CN117568235A - Bacillus subtilis for producing nitrite oxidoreductase and application thereof - Google Patents
Bacillus subtilis for producing nitrite oxidoreductase and application thereof Download PDFInfo
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- CN117568235A CN117568235A CN202311798465.1A CN202311798465A CN117568235A CN 117568235 A CN117568235 A CN 117568235A CN 202311798465 A CN202311798465 A CN 202311798465A CN 117568235 A CN117568235 A CN 117568235A
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- yxsy
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 35
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 35
- 102000004316 Oxidoreductases Human genes 0.000 title claims abstract description 28
- 108090000854 Oxidoreductases Proteins 0.000 title claims abstract description 28
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims abstract description 46
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 44
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 5
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 21
- 235000010288 sodium nitrite Nutrition 0.000 claims description 14
- 238000009360 aquaculture Methods 0.000 claims description 11
- 244000144974 aquaculture Species 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 7
- 229920002261 Corn starch Polymers 0.000 claims description 5
- 239000008120 corn starch Substances 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 5
- 239000011790 ferrous sulphate Substances 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 241000238017 Astacoidea Species 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 239000013028 medium composition Substances 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 abstract description 10
- 238000006731 degradation reaction Methods 0.000 abstract description 10
- 238000011160 research Methods 0.000 abstract description 3
- 241000238557 Decapoda Species 0.000 description 19
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 11
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 11
- 241000193830 Bacillus <bacterium> Species 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 8
- 229910002651 NO3 Inorganic materials 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 230000001546 nitrifying effect Effects 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 230000001590 oxidative effect Effects 0.000 description 6
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 238000010170 biological method Methods 0.000 description 4
- 230000000593 degrading effect Effects 0.000 description 4
- 239000007800 oxidant agent Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 235000010344 sodium nitrate Nutrition 0.000 description 3
- 239000004317 sodium nitrate Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000315694 Bacillus tequilensis Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 241000238553 Litopenaeus vannamei Species 0.000 description 1
- KEBNFKBVKVCOCL-UHFFFAOYSA-N N#CN.[Fe] Chemical compound N#CN.[Fe] KEBNFKBVKVCOCL-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012754 barrier agent Substances 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
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- 239000002360 explosive Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
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- 238000009533 lab test Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 238000003645 oxidoreductase activity assay Methods 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- 238000007873 sieving Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000008280 toxic mechanism Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
- C12Y107/01—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with NAD+ or NADP+ as acceptor (1.7.1)
- C12Y107/01004—Nitrite reductase [NAD(P)H] (1.7.1.4)
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention discloses bacillus subtilis for producing nitrite oxidoreductase and application thereof, belonging to the field of bioengineering, wherein the bacillus subtilis has a preservation number GDMCC No:64078; the invention also discloses a method for producing nitrite oxidoreductase, which comprises the steps of fermenting bacillus subtilis YXSY-01 and collecting fermentation liquor. The bacillus subtilis strain YXSY-01 provided by the invention has the capability of high-yield nitrite oxidoreductase and remarkable degradation of nitrite in water; the strain lays an important foundation for the deep research and utilization of nitrite oxidoreductase, can obviously reduce the nitrite content in the culture water body, and provides a new idea for developing safe and ecological water body purifying microbial inoculum.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to bacillus subtilis for producing nitrite oxidoreductase and application thereof.
Background
The high protein feed is excessively used in the middle and later period of the culturing of the greenhouse shrimps, so that the nitrogen element circulation is blocked, a large amount of inorganic nitrogen sources are accumulated in the water body, the nitrite content in the water body is too high, the health of aquatic animals is seriously influenced, and great loss is caused for the culturing. The effect of nitrite on aquatic animals mainly comprises the following aspects:
the toxic mechanism of nitrite can affect the oxygen delivery process of prawn. Nitrite enters the blood, inhibits the binding of oxygen to the cyanamide iron molecules, resulting in difficulty in oxygen delivery to the tissue. Nitrite in the culture environment is raised, so that oxygen deficiency in tissues is caused, respiratory metabolism of prawns is destroyed, toxicity of nitrite in pond water can reduce water quality, growth speed is reduced, oxygen consumption and ammonia concentration are increased, and even death of the prawns is caused. Exceeding the nitrite concentration severely reduces the success rate of aquaculture. Currently, the usual methods for removing nitrous acid from water are physical, chemical and biological methods.
The physical method mainly uses physical adsorption and water changing methods, and the physical adsorption method only adsorbs nitrite in the water body on the solid surface, but does not reduce the nitrite fundamentally. The water changing method is to replace part of water with high nitrite by supplementing a new water source, and has slow effect and large water changing amount, which is easy to cause the problem of stress on prawns. The chemical method for removing nitrite mainly comprises chemical blocking agent, strong oxidant and the like. The chemical barrier agent is mainly used for blocking the conversion process of ammonia nitrogen to nitrite, and can only temporarily suppress the growth of nitrite, but can cause the rise of ammonia nitrogen and the subsequent explosive growth of nitrite. The strong oxidant is mainly used for oxidizing nitrite into nitrate, the strong oxidant can cause stress reaction of the prawns, the organic matter content of the water body is very high in the middle and later period of culturing the greenhouse prawns, and the strong oxidant is difficult to achieve the effect of reducing nitrite. The biological method mainly utilizes nitrifying bacteria and denitrifying bacteria to gradually convert nitrite into nitrogen, the reaction conditions of the nitrifying bacteria and the denitrifying bacteria are mild, and the total nitrogen content in water can be reduced, so that the biological method is the safest and effective method in theory. The nitrifying bacteria and denitrifying bacteria used by the current biological method have the characteristics of low growth speed, low treatment efficiency and difficult preservation in the actual use process, so that the nitrifying bacteria and denitrifying bacteria cannot play a role in degrading nitrite in the actual culturing process of the greenhouse shrimps. Thus, there is a need to screen more safe and effective microbial species for rapid degradation of nitrite in the water body of shrimp culture.
In addition, nitrite oxidoreductase (nitrite oxidoreductase, NXR) is a key enzyme for nitrifying bacteria to oxidize nitrite to nitrate. Since it is a soluble intramembrane enzyme, its catalytic mechanism is closely linked to intramembrane electron transfer, and presents a certain difficulty in its investigation. As a key enzyme for degrading toxic substance nitrite, NXR is not only applied to comprehensive treatment of water pollution, but also used for preventing fish and shrimp poisoning in aquaculture water, and can be applied to human food and beverage for removing nitrite. However, there are relatively few reports of nitrite oxidoreductase production.
Disclosure of Invention
The invention aims to provide bacillus subtilis for producing nitrite oxidoreductase and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a bacillus subtilis YXSY-01 for producing nitrite oxidoreductase, which is preserved in the microorganism strain collection center of Guangdong province, and the preservation address is as follows: guangzhou city first middle road 100 # college 59 # building 5, deposit number: GDMCC No:64078, the preservation time is 2023, 11 and 27 days.
The invention also provides a microbial inoculum for producing nitrite oxidoreductase, which comprises the bacillus subtilis YXSY-01.
The invention also provides a method for producing nitrite oxidoreductase, which comprises the steps of fermenting the bacillus subtilis YXSY-01 and collecting fermentation liquor.
Further, the fermented culture medium comprises the following components: corn starch 5-30g/L, sodium nitrite 0.5-3g/L, dipotassium hydrogen phosphate 0.5-3g/L, magnesium sulfate 0.3-0.8g/L and ferrous sulfate 0.0001-0.01 g/L, and water is added to 1L.
Further, the fermentation conditions are as follows: shaking culture is carried out for 16-24h at 25-35 ℃ and 150 rpm.
The invention also provides application of the bacillus subtilis YXSY-01 or the microbial inoculum in reducing nitrite content in a culture water body.
Further, the bacillus subtilis YXSY-01 is inoculated into the culture water body in a form of fermentation broth.
Further, the viable count of the fermentation liquid is 1.0X10 7 ～1.0×10 9 cfu/mL。
Further, the body of aquaculture water comprises a body of crayfish aquaculture water.
The invention discloses the following technical effects:
the bacillus subtilis strain YXSY-01 provided by the invention has the capability of high-yield nitrite oxidoreductase and remarkable degradation of nitrite in water. The strain is used for producing nitrite oxidoreductase by fermentation on a fermentation medium with sodium nitrite as a unique nitrogen source, the enzyme activity is as high as 0.65U/mL, and an important foundation is laid for the deep research and utilization of nitrite oxidoreductase. The strain can degrade 1000ppm of sodium nitrite to 0ppm within 24-48 hours, and has excellent nitrite degradation capability, can obviously reduce the nitrite content in the culture water body, reduce the damage of nitrite prawns, and provide a new idea for developing safe and ecological purifying microbial inoculum.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing degradation and change of sodium nitrite of a strain YXSY-01 in a fermentation culture process;
FIG. 2 is a microscopic image of strain YXSY-01;
FIG. 3 is a colony morphology of strain YXSY-01;
FIG. 4 is a nitrite standard graph;
FIG. 5 is a nitrate standard graph;
FIG. 6 is a graph showing the results of the high yield of nitrite oxidoreductase by Bacillus subtilis YXSY-01.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
EXAMPLE 1 isolation, identification and preservation of strains
1. Isolation of strains
Microorganism screening is carried out on the sediment collected from the small shed shrimp culture pond in Taishan city, guangdong in 2023 and 5 months on a screening culture medium YX-1 flat plate by adopting a 10-time serial gradient dilution separation method.
Screening medium YX-1 used: 20g/L of corn starch, 1g/L of sodium nitrite, 0.5g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate and 0.01 g/L of ferrous sulfate; 20g/L of agar powder, regulating the pH value to 7.3, heating to dissolve the agar powder, and sterilizing at 121 ℃ for 20min.
The selected strains were transferred to YX-1 liquid medium at 30℃for 48 hours at 150r/min on YX-1 plates as screening medium.
YX-1 liquid medium: corn starch 20g/L, sodium nitrite 1g/L (1000 ppm), dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.01 g/L, water to 1L, adjusting pH to 7.3, heating to dissolve, and sterilizing at 121deg.C for 30min.
After the detection and culture are finished, the bacterial strain YXSY-01 with high viable count and strong capability of converting sodium nitrite into nitrate under the condition of taking sodium nitrite as a unique nitrogen source is finally obtained through screening by the contents of viable count (the detection culture medium is a screening culture medium YX-1) and nitrate (the detection method is GB/T35496-2017) in the fermentation broth. FIG. 1 is a graph showing the degradation change of sodium nitrite of the strain YXSY-01 in the fermentation culture process, and the result shows that the strain can degrade 1000ppm of sodium nitrite to 0ppm in a short time, and the strain has excellent nitrite degradation capability.
2. Identification of Strain YXSY-01
2.1 Morphological identification and physiological and biochemical characteristic detection
Inoculating the separated strain to a screening culture medium YX-1 plate, culturing at a constant temperature of 30 ℃ for 72 hours, observing colony growth conditions, picking a bacterial preparation smear, carrying out gram staining, and carrying out microscopic observation on bacterial morphology and spore formation conditions, thereby carrying out morphological identification.
FIG. 2 is a microscopic image of strain YXSY-01, and FIG. 3 is a colony morphology of strain YXSY-01. The results show that: the colony is round, the edge is irregular and is not transparent; single cell 0.5-0.7X12.0-2.6 microns, gram positive, no expansion of spore, 0.6-0.8X1.0-1.5 microns, central or slightly offset, columnar.
2.2 Molecular biological identification
The strain YXSY-01 was inoculated in YX-1 liquid medium at an inoculum size of 5% (V/V), a temperature of 33℃and a shaking table rotation speed of 150 rpm, and cultured for 24 hours, and the bacterial genome was extracted using a DNA extraction kit (available from Beijing Baitaike Biotechnology Co., ltd.). PCR amplification of 16SrRNA gene and specific gene GryA is carried out by taking bacterial genome as a template, and the amplified product is delivered to the Guangdong province microorganism strain collection for sequencing analysis.
The gene sequence of the 16SrRNA of the obtained strain YXSY-01 is shown as SEQ ID NO:1 is shown as follows:
CGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACAC（SEQ ID NO：1）。
the GryA gene sequence of the obtained strain YXSY-01 is shown as SEQ ID NO:2 is shown as follows:
CTCTTCCGGATGTTCGTGACGGTTTAAAACCGGTTCATAGACGGATTTTGTATGCAATGAATGATTTAGGCATGACAAGTGACAAGCCTTATAAAAAATCCGCGCGTATCGTTGGAGAAGTTATCGGGAAATACCACCCGCACGGTGATTCAGCGGTATATGAATCCATGGTCAGAATGGCTCAGGATTTCAACTACCGTTATATGCTCGTTGACGGTCACGGAAACTTCGGTTCTGTTGACGGAGACTCAGCGGCGGCCATGCGTTATACAGAAGCACGAATGTCTAAAATCTCAATGGAGATTCTTCGCGACATCACAAAAGACACAATCGATTACCAGGATAACTATGACGGGTCAGAAAGAGAACCTGTCGTTATGCCTTCAAGGTTCCCGAATCTGCTCGTGAACGGTGCTGCCGGCATTGCGGTAGGTATGGCAACAAACATTCCTCCGCACCAGCTGGGAGAAATCATTGACGGTGTACTTGCTGTTAGTGAGAATCCGGACATTACAATTCCAGAGCTTATGGAAGTCATTCCAGGGCCTGATTTCCCGACCGCGGGTCAAATCTTGGGACGCAGCGGTATCCGGAAAGCATACGAATCAGGCCGAGGCTCTATCACGATCCGGGCAAAAGCTGAGATCGAACAAACATCTTCGGGTAAAGAAAGAATTATCGTTACAGAGTTACCTTACCAAGTAAATAAGGCGAAATTAATTGAGAAAAATTGCTGATCTCGTAAGGGACAAAAAGATAGAGGGTATCACAGATCTGCGTGATGAGTCAGATCGTACAGGTATGAGAATTGT（SEQ ID NO：2）。
alignment of the 16SrRNA gene sequence at NCBI found: sample and method for preparing the sameBacillus subtilis、Bacillus tequilensis、Bacillus spizizenii、BacillusmojavensisThe homology of (2) is 100%; alignment on EZBioCloud found: sample and method for preparing the sameBacillus tequilensis、Bacillus cabrialesii、Bacillus inaquosorumThe homology of the sequences reaches 99.93 percent and the sequence is identical with that of the sequencesBacillus subtilis、Bacillus rugosusThe homology of 99.85 percent andBacillus halotolerans、Bacillus stercoris、Bacillus spizizeniithe homology of (C) is 99.78%. Identification of specific genes GryA, samples and model strainsBacillus subtilisThe homology of the bacillus strain reaches 99.88 percent, and the homology with other bacillus strains is lower than 95 percent. The strain YXSY-01 is finally identified as bacillus subtilis by combining morphological characteristics, physiological and biochemical characteristics and gene sequence comparison results of the strainBacillus subtilis）。
3. Preservation of Strain YXSY-01
Sieving bacillus subtilisBacillus subtilis) YXSY-01 was deposited at the Guangdong province microorganism strain collection at the following address: guangzhou city first middle road 100 # college 59 # building 5, deposit number: GDMCC No:64078, the preservation time is 2023, 11 and 27 days.
Example 2 Bacillus subtilis YXSY-01 high yield nitrite oxidoreductase (NXR)
The method specifically comprises the following steps:
1) Preparation of crude enzyme solution:
inoculating bacillus subtilis YXSY-01 into a liquid fermentation medium (30 g/L of corn starch, 1g/L of sodium nitrite, 0.5g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate and 0.01 g/L of ferrous sulfate, supplementing water to 1000 mL), culturing for 24 hours at the temperature of 33 ℃ at the rotation speed of 150 revolutions per minute of a shaking table, and obtaining fermentation liquor, wherein the fermentation liquor is subjected to ultrasonic disruption for 30 seconds by using a cell ultrasonic disruptor to obtain crude enzyme liquid to be detected.
And sucking the crude enzyme liquid in a water bath at 95 ℃ for 30min to obtain an inactivated crude enzyme liquid, wherein the inactivated crude enzyme liquid is used as a control group for subsequent enzyme activity measurement. 3 biological replicates were set.
2) Nitrite oxidoreductase Activity assay:
nitrite oxidoreductase (nitrite oxidoreductase, NXR) is a key enzyme for nitrifying bacteria to oxidize nitrite into nitrate, and the enzyme activity of nitrite oxidoreductase is measured and calculated by testing the process of oxidizing sodium nitrite in a substrate into sodium nitrate by crude enzyme liquid.
Standard curves of nitrite and nitrate are respectively drawn according to GB/T2367-2016 and GB/T35496-2017, wherein fig. 4 is a nitrite standard curve, and fig. 5 is a nitrate standard curve.
5mL of 500ppm sodium nitrite solution and 200 mu L of crude enzyme solution are sucked and uniformly mixed, the temperature is kept constant for 30min at 33 ℃, the temperature is kept constant for 5min, the inactivation is carried out at 95 ℃, and the nitrate content is detected by cooling. The viability units (U/mL) are defined as: the amount of enzyme required to produce 1. Mu. Mol of product (sodium nitrate/nitrate ion) per minute. The sodium nitrate/nitrate ion detection method is GB/T35496-2017. The detection results of fig. 6 show that: the activity of nitrite oxidoreductase (NXR) in the crude enzyme solution was 0.65U/mL. This example demonstrates that the fermentation of Bacillus subtilis YXSY-01 to produce nitrite oxidoreductase lays an important foundation for the in-depth research and utilization of nitrite oxidoreductase.
Example 3 laboratory test results of Bacillus subtilis YXSY-01 degrading nitrite in shrimp culture Water
Taking out greenhouse shrimp cultureWater (middle and late stage of cultivation), subpackaging the water into 300 ml triangular bottles, 100 ml each bottle, fermenting the Bacillus subtilis strain YXSY-01 obtained in example 2 (viable count is 5.0X10) 8 cfu/mL) was added to the flask in an amount of 0.01% (V/V), and the flask was incubated at 30℃for 48 hours on a shaker at 100 rpm. The nitrite in the culture water body of the greenhouse shrimp is degraded by using commercial bacillus subtilis (1000 hundred million viable bacteria, jiangsu green department biotechnology Co., ltd.) as a comparison, and the unvaccinated bacillus subtilis is used as a comparison. After the cultivation is completed, the nitrite content of the water body is detected (GB/T2367-2016). The results are shown in Table 1.
TABLE 1 Bacillus subtilis YXSY-01 results of degradation of nitrite in water of greenhouse shrimps
The experimental results in table 1 show that under laboratory conditions, the bacillus subtilis YXSY-01 has excellent nitrite degradation capability, remarkably and efficiently reduces the nitrite content in the aquaculture water body, and provides safer and more effective microorganism strains for purifying the aquaculture water body.
Example 4 application of Bacillus subtilis strain YXSY-01 in degrading nitrite in shrimp aquaculture
The test pond and the control pond are basically the same, the water body area is 320 cubes, the average water depth is 0.8 meter, the penaeus vannamei boone is mainly cultivated, 6 ten thousand seedlings are put together, the cultivation days are 60 days, (2 mouths of the test pond, 1 mouths of the control pond and 3 mouths of the pond are all arranged). The treatment mode is as follows:
and (3) a test pond: the fermentation broth of Bacillus subtilis strain YXSY-01 obtained in example 2 (viable count: 5.0X10) was used every 2 days 8 cfu/mL) 3 liters, and 5 times daily, the nitrite content was measured at 8 am and 5 pm (GB/T2367-2016).
Control pond: the use of Bacillus subtilis strain YXSY-01 broth was removed, and the other was identical to the test pond.
The results are shown in Table 2.
TABLE 2 results of degradation of nitrite in shrimp culture Water by Bacillus subtilis strain YXSY-01 fermentation broth
As can be seen from Table 2, the use of the Bacillus subtilis strain YXSY-01 fermentation broth in the test pond can significantly reduce the nitrite content of the culturing water body of the greenhouse shrimps.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (9)
1. A bacillus subtilis yxy-01 producing nitrite oxidoreductase, which is deposited in the cantonese province microorganism strain collection at the following deposit address: guangzhou city first middle road 100 # college 59 # building 5, deposit number: GDMCC No:64078, the preservation time is 2023, 11 and 27 days.
2. A nitrite oxidoreductase-producing microbial agent comprising bacillus subtilis yxy-01 according to claim 1.
3. A method for producing nitrite oxidoreductase, comprising the step of fermenting bacillus subtilis YXSY-01 according to claim 1 and collecting the fermentation liquid.
4. A method according to claim 3, wherein the fermented medium composition is: corn starch 5-30g/L, sodium nitrite 0.5-3g/L, dipotassium hydrogen phosphate 0.5-3g/L, magnesium sulfate 0.3-0.8g/L and ferrous sulfate 0.0001-0.01 g/L, and water is added to 1L.
5. A method according to claim 3, wherein the fermentation conditions are: shaking culture is carried out for 16-24h at 25-35 ℃ and 150 rpm.
6. Use of the bacillus subtilis YXSY-01 according to claim 1 or the microbial inoculum according to claim 2 for reducing nitrite content in a aquaculture water.
7. The use according to claim 6, wherein the bacillus subtilis yxy-01 is inoculated into a body of aquaculture water in the form of a fermentation broth.
8. The use according to claim 7, wherein the viable count of the fermentation broth is 1.0X10 7 ～1.0×10 9 cfu/mL。
9. The use of claim 6, wherein the body of aquaculture water comprises a body of crayfish aquaculture water.
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