JP3505570B2 - Acid and aluminum resistant bacteria - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism having acid resistance and aluminum resistance. Further, the present invention is
The present invention relates to a method of removing free aluminum ions in acidic soil and acidic water by using the above microorganism to improve soil health and water quality.

As described above, according to the present invention, aluminum ions, which are harmful to roots of plants, can be made harmless to improve the environment of soil and water system and improve the productivity of plants.
It has important meaning in the fields of agriculture, forestry, and the environment.

[0003]

2. Description of the Related Art It is said that 30 to 40% of acidic soil exists on the earth, and under such acidic conditions, aluminum is preferentially present in an ionic form. It has been pointed out that free aluminum ions (Al ³⁺ ) in acidic soil and acidic water cause growth inhibition of plant roots and are toxic to all organisms. Thus, it is known that the elution of harmful Al ³⁺ in acidic soil and acidic water adversely affects crop cultivation and the like.

Conventionally, research on aluminum-resistant bacteria under neutral conditions has been widely conducted, but there are very few reports of isolation of aluminum-resistant bacteria under acidic conditions. Konishi et al.'S report on aluminum-resistant bacteria under acidic conditions showed that the resistant strain is a bacterium, its resistance is mildew, lower than that of yeast fungi, and its growth limit is pH 3.0 (Biosci.Biotec
h. Biochem., 58 (11), 1960-1963, 1994). Also, Kanazawa
Reported that they isolated fungi from acidic soil,
Since the resistance measurement is performed on an agar medium containing phosphoric acid and the like, it is possible that the toxicity of the medium components may be mitigated, and it is necessary to further examine the extent to which it is actually resistant (Soil Sci. Plant Nutr ., 42 (1), 165-173, 1996).

[0005]

DISCLOSURE OF THE INVENTION The object of the present invention is to provide a novel acid-resistant and aluminum-resistant microorganism capable of detoxifying harmful aluminum ions under severe acidic conditions as described above. And to provide a method for deactivating and removing aluminum ions using the above microorganisms.

[0006]

The present inventors have succeeded in isolating acid-resistant / aluminum-resistant bacteria that can achieve the above objects, and have completed the present invention.

The novel microorganisms isolated according to the present invention are
(1) Aspergillus flavus Link F-6b (FERM
P-17723), (2) Penicillium sp. F-8b
(FERM P-17724), (3) Penicillium en
nthinellum Biourge F-13 (FERM P-177
25), (4) Trichoderma asperellum F-15 (F
ERM P-17726), (5) Rhodotorula glutin
is Y-2a (FERM P-17727), (6) Cry
ptococcus humicola Y-6 (FERM P-1772
8) (hereinafter, it may be abbreviated by the strain name). In addition, all strains of the present invention are Ka described in the prior art.
The species is different from the strain reported by nazawa et al.

Further, the present invention is a method for removing aluminum ions in an acidic environment, which comprises a step of growing the microorganism according to any one of (1) to (6) above in the acidic environment. Is the way.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The novel microorganism of the present invention will be described in detail below. <Isolation method> A 5-fold dilution of a commercially available nutrient medium (ordinary broth "Eiken", Eiken Chemical Co., Ltd.) was set to pH 3.5 with 1N hydrochloric acid. To this solution, add AlCl ₃ · 6H ₂ O aqueous solution to a final concentration
What was aseptically added by filter filtration so as to be ˜10 mM was used as a basic medium (the final concentration of the AlCl ₃ .6H ₂ O aqueous solution was initially 1 mM, but the concentration was gradually increased).

A suspension of tea garden soil (Soraku-gun, Kyoto Prefecture) was appropriately added to the above basic medium (preferably, a soil suspension having a volume of 2 to 3% of the basic medium was added). this,
Shaking culture was carried out at 28 to 30 ° C., and enrichment culture was repeated several times for those in which growth of microorganisms in the soil was observed. Further, the culture solution after the enrichment culture was applied to a solid plate medium in which 2% of agar was added to the above medium. Then, the appeared colonies were applied to a plate medium having the same composition. This was repeated until only the same colony was found to be a pure culture.

By the above-mentioned isolation, four new molds and two yeasts having acid resistance and aluminum resistance were obtained. The pure cultures were preserved in slant medium of the same composition.

The composition per 1000 mL of commercially available nutrient medium is shown below. Meat extract 3g Peptone 10g NaCl 5g pH 7.0

<Culture conditions> The nutrient source of the medium used for subculturing the 6 strains of the present invention is a nutrient source that is required for normal growth of microorganisms and can be assimilated by each strain. Any carbon source, nitrogen source, inorganic salts and the like may be used, and the cells can be cultured in, for example, GM medium and S-LB medium.

The compositions of GM medium and S-LB medium are shown below.

[0015] GM medium Glucose 1.0 wt% NaCl 1.0 wt% peptone 0.05 wt% yeast extract _{0.02 wt% MgSO 4 · 7H 2} O 0.02 wt% pH 3.5~7.0 S-LB medium soil extract * 1.0 L peptone 0.05 wt% yeast Extract 0.025 wt% NaCl 1.0 wt% pH 3.5-7.0 * 100 g of soil suspended in 1 L of demineralized water, stirred, filtered, and centrifuged to obtain a supernatant.

The culture temperature may be in a temperature range in which each strain can grow well, and is preferably 28 to 30 ° C. Further, the pH during the culture may be in the range where each strain can grow well, and the pH is preferably 3.5 to 7.0.

For subculture, one platinum loop (hook) is taken from the above strain-cultivated strain, inoculated into a test tube containing 4 mL of GM medium or S-LB medium, and shaken at 28-30 ° C. Incubate. Further, add 100 mL of the same medium (500
(in a shake flask with a shoulder) and the same culture is performed.

<Identification of Strains> Table 1 shows the results of identification of four new molds and two yeasts having acid resistance and aluminum resistance.

[0019]

[Table 1]

A. Molds were identified based solely on morphological observations. The morphological observation results of fungi are shown in FIGS. Figure 1 shows Aspe
rgillus flavus Link F-6b (FERM P-177
23), FIG. 2 shows Penicillium sp. F-8b (FERM
P-17724), Figure 3 shows Penicillium janthinell
um Biourge F-13 (FERM P-17725),
Figure 4 shows the Trichoderma asperellum F-15 (FERM
(P-17726) is a micrograph showing the morphology.

Since all of the four types of isolates formed hyphae with septa, they were judged to be other than algae, and aspergillus, P
It was judged to be enicillium or Trichoderma, and it was judged to be the corresponding species by referring to the following reference books. Penicillium
Only sp. F-8b could not find the corresponding species,
No species has been identified.

B. Yeasts were identified as the corresponding species by referring to the mycological properties shown below and the following reference books. The morphological observation results of yeast are shown in FIGS. 5 and 6. Figure 5 shows Rhodotorula glutinis
Y-2a (FERM P-17727 ), 6, cryp
tococcus humicola Y-6 (FERM P-1772
8 is a micrograph showing the morphology of 8).

[Reference Book] Classification and Identification of Microorganisms: Takeharu Hasegawa, Ed., University of Tokyo Press Yeast Classification and Identification Method: Hiroshi Iizuka, Shoji Goto Handbook of Food Fungi by the University of Tokyo Press: Translated by Udagawa and Matsuda Top and bottom: Shunichi Udagawa and others Kodansha

"Mycological properties" The mycological properties of the two yeast strains newly obtained in the present invention are shown below. First, Rhodotor
ula glutinis Y-2a (FERM P-17727)
About.

A) Culture temperature: 25 ° C. b) Morphological properties (1) Formation of pink colonies: + (2) Formation of lemon-shaped cells:-(3) Crest-shaped:-(4) Mitotic:-( 5) Ascospores:-(6) Budding cells: + (7) Budding on hyphae:-(8) Pseudohyphae:-(9) Ejectors:-(10) Dividing cells:-(11) With septa Mycelium :-( 12) Symmetrical ejector: -c) Sugar fermentability (1) D-glucose:-(2) D-galactose:-(3) Maltose:-(4) Sucrose:-(5) Lactose: -(6) Raffinose:-d) Utilization of carbon source (1) D-glucose: + (2) D-galactose: + (3) L-sorbose:-(4) D-glucosamine:-(5) D -Ribose :-( 6) D-Xylose :-( 7) L-Arabinose: + (8) L-Rhamnose :-( 9) Sucrose: + (10) Maltose: + (11) α, α-Trehalose: + (12) Methyl α-D-glucoside :-(13) cellobiose: + (14) melibiose:-(15) lactose:-(16) raffinose: + (17) melezitose: + (18) glycerol:-(19) erythritol:-(20) D-glucitol :-( 21) D-mannitol :-( 22) myoinositol :-( 23) 2-keto-D-gluconic acid :-( 24) D-gluconic acid :-( 25) D-glucuronic acid :-( 26 ) DL-lactic acid:-d) availability of nitrogen source (1) nitric acid: + (2) cadaverine: + (3) ethylamine:-(4) D-glucosamine:-(5) L-lysine:-e) Growth Sex (1) 0.01% cycloheximide :-( 2) Acetic acid production: -f) Growth temperature 37 ° C: + From the above properties, this strain was confirmed to belong to Rhodotorula glutinis .

Next, Cryptococcus humicola Y-6
(FERM P-17728) is shown.

A) Culture temperature: 25 ° C. b) Morphological properties (1) Pink colony formation:-(2) Lemon-shaped cell formation:-(3) Crest-like shape: + (4) Mitotic :-( 5) Ascospores:-(6) Budding cells: + (7) Budding on hyphae:-(8) Pseudohyphae: + (9) Ejectors:-(10) Dividing cells:-(11) With septa Mycelium: + (12) Symmetrical ejector: -c) Sugar fermentability (1) D-glucose:-(2) D-galactose:-(3) Maltose:-(4) Sucrose:-(5) Lactose: -(6) Raffinose: -d) Availability of carbon source (1) D-glucose: + (2) D-galactose: + (3) L-sorbose: + (4) D-glucosamine: + (5) D -Ribose: + (6) D-Xylose: + (7) L-Arabinose: + (8) L-Rhamnose: + (9) Sucrose: + (10) Maltose: + (11) α, α-Trehalose: + (12) Methyl α-D-glucoside : + (13) Cellobiose: + (14) Meribiose: + (15) Lactose: + (16) Raffinose:-(17) Merezitose: + (18) Glycerol: + (19) Erythritol: + (20) D-Glucitol : + (21) D-mannitol: + (22) myo-inositol: + (23) 2-keto-D-gluconic acid: + (24) D-gluconic acid: + (25) D-glucuronic acid: + (26 ) DL-lactic acid: + d) availability of nitrogen source (1) nitric acid :-( 2) cadaverine: + (3) ethylamine: + (4) D-glucosamine: + (5) L-lysine: + e) Growth Sex (1) 0.01% cycloheximide: + (2) Acetic acid production:-f) Growth temperature 37 ° C: -From the above properties, this strain is Cryptococcus humicola.
Was recognized as belonging to.

Next, a method for utilizing the microorganism of the present invention will be described. Any one of the microorganisms of the present invention can be used to remove aluminum ions in an acidic environment (eg, in acidic soil, acidic water, etc.). That is, by adding and growing the microorganism of the present invention in an acidic environment in which the microorganism of the present invention can grow, aluminum ions present in the acidic environment can be rendered harmless. The mechanism of detoxification may be converted into a harmless form other than aluminum ion or may be removed by precipitation, as described later in Examples. Both of the acid resistance of the microorganism of the present invention
Due to its resistance to aluminum, aluminum ions can be converted into a form harmless to living organisms.

The microorganism of the present invention can also be used for removing aluminum ions by immobilizing the cells cultured in advance on a column and circulating water containing aluminum ions.

[0030]

[Examples] The results of the acid resistance and aluminum resistance experiments of the strain of the present invention will be described below. <Example 1> The acid resistance of isolated bacteria was investigated, and the results are shown in Table 2.
Shown in.

[0031]

[Table 2]

The pH of the GM medium (pH 2.2 to pH 3.5) is
The effect on the growth of microorganisms was investigated. The strain of the present invention,
In a GM medium having each pH value shown in Table 2, 30 ° C
At 168 hours (7 days) with shaking, the growth was evaluated. Growth at pH 2.5 was observed for all 6 strains of the invention. Further, in F-13 and F-15, proliferation was clearly observed even at pH 2.2. From this result, acid resistance of the strain of the present invention was proved.

Example 2 The aluminum resistance of the isolated bacteria was investigated, and the results are shown in Table 3.

[0034]

[Table 3]

Aluminum contained in the GM medium (0
˜300 mM) was investigated for its effect on microbial growth.
In GM medium (pH 3.0), shaking culture was performed at 30 ° C. for 168 hours (7 days), and its growth was evaluated. In all 6 strains of the present invention, growth was observed up to an aluminum concentration of 100 mM. Especially in F-13, F-15 and Y-6, the proliferation was clearly observed up to an aluminum concentration of 200 mM. From this result, it was proved that all the strains of the present invention have high aluminum resistance which has never been reported before.

Example 3 Penicillium janthinellum Biourge in GM medium and S-LB medium respectively
F-13 was cultured, and changes in pH, aluminum ion (Al ³⁺ ) and total aluminum (tAl) when 1 mM aluminum ion was added to each medium were examined. The result is shown in FIG. 7.

First, in the GM medium, the amount of added aluminum ions decreased and was hardly detected on the third day, but it remained as total aluminum. From this result, it is considered that harmful aluminum ions in the culture medium were also converted into an inactive aluminum form in the culture medium. On the other hand, in the S-LB medium, the added aluminum ions and total aluminum were completely precipitated and removed from the culture solution.

As described above, although the aluminum resistance mechanism varies depending on the medium composition, it is common in that aluminum ions are removed. Further, these phenomena were similarly observed in the other strains of the present invention, and it was demonstrated that the strains of the present invention can remove aluminum ions.

[0039]

As described above, since the novel microorganism of the present invention is isolated from healthy soil, it is unlikely to exert pathogenicity.

The microorganism of the present invention can generally survive and grow in acidic soil and acidic water system environments in which the growth of the microorganism is inhibited due to its acid resistance. The microorganism of the present invention has a pH of 3.0 or less (depending on the strain, the maximum pH of 2.2
It was able to grow, and its acid resistance was higher than that of Konishi et al. And Kanazawa et al. In the prior art.

Further, the microorganism of the present invention exerts an aluminum resistance effect, and can detoxify aluminum ions harmful to plant roots from acidic soil and acidic water system. The microorganism of the present invention is resistant to aluminum up to 200 mM depending on the strain, and has higher resistance than the above-mentioned conventional techniques. Further, the prior art does not describe the detoxification / removal effect of aluminum ions by the isolated bacteria, and the detoxification / removal ability of aluminum ions by the isolated strains was shown for the first time in the present invention.

Thus, by detoxifying aluminum ions using the microorganism of the present invention, it becomes possible to improve the acidic environment and improve the productivity of plants.

[Brief description of drawings]

Figure 1: Aspergillus flavus Link F-6b (FER
Micrograph of MP-17723).

FIG. 2 Penicillium sp. F-8b (FERM P-
17724).

Figure 3: Penicillium janthinellum Biourge F-1
3 (FERM P-17725) micrograph.

FIG. 4 Trichoderma asperellum F-15 (FER
Micrograph of MP-17726).

FIG. 5: Rhodotorula glutinis Y-2a (FERM
P-17727) micrograph.

FIG. 6 Cryptococcus humicola Y-6 (FERM
P-17728) micrograph.

FIG. 7 is a view showing changes in pH, aluminum ions, and total aluminum of a medium during the course of culturing F-13.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 1/14 C12R 1:80 C12R 1:80) 1: 885 (C12N 1/14 C12N 1/16 C12R 1: 885) C12R 1: 645 (C12N 1/16 C12R 1: 645) (56) Reference JP 5-227979 (JP, A) JP 7-213279 (JP, A) JP 10-271990 (JP, A) ) FEMS Microbiol. Le tt. , Vol. 189, No. 2, pp. 143-147, 2000 Journal of Biotecnology, Vol. 27, pp. 91-116, 1993 (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 1/00 C12N 1/14 C12N 1/16 BIOSIS / WPI (DIALOG) JSTPlus file (JOIS)

Claims (7)

(57) [Claims] 1. An Aspe having acid resistance and aluminum resistance.
rgillus flavus Link F-6b (FERM P-177
23). 2. A peni having acid resistance and aluminum resistance.
cillium sp. F-8b (FERM P-17724). 3. Peni having acid resistance and aluminum resistance
cillium janthinellum Biourge F-13 (FERM
P-17725). 4. Tric having acid resistance and aluminum resistance
hoderma asperellum F-15 (FERM P-1772
6). 5. Rhod having acid resistance and aluminum resistance
otorula glutinis Y-2a (FERM P-1772
7). 6. Cryp having acid resistance and aluminum resistance
tococcus humicola Y-6 (FERM P-1772
8). 7. A method for removing aluminum ions in an acidic environment, comprising the step of growing the microorganism according to claim 1 in the acidic environment.