JP2008289379A - Method for determining putrefaction of water-soluble cutting oil utilizing detection of sulfate-reducing bacterium, and method for determining purifying ability of soil or underground water - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To establish a technique capable of paying attention to kinetics of microorganisms and roles thereof, specifying the microorganism which is to be the primary factor in putrefaction of a water-soluble cutting oil and quickly determining putrefaction of the water-soluble cutting oil through detection of the microorganism with high sensitivity, and to establish a technique capable of specifying the microorganism related to the formation of growth environments of an anaerobic microorganism, represented by an organochlorine compound-degrading microorganism and determining the purifying ability of soil or underground water through the detection of the microorganism. <P>SOLUTION: A method for determining the putrefaction of the water-soluble cutting oil comprises determining the putrefaction of the water-soluble cutting oil, with the detection of a specific sulfate-reducing bacterium as an index. A method for determining the purification ability of the soil or the underground water by anaerobic biodegradation comprises using the detection of the specific sulfate-reducing bacterium as the index. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for determining the decay of water-soluble cutting oil and the method for determining the purification ability of soil or groundwater using detection of sulfate-reducing bacteria.

  Cutting oil is used for the purpose of lubrication, cooling, prevention of welding, and rust prevention when cutting metal or ceramics using a machine tool such as a lathe or a drill. In recent years, the amount of water-soluble cutting oil that has a high cooling effect during processing and does not ignite is increasing. However, since water-soluble cutting oil tends to rot, if the same cutting oil is continuously used over a long period of time, problems such as deterioration in processing performance and generation of malodors occur, and therefore periodic replacement is necessary.

  Thus, various reports have been made on methods for treating such used water-soluble cutting oil. For example, a technique has been reported in which used water-soluble cutting oil is brought into contact with a photocatalyst such as titanium oxide to sterilize microorganisms such as general microorganisms and anaerobic microorganisms (see Patent Document 1). Such a technique exhibits an excellent bactericidal effect against sulfate-reducing bacteria that promote the generation of malodor-causing substances, and at the same time, prevents the decay of cutting oil by oxidizing hydrogen sulfide that causes malodor. . In addition, a technology that prevents the decay of water-soluble cutting oil by the action of antioxidants produced from useful microorganisms by combining porous ceramics and useful microorganisms that coexist with anaerobic and aerobic microorganisms. Has been reported (see Non-Patent Document 1). This technique is described to prevent an environment where anaerobic microorganisms such as sulfate-reducing bacteria grow by the action of antioxidants, but may not be effective depending on the use environment. . That is, it is presumed that these techniques cannot sufficiently exert their effects when there are a large number of microorganisms that cause corruption. Therefore, it is necessary to perform processing within the processing capacity range for effective processing, and establishment of a technique for accurately grasping the degree of corruption is required. However, all of the above techniques focus on the suppression of the growth of microorganisms and do not provide any technique for knowing the presence and amount of microorganisms that cause corruption.

  The detection of the presence and amount of microorganisms has been reported to be important in bioremediation technology that purifies contaminated soil and groundwater using useful microorganisms (see Patent Document 2, Non-Patent Document 2, etc.). . For example, Patent Document 2 reports a technique for estimating a period necessary for repairing organic base compound contamination by anaerobic treatment, and obtains a correlation between a quantitative value of a microorganism and a repair period, and a period necessary for repair. A technique for estimating the above is disclosed. Non-Patent Document 2 discloses an anaerobic microbial degradation technique for organochlorine compounds such as chloroethylenes. In this field, in order to induce the complete decomposition of chloroethylenes under anaerobic conditions, research has been conducted on supplying appropriate electron donors, nutrients, reducing agents, and decomposing microorganisms, and dehalogenation of microorganisms. Since hydrogen uses hydrogen as an electron donor, it is also disclosed that the competition between microorganisms producing hydrogen and other microorganisms using hydrogen affects the degradation of chloroethylenes.

  In addition, it is known that the purification of contaminated soil and groundwater by microorganisms is rarely dependent only on a single microorganism and is carried by a microorganism group in which a wide variety of microorganisms coexist. For example, it has been reported that a wide variety of microorganisms such as lactic acid bacteria coexist in a group of microorganisms that decompose dichloroethylene or vinyl chloride (Non-patent Document 3). Moreover, coexistence of a wide variety of microorganisms has also been reported for dioxin-degrading microorganism groups (Non-patent Document 4).

  However, these reports only clarify that the purification of contaminated soil and groundwater by microorganisms is carried out by a group of microorganisms in which a wide variety of microorganisms coexist, and what is the role of such a wide variety of microorganisms? It was not disclosed. In particular, it was well known that microorganisms with the ability of organochlorine compounds exert their biological activity under anaerobic conditions, but they give some information on the relationship between the formation of such anaerobic environments and coexisting microorganisms. It was not a thing.

Various methods have been reported for detecting the presence and amount of microorganisms. For example, a method for classifying sulfate-reducing bacteria using 16S ribosomal genes has been reported (Non-patent Document 5). The detection for technical group of lactic acid bacteria (non-patent document 6) and, Dehalococcoides, Desulfitobacterium, have been reported for quantitative PCR analysis technology Dehalobacter bacteria (Non-patent Document 7). However, these technologies only provide a method for detecting the presence and amount of a specific microorganism, and provide any information on the type and role of microorganisms present in cutting oil and soil and groundwater. It turned out.

JP 2006-312219 JP JP 2005-254084 Nakano Seisakusho, “Corruption delay processing technology for cutting oil and cleaning fluid”, [online], Environmental Technology Coordination Business-Business Introduction, HYPERLINK "http://www.epcc.pref.osaka.jp/reaf/index.php" Osaka Prefectural Research Institute for Environment, Agriculture, Forestry and Fisheries, [Search April 18, 2007], Internet <URL: HYPERLINK "http://www.epcc.pref.osaka.jp/center/etech/etc04/2004-2/ tech4.pdf "http://www.epcc.pref.osaka.jp/center/etech/etc04/2004-2/tech4.pdf> Akiko Suyama, Lee Tai Nabe, Ken Furukawa, “Anaerobic Microbial Degradation of Chloroethylenes”, Soil Environmental Center Technical News, “Bioremediation Special Feature”, Contribution 1, 2003 Authored by Masahiro Mizumoto, “Research and Development on Anaerobic Decomposition of Ethylene Chloride”, Commissioned by New Energy and Industrial Technology Development Organization (NEDO), “Analysis of Biodegradation and Treatment Mechanism and Development of Control Technology”, 2005 Annual Report Meeting, Lecture Summary Yoshida N., Takahashi N., Hiraishi A., “Phylogenetic characterization of a polychlorinated-dioxin-dechlorinating microbial community by use of microcosm studies.”, Applied and Environmental Microbiology., August 2005, Vol. 71, Vol. No. 8, pp. 4325-4334 Daly K., Sharp RJ., McCarthy AJ., “Development of oligonucleotide probes and PCR primers for detecting phylogenetic subgroups of sulfate-reducing bacteria.”, Microbiology. July 2000, 146, 1693–1705. Walter J., Hertel C., Tannock GW., Lis CM., Munro K., Hammes WP., “Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis ”, Applied and Environmental Microbiology. June 2001, Vol. 67, Vol. 6, pp. 2578-2585 Smits TH., Devenoges C., Szynalski K., Maillard J., Holliger C., “Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides, and Desulfitobacterium in microbial communities.”, Journal of Microbiological Methods, June 2004, volume 57, number 3, pages 369-378

  Therefore, in view of the above circumstances, the present invention focuses on the dynamics of microorganisms and their roles, identifies microorganisms that cause water-soluble cutting oil to rot, and detects water-soluble cuttings with high sensitivity and speed through detection of such microorganisms. The purpose is to establish a technology that can determine the spoilage of oil. In addition, in the same way, the establishment of a technology that can identify microorganisms related to the formation of anaerobic microorganisms such as microorganisms that decompose organochlorine compounds and determine the ability to purify soil or groundwater through detection of such microorganisms. With the goal.

  The present inventors have conducted extensive studies to solve the above-mentioned problems, and found that specific sulfate-reducing bacteria are involved in the decay of cutting oil. Furthermore, it was found that such specific sulfate-reducing bacteria play an important role in the formation of anaerobic microorganism growth environment represented by microorganisms that decompose organochlorine compounds. The present invention has been completed.

In order to achieve the above object, the present invention provides the following [1] to [3].
[1] a water-soluble cutting in oil Desulfovibrio bastinii, Desulfovibrio salexigens, Desulfovibrio caledoniensis , Desulfovibrio halophilus, Desulfovibrio gracilis, Desulfovibrio longus, Desulfovibrio gabonensis, Desulfovibrio gigas, Desulfovibrio africanus, Desulfovibrio termitidis, Desulfovibrio longreachensis, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio fairfieldensis A method for determining the decay of water-soluble cutting oil, wherein the decay of water-soluble cutting oil is determined using as an index the detection of at least one of sulfate-reducing bacteria among Desulfomicrobium baculatum , Desulfomicrobium escambiense , and Desulfovibrio acrylicus .
[2] The method according to [1], wherein the sulfate-reducing bacterium is detected using a nucleotide sequence represented by sequence recognition number 1, an oligonucleotide comprising the nucleotide sequence represented by sequence recognition number 2, or an oligonucleotide complementary to the oligonucleotide. A method for judging rot of water-soluble cutting oil.
[3] water-soluble cutting in oil Desulfovibrio bastinii, Desulfovibrio salexigens, Desulfovibrio caledoniensis , Desulfovibrio halophilus, Desulfovibrio gracilis, Desulfovibrio longus, Desulfovibrio gabonensis, Desulfovibrio gigas, Desulfovibrio africanus, Desulfovibrio termitidis, Desulfovibrio longreachensis, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio fairfieldensis Detection means for detecting at least one sulfate-reducing bacterium among Desulfomicrobium baculatum , Desulfomicrobium escambiense , and Desulfovibrio acrylicus ;
A water-soluble cutting oil management system having a cleaning means for cleaning water-soluble cutting oil based on a detection result by the detection means.

  According to the configurations [1] to [3] above, it is possible to determine the decay of water-soluble cutting oil with high sensitivity and speed through detection of specific sulfate-reducing bacteria. And since the decay of cutting oil can be detected before the decay of cutting oil progresses, the malfunction resulting from the decay of cutting oil can be avoided. Specifically, it is possible to suppress the deterioration of the machining performance of the machine tool and the metal corrosion due to the decay of the cutting oil, and it is possible to improve the life and productivity of the machine tool and the machining quality. Moreover, generation | occurrence | production of a bad odor can be suppressed and the comfortable working environment can be formed. As a result, the work efficiency can be improved, and since the rotting can be detected before the rotting proceeds, the cutting oil can be easily processed by sterilization or the like, and it is necessary to replace the cutting oil. It also leads to cost and time reduction and cost reduction such as incineration processing cost of the replaced waste liquid.

Furthermore, in order to achieve the above object, the present invention provides the following [1] to [3].
[4] of the soil or ground water, Desulfovibrio bastinii, Desulfovibrio salexigens, Desulfovibrio caledoniensis, Desulfovibrio halophilus, Desulfovibrio gracilis, Desulfovibrio longus, Desulfovibrio gabonensis, Desulfovibrio gigas, Desulfovibrio africanus, Desulfovibrio termitidis, Desulfovibrio longreachensis, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio fairfieldensis Judging the soil or groundwater purification ability by anaerobic biodegradation using the detection of sulfate-reducing bacteria of at least one of Desulfomicrobium baculatum , Desulfomicrobium escambiense and Desulfovibrio acrylicus as an index Method.
[5] The sulfate-reducing bacteria are detected using the nucleotide sequence shown in SEQ ID NO: 1, the oligonucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2, or the oligonucleotide complementary to the oligonucleotide [4] ] The soil or groundwater purification ability judgment method of].
[6] The soil or groundwater purification capacity determination method according to the above [4] or [5], using detection of lactic acid bacteria as an index.
[7] The method according to [6] above, wherein the lactic acid bacterium is detected using the nucleotide sequence shown in SEQ ID NO: 5 or the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 6 or the oligonucleotide complementary to the oligonucleotide. Method for judging the purification capacity of soil or groundwater.
[8] The soil or groundwater purification capacity determination method according to any one of the above [4] to [7], wherein detection of Dehalococcoides bacteria is used as an index.
[9] The above-mentioned [8] wherein the bacterium belonging to the genus Dehalococcoides is detected using an oligonucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 or an oligonucleotide comprising the nucleotide sequence shown in SEQ ID NO: 4 or an oligonucleotide complementary to the oligonucleotide. ] The soil or groundwater purification ability judgment method of].

According to the configuration of [4] to [9] above, anaerobic microorganisms having reductive dehalogenation ability involved in the decomposition of organochlorine compounds with high sensitivity and speed through detection of specific sulfate-reducing bacteria. It can be determined whether or not an anaerobic environment sufficient for microbial growth and its biological activity is formed. Thereby, the purification capability which the contaminated soil and groundwater used as purification object have can be determined. Then, based on the determination result, it is possible to construct a highly efficient contamination purification system, and to shorten the processing time.
In particular, according to the configuration of [6] to [9] above, by detecting the Dehalococcoides genus and lactic acid bacteria in addition to the sulfate-reducing bacteria, the purification capacity of the contaminated soil and groundwater to be purified can be determined more accurately. be able to.

  Embodiments of the present invention will be described in detail below. However, the scope of the present invention is not limitedly interpreted by these embodiments.

[Method of judging rot of water-soluble cutting oil]
The method for determining spoilage of water-soluble cutting oil according to the present invention, Microbiology, July 2000, Vol. 146, pages 1693 to 1705, is carried out by detecting bacteria classified into the sixth group of sulfate-reducing bacteria. . According to the method of the present invention, since the decay of the cutting oil can be detected before the decay of the cutting oil proceeds, it is possible to avoid a problem caused by the decay of the cutting oil. Specifically, it is possible to suppress the deterioration of the machining performance of the machine tool and the metal corrosion due to the decay of the cutting oil, and it is possible to improve the life and productivity of the machine tool and the machining quality. Moreover, generation | occurrence | production of a bad odor can be suppressed and the comfortable working environment can be formed. As a result, the work efficiency can be improved, and since the rotting can be detected before the rotting proceeds, the cutting oil can be easily processed by sterilization or the like, and it is necessary to replace the cutting oil. It also leads to cost and time reduction and cost reduction such as incineration processing cost of the replaced waste liquid.

Here, the target of detection is, for example, microorganisms classified into the sixth group of sulfate-reducing bacteria according to the classification in Microbiology, July 2000, Vol. 146, pages 1693 to 1705. Specifically, Desulfovibrio bastinii, Desulfovibrio salexigens, Desulfovibrio caledoniensis, Desulfovibrio halophilus, Desulfovibrio gracilis, Desulfovibrio longus, Desulfovibrio gabonensis, Desulfovibrio gigas, Desulfovibrio africanus, Desulfovibrio termitidis, Desulfovibrio longreachensis, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio fairfieldensis, Desulfomicrobium baculatum, Desulfomicrobium escambiense and Desulfovibrio acrylicus are included.

  The method for detecting spoilage of the water-soluble cutting oil of the present invention detects bacteria belonging to the above-mentioned sulfate-reducing bacteria group 6. Such a bacterium belonging to the sulfate-reducing bacteria group 6 can be detected by a known method. Preferably, it is used for gene analysis using a characteristic sequence of the bacterium belonging to the sulfate-reducing bacteria group 6 as an index. Detect based on.

  Therefore, the water-soluble cutting oil as the detection target sample may be directly applied to the detection method of the present invention, and for the purpose of revealing the target nucleic acid, known nucleic acid extraction and concentration are performed as necessary. Alternatively, it may be applied after a treatment such as purification. Here, examples of the nucleic acid prepared from the environmental sample include genomic DNA, total RNA, mRNA, and cDNA. Nucleic acid extraction and purification can be performed by any method. For example, it can be performed by separating microorganisms from water-soluble cutting oil and then extracting nucleic acid, and purification may be performed as necessary. Nucleic acid extraction methods include surfactant treatment such as SDS, enzyme treatment such as lysozyme and proteinase, heat treatment, ultrasonic treatment, chaotropic salt, etc. It can be performed by extracting nucleic acid based on a conventional method.

  And, when detecting bacteria belonging to the sulfate-reducing bacteria group 6 using the characteristic sequence of the bacteria belonging to the sulfate-reducing bacteria group 6 as an index, the characteristics of the bacteria belonging to the sulfate-reducing bacteria group 6 This can be done by using an oligonucleotide complementary to a typical nucleotide sequence. That is, it can be carried out by detecting a nucleic acid molecule derived from a bacterium belonging to the sulfate-reducing bacteria group 6 contained in the sample by bringing the test sample into contact with the oligonucleotide. For example, it can be performed by a hybridization method using the oligonucleotide as a probe. Moreover, it can carry out by nucleic acid amplification methods, such as PCR, using the said oligonucleotide as a primer.

  Here, complementary means that the microorganism gene to be detected and the oligonucleotide can specifically bind according to the base pairing rule to form a stable double-stranded structure. The degree of complementarity required is preferably 100%, but not only perfect complementarity, but the gene of the present invention to be detected and the oligonucleotide form a stable duplex structure with each other. As long as it is sufficient to obtain, only some nucleobases are allowed to have partial complementarity that fits along the base pairing rules. Specifically, it varies depending on the length of the complementary region, the distribution of base pair mismatch, and the like, but is generally 70% or more, preferably 90% or more, particularly preferably 95% or more.

  The length of the oligonucleotide must be long enough to specifically recognize the target nucleic acid. However, if the length is too long, a nonspecific reaction is induced, which is not preferable. Therefore, the appropriate length is determined depending on many factors such as sequence information of the target nucleic acid such as GC content, and hybridization reaction conditions such as reaction temperature and salt concentration in the reaction solution. It is 20-50 bases long.

  The oligonucleotide is prepared by a chemical synthesis method based on the phosphoramidite method or the like, and purified by means such as HPLC as necessary. In chemical synthesis, a commercially available automatic synthesizer can be used. When preparing a primer based on a chemical synthesis method, it is designed based on the sequence information of the target nucleic acid prior to synthesis and is determined by examining the sequence of the target nucleic acid in advance. Databases such as GeneBank and EBI can be suitably used for investigating the base sequence of the target nucleic acid. In addition, when a target nucleic acid has already been obtained, a restriction enzyme fragment or the like can be used as long as it has a base sequence characteristic of a microorganism.

  The oligonucleotide is not limited to DNA, RNA, or PNA, but is preferably DNA. In addition, the oligonucleotide is not limited to a single strand or a double strand, but is preferably a single strand. In addition to the region that hybridizes with the target nucleic acid, another chemical structure may be added to the oligonucleotide. For example, in the case of introducing a label for detection, or in the case of performing detection by preparing a microarray immobilized on an appropriate solid phase carrier, a form in which a functional group for solid phase is introduced Etc. are exemplified.

  In the method of the present invention, the following sequences are exemplarily shown as oligonucleotides suitable for use.

For detection of sulfate-reducing bacteria group 6
5´-GRGYCYGCGTYYCATTAGC-3´ (Sequence recognition number 1)
5´-SYCCGRCAYCTAGYRTYCATC-3´ (Sequence recognition number 2)

  In order to increase the detection sensitivity, the oligonucleotide can be introduced with a detectable label as necessary. In this case, the position at which the detectable label is introduced is not particularly limited as long as it does not significantly affect the hybridization efficiency and the efficiency of the subsequent extension reaction, but it is appropriately determined in consideration of the base sequence and length of the probe. decide. For example, a hydroxyl group part, a base part, a phosphoric acid diester part and the like at the 3 ′ end and 5 ′ end can be mentioned, and a hydrogen group part at the 5 ′ end is particularly preferable.

Since such a labeling substance is known, those skilled in the art can appropriately select and use it, and any known substance can be used as long as the stability and functionality required in the reaction can be maintained. Therefore, it does not matter whether direct labeling or indirect labeling is used. Examples include radioisotope elements, enzymes, fluorescent dyes, chemiluminescent substances, and the like. Specifically, ³² P, ³⁵ S, ¹³¹ I, ⁴⁵ Ca, ³ H and the like are exemplified as suitable radioisotope labels. Examples of the enzyme label include peroxidase and alkaline phosphatase. Fluorescent dyes include cyanine dyes such as fluorescein isothiocyanate (FITC), tetramethylrhodamine (TAMRA), tetramethylrhodamine isothiocyanate (TRICA), Texas Red, cyanine 3 (Cy3) and cyanine 5 (Cy5). Illustrated. Examples of chemiluminescent materials include acridium esters. Furthermore, haptens such as biotin and dioxygenin can also be suitably used. For example, it can be labeled with digoxigenin, visualized with a labeled anti-digoxigenin antibody, labeled with biotin, labeled with a labeled anti-biotin antibody, or visualized with labeled streptavidin or avidin. Moreover, the label | marker also using the nucleic acid, nucleic acid binding protein, etc. which couple | bond with a secondary probe specifically is illustrated. However, it is not limited to these, and a known labeling technique can be used.

The labeling substance can be introduced by a known method. For example, when the 5 ′ end is labeled with the radioisotope element ³² P, T4 polynucleotide kinase is added to the reaction solution containing the probe and ³² P-labeled nucleotide (γ- ³² P-ATP). be able to.
Be labeled.

  In addition, the oligonucleotide may be bonded to a solid phase carrier or a site capable of binding can be introduced as necessary. By observing the capture of the target nucleic acid on the solid phase carrier, rapid detection becomes possible, and by separating only the target nucleic acid captured by the solid phase carrier, cloning can be easily performed. There are no particular restrictions on the position at which the binding site or the site capable of binding is introduced to the solid phase carrier, as long as it does not significantly affect the hybridization efficiency and the efficiency of the subsequent extension reaction, but the probe base sequence, length, etc. Is determined as appropriate. For example, a hydroxyl group part, a base part, a phosphoric acid diester part and the like at the 3 ′ end and 5 ′ end can be mentioned, and a hydrogen group part at the 5 ′ end is particularly preferable. An active group of a hydrogen group part, a base part or a phosphodiester part near the 5 'end is preferred.

  As the solid phase carrier, a carrier that is insoluble in a reaction solution used in a usual hybridization method or amplification reaction can be used. For example, inorganic substances such as glass, silica gel, and bennite, synthetic polymer substances such as polystyrene, polyethylene, and polypropylene, and insoluble polysaccharides such as agarose, dextran, and polysaccharides can be preferably used. These supports can be used in the form of a sphere, rod, fine particle, or the like, or in the form of a test tube, microplate, membrane, or the like.

  Immobilization to a solid phase carrier can be performed according to a known method, and an optimal method may be selected according to the type of probe, the solid phase carrier, the functional group on the surface of the solid phase carrier, etc. Either a simple bond or a chemical bond may be used. Specifically, a method of electrostatically binding to a solid support surface-treated with a polycation such as polylysine or polyethyleneimine using the charge of DNA, various silane couplings having an amino group, an aldehyde group, an epoxy group, etc. Preferred examples include a method of covalently bonding DNA having a reactive group such as an amino group, an aldehyde group or an SH group introduced in advance as a functional group to a solid phase carrier surface-treated with an agent. It can also be immobilized using an antigen (hapten) -antibody, biotin-avidin or streptavidin, antigen-antibody, ligand-receptor, nucleic acid capable of hybridizing to each other, and the like. Furthermore, a substance capable of labeling the probe and a substance capable of binding to the substance can be immobilized using a solid phase carrier on which the substance is immobilized. For example, by using a probe labeled with biotin and a solid phase carrier on which streptavidin is immobilized, the probe can be immobilized on the solid phase carrier via biotin-streptavidin binding. In immobilization, treatments such as crosslink formation, blocking, and washing by ultraviolet irradiation can be performed as necessary to suppress nonspecific binding.

  When detecting by hybridization, for example, the sample and the oligonucleotide probe are hybridized under stringent conditions, and the presence or absence of the hybridization and the presence or absence of the microorganism in the target sample are detected. Alternatively, the abundance is detected quantitatively. Hybridization itself can be performed by a known method. Examples include Southern, Northern blot hybridization, colony hybridization, and plaque hybridization.

  Here, stringent hybridization conditions and washing conditions are appropriately selected and set empirically according to the length of the sequence to be hybridized and the content of the GC nucleotide sequence, and are homologous to the probe. It is possible to appropriately select conditions that specifically hybridize with a highly functional base sequence and exhibit sufficient detection sensitivity and specificity. Well established formulas for variations of such hybridization conditions are well known in the art, for example, Sambrook et al, "Molecular Cloning: A Laboratory Manual", 2nd edition, p. 9.47-9.51. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1989, etc.

  The method for detecting hybridization is not particularly limited. For example, it can be performed by detecting a label after hybridization with a labeled probe. The probe can be immobilized by preliminarily labeling the sample nucleic acid with a labeling substance, washing the carrier following hybridization, and detecting the label. For labeling detection of the sample nucleic acid, a general method may be used according to the label to be used. For example, when the label is a radioisotope, it can be carried out by measuring the radioactivity with a scintillation counter and exposing the X-ray film to visualize the radioactivity. If the label is a fluorescent substance, the fluorescence intensity can be measured by measuring with a fluorometer, a confocal microscope or the like. When the label is an enzyme, an enzyme substrate that develops color by an enzymatic reaction may be used to detect the color development. When a label other than the directly detectable label is introduced, a reagent for indirectly measuring the label is used. For example, when the labeled product is biotin, for example, a reaction product with fluorescently labeled avidin or streptavidin, and when the labeled product is a hapten, for example, with a fluorescent labeled antibody that specifically binds to the hapten. It can be indirectly measured by measuring the reaction product. Further, it may be combined with separation by gel electrophoresis.

  When detecting by the nucleic acid amplification method, for example, the target sample is contacted with an oligonucleotide primer, a nucleic acid amplification reaction is performed, and the presence or absence of a specific amplification product and the presence or absence of microorganisms in the target sample are determined. Detect or detect abundance quantitatively. The nucleic acid amplification method can be performed by a method known per se. For example, polymerase chain reaction (PCR), ligase chain reaction (LCR), etc. are mentioned. When an amplification reaction is performed by the PCR method, the temperature setting, time, and cycle number are appropriately selected depending on the amount of the target template nucleic acid and the primer pair. Moreover, it is preferable that the annealing conditions in the PCR reaction are adapted to the above stringent conditions.

  The specific amplification product can be detected according to the detection method of the amplification product. For example, the amplification product may be detected by subjecting the amplification product to agarose gel electrophoresis to confirm whether the target fragment is amplified. Other electrophoresis methods and chromatographic methods can also be used. When a labeled primer is used as the oligonucleotide, it can also be carried out by detecting the label. In addition, the nucleic acid is amplified by incorporating the labeled nucleotide as a substrate and detecting the label after amplification. be able to. It can also be detected by real-time PCR or the like that observes the fluorescence intensity over time. For example, it can be performed by using an oligonucleotide in which the 5 'end is modified with a fluorescent substance and the 3' end is modified with a quencher substance.

  In addition, when the amount of bacteria belonging to the sulfate-reducing bacteria group 6 is quantitatively detected, for example, a DNA standard sample of the sulfate-reducing bacteria group 6 to be detected can be used simultaneously as a control. Compare the hybridization intensity or specific nucleic acid amplification product amount obtained using the control DNA sample with the hybridization intensity or specific nucleic acid amplification product amount obtained using the detection target sample, The amount of bacteria in the subject can be calculated. In this way, by quantitatively detecting the amount of bacteria belonging to the sulfate-reducing bacteria group 6, it becomes possible to analyze the degree of spoilage.

  A management system for water-soluble cutting oil can be constructed using the method for determining corruption of the present invention. Specifically, it has a detecting means for detecting the behavior of the bacteria belonging to the sixth group of sulfate-reducing bacteria in the cutting oil, and a cleaning means for cleaning the water-soluble cutting oil based on the detection result. Configured.

  Examples of the cleaning means include means for exchanging cutting oil with fresh water-soluble cutting oil. For example, there is a means for controlling to replace all or part of the cutting oil when bacteria belonging to the sixth group of sulfate-reducing bacteria are detected in the water-soluble cutting oil. In this case, at the time of replacement of the cutting oil, it is possible to control the cleaning oil storage tank and the piping to be cleaned as necessary. The cleaning means may be a means for removing impurities such as microorganisms contained in the water-soluble cutting oil by filtering or sterilizing the cutting oil.

  In addition, various information relating to the management of cutting oil including information obtained by the detection means can be made into a database, and by using such a database, a cutting oil management system suitable for the usage state of the cutting oil can be constructed.

  By constructing a management system for water-soluble cutting oil in this way, it is possible to accurately avoid problems caused by the decay of cutting oil, and to further improve work efficiency.

[Soil or groundwater purification capacity judgment method]
Detection of bacteria classified into the above-mentioned sulfate-reducing bacteria group 6 can also be used in a contamination purification system by bioremediation. Specifically, by detecting the bacteria belonging to the sixth group of sulfate-reducing bacteria, whether or not an anaerobic environment sufficient for the growth of anaerobic microorganisms and the expression of their biological activity is formed. Judgment. This is based on the knowledge of the present inventors that bacteria belonging to the sixth group of sulfate-reducing bacteria play an important role in the formation of an anaerobic environment. And many of anaerobic microorganisms have a decomposition activity with respect to hardly decomposable harmful substances, such as an organic chlorine compound, such as having reductive dehalogenation ability. Therefore, when anaerobic microorganisms are grown and an anaerobic environment sufficient to develop their biological activity is formed, the organic chlorine compounds and the like are hardly decomposed by the reductive dehalogenation activity of the anaerobic microorganisms. The toxic harmful substances are decomposed satisfactorily. Therefore, it can be determined in advance whether or not the soil or groundwater can be satisfactorily purified from the determination result, and the purification ability of the soil and groundwater, and thus the state of bioremediation can be determined.

Therefore, the object of the present invention is soil contaminated with persistent decomposable hazardous substances, particularly organochlorine compounds, and groundwater, preferably groundwater. Examples of the organic chlorine compound to be decomposed include chloroethylenes such as tetrachloroethylene, trichloroethylene, dichloroethylene, and chloroethylene, dioxins, and PCBs. In particular, chloroethylenes that are suitably dechlorinated by bacteria belonging to the genus Dehalococcoides .

  Here, the case where the purification | cleaning capability of the soil or groundwater contaminated with the organochlorine compound is determined is illustrated. When the presence of bacteria belonging to the sixth group of sulfate-reducing bacteria is confirmed in soil and groundwater, the growth of microorganisms having reductive dehalogenation ability and anaerobic sufficient to express their biological activity It is judged that a natural environment is formed and the organic chlorine compound can be decomposed satisfactorily. On the other hand, if the detection result cannot confirm the presence of bacteria belonging to the sixth group of sulfate-reducing bacteria, the growth of microorganisms having reductive dehalogenation ability and anaerobic sufficient to express their biological activity It is judged that the environment is not formed and is not suitable for the decomposition of organochlorine compounds. And based on such determination, adding bacteria belonging to the sixth group of the sulfate-reducing bacteria that adjust the anaerobic environment, or adding a desired nutrient, for example, a nutrient for forming an anaerobic environment, A purification environment can be prepared.

  Here, the bioremediation refers to an anaerobic process, and there is no restriction on the processing form as long as the bioremediation is processed by the anaerobic process. Therefore, the present invention can be applied to general bioremediation methods including an in-situ bioremediation method in which processing is performed without excavating a contaminated site and an on-site bioremediation method in which processing is performed after excavating a contaminated site. When the treatment is performed based on the on-site bioremediation method, the treatment can be performed using a sealed or semi-sealed reaction vessel.

And preferably, it can be applied to a bioremediation composed mainly of microorganisms capable of catalyzing the reductive dehalogenation reaction of organochlorine compounds, in particular, Dehalococcoides bacteria. Bacteria belonging to the genus Dehalococcoides are anaerobic microorganisms that replace the chlorine substituent of chloroethylenes with hydrogen by a reductive dehalogenation reaction of chloroethylenes. The Dehalococcoides bacteria, Dehalococcoides ethenogenes 195 shares, Dehalococcoides sp CBDB1 share, and the like are known.

Moreover, in addition to the bacteria belonging to the sixth group of sulfate-reducing bacteria, the ability to purify soil or groundwater can be determined more accurately by detecting the genus Dehalococcoides and lactic acid bacteria.

Therefore, when coexistence of bacteria belonging to Group 6 of Dehalococcoides genus bacteria, lactic acid bacteria, and sulfate-reducing bacteria is confirmed, an anaerobic environment sufficient for the growth of Dehalococcoides genus bacteria and its biological activity is formed. Therefore, it is judged that the organic chlorine compound can be decomposed satisfactorily. On the other hand, if the presence of bacteria belonging to the sixth group of Dehalococcoides genus bacteria, lactic acid bacteria, and sulfate-reducing bacteria is not confirmed by the detection result, or the amount of any of them is small, the growth of the Dehalococcoides genus bacteria and the organisms thereof It is determined that an anaerobic environment sufficient for expressing the activity is not formed and it is not suitable for the decomposition of the organic chlorine compound. And based on such determination, addition of bacteria belonging to the sixth group of Dehalococcoides genus bacteria, lactic acid bacteria, and sulfate-reducing bacteria whose presence is not confirmed or whose abundance is small, or forms a desired nutrient such as an anaerobic environment Prepare the purification environment by adding nutrients.

Here, although the detection of Dehalococcoides genus bacteria and lactic acid bacteria can be performed by a known method, it is preferably detected based on gene analysis using the characteristic sequence of the microorganism as an index. In that case, it can be carried out by utilizing an oligonucleotide complementary to the characteristic base sequence possessed by the Dehalococcoides genus bacteria and the lactic acid bacteria group. The design and preparation of such an oligonucleotide and the gene analysis method using this are as described above.

Here, examples of oligonucleotides that can be suitably used for detection of Dehalococcoides genus bacteria and lactic acid bacteria groups are given below.

For detecting Dehalococcoides bacteria
5´-AAGGCGGTTTTCTAGGTTGTCAC-3´ (Sequence recognition number 3)
5´-CTTCATGCATGTCAAAT-3´ (Sequence recognition number 4)

For detecting lactic acid bacteria
5'-AGCAGTAGGGAATCTTCCA-3 '(Sequence recognition number 5)
5´-ATTYCACCGCTACACATG-3´ (Sequence recognition number 6)

Here, the lactic acid bacteria group is a general term for microorganisms that decompose sugars such as lactose and glucose to produce organic acids such as lactic acid. For example, Leuconostoc spp., Pediococcus spp., Streptococcus spp, Lactobcillus genus Melisscoccus genus Enterococcus spp, Lactococcus spp, Carnobacterium spp., Vagococcus sp., Tetragenococcus genus Atopobium genus Weissella spp, Lactosphaera genus Oenococcus spp, Abiotrophia genus Paralactobacillus genus, Known are Granulicatella , Atopobactor , Alkalibacterium and Olsenella .

  Based on the detection results, the environment of the purification site should be adjusted so that anaerobic microorganisms such as microorganisms having reductive dehalogenation ability involved in the decomposition of organochlorine compounds can maximize their biological activity. can do. For example, it can be configured to control a pollution purification system exemplified by control of nutrient source dosage, microorganism dosage, temperature, pH, and the like based on the detection result, thereby highly efficient environmental pollution purification. A system can be constructed, and the purification processing time can be shortened.

Therefore, based on the detection means detected at least one sulfate-reducing bacteria among the bacteria belonging to the sixth group of sulfate-reducing bacteria in soil or groundwater, and the detection result detected by the detection means, the soil or groundwater A part of the present invention can also be configured for a soil or groundwater purification capacity monitoring system comprising a control means for controlling the purification environment. Moreover, you may comprise so that the lactic acid bacteria group and Dehalococcoides genus bacteria may be detected.

Furthermore, various information about pollution purification including information obtained by using the above method can be made into a database, and a purification system suitable for the situation at the contamination site can be constructed by using such a database. For example, bacteria and lactic acid bacteria belonging to the sulfate-reducing bacteria group 6 based on information such as the type of contaminants at the contaminated site, the contamination status including the amount of contaminants, and physical properties such as the oxidation-reduction potential at the contaminated site Establish an optimum input amount of Dehalococcoides bacteria and apply the set input amount to the contaminated site. Thereby, a highly efficient environmental pollution purification system can be constructed, and the purification processing time can be shortened.

  Examples of the present invention will be described below, but the scope of the present invention is not construed as being limited by these Examples.

[Examination Example 1] Search for microorganisms present in used water-soluble cutting oil-1
The microorganisms present in the used water-soluble cutting oil were searched.

(Method)
The microorganisms present in the used water-soluble cutting oil were searched. In this examination example, Idemitsu Daphne Alpha Cool (used for cutting of iron products for 6 months) was examined as a used water-soluble cutting oil. First, the 16S ribosome gene was obtained from the microorganism contained in the used water-soluble cutting oil, and sequenced. Such sequence information is shown in the sequence listing as sequence recognition number 7. Subsequently, the base sequence of the obtained 16S ribosomal gene was analyzed by comparison with a known database. Analysis was performed by the Japan DNA Data Bank (DDBJ: http://www.ddbj.nig.ac.jp/) of the National Institute of Genetics.

(result)
The result of the analysis is shown in FIG.
As a result of this analysis, it was found that the used water-soluble cutting oil contains a microorganism having a very high homology of 99% with the KRS1 strain (sequence recognition number 8) of the genus Desulfovibrio sulfate-reducing bacterium.

[Examination Example 2] Search for microorganisms present in used water-soluble cutting oil-2
Based on the results of Study Example 1, a search was made for sulfate-reducing bacteria present in the used water-soluble cutting oil.

(Method)
Based on the results of Study Example 1, a search was made for sulfate-reducing bacteria present in the used water-soluble cutting oil. In this example, water-soluble cutting oil diluted with water was used as the water-soluble cutting oil used (iron products, Daphne Alpha Cool manufactured by Idemitsu Co., Ltd. used for 2-6 months for casting). . Specifically, the primers described in Microbiology, July 2000, Vol. 146, pp. 1693 to 1705 are synthesized by a known synthesis means and contained in the water-soluble cutting oil dilution water using a PCR method. An exhaustive search for sulfate-reducing bacteria was conducted. In addition, it describes below about the primer used here.

For first group detection
5´-TAGMCYGGGATAACRSYKG-3´ (Sequence recognition number 9)
5´-ATACCCSCWWCWCCTAGCAC-3´ (Sequence recognition number 10)
For second group detection
5'-CGCGTAGATAACCTGTCYTCATG-3 '(sequence recognition number 11)
5'-GTAGKACGTGTGTAGCCCTGGTC-3 '(sequence recognition number 12)
For third group detection
5′-CTAATRCCGGATRAAGTCAG-3 ′ (sequence recognition number 13)
5′-ATTCTCARGATGTCAAGTCTG-3 ′ (SEQ ID NO: 14)
For fourth group detection
5'-GATAATCTGCCTTCAAGCCTGG-3 '(sequence recognition number 15)
5'-CYYYYYGCRRAGTCGSTGCCCT-3 '(sequence recognition number 16)
For fifth group detection
5′-GATCAGCCACACTGGRACTGACA-3 ′ (SEQ ID NO: 17)
5'-GGGGCAGTATCTTYAGAGTYC-3 '(sequence recognition number 18)
For group 6 detection
5´-GRGYCYGCGTYYCATTAGC-3´ (Sequence recognition number 1)
5´-SYCCGRCAYCTAGYRTYCATC-3´ (Sequence recognition number 2)
Here, the group of sulfate-reducing bacteria is based on the classification of sulfate-reducing bacteria described in Microbiology, July 2000, Vol. 146, pages 1693 to 1705.

  PCR was also performed according to the method described in Microbiology, July 2000, vol. 146, pages 1693 to 1705. Specifically, a cycle comprising a denaturation step at 95 ° C. for 1 minute, an annealing step for 1 minute, and an extension step for 1 minute at 72 ° C. was repeated 30 times. The reaction solution was put into a 100 μl tube, 2 μl of each primer (10 pmol / μl), 2 μl of dNTP (10 mM each), 85 μl of distilled water, 10 μl of 10 × PCR Buffer (HT Biotech), 0.2 μl of 10% (w / w). v) Prepared with BSA, 1U SuperTaq polymerase, 100-150 ng template nucleic acid.

(result)
The results are shown in Table 1.

  From the above results, it was confirmed that bacteria belonging to the sixth group of sulfate-reducing bacteria exist in the water-soluble cutting oil according to the classification described in Microbiology, July 2000, Vol. 146, pages 1693 to 1705. It was. Since such microorganisms belong to the related species of the genus Desulfovibrio whose homology was recognized in Examination Example 1, it was found that the cause of spoilage of water-soluble cutting oil was bacteria belonging to the sixth group of sulfate-reducing bacteria. Led.

[Example 1] Detection of spoilage of water-soluble cutting oil (examination of detection sensitivity)
For the detection of rot of water-soluble cutting oil, the gene analysis of sulfate-reducing bacteria according to the present invention and the detection of rot odor were compared and examined for detection sensitivity.

(Method)
Regarding the detection of rot of water-soluble cutting oil, the genetic analysis of bacteria belonging to the sixth group of the sulfate-reducing bacteria according to the present invention and the detection of rot odor were compared and examined for detection sensitivity. Specifically, groundwater that has been confirmed to contain sulfate-reducing bacteria is collected in plastic tubes, and each is adjusted to a final concentration of 10 mM methanol, 1 mM citric acid, and 0.1% milk. The mixture was added, and sealed with a cap, and cultured at 20 ° C. for 2 weeks. The gene analysis of sulfate-reducing bacteria was performed according to the method for searching microorganisms of the sixth group of sulfate-reducing bacteria in Study Example 2. Moreover, the detection of the rotten odor was performed by detecting the hydrogen sulfide odor.

(result)
The results are shown in Table 2.

  In the case of culturing without adding nutrients and in the case of culturing with addition of methanol, the smell of hydrogen sulfide could not be confirmed. Detection of belonging bacteria was confirmed. From the above results, it was found that the spoilage of insoluble cutting oil can be detected with high sensitivity by gene analysis of the sulfate-reducing bacteria of the present invention.

[Examination Example 3] Examination of microorganisms related to reduction of oxidation-reduction potential-1
Microorganisms related to the reduction of redox potential were investigated.

(Method)
It is known that an anaerobic environment is necessary for the growth of Dehalococcoides genus bacteria known to be involved in the decomposition of organochlorine compounds and the expression of their biological activities. In addition, a group of lactic acid bacteria is known as co-flora of Dehalococcoides bacteria, may play an important role in the formation process of the anaerobic environment required by the Dehalococcoides bacteria has been pointed out. Therefore, the relationship between the abundance of the Dehalococcoides genus bacteria and lactic acid bacteria group and the redox potential was examined by adding the following nutrients and culturing. Here, the examined nutritional components are as follows.

1. No addition 2.10 mM methanol 3.1 mM citric acid 4.0.01% nonfat dry milk 5.10 mM methanol, 1 mM citric acid, 0.01% nonfat dry milk Here, the addition amount indicates the final concentration.

The ground water was filled into a 15 ml plastic tube, added with nutrients, and sealed with a cap and cultured at 20 ° C. for 2 weeks. After the culture, the abundance of Dehalococcoides genus bacteria and lactic acid bacteria group, and the redox potential were measured. Detection of bacteria belonging to the genus Dehalococcoides was performed by the method described in Journal of Microbiological Methods, June 2004, Vol. 57, No. 3, pages 369-378. Detection of lactic acid bacteria was performed by the method described in Applied and Environmental Microbiology. June 2001, Vol. 67, Vol. 6, pages 2578-2585. Here, the primers used are shown below.

For detecting Dehalococcoides bacteria
5´-AAGGCGGTTTTCTAGGTTGTCAC-3´ (Sequence recognition number 3)
5´-CTTCATGCATGTCAAAT-3´ (Sequence recognition number 4)
For detecting lactic acid bacteria
5'-AGCAGTAGGGAATCTTCCA-3 '(Sequence recognition number 5)
5´-ATTYCACCGCTACACATG-3´ (Sequence recognition number 6)

(result)
The results are shown in Table 3.

When citric acid was added alone as a nutrient component, growth of lactic acid bacteria was not observed, but it was confirmed that growth of bacteria belonging to the genus Dehalococcoides was induced. In addition, when skim milk powder was added alone as a nutritional component, sufficient growth of the lactic acid bacteria group was confirmed. However, the oxidation-reduction potential did not decrease as compared with the case where methanol, citric acid and skim milk powder were added as nutrient components, and the difference was remarkable. These results suggest the existence of microorganisms that play an important role in the formation of anaerobic environments required by bacteria belonging to the genus Dehalococcoides other than lactic acid bacteria.

  And when methanol, a citric acid, and skim milk powder were added, since the hydrogen sulfide odor was detected from the culture, the presence of sulfate-reducing bacteria was expected.

[Examination Example 4] Examination of microorganisms related to reduction in redox potential-1
Subsequent to Investigation Example 3, microorganisms related to the reduction in redox potential were examined.

(Method)
As a result of the examination in Examination Example 3, when methanol, citric acid, and skim milk powder were added, a hydrogen sulfide odor was detected from the culture.

  Specifically, ground water to which methanol, citric acid and skim milk powder were added was filled in a 15 ml plastic tube, nutrient components were added, and the mixture was cultured at 20 ° C. for 2 weeks while sealed with a cap. After culture, exhaustive detection of sulfate-reducing bacteria was performed. The detection was performed according to the method of Study Example 2.

  Moreover, it compared with the case where the said nutrient component was not added.

(result)
The results are shown in Table 4.

Proliferation of bacteria belonging to the sulfate-reducing bacteria group 5 and group 6 was confirmed. From the above results, it was suggested that the bacteria belonging to the group 5 and 6 of the sulfate-reducing bacteria may play an important role in the formation of the anaerobic environment required by bacteria belonging to the genus Dehalococcoides .

[Example 2] Relationship between sulfate-reducing bacteria and reduction in oxidation-reduction potential Based on the results of Study Examples 2 and 3, the relationship between the growth of sulfate-reducing bacteria groups 5 and 6 and the reduction in oxidation-reduction potential The sex was confirmed.

(Method)
Groundwater to which each nutrient component examined in Study Example 3 was added was cultured, and the relationship between the growth of sulfate-reducing bacteria group 5 and 6 and the reduction in redox potential was confirmed.

  Here, the examined nutrient components were the same as in Example 1, and groundwater was cultured in the same manner. After the culture, the bacteria belonging to the sulfate-reducing bacteria group 5 and group 6 were detected. The detection was performed according to the method for searching for bacteria belonging to the sulfate-reducing bacteria group 5 and group 6 in Study Example 2.

(result)
The results are shown in Table 5.

From the above results, when the redox potential was lowered, bacteria belonging to the sulfate-reducing bacteria group 5 were all detected. On the other hand, there were cases where bacteria belonging to the sulfate-reducing bacteria group 6 were not detected. From the above results, it was found that the bacteria belonging to the sixth group of sulfate-reducing bacteria play an important role in the formation of the anaerobic environment required by bacteria belonging to the genus Dehalococcoides . And it turned out that the bacteria which belong to such a sulfate-reducing bacteria group 6 are important microorganisms in soil and groundwater purification, in particular, in a pollution purification system mainly composed of bacteria belonging to the genus Dehalococcoides as decomposing microorganisms.

The figure which shows the result of the homology analysis of examination example 1

Claims (9)

Desulfovibrio Bastinii in water-soluble cutting oil, Desulfovibrio salexigens, Desulfovibrio caledoniensis, Desulfovibrio halophilus, Desulfovibrio gracilis, Desulfovibrio longus, Desulfovibrio gabonensis, Desulfovibrio gigas, Desulfovibrio africanus, Desulfovibrio termitidis, Desulfovibrio longreachensis, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio fairfieldensis, Desulfomicrobium baculatum A method for determining rot of water-soluble cutting oil, wherein the rot of water-soluble cutting oil is determined by using as an index the detection of at least one sulfate-reducing bacterium of Desulfomicrobium escambiense and Desulfovibrio acrylicus .   The water-soluble composition according to claim 1, wherein the sulfate-reducing bacterium is detected using a nucleotide sequence represented by SEQ ID NO: 1, an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2, or an oligonucleotide complementary to the oligonucleotide. Cutting oil rot judgment method. Desulfovibrio Bastinii in water-soluble cutting oil, Desulfovibrio salexigens, Desulfovibrio caledoniensis, Desulfovibrio halophilus, Desulfovibrio gracilis, Desulfovibrio longus, Desulfovibrio gabonensis, Desulfovibrio gigas, Desulfovibrio africanus, Desulfovibrio termitidis, Desulfovibrio longreachensis, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio fairfieldensis, Desulfomicrobium baculatum Detection means for detecting at least one sulfate-reducing bacterium among Desulfomicrobium escambiense and Desulfovibrio acrylicus ;
A water-soluble cutting oil management system comprising cleaning means for cleaning water-soluble cutting oil based on a detection result by the detection means. Of soil or ground water, Desulfovibrio bastinii, Desulfovibrio salexigens, Desulfovibrio caledoniensis, Desulfovibrio halophilus, Desulfovibrio gracilis, Desulfovibrio longus, Desulfovibrio gabonensis, Desulfovibrio gigas, Desulfovibrio africanus, Desulfovibrio termitidis, Desulfovibrio longreachensis, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio fairfieldensis, Desulfomicrobium baculatum A method for determining the purification capacity of soil or groundwater, wherein the purification ability of the soil or groundwater by anaerobic biodegradation is determined using detection of sulfate-reducing bacteria of at least one of Desulfomicrobium escambiense and Desulfovibrio acrylicus as an index.   The sulfate-reducing bacterium is detected by using the nucleotide sequence represented by SEQ ID NO: 1, the oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2, or the oligonucleotide complementary to the oligonucleotide. Method for judging the purification capacity of soil or groundwater. The soil or groundwater purification capacity determination method according to claim 4 or 5, wherein detection of lactic acid bacteria is used as an index. The soil according to claim 6, wherein the lactic acid bacteria are detected using the nucleotide sequence shown in SEQ ID NO: 5 or the oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 6, or the oligonucleotide complementary to the oligonucleotide. Groundwater purification capacity judgment method. The soil or groundwater purification capacity determination method according to any one of claims 4 to 7, wherein detection of Dehalococcoides genus bacteria is used as an index. The said Dehalococcoides genus bacteria are detected using the oligonucleotide which contains the base sequence shown to sequence recognition number 3, the base sequence shown to sequence recognition number 4, or the oligonucleotide complementary to the said oligonucleotide. Method for judging the purification capacity of soil or groundwater.

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