CN117757688A - Citrobacter freundii JYS, and microbial inoculum and application thereof - Google Patents
Citrobacter freundii JYS, and microbial inoculum and application thereof Download PDFInfo
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- CN117757688A CN117757688A CN202311827720.0A CN202311827720A CN117757688A CN 117757688 A CN117757688 A CN 117757688A CN 202311827720 A CN202311827720 A CN 202311827720A CN 117757688 A CN117757688 A CN 117757688A
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- citrobacter freundii
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- 241000588919 Citrobacter freundii Species 0.000 title claims abstract description 27
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 52
- 230000001580 bacterial effect Effects 0.000 claims abstract description 35
- 239000002131 composite material Substances 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000002351 wastewater Substances 0.000 claims abstract description 8
- 229920000331 Polyhydroxybutyrate Polymers 0.000 claims description 15
- 239000005015 poly(hydroxybutyrate) Substances 0.000 claims description 15
- 240000000111 Saccharum officinarum Species 0.000 claims description 14
- 235000007201 Saccharum officinarum Nutrition 0.000 claims description 14
- 239000002154 agricultural waste Substances 0.000 claims description 13
- 238000009360 aquaculture Methods 0.000 claims description 13
- 244000144974 aquaculture Species 0.000 claims description 13
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 12
- 235000013162 Cocos nucifera Nutrition 0.000 claims description 12
- 244000060011 Cocos nucifera Species 0.000 claims description 12
- 239000002861 polymer material Substances 0.000 claims description 12
- 235000010413 sodium alginate Nutrition 0.000 claims description 12
- 239000000661 sodium alginate Substances 0.000 claims description 12
- 229940005550 sodium alginate Drugs 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 11
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- 241000234295 Musa Species 0.000 claims description 10
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 10
- 239000004115 Sodium Silicate Substances 0.000 claims description 9
- 230000000813 microbial effect Effects 0.000 claims description 9
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052911 sodium silicate Inorganic materials 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
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- 238000001354 calcination Methods 0.000 claims description 4
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- 244000005700 microbiome Species 0.000 abstract description 9
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- 229910052757 nitrogen Inorganic materials 0.000 description 24
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 15
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 13
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- 239000002245 particle Substances 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 9
- 229910052799 carbon Inorganic materials 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000000725 suspension Substances 0.000 description 8
- 239000000843 powder Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
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- 239000003575 carbonaceous material Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
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- 229910052760 oxygen Inorganic materials 0.000 description 2
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- 239000002504 physiological saline solution Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000005067 remediation Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 210000003608 fece Anatomy 0.000 description 1
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- 210000003495 flagella Anatomy 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000004172 nitrogen cycle Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
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- 239000002957 persistent organic pollutant Substances 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000003516 soil conditioner Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a citrobacter freundii JYS and a microbial inoculum and application thereof, belonging to the technical field of microorganism purification wastewater, wherein the preservation number of the citrobacter freundii JYS is GDMCC No:63823; the invention also discloses an immobilized microbial inoculum for treating the culture wastewater, which contains the Citrobacter freundii JYS and an immobilized carrier. The invention screens and obtains a high-efficiency aerobic denitrifying bacterium which has both denitrification and nitrification and can be directly used in the denitrification process of the ectopic treatment of the culture water body. The invention also immobilizes the bacterial strain by preparing the composite biochar to enhance the denitrification performance of the bacterial strain in the culture tail water, combines the bacterial strain with the biochar, increases the aperture, can improve the denitrification efficiency of water treatment, accelerates the self aggregation of the citrobacter freundii thallus, promotes the formation of a biological film, and has important prospect in the field of treating the culture tail water by the biological film.
Description
Technical Field
The invention relates to the technical field of microorganism purification of wastewater, in particular to a Citrobacter freundii JYS, a microbial inoculum and application thereof.
Background
Along with the increasing living standard of people, the nutrition requirements are more comprehensive, the demand of aquatic products is also increased year by year, and thus the scale and the yield of artificial aquaculture are continuously increased. In the artificial cultivation process, a large amount of feeding is carried out to cause bait residues in a cultivation system and a large amount of accumulation of aquatic animal excreta in a water body due to overhigh density. In the current intensive cultivation mode, the utilization rate of the cultivation animals to the feed is only 20% -30%, and most of the feed is dissolved in water after the feed remains in the cultivation water body, so that serious nitrogen pollution of the cultivation water body is caused. In addition, beneficial microorganisms are killed by using the disinfectant in the cultivation process, so that outbreak diseases of the cultivated aquatic organisms are easily caused, the growth and the yield of the aquatic organisms are influenced, and the condition that the nitrogen and phosphorus content in the cultivation water body exceeds the standard is caused. In addition, with the rapid development of the aquaculture industry, a large amount of aquaculture tail water is discharged into natural water, wherein the natural water eutrophication is caused by out-of-standard pollutants, and more water environment pollution is caused.
In general, the nitrogen cycle of a water body is in a stable state, and the water quality of the water body is maintained at a normal level. However, in aquaculture, due to high-density cultivation, the ecological unbalance of water, the deterioration of water quality and the lack of oxygen in water body are caused, so that the nitrogen and phosphorus content in the aquaculture water body exceeds the standard. Total nitrogen and total phosphorus are one of important indexes for measuring eutrophication of water bodies, and a large amount of nitrogen and phosphorus produced by aquaculture tail water are discharged into the environment, so that the water bodies in lakes, rivers or offshore areas can be eutrophicated, plankton can be propagated in a large amount, dissolved oxygen in the water is reduced, the water quality is deteriorated, and fishes and other organisms die in a large amount. In the face of the more serious nitrogen pollution condition, the research on the denitrification technology of the cultivation tail water is enhanced and deepened, and the method has important significance in screening high-efficiency denitrification microorganisms and developing novel denitrification technology. At present, screening of pure-breed high-efficiency denitrification microorganisms is increasingly important for microbial remediation.
The treatment of aquaculture water can be divided into two main methods, one is in situ treatment, such as directly planting aquatic plants in aquaculture water to directly repair or reduce the pollution level of aquaculture water; the other is ex situ treatment, i.e. the treatment of the wastewater after it has been discharged, such as the most common recirculating aquaculture systems. The microbial remediation refers to a process of reducing the concentration of toxic and harmful substances in the environment by utilizing the life metabolic activity of microorganisms to make the environment harmless, so that the polluted environment can be partially or completely restored to an initial state, and has the characteristics of good effect, less investment, high safety and the like.
Biochar is a carbon material made of biomass, and has high carbon content and porosity. Biochar has a highly microporous structure, which makes it excellent in adsorption performance and microbial nutrient retention capacity. In the agricultural field, the biochar can be used as a soil conditioner to effectively promote plant growth and improve soil fertility. In the field of water treatment, biochar can be used as an adsorbent for removing various harmful substances in water, such as heavy metal ions, organic pollutants and the like, so that the efficiency of water treatment is improved. However, biochar of different raw material sources has different effects on the growth of the same microorganism. The method has important significance in screening the microbial strains which have high-efficiency denitrification effect, ecological safety and are easy to self-aggregate to form biological membranes and preparing the composite biological carbon material capable of improving the denitrification efficiency.
Disclosure of Invention
The invention aims to provide a Citrobacter freundii JYS, a bacterial agent and application thereof, so as to solve the problems in the prior art, and the bacterial strain has denitrification and nitrification functions at the same time, can be directly used in the denitrification process of ectopic treatment of a culture water body.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a strain of Citrobacter freundii (Citrobacter freundii) JYS which was deposited at the collection of microorganism strains in Guangdong province on month 21 of 2023 under the accession number GDMCC No:63823.
the invention also provides an immobilized microbial inoculum for treating the culture wastewater, which comprises the Citrobacter freundii JYS and an immobilized carrier.
Further, the immobilization carrier comprises agricultural waste, natural high polymer materials and polyhydroxybutyrate.
Further, the agricultural waste comprises sugar cane bark, banana stalks, sugar cane meal, coconut shells or banana peels; the natural polymer material comprises sodium silicate or sodium alginate.
Further, the agricultural waste is coconut shells; the natural polymer material is sodium alginate.
The invention also provides a preparation method of the immobilized microbial inoculum, which comprises the following steps:
adding water into agricultural waste, natural polymer materials and polyhydroxybutyrate, uniformly mixing, calcining at high temperature, and cooling to obtain composite biochar;
and (3) mixing and culturing the composite biochar and the Citrobacter freundii JYS, and washing and drying.
Further, the mass ratio of the agricultural waste, the natural polymer material and the polyhydroxybutyrate to the water consumption of the added water is 1:1:1:0.8; the high-temperature calcination temperature is 300 ℃ and the time is 3 hours; the cooling time was 24 hours.
Further, the Citrobacter freundii JYS is mixed and cultured with the composite biochar in a bacterial liquid form; the ratio of the composite biochar to the bacterial liquid is 25g:1L; the culture condition is 30 ℃ and 140r/min shaking culture is carried out for 12 hours.
Further, the agricultural waste comprises sugar cane bark, banana stalks, sugar cane meal, coconut shells or banana peels; the natural polymer material comprises sodium silicate or sodium alginate.
The invention also provides an application of the Citrobacter freundii JYS or the immobilized microbial inoculum in the treatment of cultivation wastewater.
The invention discloses the following technical effects:
the invention screens and obtains a high-efficiency aerobic denitrifying bacterium which has denitrification and nitrification simultaneously, can remove nitrate nitrogen and ammonia nitrogen generated in the cultivation process simultaneously, and has little nitrite accumulation in the denitrification process and no secondary pollution. After the expansion culture, the method can be directly used for the denitrification process of the ectopic treatment of the culture water body, and has great potential in practical application.
The invention also immobilizes the bacterial strain by preparing the composite biochar to enhance the denitrification performance of the bacterial strain in the culture tail water, combines the bacterial strain with the biochar, increases the aperture, can improve the denitrification efficiency of water treatment, accelerates the self aggregation of the citrobacter freundii thallus, promotes the formation of a biological film, and has important prospect in the field of treating the culture tail water by the biological film.
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 the growth rate of strain JYS in denitrification medium;
FIG. 2 is a transmission electron microscope image of strain JYS;
FIG. 3 is a phylogenetic tree of the strain JYS constructed;
FIG. 4 shows the results of preliminary denitrification capacity measurement of strain JYS;
FIG. 5 is a photograph of bacterial strain JYS grown in denitrification medium for 72h to produce floc precipitate;
FIG. 6 is a statistical chart of the removal of ammonium nitrogen from the composite biochar immobilized strain in a mixed nitrogen source medium;
FIG. 7 is a statistical chart of removal of nitrate nitrogen by the composite biochar immobilized strain in a mixed nitrogen source medium;
FIG. 8 is a statistical chart of removal of nitrite nitrogen by the composite biochar immobilized strain in a mixed nitrogen source medium;
FIG. 9 is a bar graph showing denitrification effect of the complex biochar immobilized strain in a mixed nitrogen source medium.
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.
The respective media formulations used in the following examples are shown in table 1:
table 1 Each Medium formulation
Example 1 isolation, identification and preservation of aerobic denitrifying bacteria
The inventor separates out Citrobacter freundii (Citrobacter freundii) JYS from a culture water body containing PHB slow-release carbon placed in the Zhujiang aquatic institute in Guangzhou, guangdong, 6 months, and the method is as follows:
1 isolation and purification of Strain
And collecting the added Polyhydroxybutyrate (PHB) slow-release carbon source particles from a water treatment unit of the industrial simulated culture system, mixing the Polyhydroxybutyrate (PHB) slow-release carbon source particles with a culture water body, placing the mixed particles in a sterilization conical flask, performing ultrasonic oscillation for 2 hours, and standing for 1 hour to obtain a supernatant to obtain an initial environmental sample. Directly taking 10mL of initial environmental sample, inoculating the initial environmental sample into a triangular flask containing 90mL of denitrifying bacteria enrichment medium, and placing the initial environmental sample at 30 ℃ and shaking the flask at 180r/min for shake cultivation. Diluting the enrichment culture solution, wherein the gradient dilution process of the bacterial suspension is as follows:
sucking 1mL of the bacterial suspension from the conical flask, putting the bacterial suspension into a centrifuge tube filled with 9mL of sterile water, and fully and uniformly mixing, namely diluting to 10 -1 . Then 1mL of the sterile water is sucked from the centrifuge tube and is added into another centrifuge tube filled with 9mL of sterile water for uniform mixing, and the steps are repeated to be diluted to 10 in sequence -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Eight gradients total. Then, 0.1mL of each of the dilutions was applied to the prepared BTB solid plate medium, the dilutions and the dates were indicated, and the culture was performed in a constant temperature biochemical incubator at 30℃for 2-3d. After single colonies are observed from the plate, colonies with obvious blue halos are judged to be initially screened colonies with denitrification activity, and the colonies are picked up by an inoculating loop and purified. And (3) scribing on the solid basic enrichment culture medium by adopting a plate scribing separation method, dividing the plate into four areas, fully scribing each area, placing the front surface of the plate at room temperature for 5-10min after scribing, and then culturing the plate in a constant-temperature biochemical incubator at 30 ℃ in an inverted manner. After repeated streaking and separation for 2-3 times according to the steps, single bacterial colony is streaked, no abnormal bacterial colony is observed, the bacterial strain is considered to be completely purified, and the bacterial strain JYS with the strongest denitrification reaction is selected.
Identification and preservation of strains
The biological properties of strain JYS are shown in Table 2:
TABLE 2 biological Properties of JYS strain
Bacterial strain JYS is inoculated into denitrification culture medium for culture, and is periodically sampled to measure OD 600 As shown in FIG. 1, the strain enters the logarithmic growth phase after 2 hours of culture, the growth rate is high, the biomass reaches the highest after 6 hours, and then enters the stationary growth phase. As can be seen from FIG. 2, the colony morphology of the cells was observed by a transmission electron microscope, and the cells had single-ended flagellum, no spore, no capsule, and were rod-shaped and easily self-aggregated.
The DNA extraction kit was used to extract the JYS genome of the strain. PCR amplification of the 16S rRNA gene was performed using the genome of the strain as a template, and the obtained 16S rRNA gene sequence was compared at NCBI, and found that the similarity of the 16S rRNA sequence of the strain to that of the citrobacter freundii strain was 99.9%, the 20 strains closest to each other at the species level were selected based on the 16S rRNA sequence, and a phylogenetic tree was constructed by the MEGA6.0 software selection NJ (Neighbor-Joining) method, as shown in FIG. 3. Based on the phylogenetic analysis result and physiological and biochemical characteristics of the 16S rRNA gene, the strain JYS was identified as a new strain of Citrobacter freundii.
Citrobacter freundii (Citrobacter freundii) JYS was deposited at month 21 of 2023 into the microorganism culture Collection of Guangdong province, under the accession number GDMCC No:63823.
EXAMPLE 2 determination of preliminary Denitrification Capacity of bacterial strain JYS
Inoculating the purified strain into liquid basic enrichment culture medium, culturing in a constant temperature shaking incubator at 30deg.C and 180r/min for 10 hr, centrifuging 8000g of activated bacterial liquid, pouring out supernatant, rinsing with 0.75% sterile physiological saline, repeating for 2 times, and concentrating bacterial suspension OD 600 The value is regulated to about 0.4, and the suspension is re-suspended, and the inoculum size is 1mL, and 100mL is filled with KNO 3 Culturing at 30deg.C and 180r/min in denitrification culture medium with unique nitrogen source for 0, 24, 48, and 72 hr, respectively collecting 10mL bacterial solutions, centrifuging at 8000g, collecting supernatant, and measuring NO in the culture solution 3 - Concentration of-N, NH 4 + -concentration of N to calculate nitrogen removal rate.
As shown in FIG. 4, the removal rate of nitrate nitrogen after 24 hours of culture is about 4%, the removal rate of nitrate nitrogen reaches about 97.7% at 72 hours, ammonium nitrogen rapidly accumulates at 24 hours, the removal rate reaches about 68% at 48 hours, and the removal rate is reduced. The medium in the flask was observed for 72h (fig. 5), and a clear floc precipitate was observed.
Example 3 preparation of composite biochar immobilized bacteria particles and application thereof in Mixed Nitrogen Source Medium
The preparation method of the composite biochar comprises the following steps:
(1) Drying and grinding coconut shells, sugarcane peels, banana stems and sugarcane peels, removing large particles by using a 10-mesh screen to obtain solid powder, and respectively mixing the solid powder with PHB, sodium silicate powder and deionized water according to a mass ratio of 1:1:1:0.8, uniformly mixing and agglomerating, fixing the shape by a die, calcining at 300 ℃ for 3 hours, and cooling for 24 hours to obtain the composite biochar G1-G5 (see table 3).
Drying and grinding coconut shells, sugarcane peels, banana stems and sugarcane peels, removing large particles by using a 10-mesh screen to obtain solid powder, and respectively mixing the solid powder with PHB, sodium alginate powder and deionized water according to a mass ratio of 1:1:1:0.8, uniformly mixing and agglomerating, fixing the shape by a die, calcining at 300 ℃ for 3 hours, and cooling for 24 hours to obtain the composite biochar H1-H5 (see table 3).
(2) Preparation of a suspension of Citrobacter freundii JYS: namely, 100 mu L of JYS strain is added into 100mL of LB culture medium for 8h of expansion culture, and then centrifugation is carried out, supernatant is discarded, sterile physiological saline is added after washing, and OD is adjusted 600 After a value of 0.4, a bacterial suspension was obtained.
(3) The preparation method of the immobilized particles comprises the following steps: and respectively adding the composite biochar into the JYS bacterial suspension which is subjected to expanded culture and activation in a proportion of 25g/L, culturing for 12 hours at 30 ℃ in a shaking table at 140r/min, taking out, washing with sterile water, repeatedly washing for 4-5 times, and drying to obtain composite biochar immobilized bacteria particles, wherein the drying time is 24 hours, and the drying temperature is 30 ℃.
Respectively inoculating the composite biochar immobilized bacteria particles into a mixed nitrogen source culture medium according to the inoculation amount of 0.05g/mL, and mixingThe formula of the nitrogen source culture medium is as follows: sodium citrate (C) 6 H 5 Na 3 O 7 2H 2 O)5g/L、NaCl 0.01g/L、K 2 HPO 4 0.2g/L、MgSO 4 ·7H 2 0.05g/L of O, 1mL/L of microelement solution, 0.055g/L of ammonium sulfate, 0.25g/L of potassium nitrate and 0.03g/L of sodium nitrite. Culturing at 30deg.C in a 140r/min shaker, collecting 10mL bacterial solutions respectively at culture times of 0, 4, 8, 12, 24, and 48 hr, centrifuging at 8000g, collecting supernatant, and measuring NO in the culture solution 3 - -N、NH 4 + -N、NO 2 - -concentration of N to calculate nitrogen removal rate.
The results show that: after the composite biochar immobilized bacteria particles are added into a culture medium for culturing for 48 hours, the composite biochar is molded as shown in table 3:
TABLE 3 Forming of composite biochar
And selecting G2, G3, G4, G5, H1 and H4 groups with good biochar forming to calculate the denitrification condition.
The denitrification effect of the composite biochar immobilized strain in a mixed nitrogen source culture medium (in a sterile state without strict control) is shown in fig. 6, 7 and 8, the removal capacity of the G5 group ammonium nitrogen after 48h culture is lower than that of other groups, the composite biochar immobilized strain added with sodium silicate cannot significantly remove nitrate nitrogen, the nitrite nitrogen of the other groups except the G3 group is obviously accumulated, and the nitrite nitrogen of the two groups added with sodium alginate can be removed.
As can be seen from the observation of FIG. 9, the removal rate of the tri-state nitrogen of each group is 99.08% for the G2 group ammonium nitrogen after 48 hours of culture, the removal rate of the nitrite nitrogen is only 4.01%, and the removal rate of the nitrate nitrogen is only 11.03%; the removal rate of the G3 group ammonium nitrogen reaches 94.75%, the removal rate of the nitrite nitrogen is only 27.95%, and the removal rate of the nitrate nitrogen is only 15.14%; the removal rate of the G4 group ammonium nitrogen reaches 98.04%, the nitrite nitrogen is obviously accumulated, the removal rate of the nitrite nitrogen is increased by 39.31% in an inverse way, and the removal rate of the nitrite nitrogen is only 17.63%; the removal rate of the G5 group ammonium nitrogen reaches 67.24 percent, the nitrite nitrogen is obviously accumulated, the removal rate of the nitrite nitrogen is increased by 23.31 percent in an inverse way, and the removal rate of the nitrite nitrogen is only 3.66 percent; the removal rate of H1 group ammonium nitrogen reaches 94.55%, the removal rate of nitrite nitrogen is only 90.66%, and the removal rate of nitrate nitrogen is 90.48%; the removal rate of H4 group ammonium nitrogen reaches 96.71%, the removal rate of nitrite nitrogen is only 84.53%, and the removal rate of nitrate nitrogen is 86.64%; according to the analysis of the removal rate of tri-state nitrogen, the removal capacity of the compound biological carbon immobilized bacteria added with sodium silicate on nitrite nitrogen and nitrate nitrogen is obviously weakened, the addition of sodium alginate can lead the time of removing tri-state nitrogen of the compound biological carbon immobilized bacteria to be advanced, and compared with the coconut shell and the sugarcane powder, the denitrification capacity of the compound biological carbon immobilized bacteria added with the coconut shell is stronger, so that the coconut shell and the sodium alginate are preferentially selected when the compound biological carbon immobilized bacterial strain is prepared.
In summary, the invention adopts the high-efficiency aerobic denitrifying bacteria obtained by screening in a culture system with polyhydroxybutyrate, bacterial liquid of the bacterial strain, immobilized bacterial strain and dormant cells can grow in a basic culture medium with ammonia nitrogen, nitrate nitrogen and nitrite nitrogen as nitrogen sources and mixed nitrogen sources, and the ammonia nitrogen is converted into nitrate nitrogen through nitrification and simultaneously can be converted into nitrogen through denitrification, thereby realizing synchronous nitrification-denitrification and having high degradation speed on the ammonia nitrogen. The strain does not have adverse effect on aquatic organisms in a culture system, is easy to self-aggregate, and has good effect in the direction of forming a biological film. The invention also utilizes agricultural wastes such as coconut shells and sugarcane peels, PHB and natural polymer materials such as sodium silicate or sodium alginate to prepare the composite biochar by high-temperature calcination, and the composite biochar can be directly applied to denitrification of aquaculture water after being mixed and fixed with bacterial strain JYS, the composite biochar has higher carbon content, the release speed of the composite biochar can be slowed down by adding sodium silicate, the stability of particles can be enhanced by sodium alginate, and the composite biochar has good adsorptivity due to a high-level micropore structure, so that bacterial strains can grow better to form films, the problem of non-indigenous bacteria environment inadaptation is avoided, and the composite biochar has great potential in aquaculture tail water treatment.
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 (10)
1. A strain of citrobacter freundii (jbs) deposited at 21 of month 09 of 2023 with the collection of microbiological bacterial strain at the university of guangdong, the deposit address being GDMCC No:63823.
2. an immobilized microbial inoculum for treating cultivation wastewater, which is characterized by comprising the Citrobacter freundii JYS and an immobilized carrier according to claim 1.
3. The immobilized microbial agent of claim 2, wherein the immobilization carrier comprises agricultural waste, natural polymeric materials, and polyhydroxybutyrate.
4. The immobilized microbial agent of claim 3, wherein the agricultural waste comprises sugar cane bark, banana stalks, sugar cane meal, coconut shells or banana peels; the natural polymer material comprises sodium silicate or sodium alginate.
5. The immobilized microbial agent of claim 4, wherein the agricultural waste is coconut husk; the natural polymer material is sodium alginate.
6. A method for preparing the immobilized microbial agent of any one of claims 2-5, comprising the steps of:
adding water into agricultural waste, natural polymer materials and polyhydroxybutyrate, uniformly mixing, calcining at high temperature, and cooling to obtain composite biochar;
and (3) mixing and culturing the composite biochar and the Citrobacter freundii JYS, and washing and drying.
7. The method according to claim 6, wherein the mass ratio of the agricultural waste, natural polymer material and polyhydroxybutyrate to the water consumption of the water is 1:1:1:0.8; the high-temperature calcination temperature is 300 ℃ and the time is 3 hours; the cooling time was 24 hours.
8. The preparation method of claim 6, wherein the Citrobacter freundii JYS is mixed and cultured with the composite biochar in the form of bacterial liquid; the ratio of the composite biochar to the bacterial liquid is 25g:1L; the culture condition is 30 ℃ and 140r/min shaking culture is carried out for 12 hours.
9. The method of claim 6, wherein the agricultural waste comprises sugar cane bark, banana stalks, sugar cane meal, coconut shells or banana peels; the natural polymer material comprises sodium silicate or sodium alginate.
10. Use of the Citrobacter freundii JYS according to claim 1 or the immobilized microbial agent according to any one of claims 2-3 for the treatment of aquaculture wastewater.
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