JP3933681B2 - Microorganisms capable of resolving female hormone substances and use thereof - Google Patents

Description

  The present invention relates to a microorganism having a resolution of female hormone substances and use thereof, and more particularly to a microorganism that is excreted in the environment and specifically decomposes female hormone substances that are present and a method for decomposing female hormone substances using the same. .

  Endocrine disrupting chemicals (so-called environmental hormones) that are thought to disrupt the endocrine system of living organisms have been a major social problem since the announcement of Colborn T., “Our Stolen Future” (1996). It has become. The endocrine system in the living body is a system that normally maintains and regulates the development of life forms, the development of genital organs, and the functions of various internal organs by the action of male hormones, female hormones, thyroid hormones, corticosteroids, and the like. Endocrine disrupting chemicals are a group of substances that cause damage to the endocrine system in these living organisms, and have been released in large quantities to the natural world by various human activities and have already caused many abnormalities in wildlife.

  These endocrine disrupting chemicals include organochlorine pesticides, antibacterial agents, herbicides, chemicals attributed to industry such as bisphenol A and nonylphenol, and chemicals attributed to pharmaceuticals such as hormone agents. Are known. Among these chemical substances, industrial chemical substances have strict regulations for their use due to their toxicity due to the Water Pollution Control Law and pollution-related laws and regulations. Therefore, the amount of chemical substances released into the environment is decreasing.

  However, among pharmaceutical chemical substances, female hormone substances such as 17β-estradiol, estrone, and estriol represented by the following formula (I), and synthetic female hormone substances such as ethinyl estradiol are human and animal. It is contained in urine and discharged into the environment, and its discharge increases as the population increases. For this reason, anomalies in females have been confirmed in wildlife living in rivers and lakes where large amounts of domestic wastewater are flowing in (Deborah Cadbury, supervised by Yasuzumi Iguchi, “Merifying Nature”, Shueisha (1998) ), Concerns that the balance of the ecosystem will be lost due to the inability to reproduce. Therefore, it is necessary to reduce, for example, female hormone substances in the wastewater at a stage before discharging the wastewater into a river or the like at a wastewater treatment facility.

  By the way, in a conventional wastewater treatment facility, three treatment methods of physical treatment, chemical treatment, and biological treatment are performed. Of these, the physical treatment is specifically a centrifugal separation method, a filtration method, a pressurized flotation separation method, an adsorption method or the like, and a chemical treatment is a harmful substance due to the addition of chemicals or the like. Detoxification treatment method, electrodialysis method, ion exchange method, etc. In contrast, biological treatment is a method that uses microorganisms to decompose and remove organic substances in wastewater, and is also effective for treating substances that are difficult to treat by physical and chemical treatments. It is.

  For this reason, biological treatment has been used in many wastewater treatment facilities in recent years, and is generally divided into the following three stages. That is, pretreatment, biological oxidation treatment, and sludge treatment. Among these, pretreatment includes screens, sand basins, sedimentation basins, levitation tanks, etc., and these devices remove solids with large particle sizes and inorganic suspended solids contained in wastewater, In addition, it helps to reduce the burden of organic substances on bio-oxidation facilities.

  The bio-oxidation treatment uses microorganisms, and there are a method of attaching microorganisms to the surface of a solid support and growing them, and a method of suspending microorganisms in a liquid. The former is usually carried out by a fixed bed type apparatus such as a trickling filter method, and the latter is carried out by a fluid bed type apparatus such as an activated sludge method.

  The activated sludge treatment and sprinkling filter treatment described above are very good methods for reducing pollutants in wastewater, and in recent years, these biological treatments have also been used to decompose endocrine disrupting chemicals. Various approaches have been made, and further, selection and strengthening of microorganisms have been carried out for the purpose of improving processing efficiency and decomposing endocrine disrupting chemicals that are difficult to process. For example, JP 2001-333767 describes Sphingomonas bacteria having nonylphenol resolution, and JP 11-341978 describes Fusarium bacteria having dioxin resolution. However, no microorganism has been reported so far that can effectively degrade female hormone substances.

  Accordingly, it is an object of the present invention to provide a microorganism having a resolution of female hormone substances and to provide a method for easily decomposing female hormone substances discharged into the environment from domestic wastewater and livestock farms using the microorganisms. It is what.

  As a result of screening many microorganisms by enrichment culture using a female hormone substance in order to solve the above-mentioned problems, the present inventors have found a microorganism having a particularly excellent female hormone substance resolution. Further, the present inventors have found that the female hormone substance discharged into the environment can be decomposed by the method and apparatus for decomposing the female hormone substance using the microorganism.

  That is, the present invention provides a microorganism which belongs to the genus Rhodococcus or the genus Sphingomonas and has a female hormone substance resolution.

  The present invention also provides a method for decomposing a female hormone substance, characterized by decomposing a female hormone substance using the microorganism.

  Furthermore, the present invention provides an apparatus for decomposing female hormone substances comprising means for supporting the microorganism and means for bringing the microorganism into contact with waste water or soil.

  The microorganism of the present invention is a microorganism capable of degrading female hormone substances such as 17β-estradiol, estrone, estriol or ethinyl estradiol.

  Therefore, the method and apparatus for decomposing female hormone substances using the microorganisms of the present invention can improve the environment by decomposing female hormone substances in waste water and soil contaminated with female hormone substances.

  In the present invention, the female hormone substance to be decomposed is, for example, a natural or synthetic female hormone represented by the formula (I). Of these, 17β-estradiol is a steroid hormone secreted from the ovarian follicle and is the substance with the highest physiological activity. Its secretion is controlled by the pituitary follicle stimulating hormone and luteinizing hormone. As a drug, it is used for amenorrhea, abnormal menstrual cycle, dysmenorrhea, uterine growth failure, climacteric disorder. Estrone is a kind of follicular hormone and is a metabolite of 17β-estradiol. It has strong estrogenic activity and is used as a female hormone agent for female sexual dysfunction, menopause, and prostate cancer. Furthermore, estriol is a metabolite of 17β-estradiol in vivo and is produced via estrone and appears in urine. On the other hand, ethinyl estradiol is an artificial female hormone substance and is used as a follicle hormone agonist. It is less decomposed in the liver than 17β-estradiol, and is more effective than 17β-estradiol by oral administration. It is orally administered to uterine growth disorder, amenorrhea, infertility, climacteric disorder, etc. Is also used.

  The microorganisms that decompose these female hormone substances (hereinafter simply referred to as “microorganisms of the present invention”) include, for example, microorganisms belonging to the genus Rhodococcus or Sphingomonas that are present in the activated sludge tank. It is obtained by screening with a substance.

  More specifically, the microorganism of the present invention can be obtained by subjecting the activated sludge obtained from the activated sludge tank of the sewage treatment plant to multistage screening using a female hormone substance.

  That is, the microorganisms of the present invention are cultured for a long time (approximately 10 weeks) on each activated sludge by a column accumulation culture method using a female hormone substance as a substrate, and after screening for microorganisms having a resolution of female hormone substances, the It can be obtained by culturing using a hormonal substance as a substrate and screening for microorganisms that exhibit numerically significantly higher resolution.

The following procedure can be shown as a specific example of screening for microorganisms having a resolution of female hormone substances. First, a culture apparatus including a column, a substrate inlet, and an air inlet as shown in FIG. 1 is prepared. In this culture apparatus, activated sludge and, for example, the following MDG medium (Modified DOMINIC &GRAHAM's medium) are placed in a column, into which oxygen and 17β-estradiol, estrone, estriol, ethinyl are introduced from the air inlet and the substrate inlet. A female hormone substance such as estradiol is introduced and cultured at about 25 ° C. for about 10 weeks. During this culture, the viable cell count and pH are measured at regular intervals. After completion of the culture, the strain is selected and fished using ISP medium, R ₂ A medium, YM medium or the like to obtain a microorganism having a female hormone substance resolution.

Composition of MDG medium:
K ₂ HPO ₄ 3.5
KH ₂ PO ₄ 1.5
(NH ₄ ) ₂ SO ₄ 0.5
NaCl 0.5
MgSO ₄ · 7H ₂ O 0.15
Yeast extract 0.005% (w / v)
Trace element ^* 1.0 (mL / L)
pH 7.0
The unit of the numerical value is g / L.

* Trace element composition:
NaHCO ₃ · 10H ₂ O 2.0
MnSO ₄ · _4H 2 O 0.3
ZnSO ₄ · 7H ₂ O 0.2
_{(NH 4) Mo 7 O 24} · 4H 2 O 0.02
CuSO ₄ · _5H 2 O 0.1
CoCl ₂ .6H ₂ O 0.5
CaCl ₂ · 2H ₂ O 0.05
FeSO ₄ · _7H 2 O 0.5
The unit of the numerical value is g / L.

  Next, the microorganisms selected by the above screening are cultured in, for example, a YM medium containing a female hormone substance, and the residual amount of the female hormone substance in the culture medium after the culture is measured. Microbial screening can be performed. By repeating this screening a plurality of times, microorganisms with higher resolution of female hormone substances can be obtained.

The microorganism of the present invention thus obtained is capable of biologically degrading at least one female hormone substance such as 17β-estradiol, estrone, estriol or ethinyl estradiol used in the screening, specifically, female hormone substance. As a result, when 10 ⁸ to 10 ⁹ microorganisms of the present invention are cultured for 8 hours in 1 L of MDG medium supplemented with 100 ppm of 17β-estradiol, the ability to decompose 80% or more of 17β-estradiol in the medium It is what you have.

  By the above screening, the activated sludge tank of Fushimi Sewage Treatment Plant (Kyoto City, Kyoto), the activated sludge tank of Todoroki Water Treatment Center (Kawasaki City, Kanagawa Prefecture), the activated sludge tank of Tama River Upstream Treatment Plant (Akishima City, Tokyo), The following 5 strains were obtained as microorganisms capable of degrading female hormone substances selected from microorganisms obtained from the activated sludge tank of Morigasaki Water Treatment Center (Ota-ku, Tokyo). In addition, bacterial species identification of these microorganisms is described in the literature using the 16S rDNA sequence of the microorganisms (Carl R. Woese, Bacterial Evolution., Microbiological Reviews., 51: 221-271 (1987) and E. Stackebrandt and BM Goebel, Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology., International Journal of Systematic Bacteriology, 44, (4): 846-849 (1994)) It was. Five strains of microorganisms were identified as follows by molecular evolution phylogenetic analysis.

・ Rhodococcus zopfii D-I7
・ Sphingomonas sp. C-I2
・ Rhodococcus equi A-Y2
・ Rhodococcus equi DY4
・ Rhodococcus equi E-Y5

  Among these five strains, the D-I7 strain and the C-I2 strain are the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st Higashi 1-chome, Tsukuba City, Ibaraki, Japan) )) On April 26th with deposit numbers FERM P-18839 and FERM P-18838, respectively. On the other hand, the A-2Y strain, the DY4 strain, and the EY5 strain were not deposited at the above Patent Organism Depositary Center because they were rejected due to taxonomic issues. If there is a request for sale that satisfies the conditions of Article 27-3, the applicant's Yakult Honsha Co., Ltd. guarantees the sale according to the conditions of that article.

Rhodococcus sp., Including Rhodococcus equi and Rhodococcus zopfii, which are the microorganisms of the present invention, is edited by the Japanese Society for Radiobiology, "Classification and Identification of Radioactive Bacteria" Office Center, 187-188 (2001)), Corynebacterium group which is a High-GC Gram-positive cocci. Until now, it was a group of Rhodochrous complex grouped by showing intermediate properties between Mycobacterium sp. And Nocardia sp. It was redefined with the genus name proposed by Zopf in 1891 (Tsukamura, M. A Further numerical taxonomic study of the rhodochrous group, Jpn. J. Microbiol., 18, 37-44 (1974)). . The microorganism of the present invention is aerobic and non-motile. It also does not show acidity or is very weak. Furthermore, there are a variety of mycelium-like cells and pseudo-mycotic cells, but none have aerial hyphae. In addition, mycelium-like or branch-like gonococcus generally becomes a short gonococcus or pseudococcus as cells are ruptured by long-term culture. Furthermore, the optimum growth temperature is as wide as 20 ° C. to 37 ° C. In addition, an orange to red colony is formed, and the surface thereof is both rough and smooth. In addition, cell wall peptidoglycans include A1γ-type, glycolyl type, arabinose and galactose. Furthermore, the major component of the respiratory chain quinone is MK-8 _{(H 2).} Furthermore, the microbial fatty acids are linear-monounsaturated and the content of 010 methyl fatty acids including 10Me-18: 0 varies depending on the species or strain. In addition to PE being detected as phospholipids, DPG, PG, PI, PIMs, and some glycolipids are observed. Most of these species are derived from soil, but are widely distributed in animals and plants.

  The other microorganism of the present invention, Sphingomonas sp., Is a gram-negative bacterium and is included in α-proteobacteria (Proteobacteria α-subdivision) (translated by Toshikazu Sakazaki, “Gram-negative, aerobic. And identification of facultative anaerobic bacilli ", Modern Publishing, 302-303 (1993)). In addition, there are many relatively long koji molds, and those having motility are also included. In addition, it produces an aerobic yellow insoluble pigment. Furthermore, it is usually non-growing on MacConkey agar and uses glucose, xylose, lactose, sucrose, maltose. Furthermore, mannitol is unused and non-reducing for nitrates. Moreover, it is a microorganism characterized by being negative for lysine decarboxylase and negative for arginine dihydrolase.

  The microorganisms of the present invention thus obtained were examined under suitable conditions for mass culture. The optimum culture temperature was 30 ° C. and the pH was generally 6.0 to 7.0. In addition, the examination of the optimum medium is based on the ISP medium, and the modified medium 1 and modified medium 2 shown below, in which the amount of the component is increased or decreased and the addition of inorganic salts, etc. are used, and the influence on the growth is observed. investigated. As a result, among the above five strains, except for the C-I2 strain, it grew well in the modified medium 1, and it was revealed that the C-I2 strain is the most undernourished microorganism.

Composition of modified medium 1 (in 1 L of distilled water):
Yeast Extract 5g
Peptone 10g
Glucose 5g
KH ₂ PO ₄ 1g
K ₂ HPO ₄ 2g
MgSO ₄ · 7H ₂ O 0.5g

Composition of modified medium 2 (in 1 L of distilled water):
Yeast Extract 2g
Malt Extract 10g
Dextrose 4g

  The microorganisms of the present invention described above can be used alone or in combination for decomposing female hormone substances in waste water or soil. These microorganisms may be used as they are, for example, in the case of the activated sludge method. However, it is preferable to use these microorganisms by attaching them to a solid support from the viewpoint of decomposition of female hormone substances.

  In the case of growing on a solid support, the method for immobilizing microorganisms includes a method for immobilizing microorganisms on an insoluble carrier by covalent bonds, ionic bonds, hydrogen bonds, physical adsorption, etc. There is a comprehensive immobilization method in which microorganisms are encapsulated in a polymer gel produced by polymerizing or associating the compound or changing the polymer compound from a soluble state to an insoluble state. good. However, the binding immobilization method is preferable in terms of the ability to retain microorganisms, and the solid support to which the microorganisms are attached is porous cellulose, PVA, fine powder activated carbon coated granular polyvinyl alcohol carrier (ACP carrier: Miyoshi et al. Application of decolorizing bacteria ", Journal of Sewerage Society, 39, 123-132 (2002)), etc., and the use of an ACP carrier is particularly preferable.

Specifically, when decomposing female hormone substances in wastewater, for example, when the activated sludge method is adopted, generally, about 10 ⁸ to 10 ⁹ microorganisms of the present invention are used per liter of the liquid to be treated. What is necessary is just to culture | cultivate aerobically for about 10 hours. When a method using a solid support is employed, the microorganism of the present invention on the solid support and the liquid to be treated may be brought into contact under aerobic culture conditions for about 5 to 10 hours.

Further, when decomposing the female hormone substance in the soil, for example, 10 ⁹ per 1 m ^{3 of the} microorganism of the present invention suspended in a buffer solution or physiological saline, if necessary, on the soil surface to be treated or the soil dug up. About 10 to ¹⁰ may be sprayed.

  In order to efficiently decompose the female hormone substance using the microorganism of the present invention, a means for supporting the microorganism of the present invention for decomposing the female hormone substance using the microorganism of the present invention, and draining or soiling the microorganism. Use of a device having a means for contacting with the device. Specifically, bioreactor devices such as a complete mixing type, an air lift type, a column packing type, and a lag flow type can be used. Such an apparatus can be used in the secondary treatment step during the bio-oxidation treatment. In this case, the method of using a method in which the microorganism group is adhered and grown on the surface of the solid support rather than the method of suspending the microorganism group in the waste water. Is preferred. By attaching microorganisms to the surface of the support, it is possible to prevent the microorganisms from flowing out of the bioreactor and to obtain a more concentrated bacterial solution. Can be decomposed well.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1
Screening for microorganisms capable of resolving female hormone substances by continuous enrichment culture:
A substrate inlet and an air inlet were provided at the upper part and the lower part of a cylindrical column of length 50 cm × diameter 8 cm, a tube was connected to these, and then a lid was covered with a rubber stopper to prepare a culture apparatus (FIG. 1). . 20 mL of activated sludge collected from the above-described MDG medium and the place shown in Table 1 was added to the culture apparatus. Next, 0.1 mg / L of 17β-estradiol aqueous solution was blown from the upper outlet at 60 to 100 mL / hour, and oxygen was blown from the lower outlet at 50 to 100 mL / min. In this state, batch culture was continuously performed for about 10 weeks, and the viable cell count and pH were examined every 0, 5, 24, and 72 hours and thereafter every week. In order to confirm reproducibility, the culture was performed three times. In all three cases, characteristic colonies were obtained from the culture solution at the 10th week (9th week only at Todoroki Water Treatment Center) where the growth of microorganisms was confirmed, using the ISP isolation medium, R ₂ A isolation medium and YM medium, respectively. Selected and fished. Table 1 shows the number of settlements obtained in each medium and the number of strains cultivated from them.

  As shown in this result, a total of 56 strains from Fushimi Sewage Treatment Plant, 10 strains from Todoroki Water Treatment Center, 16 strains from Tama River Upstream Treatment Plant, and 16 strains from Morigasaki Treatment Center by continuous enrichment culture. Strains of female hormone substance-degrading microorganisms were obtained.

Example 2
Screening for high-resolution microorganisms of female hormone substances:
(1) Primary screening The 56 strains of microorganisms obtained in Example 1 were cultured by a method shown below using a medium using 17β-estradiol as a substrate, and strains having a high growth rate were selected. First, 17β-estradiol was dissolved in a DMSO solution and sterilized by filtration using a 0.22 μm membrane filter. This solution was added to the MDG medium so that the 17β-estradiol concentration was 100 ppm.

  Each microorganism obtained in Example 1 stored at 25 ° C. was inoculated on a YM flat plate medium, and then lined with liquid paraffin, followed by streak culture. After 3 to 10 days, the platinum ears were scraped and inoculated into a test tube containing the medium prepared above. The inoculated test tube was vigorously shaken at 25 ° C. and cultured for 10 days. Using the test tube containing the non-inoculated substrate as a control, the turbidity of the inoculated substrate was observed. Determination of the degree of growth of microorganisms was performed according to the following criteria. The results are shown in Tables 2 and 3.

<Criteria>
-: Same as control.
+: Some turbidity is observed compared to the control, and there is growth of bacteria
is expected.
++: It is clearly turbid compared to the control, and active bacterial growth
Guessed.

  As a result of the primary screening, 25 or more of 56 strains obtained a determination of + or more.

(2) Secondary screening For 25 microorganisms selected in the primary screening, strains were selected by the method described below. First, 25 strains that showed + or more growth in the primary screening were streaked on YM plate medium and cultured in the same manner as in the primary screening. Ten days later, the entire amount of the grown medium was subjected to solid phase extraction with a C18 solid phase column (manufactured by Waters), 10 μL of the extract was spotted on a thin layer chromatograph, and a benzene-methanol (9: 1) mixture was used as a developing solvent. Development was confirmed to reduce 17β-estradiol. As a result, nine strains that effectively degraded 17β-estradiol were selected.

(3) Tertiary screening For 9 strains of microorganisms selected in the secondary screening, strains that more strongly degrade 17β-estradiol are selected, and the resolution of other female hormone substances (estrone, estriol, ethinyl estradiol) is also examined. did. The materials and methods followed the following procedure. First, female hormone substances (17β-estradiol, estrone, estriol, ethinylestradiol) were dissolved in DMSO solution and sterilized by filtration using a 0.22 μm membrane filter. The culture medium was prepared to 100 ppm.

  The culture medium was taken in a test tube, and the microorganism was inoculated in the same manner as in the secondary screening, and then cultured for a predetermined time. The concentration of the female hormone substance was measured at 5 hours, 8 hours, and 24 hours after the start of the culture, and the decrease was confirmed. The measurement of the amount of female hormone substance was carried out by retaining the total amount of the cultured medium on a C18 solid phase column (manufactured by Waters), followed by elution with methyl acetate and analysis. More specifically, the analysis was performed as follows. That is, first, an appropriate amount of the eluate was collected, and a surrogate substance (17β-estradiol-d4) was added thereto. Next, after drying with a nitrogen stream, a TMS compound was induced with N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA), dried again, and dissolved in n-hexane. Analyzed by gas chromatography under conditions.

<Measurement conditions for gas chromatography>
Gas chromatograph: GC-17A (manufactured by Shimadzu Corporation)
Capillary column: HP-1 (manufactured by Hewlett-Packard Co.)
(15m × 0.25mm; df = 0.25μm)
Column temperature: 150 ° C. → (20 ° C./min)→210° C. →
(10 ° C / min) → 300 ° C
Carrier gas: Helium injector: Splitless T = 250 ° C
Detector: FID · T = 300 ° C
Sample injection volume: 1 μL
Analysis time: 20 minutes

  As a result of the tertiary screening, 5 strains (A-Y2, E-Y5, DY4, D-I7, C-I2) were selected as microorganisms that decompose 80% or more of 17β-estradiol in 8 hours. . The change over time of the remaining amount due to the decomposition of 17β-estradiol, estrone, estriol, ethinylestradiol by the selected microorganism is shown in FIGS.

Example 3
Identification of microorganisms with female hormone substance resolution:
About five strains (A-Y2, S-Y5, D-Y4, D-I7, C-I2) of microorganisms having a female hormone substance resolution (hereinafter referred to as "female hormone substance-degrading bacteria"), Bacterial species identification using the 16SrDNA sequence was performed according to the following procedure, and a phylogenetic tree using the 16SrDNA sequence was prepared (FIGS. 6 and 7).

(1) DNA extraction from microbial cells DNA was extracted from each of the purely cultured microbial cells of the above five strains using the benzyl chloride method shown below. That is, it was cultured at 30 ° C. for 3 days using a YM slope agar medium. After culture, 250 μL of DNA extraction buffer (100 mM Tris-HCL, 40 mM EDTA mixed solution: pH 9.0), 200 μL of benzyl chloride and 50 μL of 10% SDS were added to the collected cells, and at 50 ° C. Shake vigorously for 30 minutes. After adding 150 mL of 3M sodium acetate, the mixture was centrifuged, and the supernatant was transferred to another tube, followed by isopropanol precipitation and 70% ethanol washing, followed by air-drying, and then 100 μL of TE buffer (10 mM Tris-HCl ( It was dissolved in pH 8.0) and 1 mM EDTA to obtain DNA of each bacterial cell.

(2) 16S rDNA amplification PCR reaction The following two primers were used as amplification primers.
8F: 5′-AGAGTTTGATCMMTGCTCAG-3 ′
15R: 5′-AAGGAGGTGATCCCARCGCA-3 ′
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 200 μL dNTPmixture, 1 U Taq DNA polymerase (manufactured by Takara), 50 ng bacterial cell DNA and amplification primers 8F and 15R, respectively A PCR reaction was performed with a DNA thermal cycler PTC200 (manufactured by MJ Research) on the reaction solution (0.4 μM in total) containing 0.4 μM. This PCR reaction was performed for 25 cycles at 94 ° C. for 20 seconds, 55 ° C. for 15 seconds, and 72 ° C. for 180 seconds. After the PCR reaction, the obtained amplification product was purified using Microcon-PCR (Millipore).

(3) Determination of 16S rDNA sequence The 16S rDNA base sequence was decoded using ABI PRISM 373A sequencer (manufactured by ABI) by ABI PRISM Dye Terminator Kit (manufactured by Perkin Elmer). The primers used are shown below. For alignment of sequences, AutoAssembler (manufactured by Perkin Elmer) was used.
8F: 5′-AGAGTTTGATCMMTGCTCAG-3 ′
520F: 5′-CAGGAGTGCCAGCAGCCCGCGG-3 ′
930F: 5′-CAAGCGGTGGAGCATGTGG-3 ′
1100F: 5′-CAGGAGCAACGAGCCGCAACCC-3 ′
520R: 5′-ACCGCCGCTGCTGGC-3 ′
800R: 5′-GGACTACCAGGGTATCTAAT-3 ′
1100R: 5′-AGGGTTGCCGCTCGTTQ-3 ′
15R: 5′-AAGGAGGTGATCCCARCGCA-3 ′

(4) Phylogenetic analysis of 16S rDNA sequence Molecular evolutionary phylogenetic analysis using phylogenetic analysis software (Clustul X) using the obtained 16S rDNA sequence and 16S rDNA base sequences of closely related bacteria obtained from gene databases such as DDBJ (FIG. 6). As a result, the A-Y2 strain, the EY5 strain, and the DY4 strain are strains belonging to Rhodococcus equi, the D-I7 strain is a strain corresponding to Rhodococcus zopfii, and C- It was identified as strain I2 Sphingomonas sp.

Example 4
Wastewater treatment using microorganisms capable of resolving female hormone substances:
Five female hormone-degrading bacterial strains (A-Y2, E-Y5, DY4, D-I7, and C-I2) were fixed on a carrier and treated with waste water.

(1) Preparation of Immobilized Carrier Using the three types of carriers: porous cellulose, polyvinyl alcohol (PVA), fine powder activated carbon coated granules polyvinyl alcohol (ACP) Fixed with. First, each cell of the female hormone substance-degrading bacterium to be immobilized was cultured in a large amount using a jar fermenter. In this culture, 500 mL of the logarithmic growth phase culture solution was put into a sterilized flask, and then 100 g of the three types of carriers shown in Table 4 were put in the flask and mixed well to prepare an immobilized carrier.

(2) Evaluation of microorganism-holding ability of immobilized carrier The microorganism-holding ability was evaluated by elution of bacterial cells from the immobilized carrier prepared in (1) above by the following procedure and measuring the number of viable bacteria. First, several grains of the immobilization carrier were ground and 10 g was weighed. This immobilization carrier is put in 10 mL of physiological saline (containing 0.1% Triton X-100) in a glass test tube, and this is sonicated for 10 minutes and thoroughly mixed, and then sonicated again for 10 minutes. Did. The solution thus obtained was used as an eluate for viable cell count measurement. The number of viable bacteria in the eluate was counted by a dilution plate method using CGY medium. The number of viable bacteria in the carrier was measured by collecting a part of the immobilized carrier immersed in the flask at 24 hours and 72 hours after the production of the carrier. The results are shown in Table 4. As a result, it was shown that the ACP carrier has a high ability to retain microorganisms.

(3) Efficacy test by batch aeration method Each of the five female hormone substance-degrading bacteria was immobilized on an ACP carrier in the same procedure as in (1), and an effectiveness test was conducted on synthetic sewage to which the female hormone substance was added. It was. Specifically, five strains of female hormone-degrading bacteria were cultivated in a large amount with a jar fermenter, and the obtained culture solution was autoclaved at 105 ° C. for 10 minutes and then washed with sterilized water (culture solution 500 mL). ACP carrier having a dry weight of 50 g) was immersed for 24 hours to be immobilized. Synthetic sewage was prepared by the method described in the literature (Hashimoto Sho, “High-functional activated sludge method utilizing biotechnology”, Gihodo Publishing, 267-268 (1989)). Regarding the concentration of female hormonal substances in sewage, the concentration of 17β-estradiol in inflow sewage and treatment was 0.017 to 0.068 μg / L in the actual survey at the 2000 treatment plant in 2000, and the concentration of estrone is It has been reported that it is 0.014 to 0.14 μg / L (Tokyo Sewerage Bureau Report, “Results of Survey on Endocrine Disrupting Chemical Substances in Sewerage in 2000”). This effective test was carried out by adding 17β-estradiol to 0.1 μg / L and estrone to 0.2 μg / L to the prepared synthetic sewage.

Composition of synthetic sewage (in 1 L of distilled water):
Peptone 150mg
Meat Extract 100mg
Urea 25mg
NaCl 7.5mg
KCl 3.5mg
CaCl ₂ 3.5 mg
MgSO ₄ 2.5mg
K ₂ HPO ₄ 267 mg
KH ₂ PO ₄ 33 mg

  The effectiveness test was performed by the batch type aeration method using the batch type aeration apparatus shown in FIG. That is, about 2 L of synthetic sewage is put into a batch type aeration apparatus, about 100 g of ACP carrier on which each female hormone substance-degrading bacterium is immobilized, 0.1 to 2 VVM air is sent from the air inlet, and a stirring blade The mixture was stirred at a low speed to prevent the carrier from precipitating. Table 5 shows the concentrations and decomposition rates of 17β-estradiol and estrone 8 hours after the start of aeration. Each microorganism immobilized on an ACP carrier showed the ability to degrade 80β or more of 17β-estradiol in 8 hours.

It is drawing which shows the apparatus used for continuous enrichment culture. It is drawing which shows the time-dependent change of 17 (beta) -estradiol. It is drawing which shows the time-dependent change of estrone. It is drawing which shows the time-dependent change of estriol. It is drawing which shows a time-dependent change of ethinyl estradiol. It is drawing which shows the molecular evolution phylogenetic analysis of the (Rhodococcus genus) microorganism of the present invention. It is drawing which shows the molecular evolution phylogenetic analysis of (Sphingomonas genus) microorganisms of the present invention. It is drawing which shows the apparatus used for batch type aeration culture | cultivation.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ...… Cultivation device 2…… Column 3…… Substrate introduction port 4…… Air introduction port 5…… Batch aeration device 6…… ACP carrier 7…… Synthetic sewage 8…… Stirring blade 9…… Air introduction port

more than

Claims (6)

Sphingomonas sp . C-I2 (FERM P-18838) , characterized by having a resolution of 17β-estradiol, estrone, estriol or ethinyl estradiol . When about 10 ⁸ to 10 ⁹ cells are cultured in 1 L of MDG medium (Modified DOMINIC &GRAHAM's medium) supplemented with 100 ppm of 17β-estradiol for 8 hours, they have the ability to decompose 80% or more of 17β-estradiol in the medium. Sphingomonas sp . C-I2 (FERM P-18838) according to claim 1, wherein the sphingomonas sp . Using the claim first term or the second term according Sphingomonas sp C-I2 (FERM P-18838 ), 17β- estradiol, estrone, characterized by decomposing the estriol or ethinyl estradiol 17.beta.-estradiol , Estrone, estriol or ethinyl estradiol decomposition method. 17β- estradiol in the waste water or soil, estrone, estriol or claim 3 claim of 17β- estradiol is to decompose ethinyl estradiol, estrone, estriol or decomposition method of ethinyl estradiol. 5. Decomposition of 17β-estradiol, estrone, estriol or ethinyl estradiol according to claim 3 or 4, wherein Sphingomonas sp. C-I2 (FERM P-18838) is adhered and grown on the surface of the solid support. Method. 17β- estradiol having means for contacting and drainage or soil Claim paragraph 1 or paragraph 2 wherein Sphingomonas sp C-I2 (FERM P-18838 ) means and the Sphingomonas sp C-I2 carrying the For the degradation of estrone, estriol or ethinyl estradiol .

Images