoter according to claim 4, wherein, The molded products used for pit excavation are downhole tools, downhole tool components, or temporary plugging materials.

6. A method for degrading polyglycolic acid, comprising: The contact process that brings the degradation promoter according to claim 3 into contact with polyglycolic acid.

7. The method for degrading polyglycolic acid according to claim 6, wherein, In the contacting process, the degradation promoter is contacted with the polyglycolic acid under conditions of pH 6 or higher and pH 10 or lower.

8. A method for excavating pits and wells, comprising: The process of bringing the degradation promoter according to claim 3 into contact with a molded article for pit excavation made of polyglycolic acid inside the pit.

9. The pit excavation method according to claim 8, wherein, The molded products used for pit excavation are downhole tools, downhole tool components, or temporary plugging materials.

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