JP2003159051A - Marine bacteria resistant to nickel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new marine bacteria manifesting resistance to nickel. <P>SOLUTION: The invention includes a Spongiobacter nickelotolerans 00P-Ni033-1-1-2 strain and its relative strain and a method of recovery for nickel which adds the strains to an environment including the nickel, accumulating the nickel in the environment into the fungus body and then recovering the strains. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel marine bacterium capable of growing in the presence of high-concentration nickel and having the ability to accumulate nickel in cells.

[0002]

2. Description of the Related Art Generally, heavy metals such as nickel, cobalt, zinc and copper are toxic to microorganisms, but some microorganisms are resistant to these heavy metals and survive and proliferate even in the presence of high concentrations of heavy metals. be able to. Microorganisms exhibiting such resistance include the soil bacteria Alcaligenes xylosoxidan.
s) is known. This soil bacterium is 40 mM for nickel, 20 mM for cobalt and 10 mM for zinc.
It has been reported to be resistant to mM, 1 mM to cadmium, and 1 mM to copper (J. Bacteriol.
1994, Nov., 176 (22: 7045-54).

[0003]

A microorganism resistant to heavy metals is considered to have a special gene involved in resistance. For example, the above-mentioned Alcaligenes xylose oxidans has a special plasmid called pTOM9, and the ncc region and nre region on this plasmid are considered to be involved in heavy metal resistance (J. Bacterio.
l. 1994, Nov., 176 (22): 7045-54).

Isolation of a new strain resistant to heavy metals,
It is considered that clarifying genes involved in the resistance will lead to the development of means for purifying heavy metal pollution. The present invention has been made under such a technical background, and an object thereof is to isolate a novel heavy metal-resistant bacterium.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, found a marine bacterium that can grow in the presence of high-concentration nickel, thereby completing the present invention. I arrived.

[0006] That is, the present invention provides a sponge ovactor nickelotolerance (Spongiob) resistant to nickel.
Acter nickelotolerans) OOP-Ni033-1-1-2 strain and its related strains.

Further, the present invention is characterized in that the above-mentioned strain is added to an environment containing nickel, the nickel in the environment is accumulated in the bacterial cells of the above-mentioned strain, and then the above-mentioned strain is recovered. Is the way.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

The present invention is a sponge Ovactor nickelotolerance OOP-Ni033-1-1-2 resistant to nickel.
It relates to strains and related strains.

The "closely related strain of the OOP-Ni033-1-1-2 strain" referred to herein is the same as the mycological properties of the OOP-Ni033-1-1-2 strain shown in Example 2. And strains exhibiting the same mycological properties as the OOP-Ni033-1-1-2 strain except for one or several properties.

Since the 16S rRNA gene (16S rDNA) can be used as an index for classification of microorganisms, 16S rDNA
A strain whose sequence is the same as or similar to that of the OOP-Ni033-1-1-2 strain can also be referred to as "a closely related strain of the OOP-Ni033-1-1-2 strain". The "approximation" here is usually OOP-Ni033-1-
1-2 strain 16S rDNA (SEQ ID NO: 1 shows its nucleotide sequence)
It is said that it shows 85% or more homology with
% Or more homology, more preferably 98%
It means showing the above homology.

[0012] SpongeObactor nickelotolerance OOP-Ni033-1-1-2 strain was deposited under the reference number FERM P-18596 at the Patent Microorganism Depositary Center, National Institute of Advanced Industrial Science and Technology.
Has been deposited as (deposit date: November 12, 2001).

Cultivation of the strain of the present invention can be carried out in the same manner as in ordinary marine bacteria. As the medium, for example, marine broth (Bacto Marine Broth 2216, manufactured by Difco), LB medium and the like can be used. The temperature during culture is preferably 30 ° C.

Since the strain of the present invention accumulates nickel in the cells, it can be used for recovery of nickel. That is, the strain of the present invention is added to an environment containing nickel, nickel in the environment is accumulated in the bacterial cells of the strain, and then,
Nickel can be recovered by recovering the strain. The environment containing nickel may be any environment as long as the strain of the present invention can grow,
Since the strain of the present invention is a marine bacterium, the aquatic environment such as the ocean is preferable.

[0015]

EXAMPLES The present invention will be described below with reference to examples, but it goes without saying that the present invention is not limited thereto. [Example 1] Isolation of nickel-resistant bacteria 1 g of sponge collected in Palau was put in 5 ml of sterilized seawater,
The homogenized suspension was added directly or after dilution onto selective agar medium containing 840 μM nickel (Table 1), and the colonies obtained from it were isolated. OOP-Ni033-
1-1-2 strain was obtained.

[0016]

[Table 1] [Example 2] Identification of OOP-Ni033-1-1-2 strain The morphology, growth state in a medium, and physiological properties of the OOP-Ni033-1-1-2 strain were investigated. For morphological observation, OOP-Ni03 was added to the medium.
The 3-1-1-2 strain was inoculated, cultured at 25 ° C. for 24 to 48 hours, and then subjected to an optical microscope and a transmission electron microscope. The medium is Marine Agar (Bacto Marine Agar 2216, manufactured by Difco) or Marine Broth (Bacto Marine Broth 2216,
(Manufactured by Difco) was used.

The results are shown below. (A) Morphology 1) Cell shape and size: spherical to long rod-shaped, 0.3-0.5
× 0.5-5.0 μm 2) Presence / absence of cell polymorphism: Yes 3) Presence / absence of motility, flagella epiphytic state: No, or monopolar hair. 4) Presence / absence of spores: None 5) Gram stainability: Negative 6) Presence / absence of anti-acidity: None (b) Growth condition in each medium 1) Marine agar plate culture: Good growth, colony is round, convex circle, full-edge , Wet light, milky white 2) Marine agar slope culture: good growth, filamentous, milky white 3) Marine broth culture: good growth, uniformly cloudy. 4) Marine broth gelatin stab culture: liquefy. 5) Litmus milk: No change. (C) Physiological properties 1) Reduction of nitrate: Reduce. 2) MR test: Positive 3) VP reaction: Negative 4) Indole formation: No formation. 5) Hydrolysis of starch: Decomposes. 6) Use of citric acid: Simmons medium: Use.

Cristensen: Used. 7) Pigment formation: None 8) Urease activity: Negative 9) Oxidase activity: Positive 10) Catalase activity: Positive 11) Growth pH range: pH 6-10, optimum growth pH range 7
~ 9 12) Attitude toward oxygen: facultative anaerobic 13) OF test: Glucose is decomposed fermentatively. 14) Presence or absence of acid and gas generation from sugars Baumann's BM medium is used as the basal medium After culturing for 6 weeks at 25 ° C Acid generation Gas generation L-arabinose-D-xylose-D-glucose + -D- Mannose + -D-fructose + -D-galactose-maltose-sucrose-lactose-trehalose-D-sorbit-D-mannitol-inosit-glycerin + -starch + -No production + Generate 15) Esculin decomposition: Does not decompose. 16) Decomposition of arginine: Does not decompose. 17) Degradation of DNA: Degradation. 18) Halophilic: Yes Growing salt concentration range 2-5% 19) β-galactosidase: Positive 20) GC content (mol%): 47% 21) Isoprenoid quinone: Q-9, MK-9 or higher mycological Due to the nature of the Burmese Manual of
Comparison was made with known bacterial species according to the classification criteria of determinative bacteriology. This strain is a facultative anaerobic Gram-negative bacterium with polar flagella. Glucose is fermentatively decomposed, oxidase and catalase are positive, and main quinone is
The values of Q-9, MK-9 and GC were 47 mol%. The nucleotide sequence of the 16S rRNA gene of this strain is shown in SEQ ID NO: 1. Based on the results of molecular phylogenetic analysis using this sequence and the above-mentioned mycological properties, this strain is considered to constitute a new genus that does not belong to any known bacterial species, and was identified as Spongiobacter nickelotolerans. . [Example 3] Time-dependent change of growth state in the presence of nickel To an 18 mmφ test tube, 10 ml of a normal MB liquid medium (Table 2) was added, and the OOP-Ni033-1-1-2 strain was inoculated, Preculture was performed at 30 ° C. for 24 hours. The cultured bacterial solution was centrifuged at 8,000 rpm for 15 minutes,
The supernatant was removed. Add 5 ml of sterilized artificial seawater to the cells,
After measuring the turbidity (OD = 660 nm) and adjusting to OD = 1.0,
ASG-glyce containing 42μM, 420μM, 840μM, 4.2mM NiCl ₂
100 μl of the bacterial solution was added to 10 ml of rol liquid medium (Table 3). They were subjected to shaking culture at 30 ° C. for 48 hours at 50 rpm, and the growth state was measured and recorded over time. The result is shown in FIG. The vertical axis of the graph in FIG. 1 represents the turbidity (OD) of the culture supernatant. As this figure shows, 4.2mM
The OOP-Ni033-1-1-2 strain grew well even when it contained NiCl ₂ .

[0019]

[Table 2]

[0020]

[Table 3] Example 4 Measurement of Nickel Accumulation Rate ASG-glycer containing 10 ml of nickel in an 18 mmφ test tube
The ol liquid medium was added to inoculate the OOP-Ni033-1-1-2 strain, and precultured at 30 ° C. for 24 hours. Cultured bacterial solution at 8,000 rpm
After centrifugation for 15 minutes, the supernatant was removed. Cell wet weight is 25 mg / m
ASG-glycerol medium containing 840 μM of nickel was added to the cells so that the amount became 1, and the cells were subjected to shaking culture at 30 ° C. for 24 hours. The cultured bacterial solution was centrifuged at 8,000 rpm for 15 minutes to separate into a supernatant and bacterial cells. The cells were freeze-dried, the dry mass was measured, and then H ₂ O ₂ : HNO ₃ was added at a ratio of 2: 1 at 140 ° C and 30 ° C.
After dissolving at high temperature and high pressure for 1 minute, the solution was diluted with distilled water to 1 N and then filtered with a 0.2 μm filter to obtain a sample solution. Similarly, the supernatant was also filtered with a 0.2 μm filter. The nickel concentration of each of these was measured by ICP (high-frequency plasma emission spectrophotometer). In order to determine the accumulation rate from the measured values, the ratio of the total concentration of the culture broth: the nickel concentration in the bacterial cells was determined, and the amount of nickel per 1 g of the bacterial cells was determined. As a control, Pseudoalteromona (Pseudoalteromona
s) Regarding the sp. OOP-Ni017-1 strain, the nickel concentration ratio between the supernatant and the cells was also determined. The results are shown in Table 4.

[0021]

[Table 4] As shown in Table 4, nickel in the culture broth migrated to the bacterial side at a very high rate. Therefore, it is considered that the OOP-Ni033-1-1-2 strain accumulates a high concentration of nickel in the cells.

[0022]

INDUSTRIAL APPLICABILITY The present invention provides a novel marine bacterium.
Since this marine bacterium is resistant to nickel and can accumulate nickel in the microbial cells, it can be used for recovery of nickel in the environment.

[0023]

[Sequence list]                                SEQUENCE LISTING        <110> MARINE BIOTECHNOLOGY INSTITUTE CO., LTD. <120> NOVEL MARINE BACTERIA TOLERANCE TO NICKEL <130> P01-037 <160> 1 <170> PatentIn Ver. 2.1 <210> 1 <211> 1492 <212> DNA <213> Spongiobacter nickelotolerans <400> 1 tcctggctca gattgaacgc tggcggcagg cctaacacat gcaagtcgag cggtaacaga 60 actagcttgc tagttgctga cgagcggcgg acgggtgcgt aacacgtagg aatctgcccg 120 gtagtggggg atagcccgga gaaatccgga ttaataccgc atacgcccta agggggaaag 180 caggggatct tcggaccttg cgctatcgga tgagcctgcg tcggattagc ttgttggtgg 240 ggtaaaggcc taccaaggca acgatccgta gctggtctga gaggatgatc agccacactg 300 ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt gcacaatggg 360 cgaaagcctg atgcagccat gccgcgtgtg tgaagaaggc tctagggttg taaagcactt 420 tcagcgagga ggaaagggtg taggttaata ccctgcatct gtgacgttac tcgcagaaga 480 agcaccggct aactccgtgc cagcagccgc ggtaatacgg agggtgcaag cgttaatcgg 540 aattactggg cgtaaagcgt gcgtaggcgg cttgttaagt tggatgtgaa agccccgggc 600 tcaacctggg aactgcaccc aaaactggca agctagagtg cggaagagga gtgtggaatt 660 tcctgtgtag cggtgaaatg cgtagatata ggaaggaaca ccagtggcga aggcgacact 720 ctggtctgac actgacgctg aggtacgaaa gcgtggggag caaacaggat tagataccct 780 ggtagtccac gccgtaaacg atgtctacta gtcgtcggga atcttgcatt cttggtgacg 840 cagctaacgc gataagtaga ccgcctgggg agtacggccg caaggttaaa actcaaatga 900 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 960 ccttacctgg ccttgacatc ctgcgaactt tctagagata gattggtgcc ttcgggaacg 1020 cagtgacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080 gcaacgagcg caacccttat cctcagttac cagcacttcg ggtgggcact ctggggagac 1140 tgccggtgac aaaccggagg aaggtgggga cgacgtcaag tcatcatggc ccttacggcc 1200 agggctacac acgtgctaca atggtgcata cagacggttg ccaagccgcg aggtggagct 1260 aatctgagaa agtgcatcgt agtccggatt ggagtctgca actcgactcc atgaagtcgg 1320 aatcgctagt aatcgtgaat cagaatgtca cggtgaatac gttcccgggc cttgtacaca 1380 ccgcccgtca caccatggga gtgggttgct ccagaagtgg ttagcctaac cttcgggagg 1440 gcgatcacca cggagtgatt catgactggg gtgaagtcgt aacaaggtag cc 1492

[Brief description of drawings]

FIG. 1 is a diagram showing changes over time in the growth state of the OOP-Ni033-1-1-2 strain in the presence of nickel.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI Theme Coat (reference) C12R 1:01) B09B 3/00 ZABE (72) Inventor Shuo Kano 1900 Sodeshicho, Shimizu City Shizuoka Prefecture Co., Ltd. Marine Biotechnology Research Institute Shimizu Laboratory (72) Inventor Kei Kamino 1900 Sodeshicho, Shimizu City Shizuoka Prefecture Marine Biotechnology Research Institute Shimizu Laboratory (72) Inventor Yoshiichi Shizuri 1900 Sodeshicho, Shimizu City Shizuoka Prefecture Ocean Biotechnology Research Institute Shimizu Research Institute F-term (reference) 4B065 AA01X AC20 BB02 CA56 4D004 AA41 AB03 CA17 CC07 4D040 DD03 DD20

Claims (2)

[Claims] 1. A sponge Victor Nichelotolerance OOP-Ni033-1-1-2 strain that exhibits resistance to nickel, and related strains thereof. 2. A method for recovering nickel, comprising adding the strain according to claim 1 to an environment containing nickel so that nickel in the environment is accumulated in the cells of the strain, and then the strain is recovered. .