CN117821330B - Marine heterotrophic nitrification-aerobic denitrification bacterium with biological denitrification characteristic and biological denitrification application thereof - Google Patents
Marine heterotrophic nitrification-aerobic denitrification bacterium with biological denitrification characteristic and biological denitrification application thereof Download PDFInfo
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- 239000001963 growth medium Substances 0.000 claims description 52
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Abstract
The invention relates to the technical field of microorganisms, and in particular provides a marine heterotrophic nitrification-aerobic denitrification bacterium with biological denitrification characteristics and biological denitrification application thereof. The strain is named as Pseudomonas putida (Pseudomonas putida) 4-HYK, and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.28841. The strain is separated, screened and identified from the tidal channel of the sea cucumber culture area at the mouth of the Dalian garden, and can realize high-efficiency biological denitrification in simulated wastewater.
Description
Technical Field
A novel marine heterotrophic nitrification-aerobic denitrification bacterium with biological denitrification characteristics and denitrification application thereof relate to the technical fields of microbial fishery water quality regulation and control, aquaculture tail water and wastewater treatment.
Background
The water environment pollution problem becomes a problem to be solved urgently, and particularly the water body with the nitrogen content exceeding the standard is provided. On the one hand, the exceeding of the nitrogen content can directly destroy the living environment of aquatic animals and plants, such as water eutrophication and the like; on the other hand, the exceeding of the nitrogen content also indirectly affects the health of human bodies, for example, methemoglobin and the like can occur when water with the nitrogen nitrate content exceeding 10mg/L is drunk for a long time. Therefore, in modern water pollution control systems, how to safely and efficiently treat the nitrogen content to reach the standard has become a key problem.
Among the numerous wastewater treatment technologies, there are roughly three types, physical, chemical and biological. Especially, the biological treatment method is more focused, and has the characteristics of low cost, no secondary pollution, strong sewage treatment capability, simple and stable operation and the like. However, the two stages of aerobic and anaerobic are required to be independently carried out in the traditional biological denitrification process, so that the denitrification process occupies a larger area and has higher investment cost. And heterotrophic nitrification-aerobic denitrification bacteria can realize synchronous nitrification and denitrification, so that the operation cost can be reduced. And the product of the previous step of nitrification can be applied to the subsequent denitrification so as to shorten the time of nitrification and denitrification and improve the denitrification efficiency. Therefore, the bacteria have important roles in water purification. However, since bacteria are various and have different forms and functions, the searching and culturing of heterotrophic nitrification-aerobic denitrification bacteria with high-efficiency denitrification capability on wastewater is a key link of biotechnology which is urgently needed to be solved and has application value.
Disclosure of Invention
The invention aims to provide a marine heterotrophic nitrification-aerobic denitrification bacterium with biological denitrification characteristics and application thereof in the aspect of biological denitrification of wastewater, and the strain can realize high-efficiency biological denitrification in actual seawater wastewater.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
The invention provides a marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification property, which belongs to pseudomonas putida, is named as pseudomonas putida (Pseudomonas putida) 4-HYK, is classified and named as pseudomonas putida Pseudomonas putida, is preserved in China general microbiological culture Collection center (CGMCC, address: beijing Kogyo area North Star West Leu 1 No. 3, national institute of microbiology, postal code 100101) and has a preservation number of CGMCC No.28841.
As a preferred embodiment of the present invention, the Pseudomonas putida (Pseudomonas putida)
The biological characteristics of 4-HYK are:
(1) Can grow on liquid culture medium (NH 4)2SO4、KNO3、NaNO2 is the only nitrogen source or mixed nitrogen source).
(2) White colonies with smooth surface, opaque surface, complete and regular edges and oval bulges are displayed.
(3) 16S rDNA Gene sequencing
The 16S rDNA gene sequence of the pseudomonas putida (Pseudomonas putida) 4-HYK is subjected to PCR amplification, sequencing and BLAST comparison, and the 16S rDNA gene sequence is shown as SEQ ID NO. 1, and the result shows that the 16S rDNA gene sequence of the pseudomonas putida (Pseudomonas putida) 4-HYK has higher similarity with the pseudomonas putida (Pseudomonas putida) and high similarity.
In another aspect, the present invention provides a method for screening the marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification characteristics, the screening method comprising the steps of:
1mL of 30 permillage artificial seawater sample is taken to be placed in 20mL of enrichment medium, the culture is carried out for 1 day in a shaking table at 30 ℃ and 180rpm at constant temperature, 5mL of enrichment bacterial liquid is taken to be placed in 100mL of enrichment medium for culture, and the process is repeated for three times; then, taking bacterial liquid for gradient dilution, coating the bacterial liquid on the surface of a bromothymol blue (BTB) solid culture medium, culturing at the constant temperature of 30 ℃ for 1-3 days, then selecting a strain with brightest blue halos for further purification on the BTB culture medium, selecting a single bacterial colony for streaking separation, and repeating the steps for three times to obtain a single bacterial strain; the single strain was stored in a-80℃refrigerator.
As a preferable scheme of the invention, the culture medium is prepared according to the following proportion:
(1) 30 per mill artificial seawater: 30g NaCl was dissolved in 1000mL pure water;
(2) Enrichment medium: KNO 3 0.36g·L-1、(NH4)2SO4 0.24g·L-1, sodium citrate 3.6g·L-1、KH2PO4 0.75g·L-1、K2HPO4 0.75g·L-1、MgSO4·7H2O 0.5g·L-1、FeSO4 0.02g·L-1、MnSO4·H2O 0.01g·L-1、pH 7.0～7.3;
(3) Bromothymol blue solid medium (BTB): KNO 3 1g·L-1, sodium citrate 3.6g.L -1、KH2PO40.75g·L-1、K2HPO4 0.75g·L-1, 1% bromothymol blue ethanol solution (BTB), agar 20 g.L -1, pH 7.0-7.3;
(4) Heterotrophic nitrification liquid medium (SM): (NH 4)2SO4 0.12g·L-1, sodium citrate) 3.6g·L-1、KH2PO40.75g·L-1、K2HPO4 0.75g·L-1、MgSO4·7H2O 0.5g·L-1、FeSO4 0.02g·L-1、MnSO4·H2O 0.01g·L-1,pH 7.0～7.3;
(5) Denitrification liquid medium (DM): naNO 2 0.09g·L-1/KNO3 0.18g·L-1, sodium citrate 3.6g·L-1、KH2PO4 0.75g·L-1、K2HPO4 0.75g·L-1、MgSO4·7H2O 0.5g·L-1、FeSO4 0.02g·L-1、MnSO4·H2O 0.01g·L-1,pH 7.0～7.3;
(6) Synchronous denitrification culture medium (SND)：NaNO2 0.09g·L-1/KNO3 0.18g·L-1,(NH4)2SO40.12g·L-1、 sodium citrate 7.2g·L-1、KH2PO4 0.75g·L-1、K2HPO4 0.75g·L-1、MgSO4·7H2O0.5g·L-1、FeSO4 0.02g·L-1、MnSO4·H2O 0.01g·L-1,pH 7.0～7.3.
In a third aspect of the invention, there is provided the use of a marine heterotrophic nitrification-aerobic denitrification bacterium having biological denitrification properties, the use of a marine heterotrophic nitrification-aerobic denitrification bacterium having biological denitrification properties in denitrification.
As a preferable scheme of the invention, the application of the marine heterotrophic nitrification-aerobic denitrification bacteria with biological denitrification characteristics in biological denitrification of single nitrogen source simulated wastewater or mixed nitrogen source simulated wastewater is as follows:
(1) Inoculating 1.0% of strain inoculum size to heterotrophic nitrification culture medium, wherein a single nitrogen source is ammonia nitrogen (20-25 mg/L), shake culturing at 37 ℃ and 200rpm for 24 hours, and taking water samples every 3 hours to determine ammonia nitrogen, nitrous nitrogen, nitric nitrogen concentration and solution OD 600 absorbance;
(2) Inoculating 1.0% of strain inoculum size to a denitrification culture medium, and shake culturing at 200rpm for 24 hours at 37 ℃ with nitrite nitrogen (20-25 mg/L) as a single nitrogen source, and taking a water sample every 3 hours to measure the concentration of nitrite nitrogen and nitrate nitrogen and the absorbance of a solution OD 600;
(3) Inoculating 1.0% of strain inoculum size to a denitrification culture medium, and shake culturing at 200rpm for 24 hours at 37 ℃ with nitrate nitrogen (20-25 mg/L) as a single nitrogen source, and measuring the concentration of nitrite nitrogen and nitrate nitrogen and the absorbance of a solution OD 600 every 3 hours by taking a water sample;
(4) Inoculating 1.0% of strain inoculum size to a synchronous denitrification culture medium, mixing ammonia nitrogen (20-25 mg/L) and nitrite nitrogen (20-25 mg/L) as nitrogen sources, performing shake culture at 37 ℃ and 200rpm for 24 hours, and taking a water sample every 3 hours to determine the concentration of the ammonia nitrogen, the nitrite nitrogen, the nitrate nitrogen and the absorbance of a solution OD 600;
(5) Inoculating 1.0% strain inoculum size to a synchronous denitrification culture medium, mixing ammonia nitrogen (20-25 mg/L) and nitrate nitrogen (20-25 mg/L) as nitrogen sources, performing shake culture at 37 ℃ and 200rpm for 24 hours, and taking a water sample every 3 hours to determine the concentration of the ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and the absorbance of a solution OD 600.
(6) Inoculating 1.0% of strain inoculum size to a synchronous denitrification culture medium, and carrying out shake culture for 24 hours at 37 ℃ and 200rpm under the condition that mixed nitrogen sources are nitrite nitrogen (20-25 mg/L) and nitrate nitrogen (20-25 mg/L), wherein the concentration of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen and the absorbance of a solution OD 600 are measured by taking water samples every 3 hours;
(7) Inoculating 1.0% of strain inoculum size to a synchronous denitrification culture medium, and mixing nitrogen sources of ammonia nitrogen (20-25 mg/L), nitrite nitrogen (20-25 mg/L) and nitrate nitrogen (20-25 mg/L), performing shake culture at 37 ℃ for 24 hours at 200rpm, and measuring the concentration of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen and the absorbance of a solution OD 600 every 3 hours;
On the other hand, the invention provides factors influencing the synchronous denitrification effect of the marine heterotrophic nitrification-aerobic denitrification bacteria with biological denitrification characteristics.
As a preferable scheme of the invention, the factors influencing the synchronous biological denitrification effect of the marine heterotrophic nitrification-aerobic denitrification bacteria with biological denitrification characteristics in the mixed nitrogen source simulated wastewater are explored, and the specific scheme is as follows:
(1) Effect of carbon to nitrogen ratio C/N on synchronous denitrification: the carbon nitrogen ratio C/N of the synchronous denitrification liquid culture medium is adjusted to be 3, 5, 10, 15 and 20 respectively, the synchronous denitrification liquid culture medium is inoculated into 100mL liquid culture mediums with different carbon nitrogen ratios C/N according to the inoculum size of 1.0 percent, shake culture is carried out for 24 hours at 37 ℃ and 200rpm, and water samples are taken for measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance respectively at 12 hours and 24 hours.
(2) Effect of pH on simultaneous denitrification: the pH values of the culture mediums are respectively adjusted to be 5, 6, 7, 8 and 9, 1.0 percent of strain inoculum size is inoculated into 100mL of culture mediums with different pH values, shake culture is carried out for 24 hours at 37 ℃ and 200rpm, and water samples are taken for respectively measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance at 12 hours and 24 hours.
(3) Effect of salinity on synchronous denitrification: the salinity of the culture medium is adjusted to be 5 per mill, 10 per mill, 20 per mill, 30 per mill and 40 per mill respectively, the culture medium is inoculated into 100mL culture mediums with different salinity according to the inoculum size of 1.0 percent, the culture medium is subjected to shake culture at 37 ℃ and 200rpm for 24 hours, and water samples are taken for 12 hours and 24 hours respectively to measure the concentration of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and the absorbance of solution OD 600.
(4) Influence of temperature on synchronous denitrification: the temperature of a constant temperature shaking table is adjusted to 15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃ respectively, 1.0% of strain inoculum size is inoculated into 100mL of culture medium, shake culture is carried out for 24 hours at 200rpm at the different temperatures, and water samples are taken for measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance at 12 hours and 24 hours respectively.
(5) Influence of rotational speed on synchronous denitrification: the rotation speed of a constant temperature shaking table is adjusted to be 140rpm, 160rpm, 180rpm, 200rpm and 220rpm respectively, the constant temperature shaking table is inoculated into 100mL of culture medium with the strain inoculation amount of 1.0 percent, shake culture is carried out for 24 hours at the temperature of 37 ℃ at different rotation speeds, and water samples are taken for measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance at 12 hours and 24 hours respectively.
Preferably, the strain 4-HYK has high-efficiency synchronous denitrification capability for ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in a water body with carbon nitrogen ratio C/N of 15-20 or pH value of 7-9 or salinity of 20-40 per mill or temperature of 15-35 ℃ or rotating speed of 140-220 rpm.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. The pseudomonas putida (Pseudomonas putida) 4-HYK has high-efficiency biological denitrification capability, and is a pseudomonas putida biological denitrification strain.
2. The pseudomonas putida Pseudomonas putida-HYK provided by the invention can grow by respectively using ammonia nitrogen, nitrite and nitrate as unique nitrogen sources or mixed nitrogen sources, and can realize the high-efficiency synchronous removal of the ammonia nitrogen, the nitrite and the nitrate nitrogen under the aerobic condition; in practical application, the bottleneck problem that the biological denitrification needs to be carried out by aerobic nitrification and anoxic denitrification sectional treatment in the traditional wastewater treatment is avoided.
3. The Pseudomonas putida Pseudomonas putida-HYK provided by the invention has strong environmental adaptability, can show logarithmic growth within the first 24 hours of culture, can rapidly grow the total number of living bacteria, can rapidly take over the dominant position within a certain time, and simultaneously removes nitrate nitrogen, nitrite nitrogen and ammonia nitrogen, the removal efficiency is higher than 99%, thus the invention has important application in the aspect of seawater wastewater denitrification
Drawings
The accompanying drawings, which are included to provide a further understanding of embodiments of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:
FIG. 1 is a morphological characterization of strain 4-HYK.
FIG. 2 is a graph of nitrification performance of strain 4-HYK against a single nitrogen source containing ammonia nitrogen.
FIG. 3 is a graph of denitrification performance of strain 4-HYK against a single nitrogen source containing nitrite nitrogen.
FIG. 4 is a graph of denitrification performance of strain 4-HYK against a single nitrogen source containing nitrate nitrogen.
FIG. 5 is a graph of the synchronous denitrification performance of strain 4-HYK on a mixed nitrogen source containing ammonia nitrogen and nitrite nitrogen.
FIG. 6 is a graph of the synchronous denitrification performance of strain 4-HYK on a mixed nitrogen source containing ammonia nitrogen and nitrate nitrogen.
FIG. 7 is a graph of denitrification performance of strain 4-HYK for a mixed nitrogen source containing nitrite nitrogen and nitrate nitrogen.
FIG. 8 is a graph of the synchronous denitrification performance of strain 4-HYK on a mixed nitrogen source containing ammonia nitrogen, nitrite nitrogen and nitrate nitrogen.
FIG. 9 is a graph showing the biological denitrification effect of strain 4-HYK under different carbon-nitrogen ratios C/N. a is ammonia nitrogen removal rate of 12h and 24 h; b is the nitrate nitrogen removal rate of 12h and 24 h; c is bacterial growth and nitrite nitrogen production for 12h and 24 h.
FIG. 10 is a graph showing the biological denitrification effect of strain 4-HYK at different pH values. a is ammonia nitrogen removal rate of 12h and 24 h; b is the nitrate nitrogen removal rate of 12h and 24 h; c is bacterial growth and nitrite nitrogen production for 12h and 24h.
FIG. 11 is a graph showing the biological denitrification effect of strain 4-HYK under different salinity conditions. a is ammonia nitrogen removal rate of 12h and 24 h; b is the nitrate nitrogen removal rate of 12h and 24 h; c is bacterial growth and nitrite nitrogen production for 12h and 24 h.
FIG. 12 is a graph showing the biological denitrification effect of strain 4-HYK under different temperature conditions. a is ammonia nitrogen removal rate of 12h and 24 h; b is the nitrate nitrogen removal rate of 12h and 24 h; c is bacterial growth and nitrite nitrogen production for 12h and 24 h.
FIG. 13 is a graph showing the biological denitrification effect of strain 4-HYK at different rotational speeds. a is ammonia nitrogen removal rate of 12h and 24 h; b is the nitrate nitrogen removal rate of 12h and 24 h; c is bacterial growth and nitrite nitrogen production for 12h and 24 h.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will now be described in more detail by way of examples with reference to the accompanying drawings, which are not intended to limit the invention thereto, but are illustrative only.
The invention relates to a culture medium:
(1) 30 per mill artificial seawater: 30g NaCl was dissolved in 1000mL pure water;
(2) Enrichment medium: KNO 3 0.36g·L-1、(NH4)2SO4 0.24g·L-1, sodium citrate 3.6g·L-1、KH2PO4 0.75g·L-1、K2HPO4 0.75g·L-1、MgSO4·7H2O 0.5g·L-1、FeSO4 0.02g·L-1、MnSO4·H2O 0.01g·L-1、pH 7.0～7.3;
(3) Bromothymol blue solid medium (BTB): KNO 3 1g·L-1, sodium citrate 3.6g.L -1、KH2PO40.75g·L-1、K2HPO4 0.75g·L-1, 1% bromothymol blue ethanol solution (BTB), agar 20 g.L -1, pH 7.0-7.3;
(4) Heterotrophic nitrification liquid medium (SM): (NH 4)2SO4 0.12g·L-1, sodium citrate) 3.6g·L-1、KH2PO40.75g·L-1、K2HPO4 0.75g·L-1、MgSO4·7H2O 0.5g·L-1、FeSO4 0.02g·L-1、MnSO4·H2O 0.01g·L-1,pH 7.0～7.3;
(5) Denitrification liquid medium (DM): naNO 2 0.09g·L-1/KNO3 0.18g·L-1, sodium citrate 3.6g·L-1、KH2PO4 0.75g·L-1、K2HPO4 0.75g·L-1、MgSO4·7H2O 0.5g·L-1、FeSO4 0.02g·L-1、MnSO4·H2O 0.01g·L-1,pH 7.0～7.3;
(6) Synchronous denitrification culture medium (SND)：NaNO2 0.09g·L-1/KNO3 0.18g·L-1,(NH4)2SO40.12g·L-1、 sodium citrate 7.2g·L-1、KH2PO4 0.75g·L-1、K2HPO4 0.75g·L-1、MgSO4·7H2O0.5g·L-1、FeSO4 0.02g·L-1、MnSO4·H2O 0.01g·L-1,pH 7.0～7.3.
Example 1 isolation, screening and identification of Marine heterotrophic nitrification-aerobic denitrification strains with biological denitrification Properties
1ML of 30 permillage artificial seawater sample is taken to be placed in 20mL of enrichment medium, the culture is carried out for 1 day in a shaking table at 30 ℃ and 180rpm at constant temperature, 5mL of enrichment bacterial liquid is taken to be placed in 100mL of enrichment medium for culture, and the process is repeated for three times; then, taking bacterial liquid for gradient dilution, coating the bacterial liquid on the surface of a bromothymol blue (BTB) solid culture medium, culturing at the constant temperature of 30 ℃ for 1-3 days, then selecting a strain with brightest blue halos for further purification on the BTB culture medium, selecting a single colony for streaking separation, and repeating the steps for three times to obtain a single strain; the single strain was stored in a-80℃refrigerator.
Morphological identification: the colonies were screened for white colonies with smooth, opaque, perfectly regular edges, and oval ridges. As shown in fig. 1.
Molecular biology identification: the DNA of the screened strain is extracted by adopting a bacterial genome extraction kit (Tiangen), the PCR amplification of the conserved target fragment is carried out by using the 16S rDNA gene universal primers 27F and 1492R (shown as SEQ ID NO:2 and SEQ ID NO: 3), and the amplified product is recovered by cutting gel, and then sequencing analysis is carried out. The results of PCR amplification, sequencing and BLAST comparison show that the 16S rDNA gene sequence of the strain 4-HYK has higher similarity with Pseudomonas putida (Pseudomonas putida), wherein the similarity with Pseudomonas putida strain Os _Ep_PPA_18 reaches 100%. Thus, strain 4-HYK was identified as Pseudomonas putida and designated Pseudomonas putida-HYK.
Example 2 evaluation of Nitrogen removal Effect of Pseudomonas putida 4-HYK on Single Nitrogen Source simulated wastewater
Seed liquid preparation: the strain is picked and put into a synchronous denitrification liquid culture medium, shake culture is carried out for 10 to 12 hours at 37 ℃ and 200rpm, and the absorbance of the solution OD 600 is measured by the culture medium; OD 600 reaches 0.4-0.6, which shows that the strain has entered logarithmic phase and the seed liquid can be used normally.
The evaluation scheme of the nitrogen removal effect of pseudomonas putida 4-HYK on single nitrogen source simulated wastewater is as follows:
(1) Inoculating 1.0% of strain inoculum size to heterotrophic nitrification culture medium, and shake culturing at 200rpm for 24h at 37 ℃ with ammonia nitrogen (20-25 mg/L) as single nitrogen source, and measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance every 3 h. The results are shown in FIG. 2.
(2) Inoculating 1.0% of strain inoculum size to a denitrification culture medium, and shake culturing at 200rpm for 24 hours at 37 ℃ with nitrite nitrogen (20-25 mg/L) as a single nitrogen source, and taking a water sample every 3 hours to measure the concentration of nitrite nitrogen and nitrate nitrogen and the absorbance of a solution OD 600. The results are shown in FIG. 3.
(3) Inoculating 1.0% of strain inoculum size to a denitrification culture medium, and shake culturing at 200rpm for 24 hours at 37 ℃ with nitrate nitrogen (20-25 mg/L) as a single nitrogen source, and taking a water sample every 3 hours to measure the concentration of nitrite nitrogen and nitrate nitrogen and the absorbance of a solution OD 600. The results are shown in FIG. 4.
The result shows that the pseudomonas putida 4-HYK can enter a growth stabilizing period within 12-16 h, and can remove more than 99% of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen within 24h, thereby having good biological denitrification effect.
Example 3 evaluation of biological Nitrogen removal effect of Pseudomonas putida 4-HYK on Mixed Nitrogen Source simulated wastewater
Seed liquid preparation: the strain is picked and put into a synchronous denitrification liquid culture medium, shake culture is carried out for 10 to 12 hours at 37 ℃ and 200rpm, and the absorbance of the solution OD 600 is measured by the culture medium; OD 600 reaches 0.4-0.6, which shows that the strain has entered logarithmic growth phase and the seed liquid can be used normally.
The evaluation scheme of the nitrogen removal effect of pseudomonas putida 4-HYK on the mixed nitrogen source simulated wastewater is as follows:
(1) Inoculating 1.0% strain inoculum size to a synchronous denitrification culture medium, mixing ammonia nitrogen (20-25 mg/L) and nitrite nitrogen (20-25 mg/L) as nitrogen sources, performing shake culture at 37 ℃ and 200rpm for 24 hours, and taking a water sample every 3 hours to determine the concentration of the ammonia nitrogen, the nitrite nitrogen, the nitrate nitrogen and the absorbance of a solution OD 600. The results are shown in FIG. 5.
(2) Inoculating 1.0% strain inoculum size to a synchronous denitrification culture medium, mixing ammonia nitrogen (20-25 mg/L) and nitrate nitrogen (20-25 mg/L) as nitrogen sources, performing shake culture at 37 ℃ and 200rpm for 24 hours, and taking a water sample every 3 hours to determine the concentration of the ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and the absorbance of a solution OD 600. The results are shown in FIG. 6.
(3) Inoculating 1.0% strain inoculum size to a synchronous denitrification culture medium, and mixing nitrite nitrogen (20-25 mg/L) and nitrate nitrogen (20-25 mg/L) as nitrogen sources, performing shake culture at 37 ℃ and 200rpm for 24 hours, and taking a water sample every 3 hours to determine ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance. The results are shown in FIG. 7.
(3) Inoculating 1.0% strain inoculum size to a synchronous denitrification culture medium, mixing nitrogen sources including ammonia nitrogen (20-25 mg/L), nitrite nitrogen (20-25 mg/L) and nitrate nitrogen (20-25 mg/L), shake culturing at 37 ℃ for 24 hours at 200rpm, and measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance every 3 hours by taking water samples. The results are shown in FIG. 8.
The result shows that the pseudomonas putida 4-HYK can grow and propagate rapidly, more than 99% of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen can be removed within 24 hours, and the biological denitrification effect is good.
EXAMPLE 4 Effect of C/N ratio on Pseudomonas putida 4-HYK synchronous denitrification Effect
Seed liquid preparation: the strain is picked and put into a synchronous denitrification liquid culture medium containing 40-50 mg/L ammonia nitrogen and 40-50 mg/L nitrate nitrogen, shake culture is carried out for 10-12 h at 37 ℃ and 200rpm, and the absorbance of the culture medium is measured to obtain the OD 600 absorbance of the solution; OD 600 reaches 0.4-0.6, which shows that the strain has entered logarithmic growth phase and the seed liquid can be used normally.
The carbon nitrogen ratio C/N of the synchronous denitrification liquid culture medium is adjusted to be 3, 5, 10, 15 and 20 respectively, the synchronous denitrification liquid culture medium is inoculated into 100mL liquid culture mediums with different carbon nitrogen ratios C/N according to the inoculum size of 1.0 percent, shake culture is carried out for 24 hours at 37 ℃ and 200rpm, and water samples are taken for measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance respectively at 12 hours and 24 hours. The results are shown in FIG. 9.
The result shows that the Pseudomonas putida 4-HYK has higher biological denitrification efficiency when the carbon nitrogen ratio C/N is 15-20, and no nitrite accumulation exists.
EXAMPLE 5 Effect of pH on Pseudomonas putida 4-HYK synchronous denitrification Effect
Seed liquid preparation: the strain is picked and put into a synchronous denitrification liquid culture medium containing 40-50 mg/L ammonia nitrogen and 40-50 mg/L nitrate nitrogen, shake culture is carried out for 10-12 h at 37 ℃ and 200rpm, and the absorbance of the culture medium is measured to obtain the OD 600 absorbance of the solution; OD 600 reaches 0.4-0.6, which shows that the strain has entered logarithmic growth phase and the seed liquid can be used normally.
The pH values of the culture mediums are respectively adjusted to be 5, 6, 7, 8 and 9, 1.0 percent of strain inoculum size is inoculated into 100mL of culture mediums with different pH values, shake culture is carried out for 24 hours at 37 ℃ and 200rpm, and water samples are taken for respectively measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance at 12 hours and 24 hours. The results are shown in FIG. 10.
The result shows that the Pseudomonas putida 4-HYK has higher denitrification efficiency at the pH value of 7-9 organisms, and no nitrite accumulation.
EXAMPLE 6 Effect of salinity on Pseudomonas putida 4-HYK synchronous denitrification Effect
Seed liquid preparation: the strain is picked and put into a synchronous denitrification liquid culture medium containing 40-50 mg/L ammonia nitrogen and 40-50 mg/L nitrate nitrogen, shake culture is carried out for 10-12 h at 37 ℃ and 200rpm, and the absorbance of the culture medium is measured to obtain the OD 600 absorbance of the solution; OD 600 reaches 0.4-0.6, which shows that the strain has entered logarithmic growth phase and the seed liquid can be used normally.
Salinity regulation: the salinity is regulated and controlled by the amount of NaCl, namely 5, 10, 20, 30 and 40 thousandths of NaCl is contained in each liter of water, namely 5, 10, 20, 30 and 40g of NaCl is contained in each liter of water.
The salinity of the culture medium is adjusted to be 5 per mill, 10 per mill, 20 per mill, 30 per mill and 40 per mill respectively, the culture medium is inoculated into 100mL culture mediums with different salinity according to the inoculum size of 1.0 percent, the culture medium is subjected to shake culture at 37 ℃ and 200rpm for 24 hours, and water samples are taken for 12 hours and 24 hours respectively to measure the concentration of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and the absorbance of solution OD 600. The results are shown in FIG. 11.
The result shows that the pseudomonas putida 4-HYK can grow and propagate rapidly when the salinity is 20-40 per mill, the biological denitrification efficiency is higher, and no nitrite is accumulated.
EXAMPLE 7 Effect of temperature on Pseudomonas putida 4-HYK synchronous denitrification Effect
Seed liquid preparation: the strain is picked and put into a synchronous denitrification liquid culture medium containing 40-50 mg/L ammonia nitrogen and 40-50 mg/L nitrate nitrogen, shake culture is carried out for 10-12 h at 37 ℃ and 200rpm, and the absorbance of the culture medium is measured to obtain the OD 600 absorbance of the solution; OD 600 reaches 0.4-0.6, which shows that the strain has entered logarithmic growth phase and the seed liquid can be used normally.
The temperature value of the constant temperature shaking table is adjusted to 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ respectively, 1.0% of strain inoculum size is inoculated into 100mL of culture medium, shake culture is carried out for 24 hours at the temperature and 200rpm respectively, and water samples are taken for measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance at 12 hours and 24 hours respectively. The results are shown in FIG. 12.
The result shows that the pseudomonas putida 4-HYK can grow and reproduce rapidly at the temperature of 25-35 ℃, the biological denitrification efficiency is higher, and nitrite accumulation is less.
EXAMPLE 8 Effect of rotation speed on Pseudomonas putida 4-HYK synchronous denitrification Effect
Seed liquid preparation: the strain is picked and put into a synchronous denitrification liquid culture medium containing 40-50 mg/L ammonia nitrogen and 40-50 mg/L nitrate nitrogen, shake culture is carried out for 10-12 h at 37 ℃ and 200rpm, and the absorbance of the culture medium is measured to obtain the OD 600 absorbance of the solution; OD 600 reaches 0.4-0.6, which shows that the strain has entered logarithmic growth phase and the seed liquid can be used normally.
The rotation speed values of the constant temperature shaking table are respectively 140rpm, 160rpm, 180rpm, 200rpm and 220rpm, the constant temperature shaking table is inoculated into 100mL of culture medium with the strain inoculation amount of 1.0%, the shaking table is respectively subjected to shake culture at the different rotation speeds and the temperature of 37 ℃ for 24 hours, and water samples are respectively taken for measuring ammonia nitrogen, nitrite nitrogen, nitrate nitrogen concentration and solution OD 600 absorbance at 12 hours and 24 hours. The results are shown in FIG. 13.
The result shows that the pseudomonas putida 4-HYK can grow and propagate rapidly at the rotating speed of 200-220 rpm, and the biological denitrification efficiency is higher.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. A marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification characteristics is characterized in that the strain is named as Pseudomonas putida (Pseudomonas putida) 4-HYK and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 28841.
2. A marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification as claimed in claim 1, wherein said strain 4-HYK can be grown on a liquid medium (NH 4)2SO4、KNO3、NaNO2 is the sole nitrogen source or a mixed nitrogen source.
3. The marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification properties according to claim 1, wherein the strain 4-HYK forms white colonies with smooth surface, opaque surface, complete and regular edge, and oval-shaped ridges when cultured on a culture medium.
4. The marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification characteristics as claimed in claim 1, wherein the 16S rDNA gene sequence is shown in SEQ ID NO. 1.
5. A microbial inoculum prepared by using the marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification characteristics as set forth in claim 1.
6. The use of the marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification characteristics according to claim 1 in biological denitrification of wastewater.
7. The application of the marine heterotrophic nitrification-aerobic denitrification strain with biological denitrification characteristics according to claim 6, wherein the marine heterotrophic nitrification-aerobic denitrification strain has synchronous denitrification application to ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in a water body with a carbon nitrogen ratio C/N of 15-20 or a pH value of 7-9 or a salinity of 20-40 per mill or a temperature of 15-35 ℃ or a rotating speed of 140-220 rpm.
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