JP3321592B2 - New microorganism culture method and water treatment method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism having the ability to oxidize manganese, the genus Cedesea.
GSJ / MITA24A / ASHO-RO / 1, and Cedesea GSJ / MITA24A /
ASHO-RO / 1, the genus Aeromonas (Aeromon
as) GSJ / MITA24B / ASHO-RO / 2,
Shewanella GSJ / MIT
A microbial symbiosis comprising one or more of A24C / ASHO-RO / 3 and an algae, a method of culturing the symbiosis using a diluted aqueous solution of Kester artificial seawater,
The present invention also relates to a method for removing manganese from manganese-containing water using this symbiotic organism and a method for using the manganese.

[0002]

2. Description of the Related Art Conventionally, various methods have been known for removing heavy metals, particularly manganese, from water. However, in chemical treatment, water containing manganese ions is made highly alkaline at pH 10 or more to remove manganese dioxide. The water is neutralized and then discharged after forming a precipitate and separating and removing it from the water system.The method of removing microorganisms requires not only the addition of a large amount of organic matter as a nutrient source, but also manganese. In many cases, only microorganisms exhibiting removal ability are used after being separated and purified, and these microorganisms may lose their ability to remove manganese during subculture and storage. There is no satisfactory one, and there is a demand for a method for treating manganese-containing water that removes manganese efficiently at low cost.

[0003]

DISCLOSURE OF THE INVENTION The present invention has been made to discover that a novel microorganism having an ability to efficiently remove heavy metals, particularly manganese, and a symbiotic microorganism therefor exist. It is an object to provide a method for culturing a symbiotic organism, a method for removing manganese from manganese-containing water, and a method for recycling collected manganese.

[0004]

Means for Solving the Problems The present inventors have found that a special microbial symbiosis is capable of producing heavy metals, particularly manganese, in water.
The present inventors have found that a solid substance has an action of capturing it, and a dissolved substance has an action of oxidizing and precipitating the same, thereby completing the present invention.

[0005] (1) Sedsea genus FERM P-17064 strain and
And / or a man consisting of the genus FERMP-17220
Microorganism symbiosis of cancer-oxidizing bacteria and algae
A culture obtained by culturing without inclusion. (2) In a diluted aqueous solution of artificial seawater having a dilution ratio of 0.5 to 20
The culture according to the above (1), which is cultured in

[0006] (3) Under conditions of pH 5 to 8, only with sunlight
The culture according to the above (2), which is grown. (4) The culture according to any one of the above (1) to (3), wherein the algae include cyanobacteria such as Ocillatoria, diatoms such as Navicula, and green algae such as Ulothrix. .

(5) Any one of the above (1) to (4)
Contact the described culture with water containing manganese.
By oxidizing and precipitating manganese, manganese in water
A water treatment method characterized by removing.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The source of the microbial symbiotic organism of the present invention is from water in which manganese ions are dissolved in the natural world and manganese precipitates around the same. Location. A new microorganism having the ability to oxidize manganese, the genus Cedesea (Cedec)
ea) GSJ / MITA24A / ASHO-RO / 1,
Aeromonas GSJ / MIT
A24B / ASHO-RO / 2 or Shewanella (Sh
ewanella) GSJ / MITA24C / ASHO
Microbial symbiotic organisms consisting of microorganisms containing one or more of RO / 3 and algae can be cultured in large quantities. Examples of the algae include cyanobacteria such as Ocillatoria, and Navicul.
diatoms such as a) and green algae such as Ulothrix. It is desirable that the culture conditions are acidic to weakly alkaline, preferably pH 5 to 8, and that the culture is carried out only with sunlight using water containing no organic substances. Therefore, it is not necessary to artificially add an organic substance as a nutrient source, so that a culture can be obtained by a very simple method at low cost.

[0009]

[Example 1]

 (1) Location of obtaining microorganisms Microorganisms were obtained from nature.

(2) Method of collecting microorganisms Microorganisms such as cyanobacteria such as Ocillatoria, diatoms such as Navicula, and fine algae such as green algae such as Ulothrix. Flocculants, sediments such as manganese dioxide, or aggregates composed of both,
Bacteria are obtained from samples such as the above, but as a standard, 1 ml of algae up to 3 mm from the surface of the algae was collected and added to 9 ml of water to use the whole as 10 ml. In addition, this microorganism was found to consist of three types of microorganisms from the experiments described below.
This mixed microorganism was used as GSJ-MITA-ASHORO-M
An attempt was made to deposit N-MAT-1 at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology.

(3) Growth, selection and isolation of manganese oxidizing bacteria As an artificial inorganic salt aqueous solution having a salt concentration close to that of a hot spring,
A 20% aqueous solution of Kester artificial seawater (KSW: Kester et. Al., 1967) was prepared. Suspension water obtained by serially diluting the above obtained sample with an aqueous solution of artificial inorganic salts was added to a 1/2 TZ-Mn medium (Maru
YF1-M with 20% KSW concentration in Yama et al., 1993)
It was spread on an agar plate medium prepared in n medium (Mita et al., 1994) and cultured at 20 ° C. or 37 ° C. for growth. From the emerged colonies, only those colonies having the ability to color the aqueous solution of TMBZ · HCl blue were selected, and colonies having different shapes were separated from these, and strains were isolated. Here, Mn-24 (A) is referred to as GSJ /
The MITA24A / ASHO-RO / 1 strain, Mn-24 (B) was replaced with the GSJ / MITA24B / ASHO-RO / 2 strain, Mn.
-24 (C) is GSJ / MITA24C / ASHO-RO / 3
It is called a strain.

(4) Method of identifying strain of manganese oxidizing bacteria The strain was identified by the Japan Food Research Laboratories. The strain was subjected to morphological observation, physiological property tests, and quinone-based and intracellular DNA GC content measurements.
lt (1984), Holt et al. (1994), MacDonell and Colwe
ll (1985), Lee et al. (1977), Nozue et al. (1992)
And identified. (5) Bacteriological properties and identification of GSJ / MITA24A / ASHO-RO / 1 strain The bacteriological properties of the strain are as follows. Although this strain showed properties similar to those of the Enterobacteriaceae family, Cedecea davisae, it did not have the lipase characteristic of the genus Cedecea, but it is the facultative anaerobic gram-negative bacterium that is oxidase-negative. It was similar. Therefore, this novel microorganism is referred to as Cedesea GSJ / MITA.
24A / ASHO-RO / 1
On January 25, GSJ / MITA24A / ASHO-RO / 1
Deposited as The deposit number is FERM P-1706
4.

Bacteriological properties of GSJ / MITA24A / ASHO-RO / 1 strain (Test items) (Test results) Morphology Bacillus Gram stainability-Spores-Mobility + Flagellar hair Anaerobic oxidase-catalase + OFF colony color NP (Note 1) Generation of gas from lactose + (slow) Generation of indole-Methyl red test + VP reaction + Utilization of citrate (simmons) + hydrogen sulfide Generation-Decomposition of urea-Phenylalanine deaminase-Lysine decarboxidase + (slow) Arginine dihydrolase-Ornithine decarboxylase + Liquefaction of gelatin-Utilization of malonate + Generation of acid from glucose + Generation of gas from glucose-Acid Of cellobiose + glycerin + maltose + D-mannose + -Rhamnose-salicin + trehalose + D-xylose + hydrolysis of esculin + reduction of nitrate + degradation of DNA-lipase-ONPG + GC content of intracellular DNA (mol%) 54 Quinone Q-8 (Note 1) Characteristic Does not produce colony pigment

(6) GSJ / MITA24B / ASHO
-Mycological properties and identification of the RO / 2 strain The mycological properties of the strain are as follows. Although this strain was negative for oxidase and flagella was extremely monocotyledonous, it was a facultative anaerobic gram-negative bacillus with motility,
Aeromona from the viewpoint of the CG content of the intracellular DNA and the quinone system
It was closest to the s genus. Therefore, this novel microorganism is referred to as Aeromonas GSJ / MITA.
Although named 24B / ASHO-RO / 2, this bacterium
Due to its weak energy, it could not be deposited at the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry.

Bacteriological properties of GSJ / MITA24B / ASHO-RO / 2 strain (Test items) (Test results) Morphology Bacillus Gram stainability-Spore-Mobility + Flagella poleus Anaerobic oxidase-catalase-OFF colony color tone NP (Note 1) GC content of intracellular DNA (mol%) 56 Quinone Q-8, MK-8, DMK-8 (Note 1) Not generated

GSJ / MITA24C / ASHO-RO
/ 3 Mycological properties and identification of strains Mycological properties of the strains are as follows. The strain is S
Hewanella putrefaciens. Shewanella is a polar flagellum, a gram-negative bacterium with methylmenaquinone (MMK) in its quinone system, which is mainly isolated from aquatic and marine organisms. Shewanella putrefaciens is MacD
A bacterium transferred from the genus Alteromonas by onell and Colwell (1985).

[0017] This new microorganism is used as a product of Shewanella putrefaciens.
ns) Named GSJ / MITA24C / ASHO-RO / 3 and sent to GSJ / MITA24C / A on February 17, 1999 by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology.
Deposited as SHO-RO / 3. Deposit number is FER
MP-17220.

Bacteriological properties of GSJ / MITA24C / ASHO-RO / 3 strains (Test items) (Test results) Morphology Bacillus Gram stainability-Spores-Mobility + Flagella polar pilus Attitude toward oxygen Temperary oxidase + catalase + OFO Color of colony Brown Na + requirement + salt requirement Growth in 0% NaCl medium + Growth in 1% NaCl medium + Growth in seawater medium + Degradation of DNA + Arginine dihydrolase-Ornithine Decarboxylase + lysine decarboxylase-production of hydrogen sulfide + hemolytic (sheep blood) + growth in the presence of 6% NaCl + growth at 4 ° C + growth at 37 ° C + growth at 42 ° C-on SA agar medium Growth-acid production D-ribose-maltose + L-arabinose + assimilating isoleucine-succinate + gly Serine-glucose + glucosamine-GC content (mol%) of DNA in bacterial cells 48 Quinone Q-8, Q-7, MMK-7, MK-7

Embodiment 2 a. Collection and screening of microbial symbiotic organisms (1) Manganese sulfate was added to a solution obtained by diluting Kester artificial seawater 5 times, and the manganese (II) concentration was adjusted to about 2 to 3 ppm with a sterilized filter having a pore size of 0.2 μm. Prepare a sterile filtered filtrate (А solution).

(2) Collect a green or dark green or brown or black microbial mat in a sterilized bag or bottle, and place the solution in a sterilized test tube (with a cap) containing the solution. Add a 10% microbial mat and mix well with a test tube mixer. Quickly split this solution for 2 minutes,
Separate into sterile test tubes (10 ml with cap). A fluorescent reagent, DAPI, is added to the microorganism mat and observed with a fluorescence microscope to confirm the presence of cyanobacteria and bacteria.

(3) One of the two-minute test tubes is sterilized by an autoclave at 121 ° C. and 2 atm for 15 minutes, which is referred to as a sterilized sample suspension. The other untreated tube is referred to as the untreated (raw) sample suspension. (4) Three sterilized test tubes (with cap, 50 ml) each containing 25 ml of the А solution were prepared, and a test tube 1 containing nothing and 2 ml of a sterilized sample suspension were added. Tube 2, a test tube 3 to which 2 ml of untreated (raw) sample suspension is added.

(5) Each of these three test tubes is stirred well, and then incubated at 37 ° C. for 4 days under natural light irradiation. (6) Each liquid is filtered through a filter having a pore size of 0.2 μm (sterilization is not necessary), and about 0.5 ml is dispensed into a test tube. (7) To each of these, 0.5 ml of a formaldoxime solution and 0.5 ml of a pH10 buffer solution are added, and after mixing, the mixture is allowed to stand for about 10 minutes. (8) The reaction solution from the test tube 1 turns dark red. If the color of the reaction solution from the test tube 3 is significantly weaker than the color of the reaction solution from the test tube 2, it has the desired activity (positive).
Adopted because it is determined. However, if there is little difference between the coloration of the above 2 and 3, it is judged that there is almost no activity (negative) and it is not adopted. (9) The culture adopted in this selection is effective for removing manganese.

B. Culture of microorganism symbiosis In the culture of the microorganism symbiosis of the present invention, it is not particularly necessary to artificially add an organic substance, and to a 100% solution containing 60 ppm of manganese in a 20% aqueous solution of Kester artificial seawater, or at a collection site. 100 ml of sterilized sample water in contact with the symbiotic organism
Then, 1 ml of the sample selected by the above-mentioned sorting method was collected, 0.2 ml of a microorganism symbiotic sample having a total of 10 ml was added to 9 ml of water, and the mixture was allowed to stand at 37 ° C. under natural light or artificial lighting. And cultured.

C. Treatment of Water Containing Manganese by Symbiotic Microorganisms Hot spring water containing manganese at a concentration of 2.4 ppm was filtered through a sterilizing filter and sterilized. The sterilized water was adjusted as follows, divided into three of A, B, and C, each kept at a temperature of 37 ° C., and left for 3 days in natural light.

A is one in which nothing was added to the sterilized water. B: The microbial symbiotic organism: hot spring water grown in (1) at a volume ratio of 1: 9, which was sterilized in an autoclave at 121 ° C. and 2 atm, was added to the sterilized water at 0.2%. 2% (by volume) was added. C is a mixture obtained by raising the microorganism symbiotic organism: hot spring water at a volume ratio of 1: 9 grown in 1) as it is, and adding 0.2% (by volume) to sterilized water. The materials A, B, and C were uniformly stirred and then allowed to stand still.

The result of the standing is shown in FIG. A did not change at all during the three days. B changed during the two days, and manganese ions dropped to 1.8 ppm. C is
There was a large change between the two days, and on the third day the manganese ions had dropped to near zero.

Example 3 Classification and Observation of Microalgae Unstained microbial growths with manganese dioxide precipitates being formed were placed on a slide glass, and the internal structure was observed with a phase-contrast microscope or bright-field microscope. Bacteria and cyanobacteria, which are prokaryotes without nuclei, are distinguished from other organisms, which are eukaryotes with nuclei. In addition, the same unstained sample is irradiated with ultraviolet light to observe the autofluorescence of chlorophyll that emits orange fluorescence under an epifluorescence microscope, thereby distinguishing algae such as cyanobacteria, green algae and diatoms from other microorganisms. Furthermore, the cells to which the fluorescent reagent DAPI has been added are irradiated with ultraviolet rays, and the distribution of cyanobacterial nucleic acids emitting orange fluorescence and the distribution of bacteria nucleic acids emitting blue to white fluorescence are observed together. By combining these results, the classification of algae such as cyanobacteria, green algae, and diatoms is observed from a general microbial picture book. Algae in the microbial symbiosis of the present invention include cyanobacteria such as Ocillatoria, diatoms such as Navicula, and urotrix, which are observed under a microscope.
Survival of green algae such as x) has been observed.

Example 4 Treatment of Manganese-Containing Water by Strain of Manganese-Oxidizing Bacteria An artificial seawater solution to which organic nutrients such as peptone and yeast extract were added, or a solution obtained by diluting the artificial seawater concentration to about 5 times, A system inoculated with one or two or more strains of manganese oxidizing bacteria was prepared, and together with a non-inoculated system, at 37 ° C.
Alternatively, the cells are cultured with shaking at 20 ° C. An aliquot of this solution is taken and the absorbance at 660 nm (optical density, OD 660)
The microbial load and dissolved manganese concentration (Mn ²⁺ ) are measured.

The initial concentration of manganese was set to 60 ppm, and the pH of 20 ml of a 100% artificial seawater solution added with organic nutrients was adjusted to 7.5.
And a system inoculated with one platinum loop of manganese oxidizing bacteria,
FIG. 1 shows the result of culturing the uninoculated (sterile) system liquid at 20 ° C. From this figure, it can be seen that manganese dioxide precipitates even in a liquid with a high salt concentration, and high-concentration manganese ions can be removed from an aqueous solution that does not chemically precipitate. The recovered manganese can be used as a raw material for producing batteries, glazes and the like.

[0030]

The microorganisms and symbiotic microorganisms of the present invention have never been artificially isolated or cultured so far, and the removal of heavy metals, especially manganese, using the symbiotic microorganisms. The method involves the pH adjustment of various waters containing manganese.
Since it is not necessary to make the alkalinity 10 or more, manganese can be removed at low cost by applying to wastewater treatment.

Further, by this treatment method, manganese dioxide, which is the original resource, can be regenerated from waste containing manganese, for example, used dry batteries, old iron materials and the like. Furthermore, the deposited manganese dioxide obtained by this treatment method is:
Because of its high quality, it can be effectively used as a raw material for manufacturing dry batteries, iron, glaze, glass, and the like.

[Brief description of the drawings]

FIG. 1 is a diagram showing the growth state of microorganisms and the effect of removing manganese ions.

FIG. 2 is a diagram showing the effect of removing manganese in water according to the present invention.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁷ Identification code FI C22B 47/00 C22B 47/00 // (C12N 1/12 (C12N 1/12 C12R 1:89) C12R 1:89) (C12N 1/20 (C12N 1/20 C12R 1:01) C12R 1:01) (72) Inventor Yoshishige Kato 16-3 Onogawa, Tsukuba, Ibaraki Pref., National Institute of Advanced Industrial Science and Technology (72) Inventor Akihiko Maruyama Ibaraki 1-3-1 Higashi, Tsukuba Pref., Japan Industrial Technology Research Institute Biotechnology Industrial Research Institute (72) Inventor Takanori Higashihara 1-1-3 Higashi, Tsukuba City, Ibaraki Pref. Geological Survey, 1-3-1 Higashi, Tsukuba, Ibaraki Pref., Japan (72) Inventor Akira Usui Geological Survey, 1-3-1 Higashi, Tsukuba, Ibaraki, Japan (72) Inventor Hiroyuki Miura 3-5-2, Ainosato 3-chome, Kita-ku, Sapporo-city, Hokkaido (72) Inventor Takashi 1-1, Bunkyo, Mito-shi, Ibaraki 1-303, Atago Residence 1-372 (72) Inventor Hidetoshi Tashiro, Musashino, Tokyo 1-2-2 Sherovista, Minamicho, City Border Examiner Emiko Sakazaki (56) References JP-A-59-177198 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1 / 12 C12N 1/20 C02F 3/34 C22B 47/00 BIOSIS / CA / WPIDS (ST N)

Claims (5)

(57) [Claims] 1. A strain of the genus Sedsea FERM P-17064 and / or
Or manganese consisting of the strain of Shewanella FERM P-17220
Microbial symbiosis of oxidizing bacteria and algae containing organic matter
A culture obtained by culturing without culturing. 2. Dilution of artificial seawater having a dilution ratio of 0.5 to 20.
The culture according to claim 1, which is cultured in an aqueous solution . 3. Cultivation with only sunlight under pH 5-8 conditions
3. The culture of claim 2, 4. The algae is Ocillatoria.
Which cyanobacteria, diatoms such as Navicula and corals
It contains green algae such as Ulothrix.
The culture according to any one of claims 1 to 3, wherein 5. The culture according to any one of claims 1 to 4.
By contacting the substance with water containing manganese,
A water treatment method comprising oxidizing and precipitating manganese to remove manganese in water.