CN114958669A - Klebsiella Michii and product and application thereof - Google Patents
Klebsiella Michii and product and application thereof Download PDFInfo
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Abstract
The invention discloses Klebsiella michiganensis, a product and application thereof, and is characterized in that the Klebsiella michiganensis WF-2 is named as Klebsiella micellanensis WF-2, and the preservation number of the Klebsiella michiganensis WF-2 in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 23386. The Klebsiella michigans is obtained by enriching, separating and purifying the wastewater in a pig farm, the highest degradation rate of ammonia nitrogen in the wastewater can reach 92.37%, the good degradation effect is reflected, the Klebsiella michigans has the characteristics of strong specificity, short time consumption and high removal efficiency, and can be applied to removing or reducing the residue of ammonia nitrogen in the environment, so that a safe and environment-friendly microbial degradation method is provided for the problem of ammonia nitrogen residue.
Description
Technical Field
The invention belongs to the technical field of biodegradation of ammonia nitrogen in wastewater, and particularly relates to Klebsiella Michii and a product and application thereof.
Background
The nitrogen pollution caused by untreated livestock and poultry breeding wastewater is particularly serious, wherein the ammonia nitrogen pollution is the most prominent. The livestock wastewater containing high-concentration ammonia nitrogen is directly discharged into a water body, the ammonia nitrogen in the wastewater is oxidized into nitrate nitrogen and nitrite nitrogen under the action of nitrobacteria, about 4.6mg of dissolved oxygen needs to be consumed when 1mg of ammonia nitrogen is completely oxidized, and the content of the dissolved oxygen in the water body is sharply reduced due to the consumption of a large amount of the dissolved oxygen. The excessive nitrogen in the water body can also increase the number of photosynthetic microorganisms (mainly algae), so that the eutrophication phenomenon occurs, the water body becomes black and smelly, the transparency is reduced, the water quality environment is deteriorated, and a great amount of fish and other organisms in the water are killed.
The investigation of China Taihu lake pollution sources shows that the livestock and poultry breeding wastewater is one of the important pollution sources causing nitrogen pollution and eutrophication of Taihu lake drainage basins. When livestock and poultry wastewater containing high-concentration ammonia nitrogen is used for irrigating farmlands, soil pores can be blocked, so that the air permeability and the water permeability of soil are reduced, the quality of the soil is deteriorated, and the yield of plants is reduced or even the plants die. If the excessive nitrogen element in the water can not be effectively absorbed, the excessive nitrogen element is easy to run off into surface water or be leached into underground water through air-permeable soil, so that water body pollution is caused.
The Chinese patent CN110452836A provides a microbial strain for degrading ammonia nitrogen, wherein the strain is a nutrient Psychrobacter (Psychrobacter cibarius) Z-XWW G, the nutrient Psychrobacter (Psychrobacter cibarius) Z-XWW G is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.17515 and a Bacillus zhangshuensis Z-XWW 77, the Bacillus zhangshuuensis Z-XWW 77 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.17516, and a comparison document also provides an application of the strain and a composite microorganism containing the strain in degrading ammonia nitrogen, but the efficiency of the composite microorganism group in treating waste water with ammonia nitrogen is lower.
Disclosure of Invention
In order to overcome the problems in the prior art, the application provides the Klebsiella Michii and the product and the application thereof, and the specific method is to screen out a degrading bacterium which has strong specificity, short time consumption and high removal efficiency and can degrade ammonia nitrogen in wastewater from pig farm wastewater, thereby providing abundant microbial resources for treating livestock and poultry breeding wastewater.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides Klebsiella Michii in a first aspect, the Klebsiella Michii is named as Klebsiella microtiganensis WF-2, and the preservation number of the Klebsiella Michii in the China general microbiological culture Collection center is CGMCC NO. 23386.
As an alternative embodiment, the present invention provides Klebsiella michiganensis having a nucleotide sequence as set forth in SEQ ID No. 1.
As an alternative embodiment, the Klebsiella Michii is obtained by enriching, separating and purifying the wastewater of a pig farm containing activated sludge, wherein the culture temperature in the enriching and separating process is 28-30 ℃, and the culture time is 20-24 h.
As an alternative embodiment, the basic culture medium used in the enrichment, separation and purification process of the Klebsiella michiganensis provided by the invention is LB culture medium containing ammonium chloride.
As an alternative embodiment, the screening culture medium used in the enrichment, separation and purification process of the Klebsiella Michii provided by the invention is a culture 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 enrichment, separation and purification process of the Klebsiella Michii provided by the invention 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, an 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 Klebsiella Michii provided by the invention 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 Klebsiella michiganensis provided by the invention, the specific process of enrichment, isolation and purification of Klebsiella michiganensis comprises the following steps:
the method comprises the following steps: enrichment, separation and purification of bacterial strain
Wastewater was collected from a certain pig farm and stored in a-20 ℃ refrigerator using a sterile triangular flask. Transferring 10mL of pig farm wastewater containing activated sludge into a triangular flask containing 90mL of enrichment medium, and putting the triangular flask into a shaking incubator to culture for 24h under the conditions of 28 ℃ and 160r/min to obtain enrichment bacterial liquid. Inoculating the enriched bacterial liquid into a primary screening inorganic salt culture medium according to the inoculation amount of 5 percent, culturing for 24 hours under the same condition, and repeating the operation and screening twice. The screened bacterial liquid is coated and diluted by a method of 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 And (3) coating the six concentration gradients on a solid enrichment medium, putting the solid enrichment medium into an incubator at 28 ℃ after coating, and performing inverted culture for 24 hours to complete primary screening. And (3) selecting different single colonies on the solid culture medium, separating and purifying (streaking is repeated for 2-3 times), and freezing the purified single colonies in a refrigerator at-80 ℃ for later use by using a glycerol storage method.
Step two: rescreening of bacterial strains
Performing activation culture on the primarily screened strain in LB liquid culture medium for 24h, taking an appropriate amount of bacterial liquid, centrifuging in a 10mL centrifuge tube, removing supernatant, washing the bacterial body with sterile normal saline (repeating twice), preparing into bacterial suspension, and performing OD (optical density) on the bacterial suspension 600 The values are adjusted to unity. Inoculating the strain into a re-screening inorganic salt culture medium according to the inoculation amount of 5 percent, placing the culture medium in a shaking incubator, culturing for 48 hours at the temperature of 28 ℃ at the speed of 160r/min, and then measuring the ammonia nitrogen concentration by using a multifunctional water quality tester and calculating the degradation rate. Eliminating bacteria with poor degradation capabilityThe strains with high ammonia nitrogen degradation capability are preserved by using glycerin tubes and inclined planes for later use.
Step three: identification of degrading strains
And (3) observing the morphological characteristics of the target strain and the morphology of the bacterial cells under a microscope, and comprehensively identifying the morphology and the strain type of the bacteria by combining a 16SrDNA method.
In a second aspect, the invention provides the use of klebsiella michiganensis according to any one of the embodiments above for degrading ammonia nitrogen in wastewater.
As an optional embodiment, in the application of the Klebsiella Michii in degrading ammonia nitrogen in wastewater, the pH value of the degradation solution is 6-8 and the degradation temperature is 20-40 ℃ in the process of degrading ammonia nitrogen in wastewater by the Klebsiella Michii.
As an optional implementation mode, in the application of the Klebsiella Michii in degrading ammonia nitrogen in wastewater, the initial concentration of ammonia nitrogen in wastewater is 100-900mg/L, and the inoculation amount of the Klebsiella Michii is 0.5% -10%.
The third aspect of the invention provides a microbial inoculum for degrading ammonia nitrogen in wastewater, wherein the microbial inoculum for degrading ammonia nitrogen in wastewater contains Klebsiella Michii in any one of the above embodiments.
As an optional implementation manner, in the microbial inoculum for degrading ammonia nitrogen in wastewater provided by the invention, the preparation method of the microbial inoculum for degrading ammonia nitrogen in wastewater is to perform activated culture on klebsiella michiganensis by using a liquid culture medium, and then collect a culture solution to obtain the microbial inoculum.
The invention has the following beneficial effects:
the invention obtains a strain of ammonia nitrogen high-efficiency degrading bacteria through screening, the strain is identified as Klebsiella microorganissis sp of the Klebsiella Michii through the biochemical identification and the 16S rRNA sequence analysis, and the strain is named as Klebsiella microorganissis WF-2. The preservation number is: CGMCC NO. 23386. The Klebsiella Michii has a degradation rate of 92.37% to the highest extent, shows good degradation effect, 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.
Drawings
FIG. 1 is a colony morphology map of the strain Klebsiella microtiganensis WF-2 of example 1;
FIG. 2 is a morphological diagram under a microscope of the strain Klebsiella microtiganensis WF-2 of example 1;
FIG. 3 is a phylogenetic tree of the strain Klebsiella microorganissis WF-2 according to example 2;
FIG. 4 is a graph showing the growth of the strain Klebsiella microtiganensis WF-2 of example 2;
FIG. 5 is a graph showing the results of the ammonia nitrogen concentration in the wastewater in example 3 on degradation of ammonia nitrogen by the Klebsiella microtiganensis WF-2 strain;
FIG. 6 is a graph showing the results of the ammonia nitrogen degradation by the temperature versus the strain Klebsiella microtiganensis WF-2 in example 3;
FIG. 7 is a graph showing the results of the ammonia nitrogen degradation experiment using the inoculum size of Klebsiella microorganissis WF-2 strain in example 3;
FIG. 8 is a graph showing the results of the ammonia nitrogen degradation experiment by the pH in the wastewater in example 3 using the Klebsiella microtiganensis WF-2 strain;
FIG. 9 is a graph showing the results of the experiment in example 3 for simulating the degradation of ammonia nitrogen by the carbon source strain Klebsiella microorganissis WF-2 in wastewater;
FIG. 10 is a graph showing the results of the experiment in example 3 for simulating the degradation of ammonia nitrogen in wastewater by carbon nitrogen ratio in wastewater compared to the strain Klebsiella microtiganensis WF-2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the description herein, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples" or the like 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, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. 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 equipment required in the experimental process
The reagents used in the following examples are, unless otherwise indicated, conventional in the art, and the equipment and methods are conventional in the art.
The method for configuring the culture medium used by the invention comprises the following steps:
1. enrichment medium (g/L): 0.382g of ammonium chloride; 25g of LB culture medium; 1000mL of distilled water; the pH was 7.0.
2. Primary screening of inorganic salt culture medium (g/L): 2.0g of sodium acetate; magnesium sulfate heptahydrate 0.05 g; dipotassium phosphate 0.2 g; 0.12g of sodium chloride; 0.01g of manganese sulfate; 0.01g of ferrous sulfate; 1.9g of ammonium chloride; 1000mL of distilled water; the pH was 7.0.
3. Re-screening inorganic salt culture medium (g/L): 2.0g of sodium acetate; magnesium sulfate heptahydrate 0.05 g; dipotassium phosphate 0.2 g; 0.12g of sodium chloride; 0.01g of manganese sulfate; 0.01g of ferrous sulfate; changing the content of ammonium chloride to control the concentration of ammonia nitrogen; 1000mL of distilled water; the pH was 7.0.
4. Solid enrichment medium (g/L): ammonium chloride 0.382; 25g of LB culture medium; 15g of agar; 1000mL of distilled water; the pH was 7.0.
5. Initial medium (g/L): 2.0g of sodium acetate; magnesium sulfate heptahydrate 0.05 g; dipotassium hydrogen phosphate 0.2 g; 0.12g of sodium chloride; 0.01g of manganese sulfate; 0.01g of ferrous sulfate; 0.382g of ammonium chloride; 1000mL of distilled water; the pH was 7.0.
Example 1
The application discovers a degrading bacterium capable of degrading ammonia nitrogen, wherein the degrading bacterium is Klebsiella microorganisensis sp of the genus Klebsiella Michii, is delivered to the China general microbiological culture Collection center for preservation in 10 days 09 months in 2021, and has the name: klebsiella microorganissis WF-2 with the preservation number of CGMCC NO. 23386. Address: the west road of the morning-yang district in Beijing of China.
The Klebsiella Michii (Klebsiella microorganissis WF-2) is obtained by enriching, separating and purifying the wastewater in a pig farm, and the specific steps are as follows:
the method comprises the following steps: enrichment, separation and purification of bacterial strain
Wastewater was collected from a certain pig farm and stored in a-20 ℃ refrigerator using a sterile triangular flask. Transferring 10mL of pig farm wastewater containing activated sludge into a triangular flask containing 90mL of enrichment medium, and putting the triangular flask into a shaking incubator to culture for 24h under the conditions of 28 ℃ and 160r/min to obtain enrichment bacterial liquid. Inoculating the enriched bacterial liquid into a primary screening inorganic salt culture medium according to the inoculation amount of 5 percent, culturing for 24 hours under the same condition, and repeating the operation and screening twice. The screened bacterial liquid is coated and diluted by a method of 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 And (3) coating the six concentration gradients on a solid enrichment medium, putting the solid enrichment medium into an incubator at 28 ℃ after coating, and performing inverted culture for 24 hours to complete primary screening. And (3) selecting different single colonies on the solid culture medium, separating and purifying (streaking is repeated for 2-3 times), and freezing the purified single colonies in a refrigerator at-80 ℃ for later use by using a glycerol storage method.
Step two: rescreening of bacterial strains
Activating and culturing the primarily screened strain in LB liquid culture medium for 24h, taking a proper amount of bacterium liquid, centrifuging in a 10mL centrifuge tube, removing supernatant, washing the bacterium with sterile normal saline (repeating twice) to prepare bacterium suspension,OD of the bacterial suspension 600 The values are adjusted to unity. Inoculating into a double-sieve inorganic salt culture medium according to the inoculation amount of 5%, placing in a shaking incubator, culturing at 28 ℃ and 160r/min for 48h, and measuring the ammonia nitrogen concentration by using a multifunctional water quality analyzer (LH-3BA) and calculating the degradation rate. Eliminating strains with poor degradation capability, and storing strains with high ammonia nitrogen degradation capability by using a glycerin pipe and an inclined plane for later use.
Step three: identification of degrading strains
And (4) observing the morphological characteristics of the target strain and the morphology of the bacterial cells under a microscope, and combining a 16SrDNA method to comprehensively identify the morphology of the bacteria and the species of the bacteria.
The colony morphology observation method comprises the following steps: and (2) carrying out enrichment culture on the target strain in an enrichment liquid culture medium for 24h, inoculating the target strain into a solid enrichment culture medium by using a scribing method, carrying out inverted culture on an inoculated flat plate in a constant-temperature incubator at 28 ℃ for 48h, taking out the flat plate from the incubator, and observing morphological characteristics (size, shape, transparency, color and the like) of a bacterial colony.
The microscopic observation method of the bacterial morphology comprises the following steps:
tabletting and fixing: clamping a clean glass slide by using tweezers, dropping a small drop of distilled water on the glass slide, dipping a small amount of thalli in an enrichment liquid culture medium by using an inoculating ring, uniformly coating the bacterial strain in the distilled water drop, and putting the glass slide on an alcohol lamp for heating and drying to fix the thalli.
Dyeing: after the glass slide is cooled, dripping crystal violet dye solution by using a rubber head dropper for dyeing for 1 minute, flushing the glass slide by slow water flow, washing off floating color, drying the glass slide, and dripping cedar oil.
Microscopic examination: the glass slide is fixed on an object stage, the aperture and the coarse focusing helix are adjusted, the observation is carried out by using a low power lens, and when a clear object image appears in the visual field, the bacterial form is observed by changing a high power oil lens.
After the target strain is cultured on a flat plate (LB solid medium) for 24 hours, the morphological characteristics of a bacterial colony are shown in figure 1, and the bacterial colony WF-2 is a round, moist, semitransparent, milky white bacterial colony with regular edges; the morphology of each strain observed under an oil lens is shown in FIG. 2.
Extraction of genomic DNA: the method is carried out according to the operation steps of "bacterial genome DNA extraction kit" of Tiangen. And the quality of the DNA was checked by agarose gel electrophoresis. And (3) PCR amplification: selecting a bacterial 16S rDNA universal primer, wherein the sequence of the primer is as follows:
an upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3', the sequence is shown in SEQ ID NO. 2;
a downstream primer: 5'-AAGGAGGTGATCCAGCCGCA-3', the sequence is shown in SEQ ID NO. 3;
after the PCR product was purified by the TIANGEN gel recovery kit, sequencing (two-way sequencing) was performed by Biotechnology (Shanghai) Co., Ltd., the spliced sequencing results were subjected to sequence alignment analysis in GenBank database, sequence alignment was performed with Clustal X1.8 software, and a phylogenetic evolutionary tree was constructed with MEGA7 software as shown in FIG. 3.
As a result of phylogenetic analysis, it was found that the strain had close relationship to Klebsiella microorganissis (XS3-1) and the homology was 99%, and that the strain WF-2 was Klebsiella Michii (Klebsiella microorganissis). The nucleotide sequence of Klebsiella microtiganensis WF-2 is shown in SEQ ID NO. 1.
Example 2
Drawing a growth curve of the Klebsiella microtiganensis WF-2 strain.
Inoculating the frozen strain solution of glycerol in example 1 into LB liquid culture medium containing 100mL for activation culture, culturing in a shaking incubator at 28 deg.C and 160r/min for 16-18 hours to obtain seed solutions, inoculating the seed solutions into 14 bottles of activation liquid culture medium with the inoculum size of 5%, culturing in the shaking incubator at 28 deg.C and 160r/min for 28 hours, and measuring OD of the strain solution every 2 hours by using an ultraviolet spectrophotometer 600 The control group was an activated liquid medium without inoculation. The growth curve is shown in fig. 4. Bacteria cultured for 10-14 h have vigorous life metabolic activity and stable characteristics. The bacterial liquid cultured for 10-14 h is most suitable to be used as the inoculation liquid for the subsequent example study.
Example 3
The optimal condition for degrading ammonia nitrogen by the bacterial strain Klebsiella michiganensis WF-2.
Preparing an inoculation solution; inoculating the screened target strain into liquid activation culture medium, culturing at 28 deg.C and 160r/min to logarithmic phase of strain growth, centrifuging at high speed in a centrifuge tube under aseptic condition, removing supernatant, washing thallus with sterile normal saline for 2-3 times, making into bacterial suspension, adjusting OD of bacterial solution 600 After the strains are unified, the strains are inoculated into simulated wastewater of different treatments in an inoculation amount of 5 percent to study the degradation characteristics of the ammonia nitrogen in the simulated wastewater.
The calculation method of the ammonia nitrogen degradation rate comprises the following steps: removal efficiency (%) - (C) 0 -C t )/C 0 100% wherein: c 0 And C t (mg/L) is ammonia nitrogen initial concentration and concentration at time t respectively.
(1) Influence of ammonia nitrogen concentration in wastewater on degradation of ammonia nitrogen by bacterial strain
The target strain is inoculated into simulated wastewater with the pH value of 7.0, the ammonia nitrogen concentration in the simulated wastewater is adjusted to be 100mg/L, 300mg/L, 500mg/L, 700mg/L and 900mg/L respectively, the ammonia nitrogen concentration is measured and the ammonia nitrogen degradation rate is calculated after shaking culture is carried out for 3d under the conditions of 30 ℃ and 180 r/min. The results are shown in FIG. 5. The degradation rates of the bacterial strain WF-2 in the initial ammonia nitrogen concentration of 100mg/L, 300mg/L, 500mg/L, 700mg/L and 900mg/L are 71.65%, 49.99%, 42.93%, 26.31% and 21.89% respectively. The degradation rate is obviously higher than that of the initial ammonia nitrogen concentration of 100 mg/L. Therefore, the degradation rate of the strain under different initial ammonia nitrogen concentrations is integrated, and simulated wastewater with the initial ammonia nitrogen concentration of 100mg/L is selected for subsequent tests.
(2) Influence of temperature on degradation of ammonia nitrogen by bacterial strain
Setting the initial ammonia nitrogen concentration of the simulated wastewater to be 100mg/L, adjusting the pH value to 7.0, respectively placing the simulated wastewater inoculated with the target strain in incubators at 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃, performing shaking culture for 3d under the condition of 180r/min, then measuring the ammonia nitrogen concentration and calculating the ammonia nitrogen degradation rate. The results are shown in FIG. 6. From FIG. 6, we can see that the ammonia nitrogen degradation rate of the bacterial strain WF-2 reaches 68.10% at the maximum temperature of 35 ℃, 30 ℃ and 67.16% at the second time. The strain belongs to mesophilic bacteria, when the strain is cultured at 35-40 ℃, the degradation capability of ammonia nitrogen in simulated wastewater has no obvious change, the temperature is not necessarily required to be constant in subsequent large-scale culture, but after comprehensive consideration, the culture temperature in subsequent experiments is adjusted to about 35 ℃ as appropriate.
(3) Influence of inoculation amount on degradation of ammonia nitrogen by bacterial strain
Inoculating target strains in the simulated wastewater with the initial ammonia nitrogen concentration of 100mg/L and the pH value of 7 according to the inoculation amounts of 0.5%, 1%, 2%, 3%, 4%, 5% and 10%, respectively, performing shake culture at 35 ℃ and 180r/min for 3d, then 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 bacterial strain WF-2 is inoculated into the simulated wastewater by 0.5-1%, the ammonia nitrogen degradation capability is stronger, 77.43% and 76.53% of ammonia nitrogen content in the simulated wastewater can be respectively removed, the ammonia nitrogen degradation capability of the bacterial 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 capability of the bacterial strain is only 27.1%. In consideration of practical conditions, the target strain in subsequent experiments is inoculated into the simulated wastewater at an inoculation amount of 0.5%.
(4) Influence of pH value on degradation of ammonia nitrogen by bacterial strain
Adjusting the pH value of the simulated wastewater to be 6.0, 6.5, 7.0, 7.5 and 8.0 respectively, inoculating the initial ammonia nitrogen concentration of 100mg/L into a target strain in an inoculation amount of 0.5%, performing shake culture at 35 ℃ and 180r/min for 3 days, then measuring the ammonia nitrogen concentration of the simulated wastewater and calculating the ammonia nitrogen degradation rate. The results are shown in FIG. 8. The ammonia nitrogen removal efficiency of the bacterial strain WF-2 in simulated wastewater with the initial pH value of 7 is the maximum and reaches 86.2%, and the ammonia nitrogen degradation rates are 78.7%, 85.0%, 83.8% and 81.3% 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 experiments.
(5) Influence of carbon source on degradation of ammonia nitrogen by bacterial strain
Respectively taking glucose, sucrose, starch, sodium acetate, sodium succinate and sodium bicarbonate as the only carbon source in the simulated wastewater, adjusting the initial ammonia nitrogen concentration to be 100mg/L, adjusting the pH value to be 7, inoculating a target strain with 0.5 percent of inoculum size, measuring the ammonia nitrogen concentration of the simulated wastewater after shaking culture for 3 days at 35 ℃ and 180r/min, and calculating the ammonia nitrogen degradation rate. The results are shown in FIG. 9. The degradation rate of the bacterial strain WF-2 is 80.22 percent when sodium acetate is used as a carbon source, and the effects are 63.63 percent and 50.85 percent when sodium succinate and sucrose are used as carbon sources respectively. The effect was poor with starch, sodium bicarbonate and glucose as carbon sources. In general, the utilization rate of inorganic carbon source is obviously higher than that of organic carbon source. Comprehensively considering, sodium acetate is continuously selected as the only carbon source in the simulated wastewater for research in the subsequent experiments.
(6) Influence of carbon nitrogen ratio on degradation of ammonia nitrogen by bacterial strain
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, a target strain is inoculated in an inoculation amount of 0.5%, the ammonia nitrogen concentration is measured and the ammonia nitrogen degradation rate is calculated after shaking culture is carried out for 3 days at the temperature of 35 ℃ and at the speed of 180 r/min. The results are shown in FIG. 10. The ammonia nitrogen degradation rate of the bacterial strain WF-2 is generally 57.38% and 77.5% respectively under the conditions that the carbon-nitrogen ratio is 3:1 and 5: 1. The ammonia nitrogen degradation rate is good under the conditions of 8:1 carbon nitrogen ratio and 10:1 carbon nitrogen ratio, wherein the ammonia nitrogen degradation rate is 90.06% and 89.45% respectively. The ammonia nitrogen degradation rate is optimum and is 92.37% under the condition that the carbon nitrogen ratio is 15: 1. Generally, the higher the C/N, the more favorable the growth of nitrifying bacteria, which is the same as the results of this example. Generally, the higher the C/N, the more beneficial the growth of nitrifying bacteria, but when the C/N in the simulated wastewater is gradually increased from 8:1 to 15:1, the ammonia nitrogen degradation efficiency of the strain is improved in the process, but the improvement effect is not obvious, the ammonia nitrogen degradation rate of the target strain shows a trend of being gradually stable, which indicates that in the embodiment, the larger the C/N ratio is, the better the C/N ratio is. The ammonia nitrogen degradation conditions of the strains under different C/N are integrated, and the optimal condition that the C/N is 15:1 is selected.
The optimal culture condition of the Klebsiella microorganisnsis WF-2 ammonia nitrogen degrading bacteria in simulated wastewater is that the initial ammonia nitrogen concentration is 100mg/L, the culture temperature is 35 ℃, the inoculum size is 0.5%, the pH value is 7.0, sodium acetate is used as a unique carbon source, the carbon-nitrogen ratio is 15:1, the bacteria are cultured for 3 days under the optimal condition, and the ammonia nitrogen degradation rate can reach 92.37%.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and is not intended to limit the practice of the invention to these embodiments. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
SEQUENCE LISTING
<110> Hunan university of agriculture
<120> Klebsiella Michii, and product and application thereof
<130> 2022.5.20
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1405
<212> DNA
<213> Klebsiella Michii (Klebsiella microtiganensis)
<400> 1
tgcagtcgaa cggtagcaca gagagcttgc tctcgggtga cgagtggcgg acgggtgagt 60
aatgtctggg aaactgcctg atggaggggg ataactactg gaaacggtag ctaataccgc 120
ataacgtcgc aagaccaaag agggggacct tcgggcctct tgccatcaga tgtgcccaga 180
tgggattagc tagtaggtgg ggtaacggct cacctaggcg acgatcccta gctggtctga 240
gaggatgacc agccacactg gaactgagac acggtccaga ctcctacggg aggcagcagt 300
ggggaatatt gcacaatggg cgcaagcctg atgcagccat gccgcgtgta tgaagaaggc 360
cttcgggttg taaagtactt tcagcgagga ggaaggcgat acggttaata accgtgtcga 420
ttgacgttac tcgcagaaga agcaccggct aactccgtgc cagcagccgc ggtaatacgg 480
agggtgcaag cgttaatcgg aattactggg cgtaaagcgc acgcaggcgg tctgtcaagt 540
cggatgtgaa atccccgggc tcaacctggg aactgcattc gaaactggca ggcttgagtc 600
ttgtagaggg gggtagaatt ccaggtgtag cggtgaaatg cgtagagatc tggaggaata 660
ccggtggcga aggcggcccc ctggacaaag actgacgctc aggtgcgaaa gcgtggggag 720
caaacaggat tagataccct ggtagtccac gccgtaaacg atgtcgactt ggaggttgtg 780
cccttgaggc gtggcttccg gagctaacgc gttaagtcga ccgcctgggg agtacggccg 840
caaggttaaa actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta 900
attcgatgca acgcgaagaa ccttacctac tcttgacatc cagagaactt agcagagatg 960
ctttggtgcc ttcgggaact gtgagacagg tgctgcatgg ctgtcgtcag ctcgtgttgt 1020
gaaatgttgg gttaagtccc gcaacgagcg caacccttat cctttgttgc cagcggttcg 1080
gccgggaact caaaggagac tgccagtgat aaactggagg aaggtgggga tgacgtcaag 1140
tcatcatggc ccttacgagt agggctacac acgtgctaca atggcatata caaagagaag 1200
cgacctcgcg agagcaagcg gacctcataa agtatgtcgt agtccggatt ggagtctgca 1260
actcgactcc atgaagtcgg aatcgctagt aatcgtggat cagaatgcca cggtgaatac 1320
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca aaagaagtag 1380
gtagcttaac cttcgggagg gcgct 1405
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
Claims (10)
1. The Klebsiella Michii is characterized in that the Klebsiella Michii is named as Klebsiella microtiganensis WF-2, and the preservation number of the Klebsiella Michii in the China general microbiological culture Collection center is CGMCC NO. 23386.
2. The Klebsiella michiganensis according to claim 1, wherein the Klebsiella michiganensis has the nucleotide sequence shown in SEQ ID No. 1.
3. The Klebsiella Michii according to claim 1 or 2, wherein the Klebsiella Michii is obtained by enriching, separating and purifying from the wastewater of a pig farm containing activated sludge, the culture temperature in the enriching and separating process is 28-30 ℃, and the culture time is 20-24 h.
4. The Klebsiella Michii of claim 3, wherein the basal medium used in the enrichment, isolation and purification process of Klebsiella Michii is LB medium containing ammonium chloride.
5. The Klebsiella Michii of claim 3, wherein the screening medium used in the enrichment, isolation and purification process of Klebsiella Michii is a medium comprising sodium acetate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, sodium chloride, manganese sulfate, ferrous sulfate, and ammonia nitrogen solution.
6. The Klebsiella Michii of claim 3, wherein the carbon source in the simulated wastewater used in the enrichment, isolation and purification process of Klebsiella Michii comprises at least one of glucose, sucrose, starch, sodium acetate, sodium succinate or sodium bicarbonate, and the ratio of carbon to nitrogen in the simulated wastewater is 3-15: 1.
7. Use of the Klebsiella michiganensis according to any one of claims 1-6 for degrading ammonia nitrogen in wastewater.
8. The application of Klebsiella michiganensis in degrading ammonia nitrogen in wastewater according to claim 7, wherein the pH value of the degradation solution is 6-8, the degradation temperature is 20-40 ℃, the initial concentration of ammonia nitrogen in wastewater is 900mg/L, and the inoculation amount of the Klebsiella michiganensis is 0.5% -10% in the process of degrading ammonia nitrogen in wastewater.
9. A bacterial agent for degrading ammonia nitrogen in wastewater, which is characterized in that the bacterial agent for degrading ammonia nitrogen in wastewater contains the Klebsiella michiganensis according to any one of claims 1 to 6.
10. The microbial inoculum for degrading ammonia nitrogen in wastewater as claimed in claim 9, wherein the microbial inoculum for degrading ammonia nitrogen in wastewater is prepared by performing activated culture on klebsiella michiganensis by using a liquid culture medium, and collecting a culture solution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116144531A (en) * | 2022-11-21 | 2023-05-23 | 中国水产科学研究院黑龙江水产研究所 | Phosphate-dissolving bacteria for promoting crop growth and application thereof |
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| CN104152367A (en) * | 2013-12-27 | 2014-11-19 | 井冈山大学 | Heterotrophic nitrification bacterial strain |
| CN108342339A (en) * | 2018-01-16 | 2018-07-31 | 浙江双良商达环保有限公司 | Klebsiella bacterial strain and its application of sanitary sewage containing ammonia nitrogen in river sewage and rural area |
| CN110564653A (en) * | 2019-10-09 | 2019-12-13 | 常州新东化工发展有限公司 | Klebsiella michiganensis and application thereof in production of1, 3-propylene glycol |
| CN111718867A (en) * | 2020-05-29 | 2020-09-29 | 北京理工大学 | Petroleum aromatic hydrocarbon degrading strain PB3 for producing biosurfactant and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152367A (en) * | 2013-12-27 | 2014-11-19 | 井冈山大学 | Heterotrophic nitrification bacterial strain |
| CN108342339A (en) * | 2018-01-16 | 2018-07-31 | 浙江双良商达环保有限公司 | Klebsiella bacterial strain and its application of sanitary sewage containing ammonia nitrogen in river sewage and rural area |
| CN110564653A (en) * | 2019-10-09 | 2019-12-13 | 常州新东化工发展有限公司 | Klebsiella michiganensis and application thereof in production of1, 3-propylene glycol |
| CN111718867A (en) * | 2020-05-29 | 2020-09-29 | 北京理工大学 | Petroleum aromatic hydrocarbon degrading strain PB3 for producing biosurfactant and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116144531A (en) * | 2022-11-21 | 2023-05-23 | 中国水产科学研究院黑龙江水产研究所 | Phosphate-dissolving bacteria for promoting crop growth and application thereof |
| CN116144531B (en) * | 2022-11-21 | 2023-09-12 | 中国水产科学研究院黑龙江水产研究所 | Phosphate-dissolving bacteria for promoting crop growth and application thereof |
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