JPH0426835B2 - - Google Patents

Description

[Detailed description of the invention]

(Field of Industrial Application) The present invention relates to a novel bacterium belonging to the genus Paracoccus and having the ability to decompose tetramethylammonium hydroxide (hereinafter referred to as "TMAH"), and further relates to a method for producing the bacterial cells, and further relates to a method for producing the bacterial cells. The present invention relates to a method for treating wastewater from the development of photosensitive resin (hereinafter referred to as "photoresist") discharged from a semiconductor factory using the bacteria. (Prior Art) TMAHH is used as a developer for positive photoresist in semiconductor factories. When the photoresist is placed on a substrate such as silicon and exposed to ultraviolet light, the exposed area changes to acid.
Neutralize the acid with an alkaline agent to make it easier to elute.
TMAH is used as the alkali agent. Therefore, the composition of the waste liquid from the development process of positive photoresists, which is called developer waste liquid, is
TMAH is the main component, and other than TMAH, only a small amount of exposed photoresist is included. From this, the processing of the developer waste solution is as follows:
This is nothing but TMAH processing. Conventionally, treatment of developer waste containing TMAH has been considered microbiologically difficult. The reason is,
This is because TMAH is considered to be a substance that is difficult for microorganisms to decompose. Therefore, the concentrated wastewater is combusted,
Dilute wastewater was treated using reverse osmosis membranes and ion exchange resins. However, these methods have the problem of high processing costs. In addition, there are few reports of TMAH-degrading microorganisms, and Hampton (D. Hampton) and Thattman (L.J.
Strain 5H2 (Biochemical Society Transactions) by Zatman)
Society Transactions), vol. 1, pp. 667-668,
Pseudomonas aminovorans, Protomonas extorquens, Mycobacterium sp. Nocardia sp. S-255 strain (Nocardia sp. S-255, JP-A-62-46155, and
Pollution and Countermeasures, Vol. 24, No. 3, pp. 208-212, 1988)
There is only that. Regarding the decomposition pathway of TMAH, according to the above-mentioned Zatman et al., tetramethylammonium is first decomposed into trimethylamine, and then it is decomposed through the decomposition pathway of trimethylamine. However, the enzyme that metabolizes tetramethylammonium to trimethylamine is said to be unstable and has not been elucidated biochemically. (Problems to be Solved by the Invention) As described above, in order to reduce the cost of development wastewater that has been used up to now, it is desirable to perform treatment with microorganisms. However, when using microbial treatment, there are issues such as the area required for the equipment and the stability of treatment capacity. Further, in the semiconductor manufacturing process, the developer waste liquid can be taken out without being mixed with other waste water, and it is easy and effective to use a method of treating the developer waste water alone. Therefore, it is desirable to use microorganisms that have a strong ability to assimilate or decompose TMAH and are not easily affected by contamination with other organic substances as microorganisms used for microbial treatment. In addition, when using the above-mentioned microorganisms capable of assimilating TMAH in wastewater treatment, if bacterial cells can be obtained by culturing, they can be added to microorganisms used in normal wastewater treatment, such as activated sludge. Alternatively, it is also possible to immobilize and use only the cultured bacterial cells. Therefore, it is extremely important that the culture is easy. (Means for solving the problem) The microorganism of the present invention belongs to the genus Paracoccus,
Has the ability to assimilate TMAH. Paracoccus sp. is a new bacterium that has the ability to degrade TMAH.
There is a TMA-B strain (hereinafter referred to as "TMA-B strain"). The TMA-B strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Fiber Science and Technology Research Institute No. 1959. This new bacterium is grown in a medium containing at least one selected from tetramethylammonium hydroxide, trimethylamine, dimethylamine, methylamine, trimethylamine N-oxide, formic acid, acetic acid, pyruvic acid, and L-serine as the main carbon source. It can be obtained by culturing and separating bacterial cells from the culture solution. In the method of treating a photosensitive resin development waste solution using microorganisms, it is possible to use a new bacterium according to the present invention, a bacterium belonging to the genus Paracoccus that can decompose tetramethylammonium hydroxide, or a group of microorganisms containing the bacterium. . (Function) The present inventors searched for microorganisms with a strong ability to assimilate TMAH from activated sludge and the like, which would lead to reductions in wastewater treatment costs, and isolated a plurality of microbial strains.
As a result of searching for microorganisms that have a strong ability to assimilate TMAH, we found that TMA-
Obtained B stock. The TMA-B strain used in the present invention has the following mycological properties. 1 Morphological properties Cells are rounded and rod-shaped, and the size is 0.5~
0.8 x 0.6-1.1 micrometers, usually cells singly or rarely in pairs, motile and without flagella, negative for Gram staining, non-sporulating, negative for acid-fast staining. 2. Culture properties Broth agar plate culture After culturing at 30°C for one week, small colonies with a creamy white color, round, convex lens shape, smooth, opaque, and glossy are formed. Juicy agar slant culture After 1 week of culture at 30°C, the growth is rather weak, brownish cream color, opaque, and glossy. Meat juice liquid culture After culturing at 30℃ for 2 weeks, the meat becomes slightly cloudy overall.
No precipitate or ring formation is observed. TMAH agar plate culture After culturing at 30°C for 5 days, the color is white with a pale cream color, round, convex lens-shaped, smooth and shiny.
Forms opaque colonies. TMAH agar slant culture Grows at 30℃ for 3 days. White with a pale cream color, glossy, and opaque. TMAH liquid culture The whole becomes cloudy after 1 week at 30℃. No ring formation was observed, and some precipitation was observed. Meat juice gelatin puncture culture Grows on the surface after one week of culture at 30℃. After that, it grows at the puncture site, but it hardly grows in the lower layer. Do not liquefy gelatin. Litmasque milk: No change. 3 Physiological properties Nitrate reduction: Reducing. Denitrification reaction: Although the rate is slow, denitrification occurs. MR test: negative. VP test: negative. Indole production: negative. Hydrogen sulfide formation: negative. Starch hydrolysis: negative. Citric acid availability: Negative. Availability of inorganic nitrogen sources: Use ammonium salts. Do not use nitrates. Pigment production: No pigment is produced. Urease: Negative. Oxidase: positive. Catalase: positive. Growth range: Grows at PH6-8, but PH6.9
~7.8 is preferred. Grows at 20-40℃, but 25
~37°C is preferred. Attitude towards oxygen: aerobic. O-F test: Does not break down sugar. Production of acids and gases from sugars: L-arabinose in aerobic and anaerobic cultivation,
D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, trehalose, D-sorbit, D
- Mannitol, inosyte, glycerin, and starch all produce no acid or gas. Auxotrophic requirement: Growth requires thiamin (vitamin
B ₁ ). Storage material: accumulates poly-β-hydroxybutyric acid. Utilization of various organic substances: Assimilate each of the following organic substances (final concentration 0.1% by weight) in an agar medium. Sodium formate, sodium acetate, sodium pyruvate, sodium lactate, L
-Serine, TMAH, tetramethylammonium chloride, trimethylamine, dimethylamine, methylamine, trimethylamine N-oxide, ethyltrimethylammonium, and the following organic substances are not assimilated under the same conditions as above. Sodium propionate, sodium butyrate, sodium oxalate, adipic acid, sodium malonate, sodium gluconate, sodium citrate, sodium succinate, sodium fumarate, sodium tartrate, L
-Sodium aspartate, DL-phenylalanine, L-proline, L-asparagine, L-
Arginine, L-alanine, L-valine, L-leucine, L-histidine, L-isoleucine, L
-Lysine, L-cysteine, L-glutamine, glycine, D-glucose, D-galactose, L
-arabinose, D-fructose, D-mannose, D-ribose, D-xylose, sucrose,
Lactose, melibiose, maltose, trehalose, cellobiose, L-rhamnose, L-sorbose, soluble starch, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, methyl ethyl ketone, glycerol, inosit, D-mannit, Dr sorbit, bacto-tryptone (Bacto-
Tryptone), Bacto-peptone (Bacto-
Peptone), tetramethylammonium chloride, tetrapropylammonium chloride, tetrabutylammonium chloride, choline chloride, trimethylphenylammonium chloride. Or methane cannot be assimilated. 4 Chemotaxonomic properties DNA G+C (guanine + cytosine) content:
71.5±0.2 mol%. Main quinone type: Coenzyme Q (ubiquinone)-10. Main fatty acids: C18:1. The main 3-hydroxy fatty acid is C10:0. The above properties are summarized in Birdsey's Manual of
Bergey's Manual of Systematic Bacteriology
Bacteriology), Volume 1, 1984 (hereinafter referred to as "Burgee's Manual"), it is a gram-negative, aerobic, rounded short bacillus, oxidase-positive and catalase-positive, and has denitrification ability. Therefore, it was determined that the bacterium belongs to the genus Paracoccus. In making this decision, TMA-B, which most expresses the characteristics of the genus Paracoticus,
To show the morphological characteristics of the strain, TMA
-An electron micrograph of strain B is shown in FIG. 3, and an electron micrograph of Paracoccus denitrificans (IAM12479) is shown in FIG. 3 for comparison. The morphology of these two types of cells is rounded and rod-shaped and very similar. Based on the above properties and this morphological feature, TMA
-The possibility that the B strain belongs to a genus other than Paracoticus is ruled out. Furthermore, the above-mentioned Virgie's Manual lists Paracoccus denitrificans and Paracoccus halodenitrificans.
halodenitrificans) have been described. According to this description, the G+C content of the DNA of Paracoccus halodenitrificans is 64 to 67 mol%,
The G+C content of Paracoccus denitrificans is 64-66 mol%, and 71.5± of TMA-B strain.
This is significantly different from 0.2 mol%. Moreover, Paracoccus denitrificans can assimilate sucrose, trehalose, maltose, histidine, and glucose, whereas the TMA-B strain cannot assimilate all of these. On the other hand, Paracoccus halodenitrificans requires 3% sodium chloride for growth, whereas TMA-B strain does not. Based on these findings, the TMA-B strain was determined to be a new species belonging to the genus Paracoccus. This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, in 1959.
(FERM BP-1959). The TMA-B strain of the present invention may undergo mutations in the same way as general bacteria; for example, it cannot be mutated by artificial mutation methods using ultraviolet rays, X-rays, or chemicals (e.g., nitrosoguanidine, ethyl methanesulfonic acid). Any mutant strain that has the ability to assimilate or decompose TMAH, which is the object of the present invention, can be used in the present invention. For culturing TMA-B strain, carbon sources include formic acid, formate (e.g. sodium formate), acetic acid,
acetates (e.g. sodium acetate), pyruvic acid,
pyruvate (e.g. sodium pyruvate),
Lactic acid, lactate (e.g. sodium lactate), L-serine, TMAH, tetramethylammonium salt (e.g. tetramethylammonium chloride), trimethylamine, dimethylamine, methylamine, trimethylamine N-oxide, ethyltrimethylammonium salt, etc. TMA-B strain Items that can be utilized by people are used as appropriate. When culturing using TMAH as a substrate, it may be easier to use tetramethylammonium chloride or tetramethylammonium sulfate, which are essentially neutralized with an acid, as TMAH exhibits strong alkalinity. . Ammonium salts can be used as nitrogen sources.
However, in a medium whose substrate is an organic substance containing nitrogen such as TMAH or trimethylamine, it is not necessary to add an ammonium salt as a nitrogen source. In addition, as other inorganic salts, inorganic salts normally required for culturing bacteria, such as phosphate, may be used alone or in appropriate combinations. Thiamine (vitamin) is also important for growth.
_B1 ), it is necessary to add a small amount of thiamine to the culture medium, but when culturing as a mixed microorganism such as activated sludge, for example, it is not necessary to add thiamine. The culturing method can be carried out in the same manner as the general culturing method of aerobic bacteria, and solid culture or liquid culture may be used. In the case of liquid culture, any of static culture, agitation culture, shaking culture, aeration culture, etc. may be carried out, but aeration agitation culture is particularly desirable. There are no particular limitations on the dissolved oxygen concentration in this culture solution, but it is usually desirable to have a concentration of 0.5 to 20 ppm. Culture conditions are temperature 20-40℃, preferably 25-37℃.
°C, and pH 6 to 9, preferably 6.9 to 7.8. If cultured outside of these conditions,
The growth of this bacterium becomes worse. After culturing bacteria in this manner, bacterial cells can also be obtained by separating the bacterial cells from the culture solution. The usual methods of separation include centrifuging the culture solution as it is, separating the bacteria from the culture solution by filtration, or using a flocculant to flocculate the bacteria and then centrifuging or filtering them. A method for isolating microorganisms from a culture solution can be used. In addition, when cultured cells of the TMA-B strain are used for wastewater treatment, the culture solution after cultivation is used as such, or only the cells separated from the culture solution are used to treat wastewater, such as activated sludge. It can also be used by mixing with a group of microorganisms or by immobilizing cultured microbial cells by entrapment or adsorption. One of the major characteristics of the TMA-B strain is that the range of organic matter that it can assimilate is extremely narrow.
This property has many advantages when considering adaptation to wastewater treatment. For example, even when TMAH coexists with organic substances that are easily used by common bacteria, such as sugars and alcohols, the TMA-B strain decomposes TMAH without being affected by other organic substances. this is
This shows that even if other organic matter suddenly mixes into the treatment process of wastewater containing TMAH, the treatment can be carried out without reducing the treatment capacity of TMAH. In addition, although the range of organic substances that can be assimilated is narrow, TMAH, trimethylamine, dimethylamine, methylamine, trimethylamine N-oxide, formic acid, acetic acid, pyruvic acid, and L-serine are not only available in solid media such as agar. , can also be assimilated in liquid culture. Therefore, if culture is performed using a medium containing these organic substances as a substrate,
It is easy to obtain bacterial cells of the TMA-B strain. (Example 1) Finally, 1.2 g of K ₂ HPO ₄ per liter,
KH ₂ PO ₄ 0.62g, (NH ₄ ) ₂ SO ₄ 0.5g, NaCl 0.1g,
_MgSO4・_7H2O0.2g , _CaCl2・_6H2O0.05g ,
A basal medium (PH7.1) containing 0.001 g of FeCl ₃ .6H ₂ O and 0.001 g of thiamine hydrochloride was steam sterilized, and TMAH, which had been separately sterilized by filtration, was added at a final concentration of 1.0 g/liter. One platinum loopful of TMA-B strain cells grown on an agar slant was inoculated into this medium.
Rotary shaking culture was carried out at °C for 7 days, and bacteria were collected by centrifugation. Figure 1 shows the changes in the absorbance of the culture solution at 660 nm and the TMAH concentration in the culture solution during the culture period. TMAH was analyzed by ion chromatogram. In the figure, 1 represents the growth of TMA-B strain.
The absorbance at 660 nm is 2, which indicates a decrease in TMAH concentration with proliferation. As a result, 290 mg of dry weight of bacterial cells was obtained per liter of culture solution. (Example 2) 400 mg of bacterial cells obtained by the same operation as Example 1
(dry weight) in 20 ml of pure water, and this suspension
Mix with ml 3.5% sodium alginate solution;
This mixture was added dropwise to a 0.1 mol/liter CaCl ₂ solution, and TMA− immobilized on alginate beads was added.
Obtained B stock. This immobilized bacterial cell was transferred to the medium of Example 1.
When the medium was placed in a 500 ml Erlenmeyer flask containing 100 ml and shaken rotatably at 30°C, the TMAH concentration in the medium decreased to less than 1 mg/liter in 18 hours. Similarly, 40 ml of the above suspension without immobilization was placed in a 500 ml Erlenmeyer flask containing 100 ml of the medium of Example 1, and the result was rotational shaking at 30°C.
The concentration of TMAH was below 1 mg/liter. (Example 3) The medium shown in Example 1 was further supplemented with glucose at a final concentration of 2 g/liter, casamino acid 2 g/liter,
A medium was prepared in which ethanol was added at 2 g/liter. Culture 7 was cultured in the same manner as in Example 1 in 500 ml Erlenmeyer flasks containing 100 ml of this medium and 100 ml of the medium shown in Example 1.
5 ml of the culture on day one was inoculated. As a result, it was found that when the culture medium was supplemented with organic substances such as glucose, the culture started at 50%
After 45 hours, the TMAH concentration in the culture solution was 1.
mg/liter or less. (Example 4) A medium prepared by adding trimethylamine to the basal medium of Example 1 to a final concentration of 1.3 g/liter was inoculated in the same manner as in Example 1, and cultured with rotational shaking at 30°C. After 7 days of culture, the bacteria were collected by centrifugation, and the dry weight per liter was
480mg of bacterial cells were obtained. (Example 5) Dimethylamine was added to the basal medium of Example 1 at a final concentration of 1.7 g/liter, and
Inoculation was carried out in the same manner as in Example 1, and rotational shaking culture was carried out at 30°C. After culturing for 9 days, the bacteria were collected by centrifugation, yielding 550 mg of bacterial cells per liter (dry weight). (Example 6) The final concentration of methylamine in the basal medium of Example 1
A medium containing 2.8 g/liter was inoculated in the same manner as in Example 1, and cultured with rotational shaking at 30°C. After culturing for 4 days, the bacteria were collected by centrifugation, and as a result, 430 mg of bacterial cells were obtained per liter (dry weight). (Example 7) The same inoculation as in Example 1 was carried out in a medium prepared by adding trimethylamine N-oxide to the basal medium of Example 1 at a final concentration of 1.5 g/liter.
Rotary shaking culture was performed at 30°C. After culturing for 12 days, the bacteria were collected by centrifugation, and as a result, 310 mg of bacterial cells were obtained per liter (dry weight). (Example 8) To the basal medium of Example 1, sodium formate was added to a final concentration of 2.8 g/liter.
Inoculation was carried out in the same manner as in Example 1, and rotational shaking culture was carried out at 30°C. After culturing for 7 days, the bacteria were collected by centrifugation, and as a result, 200 mg of bacterial cells were obtained per liter (dry weight). (Example 9) To the basal medium of Example 1, sodium acetate was added to a final concentration of 1.7 g/liter,
Inoculation was carried out in the same manner as in Example 1, and rotational shaking culture was carried out at 30°C. After culturing for 5 days, the cells were collected by centrifugation, and as a result, 290 mg of dry weight of cells per liter was obtained. (Example 10) The final concentration of L-serine in the basal medium of Example 1
A medium containing 1.5 g/liter was inoculated in the same manner as in Example 1, and cultured with rotational shaking at 30°C. After culturing for 10 days, the bacteria were collected by centrifugation, and as a result, 220 mg of bacterial cells were obtained per liter (dry weight). (Example 11) TMAH was treated with an apparatus consisting of an aeration tank with an effective capacity of 10 liters and a settling tank with an effective capacity of 3 liters. MLSS2500 activated sludge in the aeration tank
It was adjusted to be mg/liter. 550 mg (dry weight) of bacterial cells of the TMA-B strain obtained in the same manner as in Example 1 were added to the aeration tank together with a cationic polymer flocculant. This equipment has developer drainage.
The diluted waste liquid was diluted so that TMAH was 50 mg/liter, and the amount was 5 liters/liter for one week after the start of operation.
day, and thereafter at a flow rate of 10 liters/day.
The settled sludge was returned from the bottom of the settling tank to the aeration tank at a flow rate of 3 liters/day. As a control, the aeration tank
The treatment was carried out under the same conditions except that TMA-B strain cells were not added. Table 1 shows the TMAH concentration up to 30 days after the start of inflow.

[Table] (Effects of the invention) As described above, the TMA-B strain is a new bacterium belonging to the genus Paracoccus that has a strong ability to assimilate TMAH, and contains TMAH, trimethylamine, dimethylamine, methylamine, trimethylamine N-oxide, formic acid, Bacterial cells can be easily obtained by culturing using at least one of acetic acid, pyruvic acid, and L-serine as a substrate. Furthermore, when used in the treatment of developer waste, TMAH can be effectively decomposed or assimilated whether it is fixed or mixed with activated sludge.

[Brief explanation of drawings]

Fig. 1 is a graph showing the results of Example 1, Fig. 2 is a negatively stained transmission electron micrograph of Paracoccus SB TMA-B strain showing the bacterial morphology, and Fig. 3 is a graph showing the morphology of the bacteria. FIG. 2 is a negatively stained transmission electron micrograph of Icanth showing the morphology of bacteria. 1...Curve showing changes in absorbance indicating growth of TMA-B strain, 2...Curve showing changes in TMAH concentration.

Claims (1)

[Scope of Claims] 1. Paracoccus sp., which belongs to the genus Paracoccus and is capable of assimilating tetramethylammonium hydroxide.
A new microorganism that is the TMA-B strain (Feikoken Joyori No. 1959). 2. A medium whose main carbon source is at least one selected from tetramethylammonium hydroxide, trimethylamine, dimethylamine, methylamine, trimethylamine N-oxide, formic acid, acetic acid, pyruvic acid, and L-serine. A method for producing cells of the novel microorganism according to claim 1, which comprises culturing the novel microorganism according to claim 1 and separating the cells of the microorganism from the culture solution. 3. A method of treating a photosensitive resin developer waste solution using a microorganism, wherein the microorganism is the novel microorganism according to claim 1 or a group of microorganisms containing the novel microorganism. Method.