KR102235806B1 - Peracetic acid decomposition method and culture method of microorganism using the same - Google Patents

Abstract

The present invention relates to a method for decomposing peracetic acid and a method for culturing microorganisms using the method, and when culturing microorganisms using the method of the present invention, peracetic acid present in a medium used for sterilization can be effectively removed.

Description

Peracetic acid decomposition method and culture method of microorganism using the same}

The present invention comprises the steps of mixing peracetic acid in water; And adding and reacting iron ions, alkali metal hydroxides, ethylenediaminetetraacetic acid (EDTA), and sugar to the mixture to finally decompose the peracetic acid reactant into acetic acid, water and oxygen. It relates to a method of culturing microorganisms using.

In the case of mass production of useful metabolites using various microorganisms in the bio industry, sterilization of the medium and reactor is very important. For this, conventionally, an autoclave was mainly used to sterilize the microbial culture medium and the reactor.

However, when the medium and the reactor are sterilized using an autoclave, the sugar component of the medium reacts with one of the other components of the medium (peptone or yeast extract) at high heat, hydroxymethyl furfural (HMF), etc. There is a problem of inhibiting the growth of microorganisms by causing the generation of toxic substances that can hinder the growth of the same microorganism, and there is a limitation that the material of the microorganism reactor must be made of a material that is resistant to heat and pressure such as metal or glass.

In order to overcome the above problems and limitations, a method of filtration is used, but in the case of filtration, there is an inconvenience of having to sterilize the incubator itself separately, and there is a risk of clogging and re-contamination in the filtration process. It is large, and there is a problem that the possibility of commercial use is low because the size of the filter is limited and the cost is high.

In order to solve this problem, a sterilization method using peracetic acid (PAA) has been developed as disclosed in Korean Laid-Open Patent Publication No. 10-2015-0097295. Korean Patent Publication No. 10-2015-0097295 discloses a method of sterilizing a photobioreactor using peracetic acid in pure culture of microalgae requiring light. However, there is no disclosure of the increase in the decomposition efficiency of peracetic acid by EDTA and sugars (glucose, sucrose, waste sugar, etc.), and sterilizing a medium containing sugar and nitrogen sources (peptone, yeast extract, whey, etc.) No method is disclosed.

In addition, in the method of sterilizing the photobioreactor and the medium using peracetic acid, if the medium contains sugar and nitrogen source, a high concentration of 5 mM or more peracetic acid should be used for complete sterilization. When sterilized together, nitrogen oxides (NO3-) are generated by peracetic acid, and the generated nitrogen oxides bind with iron ions to inhibit the catalytic reaction for decomposition of peracetic acid. In this case, it takes more than one month to completely decompose the peracetic acid reactants only by the natural decomposition of peracetic acid and the catalytic reaction by iron and HEPES.

It is an object of the present invention to mix peracetic acid and water; And

To provide a method for decomposing peracetic acid comprising the step of finally decomposing the peracetic acid reactant into acetic acid, water and oxygen by adding and reacting iron ions, alkali metal hydroxides, EDTA (Ethylenediaminetetraacetic acid), and sugar to the mixture. .

Another object of the present invention is the step of sterilizing by adding peracetic acid to a medium containing sugar and a nitrogen source, and reacting; And

Iron ions, alkali metal hydroxides, and EDTA are added as peracetic acid decomposition accelerators to the sterilized medium, and the peracetic acid and peracetic acid decomposition accelerators are reacted in the medium to finally convert the peracetic acid reaction product into acetic acid, water and oxygen. It is to provide a method for sterilizing a medium comprising the step of decomposing into.

Another object of the present invention is the step of adding a sterilized medium to a bioreactor by the method of sterilizing the medium; And

It is to provide a method for culturing a microorganism comprising the step of inoculating the microorganism into the added medium and culturing it with pure water.

In order to achieve the above object, the present invention comprises the steps of mixing peracetic acid and water; And

It provides a method for decomposing peracetic acid comprising the step of finally decomposing the peracetic acid reactant into acetic acid, water and oxygen by adding and reacting iron ions, alkali metal hydroxides, ethylenediaminetetraacetic acid (EDTA), and sugar to the mixture.

In addition, the present invention is sterilized by adding peracetic acid to a medium containing sugar and a nitrogen source, and reacting; And

Iron ions, alkali metal hydroxides, and EDTA are added as peracetic acid decomposition accelerators to the sterilized medium, and the peracetic acid and peracetic acid decomposition accelerators are reacted in the medium to finally convert the peracetic acid reaction product into acetic acid, water and oxygen. It provides a method of sterilizing a medium comprising the step of decomposing into.

In addition, the present invention comprises the steps of adding a sterilized medium to a bioreactor by the method of sterilizing the medium; And

It provides a method for culturing a microorganism comprising the step of inoculating the microorganism in the added medium and culturing it with pure water.

The present invention relates to a method for decomposing peracetic acid and a method for culturing microorganisms using the method. When culturing microorganisms using the method of the present invention, the bioreactor and the medium can be completely sterilized without using heat, and sterilization It can be effectively removed by decomposing peracetic acid present in the medium used as a chemical sterilant without the use of washed water.

1 shows the observation of the aggregation state of iron ions under a microscope when EDTA is not added in the decomposition of peracetic acid.
Fig. 2 shows the observation of the aggregation state of iron ions under a microscope when EDTA is added in the decomposition of peracetic acid.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of mixing peracetic acid and water; And

It provides a method for decomposing peracetic acid comprising the step of finally decomposing the peracetic acid reactant into acetic acid, water and oxygen by adding iron ions, alkali metal hydroxides, ethylenediaminetetraacetic acid (EDTA), and sugar to the mixture and reacting.

The term "sterilization" as used in the present invention means killing the vegetative cells and spores of microorganisms.

In the method of decomposing peracetic acid of the present invention, the iron ions may be Fe ²⁺ ions or Fe ³⁺ ions, and the iron ions may be derived from salts containing Fe such as _{FeCl 3} , FeCl ₂ , and FeSO ₄ I can.

In the method of decomposing peracetic acid of the present invention, the hydroxide of an alkali metal may be LiOH, NaOH, or KOH, but is not limited thereto.

In the method of decomposing peracetic acid of the present invention, a pH value suitable for decomposition of peracetic acid (PAA) can be easily implemented by using a hydroxide of the alkali metal such as NaOH, and thus decomposition of peracetic acid can occur quickly. .

In the method of decomposing peracetic acid of the present invention, EDTA (Ethylenediaminetetraacetic acid) may increase the solubility of iron ions and prevent binding of iron and other substances to promote decomposition of peracetic acid.

In the method of decomposing peracetic acid of the present invention, the decomposition rate of peracetic acid is higher as the molar concentrations of iron ions added and EDTA are similar than when the molar concentration of iron ions added is slightly greater or less than the molar concentration of EDTA. It can be fast, and the rate of decomposition of peracetic acid can be the fastest if the molar concentrations of the added iron ions and EDTA are the same. For example, the molar ratio of iron ions to EDTA is 10:1, 9:1, 8:1. It can be 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1, and 1:2, 1:3, 1:4, 1:5, 1: It can be 6, 1:7, 1:8, 1:9, 1:10. If the amount of EDTA present during the reaction is greater than the amount of iron ions, the Fenton reaction by iron ions may be hindered and the mechanism of decomposition of peracetic acid by iron may be disturbed.

In the method for decomposing peracetic acid of the present invention, the sugar may be glucose, sucrose, or waste sugar (molasses) as a carbon source of microorganisms, but is not limited thereto.

In addition, the concentration of the sugar may be 100 g / L or less, preferably 5 to 50 g / L.

In the method for decomposing peracetic acid of the present invention, a basic buffer may be used instead of the alkali metal hydroxide or a basic buffer may be additionally added, and the basic buffer is 4-(2-hydroxyethyl)-1- Piperazineethanesulfonic acid (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEPES).

In the method of decomposing peracetic acid of the present invention, the peracetic acid reacts with water and reacts as follows to form acetic acid and hydrogen peroxide.

I) CH ₃ CO ₃ H + H ₂ O → H ₂ O ₂ + CH ₃ CO ₂ H

On the other hand, hydrogen peroxide produced by the reaction of peracetic acid and water is finally decomposed into acetic acid, water, and oxygen as shown in the following reaction equation by a Pantone reaction by iron ions.

ii) 2CH ₃ CO ₃ H → 2CH ₃ CO ₂ H + O ₂

iii) 2H ₂ O ₂ → 2H ₂ O + O ₂

The present invention is sterilized by adding peracetic acid to a medium containing sugar and a nitrogen source, and reacting; And

Iron ions, alkali metal hydroxides, and EDTA are added as peracetic acid decomposition accelerators to the medium sterilized with peracetic acid, and the peracetic acid and peracetic acid decomposition accelerators are reacted in the medium to finally convert the peracetic acid reaction product into acetic acid, It provides a method for sterilizing a medium comprising the step of decomposing into water and oxygen.

In the sterilization method of the medium of the present invention, peracetic acid and peracetic acid reactants present in the sterilized medium are finally decomposed into acetic acid, water and oxygen to remove peracetic acid and peracetic acid reactants present in the medium used for sterilization. It can be, and the medium prepared through it can be used for microbial culture.

In the sterilization method of the medium of the present invention, the medium uses a nitrogen source (peptone, yeast extract, whey, etc.) such as sugar (glucose, sucrose, waste sugar, etc.), as in the case of culturing yeast or Orantiochytrium. ) It may be the medium you need.

In the sterilization method of the medium of the present invention, the medium may be a medium that requires light, such as when culturing microalgae such as seawater species and freshwater species.

In the sterilization method of the medium of the present invention, the iron ions may be Fe ²⁺ ions or Fe ³⁺ ions, and the iron ions may be derived from salts containing Fe such as _{FeCl 3} , FeCl ₂ , FeSO ₄ have.

In the sterilization method of the medium of the present invention, the alkali metal hydroxide may be LiOH, NaOH, or KOH, but is not limited thereto.

In the method of decomposing peracetic acid of the present invention, a pH value suitable for decomposition of peracetic acid (PAA) can be easily implemented by using a hydroxide of the alkali metal such as NaOH, and thus decomposition of peracetic acid can occur quickly. .

In the sterilization method of the medium of the present invention, EDTA (Ethylenediaminetetraacetic acid) can promote the decomposition of peracetic acid by increasing the solubility of iron ions and preventing binding with other constituents.

In the sterilization method of the medium of the present invention, the decomposition rate of peracetic acid is faster as the molar concentrations of iron ions added and EDTA are similar than when the molar concentration of iron ions added is slightly higher or less than the molar concentration of EDTA. If the molar concentration of the added iron ions and EDTA are the same, the rate of decomposition of peracetic acid may be the fastest.

In the sterilization method of the medium of the present invention, the sugar may be glucose, sucrose, or waste sugar (molasses), but is not limited thereto.

In addition, the concentration of the sugar may be 100 g / L or less, preferably 5 to 50 g / L.

In the sterilization method of the medium of the present invention, a basic buffer may be used instead of the alkali metal hydroxide or a basic buffer may be additionally added, and the basic buffer is 4-(2-hydroxyethyl)-1-pipe It may be raginethanesulfonic acid (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEPES).

If the medium is sterilized as in the above method of the present invention, there is an advantage that it can be stored for a long time without being contaminated even in an open container without autoclaving until neutralization.

The present invention comprises the steps of adding a sterilized medium to the sterilized bioreactor or bioreactor by the method of sterilizing the medium; And

It provides a method for culturing a microorganism comprising the step of inoculating the microorganism in the added medium and culturing it with pure water.

In the method of culturing the microorganisms of the present invention, examples of the microorganisms are Orantiochytrium sp., Schizochytrium sp., Chlorella sp., Synechocystis sp. .), Debaryomyces sp., Yeasts, Lactic acid bacteria, Actinomycetes, Euglena, Mortierella filamentous fungi and photosynthetic bacteria. It may be one or more selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides a method of simultaneously sterilizing the bioreactor and the medium by putting a medium containing sugar and a nitrogen source into a bioreactor, adding peracetic acid, and reacting.

According to an embodiment of the present invention, when peracetic acid is added to Aurantiochytrium medium, and FeCl ₃ , NaOH, EDTA, and glucose are added to the sterilized medium by reacting for 2 hours, and reacting for 1 day or more, and It was confirmed that acetic acid was finally decomposed into acetic acid, water, and oxygen at a remarkably fast rate (Experimental Example 1).

However, according to an embodiment of the present invention, peracetic acid was added to Aurantiochytrium medium, and FeCl ₃ , NaOH, and glucose were added to the sterilized medium by reacting for 2 hours, except for EDTA, and for 1 day or more. When reacted, it was confirmed that the decomposition rate of peracetic acid was significantly reduced (Experimental Example 1).

According to an embodiment of the present invention, peracetic acid is added to Aurantiochytrium medium, and FeCl ₃ , NaOH, and EDTA are added to the sterilized medium by reacting for 2 hours, and the amount of glucose is gradually increased and added, In the case of reaction for more than 1 day, it was confirmed that the rate at which peracetic acid is finally decomposed into acetic acid, water, and oxygen gradually increased as the amount of glucose added gradually increased (Experimental Example 2).

However, according to an embodiment of the present invention, peracetic acid was added to Aurantiochytrium medium, and FeCl ₃ , NaOH, and EDTA were added to the sterilized medium by reacting for 2 hours, excluding glucose, and then adding 1 day or more. When reacted, it was confirmed that the decomposition rate of peracetic acid was significantly reduced (Experimental Example 2). When reducing sugars such as glucose are added, iron oxidized by peracetic acid is re-reduced, thereby activating the Fenton reaction by iron, thereby helping to decompose peracetic acid by iron.

According to an embodiment of the present invention, peracetic acid is added to Aurantiochytrium medium, and FeCl ₃ , NaOH, and EDTA are added to the sterilized medium by reacting for 2 hours, and the amount of sucrose instead of glucose is gradually increased. When added and reacted for more than 1 day, it was confirmed that the rate at which peracetic acid finally decomposes into acetic acid, water, and oxygen gradually increases as the amount of sucrose to be added increases gradually (Experimental Example 3).

According to an embodiment of the present invention, peracetic acid is added to Aurantiochytrium medium, and metal, NaOH, glucose, and EDTA are added to the sterilized medium by reacting for 2 hours, and when reacting for more than 1 day, FeCl ₃ +EDTA or FeSO _{When 4} +EDTA was added, peracetic acid was decomposed well, but when CaCl ₂ +EDTA, MgCl ₂ +EDTA or ZnCl ₂ +EDTA was added, peracetic acid was hardly decomposed. FeCl ₃ , FeSO ₄ , CaCl ₂ , MgCl ₂ , and ZnCl _{2 When} only the metal was added and EDTA was not added, it was confirmed that peracetic acid was hardly decomposed (Experimental Example 4).

Hereinafter, the present invention will be described in detail by examples.

However, the following examples illustrate the present invention, and the contents of the present invention are not limited by the examples.

Example 1: Confirmation of decomposition of peracetic acid

Peracetic acid (Dongmyeong ONC, 160126) was added to 1 L of Aurantiochytrium medium to be 10.52 mM, and sterilized by reacting for 2 hours. In the medium sterilized with peracetic acid 96 uM FeCl ₃ (Sigma, 7705-08-0), 5.5 mM NaOH (Sigma, 1310-73-2), 96 uM EDTA (Sigma, 60-00-4), and 20 Each component was added to make g/L glucose (Sigma, 50-99-7), and reacted for at least 1 day to finally decompose peracetic acid and the reaction product into acetic acid, water, and oxygen, and peroxide test Decomposition of peracetic acid was measured with sticks (Quantofix Peroxide 100, Macherey-Nagel GmbH & Co., Germany).

At this time, the Aurantiochytrium medium is based on 1L, yeast extract (Sigma, 8013-01-2) 2g, peptone (Sigma, 91079-40-2) 2g, D-glucose (Sigma, 50-99-7) 20g , Seawater (Namhae Sea) 500 mL and distilled water 500 mL (Table 5).

ingredient amount Yeast extract 2 g peptone 2 g D-glucose 20 g sea water 500 mL Distilled water 500 mL Total amount 1 L

Comparative Example 1

Experiments were carried out in the same manner as in Example 1, but EDTA was not added.

Comparative Example 2

The experiment was carried out in the same manner as in Example 1, but no glucose was added.

Examples 2 to 6: Difference in glucose concentration

Experiments were carried out in the same manner as in Example 1, but the amount of glucose to be added was 10 g/L (Example 2), 30 g/L (Example 3), 40 g/L or (Example 4), respectively, 50 g/L (Example 5), or 60 g/L (Example 6) were added.

Examples 7 to 12: Difference in sucrose concentration

Experiments were carried out in the same manner as in Example 1, but instead of glucose, the amount of sucrose was respectively 10 g/L (Example 7), 20 g/L (Example 8), and 30 g/L (Example 9). , 40 g/L (Example 10), 50 g/L (Example 11), or 60 g/L (Example 12) were added.

Comparative Examples 3 to 9

The experiment was carried out in the same manner as in Example 1, but without adding EDTA, _{CaCl 2} was added instead of _{FeCl 3} (Comparative Example 3), EDTA was added and _{CaCl 2} was added instead of _{FeCl 3} (Comparative Example 4), and EDTA was added. _{FeSO 4} instead of FeCl ₃ was added without adding (Comparative Example 5), _{MgCl 2} was added instead of _{FeCl 3} without EDTA (Comparative Example 6), EDTA was added and _{MgCl 2} was added instead of _{FeCl 3} (Comparative Example 7), Without adding EDTA, _{ZnCl 2} was added instead of _{FeCl 3} (Comparative Example 8), EDTA was added, and _{ZnCl 2} was added instead of _{FeCl 3} (Comparative Example 9).

Example 13

Example 1, but with carrying out experiments in the same manner, it was added to FeCl ₃ instead of FeSO4.

Experimental Example 1: Effect of EDTA

In order to confirm the effect of EDTA on the decomposition of peracetic acid, an experiment was performed in the same manner as in Example 1 and Comparative Example 1.

As a result, when EDTA is not added (Comparative Example 1, Fig. 1), the solubility of iron ions, which is a metal ion, is sharply reduced compared to the case where EDTA is added (Example 1, Fig. 2), and the decomposition rate of peracetic acid is significantly reduced. It could be confirmed that (Table 1).

In addition, it was confirmed that iron ions were condensed and bound in other polymers in the case where EDTA was not added (Comparative Example 1) compared to the case where EDTA was added (Example 1, Fig. 2) (Fig. 1). This result shows that in the presence of EDTA, the reactivity of peracetic acid decomposition increases due to the presence of iron ions in a water-soluble state. Suggests lowering.

Time (hour) Example 1 Comparative Example 1 hour PAA + N + G(20)
FeCl3+EDTA PAA + N + G(20)
FeCl3 0 >200 >200 6 100 >200 12 0 >200 18 0 >200 24 0 >200 30 0 >200

N: NaOH, G: glucose, F: FeCl ₃ , F(E): FeCl ₃ + EDTA

The numbers in the table mean the concentration of peracetic acid present, for example, >200 means that 200 mg/L or more of peracetic acid remains.

Experimental Example 2: Effect of glucose

In order to confirm the effect of glucose on the decomposition of peracetic acid, an experiment was performed in the same manner as in Examples 1 to 6 and Comparative Example 2.

As a result, when glucose was not added, peracetic acid was hardly decomposed, but it was confirmed that the decomposition rate of peracetic acid gradually increased as the amount of glucose added increased (Table 2).

Time (hour) Comparative Example 2 Example 2 Example 1 Example 3 Example 4 Example 5 Example 6 hour PAA
F(E)+N PAA
F(E)+N+G(10) PAA
F(E)+N+G(20) PAA
F(E)+N+G(30) PAA
F(E)+N+G(40) PAA
F(E)+N+G(50) PAA
F(E)+N+G(60) 0 >200 >200 >200 >200 >200 >200 >200 6 >200 >200 100 60 20 14 10 12 >200 0 0 0 0 0 0 24 >200 0 0 0 0 0 0

N: NaOH, G: glucose, F: FeCl ₃ , F(E): FeCl ₃ + EDTA

The numbers in the table mean the concentration of peracetic acid present, for example, >200 means that 200 mg/L or more of peracetic acid remains.

Experimental Example 3: Effect of sucrose

In order to confirm the effect of sucrose in decomposing peracetic acid, an experiment was performed in the same manner as in Examples 7 to 12 and Comparative Example 2.

As a result, when no sugar was added, peracetic acid was hardly decomposed, but it was confirmed that the decomposition rate of peracetic acid gradually increased as the amount of sucrose to be added increased (Table 3).

Time (hour) Comparative Example 2 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 hour PAA
F(E)+N PAA
F(E)+N+S(10) PAA
F(E)+N+S(20) PAA
F(E)+N+S(30) PAA
F(E)+N+S(40) PAA
F(E)+N+S(50) PAA
F(E)+N+S(60) 0 >200 >200 >200 >200 >200 >200 >200 6 >200 >200 >200 >200 >200 160 140 12 >200 0 0 0 0 0 0 24 >200 0 0 0 0 0 0

N: NaOH, G: Glucose, S: Sucrose, F: FeCl ₃ , F(E): FeCl ₃ + EDTA

The numbers in the table mean the concentration of peracetic acid present, for example, >200 means that 200 mg/L or more of peracetic acid remains.

Experimental Example 4: Effect of metal

In order to confirm the effect of the metal in the decomposition of peracetic acid, experiments were performed in the same manner as in Examples 1 and 13 and Comparative Examples 1 and 3 to 9.

As a result, when FeCl ₃ +EDTA or FeSO ₄ +EDTA was added, peracetic acid was decomposed well, but when CaCl ₂ +EDTA, MgCl ₂ +EDTA or ZnCl ₂ +EDTA was added, peracetic acid was hardly decomposed. , FeCl ₃ , FeSO ₄ , CaCl ₂ , MgCl ₂ , or ZnCl _{2 When} only the metal was added and EDTA was not added, it was confirmed that peracetic acid was hardly decomposed (Table 4).

N: NaOH, G: glucose, S: sucrose

The numbers in the table mean the concentration of peracetic acid present, for example, >200 means that 200 mg/L or more of peracetic acid remains.

Claims (12)

Mixing peracetic acid with water; And
A method of decomposing peracetic acid comprising adding and reacting iron ions, alkali metal hydroxides, ethylenediaminetetraacetic acid (EDTA), and sugar to the mixture to finally decompose the peracetic acid reactant into acetic acid, water and oxygen.
The method of claim 1, wherein the iron ions are Fe ²⁺ ions or Fe ³⁺ ions.
The method of claim 1, wherein the sugar is glucose, sucrose, or waste sugar.
The method of claim 3, wherein the concentration of the sugar is 100 g/L or less.
The method for decomposing peracetic acid according to claim 1, wherein a basic buffer solution is further added.
Sterilizing by adding peracetic acid to a medium containing sugar and a nitrogen source, and reacting; And
Iron ions, alkali metal hydroxides and EDTA are added as peracetic acid decomposition accelerators to the sterilized medium, and the peracetic acid and peracetic acid decomposition accelerators are reacted in the medium to finally convert the peracetic acid reaction product into acetic acid, water and oxygen. A method of sterilizing a medium comprising the step of decomposing.
The method of claim 6, wherein the iron ions are Fe ²⁺ ions or Fe ³⁺ ions.
The method of claim 6, wherein the sugar is glucose, sucrose, or waste sugar.
The method of claim 6, wherein the concentration of the sugar is 100 g/L or less.
The method for sterilizing a medium according to claim 6, wherein a basic buffer solution is further added.
Adding a medium sterilized by the method of claim 6 to a bioreactor; And
Inoculating the microorganism in the added medium, and culturing a microorganism comprising the step of pure culture.
The method of claim 11, wherein the microorganism is Orantiochytrium sp., Schizochytrium sp., Chlorella sp., Synechocystis sp., Debariomyces sp. Selected from the group consisting of Debaryomyces sp., Yeasts, Lactic acid bacteria, Actinomycetes, Euglena, Mortierella, filamentous fungi and photosynthetic bacteria. A method of culturing microorganisms, characterized in that at least one type.

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