JP3155118B2 - Method for producing sulfate-reducing magnetic bacteria strain RS-1 and magnetite magnetic fine particles - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sulfate-reducing magnetic bacterium useful for treating sewage and sewage using activated sludge, and a method for producing magnetite magnetic fine particles using the bacterium.

[0002]

2. Description of the Related Art Conventionally, bacteria capable of synthesizing magnetic fine particles made of magnetite in cells and reacting to magnetism, and bacteria having a sulfate reducing ability are known. All known magnetic bacteria were bacteria having a denitrifying ability in which the final electron acceptor was nitrate or nitrite.

Until now, no magnetic bacteria having a sulfate reducing ability have been known. The magnetite microparticles obtained by cultivation of magnetic bacteria have extremely high uniformity in shape and size so that they cannot be achieved by ordinary industrial production methods. Such magnetite microparticles are expected to be useful as a carrier for immobilizing a physiologically active substance, but have a drawback in that their practicality is low because the growth rate of conventional magnetic bacteria is low.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to produce magnetite ultrafine particles having a uniform shape and size which are useful for treating sewage, sewage, etc. using activated sludge due to its ability to reduce sulfuric acid. It is to provide a novel magnetic bacterium which is useful.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides, as a novel magnetic bacterium, a strain of the genus Desulfovibrio RS-, which has a sulfate reducing ability, a synthesizing ability for magnetic fine particles composed of magnetite in cells, and a magnetic reactivity. 1 (MICR accession number: KEK13283). Further, the present invention is exemplified by such RS-1, sulfate-reducing ability, a bacterium belonging to the genus Desulfovibrio having a magnetic particle synthesizing ability and magnetic reactivity of magnetite in cells, and cultured in the presence of sulfate ions. Another object of the present invention is to provide a method for producing magnetite magnetic fine particles, comprising separating the magnetite fine particles from the grown cells.

[0006] Mycological properties of RS-1 1) Cell morphology: slightly coiled rod. Size: length 3-5 μm, width 0.9-1.5 μm. 2) Polymorphism: None. 3) Arrangement of flagella: single flagella. 4) The cells have magnetite particles connected in a chain. Each magnetite particle has an average of 20 nm x 20 nm x 60 nm or
It is a cube or a rectangular parallelepiped of 40 nm × 40 nm × 60 nm. 5) Mobility: Moves in the magnetic direction in response to magnetism. For example, when a magnetic field is artificially applied in a certain direction, the bacteria migrate in the direction of the magnetic field.However, if the direction of the magnetic field is changed by 90 ° with respect to the initial direction on the way, the migration direction also changes to the new magnetic field direction.
° changes. Electrophoresis speed 39.4 μm / s (27.4 to 61.4 μm / s) 6) Magnetophoresis direction: North: South = 1: 1 7) Guanine-cytosine content: 65.8 ± 0.2% (HPLC) 8) Presence or absence of spores: None. 9) Gram staining: negative.

[0007] Physiological properties of RS-1 1) Sulfate reduction ability. It produces hydrogen sulfide in the presence of sulfate ions and promotes growth. 2) Correlation with oxygen: obligate anaerobic. Cannot grow in the presence of oxygen. 3) Utilization as a carbon source: Ethanol, pyruvic acid, malic acid, lactic acid, and fumaric acid can be anaerobically utilized. Acetic acid, alanine, glycine, formic acid and succinic acid cannot be assimilated. 4) Catalase positive. 5) Oxidase negative. 6) Desulfoviridin: negative. Desulfoviridine is an enzyme specific to sulfate-reducing bacteria,
It has a specific absorption wavelength around 0 nm (360 nm for an alkaline solution). Therefore, this enzyme can be detected by measuring the spectrum of the lysate of the cells with a spectrophotometer. The presence of desulfoviridine is one of the indices for identifying desulfovibrio sulfate-reducing bacteria. 7) Growth is inhibited in the presence of nitrate ions. 8) In the presence of iron quinate, the above-mentioned magnetite particles linked in a chain form in the cells are synthesized.

In general, a sulfate-reducing bacterium can dissimilarly synthesize iron sulfide by S ²⁻ generated by sulfate reduction.
It is said to contribute to the precipitation of iron and the formation of magnetic particles. In contrast, the sulfate-reducing magnetic strain RS-1 of the present invention not only produces iron sulfide catabolicly, but also synthesizes magnetite, which is a magnetic iron oxide, in the cells. In other words, not only sulfur but also oxygen is used to contribute to the precipitation of iron. Because of these properties, i.e., reducing inorganic sulphate ions to hydrogen sulfide by using them as electron acceptors, the availability of oxygen, including the inability to use acetic acid, and anaerobic, a type of sulfate-reducing bacterium Is identified as Desulfovibrio.

An example of the composition of a suitable culture medium is shown below.

[Table 1] ─────────────────── KH ₂ PO ₄ 0.2 g NH ₄ Cl 0.06 g Yeast extract (Difco) 0.3 g 0.01 M iron (III) quinate solution ^* 2 ml Wolfe mineral solution ^* 4 ml malate _{0.3g Na 2 SO 4 · 10H 2} O 0.3 g distilled water 1.0 l NaOH solution adjusted to pH 7.0 ───────────────── ── (Note) ^{* The} composition is based on ATCC1653 medium.

Means of creation Separated from mud collected from the sediment of freshwater rivers. Details are as described in the embodiments. The bacteria belonging to the genus Desulfovibrio having the sulfate reducing ability represented by the method for producing magnetite magnetic fine particles RS-1 and the like, the ability to synthesize magnetic fine particles composed of magnetite in the cells, and the magnetic reactivity are cultured in the presence of sulfate ions. To separate the magnetite magnetic particles from the grown cells, for example, centrifugal collection, washing, dissolving the cells by enzymatic treatment using lysozyme and proteinase K, separating the magnetic particles by a magnet, washing and concentrating do it. Since the growth rate of the magnetic bacteria of the present invention in the presence of sulfate ions is extremely high, they are useful for producing magnetite magnetic fine particles having a uniform shape and size.

[0011]

【Example】

(1) Separation Mud is collected from the sediment of a freshwater river.
And placed in a glass container with a lid so as to perform enrichment culture for 2 weeks. Bacteria that grew in this integrated culture were confirmed to be magnetic bacteria by changing the direction of electrophoresis in response to a magnetic field under an optical microscope (see Electrification Nos. 3 and 4). The phase water was inoculated into the separation medium.

Medium composition for separation Examples of the composition of the medium are shown below.

[Table 2] ──────────────────── KH ₂ PO ₄ 0.1 g (NH ₄ ) NO ₃ 0.05 g Succinic acid 0.02 g Wolfe mineral solution ^* 2.0 ml Quinic acid Iron (III) solution ^** 2.0 ml Yeast extract (Difco) 0.05 g Sodium thioglycolate 0.05 g Distilled water 1 l Adjust pH to 6.90 with NaOH ───────────────── ───

(Note) ^* Wolin et al., J. Biol. Chem., 28
3,2882 (1963), but without FeSO ₄ ^** FeCl ₃ 0.27 g Quinate 0.19 g Distilled water 100 ml

The magnetic bacteria grown in the separation medium take advantage of the property of magnetotacticity, and magnetically induce the magnetic bacteria by a device using a sterilized capillary tube filled with absorbent cotton and pass through the absorbent cotton to the sterile water side. Purified by collecting. Thus, a purified magnetic bacterial strain RS-1 was obtained.

(2) Culture 1) The rod-shaped magnetic bacterium RS-1 was isolated under anaerobic conditions at a concentration of 0-0.5 g / l for sodium sulfate (decahydrate) and 0.3 g / l for yeast extract in a separation medium. The cells were added and cultured at 25 ° C. As the concentration of sodium sulfate increases, the bacterial cell concentration also increases. The initial concentration of magnetic bacteria of 5.0 × 10 ⁴ cells / ml is 2.0 × after 5 days of culture.
It grew to 10 ⁷ cells / ml. The results are shown in FIG.

2) The carbon source that can be assimilated is sodium sulfate.
0.1 to 0 in a separation medium containing 3 g / l and yeast extract 0.3 g / l.
When various carbon sources were added at a concentration of 5 g / l and cultured at 25 ° C, ethanol, birubic acid, malic acid, lactic acid, and fumaric acid could be anaerobically assimilated, especially when birubic acid was used.
Proliferated to 1.0 × 10 ⁸ cells / ml. Acetic acid,
Alanine, glycine, formic acid, and succinic acid could not be assimilated. 3) When fermentation was performed using iron quinate as an iron source, magnetite was synthesized and showed magnetotactic properties.

(3) Analysis of substances produced in the culture solution When RS-1 was cultured in a medium having the composition shown in Table 1, the culture solution emitted a sulfur odor with the growth, and was analyzed using lead sugar paper (lead acetate). As a result, the lead sugar paper was blackened and lead sulfide was generated, and generation of hydrogen sulfide was confirmed, indicating that RS-1 had a sulfuric acid reducing ability. An insoluble black substance precipitated in the culture broth, and elemental analysis confirmed that it was a compound composed of iron and sulfur.

(4) Analysis of Intracellular Substances When cultured in a separation medium or a culture medium to which iron quinate has been added, an electron beam, which seems to be magnetic fine particles connected in a chain in the cells by transmission electron microscopy, is not observed. Intracellular material was observed. The cells were collected by centrifugation, washed, and dissolved by lysozyme and enzyme treatment using proteinase K, and the magnetic particles were separated by a samarium-cobalt magnet, and washed and concentrated. The separated magnetic particles are 20 × 20 × 60nm
Alternatively, it had a uniform size of 40 × 40 × 60 nm cubic or rectangular parallelepiped. As a result of elemental analysis, iron and oxygen were detected, but no sulfur was detected. In addition, it was shown to be magnetite by an electronic circuit.

[0019]

The magnetic bacterium of the present invention is useful for treating sewage and sewage using activated sludge due to its ability to reduce sulfate, and is also useful for synthesizing magnetite ultrafine particles having a uniform shape and size. .

[Brief description of the drawings]

FIG. 1 shows the effect of sulfate ion concentration on the growth of strain RS-1.

────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 1/00 C12R 1:01) (72) Inventor Noriyuki Nakamura 96968, Hawaii, Honolulu, Doll Street 2540, Holmes Hall 246, Manca (no address) (58) Fields surveyed (Int. Cl. ⁷ , DB name) BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. Desulfovibrio sp. Strain RS-1 having a sulfate reducing ability, an ability to synthesize magnetic fine particles of magnetite in cells, and a magnetic reactivity. 2. A bacterium belonging to the genus Desulfovibrio having sulfate reducing ability, ability to synthesize magnetic fine particles composed of magnetite in cells and magnetic reactivity, is cultured in the presence of sulfate ions, and magnetite fine particles are separated from the grown cells. A method for producing magnetite magnetic fine particles.