JPH0998773A - Rare earth element accumulating microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new microorganism belonging to the genus Methylobacterium and capable of accumulating rare earth elements, especially ytterbium belonging to heavy rare earth elements. SOLUTION: This microorganism is capable of accumulating rare earth elements and belongs to the genus Methylobacterium, e.g. Methylobacterium fujisawaensis 3-1 strain (FERM P-15211). The microorganism can be produced by adding agar to a diluted broth medium having a nutrient content of 1/100, sterilizing under reduced pressure, culturing an aqueous specimen in the obtained agar medium for primary screening to obtain oligotrophic bacteria, culturing the bacteria in a liquid medium for secondary screening having an ytterbium(Yb) concentration of 5ppm and selecting a strain capable of decreasing the Yb concentration in the culture liquid. The cultivation of the microorganism is preferably carried out under aerobic condition at 25-40 deg.C and pH6-8. It is preferable to add a rare earth element to the medium for accumulating rare earth elements in the microorganism in high efficiency.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel rare earth element-accumulating microorganism. More specifically, the present invention relates to a rare earth element-accumulating microorganism capable of efficiently accumulating a specific rare earth element.

[0002]

2. Description of the Related Art Rare earth elements are the largest group of elements existing in nature in the periodic table, and include 15 elements (lanthanoids) from lanthanum (La) with atomic number 57 to lutetium (Lu) with atomic number 71 to scandium. (Sc) and yttrium (Y) are a group of 17 elements, and are magnetic properties based on the behavior of 4f electrons in the incompletely packed state of ions, optical properties such as color, and rare earth-specific properties. It is used in a wide variety of fields due to its chemical nature. Specifically, as the one utilizing the property of the 4f electron, a red fluorescent substance (europium) of a cathode ray tube of a color television receiver and a magnet (samarium or neodymium) in a small electronic device such as a quartz watch or a walkman, Examples of those utilizing chemical properties include catalysts (lanthanum and cerium) used in the production of gasoline, high-temperature oxide superconductors, hydrogen storage alloys, ceramics, and reactor control materials.

Further, these rare earth elements are monazite.
It is contained in minerals such as (monazite), bastnaesite, zenotime, euxenite and gadolinite. Of these, monazite and bastnasite are mainly used as resources for light rare earth (lanthanum to europium), and xenotime is decomposed for heavy rare earth (added yttrium from gadolinium to lutetium) on an industrial scale. By refining, each rare earth element can be obtained as a resource, but since the rare earth elements are very similar in physical and chemical properties to each other, they exist in the form of coexisting in minerals. However, there is a problem that the mutual separation of the Therefore, the conventional fractional crystallization method or fractional precipitation method, which is a method for separating rare earth elements, requires repeated operations because the difference in solubility between elements is small, and mutual separation is a very difficult task. It was Therefore, in order to solve this drawback, an ion exchange method has been developed in which a rare earth element is adsorbed on a column packed with an ion exchange resin and a complexing agent is used as an eluent therethrough. The method is not suitable as a method for large-scale processing because continuous processing cannot be performed and it takes a long time to perform one operation, and the cost of resin regeneration and replacement is high. Then, what came out instead of this method is a solvent extraction method that utilizes the difference in the method of dissolving ions in solvents that do not mix with each other. However, this method has a problem that it is difficult to obtain a high-purity product in one operation, and the operation must be repeated many times in order to increase the purity. Therefore, industrially, a countercurrent multistage extraction device capable of continuously performing several tens of operations has been developed and used,
Compared with the ion exchange method, it has advantages such as a large amount of treatment, a short operation time, and an easy operation, and it is the mainstream of the present mass separation method for rare earth elements.

In addition to the above method, a method for obtaining a microorganism capable of selectively capturing a specific rare earth element and obtaining the desired rare earth element from the microorganism has been developed and proposed. Examples of such microorganisms include Japanese Patent Laid-Open No. 59-11.
There are microorganisms described in No. 8,825. However, the above-mentioned publication only discloses a method for selectively collecting yttrium using activated sludge, and has not reached the point where a microorganism is specifically specified.

[0005] As described above, the specified microorganism that efficiently accumulates rare earth elements is still under development, and the one actually used industrially does not exist yet. Is desired.

[0006]

Therefore, an object of the present invention is to provide a microorganism capable of efficiently accumulating rare earth elements.

[0007]

The above objects are achieved by a rare earth element-accumulating microorganism belonging to the genus Methylobacterium capable of accumulating rare earth elements.

The present invention shows a rare earth element-accumulating microorganism that efficiently accumulates ytterbium among rare earth elements. The present invention also shows a rare-earth element-accumulating microorganism in which the above-mentioned microorganism is Methylobacterium fusawaens. The present invention further has a deposit number of FERM.
It shows a rare earth element-accumulating microorganism, which is P-15211.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism according to the present invention belongs to the genus Methylobacterium and is characterized by being able to efficiently accumulate rare earth elements. Specific examples of this microorganism are: , Methylobacterium fujisawaen
se) 3-1 strain, and this strain is obtained by the following screening method. In the following screening method, ytterbium (Yb) having divalent and trivalent valences was used as a rare earth element for screening.

The screening is divided into two stages based on the assumption that the target strain having the ability to accumulate rare earths in the microbial group that can actively grow in an undernourished state is likely to exist, that is, After conducting a search for oligotrophic bacteria as a primary screen, a strain having the ability to accumulate ytterbium (Yb) from these bacteria, that is, a strain that reduces the Yb concentration in the culture solution was selected as a secondary screen. The screening method used in the present invention will be specifically described below.

A water-based sample collected from Lake Hamana with sterile water
Appropriately diluted 0 to 10 ³ times, 100 μl each was applied to a primary screening agar medium (pH: 7.2 to 7.4) prepared by the following method with a conradi stick, and this agar medium was heated at 30 ° C. The cells were cultured for 3 to 5 days. Next, the colonies formed on the agar medium were harvested, suspended in 10 ml of sterilized water, and further diluted 10 ² to 10 ³ times,
100 μl of each was applied to the same agar medium, and this operation was repeated several times to purify the strain. By such a primary screening, a bacterium capable of growing even in a state of having a low nutrient was obtained. Method of preparing agar medium for primary screening: 1/100 nutrient nutrient broth medium (polypeptone 0.01%, meat extract 0.01%, NaCl 0.005%, pH
7.2-7.4) (hereinafter referred to as a diluted broth medium) was prepared, and 1.5% (wt / v) of agar was added thereto and subjected to high-pressure sterilization, and about 30 ml each was added to a sterilized petri dish. Dispensed into a plate medium (hereinafter referred to as diluted broth agar medium).

Next, 5 ml of the sterilized liquid medium for secondary screening prepared by the following method was dispensed into test tubes which had been pre-sterilized under high pressure with a 5 ml aliquot. Each of the isolated bacterial cells purified by the above-mentioned primary screening was inoculated into this with one platinum loop, and cultured at 30 ° C. for 10 days with shaking. After culturing, measure the concentration of ytterbium (Yb) remaining in the culture supernatant according to the following method, select one that can be clearly determined by visual observation that the Yb concentration is decreasing, and further measure the absorbance of this sample. A bacterium in which the Yb concentration in the culture supernatant was reduced by 50% or more from the initial concentration (5 ppm) was selected as a candidate bacterium. At this time, the selected strain was inoculated again into the secondary screening medium and the same operation was repeated to confirm the reproducibility regarding the decrease in Yb concentration. In this way, strains that efficiently accumulate rare earth elements were screened. Preparation method of secondary screening medium: broth (polypeptone 1%, meat extract 1%, NaCl 0.5
%, PH 7.2-7.4) diluted 100 times with demineralized water,
A 5% (v / v) ytterbium nitrate [Yb (NO ₃ ) ₃ ] solution (1 mol / liter) was added to this diluted medium in an amount [0.1% (v / v)] equivalent to that for neutralization. A 5N sodium hydroxide solution was added in advance, and while stirring this solution, Yb of 1,000 ppm was added so that the pH was about 7.2.
(NO ₃ ) ₃ solution was gradually added dropwise to make 1 liter with demineralized water, and sterilized by filtration with a sterilized filter made of polysulfone having a pore size of 0.2 μm, and this was used as a secondary screening medium (Yb concentration: 5 ppm) ( Hereinafter referred to as Yb-containing medium). The composition of this Yb-containing medium is shown in Table 1.

[0013]

[Table 1]

The method for measuring the concentration of ytterbium (Yb) remaining in the culture supernatant will be described below. Approximately 1.4 ml of the culture solution sample after inoculating and culturing the strain to be screened was placed in an Eppendorf centrifuge tube and centrifuged (4 ° C., 12,000 rpm × 10 minutes) to obtain 1 ml of the culture supernatant for a new test. Put in a tube.
In addition, a 0.1% Arsenazo III solution prepared in advance and a hydrochloric acid / potassium chloride buffer solution (25 ml 0.2 M KCl, 59 ml) were added to the test tube.
Mix 0.2 M HCl and 16 ml demineralized water to 10
0.5 ml each of a solution having a fixed volume of 0 ml) was added, and finally 1 ml of pure water was added and well stirred by vortex. After leaving this mixed solution in the dark for 30 to 60 minutes, the absorbance of the sample solution at 655 nm was measured, and the concentration of ytterbium (Yb) contained in each sample was measured based on the standard straight line prepared as follows. I asked.
Standard straight lines are 0, 1, 2, 3, 4, 5 μl in test tubes
A Yb (NO ₃ ) ₃ solution was added thereto, and 1 ml of pure water was added thereto as a sample, and the same colorimetric operation method as described above was performed except that centrifugation was not performed. At this time, as the Yb (NO ₃ ) ₃ solution,
Ytterbium nitrate solution for atomic absorption [Yb (NO ₃ ) ₃ concentration in nitric acid solution of 1 mol / liter = 1.0 m
gYb / ml] (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

By the above screening, one strain was obtained which markedly reduced the ytterbium concentration in the Yb-containing medium as compared with other strains. The bacteriological properties of this strain are shown below.

(A) Morphology 1) Shape and size of cells: long bacilli with a length of 1.0 to
2.0 μm, diameter 3.0-4.0 μm 2) Presence or absence of cell polymorphism: None 3) Presence or absence of motility: Yes 4) Presence of spores: None (b) Culture properties 1) Meat agar plate culture (30 ° C.): Colonies are round, opaque pink and convex, and the surface is smooth and shiny. The size of the colony is about 2 mm in diameter when cultivated at 30 ° C. for 5 days, and in the diluted broth agar medium,
When cultured at 30 ° C. for 5 days, it has a diameter of about 1 mm. 2) Bennett's agar (37 ° C): + Bennett's agar (41 ° C): Weak + Bennett's agar (45 ° C):-The composition of Bennett's agar is:
Yeast extract 0.1%, beef extract 0.1%, NZ amine A 0.2%, glucose 1.0%, agar 2.0% (pH
7.3).

(C) Physiological properties (30 ° C., 14 days) 1) Gram stainability: −2) Catalase: +3) Oxidase: −4) OF test: −5) Attitude toward oxygen: Aerobic ( d) Other physiological properties 1) Use of methanol: +2) Use of glucose: -3) Use of fructose: +4) Use of acetate: +5) Use of betaine: -6) Use of methylamine: -7) Utilization of N, N-dimethylformamide: -8) Utilization of tetramethylammonium hydroxide: -9) Utilization of trimethylamine: -10) Utilization of aspartate: Slightly +11) Utilization of glutamate: + 12) Utilization of fucose: + 13) Utilization of arabinose: + 14) Utilization of citric acid: + This bacterium is Vergiz Manual of Systema Ikku Bact (Bergey's Manual of System
atic Bacteriology 8th) was not possible to classify it alone.
Of Industrial and Marine Bacteria Limited (NCIMB) (The National Collecti
ons of Industrial and Marine Bacteria Limited) (reference number: ID 2660 / NCID 4295,
Date: Strain 3 in the report document of March 23, 1995
-1) Methylobacterium Fujisawa ence (Methylobact) which has an atypical negative glucose reaction
erium fujisawaense) [Reference: Gram-negative Methylotrophic Ros and Cockey]
ds and Cocci), PN Green, Identification Methods in Applied and Environ Microbiology
mental Microbiology) 1992, RG Board
(RG Board), De Jones (D. Jones) and FA Skinner (FA Skinner), Society for Applied Bacteriology Technical Series (S
ociety for Applied Bacteriology Technical Series)
29, Blackwell Scientific (Blackwe
ll Scientific)].

Until now, there has been no report that Methylobacterium fujisawaense accumulates rare earth elements, and since this bacterium is clearly distinguished from known species, the microorganism of the present invention is a novel microorganism. This strain was designated as Methylobacterium fujisawaense 3-1 strain (hereinafter simply referred to as 3-1 strain). Also, this 3
-1 strain was deposited at the Institute of Biotechnology, Institute of Biotechnology, October 2, 1995, and its deposit number is FERM.
It is P-15211.

Although the strain 3-1 was used in the present invention, the strain is not limited to this strain and any microorganism belonging to the genus Methylobacterium capable of accumulating rare earth elements can be used.

The medium used for culturing the strain of the present invention may be either a solid medium or a liquid medium, and contains a carbon source which can be assimilated by the bacterium used, an appropriate amount of nitrogen source, inorganic salts and other nutrients. The medium may be either a synthetic medium or a natural medium.

The carbon source that can be used for culturing the strain of the present invention is not particularly limited as long as the strain used can be assimilated. Specifically, considering the assimilation of microorganisms, fructose, maltose, galactose, starch, starch hydrolysates, molasses, sugars such as molasses, meat extracts, natural products such as peptone, wheat and rice, glycerol. , Alcohols such as methanol and ethanol, fatty acids such as acetic acid, gluconic acid, pyrupic acid and citric acid,
One or more amino acids such as glycine, glutamic acid and aspartic acid can be selected and used.

Nitrogen sources that can be used in the culture of the strain of the present invention include meat extract, peptone, polypeptone, yeast extract, soybean hydrolyzate, soybean powder, milk casein, casamino acids, various amino acids, corn steep liquor, and others. Consider the assimilability of microorganisms to be used from organic nitrogen compounds such as hydrolysates of animals, plants and microorganisms, ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate and ammonium chloride, nitrates such as sodium nitrate, inorganic nitrogen compounds such as urea Then, one kind or two or more kinds are selected and used.

The inorganic salt which can be used in the present invention is one selected from phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc, hydrochlorides, sulfates and acetates. Two or more can be used. Moreover, you may add vegetable oil, surfactant, etc. to a culture medium as needed.

In order to efficiently accumulate the rare earth element in the microorganism of the present invention, it is preferable to add the rare earth element to the medium. The type and amount of added rare earth element depends on the type of rare earth source in which the microorganism used is likely to accumulate, but for example, when using strain 3-1 ytterbium, lanthanum, neodymium, terbium and thulium, It is desirable to add at a concentration of 1 to 20 ppm, more preferably 2 to 10 ppm. At this time, the rare earth element to be added may be a mixture or alloy of two or more kinds.

In the present invention, the culturing is carried out under aerobic conditions because the bacterium of the present invention is aerobic, and the culturing conditions therefor are appropriately selected depending on the composition of the medium and the culturing method.
There is no particular limitation as long as the strain can grow. Usually, the culture temperature is 20 to 70 ° C., preferably 25 to 4
It is 0 ° C., and the pH of the medium suitable for culture is 5 to
9, preferably 6-8.

In the present invention, as a method for separating and purifying rare earth elements from microorganisms, after culturing under the above-mentioned culturing conditions, the cells are collected by filtration or centrifugation, for example, freeze-thaw treatment. , Ultrasonic treatment, pressure treatment,
Osmotic pressure difference treatment, physical means such as grinding treatment, or cell wall lysing enzyme treatment such as lysozyme or chemical treatment such as contact treatment with a surfactant is performed alone or in combination to crush the cells, and the crushed cells Examples thereof include a method of purifying a target rare earth element from the body by using known methods such as a fractional crystallization method, a fractional precipitation method, an ion exchange chromatography method and a solvent extraction method, alone or in combination. As a method for purifying rare earth elements from crushed cells,
From the viewpoint of labor, time, and cost, a solvent extraction method, particularly a countercurrent multistage extraction method in which solvent extraction operations are continuously performed, is preferably used.

The microorganism of the present invention has the property of efficiently accumulating rare earth elements, but it has hitherto been the genus Methylobacterium.
There was no report on a microorganism belonging to (Methylobacterium) and having the above characteristics.

[0028]

The present invention will be described more specifically with reference to the following examples.

Example 1 The following experiment was carried out to observe the change with time of the Yb concentration by the strain 3-1 of the present invention.

Yb-containing medium sterilized by filtration through a 0.2 μm polysulfone sterilized filter (Yb concentration: 5 pp
m) 1 platinum loop of 3-1 strain was inoculated into 5 ml of the slant medium, and precultured by shaking culture at 30 ° C. for 24 hours. 5 ml of the preculture liquid thus obtained was
In the same manner, put into a 500 ml side branch Sakaguchi flask containing 95 ml of Yb-containing medium that has been sterilized by filtration.
Main culture was performed for one day. During this culture period, 3 ml of the culture solution was collected every 24 hours, and the collected culture solution was
At the same time as measuring the Yb concentration according to the measuring method described in the action, the growth of the bacteria was determined as the turbidity (OD ₆₆₀ ) at a wavelength of 660 nm, and the change in pH of the culture solution was measured with a pH meter. Note that the turbidity was measured by pouring the culture solution into the test tubular portion on the side surface of the flask and inserting a measuring instrument there.

The thus obtained culture time is 3
Changes in Yb concentration and changes in turbidity and pH of the culture solution of strain -1 are shown in FIG. From FIG. 1, the 3-1 strain showed a rapid increase in turbidity from the 3rd day (that is, rapid growth), an increase in pH and a rapid decrease in Yb concentration, and then on the 5th day. Growth was in a steady state, and the pH was constant from the 6th day, and the Yb concentration was constant from the 9th day. The decrease in Yb concentration draws an inverse S-shaped curve, and since the Yb concentration decreases with the growth of the strain, there is a correlation between the growth of the strain and the accumulation of Yb, that is, ytterbium is present in the cells during culture. Is probably accumulated.

Example 2 The following experiment was conducted to examine the selectivity of the strain 3-1 of the present invention for various rare earth elements.

The same medium composition as in Table 1 except that nitric acid solutions of various rare earth elements [lanthanoids (La to Lu) excluding promethium (Pm) and yttrium (Y)] shown in FIG. 2 are used instead of the ytterbium nitrate solution. The liquid medium having the above was sterilized by filtration with a sterilized filter made of polysulfone having a pore diameter of 0.2 μm to prepare a rare earth element-containing liquid medium.

5 ml of a diluted broth medium containing no rare earth element and sterilized by filtration with a 0.2 μm sterilized filter made of polysulfone was inoculated with 3-1 strain from a slant medium to give 1 platinum loop.
Preculture was performed by shaking culture at 0 ° C. for 24 hours. 100 μl of the thus obtained pre-culture broth was added to 5 ml of each rare earth element-containing liquid medium prepared separately in advance as described above, and main culture was carried out by shaking culture at 30 ° C. for 10 days to give a predetermined amount. The concentration of each rare earth element contained in the culture broth after the time culturing was measured, and the reduction rate with respect to the initial concentration was calculated. The method for measuring the concentration of each rare earth element is the same as the method for measuring the ytterbium concentration described in the operation except that the rare earth element used is different, but the sensitivity at the wavelength of 655 nm differs for each element, A curve was created for each element.

As a comparative control, E. coli
The JM109 strain was cultured in various rare earth element-containing media to determine the reduction rate of each rare earth element concentration.

The results of using the 3-1 strain of the present invention are shown in FIG. 2, and the results of using the E. coli JM109 strain for comparison are shown in FIG. As a result, as shown in FIG.
The strain 3-1 showed extremely high selectivity. That is, only ytterbium showed only a reduction rate of 50% or more, lanthanum and neodymium had a reduction rate of about 30%, and other elements had a low reduction rate of 10 to 20% or no reduction at all. Even things that haven't been confirmed. In addition, in comparison with the results of FIG. 3 using E. coli JM109 strain, yttrium, cerium, samarium,
It is suggested that the rate of decrease of the concentrations of europium, gadolinium, holmium and lutetium is likely to be in the margin of error.

[0037]

As described above, the microorganism belonging to the genus Methylobacterium according to the present invention is
It is a novel microorganism that can efficiently accumulate rare earth elements, especially ytterbium which is a heavy rare earth element.

[Brief description of drawings]

FIG. 1 is a graph showing changes in ytterbium (Yb) concentration in a culture solution and changes in turbidity and pH of the culture solution with respect to culture time for explaining the ability of the microorganism of the present invention to accumulate rare earth elements. Is.

FIG. 2 is a diagram showing the selectivity of the microorganism of the present invention for various rare earth elements.

FIG. 3 is a graph showing the selectivity of Escherichia coli JM109 strain for various rare earth elements.

[Procedure amendment]

[Submission date] October 12, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction contents]

Until now, there has been no report that Methylobacterium fujisawaense accumulates rare earth elements, and since this bacterium is clearly distinguished from known species, the microorganism of the present invention is a novel microorganism. This strain was designated as Methylobacterium fujisawaense 3-1 strain (hereinafter simply referred to as 3-1 strain). Also, this 3
-1 strain was deposited with the Agency of life Institute of Advanced Industrial Science and Technology dated Sep. 29, 1995, the accession number FERM
It is P-15211.

Claims (4)

[Claims] 1. A rare earth element-accumulating microorganism belonging to the genus Methylobacterium capable of accumulating rare earth elements. 2. The rare earth element-accumulating microorganism according to claim 1, which efficiently accumulates ytterbium among the rare earth elements. 3. The rare earth element-accumulating microorganism according to claim 1 or 2, wherein the microorganism is Methylobacterium fusawaens. 4. The rare earth element-accumulating microorganism according to claim 1, wherein the deposit number is FERM P-15211.