CN113249270A - Salt-resistant high-self-aggregation-capability bacillus amyloliquefaciens and application thereof in denitrification - Google Patents
Salt-resistant high-self-aggregation-capability bacillus amyloliquefaciens and application thereof in denitrification Download PDFInfo
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
The invention discloses a salt-tolerant high-self-aggregation-capability bacillus amyloliquefaciens and application thereof in denitrification. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8, which is preserved in Guangdong province collection center for microorganism strains at 6.1.2021, with the preservation numbers: GDMCC NO. 61606. The bacillus amyloliquefaciens N8 can be applied to the denitrification treatment of nitrogen-containing wastewater with certain salinity. The bacillus amyloliquefaciens N8 has the nitrification capacity of efficiently removing ammonia nitrogen and the denitrification capacity of removing nitrite and nitrate nitrogen; has certain salt tolerance and can effectively exert the denitrification capability under the condition of containing salt. The bacteria with salt-tolerant nitrification and denitrification capabilities can better solve the problem of removing ammonia nitrogen, nitrite and nitrate nitrogen in wastewater under the salt-containing condition.
Description
Technical Field
The invention belongs to the field of environmental microorganisms, and particularly relates to a salt-tolerant high-self-aggregation-capability bacillus amyloliquefaciens and application thereof in denitrification.
Background
At present, with the development of the aquaculture industry to the modernization level, a Recirculating Aquaculture System (RAS) gradually becomes a supporting technical force for the modernization of the aquaculture industry in china due to the characteristics of small occupied area, water resource saving, environmental protection and the like. Good water quality is the technical core and key advantages of RAS, with ammonia nitrogen concentration being the most critical factor affecting water quality. In addition, the harm caused by nitrite nitrogen accumulation is also serious in the culture process. Also, the lethal or near-lethal effects of high concentrations of nitrate on marine and freshwater organisms have been reported in many documents. Therefore, maintaining low concentrations of ammonia nitrogen, nitrite and nitrate is an important goal in achieving sustainable development in aquaculture industry. Biological denitrification is the most common, most effective and least-cost method for improving water quality, and is also the most popular tri-state nitrogen control method in RAS at present.
The conventional biological denitrification method requires separation of aerobic and anoxic phases, resulting in low denitrification efficiency. In contrast, heterotrophic nitrification-aerobic denitrification bacteria are of interest due to their efficient denitrification properties.
Disclosure of Invention
The invention aims to provide a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8 strain, which has good aggregation capability in the presence of tri-state nitrogen at a certain salinity and can play a role in denitrification.
The purpose of the invention is realized by the following technical scheme:
bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8, which is preserved in Guangdong province microbial culture collection center (GDMCC) at 1 st 6 th 2021 and is addressed to Guangzhou city, Michelia Tokyo 100, Dazhou 59 th, 5 th, Guangdong province microbial research institute, and the preservation numbers are as follows: GDMCC NO. 61606.
The strain N8 is obtained by enriching, separating and screening the grouper culture water body. The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is identified by combining the 16S rDNA sequence, the colony morphology and the physiological and biochemical characteristics.
A microbial denitrification agent is the above Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8, and may further comprise other microorganisms.
The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8 and the preparation thereof can be applied to denitrification treatment of nitrogen-containing wastewater with certain salinity;
the nitrogen-containing means containing ammonia Nitrogen (NH)4 +-N), nitrate Nitrogen (NO)3 --N) or nitrous Nitrogen (NO)2 --N);
the nitrogen-containing wastewater particularly refers to nitrogen-containing aquaculture water, industrial wastewater and domestic sewage;
in the nitrogen-containing wastewater, NH4Initial concentration of Cl 0.193g/L, NaNO2Has an initial concentration of 0.246g/L, KNO3The initial concentration of (3) is 0.361 g/L;
the salinity refers to NaCl concentration less than or equal to 30 per thousand (W/V), preferably less than or equal to 20 per thousand, and particularly preferably less than or equal to 10 per thousand;
preferably, 1% of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8 seed liquid is added into the nitrogen-containing wastewater at normal temperature (20-30 ℃), and the denitrification effect can be achieved after sufficient diffusion and culture;
the seed liquid is obtained by inoculating N8 to LB culture medium and culturing to OD600When the number is 1, the seed solution is used.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention screens out a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8 which has the nitrification capability of efficiently removing ammonia nitrogen and the denitrification capability of efficiently removing nitrite and nitrate nitrogen; it is further found that the bacteria have certain salt tolerance and can effectively exert the denitrification capability in the salt-containing condition. By utilizing the physiological and biochemical characteristics and the metabolic mechanism of the bacteria with salt-tolerant nitrification and denitrification capabilities, the problem of removing ammonia nitrogen, nitrite and nitrate nitrogen in wastewater under the salt-containing condition can be better solved. Meanwhile, the strain can have higher self-aggregation capability in the presence of the ternary nitrogen, and the problem of field planting of the strain in the water environment is solved under certain conditions.
2. The invention provides Bacillus amyloliquefaciensN8 for NH when the inoculum size is 1% (v/v)4 +-N、NO2 --N and NO3 -And N has higher removal rate, which indicates that the Bacillus amyloliquefaciens N8 has high efficient nitrification and denitrification performance. In addition, the salt tolerance measurement result judges that the Bacillus amyloliquefaciens N8 has certain salt tolerance, but the growth speed is slow due to overhigh salinity. Under the condition of 0-30 per mill of salinity, the Bacillus amyloliquefaciens N8 has better growth and higher denitrification performance. Meanwhile, Bacillus amyloliquefaciens N8 can have higher self-aggregation capability in the presence of tri-state nitrogen. Therefore, the strain Bacillus amyloliquefaciens N8 has good practical value and application prospect in treating high-concentration ammonia nitrogen, nitrite and nitrate wastewater.
Drawings
FIG. 1 is a colony morphology map of strain N8.
FIG. 2 is an electropherogram of PCR amplification products.
FIG. 3 is the OD of Bacillus amyloliquefaciens N8 in different salinity media600The value is obtained.
FIG. 4 is a graph showing denitrification effects of Bacillus amyloliquefaciens N8 at different salinity.
FIG. 5 shows the heterotrophic nitrification activity of Bacillus amyloliquefaciens N8 using ammonia nitrogen as a nitrogen source.
FIG. 6 shows the aerobic denitrification capacity of Bacillus amyloliquefaciens N8 using nitrate nitrogen as a nitrogen source.
FIG. 7 shows the aerobic denitrification capability of Bacillus amyloliquefaciens N8 using nitrite nitrogen as nitrogen source
FIG. 8 is the self-aggregation ability of Bacillus amyloliquefaciens N8 under nitrogen tri-state.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The strain N8 is screened according to the following steps:
(1) taking the high-density grouper culture water body to be stored in a 50ml sterile centrifuge tube at 4 ℃ for later use. Taking 1mL of water samplePutting the product into a 250mL conical flask, and adding 100mL of nitrification enriched culture medium; shaking thoroughly, 30 deg.C, 180 r.min－1Culturing under the condition, and after 2d, taking 1mL to inoculate a fresh nitrification enrichment medium again, and continuously enriching and culturing for 5 times.
(2) Under aseptic conditions, using a pipette to suck l mL of the liquid enrichment culture solution of the last generation, adding the liquid enrichment culture solution into a 20mL test tube containing 9mL of sterile water, using a pipette to blow and wash for 3 times, and fully mixing to obtain 10－1Repeating the above operations to obtain 10% of bacterial liquid－3、10－4、10－5、10－6And 10－7Bacterial solutions with different dilutions. Respectively sucking 0.1mL of bacterial liquid with different dilutions by using a pipette gun, respectively transferring into corresponding bromothymol blue (BTB) solid culture medium, uniformly coating by using a sterile glass coating rod, standing for 5min to enable the bacterial liquid to be completely adsorbed, and making 3 bacterial liquids with each dilution in parallel. And (3) placing the inoculated solid plate culture medium into an illumination incubator at 30 ℃ for inverted culture for 18-24h, and obtaining single colonies in solid plate culture media with different dilutions.
(3) Observing the conditions of the surface structure, the morphology, the color, the transparency, the edge and the like of the bacterial colony, selecting a target bacterial colony which grows well and forms a blue transparent ring, and separating and purifying by adopting a plate scribing separation method, wherein the specific operation steps are as follows: in an ultraclean workbench, an inoculating loop is burned for several minutes under an alcohol lamp to kill bacteria, after cooling, a single bacterial colony is picked up, the bacterial colony on the inoculating loop is firstly coated on a culture medium on one side of the solid culture medium to be as uniform as possible, meanwhile, the first parallel lineation is carried out for about 3-4 bacterial colonies, then, a flat plate is rotated for about 70 degrees, the residual bacterial colony on the inoculating loop is burned off, after cooling, the second parallel lineation is carried out through the first lineation part, the two parallel lineation are not overlapped, and the third and fourth parallel lineation is carried out by the same method in the same way. And finally, numbering the inoculated solid separation culture media in sequence, and carrying out inverted culture in a lighting incubator at 30 ℃. After culturing for 1-2 days, each group is inoculated. Burning the inoculating ring with outer flame of alcohol lamp to sterilize, contacting the inoculating ring with the inner wall of the test tube, and selecting single and complete colony. Each group was inoculated with 2 groups of flat BTB solid media, and another 2 groups of nitrification enriched media. In the purification process, special attention is paid to that when the bacteria on the inoculating loop are not easy to fall off, the inoculating loop is placed on the surface of a liquid culture medium and continuously rubbed with the inner wall of a test tube until the bacteria fall off into the liquid, then the inoculating loop is marked, placed in an illumination incubator for culture for 1-2 days and then stored in a refrigerator at 4 ℃ for later use. The colonies purified by the plate streaking method are preserved in a-80 refrigerator by a glycerol (25%) seed preservation method, and primary screening is completed for later use.
(4) Respectively culturing the primary screened strain in a nitrification basic culture medium taking ammonia nitrogen as a nitrogen source and a denitrification culture medium taking nitrite as a nitrogen source, and culturing with NH4 +-N and NO2 -Screening by using-N removal rate as an index, and selecting NH4 +-N and NO2 -And separating and purifying the bacterial liquid with higher-N removal rate to obtain a bacterial strain N8 with high-efficiency denitrification function, wherein the colony morphology of the bacterial strain is shown in figure 1.
Wherein the formula of the nitrifying enrichment medium is as follows: (NH)4)2SO4 0.5g/L，KH2PO4 1.0g/L，FeCl2·6H2O 0.5g/L，CaCl2·7H2O 0.2g/L，MgSO4·7H2O 1.0g/L，Na3C6H5O7·2H2O4.08 g/L, pH value of 7.0-7.5, and temperature of 121 ℃ and sterilization for 30 min;
BTB solid medium formula: KNO31.0g/L, L-asparagine 1.0g/L, 5mL of bromothymol blue (BTB) diluted with ethanol at 0.1%, Na3C6H5O7·2H2O 8.5g/L，KH2PO4 1.0g/L，MgSO4·7H2O 1.0g/L，CaCl2·6H2O 0.2g/L，FeCl2·6H20.05g/L of O, 20g/L of agar, 7.0-7.5 of pH value and 121 ℃ of high pressure for 30 min.
Wherein the formula of the nitrification basic culture medium taking ammonia nitrogen as a nitrogen source comprises the following components: NH (NH)4Cl 0.193g/L，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
Wherein, the formulation of the denitrification culture medium taking nitrite as nitrogen source is NaNO2 0.246g/L，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
The formula of the trace element solution is as follows: k2HPO4 5g/L，MgSO4·7H2O 2.5g/L，NaCl 2.5g/L，MnSO4·4H2O 0.05g/L，FeSO4·7H2O 0.05g/L。
Example 2
Characterization and characterization of Strain N8
The bacterial colony of the strain N8 is small, moist, smooth, transparent, easy to pick, neat in edge, smooth in surface and glossy. The rod shape of the bacteria is observed under a microscope, and the bacteria are gram negative bacteria. The relevant physiological and biochemical characteristics are shown in the table 1, and the V-P experiment, the gelatin liquefaction experiment and the starch hydrolysis experiment are positive; the nitrate reduction experiment is negative, citrate, propionate, L-arabinose and D-mannitol can be used as carbon sources, and D-xylose cannot be used. The growth in 7% NaCl was not possible and was possible at pH 5.7 under anaerobic conditions.
TABLE 1 physiological and biochemical characteristics of Strain N8
+: growth or positive; -: non-growth or negative
16S rDNA gene sequencing and multiplex PCR identification:
PCR amplification of strain N8 genomic DNA: taking the total DNA of the strain N8 as a template, and taking the amplification primers as a pair of universal primers;
the forward primer F is: 5'-ACTCCTACGGGAGGCAGCAG-3' (SEQ. ID. NO. 1);
the reverse primer R is as follows: 5 '-GGACTACHVGGGTWTCTAAT-3' (seq. id. No. 2).
The PCR reaction system is as follows: the PCR reaction was carried out in a 25. mu.L system. The reaction system comprises the following components: mix12uL + DNA template 2uL + primer F1uL + primer R1uL + sterile ultrapure water 9 uL.
The PCR amplification procedure was: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 60s, annealing at 55 ℃ for 60s, extension at 72 ℃ for 90s, 30 cycles in total, repair extension at 72 ℃ for 10min, termination of the reaction at 4 ℃ and sequencing of the obtained fragments.
mu.L of PCR amplification product was detected by 1% agarose gel electrophoresis. The electrophoretogram of the PCR amplification product is shown in FIG. 2.
After validation, the gel strips were excised and the PCR product was purified using a DNA gel recovery kit (Vazyme). And then sending the purified PCR product to a company for sequencing, wherein the sequencing work is finished by Shenzhen Huada gene company. The 16S rDNA gene sequence of the strain N8 is shown in SEQ ID No. 3.
Sequencing results were aligned in GenBank with 100% homology to the 16s rdna sequence of various populations of Bacillus species.
According to the manual of identifying common bacteria systems and the manual of identifying Berger bacteria, the strain N8 is identified to be Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by combining physiological and biochemical characteristics, 16S rDNA gene sequencing and multiple PCR identification results, and the named strain N8 is: bacillus amyloliquefaciens N8 with the preservation date of 2021, 6 months and 1 days is preserved in Guangdong province microorganism culture collection center (GDMCC) and the address of Guangzhou city Mielizhou 100 college No. 59 building 5 building, Guangdong province microorganism research institute, the preservation number is: GDMCC NO. 61606.
Example 3
Salt tolerance test of Bacillus amyloliquefaciens N8
Scraping 2-ring thallus Porphyrae from Bacillus amyloliquefaciens N8 stored on slant tube culture medium with inoculating loop, inoculating into 250mL Erlenmeyer flask containing 100mL sterilized LB culture medium, and culturing at 30 deg.C for 140r min－1Culturing under the condition of OD600When the number is 1, the seed solution is used.
Preparing nitrification basal culture media with different salinity gradients, wherein the NaCl concentration is 0, 10 per thousand, 20 per thousand, 30 per thousand, 40 per thousand and 50 per thousand (W/V) respectively; bacillus amyloliquefaciens N8 seed liquidInoculating 1% (v/v) into a 500mL conical flask containing 300mL of a nitrified basal medium; shaking thoroughly, 30 deg.C, 140 r.min－1Culturing for 48h under the condition of strain growth (OD)600) As the basis for judging whether the strain is resistant to corresponding salinity; media without inoculated bacteria was used as a blank, 3 replicates per group.
FIG. 3 shows the growth of Bacillus amyloliquefaciens N8 in nitrified minimal medium with different salinity gradients. After 48 hours of culture, the bacterial liquid concentration of each salinity culture medium is increased, and the growth of the strain has no significant difference (P) in the culture medium containing 0-40% of NaCl>0.05). But at a salinity of 50 per mill, the growth of the strain is obviously inhibited, and OD600Is only 0.065, significantly lower than the other groups (P)<0.05). OD at salinity of 0-30 ‰600All are more than 0.25, which proves that Bacillus amyloliquefaciens N8 has certain salt tolerance, but the growth speed is slow and even the growth is inhibited due to the excessively high salinity.
Wherein the formula of the nitrification basic culture medium is NH4Cl 0.193g/L(NH4 +-N 50mg/L)，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
The formula of the trace element solution is as follows: k2HPO4 5g/L，MgSO4·7H2O2.5 g/L, NaCl added according to the experimental requirements, MnSO4·4H2O 0.05g/L，FeSO4·7H2O 0.05g/L。
Example 4
Heterotrophic nitrification performance test of Bacillus amyloliquefaciens N8 under different salinity
Preparing nitrification basal culture media with different salinity gradients, wherein the NaCl concentration is 0, 10 per thousand, 20 per thousand, 30 per thousand, 40 per thousand and 50 per thousand respectively; the seed solution was inoculated in an amount of 1% (the same as in example 3) to sterilized medium of various salinity at 30 ℃ for 140 r.min－1Culturing for 48h under the condition; determination of NH in the Medium4 +The variation of N concentration, the nitrification performance of the strain is tested. Using culture medium without inoculated bacteria as airWhite controls, 3 replicates per group.
As shown in FIG. 4, after 48h of cultivation, Bacillus amyloliquefaciens N8 has certain NH under various salinity conditions4 +N-removing ability, but too high salinity inhibits the growth of the strain, thereby reducing its NH pair4 +-N removal capacity. Under the condition of NaCl salinity of 0-20 per mill, Bacillus amyloliquefaciens N8 has high-efficiency NH4 +The N removal capacity and the removal rate are all over 70 percent. Wherein, the removal rates of 10 per mill and 20 per mill are 74 percent and 70.39 percent respectively.
The salinity is 30 per mill, and the removal rate is 63.54 percent. Under the condition of salinity of 40-50 per mill, the excessive salinity inhibits the growth of the bacterial strain, which causes Bacillus amyloliquefaciens N8 to react with NH4 +A significant reduction in N removal capacity (P)<0.05)。
Wherein the formula of the nitrification basic culture medium is NH4Cl 0.193g/L(NH4 +-N 50mg/L)，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
The formula of the trace element solution is as follows: k2HPO4 5g/L，MgSO4·7H2O2.5 g/L, NaCl added according to the experimental requirements, MnSO4·4H2O 0.05g/L，FeSO4·7H2O 0.05g/L。
Example 5
Heterotrophic nitrification performance experiment of Bacillus amyloliquefaciens N8
Inoculating Bacillus amyloliquefaciens N8 in LB culture medium at 30 deg.C for 140r min－1Culturing under the condition of OD600When 1; inoculating the mixture into a 500mL conical flask containing 300mL of sterilized nitrification basal medium at the inoculation amount of 1%, and performing inoculation at the temperature of 30 ℃ for 140 r.min－1Culturing under the condition; taking a certain amount of culture solution every 12h to determine OD value, centrifuging, and determining NH in the culture solution4 +N, detecting the nitrification performance of the strain. Media without inoculated bacteria was used as a blank, 3 replicates per group.
As a result, as shown in FIG. 5, in culture 36After h, the NH in the basal medium is nitrified4 +-a rapid decrease in N concentration; after 72h of cultivation, Bacillus amyloliquefaciens N8 was incubated for NH4 +The removal rate of-N was 95.44%, NH in the culture broth4 +the-N concentration was reduced from the initial 48.45mg/L to 2.21 mg/L.
Wherein the LB culture medium is tryptone 10g/L, yeast extract 5g/L, NaCl 10 g/L.
Wherein the formula of the nitrification basic culture medium is NH4Cl 0.193g/L(NH4 +-N 50mg/L)，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
The formula of the trace element solution is as follows: k2HPO4 5g/L，MgSO4·7H2O 2.5g/L，NaCl 2.5g/L，MnSO4·4H2O 0.05g/L，FeSO4·7H2O 0.05g/L。
Example 6
Aerobic denitrification performance test of Bacillus amyloliquefaciens N8 by taking nitrate as nitrogen source
Inoculating Bacillus amyloliquefaciens N8 into LB culture medium for culture at 30 deg.C and 140r min－1Culturing under the condition of OD600When 1; inoculating the mixture into a 500mL conical flask containing 300mL of sterilized denitrification culture medium at the inoculation amount of 1%, and performing inoculation at the temperature of 30 ℃ for 140 r.min－1Culturing under the condition; taking a certain amount of culture solution every 12h to measure OD value, centrifuging and measuring NO in the culture solution3 -and-N, detecting the aerobic denitrification performance of the strain. Media without inoculated bacteria was used as a blank, 3 replicates per group.
As shown in FIG. 6, NO in the denitrifying medium after 72 hours of culture3 -N concentration decreased from the initial 51.89mg/L to 14.75mg/L, Bacillus amyloliquefaciens N8 to NO3 -The removal rate of-N was 71.55%.
Wherein the formula of the denitrification culture medium taking nitrate as a nitrogen source is KNO3 0.361g/L(NO3 --N50mg/L)，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
The formula of the trace element solution is as follows: k2HPO4 5g/L，MgSO4·7H2O 2.5g/L，NaCl 2.5g/L，MnSO4·4H2O 0.05g/L，FeSO4·7H2O 0.05g/L。
Example 7
Aerobic denitrification performance test of Bacillus amyloliquefaciens N8 by taking nitrite as nitrogen source
Inoculating Bacillus amyloliquefaciens N8 into LB culture medium for culture at 30 deg.C and 140r min－1Culturing under the condition of OD600When 1; inoculating the mixture into a 500mL conical flask containing 300mL of sterilized denitrification culture medium at the inoculation amount of 1%, and performing inoculation at the temperature of 30 ℃ for 140 r.min－1Culturing under the condition; taking a certain amount of culture solution every 12h to measure OD value, centrifuging and measuring NO in the culture solution2 -and-N, detecting the aerobic denitrification performance of the strain. Media without inoculated bacteria was used as a blank, 3 replicates per group.
As shown in FIG. 7, NO in the denitrifying medium after 72 hours of culture2 -N concentration decreased from the initial 53.93mg/L to 0.62mg/L, Bacillus amyloliquefaciens N8 to NO2 -The removal rate of-N was 98.85%.
Wherein the formulation of the denitrification culture medium is NaNO2 0.246g/L(NO2 --N 50mg/L)，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
The formula of the trace element solution is as follows: k2HPO4 5g/L，MgSO4·7H2O 2.5g/L，NaCl 2.5g/L，MnSO4·4H2O 0.05g/L，FeSO4·7H2O 0.05g/L。
Example 8
Self-aggregation capability of Bacillus amyloliquefaciens N8 in presence of tri-state nitrogen
BaciThe strain of llus amyloliquefaciens N8 was inoculated in LB medium and cultured at 30 ℃ for 140 r.min－1Culturing under the condition of OD600When 1; inoculating the mixture into a 500mL conical flask containing 300mL of sterilized nitrification and denitrification culture medium at the inoculation amount of 1%, and performing inoculation at the temperature of 30 ℃ for 140r min－1Culturing under the condition. Subpackaging the bacteria liquid cultured at different times into a plurality of sterile 50mL centrifuge tubes, centrifuging at 5000rpm for 10min to collect thalli, washing twice with PBS buffer solution, resuspending, respectively preparing into bacterial suspension, respectively subpackaging 6mL into 10mL sterile centrifuge tubes, oscillating on a vortex oscillator for 2min, standing at room temperature for 150min, taking 4mL of supernatant, determining absorbance at 600nm, and oscillating for 2min to obtain suspension OD600As an initial value, the self-aggregating ability of the strain N8 was characterized by changes in absorbance values before and after standing.
The calculation formula is as follows: self-aggregation ability (%) (1-supernatant OD after shaking and standing)600Initial bacterial suspension OD600)×100％。
As shown in FIG. 8, Bacillus amyloliquefaciens N8 showed self-aggregation ability in the nitrification basic medium, the denitrification medium with nitrite as nitrogen source and the denitrification medium with nitrate as nitrogen source. In the presence of NH4 +-N、NO2 --N and NO3 -And when N is a nitrogen source, the self-aggregation capability is better after 9 hours of culture. The Bacillus amyloliquefaciens N8 can keep good self-aggregation capability in both the heterotrophic nitrification process and the aerobic denitrification process, which means that the strains can be better planted in the water environment.
Wherein, the formula of the nitrification culture medium is as follows: NH (NH)4Cl 0.193g/L，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
Wherein, the formulation of the denitrification culture medium taking nitrite as nitrogen source is NaNO2 0.246g/L，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
Wherein,the formula of the denitrification culture medium taking nitrate as a nitrogen source is KNO3 0.361g/L，C4H4Na2O44.08g/L, 50mL of trace element solution, constant volume to 1L, and pH value to 7.5.
The formula of the trace element solution is as follows: k2HPO4 5g/L，MgSO4·7H2O 2.5g/L，NaCl 2.5g/L，MnSO4·4H2O 0.05g/L，FeSO4·7H2O 0.05g/L。
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of south China
<120> one strain of salt-resistant bacillus amyloliquefaciens with high self-aggregation capability and application thereof in denitrification
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Forward primer F
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> reverse primer R
<400> 2
<210> 3
<211> 456
<212> DNA
<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
<220>
<223> 16S rDNA
<400> 3
tactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc tgacggagca 60
acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg gaagaacaag 120
tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg gctaactacg 180
tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag 240
ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca 300
ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa 360
atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct gtaactgacg 420
ctgaggagcg aaagcgtggg gagcgaacag gattag 456
Claims (9)
1. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8, which is preserved in Guangdong province collection center for microorganism strains at 6.1.2021, with the preservation numbers: GDMCC NO. 61606.
2. A microbial denitrification preparation characterized by being the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8 according to claim 1.
3. The microbial denitrification formulation of claim 2, wherein: other microorganisms are also included.
4. Use of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8 according to claim 1 and the preparation according to claim 2 or 3 for denitrification of nitrogen-containing wastewater with a certain salinity.
5. Use according to claim 4, characterized in that: the salinity refers to that the NaCl concentration is less than or equal to 30 per mill (W/V).
6. Use according to claim 4, characterized in that: the nitrogen-containing means containing ammonia Nitrogen (NH)4 +-N), nitrate Nitrogen (NO)3 --N) or nitrous Nitrogen (NO)2 --N) is selected.
7. Use according to claim 4, characterized in that: in the nitrogen-containing wastewater, NH4Initial concentration of Cl 0.193g/L, NaNO2Has an initial concentration of 0.246g/L, KNO3The initial concentration of (3) was 0.361 g/L.
8. Use according to claim 4, characterized in that: under normal temperature (20-30 ℃), 1% of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) N8 seed liquid is added into nitrogen-containing wastewater, and the nitrogen removal effect can be achieved after full diffusion and culture.
9. Use according to claim 8, characterized in that: the seed liquid is obtained by inoculating N8 into LB culture medium and culturing to DO600When the number is 1, the seed solution is used.
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