CN113227346A - Method for decomposing peracetic acid and method for culturing microorganism using same - Google Patents

Method for decomposing peracetic acid and method for culturing microorganism using same Download PDF

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CN113227346A
CN113227346A CN201980087271.6A CN201980087271A CN113227346A CN 113227346 A CN113227346 A CN 113227346A CN 201980087271 A CN201980087271 A CN 201980087271A CN 113227346 A CN113227346 A CN 113227346A
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权宗喜
曺畅浩
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Ensaile Co ltd
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Abstract

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

Description

Method for decomposing peracetic acid and method for culturing microorganism using same
Technical Field
The present invention relates to a method for decomposing peroxyacetic acid and a method for culturing a microorganism using the same, the method comprising the steps of: mixing peracetic acid in water; and adding iron ions, alkali metal hydroxide, Ethylene Diamine Tetraacetic Acid (EDTA) and sugar into the mixture to react, and finally decomposing the peroxyacetic acid reactant into acetic acid, water and oxygen.
Background
In the biological industry, when a large amount of useful metabolites is to be produced using a variety of microorganisms, sterilization of a culture medium and a reactor is important. For this reason, conventionally, an autoclave (autoclave) has been mainly used for sterilizing a microbial culture medium and a reactor.
However, when the culture medium and the reactor are sterilized by an autoclave, a sugar component of the culture medium reacts with a protein (peptone or yeast extract) which is one of other components of the culture medium at a high temperature to induce the production of a toxic substance which may inhibit the growth of microorganisms such as Hydroxymethylfurfural (HMF), thereby inhibiting the growth of the microorganisms.
In order to overcome the above problems and limitations, a filtration (filtration) method is used, but in the case of filtration, there is inconvenience in that the incubator itself needs to be separately sterilized, there is a high risk of clogging and recontamination during the filtration process, the size of the filter is limited, and the required cost is high, thereby causing a problem of low industrial applicability.
To solve such problems, as disclosed in Korean laid-open patent publication No. 10-2015-0097295, a sterilization method using peracetic acid (PAA) was developed. Korean laid-open patent publication No. 10-2015-0097295 discloses a method of sterilizing a photobioreactor using peracetic acid when pure culturing microalgae requiring light. However, the above-mentioned prior art documents do not disclose that the decomposition efficiency of peracetic acid is increased by EDTA and sugars (glucose, sucrose, waste sugar, etc.), and do not disclose at all a method for sterilizing a medium containing sugars and nitrogen sources (peptone, Yeast extract, whey, etc.).
In addition, in the method of sterilizing the photobioreactor and the culture medium using peracetic acid, when the culture medium components contain sugar and a nitrogen source, it is necessary to use peracetic acid at a high concentration of 5mM or more for complete sterilization, and particularly, when the culture medium to which the nitrogen source is added is sterilized together, Nitrogen Oxide (NO) based on peracetic acid is generated3-) The nitrogen oxides formed bind to the iron ions, inhibiting the catalytic reaction for decomposing peroxyacetic acid. In this case, it takes more than one month to completely decompose the peroxyacetic acid reactant, solely by natural decomposition of peroxyacetic acid and catalytic reaction based on iron with N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid (HEPES).
Disclosure of Invention
Technical problem
The invention aims to provide a method for decomposing peroxyacetic acid, which comprises the following steps:
mixing peracetic acid in water; and
adding iron ions, alkali metal hydroxide, EDTA (ethylene diamine tetraacetic acid) and sugar into the mixture to react, and finally decomposing the peroxyacetic acid reactant into acetic acid, water and oxygen.
Another object of the present invention is to provide a method for sterilizing a culture medium, comprising the steps of:
adding peroxyacetic acid to a medium comprising a sugar and a nitrogen source, reacting and sterilizing; and
adding iron ions, an alkali metal hydroxide and EDTA as a peracetic acid decomposition accelerator to the sterilized medium, and reacting the peracetic acid and the peracetic acid decomposition accelerator in the medium to finally decompose the peracetic acid reactant into acetic acid, water and oxygen.
Another object of the present invention is to provide a method for culturing a microorganism, comprising the steps of: adding a culture medium sterilized by the method for sterilizing the culture medium to a bioreactor; and
the added culture medium was inoculated with a microorganism and subjected to pure culture.
Technical scheme
In order to achieve the above object, the present invention provides a method for decomposing peracetic acid, comprising the steps of:
mixing peracetic acid in water; and
adding iron ions, alkali metal hydroxide, Ethylene Diamine Tetraacetic Acid (EDTA) and sugar into the mixture, and reacting to finally decompose the peroxyacetic acid reactant into acetic acid, water and oxygen.
Further, the present invention provides a method for sterilizing a culture medium, comprising the steps of:
adding peroxyacetic acid to a medium comprising a sugar and a nitrogen source, reacting and sterilizing; and
adding iron ions, an alkali metal hydroxide and EDTA as a peracetic acid decomposition accelerator to the sterilized medium, and reacting the peracetic acid and the peracetic acid decomposition accelerator in the medium to finally decompose the peracetic acid reactant into acetic acid, water and oxygen.
Further, the present invention provides a method for culturing a microorganism, comprising the steps of:
adding a culture medium sterilized by the method for sterilizing the culture medium to a bioreactor; and
the added culture medium was inoculated with a microorganism and subjected to pure culture.
ADVANTAGEOUS EFFECTS OF INVENTION
The present invention relates to a method for decomposing peracetic acid and a method for culturing microorganisms using the same, in which a bioreactor and a culture medium can be thoroughly sterilized without using heat when the microorganisms are cultured by the method of the present invention, and peracetic acid used as a chemical sterilizing agent present in the culture medium can be decomposed and effectively removed without using sterilized washing water.
Drawings
FIG. 1 is a view showing the state of aggregation of iron ions under a microscope, in the case where EDTA is not added to decompose peroxyacetic acid.
FIG. 2 is a view showing the state of aggregation of iron ions observed with a microscope when EDTA is added to decompose peroxyacetic acid.
Detailed Description
The present invention will be described in detail below with reference to examples.
However, the following examples are intended to illustrate the present invention, and the contents of the present invention are not limited to the examples.
Example 1: confirmation of decomposition of Peroxyacetic acid
10.52mM peracetic acid (same name ONC, 160126) was added to 1L of Thraustochytrium (Aurantiochytrium) medium, and the reaction was performed for 2 hours for sterilization. To the medium sterilized with the above-mentioned peracetic acid was added FeCl 96uM each3(Sigma, 7705-08-0), 5.5mM NaOH (Sigma, 1310-73-2), 96uM EDTA (Sigma, 60-00-4) and 20g/L Glucose (Glucose) (Sigma, 50-99-7) were reacted for 1 day or more, whereby peracetic acid and its reaction products were finally decomposed into acetic acid, water and oxygen, and whether or not peracetic acid was decomposed was determined using a Peroxide test stick (Quantix peroxides 100, Macherey-Nagel, Germany).
In this case, the culture medium of Thraustochytrium (Aurantiochytrium) was composed of 2g of yeast extract (Sigma, 8013-01-2), 2g of peptone (Sigma, 91079-40-2), 20g of D-glucose (Sigma, 50-99-7), 500mL of seawater (seawater) (Namhae Sea) and 500mL of distilled water based on 1L (Table 5).
[ Table 5]
Composition (I) Measurement of
Yeast extractFetching article 2g
Peptone 2g
D-glucose 20g
Seawater, its production and use 500mL
Distilled water 500mL
Total amount of 1L
Comparative example 1
The experiment was performed in the same manner as in example 1 without adding EDTA.
Comparative example 2
The experiment was performed in the same manner as in example 1 without adding glucose.
Examples 2 to 6: difference in glucose concentration
An experiment was carried out in the same manner as in example 1, with addition of glucose in amounts of 10g/L (example 2), 30g/L (example 3), 40g/L or (example 4), 50g/L (example 5) or 60g/L (example 6), respectively.
Examples 7 to 12: difference in sucrose concentration
An experiment was carried out in the same manner as in example 1, except that sucrose was added in an amount of 10g/L (example 7), 20g/L (example 8), 30g/L (example 9), 40g/L (example 10), 50g/L (example 11) or 60g/L (example 12), respectively, in place of glucose.
Comparative examples 3 to 9
In the same way as in example 1The experiment was carried out without EDTA addition, with CaCl addition2To replace FeCl3Comparative example 3 EDTA and CaCl2To replace FeCl3Comparative example 4 FeSO was added without EDTA4To replace FeCl3Comparative example 5 in which MgCl was added without EDTA2To replace FeCl3Comparative example 6, EDTA and MgCl were added2To replace FeCl3Comparative example 7, in which EDTA was not added, ZnCl was added2To replace FeCl3Comparative example 8, EDTA and ZnCl were added2To replace FeCl3(comparative example 9).
Example 13
The experiment was carried out in the same manner as in example 1, with addition of FeSO4To replace FeCl3
Experimental example 1: effect of EDTA
In order to confirm the effect of EDTA in the decomposition of peroxyacetic acid, experiments were performed in the same manner as in example 1 and comparative example 1.
As a result, it was confirmed that the dissolution rate of peroxyacetic acid was significantly decreased when EDTA was not added (comparative example 1, fig. 1) compared to when EDTA was added (example 1, fig. 2) because the solubility of iron ions, which are metal ions, was rapidly decreased (table 1).
Further, it was confirmed that iron ions were condensed and bound in other polymers (fig. 1) as compared with the case where EDTA was added (example 1, fig. 2) and the case where EDTA was not added (comparative example 1). As a result, it was found that in the presence of EDTA, iron ions were present in a water-soluble state, and the reactivity of decomposition of peracetic acid was high, but in the absence of EDTA, iron ions were not present in a water-soluble state, or were combined with other components, and the reactivity of decomposition of peracetic acid was low.
[ Table 1]
Figure BDA0003140137110000051
N: NaOH, G: glucose, F: FeCl3,F(E):FeCl3+EDTA
The above-mentioned figures in the exterior and interior refer to the concentration of peracetic acid present, e.g., >200 refers to a residual amount of peracetic acid of 200mg/L or more
Experimental example 2: effect of glucose
In the decomposition of peroxyacetic acid, in order to confirm the effect of glucose, experiments were performed in the same manner as in examples 1 to 6 and comparative example 2.
As a result, it was confirmed that, although peroxyacetic acid was hardly decomposed when glucose was not added, the decomposition rate of peroxyacetic acid gradually increased as the amount of added glucose increased (table 2).
[ Table 2]
Figure BDA0003140137110000061
N: NaOH, G: glucose, F: FeCl3,F(E):FeCl3+EDTA
The above-mentioned figures in the exterior and interior refer to the concentration of peroxyacetic acid present, e.g. >200 refers to residual peroxyacetic acid of 2005mg/L or more
Experimental example 3: effect of sucrose
In the decomposition of peroxyacetic acid, in order to confirm the effect of sucrose, experiments were performed in the same manner as in examples 7 to 12 and comparative example 2.
As a result, it was confirmed that peroxyacetic acid was hardly decomposed when no sugar was added, but the decomposition rate of peroxyacetic acid was gradually increased as the amount of sucrose added was increased (table 3).
[ Table 3]
Figure BDA0003140137110000062
Figure BDA0003140137110000071
N: NaOH, G: glucose, S: sucrose, F: FeCl3,F(E):FeCl3+EDTA
The above-mentioned figures in the exterior and interior refer to the concentration of peracetic acid present, e.g., >200 refers to a residual amount of peracetic acid of 200mg/L or more
Experimental example 4: effect of Metal
In the decomposition of acetic acid, in order to confirm the effect of the metal, experiments were performed in the same manner as in examples 1 and 13 and comparative examples 1, 3 to 9.
As a result, it was confirmed that FeCl was added3+ EDTA or FeSO4In case of EDTA, although peroxyacetic acid was decomposed smoothly, CaCl was added2+EDTA、MgCl2+ EDTA or ZnCl2In the case of EDTA, peroxyacetic acid hardly decomposes, and only FeCl is added3、FeSO4、CaCl2、MgCl2Or ZnCl2The peroxyacetic acid hardly decomposed when EDTA was not added but metal (table 4).
[ Table 4]
Figure BDA0003140137110000072
N: NaOH, G: glucose, S: sucrose
The above-mentioned figures in the exterior and interior refer to the concentration of peracetic acid present, e.g., >200 refers to a residual amount of peracetic acid of 200mg/L or more
The present invention will be described in detail below.
The invention provides a method for decomposing peroxyacetic acid, which comprises the following steps:
mixing peracetic acid in water; and
iron ions, alkali metal hydroxide, edta (ethylenediaminetetraacetic acid) and sugar are added to the above mixture to react, and the peroxyacetic acid reactant is finally decomposed into acetic acid, water and oxygen.
The term "sterilization" as used herein refers to causing apoptosis of vegetative cells and spores of a microorganism.
In the method for decomposing peroxyacetic acid according to the present invention, the iron ion may be Fe2+Ions or Fe3+Ion, iron ionThe seed may be derived from FeCl containing Fe3、FeCl2、FeSO4And the like.
In the above method for decomposing peroxyacetic acid of the present invention, the alkali metal hydroxide may be LiOH, NaOH, or KOH, but is not limited thereto.
In the above-mentioned method for decomposing peroxyacetic acid of the present invention, a pH value suitable for decomposing peroxyacetic acid (PAA) can be easily achieved using the above-mentioned alkali metal hydroxide, for example, using NaOH, and thus peroxyacetic acid can be rapidly decomposed.
In the method for decomposing peroxyacetic acid according to the present invention, edta (ethylene diamine tetraacetic acid) increases the solubility of iron ions, prevents the binding between iron and other substances, and promotes the decomposition of peroxyacetic acid.
In the method for decomposing peroxyacetic acid according to the present invention, the decomposition rate of peroxyacetic acid is faster as the molar concentrations of the added iron ions and EDTA are similar to each other, as compared with the case where the molar concentration of the added iron ions is slightly more than the molar concentration of EDTA or less, and the decomposition rate of peroxyacetic acid can be fastest when the molar concentrations of the added iron ions and EDTA are the same. For example, the molar concentration ratio of ferric ion to EDTA may be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1, and may be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10. When the amount of EDTA present at the reaction is larger than the amount of iron ions, fenton reaction due to iron ions is prevented, thereby preventing the decomposition mechanism of peracetic acid due to iron.
In the method for decomposing peroxyacetic acid of the present invention, the sugar is a carbon source for the microorganism, and may be glucose, sucrose, waste sugar (molasses), or the like, but is not limited thereto.
Also, the concentration of the above-mentioned saccharide may be 100g/L or less, and preferably may be 5 to 50 g/L.
In the method for decomposing peroxyacetic acid of the present invention, an alkaline buffer such as 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) may be used instead of or in addition to the alkali metal hydroxide.
In the method for decomposing peroxyacetic acid of the present invention, the peroxyacetic acid reacts with water to form acetic acid and hydrogen peroxide as follows.
ⅰ)CH3CO3H+H2O→H2O2+CH3CO2H
On the other hand, hydrogen peroxide produced by the reaction of peracetic acid with water undergoes Fenton reaction (Fenton reaction) by iron ions, and finally decomposes into acetic acid, water, and oxygen according to the following reaction formula.
ii)2CH3CO3H→2CH3CO2H+O2
iii)2H2O2→2H2O+O2
The invention provides a method for sterilizing a culture medium, which comprises the following steps:
adding peroxyacetic acid to a medium comprising a sugar and a nitrogen source, reacting and sterilizing; and adding iron ions, an alkali metal hydroxide and EDTA as a peracetic acid decomposition accelerator to the medium sterilized with peracetic acid, and reacting the peracetic acid and the peracetic acid decomposition accelerator in the medium to finally decompose the peracetic acid reactant into acetic acid, water and oxygen.
In the above method for sterilizing a 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, and the peracetic acid and peracetic acid reactants present in the medium for sterilization can be removed, whereby the prepared medium can be used for the culture of microorganisms.
In the method of sterilizing the medium of the present invention, the medium may be a medium requiring sugar (glucose, sucrose, waste sugar, etc.) and a nitrogen source (peptone, Yeast extract (Yeast extract), whey, etc.), as in the case of culturing Yeast, Thraustochytrium (Aurantiochytrium), etc.
In the method of sterilizing the medium of the present invention, the medium may be a medium requiring light (light) as in the case of culturing microalgae such as marine species and freshwater species.
In the method for sterilizing the medium of the present invention, the iron ion may be Fe2+Ions or Fe3+Ions, the iron ions being derivable from FeCl comprising Fe3、FeCl2、FeSO4And the like.
In the method for sterilizing the medium of the present invention, the hydroxide of an alkali metal may be LiOH, NaOH, or KOH, but is not limited thereto.
In the above-mentioned method for decomposing peroxyacetic acid of the present invention, a pH value suitable for decomposing peroxyacetic acid (PAA) can be easily achieved using the above-mentioned alkali metal hydroxide, for example, using NaOH, and thus peroxyacetic acid can be rapidly decomposed.
In the method for sterilizing the medium of the present invention, edta (ethylene diamine tetraacetic acid) increases the solubility of iron ions, prevents binding with other components, and promotes decomposition of peracetic acid.
In the method for sterilizing a medium of the present invention, the decomposition rate of peracetic acid is faster as the molar concentrations of the added iron ions and EDTA are similar to each other than when the molar concentration of the added iron ions is slightly higher than or lower than the molar concentration of EDTA, and the decomposition rate of peracetic acid is fastest when the molar concentrations of the added iron ions and EDTA are the same.
In the method for sterilizing the medium of the present invention, the sugar may be glucose, sucrose or waste sugar (molasses), but is not limited thereto.
Also, the concentration of the above-mentioned saccharide may be 100g/L or less, and preferably may be 5 to 50 g/L.
In the method for sterilizing the medium of the present invention, an alkaline buffer solution may be used instead of or in addition to the hydroxide of the alkali metal, and the alkaline buffer solution may be 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid; HEPES).
In the culture medium with the method of the invention of the same sterilization, until neutralization, even without the use of autoclave, will not be in the open container pollution, has a long time storage advantage.
The invention provides a method for culturing microorganisms, which comprises the following steps:
adding a culture medium sterilized by the method for sterilizing the culture medium to a sterilized bioreactor or a bioreactor; and
the added medium was inoculated with a microorganism and pure culture was carried out.
In the method for culturing the microorganism of the present invention, the microorganism may be 1 or more selected from the group consisting of thraustochytrium (aurantichloride sp.), Schizochytrium (Schizochytrium sp.), Chlorella (Chlorella sp.), Synechocystis (Synechocystis sp.), Debaryomyces (Debaryomyces sp.), saccharomycetes (Yeast), lactobacillus (Lactobacillus), Actinomycetes (Actinomycetes), Euglena (Euglena), Mortierella (Mortierella), filamentous fungi, and photosynthetic bacteria, but is not limited thereto.
Also, the present invention provides a method of simultaneously sterilizing a bioreactor and a culture medium by placing the culture medium containing sugar and a nitrogen source in the bioreactor, adding peracetic acid, and reacting.
According to an embodiment of the present invention, it was confirmed that peracetic acid was added to a thraustochytrium (Aurantiochytrium) medium, reacted for 2 hours, and FeCl was added to a sterilized medium3NaOH, EDTA and Glucose (Glucose), which are reacted for 1 day or more, peroxyacetic acid is 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, it was confirmed that peracetic acid was added to a thraustochytrium (Aurantiochytrium) medium, the reaction was carried out for 2 hours, and FeCl other than EDTA was added to the sterilized medium3NaOH and Glucose (Glucose) were reacted for 1 day or more, and the decomposition rate of peracetic acid was significantly decreased (experimental example 1).
According to an embodiment of the present invention, it can be confirmed thatAdding peroxyacetic acid into the culture medium of the thraustochytrium (Aurantiochytrium), reacting for 2 hours, adding FeCl into the sterilized culture medium3The reaction was carried out for 1 day or more by adding Glucose (Glucose) so as to gradually increase the amount of NaOH and EDTA, and the rate of final decomposition of peracetic acid into acetic acid, water and oxygen gradually increased as the amount of Glucose added was gradually increased (experimental example 2).
However, according to an embodiment of the present invention, it was confirmed that peracetic acid was added to a thraustochytrium (Aurantiochytrium) medium, the reaction was carried out for 2 hours, and FeCl other than Glucose (Glucose) was added to the sterilized medium3NaOH and EDTA reacted for 1 day or more, and the decomposition rate of peracetic acid was significantly decreased (experimental example 2). When a reducing sugar such as glucose is added, iron oxidized by peracetic acid is reduced again, and fenton reaction due to iron is activated, thereby contributing to decomposition of peracetic acid due to iron.
According to an embodiment of the present invention, it was confirmed that peracetic acid was added to a thraustochytrium (Aurantiochytrium) medium, reacted for 2 hours, and FeCl was added to a sterilized medium3And NaOH and EDTA, Sucrose (Sucrose) was added instead of glucose to gradually increase the amount of Sucrose (Sucrose), and the rate of final decomposition of peracetic acid into acetic acid, water and oxygen gradually increased as the amount of Sucrose added was gradually increased at 1 day or more of the reaction (experimental example 3).
According to an embodiment of the present invention, it was confirmed that peracetic acid was added to a thraustochytrium (Aurantiochytrium) medium to react for 2 hours, metals, NaOH, glucose and EDTA were added to the sterilized medium, and FeCl was added to react for 1 hour or more3+ EDTA or FeSO4+ EDTA decomposition of peracetic acid is successful, but with the addition of CaCl2+EDTA、MgCl2+ EDTA or ZnCl2In the case of + EDTA, peroxyacetic acid hardly decomposed, and FeCl alone was added3、FeSO4、CaCl2、MgCl2And ZnCl2In the case of metal without addition of EDTA, peroxyacetic acid hardly decomposed (Experimental example 4).
Industrial applicability
When a microorganism is cultured by the method for decomposing peroxyacetic acid of the present invention and the method for culturing a microorganism by the above method, the bioreactor and the culture medium can be thoroughly sterilized without using heat, and thus, the method is industrially useful.

Claims (12)

1. A method for decomposing peroxyacetic acid is characterized by comprising the following steps:
mixing peracetic acid in water; and
adding iron ions, alkali metal hydroxide, ethylene diamine tetraacetic acid and sugar into the mixture for reaction, and finally decomposing the peroxyacetic acid reactant into acetic acid, water and oxygen.
2. The method of decomposing peroxyacetic acid according to claim 1, wherein the iron ion is Fe2+Ions or Fe3+Ions.
3. A process of decomposing peroxyacetic acid according to claim 1, wherein the sugar is glucose, sucrose or waste sugar.
4. The method of decomposing peroxyacetic acid according to claim 3, wherein the concentration of the sugar is 100g/L or less.
5. The method of decomposing peroxyacetic acid according to claim 1, wherein an alkaline buffer is further added.
6. A method for sterilizing a culture medium, comprising the steps of:
adding peroxyacetic acid to a medium comprising a sugar and a nitrogen source, reacting and sterilizing; and
adding iron ions, an alkali metal hydroxide and ethylenediaminetetraacetic acid as a peracetic acid decomposition accelerator to the sterilized medium, reacting the peracetic acid and the peracetic acid decomposition accelerator in the medium, and finally decomposing the peracetic acid reactant into acetic acid, water and oxygen.
7. The method for sterilizing a medium according to claim 6, wherein the iron ion is Fe2+Ions or Fe3+Ions.
8. The method for sterilizing a medium according to claim 6, wherein the sugar is glucose, sucrose or waste sugar.
9. The method of sterilizing a medium according to claim 6, wherein the concentration of the sugar is 100g/L or less.
10. The method of sterilizing a medium according to claim 6, wherein an alkaline buffer is further added.
11. A method for culturing a microorganism, comprising the steps of:
adding a culture medium sterilized according to the method of claim 6 to a bioreactor; and
the added culture medium is inoculated with microorganisms and pure culture is carried out.
12. The method of claim 11, wherein the microorganism is 1 or more selected from the group consisting of thraustochytrium, schizochytrium, chlorella, synechocystis, debaryomyces, yeast flora, lactobacillus flora, actinomycete flora, euglena, mortierella, filamentous flora, and photosynthetic bacteria.
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