KR101195900B1 - Mycobacterium sp. 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, Mycobacterium sp. SMIC-1 (Accession No. KCTC 10947 BP). ) And the use thereof to remove or reduce tetramethyl ammonium hydroxide in a sample.

Tetramethyl Ammonium Hydroxide, Mycobacterium Species SMIC-1

Description

Mycobacterium sp. SMC-1, which is resistant to tetramethyl ammonium hydroxide and grows with tetramethyl ammonium hydroxide as a carbon source, and which is used to remove or reduce tetramethyl ammonium hydroxide in a sample Method {Mycobacterium sp. 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. The figure which shows the change.

The present invention is resistant to tetramethyl ammonium hydroxide and grows using tetramethyl ammonium hydroxide as a carbon source, Mycobacterium sp. SMIC-1 and Mycobacterium sp. A method for removing or reducing tetramethyl ammonium hydroxide in a sample.

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 a control tank, an adsorption column, and an aeration tank under a dilution tank as a device for treating wastewater including 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. Further, US Patent No. 5,423,988 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.

The inventors of the present invention, while searching for a microorganism having high resistance to TMAH and TMAH as the only carbon source and energy source, and excellent in the ability to remove TMAH in the wastewater, the mycobacterium species SMIC-1 (Mycobacterium sp. SMIC-1) was discovered and the present invention was completed.

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 and energy source.

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, Mycobacterium sp. SMIC-1 (Accession No. KCTC 10947 BP). ) Provides the strain.

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 includes culturing a Mycobacterium sp. SMIC-1 (Accession No. KCTC 10947 BP) strain in a sample containing tetramethyl ammonium hydroxide. A method of removing or reducing tetramethyl ammonium hydroxide in a sample is provided.

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 in addition to the sample containing tetramethyl ammonium hydroxide, the growth of Mycobacterium sp. SMIC-1 (Accession No. KCTC 10947 BP) strain Of course, it may further include a medium component for promoting the.

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 an outer membrane solution of 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, four strains which were grown by culturing the strains in a medium containing TMAH, that is, resistant to TMAH, were isolated, and among them, the SMIC-1 strain, 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. As a result, Mycobacterium sp. The strain was identified as Mycobacterium sp. Mycobacterium sp. SMIC-1, and the Korea Biotechnology Research Institute, an international depository under the Treaty of Budapest on May 15, 2006, Deposited to Resource Center (KCTC) (Accession No. KCTC 10947 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. FeSO ₄ · 7H ₂ O and CaCl ₂ · 2H ₂ O in the composition was stored separately, and added (2ml / L) just before use, respectively.

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 solution was centrifuged at 10,000 rpm, the supernatant was discarded and the sludge was recovered. The turbidity culture was continued by turbidity (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 thickening of TMAH degradation 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, four 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 four 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 the colonies formed was observed, and the single colonies obtained therefrom were liquid cultured in minimal medium, followed by microscopic examination of cultured SMIC-1 strains. Cell morphology was observed and tested for gram staining, catalase activity and oxidase activity. The results are shown in Table 2 below.

Table 2.

characteristic SMIC-1 Mycobacterium Standard Strains Gram stain Gram positive Gram voice Catalase + + Oxidase - Unknown Cell morphology Bacillus Bacillus (up to 0.7x10㎛) Colony color Yellow-Dark (Please specify exact color ) Pink, orange, old amount ( please state exact color )

(2) Confirmation of culture characteristics

In order to characterize the purely isolated SMIC-1 in solid culture, single colonies were inoculated into an Erlenmeyer flask containing 100 ml of minimal medium, and various culture characteristics were investigated by liquid culture while stirring at 120 rpm at 30 ° C. . 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. In order to prepare a 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 degas.

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 0.8.

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. The 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.

(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 91.1% and 93.4%, 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 1276 2100 114 138 91.1 93.4

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 1453 of E. coli 16S rDNA (NCBI U00096).

In addition, a homology search for the 16S rDNA sequence of SEQ ID NO: 1 in the NCBI database revealed a mycobacterium species (eg, Mycobacterium). brisbanense strain W6743 (GenBank accession NO.AY012577), Mycobacterium cosmeticum strain LTA-388 (GenBank accession NO.AY449728), Mycobacterium It was confirmed that the homology with 99S rDNA of cosmeticum strain 2003-11-06 (GenBank accession NO. AY449729) was more than 99%.

Based on the biochemical, cultural and molecular biological properties as described above, the SMIC-1 strains resistant to TMAH purely isolated in the present invention and capable of growing using TMAH as a carbon source are belonging to the mycobacterium species. It was identified as one strain, named Mycobacterium sp. SMIC-1, and on May 15, 2006, the Korea Institute of Biotechnology and Biotechnology Center (KCTC), an international depository under the Treaty of Budapest. Deposited (Accession No. KCTC 10947 BP).

Mycobacterium sp. SMIC-1 according to the present invention is resistant to TMAH and can be grown using TMAH as a carbon 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 Co., Ltd. <120> Mycobacterium sp. SMIC-1 resistant to tetramethyl ammonium          hydroxide and capable of using it as a carbon source and a method          ofdecreasing or removing tetramethyl ammonium hydroxide in a          sample <130> PN068750 <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 1374 <212> DNA <213> Mycobacterium sp. SMIC-1 <400> 1 tgcaagtcga acggaaaggc ccttcggggt gctcgagtgg cgaacgggtg agtaacacgt 60 gggtgatctg ccctgcactt tgggataagc ctgggaaact gggtctaata ccggatagga 120 ccatggcctt catgggttgt ggtggaaagc ttttgcggtg tgggatgggc ccgcggccta 180 tcagcttgtt ggtggggtaa tggcctacca aggcgacgac gggtagccgg cctgagaggg 240 tgaccggcca cactgggact gagatacggc ccagactcct acgggaggca gcagtgggga 300 atattgcaca atgggcgcaa gcctgatgca gcgacgccgc gtgggggatg acggccttcg 360 ggttgtaaac ctctttcagc acagacgaag cgtgagtgac ggtatgtgca gaagaagcac 420 cggccaacta cgtgccagca gccgcggtaa tacgtagggt gcgagcgttg tccggaatta 480 ctgggcgtaa agagctcgta ggtggtttgt cgcgttgttc gtgaaaactc acagcttaac 540 tgtgggcgtg cgggcgatac gggcagactg gagtactgca ggggagactg gaattcctgg 600 tgtagcggtg gaatgcgcag atatcaggag gaacaccggt ggcgaaggcg ggtctctggg 660 cagtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag 720 tccacgccgt aaacggtggg tactaggtgt gggtttcctt ccttgggatc cgtgccgtag 780 ctaacgcatt aagtaccccg cctggggagt acggccgcaa ggctaaaact caaaggaatt 840 gacgggggcc cgcacaagcg gcggagcatg tggattaatt cgatgcaacg cgaagaacct 900 tacctgggtt tgacatgcac aggacgctgg tagagatatc agttcccttg tggcctgtgt 960 gcaggtggtg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1020 gagcgcaacc cttgtcctat gttgccagcg ggttatgccg gggactcgta ggagactgcc 1080 ggggtcaact cggaggaagg tggggatgac gtcaagtcat catgcccctt atgtccaggg 1140 cttcacacat gctacaatgg ccggtacaaa gggctgcgat accgtgaggt ggagcgaatc 1200 ctttcaaagc cggtctcagt tcggatcggg gtctgcaact cgaccccgtg aagtcggagt 1260 cgctagtaat cgcagatcag caacgctgcg gtgaatacgt tcccgggcct tgtacacacc 1320 gcccgtcacg tcatgaaagt cggtaacacc cgaagccggt ggcctaaccc cttt 1374 <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)

Mycobacterium sp. SMIC-1 (Accession No. KCTC 10947 BP), resistant to tetramethyl ammonium hydroxide and capable of growing with tetramethyl ammonium hydroxide as a carbon source. Tetramethyl ammonium hydroxide in the sample comprising culturing the Mycobacterium sp. SMIC-1 (Accession No. KCTC 10947 BP) strain in a sample containing tetramethyl ammonium hydroxide How to remove or reduce the lockside. 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.

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