CN114874947B - Coli and product and application thereof - Google Patents
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Abstract
The application discloses escherichia coli, a product and application thereof, which are characterized in that the escherichia coli is named as escherichia coli SQ-1A, and the preservation number of the escherichia coli in the China general microbiological culture collection center is CGMCC No. 23284. The escherichia coli is obtained by enrichment, separation and purification from pig farm wastewater, the highest degradation rate of ammonia nitrogen in the wastewater can reach 89.23%, good degradation is reflected, the method has the characteristics of high specificity, short time consumption and high removal efficiency, can be applied to removing or reducing the residue of ammonia nitrogen in the environment, and provides a safe and environment-friendly microbial degradation method for the problem of ammonia nitrogen residue.
Description
Technical Field
The application belongs to the technical field of biodegradation of ammonia nitrogen in wastewater, and particularly relates to escherichia coli, a product and application thereof.
Background
The nitrogen pollution caused by untreated livestock and poultry raising wastewater is particularly serious, wherein the ammonia nitrogen pollution is most prominent. The livestock and poultry wastewater containing high-concentration ammonia nitrogen is directly discharged into water, the ammonia nitrogen in the wastewater is oxidized and converted into nitrate nitrogen and nitrite nitrogen under the action of nitrifying bacteria, 4.6mg of dissolved oxygen is consumed for complete oxidation of 1mg of ammonia nitrogen, and the consumption of a large amount of dissolved oxygen leads to the rapid reduction of the dissolved oxygen content of the water. Excessive nitrogen in the water body can also increase the quantity of photosynthetic microorganisms (mainly algae), so that eutrophication phenomenon occurs, the water body is blackened and smelly, the transparency is reduced, the water quality environment is deteriorated, and a large amount of fishes and other organisms in the water are promoted to die.
Investigation of the pollution sources of the Taihu lake in China shows that the livestock and poultry breeding wastewater is one of important pollution sources causing nitrogen pollution and eutrophication of the Taihu lake basin. When livestock and poultry wastewater containing high-concentration ammonia nitrogen is used for irrigating farmlands, soil pores are blocked, so that the air permeability and water permeability of the soil are reduced, the quality of the soil is deteriorated, and plants are reduced in yield and even die. If the excessive nitrogen element in the water can not be effectively absorbed, the excessive nitrogen element is very easy to be lost into surface water or leached into underground water through air-permeable soil, so that water pollution is caused.
The Chinese patent No. CN110452836A provides a microbial strain for degrading ammonia nitrogen, wherein the strain is a psychrophilic bacillus (Psychrobacter cibarius) Z-XWW G, the psychrophilic bacillus (Psychrobacter cibarius) Z-XWW G is preserved in China general microbiological culture Collection center (CGMCC) No.17515, and the Bacillus Zhuzhou (Bacillus zhangzhouensis) Z-XWW 77, the Bacillus zhangzhou (Bacillus zhangzhou) Z-XWW 77 is preserved in China general microbiological collection center (CGMCC No. 17516), and the comparison document also provides application of the strain and compound microorganisms containing the strain in degrading ammonia nitrogen, but the efficiency of treating ammonia nitrogen in wastewater by the compound microorganism flora in the comparison document is lower.
Disclosure of Invention
In order to overcome the problems in the prior art, the application provides escherichia coli, and a product and application thereof, and the specific method is to screen out a strain of degrading bacteria which has strong specificity, short time consumption and high removal efficiency and can degrade ammonia nitrogen in wastewater from pig farm wastewater, so that abundant microbial resources are provided for treating livestock and poultry breeding wastewater.
In order to achieve the above purpose, the present application is realized by the following technical scheme:
the first aspect of the application provides escherichia coli, which is named Escherichia coli SQ-1A and has a preservation number of CGMCC NO. 23284 in the China general microbiological culture Collection center.
As an alternative embodiment, the application provides E.coli with the nucleotide sequence shown in SEQ ID NO. 1.
As an alternative embodiment, the application provides that the escherichia coli is obtained by enrichment, separation and purification from pig farm wastewater containing activated sludge, wherein the culture temperature in the enrichment and separation process is 28-30 ℃, and the culture time is 20-24 hours.
As an alternative embodiment, the application provides that the basic culture medium used in the enrichment, isolation and purification process of Escherichia coli is LB culture medium containing ammonium chloride.
As an alternative embodiment, the screening medium used in the process of enriching, separating and purifying the escherichia coli provided by the application is a medium containing sodium acetate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, sodium chloride, manganese sulfate, ferrous sulfate and ammonia nitrogen solution.
As an alternative embodiment, the screening culture medium used in the process of enriching, separating and purifying the escherichia coli provided by the application is a culture medium containing 2.0g of sodium acetate, 0.05g of magnesium sulfate heptahydrate, 0.2g of dipotassium hydrogen phosphate, 0.12g of sodium chloride, 0.01g of manganese sulfate, 0.01g of ferrous sulfate, ammonia nitrogen solution and 1000mL of distilled water.
As an alternative embodiment, the carbon source in the simulated wastewater used in the enrichment, separation and purification process of the escherichia coli provided by the application comprises at least one of glucose, sucrose, starch, sodium acetate, sodium succinate or sodium bicarbonate, and the carbon-nitrogen ratio in the simulated wastewater is 3-15:1.
As an alternative embodiment, in the escherichia coli provided by the application, the specific process of escherichia coli enrichment, separation and purification comprises the following steps:
step one: enrichment, isolation and purification of strains
Waste water is collected from a pig farm and stored in a-20 ℃ refrigerator by using a sterile triangular flask. Transferring 10mL pig farm wastewater containing activated sludge into a triangular flask containing 90mL of enrichment medium, and placing the triangular flask into a shaking incubator to be cultured for 24 hours at 28 ℃ and 160r/min to obtain enrichment bacterial liquid. Inoculating the enriched bacterial liquid into a preliminary screening inorganic salt culture medium according to the inoculum size of 5 percent, culturing for 24 hours under the same condition, and repeating the operation and screening twice. The bacterial liquid after screening is diluted by coating 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Six concentration gradients are coated on the solid enrichment culture medium, and after coating, the culture medium is put into an incubator at 28 ℃ for inversion culture for 24 hours to finish primary screening. Different single colonies on the solid medium were selected and isolated and purified (streak weightAnd 2-3 times), and the purified single bacteria are frozen in a refrigerator at the temperature of minus 80 ℃ for standby.
Step two: re-screening of strains
The strain obtained by primary screening is activated and cultured in LB liquid culture medium for 24 hours, a proper amount of bacterial liquid is taken and centrifuged in a 10mL centrifuge tube, the supernatant is removed, then bacterial cells are washed by sterile physiological saline (repeated twice) and prepared into bacterial suspension, and the OD of the bacterial suspension is prepared 600 The values are adjusted to unity. Inoculating into a re-screening inorganic salt culture medium according to the inoculum size of 5%, placing into a shaking incubator, culturing for 48 hours at 28 ℃ and 160r/min, measuring the ammonia nitrogen concentration by using a multifunctional water quality tester, and calculating the degradation rate. Eliminating the strain with poor degradation capability and the strain with high ammonia nitrogen degradation capability, and preserving the strain with glycerol tubes and inclined planes for standby.
Step three: identification of degradation strains
And (3) observing morphological characteristics of the target strain and morphology of bacterial cells under a microscope, and comprehensively identifying bacterial morphology and strain types by combining a 16SrDNA method.
The second aspect of the application provides the use of the escherichia coli described in any one of the embodiments above for degrading ammonia nitrogen in wastewater.
As an optional implementation mode, in the application of the escherichia coli in degrading ammonia nitrogen in wastewater, the pH value of a degradation solution is 6-8, and the degradation temperature is 20-40 ℃ in the process of degrading ammonia nitrogen in wastewater.
As an optional implementation mode, in the application of the escherichia coli provided by the application in degrading ammonia nitrogen in wastewater, the initial concentration of the ammonia nitrogen in the wastewater is 100-900mg/L, and the inoculation amount of the escherichia coli is 0.5-10%.
In a third aspect, the application provides a microbial inoculum for degrading ammonia nitrogen in wastewater, wherein the microbial inoculum for degrading ammonia nitrogen in wastewater contains escherichia coli in any one of the embodiments.
As an optional implementation mode, in the microbial inoculum for degrading ammonia nitrogen in the degradation wastewater provided by the application, the preparation method of the microbial inoculum for degrading ammonia nitrogen in the degradation wastewater comprises the steps of carrying out activation culture on the escherichia coli by using a liquid culture medium, and collecting a culture solution.
The beneficial effects of the application are as follows:
the application screens and obtains a strain of ammonia nitrogen high-efficiency degradation bacteria, and the strain is identified as escherichia coli Escherichia coli sp and named Escherichia coli SQ-1A through physiological identification and 16S rRNA sequence analysis. The preservation number is: CGMCC No. 23284. The maximum degradation rate of the escherichia coli to the ammonia nitrogen reaches 89.23%, good degradation is shown, and the method can be applied to removing or reducing the residual of the ammonia nitrogen in the environment, and provides a safe and environment-friendly microbial degradation method for the problem of ammonia nitrogen residual.
Drawings
FIG. 1 is a colony morphology of strain Escherichia coli SQ-1A of example 1;
FIG. 2 is a morphological image of strain Escherichia coli SQ-1A of example 1 under a microscope;
FIG. 3 is a phylogenetic tree of strain Escherichia coli SQ-1A of example 2;
FIG. 4 is a graph showing the growth of strain Escherichia coli SQ-1A in example 2;
FIG. 5 is a graph showing the experimental results of ammonia nitrogen concentration in wastewater in example 3 versus ammonia nitrogen degradation by strain Escherichia coli SQ-1A;
FIG. 6 is a graph showing the experimental results of ammonia nitrogen degradation by strain Escherichia coli SQ-1A at the temperature in example 3;
FIG. 7 is a graph showing the experimental results of the inoculum size versus ammonia nitrogen degradation by strain Escherichia coli SQ-1A in example 3;
FIG. 8 is a graph showing the experimental results of pH in wastewater in example 3 on degradation of ammonia nitrogen by strain Escherichia coli SQ-1A;
FIG. 9 is a graph showing experimental results of ammonia nitrogen degradation of strain Escherichia coli SQ-1A by a carbon source in simulated wastewater in example 3;
FIG. 10 is a graph showing the experimental results of degrading ammonia nitrogen by the carbon nitrogen comparison strain Escherichia coli SQ-1A in simulated wastewater in example 3.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Reagents and apparatus required in the course of experiments
The reagents used in the examples below, unless otherwise indicated, are conventional in the art, and the apparatus and methods are conventional in the art.
The preparation method of the culture medium used in the application comprises the following steps:
1. enrichment medium (g/L): ammonium chloride 0.382g; 25g of LB medium; 1000mL of distilled water; pH 7.0.
2. Primary screening inorganic salt medium (g/L): 2.0g of sodium acetate; 0.05g of magnesium sulfate heptahydrate; 0.2g of dipotassium hydrogen phosphate; sodium chloride 0.12g; 0.01g of manganese sulfate; ferrous sulfate 0.01g; 1.9g of ammonium chloride; 1000mL of distilled water; pH 7.0.
3. Re-screening inorganic salt culture medium (g/L): 2.0g of sodium acetate; 0.05g of magnesium sulfate heptahydrate; 0.2g of dipotassium hydrogen phosphate; sodium chloride 0.12g; 0.01g of manganese sulfate; ferrous sulfate 0.01g; the ammonia nitrogen concentration is controlled by changing the content of ammonium chloride; 1000mL of distilled water; pH 7.0.
4. Solid enrichment media (g/L): ammonium chloride 0.382; 25g of LB medium; 15g of agar; 1000mL of distilled water; pH 7.0.
5. Initial medium (g/L): 2.0g of sodium acetate; 0.05g of magnesium sulfate heptahydrate; 0.2g of dipotassium hydrogen phosphate; sodium chloride 0.12g; 0.01g of manganese sulfate; ferrous sulfate 0.01g; ammonium chloride 0.382g; 1000mL of distilled water; pH 7.0.
Example 1
The application discovers a strain of degrading bacteria capable of degrading ammonia nitrogen, wherein the ammonia nitrogen degrading bacteria are escherichia coli Escherichia coli sp and are delivered to China general microbiological culture Collection center for preservation in 2021 and 09 and 10 days, and the name is: escherichia coli SQ-1A with the preservation number of CGMCC NO. 23284. Address: the north-western road in the morning sun area of Beijing city of China.
The escherichia coli (Escherichia coli SQ-1A) is obtained by enriching, separating and purifying pig farm wastewater, and comprises the following specific steps:
step one: enrichment, isolation and purification of strains
Waste water is collected from a pig farm and stored in a-20 ℃ refrigerator by using a sterile triangular flask. Transferring 10mL pig farm wastewater containing activated sludge into a triangular flask containing 90mL of enrichment medium, and placing the triangular flask into a shaking incubator to be cultured for 24 hours at 28 ℃ and 160r/min to obtain enrichment bacterial liquid. Inoculating the enriched bacterial liquid into a preliminary screening inorganic salt culture medium according to the inoculum size of 5 percent, culturing for 24 hours under the same condition, and repeating the operation and screening twice. The bacterial liquid after screening is diluted by coating 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Six concentration gradients are coated on the solid enrichment culture medium, and after coating, the culture medium is put into an incubator at 28 ℃ for inversion culture for 24 hours to finish primary screening. Selecting different units on solid mediumThe colony is separated and purified (streaking is repeated for 2-3 times), and the single bacteria after purification are frozen in a refrigerator at-80 ℃ for standby by a glycerol preservation method.
Step two: re-screening of strains
The strain obtained by primary screening is activated and cultured in LB liquid culture medium for 24 hours, a proper amount of bacterial liquid is taken and centrifuged in a 10mL centrifuge tube, the supernatant is removed, then bacterial cells are washed by sterile physiological saline (repeated twice) and prepared into bacterial suspension, and the OD of the bacterial suspension is prepared 600 The values are adjusted to unity. Inoculating into a re-screening inorganic salt culture medium according to the inoculum size of 5%, placing into a shaking incubator, culturing for 48 hours at 28 ℃ and 160r/min, measuring the ammonia nitrogen concentration by using a multifunctional water quality tester, and calculating the degradation rate. Eliminating the strain with poor degradation capability and the strain with high ammonia nitrogen degradation capability, and preserving the strain with glycerol tubes and inclined planes for standby.
Step three: identification of degradation strains
And (3) observing morphological characteristics of the target strain and morphology of bacterial cells under a microscope, and comprehensively identifying bacterial morphology and strain types by combining a 16SrDNA method.
The colony morphology observation method is as follows: after the target strain is enriched and cultured in an enrichment liquid culture medium for 24 hours, the target strain is inoculated into a solid enrichment culture medium by a streaking method, the inoculated flat plate is inversely cultured in a constant temperature incubator at 28 ℃ for 48 hours, and then the flat plate is taken out of the incubator to observe the morphological characteristics (size, shape, transparency, color and the like) of the bacterial colony.
The bacterial morphology observation method under the microscope comprises the following steps:
tabletting and fixing: a clean glass slide is clamped by forceps, a small drop of distilled water is dripped on the glass slide, a small quantity of thalli is dipped in an enrichment liquid culture medium by using an inoculating loop, the thalli are uniformly coated in the distilled water drop, and the glass slide is placed in an alcohol lamp for heating and drying, so that the thalli are fixed.
Dyeing: after the glass slide is cooled, the glass slide is washed by slow water flow after being stained by dripping crystal violet staining solution for 1 minute by a rubber head dropper, the glass slide is washed off, the glass slide is dried, and the cedar oil is dripped.
And (5) microscopic examination: the slide glass is fixed on the objective table, the aperture and the coarse focusing spiral are adjusted, firstly, the low-power mirror is used for observation, and when clear object images appear in the visual field, the high-power oil mirror is used for observing the bacterial morphology.
After the target strain is cultured on a flat plate (LB solid medium) for 24 hours, the morphological characteristics of the bacterial colony are shown as a figure 1, and the bacterial strain SQ-1A is a circular, moist, semitransparent, milky and neat-edged bacterial colony; the morphology of each strain observed under the oil microscope is shown in FIG. 2.
Extraction of genomic DNA: the procedure was performed with reference to the "bacterial genomic DNA extraction kit" of the root of the heaven. And the quality of the extracted DNA was checked using agarose gel electrophoresis. And (3) PCR amplification: bacterial 16S rDNA universal primers were selected, the sequences of which were as follows:
an upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3', the sequence of which is shown as SEQ ID NO. 2;
a downstream primer: 5'-AAGGAGGTGATCCAGCCGCA-3', the sequence of which is shown as SEQ ID NO. 3;
after the PCR product is purified by a TIANGEN gel recovery kit, sequencing (two-way sequencing) is carried out by a biological engineering (Shanghai) stock company, the sequence comparison analysis is carried out on the spliced sequencing result in a GenBank database, the sequence comparison is carried out by Clustal X1.8 software, and meanwhile, a phylogenetic evolutionary tree is constructed by MEGA7 software as shown in figure 3.
As a result of phylogenetic analysis, the strain was close to Escherichia coli (CCFM 8338) in affinity, the homology was 99%, and the strain SQ-1A was determined to be Escherichia coli (Escherichia coli). The nucleotide sequence of Escherichia coli SQ-1A is shown as SEQ ID NO. 1.
Example 2
Drawing a growth curve of the strain Escherichia coli SQ-1A.
Inoculating the glycerol frozen bacterial liquid in the example 1 into LB liquid culture medium filled with 100mL for activation culture, culturing for 16-18 hours at 28 ℃ under 160r/min in a shaking incubator to prepare seed liquid, inoculating the seed liquid into 14 bottles of activation liquid culture medium respectively with 5% of inoculation amount, then placing into the shaking incubator for culturing for 28 hours at 28 ℃ under 160r/min, and measuring the OD of the bacterial liquid every 2 hours by using an ultraviolet spectrophotometer 600 Values, control, are non-inoculated activated liquid medium. The growth curve is shown in fig. 4. The bacteria liquid cultured for 10-14 h has vigorous activity of bacteria life metabolism and various characteristics. The bacterial liquid cultured for 10-14 hours is most suitable as the inoculation liquid for the subsequent examples.
Example 3
Optimal conditions for degrading ammonia nitrogen by the strain Escherichia coli SQ-1A.
Preparing an inoculation liquid; inoculating the selected target strain into liquid activating culture medium, culturing at 28deg.C under 160r/min to strain growth logarithmic phase, centrifuging at high speed under aseptic condition in centrifuge tube, removing supernatant, washing the precipitate with sterile physiological saline for 2-3 times to obtain bacterial suspension, and regulating bacterial liquid OD 600 And after unification, inoculating 5% of the strain into the simulated wastewater treated differently to study the degradation characteristics of ammonia nitrogen in the simulated wastewater.
The ammonia nitrogen degradation rate is calculated in the following way: removal efficiency (%) = (C 0 -C t )/C 0 *100% of which: c (C) 0 And C t The (mg/L) is the initial concentration of ammonia nitrogen and the concentration at the moment t respectively.
(1) Influence of ammonia nitrogen concentration in wastewater on degradation of ammonia nitrogen by bacterial strains
The target strain is inoculated into the simulated wastewater with the pH value of 7.0, ammonia nitrogen concentration in the simulated wastewater is regulated to be 100mg/L, 300mg/L, 500mg/L, 700mg/L and 900mg/L respectively, and after the target strain is subjected to shaking culture for 3 days at the temperature of 30 ℃ and the temperature of 180r/min, the ammonia nitrogen concentration is measured, and the ammonia nitrogen degradation rate is calculated. The results are shown in FIG. 5. The ammonia nitrogen degradation rate of the bacterial strain SQ-1A at the initial ammonia nitrogen concentration of 100mg/L, 300mg/L, 500mg/L, 700mg/L and 900mg/L is 71.65%, 49.99%, 42.93%, 26.31% and 21.89% respectively. The degradation rate is obviously higher than that of other concentrations when the initial ammonia nitrogen concentration is 100 mg/L. Therefore, the degradation rate of the comprehensive bacteria ammonia nitrogen under different initial ammonia nitrogen concentrations is selected to simulate the wastewater with the initial ammonia nitrogen concentration of 100mg/L for subsequent tests.
(2) Influence of temperature on degradation of ammonia nitrogen by bacterial strains
Setting the initial ammonia nitrogen concentration of the simulated wastewater to be 100mg/L, regulating the pH value to 7.0, respectively placing the simulated wastewater inoculated with the target strain into an incubator at 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃, carrying out shaking culture for 3d under the condition of 180r/min, measuring the ammonia nitrogen concentration, and calculating the ammonia nitrogen degradation rate. The results are shown in FIG. 6. From FIG. 6, it can be seen that the ammonia nitrogen degradation rate of the strain SQ-1A reaches 72.30% at the temperature of 35 ℃, belongs to mesophilic bacteria, has no significant change in the ammonia nitrogen degradation capability in simulated wastewater when the strain is cultured at the temperature of 35-40 ℃, does not necessarily require the constancy of temperature in subsequent large-scale culture, but is suitable when the culture temperature in subsequent experiments is adjusted to about 35 ℃ after comprehensive consideration.
(3) Effect of inoculum size on degradation of Ammonia by Strain
The method comprises the steps of inoculating target strains into simulated wastewater with initial ammonia nitrogen concentration of 100mg/L and pH of 7 in inoculum sizes of 0.5%, 1%, 2%, 3%, 4%, 5% and 10%, performing shake culture at 35 ℃ and 180r/min for 3 days, measuring the ammonia nitrogen concentration of the simulated wastewater, and calculating the ammonia nitrogen degradation rate. The results are shown in FIG. 7. When the strain SQ-1A is inoculated into the simulated wastewater with the inoculation amount of 0.5% -1%, the ammonia nitrogen degradation capacity is strong, 75.90% and 78.37% of the ammonia nitrogen content in the simulated wastewater can be removed respectively, the ammonia nitrogen degradation capacity of the strain is gradually weakened along with the increase of the inoculation amount, and when the inoculation amount is increased to 10%, the ammonia nitrogen degradation capacity of the strain is only 34.3%. In combination with practical considerations, the target strain in the subsequent test will be inoculated into the simulated wastewater with an inoculum size of 1%.
(4) Influence of pH value on degradation of ammonia nitrogen by bacterial strains
The pH values of the simulated wastewater are respectively 6.0, 6.5, 7.0, 7.5 and 8.0, the initial ammonia nitrogen concentration is 100mg/L, 1% of inoculation amount is used for inoculating a target strain, the ammonia nitrogen concentration of the simulated wastewater is measured after shaking culture is carried out for 3d at 35 ℃ and 180r/min, and the ammonia nitrogen degradation rate is calculated. The results are shown in FIG. 8. The ammonia nitrogen removal efficiency of the strain SQ-1A in the simulated wastewater with the initial pH value of 7 is maximum and reaches 86.17%, and the ammonia nitrogen degradation rates are 85.2%, 80.9%, 80.4% and 78.3% respectively at pH values of 6.0, 6.5, 7.5 and 8.0. The initial pH in the simulated wastewater was adjusted to 7 after comprehensive consideration for subsequent testing.
(5) Influence of carbon source on degradation of ammonia nitrogen by bacterial strains
Glucose, sucrose, starch, sodium acetate, sodium succinate and sodium bicarbonate are respectively used as the only carbon sources in the simulated wastewater, the initial ammonia nitrogen concentration is 100mg/L, the p H value is regulated to 7, 1% of inoculum size is inoculated into a target strain, the ammonia nitrogen concentration of the simulated wastewater is measured after shaking culture is carried out for 3 days under the conditions of 35 ℃ and 180r/min, and the ammonia nitrogen degradation rate is calculated. The results are shown in FIG. 9. The degradation rate of the strain SQ-1A is 77.57% when sodium acetate is used as a carbon source, and the degradation rate of the strain SQ-1A is 62.40% when sodium succinate and sucrose are used as carbon sources, and the degradation rate of the strain SQ-1A is 54.30% when sodium succinate and sucrose are used as carbon sources. The effects are worst when starch and sodium bicarbonate are used as carbon sources. In general, the utilization rate of the bacteria on inorganic carbon sources is significantly higher than that of the bacteria on organic carbon sources. In comprehensive consideration, sodium acetate is continuously selected as the only carbon source in the simulated wastewater for research in the subsequent experiments.
(6) Effect of carbon-nitrogen ratio on degradation of ammonia nitrogen by bacterial strains
Sodium acetate is used as a unique carbon source, the carbon nitrogen ratio in the simulated wastewater is adjusted to be 3:1,5:1, 8:1, 10:1 and 15:1 respectively, the concentration is set to be 100mg/L, the pH value is adjusted to be 7, 1% of inoculation amount is used for inoculating a target strain, and after shaking culture is carried out for 3 days at 35 ℃ and 180r/min, the ammonia nitrogen concentration is measured and the ammonia nitrogen degradation rate is calculated. The results are shown in FIG. 10. The ammonia nitrogen degradation rate of the strain SQ-1A under the conditions of 3:1 and 5:1 of the carbon nitrogen ratio is generally 58.45 percent and 75.65 percent respectively. The degradation rate of ammonia nitrogen is good under the conditions of the carbon nitrogen ratio of 8:1 and 10:1, and is 86.50 percent and 88.70 percent respectively. The degradation rate of ammonia nitrogen is optimal at the condition of 15:1 of carbon nitrogen ratio and is 89.23%. In general, the higher the C/N, the more favorable the growth of nitrifying bacteria, as the results of this example are. Generally, the higher the C/N, the more favorable the growth of nitrifying bacteria, but when the C/N in the simulated wastewater is gradually increased from 8:1 to 15:1, although the ammonia nitrogen degradation efficiency of the strain is improved, the improvement effect is not obvious, and the ammonia nitrogen degradation rate of the target strain shows a gradual and stable trend, which means that in the embodiment, the larger the C/N ratio is, the better the C/N ratio is. Synthesizing ammonia nitrogen degradation conditions of the strain under different C/N, and selecting a C/N of 15:1 as an optimal condition.
The optimal culture condition of the strain Escherichia coli SQ-1A ammonia nitrogen degrading bacterium in simulated wastewater is that the initial ammonia nitrogen concentration is 100mg/L, the culture temperature is 35 ℃, the inoculation amount is 1%, the pH value is 7.0, sodium acetate is used as the sole carbon source, the carbon nitrogen ratio is 15:1, and the ammonia nitrogen degradation rate can reach 89.23% after 3d culture under the optimal condition.
The foregoing is a further detailed description of the application in connection with the preferred embodiments, and it is not intended that the application be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the application, and these should be considered to be within the scope of the application.
SEQUENCE LISTING
<110> Hunan agricultural university
<120> an escherichia coli, and products and applications thereof
<130> 2022.5.18
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1350
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 1
gctgacgagt ggcggacggg tgagtaatgt ctgggaaact gcctgatgga gggggataac 60
tactggaaac ggtagctaat accgcataac gtcgcaagac caaagagggg gaccttcggg 120
cctcttgcca tcggatgtgc ccagatggga ttagcttgtt ggtggggtaa cggctcacct 180
aggcgacgat ccctagctgg tctgagagga tgaccagcca cactggaact gagacacggt 240
ccagactcct acgggaggca gcagtgggga atattgcaca atgggcgcaa gcctgatgca 300
gccatgccgc gtgtatgaag aaggccttcg ggttgtaaag tactttcagc ggggaggaag 360
ggagtaaagt taataccttt gctcattgac gttacccgca gaagaagcac cggctaactc 420
cgtgccagca gccgcggtaa tacggagggt gcaagcgtta atcggaatta ctgggcgtaa 480
agcgcacgca ggcggtttgt taagtcagat gtgaaatccc cgggctcaac ctgggaactg 540
catctgatac tggcaagctt gagtctcgta gaggggggta gaattccagg tgtagcggtg 600
aaatgcgtag agatctggag gaataccggt ggcgaaggcg gccccctgga cgaagactga 660
cgctcaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 720
aaacgatgtc gacttggagg ttgtgccctt gaggcgtggc ttccggagct aacgcgttaa 780
gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga cgggggcccg 840
cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta cctggtcttg 900
acatccacgg aagttttcag agatgagaat gtgccttcgg gaaccgtgag acaggtgctg 960
catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtcccgcaac gagcgcaacc 1020
cttatccttt gttgccagcg gtccggccgg gaactcaaag gagactgcca gtgataaact 1080
ggaggaaggt ggggatgacg tcaagtcatc atggccctta cgaccagggc tacacacgtg 1140
ctacaatggc gcatacaaag agaagcgacc tcgcgagagc aagcggacct cataaagtgc 1200
gtcgtagtcc ggattggagt ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg 1260
tggatcagaa tgccacggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1320
tgggagtggg ttgcaaaaga agtaggtagc 1350
<210> 2
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<213> artificial sequence
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agagtttgat cctggctcag 20
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aaggaggtga tccagccgca 20
Claims (5)
1. The Escherichia coli is Escherichia coli (Escherichia coli) SQ-1A, and the preservation number of the Escherichia coli is CGMCC No. 23284 in the China general microbiological culture Collection center.
2. The use of the escherichia coli according to claim 1 for degrading ammonia nitrogen in wastewater.
3. The application of escherichia coli in degrading ammonia nitrogen in wastewater according to claim 2, wherein the pH value of a degradation solution is 6-8, the degradation temperature is 20-40 ℃, the initial concentration of ammonia nitrogen in wastewater is 100-900mg/L, and the inoculation amount of escherichia coli is 0.5% -10%.
4. A microbial inoculum for degrading ammonia nitrogen in wastewater, wherein the microbial inoculum for degrading ammonia nitrogen in wastewater comprises the escherichia coli as set forth in claim 1.
5. The microbial inoculum for degrading ammonia nitrogen in wastewater according to claim 4, wherein the preparation method of the microbial inoculum for degrading ammonia nitrogen in wastewater is that after the escherichia coli is subjected to activation culture by a liquid culture medium, a culture solution is collected.
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