JPS6155952B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for decomposing styrenic compounds using specific microorganisms. Styrenic compounds, such as styrene monomer, are used as raw materials for general-purpose resins such as polystyrene and styrene-butadiene rubber, and styrene oxide is used as a raw material for β-phenethyl alcohol (fragrance), but both are highly toxic. , is a compound that poses problems in terms of pollution control and occupational health. These compounds are difficult to decompose, and to date there has only been one example of microbial decomposition of styrene monomer (Appl.
Environ.Microbiol., 35 , 124 ('78)), but the decomposition rate is extremely slow. Furthermore, no attempt has been made to microbially decompose styrene oxide. An object of the present invention is to provide a method for rapidly decomposing highly toxic styrene compounds selected from styrene monomers and styrene oxide using microorganisms that have a high ability to decompose these compounds. The present invention uses a styrene compound selected from styrene monomers and styrene oxide,
The present invention provides a method for microbial decomposition of styrenic compounds, which comprises contacting with a microorganism belonging to the genus Pseudomonas that has the ability to decompose the styrenic compounds, or a culture thereof, to decompose the styrenic compounds. Styrene monomer and styrene oxide are contained in wastewater and the atmosphere in fields such as the petrochemical industry and the fragrance industry, and from an occupational health standpoint, it is important to remove these compounds and purify the environment. It is said that In the present invention, these styrene compounds, if present in the atmosphere, are dissolved in a liquid such as water, and the solution or styrene compound-containing wastewater is further concentrated or diluted to a concentration suitable for microbial decomposition. . That is, the concentration of the styrene compound is 1 to 100,000 ppm, preferably 10 to 100,000 ppm.
After adjusting the concentration to 50,000 ppm, other nutrients are added as necessary and brought into contact with microorganisms. In order to find microorganisms that can decompose styrene compounds, the present inventors isolated microorganisms from soil etc. in various places and investigated their ability to decompose styrene compounds. As a result, it was found that microorganisms belonging to the genus Pseudomonas have this ability Pseudomonas 305-STR-1-4 strain isolated from soil in southern Chiba Prefecture,
It has been found that strains such as the 311-STWR-1-2 strain are suitable for the purpose of the present invention. Pseudomonas 305-STR-1-4 strain and
The mycological properties of the 311-STWR-1-2 strain are as shown below. Pseudomonas 305-STR-1-4 a Morphological properties (1) Shape Bacillus Size 0.7×1-1.5μ (2) Pleomorphism None (3) Motility None Flagella None (4) Spores No offspring (5) Gram stain negative (6) Anti-acidity Noneb Culture properties (1) Meat juice agar plate Good growth, internal shape, smooth surface, ridges, entire periphery, homogeneous content, pale yellowish brown , moist light (2) Meat juice agar slope Good growth, stringy (3) Meat juice liquid Good growth, slightly cloudy,
Ring formation, sediment present (4) Meat juice gelatin puncture Poor growth, no liquefaction (5) Litmus milk Alkaline, litmus reduction, unchanged Physiological properties (1) Nitrate reduction present (2) Denitrification reaction Positive (3) ) MR test Negative (4) VP test Negative (5) Indole formation None (6) Hydrogen sulfide formation Slight (7) Starch hydrolysis ability None (8) Citric acid availability Yes (9) Ammonium With salt Nitrate availability (10) Pigment production Water-soluble fluorescent dye production in King B medium (11) Urease positive (12) Oxidase positive (13) Catalase positive (14) Growth temperature (°C) 10 ~37 (optimum 30-35) PH 5.5-9.5 (optimum 6.5-8.5) (15) Attitude toward oxygen Aerobic (16) O-F test Oxidative (17) Generation of acid from sugars See the table below and gas generation No gas generation

[Table] Pseudomonas 311-STWR-1-2 a Morphological properties (1) Shape Bacillus Size 0.7-1 x 1-1.5μ (2) Pleomorphism None (3) Motile Flagella (Number) Polar hairs (1 to 3) (4) No spores (5) Gram staining negative (6) Anti-acidity Noneb Culture properties (1) Broth agar plate Good growth, round, smooth surface, Raised, entire periphery, homogeneous content, pale yellowish brown, moist light (2) Meat juice agar slope Good growth, stringy (3) Meat liquid Good growth, strongly cloudy,
Film formation, sediment present (4) Meat juice gelatin puncture Good growth, no liquefaction (5) Litmus milk Alkalinity, litmus reduction, peptonization c Physiological properties (1) Nitrate reduction present (2) Denitrification reaction Positive (3) MR test negative (4) VP test negative (5) Indole formation None (6) Hydrogen sulfide generation Yes (7) Starch hydrolysis ability No (8) Citric acid availability Yes (9) ) Ammonium salt present Nitrate availability (10) Pigment formation None (11) Urease positive (12) Oxidase positive (13) Catalase positive (14) Growth temperature (°C) 10-37 (optimal 30- 35) PH 6-9.5 (optimal 7-8.5) (15) Attitude towards oxygen Aerobic (16) O-F test Oxidative (17) Generation of acid from sugars See the table below and generation of gas No gas generation

【table】

[Table] Based on the above mycological properties, Bergey's Manual of Determinative Bacteriology
Determinative Bacteriology) 7th and 8th edition
A search of the editions revealed that these strains belong to the genus Pseudomonas. In addition, these strains are Pseudomonas 305-STR-1-4 strains (Feikoken Bacteria Collection No. 4507)
and Pseudomonas 311-STWR-1-2 strain (Fiber Science and Technology Research Institute No. 4508) are kept at the Institute of Microbial Technology, Agency of Industrial Science and Technology. In the present invention, all bacteria belonging to the genus Pseudomonas and having the ability to decompose the styrene compounds can be used, as well as the above-mentioned strains, as well as their artificial and natural mutant strains. The microorganisms used in the present invention can grow in a medium containing the styrene compound as a main carbon source. As mentioned above, the concentration of the styrene compound in the medium is 1 to 100,000 ppm, preferably 10 to 100,000 ppm.
Although the amount is adjusted to 50,000 ppm, the compound may be added at once, or may be added in several portions. The nitrogen source is not particularly limited, but potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphorus, polypeptone, etc. can be used. Further, disodium phosphate, monopotassium phosphate, etc. can be used as the inorganic salt, and magnesium sulfate, calcium chloride, ferrous sulfate, etc. can be used as the trace metal.
Furthermore, a surfactant, an antifoaming agent, etc. may be added as necessary. Culture methods include shaking culture method,
A method using a liquid medium such as a deep aeration agitation culture method is suitable. It is appropriate that the culture temperature is 25 to 35°C, the culture pH is near neutral, and the number of days of culture is 2 to 5 days. According to the present invention, a styrene compound selected from styrene monomers and styrene oxide is decomposed by contacting with the above-mentioned microbial cells or a culture thereof, and almost all of them are decomposed after about 48 hours. Therefore, in fields such as the petrochemical industry and the fragrance industry, styrene compounds present in the atmosphere and industrial wastewater can be quickly decomposed and detoxified by a simple process of contacting them with microorganisms. Therefore, the method of the present invention is very useful from the standpoint of pollution control and improvement of the working environment. Next, the present invention will be explained in detail with reference to examples. Experimental example A strain belonging to the genus Pseudomonas isolated by the present inventors was treated with NH ₄ NO ₃ 0.2%, Na ₂ HPO ₄・12H ₂ O.
0.15%, KH ₂ PO ₄ 0.15%, MgSO ₄・7H ₂ O 0.02
%, _CaCl2・_2H2O 0.001%, _FeSO4・_7H2O
0.1 in 5 ml of basal medium containing 0.0001%, yeast extract 0.01% and corn steep liquor 0.01%
One platinum loop was inoculated into a pH 7.0 medium (20 x 200 mm test tube) containing 1 ml of styrene, and cultured with shaking at 30°C for 3 days. As a result, it was found that most of the bacterial strains assimilated styrene and lowered the pH of the culture medium.
The results are shown in Table-3.

[Table] Example 1 Pseudomonas strain 305-STR-1-4 (Feikoken Bibori No. 4507) was mixed with 2% styrene, 0.5% urea, 0.15% disodium phosphate (12 hydrate), and monopotassium phosphate. 0.15%, magnesium sulfate (heptahydrate) 0.02
%, calcium chloride (dihydrate) 0.001%, ferrous sulfate (heptahydrate) 0.0001%, corn staple
The cells were inoculated into 50 ml of a pH 7.0 medium (500 ml Sakaguchi Kolben) containing 0.02% liquor, and cultured with shaking at 30°C for 3 days. The results are shown in FIG. In the figure, A indicates the degree of decomposition of the styrene monomer, and B indicates the growth of bacterial cells measured by turbidity (OD ₆₁₀ ). a and b indicate the styrene monomer decomposition degree and turbidity, respectively, when no bacteria are inoculated. The decrease in styrene monomer in this case is mainly due to volatilization. Note that the remaining amount of styrene was determined by gas chromatography. Example 2 Pseudomonas 305-STR-1-4 strain (same as Example 1) was mixed with 1% styrene and 0.1% polypeptone.
%, ammonium nitrate 0.1%, disodium phosphate (12 hydrate) 1%, monopotassium phosphate 0.55%, magnesium sulfate (7 hydrate) 0.02%, calcium chloride (dihydrate) 0.001%, ferrous sulfate ( 7 hydrate salt)
50ml (500ml) PH7.0 medium containing 0.0001% yeast extract, 0.01% corn steep liquor
Sakaguchi Kolben) and cultured with shaking at 30°C for 40 hours. The cells obtained by centrifugation from this culture solution were washed twice with 0.01M phosphate buffer (PH7.5), and then suspended in the same buffer to obtain a resting cell suspension. Prepared. This suspension of resting bacterial cells and a styrene-containing phosphate buffer were mixed in a test tube to make a total of 1 ml, and the tube was tightly stoppered. The reaction solution composition was bacterial cells (OD ₆₁₀ = 2.0), 500 μg of styrene,
Consists of 1 ml of 0.01 molar phosphate buffer. Reaction at 30℃
The test was carried out under shaking conditions. The results are shown in Table 4, and 37.6% of styrene was decomposed by microorganisms in 18 hours of reaction.

[Table] −
Remaining amount of ethylene Remaining amount of tyrene =

Claims (1)

[Claims] 1. A styrene compound selected from styrene monomers and styrene oxide is brought into contact with a microorganism belonging to the genus Pseudomonas that has the ability to decompose the styrenic compound, or a culture thereof, to decompose the styrenic compound. A microbial decomposition method for styrenic compounds characterized by the following. 2. Pseudomonas 305 is a microorganism that has the ability to decompose styrene compounds belonging to the genus Pseudomonas.
The method according to claim 1, which is the STR-1-4 strain (Feikoken Bibori No. 4507). 3. Pseudomonas 311 is a microorganism that has the ability to decompose styrene compounds belonging to the genus Pseudomonas.
The method according to claim 1, which is STWR-1-2 strain (Feikoken Bibori No. 4508).