JPH05237200A - Method for decomposing hydrophilic acylic acid-based polymer by microorganism - Google Patents

Abstract

PURPOSE:To enable the decomposition of a hydrophilic acrylic acid polymer by using bacteria by culturing the bacteria belonging to the Archrobacter which are separated from soil, etc., and are identified in a medium using the acrylic acid-based polymer as a carbon source. CONSTITUTION:The Archrobacter bacteria which are separated from the soil, sludge, factory waste water, etc., and are identified are cultured under aerobic conditions in the medium contg. the acrylic acid-based polymer. The once cultured bacterial substances are collected and are then brought into contact with the acrylic acid-based polymer in an aq. soln. As a result, the hydrophilic acrylic acid-based polymer is subjected to the biodegradation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for assimilating or degrading an acrylic acid polymer by a microorganism. More specifically, the present invention relates to a method for degrading a hydrophilic acrylic acid-based polymer by using a bacterium belonging to the genus Arthrobacter having the ability to degrade the hydrophilic acrylic acid-based polymer.

[0002]

2. Description of the Related Art In recent years, hydrophilic acrylic acid polymers have been used in a wide range of applications. For example, fiber processing, thickeners, dispersants, emulsifiers, papermaking, water treatment flocculants, soil conditioners, slurries. Applications are expanding in many fields such as inhibitors, and the amount used is increasing. Acrylic acid-based polymers are usually harmless, but if a large amount of the polymers are discarded, it is not preferable in terms of environmental pollution, and development of an appropriate treatment method is desired.

Since the environmental impact of synthetic polymer waste has come to be regarded as a problem, various studies have been conducted with the aim of searching for degrading microorganisms and developing microbial treatment methods. Microorganisms that decompose polymers such as polyester, nylon, polyethylene glycol, and polyvinyl alcohol have been discovered. However, to date, there have been few reports of microbial degradation of hydrophilic acrylic acid-based polymers.

Matsumura et al. Investigated the biodegradability of sodium polyacrylate using general activated sludge and found that the biochemical oxygen demand (BOD ₅ ) ÷ sodium polyacrylate having an average molecular weight of 520
Theoretical oxygen demand (TOD) x 100 is 8%, average molecular weight is 1070
Biological oxygen demand (BOD ₅ ) of sodium polyacrylate in
Theoretical oxygen demand (TOD) x 100 has been obtained as 1.3% (Proceedings of Polymer, Volume 45, 317 pages, 1988)
However, there were no detailed reports on the degradation of acrylic acid-based polymers using bacteria, which have excellent ability to degrade polymers.

[0005]

Accordingly, the present invention is intended to provide a method for degrading a hydrophilic acrylic acid type polymer by using a specific bacterium.

[0006]

[Means for Solving the Problems] As a result of broadly searching the natural world for microorganisms capable of decomposing acrylic acid-based polymers, the present inventors newly isolated and identified Arthrobacter from the soil. It was found that bacteria belonging to the genus are decomposed by culturing them in a medium containing an acrylic acid-based polymer as a carbon source, and completed the present invention.

[0007]

[Detailed Description] In the acrylic acid-based polymer of the present invention, the basic main chain is represented by the following formula:

[Chemical 1] (Where X represents hydrogen, a monovalent metal, a divalent metal, a trivalent metal, an ammonium group, or an organic ammonium group), an acrylic acid homopolymer or a monovalent metal salt thereof, Included are divalent metal salts, trivalent metal salts, ammonium salts, or organic amine salts that are hydrophilic. The acrylic acid-based polymer referred to in the present invention further includes those modified by being treated by the method as described herein.

Further, a hydrophilic polymer obtained by copolymerizing with acrylic acid using a compound copolymerizable with acrylic acid can be a target polymer of the decomposition method according to the present invention. The compound copolymerizable with acrylic acid used in the present invention, various compounds can be used within a range in which the polymer copolymerized with acrylic acid does not lose water solubility, and specific examples thereof include, for example,

Methacrylic acid, maleic acid, crotonic acid,
Monoethylenically unsaturated carboxylic acids such as fumaric acid, itaconic acid, citraconic acid and aconitic acid, and their monovalent metal salts, divalent metal salts, trivalent metal salts, ammonium salts and organic amine salts; ethylene, propylene , Isobutylene, n-butylene and other α-olefins having 2 to 4 carbon atoms;

Acrylic acid and methacrylic acid such as methyl (meth) acrylate, ethyl (meth) acrylate, isopropyl (meth) acrylate, n-propyl (meth) acrylate, n-butyl (meth) acrylate and isobutyl (meth) acrylate. Carbon number of 4-8
An alkyl ester of hydroxyethyl (meth) acrylate, hydroxy-n-propyl (meth) acrylate, hydroxyisopropyl (meth) acrylate, hydroxy-n-butyl (meth) acrylate, hydroxyisobutyl (meth) acrylate, etc. C5-C8 hydroxyalkyl ester of methacrylic acid;

Polyether mono (meth) acrylate having 6 to 104 carbon atoms such as polyethylene glycol mono (meth) acrylate, polypropylene glycol mono (meth) acrylate, polyethylene glycol polypropylene glycol mono (meth) acrylate; 2-sulfoethyl (meth) Acrylate, 2-sulfopropyl (meth) acrylate, 3-sulfopropyl (meth) acrylate, 1-sulfopropan-2-yl (meth) acrylate, 2-sulfobutyl (meth) acrylate, 3-
Sulfobutyl (meth) acrylate, 4-sulfobutyl (meth) acrylate, 1-sulfobutan-2-yl (meth) acrylate, 1-sulfobutan-3-yl (meth) acrylate, 2-sulfobutan-3-yl (meth) acrylate, 2-methyl-2-sulfopropyl (meth) acrylate, 1,1-dimethyl-2-sulfoethyl (meth) acrylate and the like, sulfoalkyl (meth) acrylates having 4 to 10 carbon atoms;

Sulfoethoxy polyethylene glycol mono (meth) acrylate, sulfopropoxy polyethylene glycol mono (meth) acrylate, sulfobutoxy polyethylene glycol mono (meth) acrylate,
Sulfoethoxy polypropylene glycol mono (meta)
Sulfoalkoxypolyalkylene glycol mono (meth) acrylates having 7 to 197 carbon atoms such as acrylate, sulfopropoxy polypropylene glycol mono (meth) acrylate, sulfobutoxy polypropylene glycol mono (meth) acrylate, and monovalent metal salts thereof, Divalent metal salts, trivalent metal salts, ammonium salts and organic amine salts;

Polyethylene glycol monoallyl ether, polypropylene glycol monoallyl ether, polyethylene glycol polypropylene glycol monoallyl ether, polyethylene glycol monomethallyl ether, polypropylene glycol monomethallyl ether, polyethylene glycol polypropylene glycol monomethallyl ether, etc., having 5 to 104 carbon atoms Polyalkylene glycol mono (meth) allyl ethers; vinyl acetate, propenyl acetate, and other alkenyl acetates having 4 to 7 carbon atoms (alkenyl acetates are hydrolyzed after polymerization and part or all of them are vinyl). Can be alcohol);

Aromatic vinyls having 8 to 10 carbon atoms such as styrene and p-methylstyrene; aminoethyl (meta) having 5 to 10 carbon atoms such as dimethylaminoethyl (meth) acrylate and diethylaminoethyl (meth) acrylate. ) Acrylates; (meth) acrylamides, dimethylaminopropyl (meth) acrylamides, diethylaminopropyl (meth) acrylamides, and the like, (meth) acrylamides having 3 to 12 carbon atoms; 2-acrylamide-2-
C2-C4 monoethylenically unsaturated sulfonic acids such as methyl sulfonic acid, vinyl sulfonic acid, and allyl sulfonic acid; C2-C4 monoethylenically unsaturated phosphones such as vinylphosphonic acid and allylphosphonic acid Acid; (meta)
Examples thereof include acrylonitrile, acrolein, and allyl alcohol, and one or more of these can be used.

Further, a polymer modified with a compound capable of reacting with a carboxyl group contained in the above-mentioned polymer can be an object of the decomposition method according to the present invention as long as it does not lose water solubility. The following are specific examples of compounds used for modification,

Ethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, glycerin, polyglycerin, propylene glycol, polyoxypropylene, oxyethyleneoxypropylene block copolymer, diethanolamine, triethanolamine, pentaerythritol, sorbitol, sorbitan Fatty acid ester, hydroxyacetic acid glycol monoester, lactic acid glycol monoester, hydroxypivalic acid neopentylene glycol ester,
Polyhydric alcohols such as polyvinyl alcohol and saponified polyvinyl acetate;

Poly δ-containing hydroxyl groups at both ends
Lactone polymers containing hydroxyl groups at both terminals such as caprolactone; ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, glycerol polyglycidyl ether, diglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, sorbitol polyglycidyl ether, pentaerythritol Polyglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, resorcin diglycidyl ether, 1,6-hexanediol diglycidyl ether, adipic acid diglycidyl ester, o-phthalic acid diglycidyl ester, terephthalic acid diglycidyl ester , Glycidyl ester ethers of p-hydroxybenzoic acid, etc. Rishijiru compounds;

Ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, polyethyleneimine,
Polyvalent amines such as phenylenediamine; 2,2-bishydroxymethylbutanol-tris [3- (1-aziridinyl) propionate), 1,6-hexamethylenediethyleneurea, diphenylmethane-bis-4,4'-
Polyhydric aziridines such as N, N'-diethyleneurea; polyhydric aldehydes such as glutaraldehyde and glyoxal; polyhydric isocyanates such as 2,4-toluylene diisocyanate and hexamethylene diisocyanate. Alternatively, two or more kinds are used.

Bacteria of the genus Arthrobacter used as microorganisms capable of degrading acrylic acid polymers in the present invention can be widely collected from the natural world such as soil, sludge and industrial wastewater. Specific examples of the microorganisms include the present invention. Arthrobacter sp. NO-18, which was newly isolated and identified by the present inventors from the Himeji soil, is mentioned.

Table 1 shows the mycological properties of this strain. In addition,
The mycological tests described below are performed based on the methods described in "Classification and Identification of Microorganisms" (1975) edited by Takeharu Hasegawa and "Chemical Classification Experimental Method for Microorganisms" (1982) edited by Kazuo Komagata. It was

Table 1 NO-18 strain (1) Morphology a) Bacterial shape and size Bacilli, 0.6 × 1.4 μm b) Motility c) No spores d) Gram stain positive

(2) Growth conditions in medium a) Meat broth agar plate medium 1. Shape, color and size of colony Round, approx. 1 mm, orange 2. Surface morphology Smooth 3. Uplift convex circle 4. Clear and opaque b) Broth agar slope medium 1. Good growth condition, filamentous c) Broth liquid medium 1. Good growth condition d) Broth gelatin medium 1. Good growth 2. Liquefaction of gelatin Negative

(3) Physiological properties a) Nitrate reduction negative b) Indole production negative c) Hydrogen sulfide production negative d) Pigment production positive e) Oxidase negative f) Catalase positive g) Attitude toward oxygen Aerobic h ) Use of citric acid Positive i) VP test Negative j) Methyl red test Negative k) Use of inorganic nitrogen source Positive 1) Salt tolerance up to 7% m) Growth pH 6-10 n) Growth temperature optimum 30-40 ° C o ) OF test Negative p) Sugar utilization test (+ acid is produced, -acid is not produced)

Based on the above-mentioned mycological properties, Bergey's Manual of Systematic Bacteriology No. 2
When the strain was searched based on the volume (1986), it was concluded that the strain, which is a Gram-positive bacillus, belongs to the genus Arthrobacter, and the strain was named Arthrobacter genus NO-18. did. This strain was deposited on March 20, 1991, at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and the deposit number is Micromachine Lab. No. 12115 (FERM P-12115).

In the present invention, not only the above-mentioned bacterial cells or mutants thereof, but also any bacteria belonging to the genus Arthrobacter and capable of degrading acrylic acid polymers can be effectively used. You can

The medium used for culturing the present bacterium may be either solid or liquid medium. As long as it contains a carbon source that can be assimilated by the bacterium, a suitable amount of nitrogen source, inorganic salts and the like, natural medium and synthetic medium can be used. The bacterium can grow on any of the media. This bacterium has bouillon, peptone,
Although it grows in a medium containing a large amount of a rich nutrient source such as yeast extract, it is not preferable because repeated decomposition of subculture may cause a decrease in polymer decomposing ability, and rather, an appropriate amount of yeast extract, for example, It is preferable to culture using a medium containing about 0 to 0.1% by weight of yeast extract.

The hydrophilic polymer serving as a carbon source may be the above-mentioned acrylic acid-based polymers alone or in combination of two or more, and is usually added in an amount of 0 to 10% by weight, and preferably.
It is used by adding it to the medium to a concentration of 0.001 to 2% by weight or less. Ammonium sulfate as a nitrogen source,
Inorganic nitrogen sources such as ammonium nitrate and sodium nitrate, as well as organic nitrogen sources such as peptone and casamino acid can be used if they are used in appropriate amounts. In addition, inorganic salts such as potassium salt, magnesium salt, iron salt and calcium salt may be added.

The culture is carried out under aerobic conditions and the culture temperature is 25
.About.37.degree. C. and pH of 6 to 8 are suitable. In the degradation of the acrylic acid-based polymer according to the present invention, under the culture conditions as described above, Arthrobacter bacteria capable of degrading the acrylic acid-based polymer are aerobically aerated in the culture containing the acrylic acid-based polymer. It may be cultured under the conditions.
Alternatively, the acrylic acid-based polymer can be decomposed by collecting the once-cultured bacterial cells and then contacting them with the acrylic acid-based polymer in an aqueous solution.
The concentration of acrylic acid-based polymer in these cases is
For example, it decomposes well at 0 to 10% by weight or less, and preferably 0.001 to 2% by weight or less.

Specific methods for culturing or contacting under the aerobic conditions described above include methods such as aeration and stirring, as well as methods conventionally used in aerobic wastewater treatment, such as sprinkler filtration. It is also possible to use a bed method, an immersion filter bed method, a bacterial cell immobilization method, or the like.

In addition to the batch treatment, the polymer can be continuously added by adding the polymer to the continuous system. The period required for the decomposition of the polymer varies depending on factors such as the type of acrylic acid-based polymer, the concentration, and the degree of polymerization, but usually the polymer is significantly decomposed within one month. Also, the period required for decomposition varies depending on the concentration of the decomposing bacteria in the system, and the period required for decomposition can be significantly shortened by adjusting the initial addition amount of the decomposing bacteria.
It is also possible to carry out significant polymer degradation within 5 days.

[0031]

EXAMPLES The present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to these Examples.

Example 1. Distilled water was added to soil samples collected from various places to suspend the soil, and this suspension was used in 0.1-0.2.
Add ml to 5 ml of the culture solution having the composition shown in Table 2, and add 30 ° C.
The cells were shake-cultured for about 20 days. Table 2 Sodium polyacrylate (Average molecular weight 1000) 2g Ammonium sulfate 1g 1 Potassium phosphate 0.5g Dipotassium phosphate 0.5g Magnesium sulfate heptahydrate 0.2g Sodium chloride 0.1g Yeast extract 0.1g Calcium chloride 2 Hydrate 2 mg Ferrous sulfate heptahydrate 2 mg Manganese (II) sulphate 4-5 hydrate 2 mg Zinc sulphate heptahydrate 7 mg Distilled water 1000 ml Adjust the pH to 7.0 with sodium hydroxide. ..

After culturing, 0.5 ml of the culture broth was aseptically added to 5 ml of the above-mentioned medium which had been sterilized in a conventional manner, and shake culture was carried out at 30 ° C. in the same manner. After this repeated cultivation method was repeated 4 times, the resulting culture solution was inoculated on a polymer plate medium prepared by adding 2% by weight of agar to the above medium, spread, spread, and cultured at 30 ° C for 20 days to colony. Were formed on an agar medium. Then, the generated colonies are separated into single cells,
The Arthrobacter NO-18 strain as a pure strain was obtained.

Example 2. 50 ml of the culture solution having the same composition as shown in Example 1 was aseptically placed in a 500 ml capacity Sakaguchi flask that had been sterilized beforehand.
No. 12115) (FERM P-12115) was aseptically inoculated with 5 ml of a preculture liquid, and shake culture was performed at 30 ° C. After culturing, the amount of bacteria in the culture was measured by the turbidity (OD ₆₆₀ nm) of the culture at ₆₆₀ nm. The decomposition rate of the polymer is measured by analyzing the polymer by gel permeation chromatography (GPC) using ultraviolet rays (UV210 nm) or a differential refractometer (RI) as a detector, or measuring the peak area of the polymer, or The measurement was performed by measuring the total organic carbon content (TOC) in the medium after removing the cells.

The decomposition rate of the polymer was determined by shaking the sample in which 5 ml of sterile distilled water was added in advance in the medium in place of the preculture liquid in the medium as the blank sample and shaking the medium under the same conditions. From the change, it was calculated by the following formula.

[Equation 1]

The GPC analysis was conducted under the following conditions. Column: SHODEX OHpak KB-804,803,802.5 (manufactured by Showa Denko KK) Column temperature: 40 ° C Eluent: 0.1M NaH ₂ PO ₄ + 0.2M NaCl (adjusted to pH 7.0 with NaOH) Flow rate: 1 ml / min A TOC measuring device TOC-500 (manufactured by Shimadzu Corporation) was used for the analysis. The results are shown in Table 3.

Table 3 Number of days of culture (days) Growth rate Degradation rate of polymer (%) (OD ₆₆₀ nm) GPC analysis TOC analysis 20 0.453 38 44 40 0.620 53 60

Example 3. In the culture described in Example 2, the same test was performed using polyacrylic acid soda having an average molecular weight of 2000 as the acrylic acid-based polymer, and the results are shown in Table 4. Table 4 Number of days of culture (days) Growth rate Degradation rate of polymer (%) (OD ₆₆₀ nm) GPC analysis TOC analysis 20 0.228 15 20 40 0.303 25 28

Example 4. In the culture described in Example 2, a copolymer of acrylic acid having an average molecular weight of 1000 and hydroxyethyl acrylate (composition ratio 6.1: 1 (molar ratio)) was used as the acrylic acid-based polymer at 30 ° C. After shaking culture for 30 days, the degradation rate of the polymer was 40% (TOC
Measurement).

Example 5. In the culture described in Example 2, a copolymer of acrylic acid having an average molecular weight of 1000 and hydroxyethyl methacrylate (composition ratio 3.4: 1 (molar ratio)) was used as the acrylic acid-based polymer at 30 ° C. After 30 days of shaking culture, the degradation rate of the polymer was 45%.
(TOC measurement).

Example 6. In the culture described in Example 2, a copolymer of acrylic acid and maleic acid having an average molecular weight of 1000 (composition ratio 1: 1 (molar ratio)) was used as an acrylic acid-based polymer and shaken at 30 ° C. for 30 days. When culturing was performed, the decomposition rate of the polymer was 33% (TOC measurement value).

Example 7. In the culture described in Example 2, a copolymer of acrylic acid having an average molecular weight of 1000 and polyethylene glycol monoacrylate (composition ratio 1.6: 1 (molar ratio)) was used as an acrylic acid-based polymer at 30 ° C.
After shaking culture for 30 days, the degradation rate of the polymer was
It was 46% (TOC measurement value).

Example 8. In the culture described in Example 2, a copolymer of acrylic acid having an average molecular weight of 2000 and 2-hydroxyacrylic acid (composition ratio 1: 1 (molar ratio)) was used as an acrylic acid-based polymer at 30 ° C. at 30 ° C. After shaking culture for a day, the degradation rate of the polymer was 35% (TOC measurement value)
Met.

Example 9. In the culture described in Example 2, a copolymer of acrylic acid and vinyl alcohol having an average molecular weight of 2000 as an acrylic acid-based polymer (composition ratio 9:
1 (molar ratio)) and shaking culture was carried out at 30 ° C. for 30 days, the polymer degradation rate was 27% (TOC measurement value).

Example 10. In the culture described in Example 2, a copolymer of acrylic acid and vinyl sulfonic acid having an average molecular weight of 2000 as an acrylic acid-based polymer (composition ratio 9: 1
(Mole ratio)), shaking culture was carried out at 30 ° C. for 30 days, and the polymer degradation rate was 25% (TOC measurement value).

Example 11. In the culture described in Example 2, a copolymer of sodium acrylate having an average molecular weight of 2000 and a glycoside having the following structure (composition ratio 9: 1 (molar ratio)) was used as the acrylic acid-based polymer. After shaking culture for 30 days at ℃, the degradation rate of polymer is 33% (TOC measurement value)
Met.

[0047]

[Chemical 2]

Example 12 In the culture described in Example 2, the same test was conducted for 20 days while changing the concentration of sodium polyacrylate to 0.1 to 2%. The results are shown in Table 5. Table 5 Polymer concentration (%) Growth rate Polymer degradation rate (%) OD ₆₆₀ nm GPC analysis TOC analysis 0.1 0.404 68 81 0.2 0.445 40 50 0.5 0.615 29 31 1.0 0.531 20 23 2.0 0.494 13 15

Example 13 In the culture described in Example 2, 50 ml of the preculture liquid of Arthrobacter NO-18 was added to 10000 rp.
The cells were collected by centrifugation at m for 15 minutes. The collected bacterial cells were washed 3 times with 10 mM phosphate buffer (pH 7), suspended in 5 ml of the buffer, added to 50 ml of the prepared medium, and cultured in the same manner as in Example 2 below. went. The obtained results are shown in Table 6. Table 6 Number of days of culture (days) Polymer degradation rate (%) GPC analysis TOC analysis 4 60 65 7 78 80

Comparative Example 1. In the culture described in Example 2, 5 ml of standard activated sludge was inoculated instead of inoculating Arthrobacter NO-18 cells, and shake culture was performed at 30 ° C. for 30 days. The decomposition rate was 7% (TOC (Measured value), and almost no biodegradation occurred.

[0051]

As described above, the bacterium NO-18 belonging to the genus Arthrobacter according to the present invention has the ability to satisfactorily decompose hydrophilic acrylic acid-based polymers. That is, the polymer can be effectively biodegraded.

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Claims (1)

[Claims] 1. A method for degrading an acrylic acid polymer by a microorganism, which comprises degrading a hydrophilic acrylic acid polymer using a bacterium belonging to the genus Arthrobacter .