JP5165342B2 - New microorganism, selenium oxyanion adsorbent and method for removing selenium oxyanion - Google Patents

Description

  The present invention relates to a novel microorganism, a selenium oxyanion adsorbent containing the microorganism as an active ingredient, and a method for removing selenium oxyanion using the microorganism.

Selenium is known to be useful as a semiconductor material and a photosensitive material, and is also an element having high utility value in the industry, such as being used as a colorant and decoloring agent for glass.
On the other hand, although a trace amount of selenium is an essential element for the human body, it is known that it shows toxicity when ingested in excess of the necessary amount. Therefore, in order to prevent water pollution and soil contamination, it has been designated as an environmental standard.
Under such circumstances, in the industry as described above, there is a possibility that selenium may be mixed in factory effluent, and since selenium is also contained in coal in trace amounts, Therefore, the removal of selenium in the wastewater is an important issue.

Selenium, which is a problem in industrial wastewater, is selenic acid (H ₂ SeO ₄ ), which is a selenium (hexavalent) compound, or a salt thereof, and selenious acid (H ₂ SeO ₃ ), which is a selenium (tetravalent) compound. Or a salt thereof. Usually, these selenate compounds are in the form of selenate ions (SeO ₄ ²⁻ ) and only a part of them in the form of selenite (SeO ₃ ²⁻ ) ions in an aqueous solution such as factory effluent, that is, seleniumoxy. It is thought that it exists as an anion.

Incidentally, it is known that some microorganisms in the natural world adsorb these selenium oxyanions (see Patent Document 1). Specifically, by allowing a specific microorganism to coexist with a selenium oxyanion in a liquid, the microorganism adsorbs the selenium oxyanion to an extracellular polymer such as a polysaccharide or a lipid present on the cell surface. This phenomenon is known as bioadsorption (biosorption). By utilizing this phenomenon, it is considered that the selenium oxyanion can be removed from the liquid, and the use for removing other anion species is also expected.
Thus, it can be said that the method of removing a selenate compound from waste water using microorganisms is an excellent method with little environmental load.
JP 7-506006 A

However, a practical method has not yet been established for removing selenium oxyanions using conventional bioadsorption, including Patent Document 1.
The present invention has been made in view of the above circumstances, and includes a novel microorganism excellent in adsorption ability of selenium oxyanion, a selenium oxyanion adsorbent containing the microorganism as an active ingredient, and a method for removing selenium oxyanion using the microorganism. The issue is to provide.

To solve the above problem,
The invention described in claim 1 is Cryptococcus sp. JPCCY0075 strain (NITE P-414).
The invention according to claim 2 is a selenium oxyanion adsorbent comprising the microorganism according to claim 1 as an active ingredient.
The invention of claim 3 is a method for removing selenium oxyanions using microorganisms of claim 1.

  According to the present invention, selenium oxyanions in a sample can be efficiently removed.

The present invention will be described in detail below.
In the following, “Cryptococcus genus JPCCY0075 strain (NITE P-414)” may be abbreviated as “JPCCY0075”.
In the present invention, “selenium (hexavalent) oxyanion” means “selenate ion (SeO ₄ ²⁻ )”, and “selenium (tetravalent) oxyanion” means “selenite ( “SeO ₃ ²⁻ ) ion”. The “selenium oxyanion” refers to “selenium (hexavalent) oxyanion and / or selenium (tetravalent) oxyanion”.

<Acquisition of JPCCY0075>
A separation medium was prepared by the following procedure.
Separation medium: 5 g of yeast extract, 20 g of glucose, 10 g of peptone, 37 g of artificial seawater, 0.3 g of chloramphenicol, 0.15 g of streptomycin, 0.1 g of ampicillin and 12 g of powdered agar are added to 1 L of pure water, and 10 at 121 ° C. After sterilizing for a minute, an appropriate amount was dispensed onto a sterilized flat plate to prepare an agar flat plate.
Next, mud collected from a mangrove forest in the Nakagawa River on Iriomote Island (surface mud in the brackish water area) was diluted 100-fold with physiological saline, applied to a flat plate, inoculated, and incubated at 25 ° C. The colonies confirmed on the third day after the inoculation were fished with a sterilized toothpick, inoculated into a new yeast medium, and were singulated by repeating growth to obtain JPCCY0075.

<Identification of JPCCY0075>
JPCCY0075 was identified by entrusting Technosuruga Corporation. Then, using the obtained specimen, morphological properties, culture properties, and physiological properties were confirmed, and the base sequence of 18S rDNA was identified.

[Morphological properties, culture properties, physiological properties]
(Test method)
Morphological observation and biochemical property tests were conducted by the following methods.
1. Culture conditions A strain cultured under the following conditions was used as a test cell.
(1) medium;
2% Malt extract liquid medium (Kurtzman, 1998) (Becton Dickinson, MD, USA)
・ Yeast extract-Mal extract (YM) plate medium (Becton Dickinson, MD, USA)
・ Potato dextrose agar medium (PDA plate medium) “Digo” (Nippon Pharmaceutical, Tokyo)
2% Malt extract plate medium (Kurtzman, 1998)
1/5 Malt extract plate medium (Kurtzman, 1998)
(2) Culture temperature: 25 ° C
(3) Culture period: 2 days to 1 month Morphological Observation Macroscopic observation (colony observation) and microscopic observation (morphological property observation) were performed using the following.
(1) Microscope: Optical microscope BX50F4 (Olympus, Tokyo) (differential interference observation)
(2) Mount wave; sterilized distilled water Biochemical property test The following two items were tested, and the method was based on Yarrow (1998).
(1) Diazonium blue B (DBB) reaction (2) Urea hydrolysis test (urease activity)

(Test results)
The results are shown below.
(1) Macroscopic observation (colony observation)
The colonies showed the following properties on YM plate medium, PDA plate medium and 1/5 MA plate medium at 25 ° C./culture for 7 days, and on YM plate medium at 25 ° C./culture for 30 days. The images taken together are shown in FIG.
(A) YM plate medium (25 ° C., culture for 7 days)
・ Rear shape All edges ・ Rising state Flat and slightly central projection ・ Surface shape Smooth ・ Gloss and properties Glossy and wet ・ Color tone Cream (B) PDA flat plate medium (25 ° C., 7 days culture)
・ Rear shape Full edge ・ Rising state Flat and slightly semi-lens shape ・ Surface shape Smooth ・ Gloss and properties Glossy and wet ・ Color tone Cream (C) 1/5 MA flat plate medium (25 ° C., 7 days in culture)
-Peripheral shape Turbulent shape-Raised state Flatness-Surface shape Smooth-Glossy and properties Glossy and properties Glossy and moist in the center-Color tone Cream (D) YM flat plate medium (25 ° C, 30 days culture)
・ Rim shape Whole edge ・ Rising state Flat central projection ・ Surface shape Smooth, ring-shaped ・ Gloss and properties Glossy, butter-like, wet ・ Color tone Yellowish cream

(2) Microscopic observation (morphological property observation)
On the 5th day from the start of culture at 25 ° C. in a 2% Malt extract liquid medium, the vegetative cells are polymorphs including (3-7) × (2.8-3.5) μm of inverted pear shape, spherical to elliptical, etc. It was confirmed that the growth was due to extreme budding. A photographed image at this time is shown in FIG.
The fluctuation characteristics were also exhibited in the YM plate medium. A photographed image at this time is shown in FIG.
In 1/5 MA plate medium, vegetative cells showed polymorphism at 25 ° C./day 4 after the start of culture, but many spherical cells were observed. A photographed image at this time is shown in FIG.
Mycelia and pseudomycelia were confirmed on the YM plate medium at 25 ° C. and on the 6th to 10th day after the start of the culture. A photographed image at this time is shown in FIG.
In the 1/5 MA plate medium, mycelia in which racket-shaped cells were connected were confirmed at 25 ° C. and 4 days after the start of culture. A photographed image at this time is shown in FIG.
In addition, formation of sexual reproductive organs was not observed on the plate after one month of culture.

(3) Biochemical property test As a result of the diazonium blue B (DBB) reaction and urea hydrolysis test (urease activity), both were positive.

(4) Consideration JPCCY0075 is a creamy to yellowish, the peripheral part forms a turbulent colony from the whole edge, its vegetative cells are polymorphic, proliferate by budding, and hyphae and pseudohyphae Formed, and no formation of sexual reproductive organs was observed. These morphological features are described in the genus Cryptococcus described in “The Yeasts, a taxonomic study, 4th edition, Kurtzman and Fell, 1998” and “Yeasts, 3rd edition, Barnett et al., 2000”. (Hereinafter abbreviated as Cryptococcus).
In addition, the diazonium blue B (DBB) reaction is negative (pale yellow or colorless) in ascomycete yeast and positive (red) in basidiomycetous yeast, and the hydrolysis test of urea (urease activity) is It has already been reported that most of the Ascomycete yeasts are negative and positive in basidiomycete yeasts (Yarrow, 1998). JPCCY0075 is a diazonium blue B (DBB) reaction and urea hydrolysis test ( JPCCY0075 was determined to be a basidiomycete-based yeast since both showed positive results in urease activity.

(Test method)
A physiological property test was performed on the following items.
The test method is described in Barnett et al. (2000) and Kurtzman and Fell (1998), the culture was performed at 25 ° C. except for the temperature tolerance test. In the saccharide fermentability test, carbon source utilization test, and various resistance tests, the cells were cultured for up to 4 weeks, and the presence or absence of growth was confirmed.
Test items: saccharide fermentability test, carbon source utilization test, nitrogen source utilization test, vitamin requirement test, temperature tolerance test (30 ° C and 35 ° C), drug resistance test

(Test results)
The results are shown in Tables 1-5. The symbols in Tables 1 to 5 have the following meanings.
+ ... Reaction is positive -... Reaction is negative W ... Reaction is weak positive (weak)
S: Slowly positive reaction was observed over 2 to 3 weeks after the start of the test (slow)
L: A positive reaction was rapidly observed after 2 weeks from the start of the test (latent)
In addition, the culture | cultivation in a saccharide | sugar fermentation medium produced the film | membrane (ring) and precipitation (sediment).

(Discussion)
The physiological properties of JPCCY0075 were compared with the characteristics of the genus Cryptococcus described in “The Yeasts, a taxonomic study, 4th edition, Kurtzman and Fell, 1998”. As a result, as shown in Tables 1 to 5, JPCCY0075 shows urease activity and coloration of colonies by diazonium blue B (DBB), does not show saccharide fermentability, and these characteristics are consistent with those of the genus Cryptococcus did.
Further, among the known species of the genus Cryptococcus, it showed characteristics similar to Cryptococcus podzolicus. It showed a difference in growth from Cryptococcus podzolicus in that it showed moderate growth in 10% NaCl / 5% glucose medium and weak growth in vitamin-deficient medium.
From the above, JPCCY0075 was determined to be a new species belonging to the genus Cryptococcus.

(Base sequence)
As a result of identifying JPCCY0075 18S rDNA by a known method, it was confirmed to have the base sequence shown in SEQ ID NO: 1.

  The JPCCY0075 of the present invention is entrusted to the Patent Microorganism Depositary, National Institute of Technology and Evaluation, under the accession number NITE P-414 on September 25, 2007.

<New species>
The microorganism of the present invention is Cryptococcus sp. JPCCY0075. The present invention also relates to microorganisms belonging to the species to which the Cryptococcus genus JPCCY0075 belongs, and the base sequence of 18S rDNA has 96% or more homology with the base sequence shown in SEQ ID NO: 1, and has the ability to adsorb selenium oxyanions. It includes microorganisms belonging to the genus Cryptococcus. Such a microorganism is considered to be the same type of microorganism as the genus Cryptococcus JPCCY0075.

The microorganism of the present invention captures selenium oxyanion reversibly or irreversibly, and “selenium oxyanion adsorption ability” refers to such an ability to capture.
The microorganisms of the present invention have the ability to adsorb selenium oxyanions because selenium oxyanions are captured by extracellular macromolecules such as polysaccharides and lipids existing in the cell surface of the microorganisms, or are taken into cells. This is presumed to be due to
In addition, the microorganism of the present invention can adsorb selenium oxyanion even in a sample containing a high concentration of ionic species other than selenium oxyanion. In particular, selenium oxyanions can be adsorbed even in the presence of anions, and the ability to selectively adsorb selenium oxyanions is high.

The microorganism of the present invention is preferably used after being separated in the separation medium and then pre-cultured and main-cultured in a growth medium. The growth medium only needs to contain components suitable for growth, such as yeast extract, glucose, and peptone, and may be either a liquid medium or a solid medium, but a liquid medium is preferable. When using a liquid medium, it is preferable to culture by shaking or stirring, and more preferably to culture by shaking.
The medium used for the culture is preferably sterilized at 100 to 130 ° C. for 5 to 15 minutes, for example.
Moreover, you may make a culture medium contain another component in the range which does not impair the effect of this invention.
The culture conditions such as the pH of the medium, the culture temperature, the culture time, and the amount of inoculated microorganisms can be appropriately adjusted according to the purpose, but are generally as follows.
The pH of the medium is preferably 2.0 to 8.0, and more preferably 3.5 to 7.5, for example, if it is a liquid medium for growth.
The culture temperature is preferably 15 to 38 ° C, more preferably 20 to 30 ° C.
The culture time may be appropriately selected according to the growth situation, but is usually preferably 20 hours or longer, more preferably 30 hours or longer.
There is no particular limitation on the amount of inoculated microorganisms during culturing before adding to the sample.

<Selenium oxyanion adsorbent>
The selenium oxyanion adsorbent of the present invention contains the microorganism of the present invention as an active ingredient. And the microorganisms of this invention contained may be 1 type individual, and 2 or more types may be sufficient as them. In the case of two or more kinds, the combination and ratio can be arbitrarily selected according to the purpose.

As the selenium oxyanion adsorbent, for example, a culture solution obtained by culturing the microorganism of the present invention may be used as it is, or a filtrate obtained by filtering the culture solution may be used. You may dry and use a thing. Or what refine | purified the microorganisms of this invention after culture | cultivation may be used. What is necessary is just to dry by well-known methods, such as ventilation drying and freeze-drying, when drying.
As described above, in the selenium oxyanion adsorbent of the present invention, not only living microorganisms (viable bacteria) but also those obtained by drying (drying cells) or purifying the microorganisms of the present invention may be used. Yes, it does not matter whether microorganisms that are active ingredients are alive or dead.

  Various additives may be added to the selenium oxyanion adsorbent as needed within a range not impeding the effects of the present invention. Furthermore, the microorganisms of the present invention are used by immobilizing them on, for example, natural polymer gels such as agar and gellan gum; acrylamide; polymer resins such as ultraviolet curable resins; fibers such as carbon fibers, hollow film, and nonwoven fabrics; Also good. Immobilization may be either internal encapsulation or surface fixation.

  The content of the microorganism of the present invention in the selenium oxyanion adsorbent is not particularly limited, and the form of the sample to be added, the selenium oxyanion content in the sample, the form of the selenium oxyanion adsorbent It can be appropriately selected according to the like. Usually, the higher the content of the microorganism of the present invention, the more preferable is the point that the adsorptive power of selenium oxyanion increases.

<Method for removing selenium oxyanion>
The method for removing selenium oxyanion of the present invention uses the microorganism of the present invention.
Since the microorganism of the present invention has the ability to adsorb selenium oxyanion, the selenium oxyanion in the sample is adsorbed by the microorganism by adding the microorganism to the sample containing selenium oxyanion. Thereafter, the seleniumoxyanion can be removed from the sample by separating the microorganism adsorbed with the seleniumoxyanion from the sample.

Here, the sample is not particularly limited as long as it contains selenium oxyanion, and specific examples thereof include drainage, soil, sludge, ground water, and water in a reservoir. Among them, those having a high water content are preferable.
In a sample, selenic acid (H ₂ SeO ₄ ) which is a selenium (hexavalent) compound or a salt thereof, or selenious acid (H ₂ SeO ₄ ) which is a selenium (tetravalent) compound or a salt thereof (hereinafter, When these are abbreviated as selenate compounds), some of them become free selenium oxyanions in the presence of water and are adsorbed by the microorganism of the present invention. If the amount of free selenium oxyanion in the sample decreases, it is estimated that a part of the remaining selenate compound further becomes free selenium oxyanion and is similarly adsorbed by the microorganism of the present invention. The amount of free selenate compound in the sample can also be reduced.

What is necessary is just to add the selenium oxyanion adsorbent of this invention when adding microorganisms to a sample.
The addition amount of the microorganism is not particularly limited, and can be appropriately selected according to the form of the sample to be added, the selenium oxyanion content of the sample, the form of the selenium oxyanion adsorbent, and the like. For example, when adding viable bacteria to an aqueous solution containing 10 ppm or less of selenium oxyanion, the concentration (cell concentration) of the microorganism after addition is 1.0 × 10 ^{7 to} 1.0 × 10 ¹¹ cells / mL. It is preferable to add so that it may become, and when adding a dry microbial cell, it is preferable to add so that the density | concentration of the microorganisms after addition may be set to 1-100 mg / mL.

  As described in the explanation of the selenium oxyanion adsorbent, the microorganism to be added may be either a living state or a non-viable state. However, in a non-viable state, for example, those in which the cell structure, particularly the cell surface structure is not significantly impaired, are preferable. What was processed can be illustrated.

  When a living microorganism is added, the composition of the sample is preferably adjusted so that the microorganism can grow in the sample after the addition. For this purpose, the composition of the sample may be adjusted with reference to the microorganism acquisition conditions and culture conditions described above. For example, a medium prepared by appropriately adding the above-mentioned components suitable for the growth of the microorganism of the present invention to the sample Otherwise, it is better to culture with reference to the culture conditions described above. Such a method can be applied even if the selenium oxyanion removal target is, for example, drainage, soil, sludge, ground water, water in a reservoir, or the like.

After adding the microorganism, it is preferable to mix the sample. By uniformly dispersing the microorganisms in the sample, the adsorption efficiency of the selenium oxyanion can be increased. The mixing method of the sample can be arbitrarily selected from known methods such as shaking and stirring, but shaking is preferable.
Various conditions such as the temperature of the sample after addition of the microorganism, the mixing method, and the mixing time may be appropriately selected according to the purpose. For example, when removing the selenium oxyanion in an aqueous solution, the same conditions as when the microorganism is cultured in a medium can be applied regardless of whether the microorganism is in a living state or a non-viable state.

  The method for separating the microorganisms adsorbed with selenium oxyanion from the sample can be arbitrarily selected from known solid-liquid separation methods depending on the state of the sample at the time of adsorption. Specifically, for example, filtration may be performed using a filter, or solid-liquid separation may be performed using a centrifuge. Before solid-liquid separation, pretreatment such as dilution and washing may be performed as necessary.

  Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.

[Example 1]
<Removal of selenium (hexavalent) oxyanion in pure water using viable JPCCY0075>
(Preparation of bacterial cells)
Dispense 10 mL of growth yeast medium consisting of 5 g of yeast extract, 20 g of glucose, 10 g of peptone and 1 L of pure water into an L-shaped test tube, inoculate the obtained JPCCY0075 with a platinum wire, and pre-culture at 25 ° C. for 2 days. Went. Next, 5 mL of the obtained preculture was dispensed and inoculated into a 1 L Erlenmeyer flask into which 250 mL of the yeast for growth was dispensed. Then, after shaking culture at 150 rpm for 2 days at 25 ° C., centrifugation was performed using a centrifuge at 8000 rpm for 10 minutes to recover viable bacteria of JPCCY0075.

(Removal of selenium (hexavalent) oxyanion in pure water)
High purity sodium selenate was mixed with pure water to adjust the final concentration of selenium (hexavalent) oxyanion to 2.1 ppm, and then sterilized by filter. Next, in 10 mL of the obtained sterilized solution, the collected JPCCY0075 cells were 2.9 × 10 ⁹ cells, 1.4 × 10 ¹⁰ cells, 2.3 × 10 ¹⁰ cells, 2.9 × 10 ¹⁰ cells. The cells were mixed to form cells and incubated with shaking for 24 hours. Twenty-four hours later, the mixture was centrifuged at 8000 rpm for 10 minutes using a centrifuge to collect the total amount of cells, and the supernatant was filtered through a filter and nitric acid was added. Valence) Oxyanion was used for measurement.

(Measurement result)
The measurement results are shown in FIG. The vertical axis of the graph shown in FIG. 7 indicates the concentration of selenium (hexavalent) oxyanion in the supernatant. As is clear from FIG. 7, it was confirmed that the removal amount of selenium (hexavalent) oxyanion increases as the number of cells increases, and in the number of cells of 2.3 × 10 ¹⁰ cells or more, selenium (hexavalent) ) The concentration of oxyanion became 0.1 ppm or less. From this, it was confirmed that selenium (hexavalent) oxyanion can be removed by adsorption with viable JPCCY0075.

[Example 2]
<Removal of selenium (hexavalent) oxyanion in pure water using dried JPCCY0075 cells>
(Preparation of bacterial cells)
Dispense 10 mL of growth yeast medium consisting of 5 g of yeast extract, 20 g of glucose, 10 g of peptone and 1 L of pure water into an L-shaped test tube, inoculate the obtained JPCCY0075 with a platinum wire, and pre-culture at 25 ° C. for 2 days. Went. Subsequently, 5 mL of the obtained preculture solution was dispensed and inoculated into a 1 L Erlenmeyer flask into which 250 mL of the yeast for growth was dispensed. Then, after culturing at 25 ° C. for 2 days, the mixture was centrifuged using a centrifuge at 8000 rpm for 10 minutes, and the total amount of JPCCY0075 cells was recovered. Next, the collected cells were stored frozen at −80 ° C., dried in a freeze dryer, and crushed in a mortar to obtain dried cells.

(Removal of selenium (hexavalent) oxyanion in pure water)
High purity sodium selenate was mixed with pure water to adjust the final concentration of selenium (hexavalent) oxyanion to 2.1 ppm, and then sterilized by filter. Next, 10 mL of the obtained sterilizing solution was mixed with 0.1 g, 0.3 g, and 0.5 g of the dried cells, and the whole amount was transferred to an L-shaped test tube, and cultured with shaking on a shaker for 24 hours. Went. Twenty-four hours later, the mixture was centrifuged at 8000 rpm for 10 minutes using a centrifuge to collect the total amount of cells, and the supernatant was filtered through a filter and nitric acid was added. Valence) Oxyanion was used for measurement.

(Measurement result)
The measurement results are shown in FIG. The vertical axis of the graph shown in FIG. 8 indicates the concentration of selenium (hexavalent) oxyanion in the supernatant. As is clear from FIG. 8, even when dry cells were used, it was confirmed that the selenate concentration in the solution decreased, and when the dry cell addition amount was 0.5 g, which was the largest, about 1.3 ppm. Selenium (hexavalent) oxyanion was removed. From this, it was confirmed that the selenium (hexavalent) oxyanion can be removed by adsorption with the dry cells of JPCCY0075.

[Example 3]
<Removal of selenium (hexavalent) oxyanion in actual waste water using viable JPCCY0075 (1)>
(Preparation of bacterial cells)
Dispense 10 mL of growth yeast medium consisting of 5 g of yeast extract, 20 g of glucose, 10 g of peptone and 1 L of pure water into an L-shaped test tube, inoculate the obtained JPCCY0075 with a platinum wire, and pre-culture at 25 ° C. for 2 days. Went. Subsequently, 5 mL of the obtained preculture solution was dispensed and inoculated into a 1 L Erlenmeyer flask into which 250 mL of the yeast for growth was dispensed. And after culture | cultivating for 2 days at 25 degreeC, the quantity required for a measurement was fractionated, it centrifuged on condition of 8000 rpm and 10 minutes using the centrifuge, and the living microbe of JPCCY0075 was collect | recovered.

(Removal of selenium (hexavalent) oxyanion in actual waste water)
High-purity sodium selenate is mixed into the nitrification tank drainage of the power plant, the final concentration of selenium (hexavalent) oxyanion is adjusted to 2.3 ppm, and the pH is adjusted to 4 with 5N hydrochloric acid, followed by filter sterilization. did. Then, 10 mL of the obtained sterilized solution was collected from the collected JPCCY0075 cells 2.0 × 10 ⁹ cells, 6.0 × 10 ⁹ cells, 1.0 × 10 ¹⁰ cells, 1.6 × 10 ¹⁰ cells, 2. Each was mixed in 0 × 10 ¹⁰ cells, transferred to an L-shaped test tube, and cultured with shaking on a shaker for 24 hours. Twenty-four hours later, the mixture was centrifuged at 8000 rpm for 10 minutes using a centrifuge to collect the total amount of cells, and the supernatant was filtered through a filter and nitric acid was added. Valence) Oxyanion was used for measurement.

(Measurement result)
The measurement results are shown in FIG. The vertical axis of the graph shown in FIG. 9 indicates the concentration of selenium (hexavalent) oxyanion in the supernatant. As is clear from FIG. 9, selenium (hexavalent) oxyanion was confirmed to be able to be removed even in actual waste water, and the removal effect was highest in the number of cells of 1.6 × 10 ¹⁰ cells, and selenium (hexavalent) oxyanion. Concentration was 1.7 ppm, and the amount of selenium (hexavalent) oxyanion removed was 0.5 mg. Although the removal amount is smaller than that in the case of pure water, it is possible to remove all the amount of selenium (hexavalent) oxyanion in the actual waste water by performing the removal in multiple stages and increasing the number of removal treatments.
In addition, the used nitrification tank drainage contains about 1500 ppm of calcium ions, about 4000 ppm of chloride ions, about 2500 ppm of sodium ions and about 1500 ppm of sulfate ions. It was confirmed that selenium oxyanions can be removed even in the presence of chloride ions and sulfate ions, which are contained in high concentrations and are anions.

[Example 4]
<Removal of selenium (tetravalent) oxyanion in pure water using JPCCY0075 live bacteria>
(Preparation of bacterial cells)
Dispense 10 mL of growth yeast medium consisting of 5 g of yeast extract, 20 g of glucose, 10 g of peptone and 1 L of pure water into an L-shaped test tube, inoculate the obtained JPCCY0075 with a platinum wire, and pre-culture at 25 ° C. for 2 days. Went. Subsequently, 5 mL of the obtained preculture solution was dispensed and inoculated into a 1 L Erlenmeyer flask into which 250 mL of the yeast for growth was dispensed. Then, after shaking culture at 150 rpm for 2 days at 25 ° C., centrifugation was performed using a centrifuge at 8000 rpm for 10 minutes to recover viable bacteria of JPCCY0075.

(Removal of selenium (tetravalent) oxyanion in pure water)
High purity sodium selenite was mixed with pure water to adjust the final concentration of selenium (tetravalent) oxyanion to 1.6 ppm, and then sterilized by filter. Next, in 10 mL of the obtained sterilizing solution, the collected cells of JPCCY0075 are 2.0 × 10 ⁹ cells, 1.0 × 10 ¹⁰ cells, 1.6 × 10 ¹⁰ cells, and 2.0 × 10 ¹⁰ cells. And incubated for 24 hours with shaking. Twenty-four hours later, the mixture was centrifuged at 8000 rpm for 10 minutes using a centrifuge to collect the whole amount of cells, and the supernatant was filtered through a filter and nitric acid was added. Valence) Oxyanion was used for measurement.

(Measurement result)
The measurement results are shown in FIG. The vertical axis of the graph shown in FIG. 10 indicates the concentration of selenium (tetravalent) oxyanion in the supernatant. As is apparent from FIG. 10, 1.5 ppm or more of selenium (tetravalent) oxyanion was removed at a cell number of 2.0 × 10 ⁹ cells, and selenium (tetravalent) was also obtained at a cell number of more than that. The oxyanion concentration was 0.1 ppm or less. From this, it was confirmed that the selenium (tetravalent) oxyanion can be efficiently removed by adsorption by viable bacteria of JPCCY0075.

[Example 5]
<Removal of selenium (tetravalent) oxyanion in actual waste water using viable JPCCY0075>
(Preparation of bacterial cells)
Dispense 10 mL of growth yeast medium consisting of 5 g of yeast extract, 20 g of glucose, 10 g of peptone and 1 L of pure water into an L-shaped test tube, inoculate the obtained JPCCY0075 with a platinum wire, inoculate it at 25 ° C. Pre-culture was performed for days. Subsequently, 5 mL of the obtained preculture solution was dispensed and inoculated into a 1 L Erlenmeyer flask into which 250 mL of the yeast for growth was dispensed. Then, after culturing at 25 ° C. for 2 days, centrifugation was performed using a centrifuge at 8000 rpm for 10 minutes, and viable JPCCY0075 was collected.

(Removal of selenium (tetravalent) oxyanion in actual waste water)
After mixing high-purity sodium selenite into the power plant nitrification tank wastewater, the final concentration of selenium (tetravalent) oxyanion is adjusted to 2.2 ppm, and the pH is adjusted to 4 with 5N hydrochloric acid, and then the filter Sterilized. Next, in 10 mL of the obtained sterilizing solution, the collected cells of JPCCY0075 are 2.0 × 10 ⁹ cells, 1.0 × 10 ¹⁰ cells, 1.6 × 10 ¹⁰ cells, and 2.0 × 10 ¹⁰ cells. And incubated for 24 hours with shaking. Twenty-four hours later, the mixture was centrifuged at 8000 rpm for 10 minutes using a centrifuge to collect the whole amount of cells, and the supernatant was filtered through a filter and nitric acid was added. Valence) Oxyanion was used for measurement.

(Measurement result)
The measurement results are shown in FIG. The vertical axis of the graph shown in FIG. 11 indicates the concentration of selenium (tetravalent) oxyanion in the supernatant. As is clear from FIG. 11, it was confirmed that 2.1 ppm of selenium (tetravalent) oxyanion can be removed with 2.0 × 10 ⁹ cells even in actual waste water. It was confirmed that the concentration of selenium (tetravalent) oxyanion was 0.1 ppm or less even when the number of cells was more than that. From this, it was shown that selenium (tetravalent) oxyanion can be removed by adsorption at a level equivalent to that in pure water by virtue of JPCCY0075.
Moreover, the nitrification tank waste water used is the same as that used in Example 3. Therefore, it contains ionic species other than selenium oxyanion at a high concentration, and selenium oxyanion can be removed even in the presence of anions. Was confirmed.

  The present invention can be used to remove selenate compounds in waste water, soil, and sludge.

It is a photographed image of a colony of JPCCY0075, (a) is a YM plate medium (25 ° C., 7 days of culture), (b) is a PDA plate medium (25 ° C., 7 days of culture), (c) is a 1/5 MA plate medium. (D) It is a photographed image of colonies when cultured in a YM plate medium (25 ° C., culture for 30 days). It is a photographed image (1500 times) of vegetative cells on a 2% Malt extract liquid medium (25 ° C., culture for 5 days). It is the picked-up image (1500 times) of a vegetative cell on a YM flat plate culture medium (25 degreeC and culture | cultivation 4 days). It is the picked-up image (1500 times) of the vegetative cell on 1 / 5MA flat plate culture medium (25 degreeC and culture | cultivation 4 days). It is a photographed image of a vegetative cell on a YM plate medium, (a) is a photographed image (600 times) at 25 ° C./culture for 10 days, and (b) is a photographed image (1500 times) at 25 ° C./culture for 4 days. . It is the picked-up image (1500 times) of the mycelium on 1 / 5MA flat plate culture medium (25 degreeC and culture | cultivation 4 days). 2 is a graph showing the removal activity of selenium (hexavalent) oxyanion in Example 1. 4 is a graph showing the removal activity of selenium (hexavalent) oxyanion in Example 2. 4 is a graph showing the removal activity of selenium (hexavalent) oxyanion in Example 3. 4 is a graph showing the removal activity of selenium (tetravalent) oxyanion in Example 4. 6 is a graph showing the removal activity of selenium (tetravalent) oxyanion in Example 5.

Claims (3)

  Cryptococcus genus JPCCY0075 strain (NITE P-414). A selenium oxyanion adsorbent comprising the microorganism according to claim 1 as an active ingredient. The removal method of the selenium oxyanion using the microorganisms of Claim 1 .

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