KR101104913B1 - Bacillus nitroreducens PLC9 KACC91464P and its catalase with high decomposing-activity of hydrogen peroxide and survival rate in strong concentration of hydrogen peroxide - Google Patents

Abstract

The present invention relates to an excellent novel microorganism Bacillus nitroreducens PLC9, which has excellent hydrogen peroxide resistance and has a hydrogen peroxide decomposition ability in a wide temperature range.

The present invention relates to a novel microbial Bacillus nitroreducens PLC9 that exhibits a linear survival curve with respect to the concentration of hydrogen peroxide so that it can be used to set the criteria for sterilization with hydrogen peroxide.

The invention microorganism Bacillus to process hydrogen peroxide nitroreducens relates to catalase of PLC9.

The present invention is a novel microorganism Bacillus The nitroreducens PLC9 has the effect of providing a method for treating hydrogen peroxide in a short time.

According to the present invention, the microorganism Bacillus can be used as an indicator microorganism essential for the verification of bactericidal effect by showing a constant resistance mechanism and a linear survival curve for hydrogen peroxide water. This has the effect of providing nitroreducens PLC9.

Hydrogen Peroxide, Wastewater Treatment, Catalase, Bacillus nitroreducens PLC9

Description

New microorganisms with excellent ability to decompose and resist hydrogen peroxide, bacillus nitroreducens PLC9 (KACC91464P) and its catalase with high decomposing-activity of hydrogen peroxide and survival rate in hydrogen concentration

As environmental pollution caused by the use of chlorine bleach has become a problem, hydrogen peroxide has become an important substitute for it (Neyens et. al . 2003). Unlike washing water with chlorine bleach, which produces other ions that cause corrosion, hydrogen peroxide is less corrosive, and when dried, does not leave solid residual contaminants such as salts and decomposes into water and oxygen when completely decomposed. It is an oxidant that is used in many processes such as precision process and dyeing process, ultrapure water piping and surface cleaning of aircraft, sterilization and cleaning of contact lens. However, residual hydrogen peroxide contained in waste water and washing water may cause environmental pollution and may cause serious skin problems if left after washing of contacting products such as contact lenses. It can also have a serious impact on post-treatment processes, especially biological processes. Therefore, there is a need for a method for decomposing and removing residual hydrogen peroxide.

Currently, a general method for removing hydrogen peroxide remaining in wastewater and washing water is to use a reducing agent such as sodium bisulfite or hydrosulfite. Such a reducing agent requires a large amount or more equivalent to the hydrogen peroxide contained in the bleaching wash water, and the reducing agent that does not react completely with the hydrogen peroxide is consequently present as sulfate, making it difficult to reuse the washing water. In addition, in the case of products such as fibers or fabrics that directly contact the human body, residual hydrogen peroxide or unreacted reducing agent may have a serious adverse effect on the quality of the product. Therefore, there is a demand for a process that can reuse the treated water while effectively decomposing and removing hydrogen peroxide without generating harmful by-products.

Another method of removing hydrogen peroxide remaining in wastewater and washing water is through the use of microorganisms. In Korea, most wastewater treatment plants are exposed to the external environment, which is very environmentally affected. In Korea, seasonal temperature fluctuates so much that when microorganisms are used for wastewater treatment, the efficiency of wastewater treatment is very low in winter when the growth and activity of microorganisms is low. This requires the use of microorganisms with low temperature variation and high survival rates at high concentrations of hydrogen peroxide. In addition, to date, most of the microbial treatment agents used to treat residual hydrogen peroxide in waste water or washing water are dependent on imports. However, these imported microbial agents lack data on their constituent microorganisms and may cause secondary pollution. In addition, in the residual hydrogen peroxide treatment, there are disadvantages of severe temperature variation, low efficiency, and very long treatment time. Therefore, it is necessary to develop microorganisms capable of high processing efficiency and short processing time.

Therefore, the present invention is to develop a microorganism that decomposes hydrogen peroxide, using the developed microorganisms directly to remove the hydrogen peroxide contained in the washing water at a low cost, and to reduce the washing water discharge through the reuse of the washing water I want to develop. Hydrogen peroxide decomposition process using the microorganism developed in the present invention will be a method that can reduce the amount of washing water discharged in a large amount in the semiconductor production process, fiber dyeing process, etc., and at the same time increase the reuse. In addition, the catalase of the microorganism developed in the present invention will be a method of removing various residual hydrogen peroxide alone.

Conventional techniques for removing hydrogen peroxide remaining in wastewater and washing water are generally a reduction process using a reducing agent such as sodium bisulfate (NaHSO 4) or hydrosulfite (Na ₂ S ₂ O ₂ H ₂ O). In the case of using such a reducing agent, since the reducing agent reacts with hydrogen peroxide to form a sulfate, the pH is changed to acidic, and thus an alkali agent for pH adjustment is required. In addition, it is pointed out that the accuracy of control, difficulty in maintenance and management, such as measuring the redox potential and measuring and treating the unreacted reducing agent, to confirm the end of decomposition of hydrogen peroxide.

Another method of removing hydrogen peroxide is using activated carbon. It is a method to remove hydrogen peroxide using the reducing power of activated carbon by passing wastewater or washing water containing hydrogen peroxide through an activated carbon packed column. However, before passing the activated carbon packed column, the pH of the wastewater must be adjusted to 10 or more, and the cost increase is caused by the addition of alkaline agents for the pH adjustment and the expensive pH adjusting equipment and control equipment.

Similarly, there is a method of removing hydrogen peroxide by passing wastewater containing hydrogen peroxide through granular activated carbon through an upflow fluidized bed method. This technique results in the reduction of hydrogen peroxide along the new surface of the activated carbon, so the activity of the activated carbon is slowed down and no pH adjustment is required. However, this technology requires that activated carbon particles break during the movement of wastewater, including hydrogen peroxide, to be mixed and discharged into the wastewater and washing water. In addition, the inflow of the damaged activated carbon deteriorates the water quality of the waste water and the washing water and causes a problem of increasing the fine particles.

Another method is to decompose hydrogen peroxide by high temperature treatment with heat. However, this method is disadvantageous in that a large amount of energy is consumed to treat excess waste water and washing water at high temperatures.

Recently, in order to compensate for these disadvantages, a method of passing wastewater containing hydrogen peroxide through fiber activated carbon using metals such as iron, nickel, manganese and silver has been proposed. However, these methods are inadequate for the treatment of large volumes of wastewater and washing water and have complicated maintenance and management.

The present invention is to provide a novel microorganism with excellent hydrogen peroxide resistance.

In addition, the present invention is to provide a novel microorganism with excellent hydrogen peroxide decomposition ability.

The present invention also provides a novel microorganism having excellent hydrogen peroxide decomposition ability in a wide temperature range.

It is also an object of the present invention to provide a novel microorganism producing a new catalase.

The present invention also provides a new catalase.

As mentioned above, the bacterial PLC9 isolated from the wastewater of the present invention is excellent in the ability to decompose high concentrations of hydrogen peroxide and the resistance of the resistor. In addition, the activity is maintained at a wide temperature can be used as wastewater treatment agent. In particular, when the bacterium of the present invention is immobilized on a carrier, higher efficiency can be expected. The novel catalases they produce can also be used to remove residual hydrogen peroxide after sterilization with various hydrogen peroxides.

Example  1: Isolation and Identification of Strains

* After sampling the waste water and the wash water of the plant, it was filtered through Whatman filter paper and treated with a concentration of 3% hydrogen peroxide and reacted for 10 minutes. 100 ul of filtered washing water reacted with hydrogen peroxide was inoculated in Muller-Hinton solid medium and incubated overnight at 37 ° C., and microbial colonies of different shapes were isolated and recultured to isolate strain PLC9 that decomposes hydrogen peroxide. Gram staining, biochemical testing according to Bergey's Systematic Bacteriology and 16S rRNA sequencing were performed. As a result of comparison and analysis of 16S rRNA sequences using biochemical tests and NCBI blast (http://www.ncbi.nlm.nih.gov/BLAST/), PLC9, a hydrogen peroxide degradation strain, was recently reported as Bacillus nitroreducens (IJS). / 2008/002089) and the 16S rRNA gene sequence was found to be 100% identical. The name of this bacterium is Bacillus It was named nitroreducens PLC9 and deposited with the Korea Agricultural Culture Collection (KACC) and received the accession number KACC91464.

(1) microscopic observation

* According to the new microbial identification and reporting procedure, morphology and flagella were observed using an optical microscope (Heimbrook et. al ., 1989), the surface morphology of microorganisms was observed using scanning electron microscopy (SEM) (Sambrook et al. al ., 1989). The results are shown in FIGS. 1 and 2.

(2) cell wall and lipid assay

Cell wall and lipid analysis according to the Schleifer and Kandler method (Schleifer and Kandler, 1972) are shown in Table 1 below.

Table 1. Cell Wall and Lipid Analysis Test Results of Hydrogen Peroxide Degrading Microorganism PLC9

Fatty acid One 2 3 4 5 average STDEV Final result 10: 0 0.14 0.09 0.1 0.0 0.1 tr 11: 0 ANTEISO 0.16 0.2 0.2 tr 13: 0 ISO 0.15 0.2 0.2 tr 14: 0 ISO 0.78 1.17 0.98 0.90 1.28 1.0 0.2 1.0 ± 0.2 1.0 ± 0.2 14: 0 0.54 0.55 0.49 0.18 0.35 0.4 0.2 0.4 ± 0.2 tr 15: 0 ISO 24.17 30.47 26.76 35.99 27.85 29.0 4.5 29.0 ± 4.5 29.0 ± 4.5 15: 0 ANTEISO 17.66 18.20 19.16 15.45 17.01 17.5 1.4 17.5 ± 1.4 17.5 ± 1.4 15: 0 2.12 4.47 1.70 3.65 3.95 3.2 1.2 3.2 ± 1.2 3.2 ± 1.2 16: 1 w7c alcohol 3.44 5.56 3.56 5.50 6.13 4.8 1.2 4.8 ± 1.2 4.8 ± 1.2 16: 0 ISO 9.48 8.29 11.12 5.97 8.33 8.6 1.9 8.6 ± 1.9 8.6 ± 1.9 16: 1 w11c 2.30 3.40 2.49 2.98 3.79 3.0 0.6 3.0 ± 0.6 3.0 ± 0.6 16: 0 4.31 2.21 4.63 0.91 1.92 2.8 1.6 2.8 ± 1.6 2.8 ± 1.6 ISO 17: 1 w10c 3.78 6.16 2.67 9.64 8.14 6.1 2.9 6.1 ± 2.9 6.1 ± 2.9 Sum In Feature 4 * 1.77 1.26 0.72 2.15 2.13 1.6 0.6 1.6 ± 0.6 1.6 ± 0.6 17: 0 ISO 10.43 6.24 9.42 7.44 7.16 8.1 1.7 8.1 ± 1.7 8.1 ± 1.7 17: 0 ANTEISO 16.05 10.60 14.83 8.00 10.03 11.9 3.4 11.9 ± 3.4 11.9 ± 3.4 17: 0 1.21 1.26 0.82 0.74 0.82 1.0 0.2 1.0 ± 0.2 1.0 ± 0.2 16: 0 2OH 0.34 0.3 0.3 tr 17: 0 2OH 0.18 0.2 0.2 tr 18: 1 w9c 0.38 0.4 0.4 tr 18: 0 1.11 0.65 0.38 0.7 0.4 0.7 ± 0.4 0.7 ± 0.4 17: 0 2OH 0.21 0.2 0.2 tr 19: 0 ISO 0.32 0.27 0.3 0.0 0.3 tr

(3) biochemical characterization

Biochemical assays for hydrogen peroxide-decomposing microorganism PLC9 are described by Gordon et al. (1973) and Rheims et al. (1999), Microbiological applications (1990), and assayed using the API 50CH system (bioMerieux). The biochemical assay results are shown in Table 2 below. PLC9 grew best at temperature of 10-45C and weakly at 4C. It also survived in an environment of pH 5.0-11.5. Biochemical assay showed that PLC9 was most similar to Bacillus silvestris ATCC BAA-269, but the oxidase test results were different.

Table 2. Biochemical Assay of Hydrogen Peroxide Degrading Microorganism PLC9 and Control Strains

Bacillus silvestris
ATCC BAA-269 PLC9 Catalase reaction + + Oxidase reaction - + Voges-Proskauer (VP) test - - Temperature range for growth 15-40C 10-45C Presence of lecithinase - - Resistance to lysozyme - - Growth in the presence of NaCl in TSB 0-5% 0-6% Growth at pH 5 · 7 pH 5 - + pH 7 + + Formation of acid from sugar D-glucose - - L-arabinose - - D-xylose - - D-mannitol - - D-fructose - - Formation of gas from glucose - - Hydrolysis of starch - - Decomposition of Tween 80 + + Use of citrate and propionate - - Reduction of nitrate - - Production of indole - - Deamination of phenylalanine - - Decomposition of casein and tyrosine - - Liquefaction of gelatin - - Presence of arginine dihydrolase - - Hydrolysis of aesculin - - Utilization of glycerol - -

(4) G + C content measurement

Genomic DNA was isolated by Enzyme lysis method and G + C value was determined by HPLC method (Mesbah et. al . , 1989 & Marmur et al . 1962). As a result, the G + C value of the hydrogen peroxide decomposing microorganism PLC9 was 36 mol%.

(5) 16S rRNA gene sequence analysis

16S rRNA sequencing was performed to confirm the genetic hierarchy of the hydrogen peroxide degrading microorganism PLC9 and 16S rRNA sequences were compared and analyzed using NCBI blast (http://www.ncbi.nlm.nih.gov/BLAST/). 16s rRNA sequences of the hydrogen peroxide degrading microorganism PLC9 are shown in FIG. 3. 16S rRNA sequencing results Bacillus Odysseyi was identified as the genetically closest species with a similarity of 96.8% (Table 3), and showed similarity of less than 97% with the type strains of all species of Bacillus so far registered (Figure 4). In particular, it showed 100% similarity with Bacillus nitroreducens (ACC No. EU391158, IJS / 2008/002089) recently registered. Bacillus 16s rRNA sequences of nitroreducens PLC9 were registered in NCBI GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) (Accession No. FJ973430).

Table 3. 16s rRNA sequence similarity analysis of hydrogen peroxide-degrading microorganism PLC9

Strain Acc No % Sim nt diff / comp Bacillus nitroreducens b04i-3, KCTC 13219 ^T EU391158 100.00 0/1383 Bacillus odysseyi 34hs1T AF526913 96.75 45/1384 Bacillus sphaericus NCTC 10338T X60639 96.12 51/1314 Bacillus silvestris HR3-23T AJ006086 96.10 54/1384 Bacillus fusiformis ATCC 7055T 95.48 61/1351 Bacillus tequilaensis 10b AY197613 95.06 27/547 Bacillus massiliensis 4400831 AY677116 94.54 76/1391 Bacillus insolitus DSM 5T X60642 94.09 78/1319 Bacillus psychrodurans DSM 11713T AJ277984 93.91 84/1380 Escherichia coli J01695 78.07 298/1359

Example 2 : Hydrogen Peroxide Degradation Rate Measurement Experiment

(1) Determination of hydrogen peroxide decomposition rate of microorganisms that decompose hydrogen peroxide

Hydrogen peroxide decomposition rate was measured by spectrophotometer assay and potassium permanganate assay using hydrogen peroxide absorbance at 240 nm. Hydrogen peroxide digestion microbial PLC9 suspension with OD set to 1 was added to 50 mM phosphate buffer (pH 7.0), 100 mM hydrogen peroxide, and then absorbance at 240 nm was measured every minute to calculate absorbance reduction (spectrophotometer assay , 1984 & Katsuwon et al . 1989), hydrogen peroxide decomposition rate was measured. Also, add 10 ml of 1% hydrogen peroxide to a 50 ml beaker, add 1 ml of a PLC9 suspension with an OD of 1, mix, take 5 ml samples over time, transfer to a new beaker, add 10 ml of 1M H2SO4, and add 2% potassium. Permanganate (KMnO4) was titrated dropwise until it developed pink or brown color (potassium permanganate assay, http://www.h2o2.com/intro/iodometric.html) to determine the rate of hydrogen peroxide degradation. At this time, the hydrogen peroxide standard curve was prepared to measure the residual hydrogen peroxide. Hydrogen peroxide decomposition rate was measured by spectrophotometer assay and potassium permanganate assay, and 72% hydrogen peroxide was decomposed for 120 minutes and 91.2% was decomposed for 180 minutes (FIG. 5).

(2) Determination of hydrogen peroxide decomposition rate of microorganisms decomposing hydrogen peroxide fixed on agar

PLC9, a hydrogen peroxide-decomposing microorganism, After culturing to a value of 0.6, mix with agar so that it is 1/50 of the agar volume, and make and fix agar blocks having a size of 1 cm. The amount of remaining hydrogen peroxide is measured by reacting 10 agar blocks with 100 ml of 1% hydrogen peroxide. Degradation rate measurement resulted in decomposition of 71% hydrogen peroxide for 120 minutes. The strain itself did not show a difference in the decomposition rate and the hydrogen peroxide decomposition rate (Fig. 6).

Example 3 : Experiment for measuring survival rate of microorganisms that decompose hydrogen peroxide

Since the microorganisms decomposing hydrogen peroxide can be killed within a short time after the decomposition of hydrogen peroxide, the survival rate of the microorganisms decomposing hydrogen peroxide was measured. Hydrogen peroxide decomposed microorganisms were dissolved in MH liquid medium to 10 ⁸ CFU / ml, and 100 μl of the cells were taken per hour, smeared in MH solid medium, and placed in a 37 ° C. incubator overnight to determine the number of colonies. PLC9 showed 100% survival rate after exposure to 1% hydrogen peroxide for 6 hours (360 minutes), and 10 ² / ml of bacteria were survived at the same time in 3% hydrogen peroxide. As shown in FIG. 7, Bacillus cereus ATCC 9634 used as a control group was killed within 1 hour (60 minutes) when exposed to 1% hydrogen peroxide.

Example 4 Isolation and Characterization of Catalase

(1) Separation of Catalase by Column Chromatography

Catalase, a hydrogen peroxide degrading enzyme of microorganism PLC9, which decomposes hydrogen peroxide, was modified by Kuusk et al. (Kuusk et al. al . 2001). Hydrogen peroxide degrading microorganism PLC9 was cultured overnight in liquid nutrient medium, followed by centrifugation to collect only microbial cells and 100 ml buffer A (100 mM KH ₂ PO ₄ , 5 mM EDTA, pH 6.0) with protease inhibitor and DNase, 0.5 mM MgCl ₂ . ) Was made into a suspension and crushed by bead-beater. Centrifuged at 15,000 X g for 20 minutes to remove unbroken microbial cells, only the supernatant was taken and again centrifuged at 144,000 X g for 2 hours to take a supernatant. After centrifugation, the final concentration was 0.5 M (NH ₄ ) ₂ SO ₄ was treated and placed on a Phenyl-sepharose column with buffer A containing 0.5 M (NH ₄ ) ₂ SO ₄ . 500 ml of buffer A and buffer B (5 mM KH ₂ PO ₄ , 5 mM EDTA, pH 6.8) were eluted in that order. Then eluted again with 600 ml of buffer B and buffer C (50% ethylene glycol, 5 mM KH ₂ PO ₄ , 5 mM EDTA, pH 6.8). The highest catalase activity among the eluted fractions was selected and concentrated to a final volume of 1 ml with a centrifugal concentrator. The concentrated sample was placed on a Sephacryl-300 HR (2.6 × 100 cm) column and eluted with buffer B. Among the eluted fractions, the best catalase activity was added to the DEAE-cellulose DE52 (2.6 × 20 cm) column with buffer B, eluted with the same buffer, and the final concentration of 0.5 M NaCl was added to buffer B. The fraction with the highest catalase activity was put on the Phenyl-sepharose column once again and eluted with buffer C to obtain the fraction with the highest catalase activity. The catalase separation process is summarized in Table 4. The isolated catalase was measured by spectrophotomter assay. The active unit of the enzyme was set to 1 unit of the amount of enzyme to decompose 1 umole of hydrogen peroxide per minute.

The isolated catalase was confirmed by protein electrophoresis (SDS-PAGE) (Bollag et al. 1991 & Mutsuda et al . 1996). A 15% polyacrylamide gel is prepared, and the separated catalase is electrophoresed and fixed with methanol. Gels electrophoresed with 0.1% Coomassie brilliant blue staining reagent, and stained with 50% methanol and 10% acetic acid solution were observed. Electrophoresis revealed that the catalase produced by PLC9 was about 66 kDa protein (FIG. 8).

 Table 4. Protein Separation Yield of Catalase Produced by PLC9

step protein
(ug / ml) Tot.protein
(mg) Tot. activity x 10 ⁵
(U) Spec. activity
(U / mg) Yield
(%) Purification
(fold) crude extract 3650.7 182.5 102.2 56,000 100.0 1.00 phenyl-agarose 803.0 40.2 85.1 212,000 83.3 3.79 sephacryl S-300 673.7 3.4 17.0 503,283 16.6 8.99 DEAE-sepharose 62.3 3.1 16.5 529,947 16.1 9.46 phenyl-agarose 50.0 1.5 13.7 915,000 13.4 16.34

(2) Confirmation of enzyme activity of catalase using ferricyanide negative staining

7% native acrylamide gel (non-denaturing gel) of the isolated catalase was used to perform ferricyanide negative staining after electrophoresis (Wayne et al . 1986) to confirm the activity of catalase (Fig. 9). The electrophoretic native acrylamide gel was reacted with 4 mM hydrogen peroxide for 10 minutes and then rinsed with distilled water. Gels washed with distilled water were developed with 0.5% potassium ferricyanide (K3Fe (CN) 6) and 0.5% ferric chloride (FeCl ₃ H ₂ O) until the gel turned blue. Where there is catalase activity, it is not bluish and transparent.

(3) Analysis of Amino Acid Sequence of Catalase

Catalase isolated for N-terminal amino acid sequence analysis of catalase is subjected to protein electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore, Beford, MA) by electroblotting. The protein-transferred membrane was stained with 0.1% Coomaji Blue staining reagent as in protein electrophoresis gel, and dried by decolorizing in 50% methanol and 10% acetic acid solution. The dried membrane was subjected to N-terminal amino acid sequencing by Procise 492 clc protein sequencer (Applied Biosystems, USA). The analyzed N-terminal amino acid sequence is shown in FIG. The analyzed amino acid sequences were compared and analyzed by NCBI protein blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi). And 62% similar to catalase isolated from Yersinia species (FIG. 11). Therefore, catalase isolated from microorganism PLC9, which degrades hydrogen peroxide, is thought to be a new kind of catalase.

(4) Determination of Catalase Activity According to Temperature and pH

100 mM of catalase was treated with 30 mM hydrogen peroxide and reacted at each temperature and pH for 10 minutes, and then the amount of remaining hydrogen peroxide was measured using a spectrophotometer to determine the decomposition rate. Catalase produced by PLC9 as a result of the decomposition rate was confirmed to decompose hydrogen peroxide very stably at 4-50C (Fig. 11). Catalase of PLC9 also decomposed hydrogen peroxide very stably under conditions ranging from pH 4 to 10. Hydrogen peroxide decomposition rate was reduced by 35.3% at pH 3 condition.

1 is a photograph of observation of optical microscope after Gram staining of hydrogen peroxide decomposed microorganism PLC9

2 is a SEM image of PLC9

Figure 3 16S rRNA sequence of the hydrogen peroxide decomposition microorganism PLC9

[Figure 4] Schematic analysis of hydrogen peroxide decomposition microorganism PLC9

5 is hydrogen peroxide decomposition rate of PLC9

FIG. 6 Hydrogen peroxide decomposition rate of agar block on which PLC9 is fixed

[Figure 7] Hydrogen peroxide decomposition microorganism PLC9 and control Bacillus survival rate of cereus ATCC 9634

[Figure 8] Electrophoresis of catalase produced by the hydrogen peroxide decomposition microorganism PLC9

9 is catalase electrophoresis of PLC9 isolated using column chromatography

10 is the amino acid sequence of the catalase produced by the hydrogen peroxide decomposition microorganism PLC9

11 is a similarity of the amino acid sequence of the catalase produced by the hydrogen peroxide decomposition microorganism PLC9

12 is checking the stability of the catalase produced by the hydrogen peroxide decomposition microorganism PLC9

Claims (6)

Bacillus nitroreducens KACC91464P strain with the ability to decompose hydrogen peroxide. Bacillus nitroreducens ( Bacillus nitroreducens ) of claim 1 composition for the decomposition of hydrogen peroxide containing KACC91464P strain. The composition of claim 2, wherein the strain has a resistance to hydrogen peroxide and the mortality of the strain is linear with respect to the concentration of hydrogen peroxide. Claim 1, characterized in that the Bacillus nitroreducens (Bcillus nitroreducens ) KACC91464P strain is used as an indicator microorganism, a method for setting the pipe sterilization criteria by hydrogen peroxide. delete delete

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