JP4088690B2 - New microorganisms and methods for removing arsenic by microorganisms - Google Patents

Description

  The present invention relates to a novel microorganism and a method for removing arsenic by a microorganism, and more particularly, to a method for removing arsenic by microbial treatment of an arsenic-containing aqueous solution.

For removing arsenic in the waste liquid, for example, a method of adsorbing with arginine gel (Non-patent Document 1) or iron particles (Non-patent Document 2) coated with iron (3), activated red mud (Non-patent Document) 3) and a method of adsorbing to fly ash (Non-Patent Document 4), a chemical precipitation method (Non-Patent Document 5), adsorption with molybdenum-impregnated chitosan beads (Non-Patent Document 6), a method using a carbon-based adsorbent (Non-Patent Document) 7) etc. are generally known. Further, as a biological adsorption method, a method of removing using chemically modified biomass bacteria is also known in the laboratory (Patent Document 8). Iron (3) and Fe (3) indicate trivalent iron (Fe), As (3) indicates trivalent arsenic (As), As (5) and Arsenic (5) It represents pentavalent arsenic (As).
Min JH et al: Arsenate sorption by Fe (3) -doped alginate gels.Water Res 32 [1544] 52 (1998) Matis KA et al: Sorption of As (5) by goethite particles and study of their flocculation.Water Air Soil Pollut 111 [297] 316 (1999) Bildik M. et al: Arsenic removal from aqueous solutions by adsorption on red mud.Waste Manage 22 [357] 63 (2002) Diamantopoulos E et al: As (5) removal from aqueous solution by fly ash.Water Res 27 (12) [1773] 7 (1993) Harper TR et al: Removale of Arsenic from wastewaters using chemical precipitation methods. Water Environ Res 64 [200] 3 (1992) Dambies l et al: Arsenic (5) sorption on molybdate-impregnated chitosan beads.Colloids Surf A 170 [19] 31 (2000) Pattanayak J et al: A parametric evaluation of the removal of As (5) and As (3) by carbon-based adsorbents. Carbon 38 [589] 96 (2000) Maria X et al: Removal of As (5) from wastewaters by chemically modified fungal biomass.Water Res 37 [4544] 52 (2003)

On the other hand, regarding the removal of heavy metals by biological adsorption, immobilized M.P. A method using a biosorption column with rouxii biomass (Non-patent Document 9), a method using Aspergillus niger (Non-patent Document 10), and the like are known.
Yan G et al. Heavy metal removal in a biosorption column by immobilized M. rouxii biomass. Bioresource Technology 78 [243] 249 (2001) Kapoor A et al. Removal of heavy metals using the fungus Aspergillus niger. Bioresource Technology 70 [95] 104 (1999)

  The biosorption of heavy metals is widely known without waiting for Non-Patent Documents 9 and 10, but there are few examples of using microorganisms to remove arsenic from arsenic-containing wastewater. Non-patent document 8 such as Maria is one of few examples. However, the fungus actually used in the adsorption method of arsenic such as Maria is Penicillium chrysogenum biomass. The arsenic to be removed is As (5). Moreover, the recovery rate of As (5) is low with the fungus biomass itself, and the intended purpose is achieved unless the fungus biomass is pretreated with a normal surfactant and a cationic electrolyte and chemically modified. do not do. Although the pretreatment is relatively simple, it is a significant cost obstacle for industrial implementation. So far, there is no known example in which microorganisms are used for removing arsenic, in particular, removal techniques by adsorption of As (3) by microorganisms.

  An object of the present invention is to provide a novel microorganism having an ability to adsorb arsenic to solve the above-described problems.

  Another object of the present invention is to provide a method for removing arsenic that can be increased in large quantities by culturing and that is easy to recover arsenic from an arsenic or arsenic compound-containing aqueous solution using a biodegradable microorganism. Yes.

  The novel microorganism which is the first invention of the present invention which has solved the above problems is a new strain Bacillus megaterium UM-123 (Bacillus megaterium UM-123, February 12, 2004) having an arsenic adsorption ability isolated from soil. And the deposit number [FERM P-19682]) at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology.

  The method for removing arsenic by a microorganism according to the second invention of the present invention is characterized in that an arsenic-containing aqueous solution is treated using a new strain, Bacillus megaterium UM-123.

  The second aspect of the present invention is particularly effective in removing As (3) arsenic. In this case, the pH of the aqueous solution to be treated is preferably 2 to 11, desirably 2 to 8, and more desirably 6 to 8 from the viewpoint of removal rate. The treatment temperature is preferably in the range of about 30 to 50 ° C. from the viewpoint of removal rate.

  Hereinafter, the arsenic in the present invention refers to trivalent or pentavalent arsenic, As (3) As (5) or an arsenic compound containing them.

  The present invention has the following industrial effects and advantages.

(1) According to the first and second inventions of the present invention, by using the new strain Bacillus megaterium UM-123 belonging to the genus Bacillus, As (3), which has never been seen before, under mild conditions, Arsenic can be removed from an arsenic-containing aqueous solution with a high recovery rate of about 85%.

(2) According to the third invention of the present invention, by using a bacterium belonging to the genus Bacillus and Escherichia among microorganisms, a filamentous fungus belonging to the genus Aspergillus, or a yeast belonging to the genus Saccharomyces, arsenic is removed from an arsenic-containing aqueous solution. Can be removed.

  Each of the present inventions has an advantage that the environmental load of waste after arsenic removal is small compared to chemical adsorption by adsorbing arsenic by a biodegradable or self-digestible microorganism.

  The novel strain Bacillus megaterium UM-123, which is the first invention of the present invention, is obtained by suspending soil in sterilized water and then culturing the supernatant by applying it to, for example, an arsenic-containing agar plate medium. Can be obtained. The medium used for the culture is not particularly limited, but the use of the arsenic-containing medium is due to consideration of selection of bacteria resistant to arsenic.

  The bacteriological properties of the obtained new strain Bacillus megaterium UM-123 and the differences from the known homologous bacteria are as shown in Table 1 based on Example 1 and Example 1 described later.

Arsenic compounds that can be adsorbed and removed in the second and third inventions of the present invention are not particularly limited, such as arsenic alone, trivalent As (3) and pentavalent As (5), and arsenic compounds thereof. However, in the second invention using the new strain Bacillus megaterium UM-123, as is clear from FIG. 1, the removal rate of the trivalent arsenic compound, diarsenic trioxide (As ₂ O ₃ ) is pentavalent. The arsenic compound, disodium arsenate heptahydrate (Na ₂ HAsO ₄ · 7H ₂ O), is considerably higher than that. Therefore, the second invention is particularly effective in As (3).

  Examples of the bacterium used in the third invention of the present invention include Bacillus megaterium and Bacillus subtilis having the ability to adsorb arsenic belonging to the genus Bacillus and Escherichia. In particular, the use of the new strain Bacillus megaterium UM-123 described below shows a high As (3) removal rate. In addition, Escherichia coli having an ability to adsorb arsenic belonging to the genus Escherichia is also useful. Examples of filamentous fungi include Aspergillus niger, and examples of yeast include Saccharomyces cerevisiae. Bacillus subtilis (IFO03335), Escherichia coli (IFO3301), Aspergillus niger (IFO4414), and Saccharomyces cerevisiae (IFO2044) are all known bacteria that can be purchased from the Fermentation Research Institute.

  For culturing the microorganisms used in the present invention, for example, meat extract, peptone, sodium chloride, phosphoric acid-potassium hydrogen phosphate, etc. are used as the medium, and the pH is adjusted to about 7. The bacteria are inoculated in a medium sterilized by autoclaving, and cultured at 30 ° C. for 24 hours with shaking at 110 strokes / minute. After culturing, the cells are separated from the culture solution by a centrifuge, washed with physiological saline, and lyophilized to obtain the cells.

  The removal of arsenic of the present invention generally depends on pH adjustment of the aqueous solution to be treated, temperature, pressure, added amount of fungus, mixing stirring time and the like.

  Although pH cannot be specified because it varies depending on the type of cells used, in the second invention of the present invention, as shown in FIG. 1, from the viewpoint of the removal rate of As (3), pH is 2 to 11, preferably 8 or less. More preferably, the neutral vicinity of 6 to 8 is suitable. As shown in FIG. 2, the temperature is preferably 30 to 50 ° C., particularly 30 to 45 ° C.

  The amount of fungus added varies depending on the concentration of arsenic and the type of fungus. For example, if the arsenic concentration is 1 mg / L, the amount of fungus may be about 0.2 to 2.0 w / v.

  A stirring time of about 2 hours is sufficient. There is no significant change in the removal rate even over 2 hours or longer. After the treatment, clean water from which arsenic has been removed can be obtained by separating the cells from the supernatant using a centrifuge and removing the cells.

(Acquisition and identification of a new strain according to the first invention of the present invention)
[Acquisition of new strain]
The field soil in Miyazaki Prefecture was suspended in sterilized water, and the supernatant was applied to a plate medium and cultured at 30 ° C. The plate medium used was a trade name “Nutrient Agar” (manufactured by Nissui Pharmaceutical Co., Ltd.) with diarsenic trioxide [As ₂ O ₃ : As (3)] added to 1 mg / L. The main ingredients are 0.5% meat extract, 1.0% peptone, 0.5% sodium chloride, 1.5% agar and pH is 7. Colonies on the plate medium were made single by fractionation. The resulting strain was inoculated into the medium. The medium used was a trade name “Nutrient Broth” (manufactured by Nissui Pharmaceutical Co., Ltd.) with diarsenic trioxide [As ₂ O ₃ : As (3)] added to 1 mg / L. The main components are 0.5% meat extract, 1.5% peptone, 0.5% sodium chloride, 0.5% potassium hydrogen phosphate and pH is 7. After inoculation, shaking culture was performed at 30 ° C. for 24 hours at 110 strokes / minute. Subsequently, the cells were separated from the culture solution by a centrifuge, and the arsenic concentration of the supernatant was measured. The arsenic concentration was measured by an arsenic morphology analysis system (arsenic morphology pretreatment device-atomic absorption spectrophotometer) in the instrumental analysis field of the Miyazaki University Frontier Science Experimental Center. A strain having a remarkable decrease in the arsenic concentration in the culture supernatant was designated as a novel strain.

[Identification of new strain 1: First stage microorganism test]
For new strain UM-123 (registration number SIID2588), in order to estimate a classification group showing similar properties to SIID2588 from the results of morphological and physiological biochemical tests at NMC Japan, Inc. The test was conducted.

  The cell morphology, Gram stainability, the presence or absence of spores, and the presence or absence of flagellar motility were observed with an optical microscope U-LH1000 (Olympus, Japan). Colony morphology was observed on Nutrient Agar (Oxoid, England, UK) plate media. Catalase reaction, oxidase reaction, acid / gas production from glucose, glucose oxidation / fermentation (O / F) were tested. The results are shown in Table 1.

As is clear from Table 1, SIID2588 shows properties such as Gram staining positive, Neisseria gonorrhoeae, spore formation, growth under aerobic conditions, catalase reaction requirement, etc., BARROW, GI et al: Cowan and Steel's Manual for the Identification of Medical Bacteria. 3 ^rd . Ed. 1993, Cambridge University Press and SNEATH, PHA et al: Bergey's Manual of Systematic Bacteriology. 2 1984, Williams and Wilkins, Baltimore .
[Identification of new strain 2: 16S rDNA-500 nucleotide sequence analysis]

  Next, for the registration number SIID2588, in order to estimate the belonging taxonomic group of the specimen using a partial base sequence 500 bp of 16S rDNA (16S rRNA gene) at NMC Japan Co., Ltd., nucleotide sequence analysis was performed.

  SIID2588 was inoculated into Nutrient Agar (Oxoid, England, UK) and cultured at 30 ° C. for 2 days. Thereafter, this microbial cell was used as a test microbial cell for DNA extraction.

  PrepMan Method (Applied Biosystems, CA, USA) was used for genomic DNA extraction. The extracted genomic DNA was used as a template to amplify a region of about 500 bp at the 5 ′ end of the 16S Ribosomal RNA gene (16S rDNA) by PCR. Thereafter, the amplified base sequence was sequenced to obtain a 16S rDNA partial base sequence of the specimen. For purification of PCR products and cycle sequence, MicroSeq500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems, CA, USA) was used. A GeneCycle PCR System 9600 (Applied Biosystems, CA, USA) was used for the thermal cycler, and an ABI PRISM 3100 DNA Sequencer (Applied Biosystems, CA, USA) was used for the DNA sequencer. The basic operation from the extraction of genomic DNA to the cycle sequence was in accordance with the protocol (P / N4308132 Rev. A) of Applied Biosystems.

In the analysis, a homology search was performed using the obtained 16S rDNA base sequence, and the top 10 strains with the highest homology were determined. Furthermore, using the searched top 10 strains and 16S rDNA of the specimen, the neighbor-joining method (anew method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4 406-425 (1987) ) To create a molecular phylogenetic tree, and examined closely related species and assigned taxonomic groups. For homology search and phylogenetic tree construction, MicroSeq Microbial Identification System Software V. As a database for performing homology search, MicroSeq Bacterial 500 Library v. 0023 (Applied Biosystems, CA, USA) was used. In addition, in the homology search for MicroSeq Bacterial 500 Library, when no matching strain was found with a homology rate of 100%, BLAST contains ALTDCHUL, SF et al: Gapped BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25 3389-3402 (1997) and SKERMAN VBD et al: Approved lists of Bacterial Names. INt. J. Syst. Bacteriol., 30 225-420 (1980), DNA base sequence database (GenBank / A homology search was performed on DDBJ / EMBL).

The 16S rDNA nucleotide sequence analysis result of the sample [UM-123] (SIID2588) strain by Microseq is shown in the free text of the following sequence listing.

(Free text of sequence listing)

Strains related to this specimen and their differences Library: 500 0023 0.9 1286/1286
BLAST: 536 SID2588
0.37% 536 Bacillus megaterium
3.54% 536 Bacillus flexus
10.26% 536 Bacillus cohni
10.75% 530 Bacillus psychosaccharolyticus
11.57% 536 Bacillus horikoshii
12.03% 532 Bacillus circulans
12.15% 535 Bacillus azotoformans
12.17% 534 Bacillus fastidiosus
12.73% 534 Bacillus marinus
12.87% 537 Bacillus cereus

Differences between this sample and related upper strains
4
7
7
SIID2588 Y
Bacillus megaterium T

  FIG. 3 shows a phylogenetic tree based on the neighborhood binding method between this specimen and a related strain.

  Results of homology search performed on BLAST Result DNA base sequence database: Table 2 shows a comparison of identities between the top 5 base sequences of the searched top 20 entries and the SIID2588 base sequence.

  Furthermore, the sequence comparison of the base sequences of SIID2588 rDNA and the entry topmost DNA was carried out. The results are shown in FIG.

  As a result, in the analysis using MicroSEq, the 16S rDNA partial nucleotide sequence of SIID2588 has a homology of 99.63%, and Baccillus megaterium (STACKEBRANDT, E. et al: Report of the ad hoc committee for the re-evaluation of the Species definition in bacteriology. Int. J. Syst. Evol. Microbiol., [52] 1043-1047 (2002)), showing a high homology rate, and the difference between 16S rDNA of two strains is the IUB code ( Y = C or T) and only one base. In the molecular phylogenetic tree, 16S rDNA of SIID2588 was located at the same position as 16S rDNA of Bacillus megaterium. As a result of homology search for GenBank / DDBJ / EMBL using BLAST, 16S rDNA of SIID2588 showed the highest homology to 16S rDNA of Bacillus megaterium strain QMB1551 with a homology of 99.4%. From the above results, SIIDD2588 was presumed to be a novel strain belonging to Bacillus megaterium and named Bacillus megaterium UM-123.

(Removal of arsenic according to the second invention)
The medium was inoculated with Bacillus megaterium UM-123 obtained from soil. The brand name “Nutrient Broth” (manufactured by Nissui Pharmaceutical Co., Ltd.) was used as the medium. The main components are 0.5% meat extract, 1.5% peptone, 0.5% sodium chloride, 0.5% potassium hydrogen phosphate and pH is 7. After inoculation, shaking culture was performed at 30 ° C. for 24 hours at 110 strokes / minute. Subsequently, the bacterial cells were separated from the culture solution by a centrifuge, washed with physiological saline, and lyophilized to obtain bacterial cells.

In a buffer solution (Mclvaine, Carmody) adjusted as follows, diarsenic trioxide [As ₂ O ₃ : As (3)] and disodium arsenate heptahydrate [Na ₂ HAsO ₄ · 7H ₂ O: As (5)] was added to 1 mg / L. The following amounts of bacterial cells, Bacillus megaterium UM-123, were added to this arsenic aqueous solution, stirred for the following time, allowed to stand, and each supernatant was collected.
pH adjustment; pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
(Bacteria added amount 2.0 w / v, stirring time 2 hours)
Bacterial cell addition amount: 0.2, 0.5, 1.0, 1.5, 2.0% (w / v)
(PH 7, stirring time 4 hours)
Stirring time: 2, 4, 6 hours
(PH 7, added amount of bacterial cells 2.0% (w / v))

  The arsenic concentration of each supernatant was measured. However, in each test, the initial concentration of arsenic was As (3) 1 mg / L. In the pH dependence test, the added amount of cells: 2.0% (w / v), stirring time: 2 hours, and in the added amount dependency test of pH: 7, stirring time: 4 hours, In the stirring time dependency test, the pH was 7, and the amount of added bacterial cells was 2.0% (w / v). The arsenic concentration was measured by an arsenic morphology analysis system (arsenic morphology pretreatment device-atomic absorption spectrophotometer) in the laboratory analysis field of the Miyazaki University Frontier Science Experimental Center. The results of pH dependency are shown in Table 3 and FIG. 1, the results of dependency on the amount of added cells are shown in Table 4, and the stirring time dependency is shown in Table 5.

  As is apparent from Table 3 and FIG. 1, as for pH dependence, As (3) showed a high removal rate of 84.5% near pH 7, but As (5) had no pH dependence and 5% Only the following removal rates were shown. From this, it was found that in the microbial treatment with Bacillus megaterium UM-123, extremely high selective adsorption occurs at pH 8 or lower, preferably in the range of pH 6-8, with respect to As (3), which has never been seen before. .

  As is clear from Table 4, it was found that the removal rate was higher as the amount of added bacterial cells was larger. Further, as is apparent from Table 5, no change is observed if the stirring time is 2 hours or longer.

(Removal of arsenic according to the third invention)
Arsenic trioxide [As ₂ O ₃ : As (3)] was added to a buffer solution (McIlvaine, Carmody) adjusted to pH 7 to 1 mg / L. The following cells were added to this arsenic aqueous solution at 2.0% (v / w), stirred for 4 hours, and allowed to stand, and each supernatant was collected.
・ Bacillus megaterium UM-123
・ Bacillus subtilis
・ Escherichia coli
・ Aspergillus niger
・ Saccharomyces cerevisiae

  The removal rate of As (3) was observed for the supernatant of each specimen in the same manner as in Example 1. The results are shown in Table 6.

  As is apparent from Table 6, it was found that As (3) can be removed at an effective rate of 50% or more by using an arsenic-adsorbable bacterium of the genus Bacillus and Escherichia. Although the removal rate is not so high, it has been found that arsenic can be removed even by filamentous fungi and yeast.

  The novel strain Bacillus megaterium UM-123 having the ability to adsorb arsenic of the present invention and an arsenic removal method using the same, or a bacterium having the ability to adsorb arsenic belonging to the genus Bacillus, and having the ability to adsorb arsenic belonging to the genus Escherichia Any method of removing arsenic using bacteria can be effectively used to remove arsenic from groundwater due to arsenic contamination of soil. Furthermore, filamentous fungi belonging to the genus Aspergillus and yeast belonging to the genus Saccharomyces can also be used for the removal of arsenic.

It is a graph which shows the pH dependence in the arsenic removal method in 2nd invention of this invention. It is a graph which shows the temperature dependence in the arsenic removal method in 2nd invention of this invention. It is a phylogenetic tree by the neighborhood joint method of this specimen and related strain in new strain identification in the 1st invention of the present invention. It is a sequence comparison figure of the base sequence of SIID2588 rDNA by the new strain identification in 1st invention of this invention, and DNA of the highest entry.

Claims (7)

  New strain Bacillus megaterium UM-123 characterized by having an arsenic adsorption ability.   A method for removing arsenic by a microorganism, comprising treating an arsenic-containing aqueous solution with the new strain Bacillus megaterium UM-123 according to claim 1.   The method for removing arsenic by a microorganism according to claim 2, wherein the arsenic is trivalent arsenic.   The method for removing arsenic by microbial treatment according to claim 2, wherein the pH of the aqueous solution is 2-11.   The method for removing arsenic by microbial treatment according to claim 2, wherein the pH of the aqueous solution is 2-8.   The method of removing arsenic by microbial treatment according to claim 2, wherein the pH of the aqueous solution is 6-8.   The method for removing arsenic by a microorganism according to claim 2, wherein the treatment temperature is 30 to 50 ° C.

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