CN117757688B - 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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- CN117757688B CN117757688B CN202311827720.0A CN202311827720A CN117757688B CN 117757688 B CN117757688 B CN 117757688B CN 202311827720 A CN202311827720 A CN 202311827720A CN 117757688 B CN117757688 B CN 117757688B
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- 241000588919 Citrobacter freundii Species 0.000 title claims abstract description 28
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 10
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- 230000001580 bacterial effect Effects 0.000 claims abstract description 34
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- 238000000034 method Methods 0.000 claims abstract description 17
- 244000005700 microbiome Species 0.000 claims abstract description 11
- 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
- 238000009360 aquaculture Methods 0.000 claims description 13
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- 239000002154 agricultural waste Substances 0.000 claims description 11
- 239000002861 polymer material Substances 0.000 claims description 11
- 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 10
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- 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 13
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- 230000000694 effects Effects 0.000 description 9
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- 239000004115 Sodium Silicate Substances 0.000 description 7
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- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 7
- 229910052911 sodium silicate Inorganic materials 0.000 description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 description 6
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- 239000007787 solid Substances 0.000 description 6
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- 150000001875 compounds Chemical class 0.000 description 4
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- 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
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
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- 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
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- 229910001385 heavy metal Inorganic materials 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 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
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Classifications
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- 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
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a citrobacter freundii JYS and a microbial inoculum and application thereof, belonging to the technical field of microorganism purification of 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. But 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 (Citrobacterfreundii) JYS which is preserved in the microorganism strain collection of Guangdong province at the year of 2023, 09 and 21, wherein the preservation address is the institute of microorganisms, the academy of sciences of Guangdong province, and the preservation number is 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 the JYS of the Citrobacter freundii (Citrobacterfreundii) from the culture water body of the PHB slow-release carbon source placed in the Zhujiang aquatic products research institute in Li Gulf area of Guangzhou, guangdong in 2022, and the concrete steps are 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:
1mL of the bacterial suspension is sucked from the conical flask and is put into a centrifuge tube filled with 9mL of sterile water, and the bacterial suspension is fully and uniformly mixed, namely diluted to 10 -1. Then 1mL of the solution is sucked from the centrifuge tube and is added into another centrifuge tube filled with 9mL of sterile water for uniform mixing, and the solution can be diluted to 10 -2、10-3、10-4、10-5、10-6、10-7、10-8 gradients in sequence by repeating the step. 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
The strain JYS is inoculated into a denitrification culture medium for culture, OD 600 is periodically sampled and measured, as shown in figure 1, the strain enters a logarithmic growth phase after 2 hours of culture, the growth speed is high, biomass reaches the highest after 6 hours, and then the strain enters a 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 16S rRNA gene is carried out by taking a strain genome as a template, the obtained 16S rRNA gene sequence is compared on NCBI, the similarity of the 16S rRNA gene sequence of the strain and the 16S rRNA sequence of Citrobacterfreundii bacteria is high and is 99.9%, 20 strains closest to the species level are selected based on the 16S rRNA sequence, and a phylogenetic tree is constructed by a MEGA6.0 software selection NJ (Neighbor-Joing) method, as shown in figure 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 (Citrobacterfreundii).
Citrobacter freundii (Citrobacterfreundii) JYS was deposited at 2023, month 09, and 21 to the microorganism strain collection, division of Cantonese, accession number GDMCC No:63823.
EXAMPLE 2 determination of preliminary Denitrification Capacity of bacterial strain JYS
Inoculating the purified strain into a liquid basic enrichment culture medium, placing the culture medium into a constant-temperature shaking incubator at 30 ℃ and 180r/min for culturing for 10 hours, centrifuging 8000g of activated bacterial liquid, pouring out supernatant, rinsing with 0.75% sterile physiological saline, repeating for 2 times, adjusting the OD 600 value of bacterial suspension to about 0.4 for resuspension, inoculating the bacterial suspension into 100mL of denitrification culture medium with KNO 3 as a unique nitrogen source according to the inoculation amount of 1mL, culturing for 0, 24, 48 and 72 hours at 30 ℃ and 180r/min, respectively taking 10mL of bacterial liquid, centrifuging at 8000g, taking supernatant, and measuring the concentration of NO 3 - -N and the concentration of NH 4 + -N in the culture liquid to calculate the 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: 100 mu L of JYS strain is added into 100mL of LB culture medium for 8h expansion culture, centrifugation is carried out, supernatant is discarded, sterile physiological saline is added after washing, and OD 600 value is adjusted to 0.4, thus obtaining bacterial suspension.
(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 ℃.
According to the inoculation amount of 0.05g/mL, the composite biochar immobilized bacteria particles are respectively inoculated into a mixed nitrogen source culture medium, and the formula of the mixed nitrogen source culture medium is as follows: sodium citrate (C6H5Na3O72H2O)5g/L、NaCl 0.01g/L、K2HPO40.2g/L、MgSO4·7H2O 0.05g/L、 microelement solution 1mL/L, ammonium sulfate 0.055g/L, potassium nitrate 0.25g/L, sodium nitrite 0.03g/L. Culturing at 30deg.C in a 140r/min shaker, collecting 10mL bacterial solutions at 0, 4, 8, 12, 24, 48h, centrifuging at 8000g, collecting supernatant, and measuring concentration of NO 3 --N、NH4 +-N、NO2 - -N in the culture solution 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 inversely increased by 39.31%, 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 nitrate 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 (3)
1. An immobilized microbial inoculum for treating cultivation wastewater, which is characterized by comprising Citrobacter freundii (Citrobacter freundii) JYS and an immobilized carrier;
The immobilized carrier comprises agricultural wastes, natural high polymer materials and polyhydroxybutyrate;
The agricultural waste is coconut shells; the natural polymer material is sodium alginate;
The Citrobacter freundii JYS was deposited at the collection of microorganisms and strains of Guangdong province at the location of the national institute of microbiology, university of Guangdong, and accession number GDMCC No at month 21 of 2023: 63823;
the preparation method of the immobilized microbial inoculum comprises the following steps: the method 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;
Mixing and culturing the composite biochar and the Citrobacter freundii JYS, and washing and drying;
The mass ratio of the agricultural waste, the natural polymer material and the 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.
2. The immobilized microbial agent of claim 1, wherein 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.
3. Use of the immobilized microbial agent of claim 1 for treating aquaculture wastewater.
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