JP2001128665A - Decomposition of benzophenone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new means for decomposing benzophenone or hydroxybenzophenone. SOLUTION: A bacterium belonging to Bacillus capable of decomposing benzophenone or hydroxybenzophenone, e.g. Bacillus brevis bacterium is provided. A method for decomposing benzophenone or hydroxybenzophenone which uses the same bacterium is provided.

Description

Description: TECHNICAL FIELD The present invention relates to a Bacillus (Bacillu).
s) A novel bacterium belonging to the genus and capable of degrading benzophenone or oxybenzophenone, and a method for biologically decomposing and removing benzophenone or oxybenzophenone using the same. BACKGROUND OF THE INVENTION Benzophenone and oxybenzophenone have been generally considered to be hardly decomposable substances because they have a benzene ring. In addition, benzophenone was also designated as an endocrine disrupting substance, and there was concern about environmental pollution. However, microorganisms and treatment methods that rapidly degrade benzophenone and oxybenzophenone are not known. [0003] Accordingly, an object of the present invention is to provide a microorganism capable of decomposing benzophenone or oxybenzophenone, and a method for decomposing benzophenone or oxybenzophenone using the same. [0004] In order to achieve the above object, the present inventor has developed a microorganism which is capable of decomposing benzophenone and oxybenzophenone and which does not die during storage from nature. Thus, a bacterial strain having these capabilities was isolated from seawater, and the bacterial strain was identified, thereby completing the present invention. That is, the present invention provides a bacterium belonging to the genus Bacillus, which has the ability to degrade benzophenone or oxybenzophenone. The present invention also provides a method for decomposing benzophenone or oxybenzophenone, which comprises causing a bacterium belonging to the genus Bacillus having the ability to degrade benzophenone or oxybenzophenone or a processed product thereof to act on benzophenone or oxybenzophenone. [0006] The present invention also provides a method for treating wastewater containing benzophenone or oxybenzophenone, wherein a culture of a bacterium belonging to the genus Bacillus having the ability to degrade benzophenone or oxybenzophenone is added to the treatment tank. . The present invention also relates to a method of treating wastewater containing benzophenone or oxybenzophenone using activated sludge, wherein the microorganisms constituting the activated sludge contain bacterium belonging to the genus Bacillus having the ability to degrade benzophenone or oxybenzophenone. Provide a way. [0007] The present invention further provides a method for treating wastewater using a biofilm, wherein the microorganisms constituting the biofilm include a bacterium belonging to the genus Bacillus having the ability to degrade benzophenone or oxybenzophenone. provide. As the above Bacillus bacteria,
Examples include Bacillus brevis bacteria, and specific examples include Bacillus brevis GU-1.
(FERM P-17615). BEST MODE FOR CARRYING OUT THE INVENTION A microorganism capable of decomposing benzophenone or oxybenzophenone may be any microorganism belonging to the genus Bacillus and having the above-mentioned activity, and is preferably a bacterium belonging to the species Bacillus brevis. It is. Such bacteria have already been isolated and
C, may be selected from the bacterial species retained in IFO and the like by the method described herein, or may be isolated from the natural world such as soil, drainage, and seawater. Next, examples isolated in the present invention will be specifically described. The separation was performed by culturing a sample as a separation source in a medium containing benzophenone or oxybenzophenone as a sole carbon source, and collecting the growing microorganism. Examples of a medium containing benzophenone or oxybenzophenone as a sole carbon source include, for example, benzophenone or oxybenzophenone 0.01%, ammonium nitrate
0.2%, dipotassium phosphate 0.2%, monosodium phosphate
0.1%, magnesium sulfate 0.05%, calcium chloride 0.00
Liquid medium containing 3% and ferrous sulfate 0.003% (pH 6.8)
Alternatively, add agar medium containing 1.5% agar to this at 121 ° C for 15 minutes.
For example, those sterilized by autoclave for one minute can be exemplified. In order to isolate microorganisms from seawater, 1 mL of seawater collected from the natural world was dropped into 100 mL of the above liquid medium, followed by shaking culture at a culture temperature of 30 ° C. for 7 days. Next, apply 0.1 mL of the diluted solution (100-fold dilution) after shaking culture to 15 mL of the above agar medium on the surface of the medium.
The colonies that grew after cultivation in the above were picked. [0011] The strain obtained by inoculation is inoculated into the liquid medium,
After shaking culture at 30 ° C., benzophenone or oxybenzophenone remaining in the medium was extracted with methanol, and the ability of the strain to degrade oxybenzophenone was measured. As a result, one strain of Bacillus brevis GU-1 (Bacillus brevi) isolated from seawater in Kanazawa Ward, Yokohama City, Kanagawa Prefecture
s GU-1) strain had the resolution of benzophenone and oxybenzophenone. Next, the Bacillus brevis GU-1 of the present invention is used.
The bacteriological properties of the (Bacillus brevis GU-1) strain will be described. The test and classification method for mycological properties are described in Bergey's Manual of Systematic Bacteriolog.
y) (1984). (1) Morphology 1. Morphology and size of cells: 0.5 μm in width and 1 μm in length
2. Spores: formation 3. Motility: no 4. Gram stain: negative [2] Physiological properties 1. Oxidase: negative 2. Catalase: positive 3. VP reaction: positive 4. Nitrate reduction: positive 5. Use of Glucose: Positive 6. Use of Fructose: Positive 7. Use of Mannitol: Positive 8. Use of Sorbitol: Positive 9. Use of L-Arabitol: Positive As a result of identification based on the classification method, it was determined to be a bacterium belonging to the genus Bacillus. Compared to known strains of the genus Bacillus, the strain of the present invention is very similar to Bacillus brevis. But,
The strain of the present invention differs from Bacillus brevis in that it does not have the biodegradability of benzophenone or oxybenzophenone. As described above, since the strain of the present invention is distinguished from known strains, Bacillus b.
revis) and named Bacillus brevis GU-1 strain in 1999.
On October 19, they were deposited with the Ministry of International Trade and Industry at the Institute of Biotechnology and Industrial Technology under the microorganism accession number FERM P-17615. The Bacillus brevis GU-1 (Ba
Cillus brevis GU-1) The nutrient source of the medium used for culturing the strain is necessary for the growth of microorganisms such as a carbon source, a nitrogen source, and an inorganic salt. Any material can be used as long as it can be cultured by a usual culture method. Preferably, peptone or meat extract as a source of organic nitrogen, yeast extract, etc., ammonium nitrate or sodium nitrate as a source of inorganic nitrogen, dipotassium phosphate, monosodium phosphate, magnesium sulfate, calcium chloride, ferrous sulfate as an inorganic salt It is desirable to culture aerobically under conditions of pH of the medium of 4 to 9, preferably 5 to 7, and culture temperature of 15 to 40 ° C, preferably 25 to 37 ° C. It is desirable to continue subculture using benzophenone or oxybenzophenone as a carbon source in order to maintain excellent biodegradability of benzophenone and oxybenzophenone of this strain. Also, when producing cells for degrading benzophenone and / or oxybenzophenone, it is preferable to add benzophenone and / or oxybenzophenone to the medium in order to obtain cells having high resolution. The addition amount is preferably 0.001 to 1.0%. The Bacillus brevis GU-1 (Ba of the present invention)
The cillus brevis GU-1) strain has the ability to degrade benzophenone and oxybenzophenone. Therefore, these compounds can be decomposed by allowing the bacterium itself or a processed product thereof to act on benzophenone and / or oxybenzophenone. Examples of the processed product include crushed cells, immobilized cells, and the like. As an example of an embodiment in which the above-mentioned cells and the like are allowed to act on benzophenone and / or oxybenzophenone, bacterium itself or a culture thereof is mixed with activated sludge to give the activated sludge itself benzophenone and oxybenzophenone decomposability. can do. Further, by using this strain for a biofilm, the biofilm itself can be imparted with the ability to degrade benzophenone and oxybenzophenone. In addition, as a wastewater treatment method using a biofilm, a trickling filter method, a rotating plate biological method, a contact detonation method, and the like can be exemplified. The Bacillus brevis GU-1 (Ba
cillus brevis GU-1) has an excellent ability to degrade benzophenone and oxybenzophenone.For example, a microorganism-containing agent obtained by mixing a culture of this bacterium and a microbial carrier such as bran is transported to a wastewater treatment facility and directly By introducing the wastewater into the detonation tank, an excellent treatment effect of wastewater containing benzophenone or oxybenzophenone can be easily achieved with the existing facility. In the present invention, Bacillus brevis
In addition to the GU-1 (Bacillus brevis GU-1) strain, this Bacillus brevis GU-1 strain is exposed to ultraviolet light or the like as long as it has the same or better benzophenone and oxybenzophenone resolution as the Bacillus brevis GU-1 strain. Mutants that have been mutated can also be used. EXAMPLES Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. Example 1 0.01% benzophenone, dipotassium hydrogen phosphate
0.65%, potassium dihydrogen phosphate 0.26%, disodium hydrogen phosphate dodecahydrate 1.3%, ammonium chloride 0.051%, magnesium sulfate heptahydrate 0.675%, calcium chloride 0.875%
100 mL of a sterile liquid medium (pH 7.2) containing 0.0075% of ferrous chloride hexahydrate and a fermented chloride in a 500 mL shake flask, sterilized in an autoclave at 121 ° C for 15 minutes, cooled to room temperature, and then cooled to room temperature.・ Brevis GU-1 (Bacill
us brevis GU-1) strain (FERM P-17615), and cultured in a shaking incubator at 30 ° C. for 28 days. Sampling was performed after 1 day, 3 days, 7 days, 14 days, 21 days and 28 days of culture, and the residual amount of benzophenone was measured. In the control group, those to which no bacteria were inoculated were measured. For the measurement of benzophenone, 9 mL of methanol was added to 1 mL of the culture solution, and the mixture was stirred well, and then the residual amount of benzophenone was measured by liquid chromatography at an ultraviolet absorption wavelength of 254 nm. The residual ratio of benzophenone over time was determined with the measured value at the start of culture as 100%. FIG. 1 shows the measurement results of the residual amount of benzophenone. As is apparent from FIG. 1, the Bacillus brevis GU-1 strain (FERM P-176) of the present invention is used.
15) Compared with the control test group, the residual ratio of benzophenone decreased over time in the added test group, and became almost 0% in 10 days.
Considering the above results, the Bacillus brevis GU-1 strain (FERM P-1761) of the present invention is considered.
It is clear that 5) biodegrades benzophenone. EXAMPLE 2 Inflow of actual wastewater (raw water) containing 100 ppm of benzophenone, 1 L / day, activated sludge suspended matter (MLS
S) (Mixed Liquor Suspended Solid) 5000ppm, room temperature 2
At 5 ° C, the Bacillus brevis GU of the present invention is installed in a culture device in which an aeration tank (capacity 2 L) and a precipitation tank (capacity 0.5 L) are separated.
-1 (Bacillus brevis GU-1) strain (FERM P-17615) was inoculated and benzophenone was measured. In the control test group, Bacillus brevis GU-1 (Bacillus
brevis GU-1) strain (FERM P-17615), except that the inoculation was not performed. Raw water adjusted to pH 7 was used. For the measurement of benzophenone, 9 mL of methanol was added to 1 mL of the culture solution, stirred well, and the residual amount of benzophenone was measured by liquid chromatography at an ultraviolet absorption wavelength of 254 nm. The residual ratio of benzophenone over time was determined with the measured value at the start of culture as 100%. FIG. 2 shows the measurement results of the detected amount of benzophenone. As can be seen from FIG. 2, the Bacillus brevis GU-1 strain (FERM P-176) of the present invention.
15) The benzophenone content of the added test group decreased over time compared to the control test group and raw water. Considering the above results, the Bacillus brevis GU-1 of the present invention is considered.
It is clear that the (Bacillus brevis GU-1) strain (FERM P-17615) biodegrades benzophenone. Example 3 Oxybenzophenone 0.01%, dipotassium hydrogen phosphate 0.65%, potassium dihydrogen phosphate 0.26
%, Disodium hydrogen phosphate dodecahydrate 1.3%, ammonium chloride 0.051%, magnesium sulfate heptahydrate 0.675%, calcium chloride 0.875% and ferrous chloride hexahydrate 0.0075%
100 mL of a sterilized liquid medium (pH 7.2) containing the mixture was placed in a 500 mL shake flask, sterilized in an autoclave at 121 ° C. for 15 minutes, cooled to room temperature, and then cooled to the room temperature of Bacillus brevis GU-1 of the present invention. ) Strain (FERM P-17615)
And cultured at 30 ° C. for 28 days in a shaking incubator. Sampling was performed after 1 day, 3 days, 7 days, 14 days,
After 21 days and 28 days, the residual amount of oxybenzophenone was measured. In the control group, those to which no bacteria were inoculated were measured. The measurement of oxybenzophenone was performed using 1 m of the culture solution.
9 mL of methanol was added to L, and the mixture was stirred well, and then the residual amount of oxybenzophenone was measured by liquid chromatography at an ultraviolet absorption wavelength of 254 nm. With respect to oxybenzophenone, the residual value over time was determined with the measured value at the start of culture as 100%. FIG. 3 shows the measurement results of the residual amount of oxybenzophenone. As is clear from FIG. 3, the Bacillus brevis GU-1 strain (FERM) of the present invention was used.
P-17615) In the test group, the residual ratio of oxybenzophenone decreased over time compared to the control test group.
%Became. From the above results, it is clear that the Bacillus brevis GU-1 strain (FERM P-17615) of the present invention biodegrades oxybenzophenone. Example 4. Inflow of actual wastewater (raw water) containing 100 ppm of oxybenzophenone (1 L / day), activated sludge suspended matter (MLSS) 5000 ppm, room temperature 25 ° C., aeration tank (capacity 2 L) and sedimentation tank (capacity) (0.5 L) was inoculated with the Bacillus brevis GU-1 strain (FERM P-17615) of the present invention, and oxybenzophenone was measured. In the control test plot, the Bacillus brevis GU-1 strain (FERM) of the present invention was used.
The same operation was performed except that the inoculation of P-17615) was not performed. Raw water adjusted to pH 7 was used. The measurement of oxybenzophenone was performed by
9 mL of methanol was added to L, and the mixture was stirred well, and then the residual amount of oxybenzophenone was measured by liquid chromatography at an ultraviolet absorption wavelength of 254 nm. With respect to oxybenzophenone, the residual value over time was determined with the measured value at the start of culture as 100%. FIG. 4 shows the measurement results of the detected amount of oxybenzophenone. As can be seen from FIG. 4, the Bacillus brevis GU-1 strain (FERM) of the present invention was used.
P-17615) The oxybenzophenone content of the added test group decreased over time as compared with the control test group and raw water. Considering the above results, the Bacillus brevis GU-1 strain (FERM P-1) of the present invention is considered.
7615) clearly degrades oxybenzophenone. Example 5. Benzophenone 0.01%, dipotassium hydrogen phosphate 0.65%, potassium dihydrogen phosphate 0.26%, disodium hydrogen phosphate dodecahydrate 1.3%, ammonium chloride 0.051%, magnesium sulfate heptahydrate 100 mL of a sterilized liquid medium (pH 7.2) containing 0.675%, 0.875% calcium chloride, and 0.0075% ferrous chloride hexahydrate in a 500mL shake flask, autoclave sterilization at 121 ° C for 15 minutes, and then to room temperature. After cooling, 0.1 mL of the culture solution on the 28th day of Example 1 was inoculated and cultured at 30 ° C. for 28 days in a shaking incubator. Sampling was performed after 1 day, 3 days, 7 days, and 14 days of culture.
After 21 days and after 28 days, the residual amount of benzophenone was measured. In the control group, the same operation was performed except that the culture solution of Example 1 was not inoculated. For the measurement of benzophenone, 9 mL of methanol was added to 1 mL of the culture solution, stirred well, and the remaining amount of benzophenone was measured by liquid chromatography at an ultraviolet absorption wavelength of 254 nm. The residual ratio of benzophenone over time was determined with the measured value at the start of culture as 100%. FIG. 5 shows the measurement results of the residual amount of benzophenone. As can be seen from FIG. 5, the residual ratio of benzophenone decreased over time in the test group with the culture solution added 28 days after Example 1 of the present invention as compared with the control test group, and became almost 0% in 10 days. From the above results, it is clear that benzophenone biodegrades even when a culture solution in which the decomposition of benzophenone is once completed in Example 1 is newly inoculated into a culture solution containing benzophenone. Example 6 Inflow rate of actual wastewater (raw water) containing 100 ppm of benzophenone, 1 L / day, MLSS 5000 ppm, room temperature
At 25 ° C., 10 mL of the culture solution 28 days after the addition test in Example 2 was inoculated into a culture apparatus in which an aeration tank (capacity: 2 L) and a precipitation tank (capacity: 0.5 L) were separated, and benzophenone was measured. In addition, the control test group used the same operation except that the culture solution of Example 2 was not inoculated. Raw water used was adjusted to pH 7. For the measurement of benzophenone, 9 mL of methanol was added to 1 mL of the culture solution, stirred well, and the residual amount of benzophenone was measured by liquid chromatography at an ultraviolet absorption wavelength of 254 nm. The residual ratio of benzophenone over time was determined with the measured value at the start of culture as 100%. FIG. 6 shows the measurement results of the detected amount of benzophenone. As can be seen from FIG. 6, the culture solution addition test group 28 days after Example 2
The benzophenone content decreased over time as compared to the control test group and raw water. Considering the above results, even if the culture solution in which the decomposition of benzophenone was once completed in Example 2 was newly inoculated into the culture solution containing benzophenone,
It is clear that benzophenone biodegrades. Example 7. Oxybenzophenone 0.01%, dipotassium hydrogen phosphate 0.65%, potassium dihydrogen phosphate 0.26
%, Disodium hydrogen phosphate dodecahydrate 1.3%, ammonium chloride 0.051%, magnesium sulfate heptahydrate 0.675
%, Calcium chloride 0.875%, ferrous chloride hexahydrate 0.00
500 mL of sterile liquid medium (pH 7.2) containing 75%
L The mixture was placed in a shake flask, sterilized in an autoclave at 121 ° C. for 15 minutes, cooled to room temperature, then inoculated with 0.1 mL of the culture solution 28 days later in Example 3, and cultured in a shake incubator at 30 ° C. for 28 days. Sampling is 1 day after culture, 3 days after, 7 days after,
After 14 days, 21 days and 28 days, the remaining amount of oxybenzophenone was measured. In the control group, those to which no bacteria were inoculated were measured. The measurement of oxybenzophenone was performed using the culture solution 1
9 mL of methanol was added to the mixture, and the mixture was stirred well. After that, the residual amount of oxybenzophenone was measured by liquid chromatography at an ultraviolet absorption wavelength of 254 nm. With respect to oxybenzophenone, the residual value over time was determined with the measured value at the start of culture as 100%. FIG. 7 shows the measurement results of the residual amount of oxybenzophenone. As can be seen from FIG.
The residual ratio of oxybenzophenone decreased over time in the culture solution-added test group after the day compared to the control test group, and became almost 0% in 10 days. From the above results, it is clear that oxybenzophenone biodegrades even when a culture solution in which the decomposition of oxybenzophenone has been once completed in Example 3 is newly inoculated into a culture solution containing oxybenzophenone. The Bacillus brevis GU-1 of the present invention
By adding (Bacillus brevis GU-1) strain (FERM P-17615) to a biological wastewater treatment facility, it can be included in wastewater as it is at the current wastewater treatment facility without the need for special technology at low cost. Benzophenone can be efficiently and rapidly decomposed and removed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the time course of benzophenone when the bacteria of the present invention are cultured in a medium containing 0.01% of benzophenone. FIG. 2 is a graph showing the time course of benzophenone when the bacteria of the present invention are inoculated into an aeration tank when treating actual wastewater containing benzophenone in an aeration tank. FIG. 3 is a graph showing changes over time of oxybenzophenone when the bacteria of the present invention are cultured in a medium containing 0.01% of oxybenzophenone. FIG. 4 is a graph showing the change over time of oxybenzophenone when the bacteria of the present invention are inoculated into an aeration tank when treating actual wastewater containing oxybenzophenone in an aeration tank. FIG. 5 is a graph showing the time-dependent change (decomposition) of benzophenone when a culture solution collected after culturing for 28 days to decompose benzophenone in Example 1 was inoculated into a new medium containing benzophenone. It is. FIG. 6 is a graph showing the time-dependent change (decomposition) of benzophenone when a culture solution collected after decomposing benzophenone by culturing for 28 days in Example 2 was inoculated into a new medium containing benzophenone. It is. FIG. 7 shows a time-dependent change (degradation) of oxybenzophenone when a medium collected after decomposing oxybenzophenone by culturing for 28 days in Example 3 was inoculated into a new medium containing oxybenzophenone. FIG.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 11/00 C12N 11/00 // (C12N 1/20 (C12N 1/20 A C12R 1:08) C12R 1:08) F term (reference) 4B033 NA02 NA12 NA19 NC04 ND04 NF06 4B065 AA15X AC20 BA23 BB13 BC01 BC41 CA56 4D040 DD03 DD12 DD31

Claims (1)

Claims 1. A bacterium belonging to the genus Bacillus having the ability to degrade benzophenone or oxybenzophenone. 2. Bacillus blevis (Bacillus) having benzophenone or oxybenzophenone resolution.
brevis) bacteria. 3. Bacillus brevis GU-1 (Bacillus)
The bacterium according to claim 2, which is a brevis GU-1) strain (FERM P-17615). 4. A method for decomposing benzophenone or oxybenzophenone, which comprises causing a bacterium belonging to the genus Bacillus having the ability to degrade benzophenone or oxybenzophenone or a processed product thereof to act on benzophenone or oxybenzophenone. 5. The method according to claim 4, wherein the Bacillus bacterium is Bacillus brevis. 5. The method according to claim 5, wherein the Bacillus brevis bacterium is Bacillus brevis GU-1 (FERM P-17615). 7. A method for treating wastewater containing benzophenone or oxybenzophenone, wherein a culture of a bacterium belonging to the genus Bacillus having the ability to degrade benzophenone or oxybenzophenone is added to the treatment tank. 8. The method according to claim 7, wherein the Bacillus bacterium is Bacillus brevis. 9. The method according to claim 8, wherein the Bacillus brevis bacterium is Bacillus brevis GU-1 (FERM P-17615). 10. A method for treating wastewater containing benzophenone or oxybenzophenone using activated sludge,
Bacillus (Bacill) with benzophenone or oxybenzophenone decomposability
us) A method characterized by including a genus bacterium. 11. The method according to claim 10, wherein the Bacillus bacterium is Bacillus brevis. 12. The method according to claim 11, wherein the Bacillus brevis bacterium is Bacillus brevis GU-1 (FERM P-17615). 13. A method for treating wastewater using a biofilm, wherein the microorganisms constituting the biofilm are treated with Bacillus (Baci) having benzophenone or oxybenzophenone decomposability.
llus) A method characterized by including bacteria of the genus. 14. The method according to claim 13, wherein the Bacillus bacterium is Bacillus brevis. 15. The method according to claim 14, wherein the Bacillus brevis bacterium is Bacillus brevis GU-1 (FERM P-17615).