JPH0870851A - Rare earth element-accumulating microorganism - Google Patents

Abstract

PURPOSE: To allow a cell body to accumulate rare earth elements, especially yttrium, lanthanum, cerium, praseodymium and neodymium in it by culturing a microorganism capable of accumulating rare earth elements and belonging to the genus Variovorax. CONSTITUTION: A microorganism belonging to the genus Variovorax e.g. Variovorax paradoxus R-1 (FERM P-14492), which is capable of accumulating rare earth elements, is cultured under aerobic conditions. A culture temperature and a medium pH are preferably 20-70 deg.C and 5-9, respectively.

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 having an ability to efficiently accumulate rare earth elements.

[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 added to the 17 element groups, and are monazite and bastnaesi.
te), Zenotime, euxenit
It is contained in minerals such as e) and gadolinite. Of these, monazite and bastnasite are mainly used as resources for light rare earth (lanthanum to europium), and xenotime as resources for heavy rare earth (gadolinium to ruthenium). The rare earth element of is obtained as a resource.

Rare earth elements are used in a wide variety of fields due to their magnetic properties and optical properties such as color based on the behavior of 4f electrons in an incompletely packed state of ions, and their unique chemical properties. ing. Specifically, the one utilizing the property of 4f electrons is a red fluorescent substance (europium) of a cathode ray tube of a color television receiver.
Magnets (samarium and neodymium) in small electronic equipment such as quartz and quartz wristwatches and walkmans, and also those that use chemical properties, include catalysts (lanthanum and cerium) used in the production of gasoline, and high temperature oxides. Examples include conductors, hydrogen storage alloys, ceramics, and reactor control materials.

Conventionally, as a microorganism capable of collecting a rare earth element, there is a microorganism described in JP-A-59-118,825. However, JP-A-59-
No. 118,825 only discloses a method for selectively collecting yttrium using activated sludge, and has not yet specifically identified microorganisms.

For this reason, there have been no reports to date of the specified microorganisms that efficiently accumulate rare earth elements.

[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 Variovorax capable of accumulating rare earth elements.

The present invention shows a rare earth element-accumulating microorganism which efficiently accumulates yttrium, lanthanum, cerium, praseodymium and neodymium among rare earth elements. The present invention further has a deposit number of FERM P-1.
4492 shows a rare earth element-accumulating microorganism.

[0009]

The microorganism of the present invention is a microorganism belonging to the genus Variovorax and capable of efficiently accumulating rare earth elements. Specific examples of this microorganism include the Variovorax paradox. (Variovorax paradoxus) R-1 strain is mentioned, and this strain is obtained by the following screening method. In the following screening method, yttrium (Y), which is most abundant in nature, was used as a rare earth element for screening.

From the speculation that there is a high possibility that the target strain having the ability to accumulate rare earths in microorganisms that can grow even under malnutrition is likely to exist, the screening should be divided into two stages, that is, the primary screening should be carried out under malnutrition. After conducting a search for sexual bacteria, a strain that reduces the yttrium concentration in the culture was selected as a secondary screen. The screening method according to the present invention will be specifically described below.

Samples collected from the soil of Tanigumi Shrine (Tanigumi village, Gifu Prefecture) or diluted 10 to 10 ³ times with sterilized water, 100 μl each, using a conradi rod, were prepared by the following method for primary screening agar. Medium (p
H: 7.2-7.4) and this agar medium at 30 ° C.
The cells were cultured for 7 to 10 days. Next, the colonies formed on the agar medium were picked up, suspended in 10 ml of sterilized water and further diluted 10 ² to 10 ⁴ times, and then 100 μl each was spread on the same agar medium, and this operation was further performed. The above procedure 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 1/100 diluted broth medium), 1.5% (wt / v) of agar was added thereto and sterilized under high pressure, and about 25 ml of each was added to a petri dish. Dispensed into a plate medium.

Next, in a liquid medium (5 ml) for secondary screening, which had been subjected to aseptic treatment and was prepared by the following method,
Each of the isolated strains purified by the above-mentioned primary screening was inoculated with a platinum loop, one platinum loop, and cultured at 30 ° C. for 7 days with shaking. Furthermore, the concentration of yttrium (Y) remaining in the culture supernatant was measured according to the following method, and a bacterium in which the Y concentration in the culture supernatant was less than 50% of the initial concentration (5 ppm) was selected as a candidate bacterium. . At this time, the selected strain was inoculated again into a secondary screening medium (hereinafter referred to as Y-containing medium) and the same operation was repeated to confirm reproducibility regarding Y concentration decrease. 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) was diluted 1/100 times with demineralized water, and an amount corresponding to neutralizing a 5% (v / v) nitric acid aqueous solution (1 mol / liter) in this diluted medium [0. 1% (v /
v)] 5N sodium hydroxide solution was added in advance, and a yttrium nitrate solution was gradually added dropwise to the solution while stirring to adjust the pH to about 7.2. Filter sterilized with a sterilized filter, and use this as a secondary screening medium (Y concentration: 5 pp
m). The medium for secondary screening has the composition shown in Table 1.

[0013]

[Table 1]

The method for measuring the concentration of yttrium (Y) remaining in the culture supernatant will be described below. 1.5 ml of Y-containing medium after inoculating and culturing the strain to be screened
Place in an Eppendorf centrifuge tube and centrifuge (12,000
0.5 ml of 0.1% Arsenazo II was added to 1 ml of the culture supernatant obtained by applying 0 rpm, 4 ° C., 10 minutes).
I (Arsenazo III) solution, 0.5 ml hydrochloric acid / potassium chloride buffer solution (30 ml 0.2 M KCl, 20 ml
Mix 0.2 M HCl and 50 ml demineralized water to 10
The concentration of yttrium (Y) was calculated based on the standard straight line by adding 1 ml of demineralized water and a solution having a pH of about 1.5) and measuring the absorbance at 655 nm. It was At this time, the standard line of yttrium is the yttrium nitrate solution for atomic absorption [Y (NO ₃ ) in a nitric acid solution of 1 mol / liter]
₃ concentration = 1.0 mgY / ml] (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

The mycological properties of the strain obtained by the above screening are shown below.

(A) Morphology 1) Cell shape and size: In bacilli (0.6-
0.9) μm × (3.0 to 4.0) μm 2) Presence or absence of polymorphism in cells: None 3) Presence or absence of motility: None 4) Presence or absence of spores: None (b) Culture properties 1) Meat broth agar plating (30 ° C., 24 hours): Colonies are round and have a smooth surface with a glossy yellow color.
The size of the colony is about 2 mm in diameter when cultured at 30 ° C. for 2 days. 2) Meat juice agar medium (37 ° C):-Meat juice agar medium (45 ° C) :-( c) Physiological properties (30 ° C, 72 hours) 1) Gram stain: -2) Catalase: +3) Oxidase: +4 ) OF test: + (slightly oxidative fermentation) 5) Reduction of nitrate: + 6) Production of indole: -7) Production of acid from glucose: -8) Arginine dehydrolase: -9) Urease: -10) Esculin hydrolysis: -11) Gelatin hydrolysis: -12) β-galactosidase: -13) Glucose assimilation: -14) Arabinose assimilation: -15) Mannose assimilation: -16) Mannitol assimilation: -17) N-acetylglycosamine. Assimilation: -18) Maltose assimilation: -19) Gluconate assimilation: +20) Caprate assimilation: +21) Adipate Assimilation: +22) Malate assimilation: +23) Citrate assimilation: -24) Phenylacetate assimilation: -25) Cytochrome oxidase: + (d) Supplement of physiological properties (30 ° C, 7 days) 1) Tween80 hydrolysis: + 2) Acid production from glucose OF: + (slightly) 3) Acid production from fructose OF: + 4) Acid production from xylose OF: -5) Growth in 3.5% NaCl:- 6) NO ₂ -N ₂ : -7) Alkalization of acetamide: +8) Alkalization of allantoin: +9) Alkalation of tartaric acid: +10) Urease: + (slightly) 11) Gelatin decomposition: -12) Deoxyribonuclease: -13) Lysine decarboxylase: -14) Use of citrate: +15) Use of glucose: + (minor Crab) is a Bergey's Manual of System
Since it could not be classified by atic Bacteriology 8th), the identification of this bacterium was performed by the International Collection of Industrial and Marine Bacteria Limited (NCIMB) (The National Col
lection of Industrial and Marine Bacteria Limited)
Request (reference number: ID 2591 / NCID 341)
5, date: Strain F in the report document of July 18, 1994), Variovorax paradox (Va
riovorax paradoxus) (reference: International Journal of
Systematic Bacteriology (International
Journal of systematic Bacteriology) 1991 41
(3), pp. 445-450, International Journal of systematic Bacteriolog
y) 1991 41 (3), pp. 427-444, Journal of Clinical Microbiology (Jour
nal of Clinical MicroBaiology), Vol. 23, 1986,
920-923).

Until now, there has been no report that Variovorax paradoxus accumulates rare earth elements, and since this bacterium is clearly distinguished from known bacterial species, the bacterium of the present invention is a novel microorganism. And judge
This strain is called Variovorax Paradox R-1 (Variov
orax paradoxus R-1) (hereinafter simply referred to as R-1 strain)
I named it. In addition, this R-1 strain was purchased on August 26, 1994.
Deposited at Institute of Biotechnology, Institute of Biotechnology, AIST, on the date,
The accession number is FERM P-14492.

The medium used for culturing the strain of the present invention may be either a solid or liquid medium, and a medium containing a carbon source that can be assimilated by the bacterium, an appropriate amount of nitrogen source, an inorganic salt and other nutrients. So long as it is a synthetic medium or a natural medium.

The carbon source that can be used in culturing the strain of the present invention is not particularly limited as long as it is a carbon source that can be assimilated by the strain. Specifically, considering the assimilation of microorganisms, glucose, fructose, maltose, galactose, starch, starch hydrolysates, sugars such as molasses and molasses, natural products such as meat extract, peptone, wheat and rice. ,
One or more kinds of alcohols such as glycerol, methanol and ethanol, fatty acids such as acetic acid, gluconic acid, pyrupic acid and citric acid, 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 and inorganic nitrogen compounds such as urea. Then, one kind or two or more kinds are selected and used.

The inorganic salt that 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, or the like. 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 rare earth elements in the microorganism of the present invention, rare earth elements, more preferably yttrium, lanthanum, cerium, praseodymium and neodymium, are contained in the medium at 1 to 20 ppm, more preferably 2-1.
It is preferable to add it at a concentration of 0 ppm. On this occasion,
The rare earth element to be added may be a mixture of two or more kinds or an alloy such as misch metal.

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 at that time 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, the solvent extraction method, particularly the countercurrent multistage extraction method in which the solvent extraction operation is continuously performed, is preferably used.

The microorganism of the present invention has a characteristic of efficiently accumulating rare earth elements, but conventionally, it belongs to the genus Variovorax, and there has been no report regarding the microorganism having the above characteristic.

[0026]

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

Example 1 5 ml of Y-containing medium (Y concentration: 5 ppm) sterilized by filtration through a 0.2 μm polysulfone sterilized filter was inoculated with 1 platinum loop of the R-1 strain from the slant medium, and the mixture was incubated at 30 ° C. for 24 hours. Pre-culture was performed by shaking culture. 5 ml of the thus obtained culture broth was placed in a 500 ml side branch Sakaguchi flask containing 100 ml of Y-containing medium that had been sterilized in the same manner, and main culture was carried out at 30 ° C. for 15 days. During this culturing period, the culture solution is added to the medium every 24 or 48 hours.
The collected culture broth was measured for Y concentration according to the measuring method described in the action, and at the same time, the turbidity at a wavelength of 660 nm (OD ₆₆₀ ) and the pH of the culture broth were measured. 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.

R for the culture time thus obtained
The change in Y concentration, the change in turbidity and the change in pH of the culture solution of strain -1 are shown in FIG. As shown in FIG. 1, the Y concentration of the R-1 strain cultivated reached a value near 0 within a short period of time (6 days) after the start of culturing, and thereafter remained almost constant. It can be seen that the change draws a shape close to an exponential curve. In addition, bacteria grow exponentially and the decrease curve of Y concentration by the R-1 strain is symmetrical with the above growth curve. Therefore, the decrease of Y concentration is due to the biological activity of the R-1 strain. It is believed that there is.

Example 2 R-1 was added to 5 ml of Y-containing medium (Y concentration: 5 ppm) sterilized by filtration through a 0.2 μm polysulfone sterilized filter.
The strain is inoculated with 1 loop of loops from the slant medium and cultured at 30 ° C. for 7 days with shaking. 1.5 ml of Y-containing culture solution after culturing for 7 days
Place in an Eppendorf centrifuge tube and centrifuge (12,000
At 0 rpm, 4 ° C. for 10 minutes), a culture supernatant was obtained. Subsequently, 1 ml of 150 mM NaCl was added to the precipitate (bacteria) obtained by the above centrifugation to suspend it, and this was centrifuged again to obtain a supernatant. This operation was repeated 3 times in total, and the supernatants were combined to form a washing solution.

The precipitate (bacteria) washed in this way was suspended by adding 1 ml of 150 mM NaCl. This suspension is ultrasonic crusher (Branson (BRANSO
N), Duty: 30, Output: 3, 30
The cells were crushed by performing a crushing treatment for 5 seconds, and 0.5 ml of 150 mM NaCl was further added and the mixture was centrifuged to obtain a cell extract. Then, 0.5 ml of concentrated sulfuric acid and 0.5 ml of concentrated nitric acid are added to the residue of the cells after centrifugation to heat and decompose the acid, and 150 mM of NaCl is added to this.
1 ml was added to obtain a residual liquid of bacterial cells.

As a comparison, Escherichia coli (Escherichia
The same evaluation was carried out using E. coli IFO 1041).

The Y concentration contained in each of the obtained fractions was measured using inductively coupled plasma ICP. The result is shown in FIG.

As is clear from this result, Y, which was not accumulated at all in E. coli, was accumulated in the bacterial cells in the R-1 strain at about 70% of the initial concentration.

Example 3 Each rare earth element [lanthanoid (La-L
u) and yttrium (Y)] nitrate solution is used instead of the yttrium nitrate solution, and the rare earth element-containing liquid medium having the same medium composition as in Table 1 has a pore size of 0.2 μm.
It was sterilized by filtration with a sterilized filter made of polysulfone. This rare earth element-containing liquid medium (concentration of each rare earth element: 5 pp
m) Inoculate 5 ml of R-1 strain from a slant medium one platinum loop at a time, shake culture at 30 ° C. for 7 days, and measure each rare earth element concentration contained in the culture solution to determine the reduction rate relative to the initial concentration. It was calculated. The method of measuring the concentration of each rare earth element is the same as the method of measuring the concentration of yttrium described in the operation except that the rare earth element used is different.
Since the sensitivity at the wavelength of nm differs for each element, a standard curve was created for each element.

The results are shown in FIG. As shown in FIG. 3, substances with a high reduction rate are biased toward the one with the smaller atomic weight (left side in FIG. 3), and praseodymium (Pr) is slightly lower (
2%) shows a relatively high reduction rate of about 40% from yttrium to neodymium. In addition, since the valences of all elements from lanthanum to neodymium, which show a high reduction rate, are limited to 3 or more, the reduction rate is reduced when yttrium or light rare earth elements whose valences are limited to 3 or more are used. Is considered to be high.

[0036]

INDUSTRIAL APPLICABILITY As described above, the microorganism belonging to the genus Variovorax according to the present invention is a novel microorganism and can efficiently accumulate rare earth elements, particularly yttrium, lanthanum, cerium, praseodymium and neodymium. Is.

[Brief description of drawings]

FIG. 1 is a graph showing changes in yttrium (Y) concentration in a culture solution with respect to a culture time, and turbidity and pH of the culture solution for explaining the ability of the microorganism of the present invention to accumulate rare earth elements.
It shows the change of.

FIG. 2 is a diagram showing a distribution state of Y.

FIG. 3 is a diagram showing the selectivity of the microorganism of the present invention for lanthanoids.

Claims (3)

[Claims] 1. A rare earth element-accumulating microorganism belonging to the genus Variovorax capable of accumulating rare earth elements. 2. The rare earth element-accumulating microorganism according to claim 1, which efficiently accumulates yttrium, lanthanum, cerium, praseodymium, and neodymium among the rare earth elements. 3. The rare earth element-accumulating microorganism according to claim 1, wherein the deposit number is FERM P-14492.