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 PDFInfo
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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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- Prior art keywords
- acid
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- decomposing
- sugar
- peracetic acid
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- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 title claims abstract description 260
- 238000000034 method Methods 0.000 title claims abstract description 69
- 244000005700 microbiome Species 0.000 title claims abstract description 26
- 238000012258 culturing Methods 0.000 title claims abstract description 14
- 230000001954 sterilising effect Effects 0.000 claims abstract description 37
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 65
- 229960001484 edetic acid Drugs 0.000 claims description 61
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 52
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 48
- 239000002609 medium Substances 0.000 claims description 45
- 229910052742 iron Inorganic materials 0.000 claims description 39
- 238000000354 decomposition reaction Methods 0.000 claims description 32
- -1 iron ions Chemical class 0.000 claims description 32
- 239000001963 growth medium Substances 0.000 claims description 31
- 235000000346 sugar Nutrition 0.000 claims description 29
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 239000008103 glucose Substances 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 18
- 229930006000 Sucrose Natural products 0.000 claims description 18
- 239000005720 sucrose Substances 0.000 claims description 18
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 15
- 239000001301 oxygen Substances 0.000 claims description 15
- 229910052760 oxygen Inorganic materials 0.000 claims description 15
- 150000008044 alkali metal hydroxides Chemical class 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000000376 reactant Substances 0.000 claims description 12
- 241000233675 Thraustochytrium Species 0.000 claims description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 150000002500 ions Chemical class 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 6
- 239000002699 waste material Substances 0.000 claims description 6
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 5
- 229910001447 ferric ion Inorganic materials 0.000 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 241000195620 Euglena Species 0.000 claims description 3
- 241000186660 Lactobacillus Species 0.000 claims description 3
- 241000235575 Mortierella Species 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 229940039696 lactobacillus Drugs 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims description 2
- 241000235035 Debaryomyces Species 0.000 claims description 2
- 241000233671 Schizochytrium Species 0.000 claims description 2
- 241000192584 Synechocystis Species 0.000 claims description 2
- 230000000243 photosynthetic effect Effects 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 2
- 241001446247 uncultured actinomycete Species 0.000 claims 1
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 43
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 241001306132 Aurantiochytrium Species 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 9
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- XDIYNQZUNSSENW-UUBOPVPUSA-N (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O XDIYNQZUNSSENW-UUBOPVPUSA-N 0.000 description 4
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 4
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 4
- 239000001110 calcium chloride Substances 0.000 description 4
- 229910001628 calcium chloride Inorganic materials 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 description 4
- 239000011592 zinc chloride Substances 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 3
- 239000013535 sea water Substances 0.000 description 3
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 2
- 229910021577 Iron(II) chloride Inorganic materials 0.000 description 2
- 239000005862 Whey Substances 0.000 description 2
- 102000007544 Whey Proteins Human genes 0.000 description 2
- 108010046377 Whey Proteins Proteins 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 2
- 229910052783 alkali metal Inorganic materials 0.000 description 2
- 150000001340 alkali metals Chemical class 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 2
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000013379 molasses Nutrition 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000195651 Chlorella sp. Species 0.000 description 1
- 241001030162 Debaryomyces sp. Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- ODUCDPQEXGNKDN-UHFFFAOYSA-N Nitrogen oxide(NO) Natural products O=N ODUCDPQEXGNKDN-UHFFFAOYSA-N 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 241000598397 Schizochytrium sp. Species 0.000 description 1
- 241000192581 Synechocystis sp. Species 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000003206 sterilizing agent Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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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
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]
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]
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]
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]
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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