CN113308410B - Denitrifying bacterium for efficient denitrification and application thereof - Google Patents
Denitrifying bacterium for efficient denitrification and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of environmental microorganisms and sewage treatment, and discloses denitrifying bacteria for efficient denitrification and application thereof. The denitrifying bacteria capable of efficiently denitrifying under anoxic and aerobic conditions are separated from sewage, have strong reproductive capacity and high denitrifying activity, have high nitrate nitrogen and nitrite nitrogen tolerance, and improve the denitrification efficiency.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms and water body treatment, in particular to denitrifying bacteria for efficient denitrification and application thereof.
Background
With the rapid development of human production and life, high nitrogen pollution becomes a great concern all over the world, the total nitrogen emission far exceeds the environmental capacity of a receiving water body, the excessive total nitrogen causes eutrophication of the water body, abnormal proliferation of algae is induced, and water quality is deteriorated. Therefore, how to reduce the nitrogen pollution of the reservoir water body becomes a hotspot problem in the water environment field.
In recent years, annual discharge of sewage in China is continuously increased, annual discharge of sewage in 2015 is only 466.62 billion cubic meters, annual discharge of sewage in 2018 breaks through 500 billion cubic meters, annual discharge of sewage in 2019 is increased to 554.65 billion cubic meters, the annual discharge of sewage is increased by 6.4%, a sewage treatment plant is an important link for sewage treatment and is an important place for removing organic matters and nitrogen in wastewater, nitrate (nitrate nitrogen) is a main form of nitrogen in tail water of the sewage treatment plant, and deep denitrification is in the forefront.
Biological denitrification is an efficient, economic and environment-friendly denitrification method, and denitrification bacteria are often used for removing nitrate in the water body remediation and sewage treatment processes. Numerous researches indicate that denitrifying bacteria can achieve excellent denitrification effect under the anoxic condition (dissolved oxygen is 0.2 mg/L-0.5 mg/L), and in recent years, researches find that part of denitrifying bacteria can perform denitrification under the aerobic condition, but the denitrification effect is not ideal, the tolerance concentration is obviously low, and the treatment efficiency is low, for example, a Zubel bacterium disclosed in patent 201910234181.7 (application 2019.03.26) can remove nitrate and nitrite in a water body under the aerobic condition, but the tolerance concentration is low (<200mg/L), the treatment speed is low, and nitrate and nitrite with low concentration can be removed within 48 hours, which indicates that a microbial inoculum with high-efficiency denitrification effect is not discovered under the aerobic condition; meanwhile, the discovery of aerobic denitrifying bacteria provides possibility for realizing synchronous nitrification and denitrification, the nitrification process and the denitrification process can be simultaneously carried out in the aerobic tank, the time, the operating cost, the carbon source usage amount, the alkali liquor adding amount and the like of the whole reaction process are saved, the capital construction cost of the anoxic tank is reduced, and the method has obvious advantages compared with the traditional denitrification process.
In addition, nitrite nitrogen is a toxic substance for microorganisms, high-concentration nitrite nitrogen has an inhibiting effect on the growth and reaction of denitrifying bacteria, and the existing denitrifying bacteria have no effect of high tolerance to nitrite nitrogen, so that the efficiency is poor in removing the high-concentration nitrite nitrogen.
Therefore, it is one of the important researches to select a microorganism strain which can improve the level of tolerant nitrate nitrogen from nature, has higher denitrification efficiency (nitrate nitrogen + nitrite nitrogen + total nitrogen), and can denitrify under anoxic and aerobic conditions, and is suitable for different condition treatment systems.
Disclosure of Invention
In view of the above, the present invention aims to provide a denitrifying bacterium with high denitrification efficiency, which not only has higher tolerance of nitrate nitrogen and nitrite nitrogen, but also can denitrify under aerobic and anoxic conditions with high efficiency;
the invention also aims to provide the application of the denitrifying bacteria in the relevant fields of water body treatment, microbial agents and the like.
In order to achieve the above purpose, the invention provides the following technical scheme:
the denitrifying bacterium with high denitrification efficiency is Zobellella densificans LBS W21-01 with the preservation number of CGMCC No. 22730.
Through long-term research, the invention obtains a microbial strain which can efficiently denitrify under the anoxic and aerobic conditions by screening from the sewage, the strain is named as LBS W21-01, the strain has fast propagation and strong activity, and tests show that the strain can be applied to various sewage treatment systems, natural water bodies and aquaculture water bodies, and can effectively remove nitrate nitrogen, nitrite nitrogen and total nitrogen in the water.
The strain LBS W21-01 is subjected to 16SrDNA sequencing, the nucleotide sequence is shown as Seq ID No. 1, the sequence is the complete sequence of 16S rDNA of the strain, and BLAST alignment is carried out on the determined 16SrDNA sequence. The strain has the morphological characteristics that bacterial colonies are milky white, the edges are neat, the bacterial colonies can grow rapidly under the wet state and the anoxic and aerobic conditions. The identification proves that the strain is determined to be Zobellella densificans (Zobenella densificans) on the molecular level, and the preservation number of the strain preserved in the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No. 22730.
The separated and obtained Zolbert cell-bacter LBS W21-01 is respectively inoculated into a culture medium with nitrate nitrogen and nitrite nitrogen as unique nitrogen sources, static culture is carried out at 30 ℃, 2mL of culture solution is taken out at 0h and 24h respectively, centrifugation is carried out, nitrate nitrogen, nitrite nitrogen and ammonia nitrogen in supernatant are measured, and the result shows that the Zolbert cell-bacter LBS W21-01 respectively has quick and efficient removal effects on potassium nitrate and sodium nitrite in a water body. In a culture medium taking nitrate nitrogen as a unique nitrogen source, the strain is cultured for 24 hours and then NO is added3 -The removal rate of N and total nitrogen is 100%, wherein the highest tolerance concentration of nitrate nitrogen is as high as 1500 mg/L; in a culture medium taking nitrite nitrogen as a unique nitrogen source, the strain is cultured for 24 hours and then NO is added2 -The removal rate of-N and total nitrogen is 100%, wherein the highest tolerance concentration of nitrite nitrogen is as high as 600 mg/L.
Meanwhile, the Zubelia bescens LBS W21-01 can still perform denitrification at the low temperature of 15 ℃; under aerobic and anoxic conditions, the debaryomyces superbus LBS W21-01 can efficiently complete denitrification within 16 hours.
In view of the excellent effects of the Znobel bacterium LBSW21-01, the invention provides the application of the Znobel bacterium in denitrification treatment of water bodies and preparation of microbial agents for denitrification of water bodies, wherein the water bodies comprise but are not limited to various sewage, natural water bodies and aquaculture water bodies needing denitrification.
The invention also provides a preparation method of the microbial agent for water denitrification, and the method comprises the steps of inoculating the Zubelia debaryi LBSW21-01 with the preservation number of CGMCC No.22730 into a denitrification liquid culture medium for activation, then replacing the denitrification liquid culture medium to prepare a seed solution, and inoculating the seed solution into a fermentation culture medium for fermentation to obtain the liquid microbial agent.
Wherein the denitrification liquid medium comprises KNO3Sodium citrate, NaHPO4、MgSO4·7H2O、K2HPO4Trace elements including EDTA and ZnSO, and water4、CaCl2、MnSO4、FeSO4、Na6MoO2、CuSO4And CoCl2。
Preferably, the formulation of the denitrification liquid culture medium is as follows: KNO30.1-1.2 percent of sodium citrate, 0.15-2.0 percent of NaHPO40.05-0.2％，MgSO4·7H2O 0.002-0.005％，K2HPO40.05-0.2 percent of the total weight of the feed additive, the balance of distilled water, pH7.0-7.4, 2-4ml/L of trace elements and sterilization for 20min at 121 ℃. The formula of the trace elements is as follows: EDTA 2-8%, ZnSO4 0.1-0.3％、CaCl2 0.3-1.0％、MnSO4 0.2-1.0％、FeSO4 0.2-1.0％、Na6MoO2 0.05-0.2％、CuSO4 0.1-0.2％、CoCl20.1-0.2 percent, and the solution is prepared for use at present after being fixed in a volumetric flask of 100 ml.
Preferably, the fermentation medium comprises corn steep liquor dry powder, sodium citrate, potassium nitrate, potassium dihydrogen phosphate, disodium hydrogen phosphate and trace elements. More preferably, the corn steep liquor comprises 0.5-1% of corn steep liquor dry powder, 0.5-1.2% of sodium citrate, 0.2-0.6% of potassium nitrate, 0.1-0.3% of monopotassium phosphate, 0.05-0.2% of disodium hydrogen phosphate and 2-4ml/L of trace elements (described above), the pH value is 7.0-7.4, and the corn steep liquor is sterilized at 121 ℃ for 20 min.
Preferably, the fermentation can be divided into two steps of seed fermentation tank fermentation and fermentation tank fermentation, the two steps are different in fermentation specification, and the fermentation can be selected according to actual needs. In a specific embodiment of the present invention, the liquid microbial agent is prepared as follows:
(1) activating strains: inoculating 1-5uL of frozen Zolbertymella into a denitrification liquid culture medium containing 5mL, and standing and culturing in an incubator at 28-37 ℃ for 8-20 h;
(2) preparing liquid seeds: transferring the activated strain into 200mL of denitrification liquid culture medium, and placing the culture medium in an incubator at 28-37 ℃ for static culture for 8-20 h;
(3) seed fermentation tank: inoculating the prepared liquid seeds into a 30L seed tank with the liquid loading amount of 60-70% according to the inoculation amount of 5-10% v/v for amplification culture, wherein the stirring speed is 50-140rpm, the culture temperature is 28-37 ℃, the pressure of a fermentation tank is 0.01-0.2MPa, and the fermentation period is 6-20 h;
(4) fermentation in a fermentation tank: inoculating the obtained seed fermentation tank bacterial liquid into a culture medium of a 300L fermentation tank with the liquid loading amount of 60-70% according to the inoculation amount of 5-10% v/v for culture, wherein the culture medium and the culture conditions are the same as those of seed tank fermentation; the number of the thalli after the fermentation is finished reaches 2 multiplied by 109-4×1010cfu/mL, and taking the culture solution out of the tank after fermentation is finished to obtain the liquid microbial inoculum.
In addition, the solid microbial agent can be prepared by adding glycerol, diatomite and starch on the basis of preparing the liquid microbial agent; the fermented liquid microbial inoculum is prepared according to the proportion of 1 per thousand-1% v/vAdding glycerol, adding 2-4% of diatomite and 1-3% of corn starch according to the mass ratio for adsorption treatment of liquid microbial inoculum fermentation liquor, centrifuging and separating the liquid microbial inoculum fermentation liquor to obtain solid thalli, drying to obtain bacterial powder, controlling the water content of the bacterial powder to be below 10%, and detecting that the effective viable count is 3 multiplied by 1010-2×1011cfu/g。
According to the application, the invention also provides a water body denitrification treatment method, which comprises the steps of putting the Zubelia Zubelii LBSW21-01 with the preservation number of CGMCC No.22730 or a microbial agent prepared from the Zubelia Zubelii LBSW21-01 into pretreated water body for domestication, and then carrying out denitrification treatment under the aerobic and/or anoxic conditions. The reference usage amount of the liquid microbial agent is 0.5-1%; the reference usage amount of the solid microbial agent is 0.5-1 per mill. When the Zubeiella or the microbial agent prepared from the Zubeiella is used for anoxic/aerobic denitrification, the dissolved oxygen in water is adjusted to be 0.1-5mg/L (the dissolved oxygen is 0.2-0.5 mg/L under an anoxic condition), the pH is 6.5-8.0, and the temperature is controlled to be 15-35 ℃.
When the denitrification of the water body is carried out, the consumption condition of the carbon source of the water body needs to be monitored, the debaryomyces nobilis can ensure that the currently specified denitrification effect can be achieved when the C/N ratio reaches 4, and 100% can be removed when the C/N ratio reaches 6. Meanwhile, the influence of different carbon sources such as glycerol, sodium succinate, super carbon, alcohol, glucose, sodium citrate and the like on denitrification is compared, and the sodium citrate and the sodium succinate have better promotion effects compared with other carbon sources. Therefore, during the denitrification treatment process, sodium citrate and/or sodium succinate are used as carbon sources for carbon source supplement, and the C/N ratio is maintained to be not less than 4.
According to the technical scheme, the denitrifying bacteria capable of efficiently denitrifying under anoxic and aerobic conditions are separated from sewage, the breeding capability is strong, the denitrifying activity is high, and the capability of high tolerance to nitrate nitrogen and nitrite nitrogen is realized.
Biological preservation Instructions
LBSW21-01, named after classification: the Zobenella dentiricans are preserved in the China general microbiological culture Collection center on 17 th 6 th 2021, with the address of No. 3 Xilu-1 of Beijing university facing Yang district, and the preservation number of CGMCC No.22730 at the institute of microbiology of China academy of sciences.
Drawings
FIG. 1 shows a case of Zoerella in a solid medium according to the present invention;
FIG. 2 is a schematic diagram showing the influence of denitrification effect of the Zubel bacterium of the present invention under different carbon sources; the ordinate is the concentration mg/L of nitrogen;
FIG. 3 is a schematic diagram showing the effect of the Zubeliella on denitrification by comparison of different carbon sources; the ordinate is the concentration mg/L of nitrogen;
FIG. 4 is a schematic diagram showing the tolerance of the Zubel bacterium to nitrite nitrogen at different concentrations; the ordinate is the concentration mg/L of nitrogen; legends 200, 400, 600 represent NO2 --N concentration in mg/L;
FIG. 5 is a schematic diagram showing the degree of tolerance of the bacterium of the present invention to nitrate nitrogen at various concentrations; the ordinate is the concentration mg/L of nitrogen; legend series 1-7 indicates NO in sequence3 --N concentration of 200, 400, 600, 800, 1200, 1500, 1800mg/L, respectively;
FIG. 6 is a schematic diagram of denitrification effect of the Zubeniella terniformis at 15 ℃;
FIG. 7 is a schematic diagram of denitrification effect of the Zubel bacterium under aerobic conditions;
FIG. 8 is a schematic diagram of denitrification effect of the Zubel bacterium under anoxic conditions.
Detailed Description
The invention discloses denitrifying bacteria for efficient denitrification and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the denitrifying bacteria as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the denitrifying bacteria and their uses of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately modifying or combining the denitrifying bacteria and their uses described herein without departing from the spirit, scope and spirit of the invention.
In the denitrification process of the water body, corresponding nutrient salt can be added for activation before the solid microbial inoculum is added, so that the rapid growth and propagation of the debaryomyces nobilis can be promoted; the nutrient salt comprises: NaHPO4，MgSO4·7H2O，K2HPO4，CaCl2，MnSO4And FeSO4；
The specific activation method comprises the following steps: inoculating the solid microbial inoculum into the nutrient salt liquid according to the dosage, wherein the solid microbial inoculum is inoculated according to the inoculation quantity of 1-5 per mill of the solid microbial inoculum, and the activation condition is that dissolved oxygen is 0.1-5mg/L, the pH value is 7.0-7.5, the temperature is 28-35 ℃, and the activation is carried out for 6-8 h;
the nutrient salt solution during activation had the following composition: NaHPO4 0.05-0.2％，MgSO4·7H2O 0.002-0.005％，K2HPO4 0.05-0.2％，CaCl2 0.3-1.0％、MnSO4 0.2-1.0％、FeSO4 0.2-1.0％。
More preferably, after the microbial inoculum is added for the first time, the microbial inoculum is added every 7-10 days, the adding amount is the same as the adding amount for the first time, and the effect is better after the microbial inoculum is continuously added for 3 times. The denitrifying bactericide disclosed by the invention can be quickly adapted to the water environment, so that a system can stably run.
In addition, the total nitrogen referred to in the present invention refers to the sum of nitrate nitrogen, nitrite nitrogen and ammonia nitrogen.
The invention is further illustrated by the following examples.
Example 1: strain screening process
(1) Domesticating strains: taking anoxic pond sludge from a certain sewage treatment plant in Maxingxian county of Binzhou, Shandong province, placing the anoxic pond sludge into a denitrification liquid culture medium, setting the initial nitrate nitrogen concentration to be 100mg/L, standing and culturing at 35 ℃, sampling and detecting nitrate nitrogen, ammonia nitrogen and nitrite nitrogen every 24 hours, and continuously supplementing KNO when the sum of the three nitrogen is lower than 20mg/L3To gradually increase the concentration of nitrate nitrogen at 200, 300, 400, 500 and 600mg/L,so as to gradually acclimatize and improve the degradation capability and tolerance of the strain;
(2) and (3) strain separation and purification: and (3) performing strain separation by adopting a double-layer plate method. Inverting the denitrification solid culture medium (liquid culture medium, and 1.5-2% agar and 1% bromothymol blue alcoholic solution) to solid plate, cooling, and diluting according to gradient 10-1、10-2……10-7Coating a flat plate, inverting a thin layer of sterilized agar with the concentration of 1.5% which is cooled to about 40 ℃ above the flat plate, covering the coated strain to play a role in isolating oxygen, placing the coated strain in a constant-temperature incubator at 35 ℃ for culture, after the strain grows out, selecting a bacterial colony at the position where the culture medium turns blue, and carrying out streak purification to obtain the denitrifying strain, as shown in figure 1.
Example 2: preparation of microbial Agents
(1) Activating strains: inoculating 1-5uL of frozen Zolbertymella into a denitrification liquid sterile culture medium containing 5mL, and standing and culturing in an incubator at 28-37 ℃ for 8-20 h;
the formula of the denitrification liquid culture medium is as follows: KNO30.1 percent, 0.15 percent of sodium citrate and NaHPO4 0.1％，MgSO4·7H2O 0.005％，K2HPO40.1 percent, the balance of distilled water, pH7.0-7.4, 2ml/L of trace elements and sterilization for 20min at 121 ℃.
The formula of the trace elements is as follows: EDTA 5%, ZnSO4 0.2％、CaCl2 0.5％、MnSO4 0.3％、FeSO40.4％、Na6MoO2 0.1％、CuSO4 0.15％、CoCl20.15 percent, and the solution is prepared for use at present after being fixed in a volumetric flask of 100 ml.
(2) Preparing liquid seeds: transferring the activated strain to 200mL of denitrification liquid culture medium, and placing the culture medium in an incubator at 28-37 ℃ for static culture for 8-20 h;
(3) seed fermentation tank: inoculating the prepared liquid seeds into a 30L seed tank with the liquid loading amount of 60% according to the inoculation amount of 10% v/v for amplification culture, wherein the stirring speed is 50-140rpm, the culture temperature is 28-37 ℃, and the fermentation period of a fermentation tank is 12h under the condition of sub 0.05 MPa;
the formula of the culture medium in the seeding tank is as follows: 0.7% of corn steep liquor dry powder, 0.1% of sodium citrate, 0.4% of potassium nitrate, 0.2% of monopotassium phosphate, 0.1% of disodium hydrogen phosphate and 2ml/L of trace elements, wherein the pH value is 7.0-7.4, and the sterilization is carried out for 20min at 121 ℃.
(4) Fermentation in a fermentation tank: inoculating the obtained seed solution into a culture medium of a 300L fermentation tank with the liquid loading amount of 60-70% according to the inoculation amount of 5-10% v/v for culture, wherein the culture medium and the culture conditions are the same as those of seed tank fermentation; the number of the thalli after the fermentation is finished reaches 2 x 1010cfu/mL, and taking the culture solution out of the tank after fermentation is finished to obtain a liquid microbial inoculum;
after the liquid microbial inoculum is prepared, the microbial inoculum can be obtained on the basis of the liquid microbial inoculum, and the preparation method comprises the following steps:
(5) preparing a solid microbial inoculum: after fermentation, adding glycerol into the fermented bacteria liquid according to the ratio of 1 per thousand-1% v/v, adding 2-4% of diatomite and 1-3% of corn starch according to the mass ratio to adsorb the fermentation liquid, centrifuging and separating the fermentation liquid to obtain solid bacteria, drying to obtain bacteria powder, controlling the water content of the bacteria powder to be below 10%, and detecting that the effective viable count is 7 x 1010cfu/g。
Example 3: research on Denitrification
1. Effect of different carbon sources on denitrification of denitrifying Zoebiella denitrificans
The culture medium for verification is KNO3 3.16g(NO3 -N is about 300mg/L), KH2PO41g, carbon source (setting carbon-nitrogen ratio to be 8:1), 2ml of trace elements, 1000ml of distilled water, pH7.0, 1ml of 0.1% bromothymol blue indicator and 20nin for sterilization at 121 ℃, wherein glycerol, sodium succinate, super carbon, alcohol, glucose and sodium citrate are respectively used as unique carbon sources for denitrification effect verification, and the results of static culture at 30 ℃ for 24 hours show that the treatment effect is poor when the ethanol and the glucose are used as the unique carbon sources by using the tobermella cultured by using a denitrification liquid medium, the total nitrogen removal rate is 0, the treatment effect is general when the glycerol and the super carbon are used as the unique carbon sources, the total nitrogen removal rate is only about 50%, nitrous nitrogen accumulation occurs, and when the sodium citrate and the sodium succinate are used as the carbon sources, the total nitrogen removal rate is 0The removal rate can reach 100%, no nitrite nitrogen is generated, and reference can be made to the attached figure 2 specifically; the results show that in the denitrification process of the water body, the carbon sources capable of realizing high-efficiency denitrification within 24 hours are sodium citrate and sodium succinate.
2. Effect of different carbon-nitrogen ratios on denitrification of Zoerella
To verify the culture medium KNO3 2.16g(NO3 -N is about 300mg/L), KH2PO41g, sodium citrate (carbon source), 2ml of trace elements, 1000ml of distilled water, pH7.0, 1ml of 0.1% bromothymol blue indicator and 20nin of sterilization at 121 ℃, wherein the content of sodium citrate is adjusted to ensure that the C/N ratio is 2, 4, 6, 8, 10, 12 and 14, the Zolbert buergerii cultured in a denitrification liquid culture medium is inoculated into the culture medium according to the inoculation amount of 1%, standing and culturing is carried out for 24 hours at 30 ℃, sampling is carried out, centrifugation is carried out at 8000rpm for 5min, and supernatant is taken to measure the content of total nitrogen in the culture solution before and after culture, and the result shows that along with the increase of C/N, the denitrification effect of denitrifying bacteria is gradually enhanced, when the C/N is more than 6, the removal rate of the total nitrogen can basically reach 100%, and particularly, the attached figure 3 can be referred to
3. Effect of nitrite nitrogen in different concentrations on denitrification of Zoerella
To verify the culture medium NaNO2 0.99g(NO2 --N200 mg/L), sodium citrate 1.62g, KH2PO41g, 2ml of trace elements, 1000ml of distilled water, pH7.0, sterilization at 121 ℃ for 20nin, wherein NaNO is adjusted2And sodium citrate in such an amount that NO is present2 -the-N is 200, 400 and 600mg/L respectively, the C/N is 6, the Zubeiella cultured by the denitrification liquid culture medium is inoculated in the culture medium according to the inoculation amount of 1 percent, standing culture is carried out for 48h at the temperature of 30 ℃, sampling is carried out, centrifugation is carried out for 5min at 8000rpm, the supernatant is taken to measure the content of total nitrogen in the culture solution before and after culture, and the result shows that the degradation time is increased when the total nitrogen removal rate reaches 100 percent along with the increase of the concentration of nitrite nitrogen, NO is increased, and2 -the total nitrogen removal rate of 24h can reach 100 percent when-N is 200mg/L, and NO2 -The total nitrogen removal rate of 48h at 400mg/L of N can reach 100 percent, and the nitrogen-removing agent can tolerate the concentration of 600mg/L, but the degradation rate of the nitrogen-removing agent is influenced (seeFIG. 4), concentrations above 600mg/L are subject to the toxic effects of nitrous nitrogen, with a significant reduction in the rate of total nitrogen degradation.
4. Effect of nitrate nitrogen of different concentrations on denitrification of Bessella zerumbet
The denitrification effect of nitrate nitrogen of different concentrations on the debaryomyces terniformis: to verify the culture medium KNO3 1.443g(NO3 --N200 mg/L), sodium citrate 1.62g, KH2PO41g, 2ml of trace elements, 1000ml of distilled water, pH7.0, sterilization at 121 ℃ for 20nin, wherein KNO is adjusted3And sodium citrate in such an amount that NO is present3 -the-N is 200, 400, 600, 800, 1200, 1500 and 1800mg/L respectively, the C/N is 6, the Zubeiella cultured by the denitrification liquid culture medium is inoculated in the culture medium according to the inoculation amount of 1 percent, standing culture is carried out at 30 ℃, each sample is taken for 24 hours, centrifugation is carried out at 8000rpm for 5 minutes, the supernatant is taken to measure the content of total nitrogen in the culture solution before and after culture, and the result shows that the degradation time is increased when the total nitrogen removal rate reaches 100 percent and the NO is increased along with the increase of the concentration of nitrate nitrogen, so that the NO is subjected to the degradation reaction3 -The total nitrogen removal rate can reach 100 percent in 24 hours under the condition of-N800 mg/L, and NO3 -The total nitrogen removal rate of 48h at-N1200 mg/L can reach 100 percent, and NO3 -The total nitrogen degradation rate at 1500mg/L is obviously reduced, but the degradation rate is still in a trend of continuing to decrease, which shows that the nitrate nitrogen concentration at 1500mg/L can be tolerated, but the degradation rate is influenced, and particularly, refer to the attached figure 5.
5. Effect of Low temperature on Denitrification of denitrifying bacteria
Culture medium KNO3 4.32g(NO3 -N is about 600mg/L), KH2PO41g, sodium citrate 6.55g, trace elements 2ml, distilled water 1000ml, pH7.0, 121 ℃ sterilization 20 nin. The method comprises the steps of inoculating 1% of the Zoebiella in a denitrification liquid culture medium into the culture medium, standing and culturing for 48 hours at 15 ℃, sampling, centrifuging for 5 minutes at 8000rpm, and taking supernatant to measure the content of total nitrogen in the culture solution before and after culture, wherein the results show that the Zoebiella can still carry out denitrification at the low temperature of 15 ℃, and the denitrification rate is limited to a certain extent (figure 6).
6. Research on denitrification effect of denitrifying bacteria under aerobic/anoxic conditions
Culture medium KNO3 4.32g(NO3 -N is about 600mg/L), KH2PO41g, sodium citrate 6.55g, trace elements 2ml, distilled water 1000ml, pH7.0, 121 ℃ sterilization 20 nin. The method comprises the steps of inoculating 1% of the Zolbertymella in a denitrification liquid culture medium into the culture medium, respectively culturing at 30 ℃ and 170r/min for 24h (aerobic) and standing at 30 ℃ for 24h (anoxic), sampling, centrifuging at 8000rpm for 5min, and taking supernatant to measure the total nitrogen content in the culture solution before and after culture, wherein the result shows that the denitrification efficiency of denitrifying bacteria under aerobic conditions is slightly lower than that under anoxic conditions, the 16h removal rate is 86.88%, mainly a small amount of nitrite nitrogen is accumulated under aerobic conditions, while no nitrite nitrogen is accumulated under anoxic conditions, and the 16h removal rate is 100% (fig. 7 and 8).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shandong Lvbang Biotechnology Ltd
<120> denitrifying bacterium for efficient denitrification and application thereof
<130> MP21012450
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1478
<212> DNA
<213> Zobellella denitrificans LBSW21-01
<400> 1
ggctcagatt gaacgctggc ggcaggccta acacatgcaa gtcgagcggt aacagagggt 60
agcttgctac tttgctgacg agcggcggac gggtgagtaa ggcttgggta tctgcccagt 120
cgagggggat aaccgttgga aacgacggct aataccgcat acgccctacg ggggaaagca 180
ggggaccttc gggccttgcg cgattggatg agcccaagtg agattagcta gttggtaggg 240
taatggccta ccaaggcgac gatctctagc tggtctgaga ggatgaccag ccacactggg 300
actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatggggg 360
aaaccctgat gcagccatgc cgcgtgtgtg aagaaggcct tcgggttgta aagcactttc 420
agtggggagg aaaggtgagt ggcgaatacc cacttactgt gacgttaccc acagaagaag 480
caccggctaa ctccgtgcca gcagccgcgg taatacggag ggtgcaagcg ttaatcggaa 540
tgactgggcg taaagcgcac gcaggcggtc tgttaagcca gatgtgaaag ccccgggctc 600
aacctgggaa ctgcatttgg aactggcagg ctagagtctt ggagaggggg gtagaatttc 660
cggtgtagcg gtgaaatgcg tagagatcgg aaggaatacc agtggcgaag gcggccccct 720
ggccaaagac tgacgctcag gtgcgaaagc gtggggagca aacaggatta gataccctgg 780
tagtccacgc tgtaaacgat gtcaacttgg agtctgtgcc atttgagcgc gggttccgga 840
gctaacgcgt taagttgacc gcctggggag tacggccgca aggttaaaac tcaaatgaat 900
tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgatgcaac gcgaagaacc 960
ttacctaccc ttgacatagt aagaacttgc cagagatggc tgggtgcctt cgggaactta 1020
catacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1080
aacgagcgca acccttgtcc tttgttgcca gcgattcggt cgggaactca aaggagactg 1140
ccggtgataa accggaggaa ggtggggacg acgtcaagtc atcatggccc ttacgggtag 1200
ggctacacac gtgctacaat ggcgcgtaca gagggcggcg aaccagcgat ggtaagcgaa 1260
tcccaaaaag cgcgtcgtag tccggatcgg agtctgcaac tcgactccgt gaagtcggaa 1320
tcgctagtaa tcgcaaatca gaatgttgcg gtgaatacgt tcccgggcct tgtacacacc 1380
gcccgtcaca ccatgggagt gggttgctcc agaagtagat agcttaacct tcgggagggc 1440
gtttaccacg gagtgattca tgactggggt gaagtcgt 1478
Claims (9)
1. The denitrifying bacterium with high denitrification efficiency is characterized by being the bacterium (Zubel) bacteriumZobellella denitrificans) LBSW21-01 with preservation number of CGMCC No. 22730.
2. The application of the Zubelia nobilis LBSW21-01 with the preservation number of CGMCC No.22730 in the denitrification treatment of water and the preparation of microbial agents for denitrification of water.
3. The microbial agent for denitrification of the sewage is characterized by comprising Znobel bacterium LBSW21-01 with the preservation number of CGMCC No. 22730.
4. The microbial agent according to claim 3, which is a liquid microbial agent or a solid microbial agent, wherein the number of the liquid microbial agent is 2 x 109-4×1010cfu/mL, the number of the solid microbial inoculum is 3 multiplied by 1010-2×1011cfu/g。
5. A preparation method of a microbial agent for water denitrification is characterized in that a Zubelia nobilis LBSW21-01 with the preservation number of CGMCC No.22730 is inoculated into a denitrification liquid culture medium for activation, then the denitrification liquid culture medium is replaced to prepare a seed solution, and the seed solution is inoculated into a fermentation culture medium for fermentation to obtain the liquid microbial agent.
6. The preparation method according to claim 5, further comprising adding glycerol, diatomaceous earth and starch to the prepared liquid microbial agent to prepare a solid microbial agent.
7. The method according to claim 5, wherein the fermentation medium comprises corn steep liquor dry powder, sodium citrate, potassium nitrate, potassium dihydrogen phosphate, disodium hydrogen phosphate and trace elements.
8. A water body denitrification treatment method is characterized in that Zubel bacterium LBSW21-01 with the preservation number of CGMCC No.22730 or a microbial agent prepared from the Zubel bacterium LBSW21-01 are put into pretreated water body for domestication, and then denitrification treatment is carried out under the aerobic or anoxic condition.
9. The treatment method according to claim 8, wherein the carbon source is supplemented with sodium citrate and/or sodium succinate as a carbon source during the denitrification treatment while maintaining the C/N ratio at not less than 4.
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