JPH06327467A - New microorganism - Google Patents

Abstract

PURPOSE:To provide a microorganism having a function to decompose and assimilate N-(2-hydroxyethyl)iminodiacetic acid. CONSTITUTION:Achromobacter sp. H1 (CDC Vd), Achromobacter sp. H2 (CDC Vd) and Pseudomonas pickettii H3. These bacteria have a function to completely decompose and assimilate N-(2-hydroxyethyl) iminodiacetic acid. Microorganisms H1, H2 and H3 can be effectively used for the disposition of waste material or drainage containing N-(2-hydroxyethyl)iminodiacetic acid by biodegradation or the Bio-Remediation targeting the substance.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel microorganism,
More specifically, N- (2-hydroxyethyl) imino 2
The present invention relates to a novel bacterium having an ability to decompose and assimilate acetic acid.

[0002]

2. Description of the Related Art In the natural world, substance circulation is carried out by the purifying action of an ecosystem that is stably supported by the biological activities of various microbial communities. In recent years, a large amount of waste,
Persistent substances, harmful substances, etc. are released into the natural world, exerting a load on the purifying effect of the natural world, gradually but clearly,
Environmental destruction is progressing and it is a big social problem.

Under such a background, various chemical substances are required to have biodegradability, and it is necessary to treat the various chemical substances with microorganisms that biodegrade them when they are discarded. ing. In addition, the development of bio-remediation, in which various chemical substances released into the environment are decomposed and removed by utilizing their biological functions to protect the environment, is also under way.

N- (2-hydroxyethyl) iminodiacetic acid has the following structural formula:

[0005]

[Chemical 1]

[0006] It is a compound represented by, and its biodegradability has been reported (Journal of American Oil
Chemists' Society 52 , 41-43 (197)
5)), but there is no report on the degrading microorganisms.

[0007]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention A substance containing N- (2-hydroxyethyl) iminodiacetic acid for decomposing a substance or wastewater for biodegradation treatment or bioremediation targeting the substance. Utilization of microorganisms having the ability to transform becomes effective. Therefore, an object of the present invention is to provide a microorganism having the ability to decompose and assimilate N- (2-hydroxyethyl) iminodiacetic acid.

[0008]

The present inventors have extensively searched for N- (2-hydroxyethyl) iminodiacetic acid-degrading assimilating microorganisms from the natural world in order to achieve the above-mentioned object, and as a result, Hyogo Prefecture The present invention was completed by recognizing that two strains belonging to the genus Achromobacter and one strain belonging to the genus Pseudomonas obtained from soil in Itami City achieve the above object.

The N- (2-hydroxyethyl) iminodiacetic acid-degrading assimilating microorganism of the present invention is a bacterium belonging to the genus Achromobacter, particularly Achromobacter sp.
bacterium sp.) H1 (CDC group V _d ) and achromobacter sp. (Achromobacter sp.) H2
(CDC group V _d ), and bacteria belonging to the genus Pseudomonas, in particular Pseudomonas picketti (Pseu)
domonas pickettii) H3.

The above-mentioned specific strains have the following mycological properties.

The test and classification method for the mycological properties are described in "Bergey's Manual of Determinate Bacteriology".
minative Bacteriology) 8th edition (1974) ",
The Burgers Manual of Systematic
Bact (Bergey ^'s Manual of Systematic
Bacteriology (1986), "and American Diseases Control (American Disease Cot.
rol) CDC group (Centers for Disease C
ontrol).

(1) Achromobacter sp. H1 (CDC grou p V _d ) A. Form (1) Cell form Bacillus, straight or slightly curved (2) Cell size 0.3 × 1.0 -2.0 (μm) (3) Motility + (Yamaburana lateral hair, wavy) (4) Presence of spores-(5) Gram stain-(6) Anti-acid stain-B. Growth condition in each medium (1) Standard agar plate culture Light brown, gloss ant, round, smooth (2) Standard agar slope culture Light brown, gloss ant, smooth, condensed water Sanko color (3) Standard liquid culture Almost uniformly turbid (4) Standard gelatin puncture Slight turbidity only in the upper layer of culture (5) No change in litmus milk C. Physiological properties (1) Reducibility of nitrate + (2) Denitrification reaction + (3) Methyl red test − (4) Formation of acetylmethylcarbinol -(5) Indole formation- (6) Hydrogen sulfide formation- (7) Starch hydrolysis − (8) Use of citric acid − (9) Use of inorganic nitrogen source Nitrate: − Ammonium salt: − (10) Dye formation − (11) Urease activity + (12) Oxidase activity + (13) Catalase activity + ( 14) Range of growth pH 9.0 + 6.0 + 5.5 − 5.0 − Temperature 10 ° C + 30 ° C + 35 ° C − 40 ° C − (15) Attitude toward oxygen Aerobic (16) Formation of dioxyacetone − (17) Decomposition- (18) Decomposition of amino acid Lysine-Arginine-Ornithine- (19) Deamination of phenylalanine- (20) Temperature resistance (85 ° C, 10 minutes)-(21) Resistance to sodium chloride 2.0% + 5.0% -7.0 % -10%-(22) Sabouraud agar growth- (23) 0.001% lysozyme growth + (24) Tyrosine degradation + (25) Citrate / ammonium agar production on agar- (26) Casein degradation -(27) Degradability of gelatin- (28) Growth in anaerobic medium (29) MacConkey medium viability + ^W (30) lecithinase reaction - (31) an alkali-producing ability + ^W in VP medium (32) sugars available raw acidic L- arabinose + D-xylose + D-glucose + D-mannose + D-fructose + D-galactose + ^W maltose + sucrose + ^W lactose-trehalose + D-sorbit + D-mannitol + inosit-glycerin + starch-melibiose-salicin-ethanol- (33) esculin hydrolysis- (34) Oxidation of gluconic acid- (w = weak)

(2) Achromobacter sp. H2 (CDC grou p V _d ) A. Form (1) Cell form Bacillus, straight or slightly curved (2) Cell size 0.3 × 1.0 -2.0 (μm) (3) Motility + (Yamaburana lateral hair, wavy) (4) Presence of spores-(5) Gram stain-(6) Anti-acid stain-B. Growth condition in each medium (1) Standard agar plate culture Light brown, gloss ant, round, smooth (2) Standard agar slope culture Light brown, gloss ant, smooth, condensed water Sanko color (3) Standard liquid culture Almost uniformly turbid (4) Standard gelatin puncture Slight turbidity only in the upper layer of culture (5) No change in litmus milk C. Physiological properties (1) Reducibility of nitrate + (2) Denitrification reaction + (3) Methyl red test − (4) Formation of acetylmethylcarbinol -(5) Indole formation- (6) Hydrogen sulfide formation- (7) Starch hydrolysis − (8) Use of citric acid − (9) Use of inorganic nitrogen source Nitrate: − Ammonium salt: − (10) Dye formation − (11) Urease activity + (12) Oxidase activity + (13) Catalase activity + ( 14) Range of growth pH 9.0 + 6.0 + 5.5 − 5.0 − Temperature 10 ° C + 30 ° C + 35 ° C − 40 ° C − (15) Attitude toward oxygen Aerobic (16) Formation of dioxyacetone − (17) Decomposition- (18) Decomposition of amino acid Lysine-Arginine-Ornithine- (19) Deamination of phenylalanine- (20) Temperature resistance (85 ° C, 10 minutes)-(21) Resistance to sodium chloride 2.0% + 5.0% -7.0 % -10%-(22) Growth of Sabouraud agar- (23) Growth of 0.001% lysozyme medium + (24) Degradation of tyrosine + (25) Alkaline production on citric acid / ammonium agar- (26) Degradation of casein -(27) Degradability of gelatin- (28) Growth in anaerobic medium (29) MacConkey medium viability + ^W (30) lecithinase reaction - (31) an alkali-producing ability + ^W in VP medium (32) sugars available raw acidic L- arabinose + D-xylose + D-glucose + D-mannose + D-fructose + ^W D-galactose + ^W maltose + ^W sucrose + ^W lactose-trehalose + D-sorbit + D-mannitol + inosit-glycerin + starch-melibiose-salicin-ethanol- (33) esculin hydrolysis- (33 34) Oxidation of gluconic acid- (w = weak)

(3) Pseudomonas picekettii H3 A. Form (1) Cell shape Bacillus, straight or slightly curved (2) Cell size 0.5-0.7 × 1.0-1.5 (μm) (3) Motility + (Monopolar hair) (4) Presence of spores-(5) Gram stain-(6) Acid-fast stain-B. Growth state in each medium (1) Standard agar plate culture Light brown, glossy ant, Round, smooth (2) Standard agar slant culture Light brown, glossy ant, smooth (3) Standard liquid culture Almost uniformly turbid (4) Standard gelatin stab culture Slightly turbid only in the upper layer (5) Litmus milk No change C. Physiological properties (1) Reducibility of nitrate + (2) Denitrification reaction + (3) Methyl red test − (4) Formation of acetylmethylcarbinol − (5) Formation of indole − (6) Formation of hydrogen sulfide − (7) Starch hydrolysis- (8) Use of citric acid + (9) Use of inorganic nitrogen source Nitrate + Ammonium salt :-( 10) Pigment formation- (11) Urease activity + (12) Oxidase activity + (13) Catalase activity + (14) Growth range pH 9.0 + 5.0 + 4.5 + 4.0-Temperature 10 ° C + 30 ℃ + 35 ℃ + 40 ℃ − (15) Attitude toward oxygen Aerobic (16) Generation of dioxyacetone − (17) Decomposition of hippuric acid − (18) Decomposition of amino acid Lysine-Arginine-Ornithine − (19) Phenylalanine Deamination- (20) Temperature resistance (85 ° C, 10 minutes)-(21) Sodium chloride tolerance 2.0% + 5.0%-7.0% -10%-(22) Growth of Sabouraud agar- (23) 0.001% Growth of lysozyme medium + (24) Tyrosine degradation − (25) Citrate / ammonium production by alkali in agar + (26) Casein degradability − (27) Gelatin degradability − (28) Growth in anaerobic medium - (29) McConkey medium growth of + ^W (30) lecithinase anti - (31) an alkali-producing ability + ^W in VP medium (32) sugars available raw acidic L- arabinose + D-xylose + ^W D-glucose + D-mannose + D-fructose + D-galactose + Maltose - Sucrose - Lactose-trehalose + D-sorbit + D-mannitol + ^W inosit + glycerin-starch-melibiose-salicin-ethanol- (33) esculin hydrolysis- (w = weak)

From the above-mentioned mycological properties, it is considered that the H1 and H2 strains belong to the genus Achromobacter, but most of the genus Achromobacter are currently genus Pseudomonas, Alcaligenes or Acinetobacter. It has been transferred to the genus, and undecided ones are classified as CDC groups. The H1 and H2 strains show intermediate properties between Pseudomonas and Alcaligenes, and no corresponding bacterial species were found. Therefore, the strains were judged to be CDC group V _d with the closest properties, and Achromobacter sp. (C
Was designated DC Group V _d) and Achromobacter sp H2 (CDC group V _d).

The H3 strain was determined to be one strain of Pseudomonas pickettii from the mycological properties, and was named Pseudomonas picketti H3.

The above strain was deposited at the Institute for Microbial Industry, Institute of Industrial Technology, as follows. Achromobactor SP H1 (CDC group
V _d ) (Microtech Lab, No. 13521) Achromobacter sp. H2 (CDC group)
V _d ) (Ministry of Microbiological Research No. 13522) Pseudomonas Picketti H3 (No. 1
No. 3523)

The microorganisms of the present invention include the above-mentioned Achromobacter strains H1 and H2 and Pseudomonas picketti.
In addition to the H3 strain, a microorganism belonging to the genus Achromobacter or the genus Pseudomonas and having the ability to decompose and assimilate N- (2-hydroxyethyl) iminodiacetic acid is included. Further, natural or artificial mutant strains of these strains or gene fragments necessary for metabolic activity for degrading assimilation of N- (2-hydroxyethyl) iminodiacetic acid were artificially extracted, and other microbial strains incorporating them Even belonging to the microorganism of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. In addition,% is w / v% unless otherwise specified.

Example 1 Isolation of N- (2-hydroxyethyl) iminodiacetic acid-degrading assimilating strain: about 1 g of a soil sample collected in Itami City, Hyogo Prefecture, was sterilized with 50 mM phosphorus containing 0.85% NaCl. Disperse in 10 ml of acid buffer (pH 7.4), add 0.1 ml of the supernatant to 10 ml of liquid medium containing N- (2-hydroxyethyl) iminodiacetic acid, and shake aerobically at 25 ° C. Cultured. The composition of the liquid medium used is shown below. N- (2-hydroxyethyl) iminodiacetic acid 1 mM Ammonium chloride 1 mM Magnesium sulfate heptahydrate 0.02% Calcium chloride dihydrate 0.002% Phosphate buffer (pH 7.0) 50 mM (with HCl or NaOH pH adjusted to 7.0)

After the start of the culture, a part of the culture solution was sampled every 7 days, and the total amount of organic carbon (TOC) in the culture solution was measured by J
22. of IS K 0102. It was measured according to the method described in. A TOC-5000 total organic carbon meter manufactured by Shimadzu Corporation was used to measure the TOC amount. The usage of this measuring instrument was in accordance with the instruction manual.

As the culture progressed, one platinum loop of the culture solution in which the TOC amount was found to be reduced was streaked onto ordinary agar medium (Code 05514 manufactured by Nissui Pharmaceutical Co., Ltd.),
The grown colonies were classified by visual observation and purified by the same medium 2 to 3 times to obtain 36 homogenized strains.

The thus obtained strain was inoculated into the above liquid medium containing N- (2-hydroxyethyl) iminodiacetic acid, and 25
The cells were aerobically shake-cultured at 0 ° C., the TOC amount in the culture solution was measured over time by the above-mentioned method, 3 strains giving a remarkable decrease in the TOC amount were obtained, and a strain identification experiment was performed. As a result, as described above, these strains were named Achromobacter sp. H1 (CDC group V _d ), Achromobacter sp. H2 (CDC group V _d ), and Pseudomonas picketti H3.

Example 2 Examination of the substance assimilating and assimilating ability of the H1, H2 and H3 strains when N- (2-hydroxyethyl) iminodiacetic acid was used as the sole C source: obtained in Example 1. H1, H2 and H3 strains were added with N- (2-hydroxyethyl) iminodiacetic acid shown in Table 1 as a single C source to inoculate a liquid medium and as a control, the substance was inoculated into a liquid medium without addition, 25 Aerobic shake culture at ℃, TOC
Changes in quantity, viable cell count and pH were measured. The TOC amount was measured in the same manner as in Example 1, and the viable cell count was measured by diluting the sampled culture broth with 50 mM phosphate buffer (pH 7.4) containing 0.85% NaCl as appropriate, and culturing on an ordinary agar medium. (Nissui Pharmaceutical Co., Ltd. Code 05514)
It was calculated by the number of grown colonies after aerobically stationary culture at 5 ° C. for 2 days. In addition, the blank test was classified as a category in which no microorganism was inoculated.

[0025]

The results are shown in FIG. From this result, H
1, H2 and H3 strains grow only in the test plots, and
Since the number of viable bacteria increases with the decrease in OC amount,
These strains are N- (2-hydroxyethyl) imino 2
When acetic acid is used as the sole C source, it decomposes and assimilates the substance. In addition, these strains are N- (2-
Hydroxyethyl) iminodiacetic acid was completely decomposed in 5 to 6 days.

Example 3 N- (2-hydroxyethyl) iminodiacetic acid was used as a single C,
Examination of the substance-degrading and assimilating ability of the H1, H2, and H3 strains when used as the N source: The H1, H2, and H3 strains obtained in Example 1 are shown in Table 2 as N- (2-hydroxyethyl) imino. 2 acetic acid was inoculated into a liquid medium containing acetic acid as a single C and N source and a liquid medium containing no added substance as a control, and 2
Cultured aerobically at 5 ° C, and TOC as the culture progressed
Changes in the amount, viable cell count and pH were measured in the same manner as in Example 2. In addition, the blank test was classified as a category in which no microorganism was inoculated.

[0028]

The results are shown in FIG. From this result, H
1, H2 and H3 strains grow only in the test plots, and
Since the number of viable bacteria increases with the decrease in OC amount,
These strains are N- (2-hydroxyethyl) imino 2
When acetic acid is used as the sole C, N source, it decomposes and utilizes the substance. In addition, these strains were
-(2-Hydroxyethyl) iminodiacetic acid was completely decomposed in 3 to 4 days.

[0030]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, a microorganism having the ability to decompose and assimilate N- (2-hydroxyethyl) iminodiacetic acid was obtained. By using these microorganisms, N- (2-hydroxyethyl)
It is possible to effectively perform treatment by biodegradation of waste or wastewater containing iminodiacetic acid, or bioremediation targeting the substance.

[Brief description of drawings]

FIG. 1 is a graph showing the progress of culture of H1, H2 and H3 strains when N- (2-hydroxyethyl) iminodiacetic acid was used as a single C source.

FIG. 2: H1, H2 and H3 when N- (2-hydroxyethyl) iminodiacetic acid was used as the sole C, N source.
It is a graph which shows the culture progress of a strain.

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Claims (5)

[Claims] 1. N- (2-hydroxyethyl) imino 2
A microorganism belonging to the genus Achromobacter having the ability to decompose and assimilate acetic acid. 2. N- (2-hydroxyethyl) imino 2
A microorganism belonging to the genus Pseudomonas having the ability to decompose and assimilate acetic acid. 3. The microorganism is Achromobacter sp.
The microorganism according to claim 1, which is H1 (CDC V _d ) Microbiology Research Institute No. 13521. 4. The microorganism is Achromobacter sp.
The microorganism according to claim 1, which is H2 (CDC V _d ) Microbiology Research Institute No. 13522. 5. The microorganism according to claim 2, wherein the microorganism is Pseudomonas piketti H3 Microindustrial Research Institute No. 13523.