CN113789275A - Kosakonia oryzae CH-5 strain and application thereof - Google Patents

Kosakonia oryzae CH-5 strain and application thereof Download PDF

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CN113789275A
CN113789275A CN202111064471.5A CN202111064471A CN113789275A CN 113789275 A CN113789275 A CN 113789275A CN 202111064471 A CN202111064471 A CN 202111064471A CN 113789275 A CN113789275 A CN 113789275A
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oryzae
kosakonia
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ammonia nitrogen
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CN113789275B (en
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陈步东
孙筱君
吴启军
曹飞飞
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Hangzhou Chuhuan Science And Technology Co ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/34—Chemical or biological purification of waste gases
    • B01D53/46—Removing components of defined structure
    • B01D53/54—Nitrogen compounds
    • B01D53/58—Ammonia
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/34—Chemical or biological purification of waste gases
    • B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
    • B01D53/84—Biological processes
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2251/00—Reactants
    • B01D2251/95—Specific microorganisms
    • 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
    • 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
    • C02F2101/30—Organic compounds
    • C02F2101/38—Organic compounds containing nitrogen
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters

Abstract

The invention provides a Kosakonia oryzae CH-5 strain and application thereof, wherein the preservation number of the Kosakonia oryzae CH-5 strain is CCTCC M2021786. The Kosakonia oryzae CH-5 strain of the invention can survive in the environment with pH 4-11; the high-pH-value-tolerance ammonia nitrogen degradation agent has high pH tolerance and high salt tolerance, can grow at 10-45 ℃, has high ammonia nitrogen degradation efficiency, can be applied to ammonia nitrogen degradation in polluted environment, can be applied to preparation of a total nitrogen remover, an ammonia nitrogen remover, a COD remover and preparation of an ammonia gas degradation agent in industrial waste gas, and has the advantages of simultaneously and efficiently removing ammonia nitrogen, total nitrogen and OD in sewage and efficiently degrading ammonia gas components in industrial waste gas.

Description

Kosakonia oryzae CH-5 strain and application thereof
Technical Field
The invention belongs to the technical field of biology, relates to a Kosakonia oryzae CH-5 strain capable of removing ammonia nitrogen and total nitrogen in sewage simultaneously, and application thereof in ammonia nitrogen degradation.
Background
With the continuous improvement of social economy and living standard and the continuous increase of population, the sewage and waste gas treatment capacity of each region is also continuously increased, and ammonia gas as a typical stink substance is an important work content for sewage and waste gas treatment. Compared with the traditional physical and chemical methods, the biological deodorization method which is started in recent years has the advantages of low price, high efficiency, simple equipment, low energy consumption, no secondary pollution and the like. The key point of the biological method for removing ammonia nitrogen is to obtain the ammonia removing microorganism with high efficiency and strong stress resistance, and the ammonia removing microorganism is applied to the actual waste gas and wastewater treatment after the expanded culture.
Microbial activity is generally greatly affected by ambient temperature, pH and osmotic pressure. Wherein the temperature has the largest influence on the metabolism of the microorganism, and the metabolic activity of the microorganism is influenced by over-high or under-low temperature. The optimal temperature range of the conventional denitrification strain is 28-37 ℃, when the temperature is reduced by 10 ℃, the total amount and the enzyme activity of denitrification microorganisms are seriously influenced, and the pollutant degradation efficiency is reduced by 1-2 times. Therefore, in cold environment in winter, low temperature can generate strong inhibition effect on microbial activity, so that the biochemical performance of a sewage system and a waste gas treatment system is deteriorated, and the efficiency of sewage treatment and waste gas treatment is obviously reduced. The growth rate and the ammonia removal efficiency of general ammonia removal microorganisms such as bacillus at 15 ℃ are greatly reduced.
High salinity wastewater generally refers to wastewater with a total salt content of more than 1 percent and contains higher concentration of Cl–、SO4 2–、Na+、Ca2+And inorganic ions. The high-salinity wastewater has wide sources and mainly comprises chemical production such as printing and dyeing, pickling, papermaking and the like and direct utilization of seawaterThe waste water produced in the process. The biological method for removing ammonia nitrogen is gradually applied to the treatment of high-salt nitrogen-containing wastewater, however, when the high-salt wastewater is treated by common microorganisms, the problems of reduction of the number of floras in a treatment system, reduction of the removal rate of organic matters and ammonia nitrogen, high concentration of suspended matters in effluent and the like exist all the time due to overhigh salinity. Therefore, the screening of the denitrification microorganisms with salt tolerance and halophilic performance gradually becomes a hotspot of research in the field of high-salt nitrogen-containing wastewater treatment. The salt concentration of the Bacillus methylotrophicus L7 obtained from domestic wastewater is only 3.5%.
The common deammoniation nitrogen bacterial strain tends to grow in neutral or slightly alkaline environment, and when the pH value exceeds the adaptive range of the bacterial strain, the absorption of the bacterial strain to nutrient substances and the enzyme activity are reduced, so that the growth of microorganisms is inhibited, and the denitrification performance of the bacterial strain is influenced. The Yang et al separate Acinetobacter sp.JR1 from acidic pharmaceutical wastewater and study the denitrification performance under acidic condition (pH 4.5), and the results show that when the initial pH is 4.5 and the ammonia nitrogen is 103mg/L, the total ammonia nitrogen removal rate is up to 97.67%. But its ammonia nitrogen removal effect under high pH conditions is not ideal.
When the ammonia nitrogen removing microorganism is applied to the waste gas treatment device, ammonia gas or other malodorous gases are firstly dissolved in nutrient solution or circulating water in a biological filter of the waste gas treatment device, and when the ammonia gas is converted from a gas phase to a liquid phase, the microorganism in the biological filter plays a role
Therefore, the microorganism strains which have high activity and high pH tolerance under the low-temperature condition can play a positive role in enhancing the effects of low-temperature water treatment and low-temperature weather treatment of waste gas, while the microorganism strains which have high pH tolerance and high salt tolerance have a positive role in controlling the cost of sewage and waste gas treatment, and the mode of improving and promoting ammonia nitrogen in the waste gas and waste water treated by the active sludge in the low-temperature environment, high-pH environment and high-osmotic pressure environment by utilizing the ammonia nitrogen degrading bacteria can obviously improve the running stability of a sewage system and a waste gas treatment system under the conditions of low temperature, high pH and high osmotic pressure.
Disclosure of Invention
The invention aims to provide a Kosakonia oryzae CH-5 strain for solving the defect that the ammonia nitrogen removing effect of the existing ammonia nitrogen degrading bacteria is not ideal under the conditions of low temperature, high pH and high permeability, wherein the Kosakonia oryzae CH-5 strain can rapidly degrade ammonia nitrogen at a lower temperature and has high pH tolerance and high salt tolerance.
The invention also provides application of the Kosakonia oryzae CH-5 strain in ammonia nitrogen degradation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a Kosakonia oryzae CH-5 strain is preserved in China center for type culture Collection at 28.06.2021, the preservation address is Wuhan university in Wuhan, China, and the classification name is as follows: kosakonia oryzae CH-5 with the deposit number: CCTCC M2021786.
In the invention, the coxsackiella has no report of ammonia nitrogen removal function before the application date, and has the advantages of short thallus lag phase, rapid growth and strong ammonia nitrogen removal capability.
The Kosakonia oryzae CH-5 strain is applied to ammonia nitrogen degradation.
The Kosakonia oryzae CH-5 strain is applied to the preparation of industrial waste gas ammonia nitrogen degradation preparations.
As a preferable scheme of the invention, the preparation method of the ammonia nitrogen degradation preparation comprises the following steps: selecting Kosakonia oryzae CH-5 single bacteria, inoculating the single bacteria into an LB liquid culture medium, culturing for 10-12h under the conditions of 30 ℃ and 200r/min to obtain Kosakonia oryzae CH-5 seed liquid, respectively inoculating the Kosakonia oryzae CH-5 seed liquid into the LB liquid culture medium according to the inoculation amount of 1-3% of the volume ratio, and culturing for 12-14h under the conditions of 10-37 ℃ and 200r/min to obtain an ammonia nitrogen degradation preparation;
in a preferred embodiment of the present invention, the LB liquid medium has a pH of 4 to 11.
In a preferred embodiment of the present invention, the LB liquid medium has a NaCl concentration of 1% to 10%.
The Kosakonia oryzae CH-5 strain is applied to the preparation of a total nitrogen removal agent in sewage.
The Kosakonia oryzae CH-5 strain is applied to the preparation of ammonia nitrogen removal agents in sewage.
The Kosakonia oryzae CH-5 strain is applied to the preparation of COD remover in sewage.
The Kosakonia oryzae CH-5 is applied to the preparation of ammonia degradation agent in industrial waste gas.
Compared with the prior art, the invention has the following beneficial effects:
1) the Kosakonia oryzae CH-5 strain has no report of ammonia nitrogen removal function before the application date, and has short lag phase of the strain, rapid growth and strong ammonia nitrogen removal capability;
2) the Kosakonia oryzae CH-5 strain disclosed by the invention has the advantages that the thallus grows faster at a low temperature, the removing capacity is higher, the ammonia nitrogen degradation rate at 15 ℃ for 24 hours can reach 41.8%, the Kosakonia oryzae CH-5 strain has high pH tolerance, the tolerable pH range is 3-12, the tolerance of NaCl is high, the salt concentration can be tolerated by 10% at most, the Kosakonia oryzae CH-5 strain has strong tolerance in an extreme environment, and the Kosakonia oryzae CH-5 strain has strong adaptability when being applied to sewage treatment and waste gas treatment, can better improve the removal stability of ammonia nitrogen in waste gas and waste water, can efficiently remove ammonia nitrogen, total nitrogen and COD in sewage containing inorganic nitrogen, and can efficiently remove ammonia nitrogen and COD in domestic sewage containing organic nitrogen, and efficiently degrade industrial waste gas containing ammonia, and the application range is wide.
Drawings
FIG. 1 is a graph showing the culture characteristics of Kosakonia oryzae CH-5 strain.
FIG. 2 is a microscopic morphological view of Kosakonia oryzae CH-5 strain.
FIG. 3 is a growth curve of Kosakonia oryzae CH-5 strain.
FIG. 4 is a standard curve for ammonia nitrogen determination.
FIG. 5 is a graph showing the degradation of ammonia nitrogen, COD and total nitrogen in water by Kosakonia oryzae CH-5 strain.
FIG. 6 is a graph showing the degradation profile of ammonia nitrogen in the degradation of Kosakonia oryzae CH-5 strain.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The technical scheme of the invention is a conventional technology if not particularly specified; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1
Isolation and characterization of Kosakonia oryzae CH-5 Strain:
1) taking 10g of filler in a biological filter in a certain sewage treatment plant, and adding the filler into a triangular flask containing 90mL of sterile physiological saline and glass beads; and shaking at 30 ℃ and 200rmp for 1-2h until attachments on the filler are basically peeled off and the liquid becomes turbid.
2) And (3) sucking the culture obtained in the step (1), taking 10mL of suspension after shaking, and culturing for 48h at the temperature of 30 ℃ and the rpm of 200 in 90mL of liquid ammonia-removing microorganism screening culture medium.
3) Taking the liquid culture obtained in the step 2, diluting the liquid culture by multiple times, and coating the diluted liquid culture on a solid plate of an LB (Luria Bertani) culture medium to obtain ammonia nitrogen degrading bacteria CH-5;
wherein the components of the ammonia removal microorganism screening culture medium are as follows: each 1L deionized water contains glucose 5.0g, ammonium sulfate 0.75g, and dipotassium hydrogen phosphate (KH)2PO4)0.5g, magnesium sulfate heptahydrate (MgSO)4·7H20.25g of O), 1g of sodium chloride and ferrous sulfate heptahydrate (FeSO)4·7H2O)0.05g，pH7.0-7.2；
The LB solid medium comprises the following components: 10g of peptone, 5g of yeast extract powder, 10g of sodium chloride, 15g of agar powder, pH7.0-7.2, and the volume is made up to 1L by using distilled water.
(1) Morphological characteristics
The colony morphology of the isolated Kosakonia oryzae (Kosakoniaooryzae) CH-5 on LB solid medium is shown in FIG. 1, the colony morphology is regular, the edge is clear, the colony is rough and opaque, the surface is moist and smooth, and the colony is light yellow or beige yellow. After safranin staining, no spores were seen microscopically, and the cells were in the form of small short rods (as shown in FIG. 2).
(2) Gene identification of Kosakonia oryzae CH-5 Strain
Extracting the genome DNA of the strain to be detected by using a bacterial DNA extraction kit, and referring to the instruction for the specific operation steps. And (3) amplifying the bacteria by using the extracted bacteria genome DNA as a template, wherein a primer for amplifying 16S rRNA:
27F:5'-gtttgatcctggctcag-3'；
1492R:5'-tacggctaccttgttacgactt-3'；
the 16s rDNA nucleotide sequence of Kosakonia oryzae CH-5 is shown in SEQ ID NO: 1, and the following components:
ccactcccatggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgtgacattctgattcacgattactagcgattccgacttcatggagtcgagttgcagactccaatccggactacgacgcactttatgaggtccgcttgctctcgcgaggtcgcttctctttgtatgcgccattgtagcacgtgtgtagccctggtcgtaagggccatgatgacttgacgtcatccccaccttcctccagtttatcactggcagtctcctttgagttcccggccggaccgctggcaacaaaggataagggttgcgctcgttgcgggacttaacccaacatttcacaacacgagctgacgacagccatgcagcacctgtctcacagttcccgaaggcaccccggcatctctgccaggttctgtggatgtcaagaccaggtaaggttcttcgcgttgcatcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcatttgagttttaaccttgcggccgtactccccaggcggtcgatttaacgcgttagctccggaagccacgcctcaagggcacaacctccaaatcgacatcgtttacggcgtggactaccagggtatctaatcctgtttgctccccacgctttcgcacctgagcgtcagtcttcgtccaggaggccgccttcgccaccggtattcctccagatctctacgcatttcaccgctacacctggaattctacctccctctacgagactccagcctgccagtttcgaatgcagttcccaggttgagcccggggatttcacatccgacttgacagaccgcctgcgtgcgctttacgcccagtaattccgattaacgcttgcaccctccgtattaccgcggctgctggcacggagttagccggtgcttcttctgcgggtaacgtcaatcggcacggttattaaccgtaccgccttcctccccgctgaaagtgctttacaacccgaaggccttcttcacacacgcggcatggctgcatcaggcttgcgcccattgtgcaatattccccactgctgcctcccgtaggagtctggaccgtgtctcagttccagtgtggctggtcatcctctcagaccagctagggatcgtcgcctaggtgggccgttaccccgcctactagctaatcccatctgggcacatctgatggcaagaggcccgaaggtccccctctttggtcttgcgacgttatgcggtattagctaccgtttccagtagttatccccctccatcaggcagtttcccagacattactcacccgtccgccactcgtcacccgagagcaagctctctggctaccgttcgactg。
the splicing sequence is found to belong to the Kosakh bacteria by Blast homologous sequence search, the strain is preserved in China center for type culture Collection at 28 th 06 th 2021 at the preservation address of Wuhan university in Wuhan, China, and the classification is named as follows: kosaknia oryzae (Kosakonia oryzae) CH-5 with the deposit number: CCTCC M2021786.
Example 2
Growth Performance test of Kosakonia oryzae CH-5 Strain:
(1) growth Curve assay of Kosakonia oryzae CH-5 Strain
A single colony of the Kosakonia oryzae CH-5 strain was picked from an LB plate and cultured in 100mL of liquid LB medium at 30 ℃ for 12 hours at 200r/min to obtain a seed solution of the Kosakonia oryzae CH-5 strain.
Inoculating seed liquid of Kosakonia oryzae CH-5 strain into LB liquid culture medium according to the inoculum size of 2% (volume ratio), culturing at 30 deg.C and 200r/min, sampling every 2h for OD600Measurement until OD600Reaches a maximum and tends to fall.
The growth curve is shown in FIG. 3, and it can be seen from FIG. 3 that the strain reaches the plateau stage when it grows for 12-14h, the growth cycle of the strain is short, and the growth is fast.
(2) Growth status of Kosakonia oryzae CH-5 strain at different pH:
inoculating seed liquid of Kosakonia oryzae CH-5 strain into LB culture medium with different initial pH according to 2%, culturing at 30 deg.C and 200r/min, sampling for OD after culturing for 14 hr600The results of the measurements are shown in Table 1. The initial pH gradients of LB medium were 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, respectively. Kosakonia oryzae CH-5 has an optimum pH of 7 and is tolerant to a pH range of 3 to 12. Under the condition of high pH11, the growth activity is still higher.
TABLE 1 OD at different pH600
pH 3 4 5 6 7 8 9 10 11 12
OD600 0.0765 1.938 2.4125 2.49 2.665 2.0775 1.79 0.54 0.1805 0.057
(3) Growth status of Kosakonia oryzae CH-5 Strain at different salt concentrations
Inoculating seed liquid of Kosakonia oryzae CH-5 strain into LB culture medium with different initial salt concentration according to the inoculation amount of 2%, respectively, at 30 ℃,Culturing at 200r/min and pH7 for 14 hr, sampling, and measuring OD600The results are shown in Table 2. The NaCl concentration gradient of LB medium was set at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, respectively. The optimum salt concentration of the Kosakonia oryzae CH-5 strain was 1% and the highest salt concentration tolerated 10% NaCl.
TABLE 2 OD at different NaCl concentrations600
Figure BDA0003257675340000061
(4) Growth temperature experiment:
inoculating seed liquid of Kosakonia oryzae CH-5 strain into LB culture medium according to the inoculation amount of 2%, culturing at different temperatures and under the conditions of 200r/min and pH7, sampling after culturing for 14h, and measuring OD600The results are shown in Table 3. The temperature gradient of the culture medium is set at 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 28 deg.C, 37 deg.C, 40 deg.C, and 45 deg.C. The optimal temperature of the strain is 30 ℃, the strain still grows vigorously at 10 ℃, and the strain has good low-temperature resistance.
TABLE 3 OD at different temperatures600
Temperature/. degree.C 10 15 20 25 28 30 37 40 45
OD600 0.943 1.252 1.539 2.361 2.867 3.021 2.319 0.537 0.113
Example 3
Determination of ammonia nitrogen degradation efficiency of Kosakonia oryzae CH-5 strain
By adopting a (HJ 535-2009 ammonia nitrogen determination nano-reagent spectrophotometry), ammonia nitrogen existing in the form of free ammonia or ammonia ions and the like reacts with a nano-reagent to generate a light red brown complex, the absorbance of the complex is in direct proportion to the content of ammonia nitrogen, and the absorbance is measured at the wavelength of 420 nm.
Drawing of standard curve
(1) A nano-grade reagent: mercuric iodide-potassium iodide-sodium hydroxide (HgI)2KI-NaOH) solution 16.0g of sodium hydroxide (NaOH) was weighed, dissolved in 50mL of water, and cooled to room temperature. 7.0g of potassium iodide (KI) and 10.0g of mercuric iodide (HgI) were weighed out2) Dissolved in water, and this solution is then slowly added to the above 50mL sodium hydroxide solution with stirring and diluted to 100mL with water. Storing in polyethylene bottle, covering tightly with rubber plug or polyethylene cap, storing in dark place, and prolonging the shelf life by one year.
(2) Sodium potassium tartrate solution,. rho.500 g/L: 50.0g of potassium sodium tartrate (KNaC) was weighed4HgO6·4H2O) was dissolved in 100mL of water, heated to boil to drive off ammonia, cooled sufficiently, and diluted to 100 mL.
(3) Ammonia nitrogen standard stock solution, rhoN1000 ug/mL. 3.8190g of ammonium chloride (NH) were weighed out4Cl, superior purity, drying for 2h at 100-105 ℃, dissolving in water, transferring into a 1000mL volumetric flask, diluting to a marked line, and storing for 1 month at 2-5 ℃.
(4) Ammonia nitrogen standard working solution, rhoN10 μ g/mL. 5.00mL of ammonia nitrogen standard storage solution is sucked into a 500mL volumetric flask and diluted to the scale. It is prepared before use.
(5) 0.00mL, 0.50mL, 1.00mL, 2.00mL, 4.00mL, 6.00mL and 8mL of ammonia nitrogen standard working solution are respectively added into 8 colorimetric tubes, and water is added until the marked line is formed. 0.50mL of sodium potassium tartrate solution is added, and after shaking up, 0.50mL of Narse reagent is added and shaking up. After standing for 10min, the absorbance was measured at a wavelength of 420nm using a 1cm cuvette with water as a reference.
And (3) taking the absorbance after blank correction as an abscissa and the corresponding ammonia nitrogen content (mug) as an ordinate, and drawing a calibration curve, wherein the standard curve is shown as a graph 4 and the curve is as follows: 61.448x +0.0711, R2＝0.9971。
Second, sample measurement
Taking a proper amount of cultured bacterial suspension, and centrifuging at 10000rpm for 10 min. The appropriate volume of supernatant was taken and absorbance was measured in the same procedure as the calibration curve.
Blank test, the non-inoculated culture medium is taken, and pre-treatment and measurement are carried out according to the same steps as the sample.
Thirdly, calculating the result
Figure BDA0003257675340000071
In the formula:
ρNthe mass concentration of ammonia nitrogen in the water sample is mg/L in terms of nitrogen;
AS-absorbance of a water sample;
Ab-absorbance of blank test;
a-intercept of the calibration curve;
b-the slope of the calibration curve;
v-sample volume, mL.
Fourthly, determining the degradation rate of ammonia nitrogen degrading bacteria to ammonia nitrogen
Culturing ammonia nitrogen degrading bacterium Kosakonia oryzae CH-5 strain in LB liquid culture medium, centrifugally collecting thallus, re-suspending with sterile water, and diluting to OD 6001, converting OD600Inoculating 5mL of the bacterial solution which is 1 into 100.0mL of an ammonia removal determination culture medium, placing the culture medium in a constant temperature shaking table at 200r/min, culturing at 30 ℃, and determining the ammonia nitrogen degradation rate after 24 hours.
Wherein the components of the ammonia removal determination culture medium are as follows: each 1L of deionized water contains 5.0g of sodium citrate, 0.5g of ammonium sulfate, 50mL of Vickers salt solution and pH of 7.0-7.2;
the Vickers salt solution contains dipotassium hydrogen phosphate (K) in each L of Vickers salt solution2HPO4)0.5g, magnesium sulfate heptahydrate (MgSO)47H2O)2.5g, sodium chloride (NaCl)1g, ferrous sulfate heptahydrate (FeSO)4·7H2O)0.05g, tetrahydrate and manganese sulfate (MnSO)4·4H2O)0.05g。
Wherein, the Kosakonia oryzae CH-5 strain is obtained by screening, and the degradation rate of the strain to ammonia nitrogen in 24 hours is respectively as follows: 99.8 percent; the ammonia nitrogen degradation efficiency is obvious.
Fifthly, culturing the ammonia nitrogen degrading bacterium Kosakonia oryzae CH-5 strain and the alcaligenes pseudomonad in an LB liquid culture medium, centrifugally collecting the thalli, resuspending the thalli with sterile water, and diluting the thalli to OD600The dilution of each of the two selected strains (5 mL) was inoculated into 100.0mL of an ammonia removal assay medium, incubated at 10 ℃ in a shaker at 200r/min, and the degradation rate was measured after 24 hours.
Wherein the screened Kosakonia oryzae CH-5 strain and the pseudomonas alcaligenes have the degradation rates to ammonia nitrogen for 24 hours at 15 ℃ as follows: 41.8 percent and 8.8 percent, and the ammonia nitrogen degradation effect is obvious at low temperature.
Example 4:
determination of COD and total nitrogen degradation efficiency of Kosakonia oryzae CH-5 strain
The Kosakonia oryzae CH-5 bacterial strain degrades ammonia nitrogen, total nitrogen and COD in the artificial sewage containing inorganic nitrogen: preparing 3L-5L sewage from glucose and ammonium chloride according to the ratio of C to N being 100 to 5, and adding a small amount of K2HPO4The pH value is adjusted to 6.5-8.0, and the concentration of ammonia nitrogen, total nitrogen and COD in the prepared artificial sewage are respectively about 40mg/L, 40mg/L and 300 mg/L. The reactor is provided with an aeration device, the dissolved oxygen is about 4mg/L, and the top of the reactor is sealed by a sterile sealing film to prevent the entry of mixed bacteria; then carrying out aeration treatment, sampling every 4 hours, and determining that the pH values are all about 6.5-8.0 to reach the range of the discharge standard; and detecting the concentration changes of ammonia nitrogen, total nitrogen and COD in the sample, wherein the data are the mean values of parallel sample determination.
The experiment was run for 2d and the data is as follows: and taking a sample for 4 hours, and measuring ammonia nitrogen, total nitrogen and COD.
Referring to fig. 5, it can be found that ammonia nitrogen in the artificial sewage is basically removed at 24 hours, and the removal rate can reach more than 90%. As for COD in water, the removal of the bacteria is slow, and the bacteria is reduced to 40mg/L within about 40 hours, thereby reaching the national emission standard. However, the total nitrogen in the water still does not reach the discharge standard after being treated for 48 hours, the difference between the total amount of the total nitrogen and the total amount of the total nitrogen is not large, the bacteria do not have the denitrification function, and the ammonia nitrogen cannot be denitrified into N2。
The Kosakonia oryzae CH-5 strain has good treatment effect on sewage, can treat ammonia nitrogen and COD in water within a certain time, and enables the sewage to reach the standard and be discharged.
Example 5
Determination of ammonia nitrogen removal effect of Kosakonia oryzae CH-5 strain in biological spray tower
In the biological spray tower of certain gas treatment equipment in Hubei, the air quantity is about 1000m3H, filling bamboo charcoal filler in the biological tower, wherein the filling height is 1.5m in total, and the volume is 4.71m3The gas retention time is 16.97s, and the spray flow of the circulating liquid is about 2m3And h, continuously spraying. And collecting the field waste gas, and discharging the field waste gas into the biological spray tower.
And lifting the nutrient solution containing Kosakonia oryzae CH-5 strain to the top of the biological spraying tower through a circulating water pump to spray downwards, and hanging a film 7d on the filler under the circulating spraying. And after the film formation is finished, the circulating liquid is directly emptied from the tower. And (3) starting to operate the biological spray tower, discharging the gas into an empty bed, keeping the EBRT for 15s, detecting the concentration of the ammonia gas at the gas inlet and the gas outlet 1 time every day, and operating and measuring for 14 days.
As can be seen from FIG. 6, the removal rate of the Kosakonia oryzae CH-5 strain to ammonia nitrogen in the spray tower reaches more than 94%, the strain has good ammonia nitrogen removal effect, and ammonia gas can be efficiently treated even under the condition of high ammonia gas concentration and can be discharged up to the standard.
In conclusion, the Kosakonia oryzae CH-5 strain can be applied to removing ammonia nitrogen and COD in water and can also be applied to treating industrial waste gas containing ammonia gas in a biological trickling filter.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Sequence listing
<110> Hangzhou Chuhuan technology corporation
<120> Kosakonia oryzae CH-5 strain and application thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gtttgatcct ggctcag 17
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tacggctacc ttgttacgac tt 22
<210> 3
<211> 1361
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccactcccat ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgtgacat 60
tctgattcac gattactagc gattccgact tcatggagtc gagttgcaga ctccaatccg 120
gactacgacg cactttatga ggtccgcttg ctctcgcgag gtcgcttctc tttgtatgcg 180
ccattgtagc acgtgtgtag ccctggtcgt aagggccatg atgacttgac gtcatcccca 240
ccttcctcca gtttatcact ggcagtctcc tttgagttcc cggccggacc gctggcaaca 300
aaggataagg gttgcgctcg ttgcgggact taacccaaca tttcacaaca cgagctgacg 360
acagccatgc agcacctgtc tcacagttcc cgaaggcacc ccggcatctc tgccaggttc 420
tgtggatgtc aagaccaggt aaggttcttc gcgttgcatc gaattaaacc acatgctcca 480
ccgcttgtgc gggcccccgt caattcattt gagttttaac cttgcggccg tactccccag 540
gcggtcgatt taacgcgtta gctccggaag ccacgcctca agggcacaac ctccaaatcg 600
acatcgttta cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc 660
acctgagcgt cagtcttcgt ccaggaggcc gccttcgcca ccggtattcc tccagatctc 720
tacgcatttc accgctacac ctggaattct acctccctct acgagactcc agcctgccag 780
tttcgaatgc agttcccagg ttgagcccgg ggatttcaca tccgacttga cagaccgcct 840
gcgtgcgctt tacgcccagt aattccgatt aacgcttgca ccctccgtat taccgcggct 900
gctggcacgg agttagccgg tgcttcttct gcgggtaacg tcaatcggca cggttattaa 960
ccgtaccgcc ttcctccccg ctgaaagtgc tttacaaccc gaaggccttc ttcacacacg 1020
cggcatggct gcatcaggct tgcgcccatt gtgcaatatt ccccactgct gcctcccgta 1080
ggagtctgga ccgtgtctca gttccagtgt ggctggtcat cctctcagac cagctaggga 1140
tcgtcgccta ggtgggccgt taccccgcct actagctaat cccatctggg cacatctgat 1200
ggcaagaggc ccgaaggtcc ccctctttgg tcttgcgacg ttatgcggta ttagctaccg 1260
tttccagtag ttatccccct ccatcaggca gtttcccaga cattactcac ccgtccgcca 1320
ctcgtcaccc gagagcaagc tctctggcta ccgttcgact g 1361

Claims (10)

1. A Kosakonia oryzae CH-5 strain is characterized in that the strain is preserved in China center for type culture Collection at 28 th 06 months 2021, the preservation address is Wuhan university in Wuhan, China, and the classification name is as follows: kosakonia oryzae CH-5 with the deposit number: CCTCC M2021786.
2. The Kosakonia oryzae CH-5 strain of claim 1, for use in ammonia nitrogen degradation.
3. Use of the Kosakonia oryzae CH-5 strain of claim 1 for the preparation of industrial waste gas ammonia nitrogen degradation preparations.
4. The application of the Kosakonia oryzae CH-5 strain in preparing an industrial waste gas ammonia nitrogen degradation preparation according to claim 3, wherein the preparation method of the ammonia nitrogen degradation preparation comprises the following steps: selecting Kosakonia oryzae CH-5 single bacteria, inoculating the single bacteria into LB liquid culture medium, culturing for 10-12h under the conditions of 30 ℃ and 200r/min to obtain Kosakonia oryzae CH-5 seed liquid, respectively inoculating the Kosakonia oryzae CH-5 seed liquid into the LB liquid culture medium according to the inoculation amount of 1-3% of the volume ratio, and culturing for 12-14h under the conditions of 10-37 ℃ and 200r/min to obtain the ammonia nitrogen degradation preparation.
5. The use of Kosakonia oryzae CH-5 in the preparation of ammonia nitrogen degrading formulations in industrial waste gas according to claim 4, wherein the LB liquid medium has a pH of 4-11.
6. The use of Kosakonia oryzae CH-5 in the preparation of ammonia nitrogen degradation preparation in industrial waste gas according to claim 4, wherein the NaCl concentration of the LB liquid medium is 1% -10%.
7. Use of the Kosakonia oryzae CH-5 strain according to claim 1 for the preparation of a total nitrogen removal agent in wastewater.
8. The use of the Kosakonia oryzae CH-5 strain as claimed in claim 1 for the preparation of ammonia nitrogen removal agents in sewage.
9. The use of the Kosakonia oryzae CH-5 strain of claim 1 for the preparation of a COD-removing agent in sewage.
10. Use of Kosakonia oryzae CH-5 according to claim 1 for the preparation of ammonia degradation agents in industrial waste gases.
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