KR101195899B1 - Methylobacterium extorquens SMIC-1 resistant to tetramethyl ammonium hydroxide and capable of using it as a carbon source and a method of decreasing or removing tetramethyl ammonium hydroxide in a sample - Google Patents

Abstract

The present invention is resistant to tetramethyl ammonium hydroxide and can be grown using tetramethyl ammonium hydroxide as a carbon source. Methylobacterium extorquens SMIC-1) (Accession No. KCTC 10946 BP) and methods thereof are used to remove or reduce tetramethyl ammonium hydroxide in a sample.

Tetramethyl Ammonium Hydroxide, Methylobacterium Extoquens SMIC-1

Description

It is resistant to tetramethyl ammonium hydroxide and can be grown using tetramethyl ammonium hydroxide as a carbon source to remove tetramethyl ammonium hydroxide in a sample using Methylobacterium extorquens SMIC-1 resistant to tetramethyl ammonium hydroxide and capable of using it as a carbon source and a method of decreasing or removing tetramethyl ammonium hydroxide in a sample}

1 is a view showing the change in TOC during the inoculation of activated sludge obtained in the process of processing semiconductor factory wastewater in the minimum medium and concentrated culture.

FIG. 2 is a diagram illustrating a ratio TOC reduction ratio in each pass step in FIG. 1.

3 is a view showing the results of monitoring the ammonia concentration during the concentration culture.

Figure 4 shows the growth curve during the liquid culture of SMIC-1 strain.

5A and 5B show growth curves and concentrations of pH (A in FIG. 5A), ammonia (B in FIG. 5A), TOC (A in FIG. 5B) and TMAH (B in FIG. 5B) during liquid culture of SMIC-1 strains. It is a figure which shows the change.

FIG. 6 is a diagram showing the results of culturing SMIC-1 strains in a minimum medium and a minimum medium (control) without (NH ₄ ) ₂ SO ₄ .

FIG. 7 shows the results of observing the ammonia concentration theoretically estimated to occur from TMAH and the concentration of ammonia generated during the culture of SMIC-1 strains in a minimal medium without (NH ₄ ) ₂ SO ₄ .

The present invention is resistant to tetramethyl ammonium hydroxide and can be grown using tetramethyl ammonium hydroxide as a carbon source. Methylobacterium extorquens SMIC-1) and methods for removing or reducing tetramethyl ammonium hydroxide in a sample in the sample using the same.

Tetramethyl ammonium hydroxide (hereinafter also referred to as "TMAH") is a material used as a developer and a stripper in manufacturing processes such as semiconductors and LCDs. TMAH is known to be highly toxic to microorganisms, difficult to decompose and to inhibit nitrification. In addition, since TMAH is contained at a high concentration in the developer and the stripper, it is difficult to remove TMAH using microorganisms.

U.S. Patent No. 5,423,988 discloses a wastewater treatment apparatus including an adjustment tank, an adsorption column, and an aeration tank under a dilution tank as a device for treating wastewater containing a developer of a semiconductor or LCD factory (see FIG. 1). ). The surfactant in the developer is removed by an adsorption column, and then the developer containing TMAH is diluted by biological treatment and then treated by aerobic microorganisms. In this case the volume of TMAH volume per vent volume is very low, since it usually has to be diluted 10 times. Therefore, there is a disadvantage that the processing efficiency is low. Since the TMAH concentration in the developer is generally known to be 2000 ppm to 10000 ppm, when diluting about 10 times, it means that it can be biologically treated only when diluted to 200 ppm to 1000 ppm. U.S. Patent No. 5,423,988 also discloses a method for treating the developer-containing wastewater by maintaining the concentration of Bacillus subtilis at a high density.

However, even in the above-described conventional techniques, microorganisms that have a high resistance to TMAH and can grow using TMAH as the only carbon and energy source are not known.

Therefore, the present inventors can grow the TMAH as the only carbon and energy source with high resistance to TMAH, and search for a microorganism having excellent ability to remove TMAH in the wastewater, while the Methylbacterium extocuens SMIC of the present invention. -1 (Methylobacterium extorquens SMIC-1) discover and completed the present invention.

An object of the present invention is to provide a microorganism having high resistance to TMAH and capable of growing with TMAH as the only carbon source and energy support.

Another object of the present invention is to provide a method for removing or reducing TMAH in a sample using the microorganism.

The present invention is resistant to tetramethyl ammonium hydroxide and can be grown using tetramethyl ammonium hydroxide as a carbon source. Methylobacterium extorquens SMIC-1) (Accession No. KCTC 10946 BP) strain is provided.

The strain of the present invention is resistant to tetramethyl ammonium hydroxide and can be grown using tetramethyl ammonium hydroxide as a carbon source. The degree of resistance is at least 2000 ppm TMAH. Therefore, the strain of the present invention can be used for the purpose of removing or reducing TMAH in a sample because TMAH can be grown as a carbon source even in a solution in which high toxicity TMAH is present.

Accordingly, the present invention also relates to a sample containing Methylobacterium Methylobacterium in a sample containing tetramethyl ammonium hydroxide. Provided are methods for removing or reducing tetramethyl ammonium hydroxide in a sample, comprising culturing extorquens SMIC-1) (Accession No. KCTC 10946 BP) strain.

In the method of the present invention, the strain may be in a free state or immobilized on a carrier. When the strain is immobilized on a carrier, the carrier may be any carrier known in the art, for example, collagen, gelatin, agar, alginic acid, color quinone, cellulose, acetate, polyacrylic It may be selected from the group consisting of amide, amide, polyurethane, polyvinyl alcohol, and polyethylene glycol, but is not limited to these examples.

In the method of the present invention, the culture may be any type of culture method known in the art. For example, it may be selected from the group consisting of batch culture, fed-batch culture and continuous culture, but is not limited to these examples. In the case of continuous culturing, the strain of the present invention is first inoculated into a fermenter containing a nutrient medium, and then batch cultured to a predetermined density (for example, OD = 0.2) or more, followed by tetramethyl ammonium hydroxide. The sample (eg, semiconductor factory wastewater) may be cultured while flowing at a constant rate.

 In addition, the culturing may be made in a stirred or non-banned state, preferably culturing in a stirred state. In addition, the culturing is carried out under aerobic conditions.

In the method of the present invention, the culture is tetramethylammonium hydroxide in addition to the sample, which contains the side, bacterium methyl Solarium X Toku Enschede SMIC-1 (Methylobacterium extorquens SMIC- 1) ( accession No. KCTC 10946 BP) of the strain Naturally, it may further include a medium component for promoting growth.

In the method of the present invention, any sample containing tetramethyl ammonium hydroxide may be used as long as it contains tetramethyl ammonium hydroxide, for example, containing tetramethyl ammonium hydroxide and Wastewater stock solution or diluted wastewater. The wastewater includes, for example, a developer or stripper in a semiconductor or LCD factory.

The present inventors inoculated the activated sludge directly to the TMAH minimum medium in an amount of about 5% from a wastewater treatment plant treating wastewater from a semiconductor manufacturing plant in Korea. Next, the strains were grown by culturing in a medium containing TMAH, that is, six strains resistant to TMAH were isolated. Among them, the strain SMIC-1, the strain having the highest TMAH resolution, was used as the final strain. Selected. Next, the base sequence of the 16S RNA of the SMIC-1 strain was determined, and the base sequence of the 16S RNA was identified by comparison with the base sequence of the 16S RNA of the conventionally known standard strain. Methylobacterium Verify that the new owners belonging to extorquens), and the strain of bacterium methyl Solarium X Tokugawa Enschede SMIC-1 (named Methylobacterium extorquens SMIC-1), and May 15, 2006 an international deposit under the Budapest Treaty organization, Korea Research Institute of Bioscience and Biotechnology Deposited to KCTC (Accession No. KCTC 10946 BP).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example  One: TMAH Isolation of strains resistant to

In this example, wastewater activated sludge was collected from a wastewater treatment plant of a semiconductor manufacturing plant in Korea, and TMAH resistant strains were isolated therefrom.

First, inoculated the activated sludge of the semiconductor plant wastewater treatment plant to about 5% in an Erlenmeyer flask containing 100 ml of the minimum medium having the composition of Table 1 including TMAH, and incubated with stirring at 30 ° C. at 120 rpm.

Table 1. Composition of Minimal Media Containing TMAH

function ingredient density Carbon source TMAH (pentahydrate) 4g / l Nitrogen source (NH ₄ ) ₂ SO ₄ 0.1g / L Phosphate Sources and Buffers
K ₂ HPO ₄ 0.7g / L KH ₂ PO ₄ 0.3g / L Trace element Trace element solution * 2ml / L Etc MgSO ₄ 0.5g / L pH 7.0

* Trace elements _{solution: H 3 BO 3 0.3g / L} , CoSO 4 and _{7H 2 O 0.2g / L, ZnSO} 4 and _{7H 2 O 0.16g / L, MnCl} 2 and _{4H 2 O 0.041g / L, Na} 2 MoO ₄ 2H ₂ O 0.0324 g / L, NiCl ₂ 6H ₂ O 0.032 g / L, CuSO ₄ 5H ₂ O 0.014 g / L, FeSO ₄ 7H ₂ O 0.25 g / L, CaCl ₂ 2H ₂ O 100 g / L. In the composition, FeSO ₄ · 7H ₂ O and CaCl ₂ · 2H ₂ O were stored separately, and added (2ml / L) immediately before use.

Total organic carbon (hereinafter referred to as "TOC") concentration was monitored during the culture. The initial TOC was about 1200 ppm, and when the TOC concentration was reduced to about 200 ppm, the culture medium was centrifuged at 10,000 rpm, the supernatant was discarded and only the sludge was recovered, and the recovered sludge was added to an Erlenmeyer flask containing 100 ml of the minimum medium. Resuspension continued the thickening culture (hereinafter also referred to as "passage culture"). TOC was analyzed using a TOC analyzer (TOC-5000A, SHIMADZU) according to the user manual. As a result, it was subcultured up to seven times.

The concentration of TMAH contained in the medium is about 2000 ppm, and whether or not TMAH is decomposed can be estimated from the decrease in the concentration of TOC.

1 is a view showing the change in TOC during the inoculation of activated sludge obtained in the process of processing semiconductor factory wastewater in the minimum medium and concentrated culture. As shown in FIG. 1, the degradation rate of TMAH was relatively low at the early stage of the thickening culture, but the consumption rate of TMAH increased as the passage was progressed. In Figure 1, the empty circle is thickening culture 1, the full circle is thickening culture 2.

FIG. 2 is a diagram illustrating a ratio TOC reduction ratio in each pass step in FIG. 1. As shown in FIG. 2, it can be seen that as the number of passages increases, the ratio of decreasing ratio TOC increases.

3 is a view showing the results of monitoring the ammonia concentration during the concentration culture. As shown in Figure 3, it can be seen that the ammonia concentration in the culture medium increases with decreasing TOC. It is believed that nitrogen in the molecular structure is liberated in ammonia form upon decomposition of TMAH, but the present invention is not limited to any particular mechanism. Therefore, it can be seen that mineralization proceeds as the concentrated strain degrades TMAH.

From the above results, it can be concluded that the concentration of TMAH decomposed strains in the minimal medium was successful.

Example  2 : thickening  From culture TMAH  Pure separation of strains with high resolution

In the present Example, the TMAH obtained in Example 1 was purely isolated from the concentrated culture medium in which the degradation strain was concentrated.

First, the cells obtained by centrifuging the concentrated culture broth obtained after passage 7 times in Example 1 were plated in a solid medium consisting of agar 15 g / L minimum medium, and cultured at 30 ° C. until colonies were formed. Next, six of the colonies formed on the solid medium were taken and inoculated into vials containing 5 mL of minimum medium, respectively, and incubated at 30 ° C. for 96 hours to reconfirm growth in a minimum medium containing TMAH. The fastest growing strain among the six strains was named SMIC-1 and used for the following experiment.

The colonies grown by culturing the SMIC-1 strain in the solid medium were visually confirmed, and then grown colonies were randomly selected and passaged several times in the same solid medium to obtain purely cultured SMIC-1 strains.

Example  3: SMIC Characterization of -1 strain

In this example, the biochemical and culture properties of SMIC-1 isolated in Example 2 were investigated and identified.

(1) Identification of biochemical properties

SMIC-1 strains were plated in solid medium consisting of agar 15 g / L minimal medium, and the shape of colonies formed was observed. Single colonies obtained therefrom were liquid cultured in minimal medium, and then cultured SMIC-1 strains were cultured. The cell morphology was observed under a microscope, and a test for confirming gram staining, caltalase activity and oxidase activity was performed. The results are shown in Table 2 below.

Table 2.

characteristic SMIC-1 Methylbacterium standard strain Gram stain Gram voice Gram voice Catalase +++ + Oxidase + + (Weak) Cell morphology Bacillus Bacillus (up to 1x8㎛) Colony color Light pink, orange-red pink

(2) Confirmation of culture characteristics

In order to characterize the purely isolated SMIC-1 in solid culture, a single colony was inoculated into an Erlenmeyer flask containing 100 ml of minimal medium, and various culture characteristics were investigated by liquid culture with stirring at 30 ° C at 120 rpm. Culture characteristics were examined for growth characteristics, TMAH degradation activity, pH of the culture, changes in ammonia and TOC concentrations, and growth in medium excluding (NH ₄ ) ₂ SO ₄ in the minimum medium composition.

TOC was analyzed as shown in Example 1, TMAH was analyzed using IC (650 conductivity dectector, Althech). The column used was reverse phase C18 (250 mm × 4.6 mm), column temperature 30 ° C., mobile phase 2.25 mM nonafluoropentanoic acid (NFPA) / distilled water / acetonitrile (90/10), flow rate 0.8 mL / min. It was. To prepare the mobile phase, 660 μL of NFPA and 100 mL of acetonitrile were added to the tertiary distilled water, and the final volume was 1 L with distilled water, followed by filtration through a 0.2 μm membrane filter for 1 hour to degassing.

Figure 4 shows the growth curve during the liquid culture of SMIC-1 strain. As shown in FIG. 4, the SMIC-1 strain could grow to OD ₆₀₀ = about 0.3.

5A and 5B show growth curves and concentrations of pH (A in FIG. 5A), ammonia (B in FIG. 5A), TOC (A in FIG. 5B) and TMAH (B in FIG. 5B) during liquid culture of SMIC-1 strains. It is a figure which shows the change. As shown in FIGS. 5A and 5B, as the culture proceeded, the pH was lowered, the ammonia concentration was increased, and the TOC and TMAH concentrations were decreased. It can be seen that the decrease in the TOC concentration, the increase in the ammonia concentration and the decrease in pH correspond to the decomposition of TMAH.

As shown in Example 1, since an increase in ammonia was observed in accordance with the decomposition of TMAH in the concentrated culture, it was estimated that isolated strains could be grown even in a medium without (NH ₄ ) ₂ SO ₄ . To confirm this, a medium without (NH ₄ ) ₂ SO ₄ was prepared in a minimal medium, and the SMIC-1 strains were cultured in the same conditions as described above.

FIG. 6 is a diagram showing the results of culturing SMIC-1 strains in a minimum medium and a minimum medium (control) without (NH ₄ ) ₂ SO ₄ . As shown in FIG. 6, SMIC-1 strains grow in minimal medium without (NH ₄ ) ₂ SO ₄ but grow in minimal medium (ie, medium containing (NH ₄ ) ₂ SO ₄ ). It can be seen that the growth rate is slower than that. This may be due to the use of ammonia from TMAH decomposition as a nitrogen source.

FIG. 7 shows the results of observing the ammonia concentration theoretically estimated to occur from TMAH and the concentration of ammonia generated during the culture of SMIC-1 strains in a minimal medium without (NH ₄ ) ₂ SO ₄ . As shown in FIG. 7, it can be seen that the amount of NH ₃ —N generated as the TMAH is decomposed almost matches the theoretical amount. That is, since the equivalent amount of ammonia was released from the decomposition of TMAH, it was confirmed indirectly that this strain mineralized TMAH.

(3) Measurement of TMAH Degradation Activity of SMIC-1 Strains

SMIC-1 strains were inoculated at 5% in an SMIC-1 strain preculture with a microorganism concentration of 0.3 OD600 at 5% in an Erlenmeyer flask containing 100 ml of minimal medium (TMAH concentration was 2000 ppm) and incubated for 84 hours with stirring at 120 ° C at 30 ° C. After that, the TMAH concentration was measured and the decomposition rate of TMAH by SMIC-1 was investigated. As a result, TOC and TMAH degradation rates were 88.2% and 86.0%, respectively (see Table 3). As a control test, the cells were cultured under the same conditions without the SMIC-1 strain.

Table 3. TMAH Degradation Rate of SMIC-1

Strain Control group Experimental group Decomposition Compound class TOC (mg / L) TMAH (mg / L) TOC (mg / L) TMAH (mg / L) TOC (%) TMAH (%) density 1186 1720 140.2 241 88.2 86.0

Example 4 Confirmation of Molecular Biology of SMIC-1 Strains

In this example, the SMIC-1 strain was identified molecularly by analyzing the sequence of 16S rDNA of the SMIC-1 strain and comparing it with the sequence of a known strain.

First, 16S rDNA was amplified by PCR using the genomic DNA of the SMIC-1 strain as a template and primers with the universal primers 27F (SEQ ID NO: 2) and 1492R (SEQ ID NO: 3), and the amplified PCR product was 0.8%. After checking the size through agarose gel electrophoresis, it was subjected to sequencing by Macrogen (Korea). As a result, the sequence of 16S rDNA of the identified SMIC-1 strain is as shown in SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 1 corresponds to Nos. 56 to 1434 of E. coli 16S rDNA (NCBI U00096).

In addition, the homology search of the 16S rDNA sequence of SEQ ID NO: 1 in the NCBI database confirmed that it showed 100% homology with the 16S rDNA of the methyllobacterium ectococcus species.

Based on the biochemical, culture and molecular biological properties as described above, the SMIC-1 strain resistant to TMAH purely isolated in the present invention and capable of growing using TMAH as a carbon source is methyllobacterium extocuens species. in was found to be a novel strain belonging, tumefaciens X Tokugawa Enschede SMIC-1 (Methylobacterium extorquens SMIC- 1) naming, and May 15, 2006 an international deposit under the Budapest Treaty organization, Korea Research Institute of Bioscience and biotechnology biological resource Center of methyl (KCTC) was deposited (Accession Number KCTC 10946 BP).

Methyl tumefaciens X Toku Enschede SMIC-1 (Methylobacterium extorquens SMIC- 1) in accordance with the present invention can be grown to a TMAH TMAH has a resistance to a carbon source and energy source. Thus, it can be used to remove TMAH from samples containing TMAH.

According to the method for removing or reducing tetramethyl ammonium hydroxide in a sample according to the present invention, it is possible to efficiently remove or reduce tetramethyl ammonium hydroxide in a sample.

<110> Samsung Engineering., Ltd. <120> Methylobacterium extorquens SMIC-1 resistant to tetramethyl          ammonium hydroxide and capable of using it as a carbon source and          a method of decreasing or removing tetramethyl ammonium hydroxide          in a sample <130> PN068749 <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 1321 <212> DNA <213> Methylobacterium extorquens SMIC-1 <400> 1 tgcaagtcga acgggcacct tcgggtgtca gtggcagacg ggtgagtaac acgtgggaac 60 gtacccttcg gttcggaata actcagggaa acttgagcta ataccggata cgcccttttg 120 gggaaaggtt tactgccgaa ggatcggccc gcgtctgatt agcttgttgg tggggtaacg 180 gcctaccaag gcgacgatca gtagctggtc tgagaggatg atcagccaca ctgggactga 240 gacacggccc agactcctac gggaggcagc agtggggaat attggacaat gggcgcaagc 300 ctgatccagc catgccgcgt gagtgatgaa ggccttaggg ttgtaaagct cttttgtccg 360 ggacgataat gacggtaccg gaagaataag ccccggctaa cttcgtgcca gcagccgcgg 420 taatacgaag ggggctagcg ttgctcggaa tcactgggcg taaagggcgc gtaggcggcc 480 gattaagtcg ggggtgaaag cctgtggctc aaccacagaa ttgccttcga tactggttgg 540 cttgagaccg gaagaggaca gcggaactgc gagtgtagag gtgaaattcg tagatattcg 600 caagaacacc agtggcgaag gcggctgtct ggtccggttc tgacgctgag gcgcgaaagc 660 gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gaatgccagc 720 cgttggcctg cttgcaggtc agtggcgccg ctaacgcatt aagcattccg cctggggagt 780 acggtcgcaa gattaaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 840 tggtttaatt cgaagcaacg cgcagaacct taccatccct tgacatggca tgttacctcg 900 agagatcggg gatcctcttc ggaggcgtgc acacaggtgc tgcatggctg tcgtcagctc 960 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccacgtcct tagttgccat 1020 cattcagttg ggcactctag ggagactgcc ggtgataagc cgcgaggaag gtgtggatga 1080 cgtcaagtcc tcatggccct tacgggatgg gctacacacg tgctacaatg gcggtgacag 1140 tgggacgcga agccgcgagg tggagcaaat ccccaaaaac cgtctcagtt cggattgcac 1200 tctgcaactc gggtgcatga aggcggaatc gctagtaatc gtggatcagc acgccacggt 1260 gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagttg gtcttacccg 1320 a 1321 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tacggytacc ttgttacgac tt 22

Claims (6)

Tetramethylammonium hydroxide, and the resistance to the lock side, in which methyl for growth by a tetramethylammonium hydroxide as a carbon source tumefaciens X Toku Enschede SMIC-1 (Methylobacterium extorquens SMIC-1) (Accession No. KCTC 10946 BP). Methylobacterium Methylobacterium in samples containing tetramethyl ammonium hydroxide extorquens SMIC-1) (Accession No. KCTC 10946 BP) A method of removing or reducing tetramethyl ammonium hydroxide in a sample comprising culturing. The method of claim 2, wherein the strain is immobilized on a carrier. The method of claim 2, wherein the culture is selected from the group consisting of batch culture and continuous culture. The method of claim 3, wherein the carrier is selected from the group consisting of collagen, gelatin, agar, alginic acid, color quinone, cellulose, acetate, polyacrylamide, amide, polyurethane, polyvinyl alcohol, and polyethylene glycol Wherein the method removes or reduces tetramethyl ammonium hydroxide in the sample. The method of claim 2, wherein the sample is a wastewater stock or diluted wastewater containing tetramethyl ammonium hydroxide.

Images