Pseudomonas fluorescens as well as application and preparation thereof

Abstract

The invention provides pseudomonas fluorescens MPEB0009802 which is preserved in Guangdong Province Microbial Culture Collection Center (GDMCC), the preservation address is the 5th floor of the building 59, No.100 courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 65112. The pseudomonas fluorescens MPEB0009802 is named as Pseudomonas fluorescens MPEB0009802. The pseudomonas fluorescens MPEB0009802. The invention further provides a preparation, and the preparation comprises the pseudomonas fluorescens. The invention also provides an application of the pseudomonas fluorescens in sewage treatment.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and more particularly to Pseudomonas fluorescens and applications and preparations thereof. Background Art

[0002] In recent years, the livestock and poultry farming industry has developed rapidly, and intensive and high-density farming models have been vigorously promoted. Trace elements are essential nutrients for the growth and development of livestock and poultry, but they are also the main source of pollution in the livestock industry. The wastewater generated by the livestock and poultry farming industry is mainly composed of livestock and poultry manure, feed residues and farm flushing water. Livestock and poultry farming wastewater is an organic polluted wastewater with high pollutant concentration and strong complexity, and its discharge seriously threatens the water ecological environment. At present, the carbon-nitrogen ratio of some water bodies in water pollution is extremely unbalanced (COD concentration 400-20000mg / L, total nitrogen concentration 200-16000mg / L), heavy metal content exceeds the standard (copper and zinc are the most serious), and it is difficult to treat. It is rich in organic matter, nitrogen, phosphorus and other nutrients and energy substances. Directly discharging livestock and poultry wastewater into water bodies will lead to eutrophication of water bodies, destroy the ecological balance of water bodies, and pose a fatal threat to organisms. Therefore, it is imperative to treat aquaculture wastewater and make it meet the discharge standards.

[0003] At present, the technologies used for sewage treatment at home and abroad can be mainly divided into three categories: physical, chemical and biological methods. Among them, the biological method refers to the use of microbial agents for sewage treatment. It has good treatment effects, low probability of rebound in the later period, and will not cause secondary pollution. Therefore, it is currently the most important treatment method. It uses the metabolic activities of microorganisms to transfer, transform and degrade pollutants in water, improve water quality, and at the same time build a water ecosystem with a complete trophic level structure, fundamentally restoring the health of the water system. Summary of the invention

[0004] The invention provides a Pseudomonas fluorescens MPEB0009802, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with a deposit address of 5th floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a deposit number of GDMCC No: 65112.

[0005] The present invention also provides a preparation, comprising the above-mentioned Pseudomonas fluorescens.

[0006] The present invention also provides application of the fluorescent pseudomonas or the preparation in sewage treatment.

[0007] Compared with the prior art, this application has the following beneficial effects:

[0008] (1) The Pseudomonas fluorescens MPEB0009802 of the present application has strong phosphorus-solubilizing ability, can dissolve insoluble phosphates in inorganic phosphorus medium, and can be applied to the treatment of phosphorus-containing sewage and manure pollution, etc., to reduce the harms caused by high-concentration phosphorus to the health of humans and livestock, such as organ tissue lesions, metabolic disorders, reduced immunity and livestock production performance.

[0009] (2) The Pseudomonas fluorescens MPEB0009802 of the present application can degrade ammonia nitrogen. Due to the influence of factors such as livestock and poultry manure and feed residues in livestock and poultry breeding wastewater, the contents of pollutants such as COD, ammonium nitrogen (NH 4+ -N), nitrite nitrogen (NO 2- -N) and hydrogen sulfide are greatly increased, resulting in water eutrophication. This strain can degrade ammonia nitrogen to reduce the pollution caused by breeding wastewater.

[0010] (3) The Pseudomonas fluorescens MPEB0009802 of the present application has the ability to produce siderophores. Siderophores are an important way for microorganisms to absorb iron. In addition, siderophores not only play an important role in microbial nutrition, but also play an important role in environmental applications. Siderophores have an impact on the mobility of some metal ions in the environment, so they can be used to repair heavy metal pollution.

[0011] (4) The Pseudomonas fluorescens MPEB0009802 of the present application has the ability to produce lipase. There are a large amount of organic matter and inorganic mineral elements in the wastewater of the breeding farm. The treatment efficiency of ordinary treatment methods is low, the treatment is not thorough, and secondary pollution is easily formed. In addition, the large discharge of wastewater will also cause water eutrophication and heavy metal exceeding the standard. Using enzyme preparations such as lipase to treat wastewater not only improves the degradation effect of organic matter, but also can recycle livestock and poultry wastewater. Brief Description of the Drawings

[0012] Figure 1 is the colony morphology of the Pseudomonas fluorescens MPEB0009802 of the present application on the solid medium.

[0013] Figure 2 is the Gram-stained cell morphology of the Pseudomonas fluorescens MPEB0009802 of the present application.

[0014] Figure 3 is the phosphorus-solubilizing circle formed by the Pseudomonas fluorescens MPEB0009802 of the present application on the inorganic phosphorus medium.

[0015] Figure 4It is a phosphorus standard curve.

[0016] Figure 5 It is the change curve of the soluble phosphorus content in the supernatant of Pseudomonas fluorescens MPEB0009802 of the present application with the number of culture days.

[0017] Figure 6 It is the growth situation of Pseudomonas fluorescens MPEB0009802 of the present application on the ammonia nitrogen medium.

[0018] Figure 7 It is an ammonia nitrogen standard curve.

[0019] Figure 8 It is the change curve of the ammonia nitrogen content in the supernatant of Pseudomonas fluorescens MPEB0009802 of the present application with the number of culture days.

[0020] Figure 9 It is the growth situation of Pseudomonas fluorescens MPEB0009802 of the present application on the CAS medium.

[0021] Figure 10 It is the growth situation of Pseudomonas fluorescens MPEB0009802 of the present application on the lipase screening medium.

[0022] The Pseudomonas fluorescens MPEB0009802 provided by the present invention, with the taxonomic name of Pseudomonas fluorescens, was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms (GDMCC) on September 9, 2024, with the deposit number: GDMCC No: 65112; the deposit address is: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou. Specific Embodiments

[0023] The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0024] Reagents and media involved in the examples:

[0025] MH broth (1L): Weigh 21.00 g of MH broth, dissolve it in 1000 mL of ddH2O, and autoclave at 121°C for 20 min for standby.

[0026] MH(A) medium (1L): Weigh 36.50 g of MH(A) medium (Mueller-Hinton Agar), add 1000 mL of ddH2O, autoclave at 121°C for 20 min, cool to 50°C, pour the plate, and after cooling and solidifying, invert it for standby.

[0027] Phosphate-solubilizing liquid medium (1 L): Glucose 10.00 g, magnesium chloride hexahydrate 5.00 g, magnesium sulfate heptahydrate 0.25 g, potassium chloride 0.20 g, ammonium sulfate 0.10 g, tricalcium phosphate 5.00 g. Add 1000 mL of ddH2O, adjust the pH to 6.8 - 7.0, and sterilize at 121 °C for 20 min for standby use.

[0028] Phosphate-solubilizing solid medium (1 L): Add 15.00 g of agar to 1 L of phosphate-solubilizing liquid medium. After sterilizing at 121 °C for 20 min, cool to 50 °C, pour the medium into plates. After cooling and solidifying, invert for standby use.

[0029] Ammonia nitrogen liquid medium (1 L): Glucose 10.00 g, dipotassium hydrogen phosphate 1.00 g, potassium dihydrogen phosphate 0.25 g, magnesium sulfate 0.50 g, ferrous sulfate 0.02 g, ammonium sulfate 3.54 g (initial ammonia nitrogen concentration is 750 mg / L), and 1 mL of trace element solution. Add 1000 mL of ddH2O, adjust the pH = 7.0, and sterilize at 121 °C for 20 min for standby use.

[0030] Trace element solution: Weigh 3.90 g of zinc sulfate, 7.00 g of calcium chloride, 5.10 g of manganese sulfate, 1.10 g of ammonium molybdate, 1.60 g of copper sulfate, and 1.60 g of cobalt chloride. Dissolve them in ddH2O, transfer to a 1000 mL volumetric flask, and dilute to the mark.

[0031] Ammonia nitrogen solid medium (1 L): Add 15.00 g of agar to 1 L of ammonia nitrogen liquid medium. After sterilizing at 121 °C for 20 min, cool to 50 °C, pour the medium into plates. After cooling and solidifying, invert for standby use.

[0032] Iron-free solid medium (1 L): Glycerol 15 mL, iron-free casein amino acids 5 g, dipotassium hydrogen phosphate 2.5 g, magnesium sulfate heptahydrate 2.5 g, agar 15.00 g. Add 1000 mL of ddH2O, adjust the pH to 7.4, and sterilize at 121 °C for 20 min for standby use.

[0033] CAS solid medium (1 L): Weigh 10.87 g of CAS detection medium and dissolve it in 1000 mL of ddH2O. Sterilize at 115 °C for 30 min for standby use.

[0034] Lipase screening medium (1 L): Peptone 10.00 g, yeast extract 5.00 g, sodium chloride 5.00 g, calcium chloride 0.10 g, Tween-80 10.00 mL, agar 15.00 g. Add 1000 mL of ddH2O. After sterilizing at 121 °C for 20 min, cool to 50 °C, pour the medium into plates. After cooling and solidifying, invert for standby use.

[0035] Example 1

[0036] Isolation, purification and identification of Pseudomonas fluorescens MPEB0009802

[0037] The Pseudomonas fluorescens MPEB0009802 of the present application was isolated from a soil sample in Songshan Park. The specific steps are as follows:

[0038] Collect the soil sample from Songshan Park, put it into a 15 mL centrifuge tube containing 9 mL of sterile distilled water, mix it with an oscillator, aspirate 10 μL of the mixed liquid onto the MH(A) medium, streak plate, and culture it in an incubator at 35°C for 2 days. Observe the growth status of the colonies regularly. When single colonies grow on the plate, pick a single colony for three consecutive streak separation and purification. Then pick a single colony and inoculate it into the MH broth medium, place it in a shaker for constant temperature culture for 24 h. Part is mixed with glycerol and stored in a cryotube, part is aliquoted and stored in a 1.5 ml centrifuge tube, and part is subjected to 16s rRNA sequencing.

[0039] The Pseudomonas fluorescens MPEB0009802 described in the present application is Gram-negative, forms opaque, grayish-white, slightly convex, round, smooth-surfaced colonies on the plate. The colony morphology is shown in Figure 1 . The scanning electron microscopy morphology of strain MPEB0009802 is shown in Figure 2 , and the bacterium is short rod-shaped with polar flagella.

[0040] Example 2

[0041] Qualitative and quantitative analysis of the phosphorus-solubilizing ability of strain MPEB0009802

[0042] 2.1 Qualitative analysis of the phosphorus-solubilizing ability of strain MPEB0009802

[0043] The phosphorus-solubilizing ability of Pseudomonas fluorescens MPEB0009802 to insoluble phosphorus was preliminarily evaluated using a phosphorus-solubilizing solid medium: Aspirate 10 μL of the overnight culture of MPEB0009802 and spot-inoculate it on the surface of the phosphorus-solubilizing solid medium, place it in an incubator at 35°C for 7 days, then measure the diameter (D) of the phosphorus-solubilizing circle and the diameter (d) of the colony, and calculate the ratio of the phosphorus-solubilizing circle to the colony diameter as 2.36 ± 0.02. The phosphorus-solubilizing phenomenon of MPEB0009802 in the phosphorus-solubilizing solid medium is shown in Figure 3 . According to the ratio, it is judged that Pseudomonas fluorescens MPEB0009802 has strong phosphorus-solubilizing ability.

[0044] 2.2 Quantitative analysis of the phosphorus-solubilizing ability of strain MPEB0009802

[0045] The phosphorus solubilization ability of Pseudomonas fluorescens MPEB0009802 was quantitatively analyzed by the ammonium molybdate spectrophotometric method. Preparation of ammonium molybdate solution: Measure 150 mL of concentrated sulfuric acid and slowly add it to 150 mL of distilled water, stirring continuously and cooling. Weigh 13 g of ammonium molybdate and dissolve it in 100 mL of water. Weigh 0.35 g of potassium antimonyl tartrate and dissolve it in 100 mL of water. While stirring continuously, slowly add the ammonium molybdate solution to 300 mL of sulfuric acid, add the potassium antimonyl tartrate solution and mix well, and store it in a brown reagent bottle. Preparation of ascorbic acid solution: Weigh 10 g of ascorbic acid and dissolve it in water, and make up the volume to 100 mL, and store it in a brown reagent bottle. Preparation of potassium persulfate solution: Weigh 5 g of potassium persulfate, dissolve it in water, and make up the volume to 100 mL. Plotting of the phosphate standard curve: Accurately pipette 0 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL of 2 mg / L phosphate standard working solution (prepared freshly before use) into 25-mL colorimetric tubes respectively, add water to 12.5 mL, and the standard solutions with phosphorus contents of 0.00, 0.08, 0.16, 0.32, 0.48, 0.64, 0.80 mg / L respectively. Add 2 mL of potassium persulfate solution, stopper tightly, and place it in an autoclave for digestion at 121 °C for 30 min. After the autoclaving is completed, take it out and cool, add 0.5 mL of ascorbic acid solution and mix well. After 30 s, add 1 mL of molybdate solution, mix well thoroughly, let it stand at room temperature for 15 min, and measure its absorbance at a wavelength of 700 nm. Taking the absorbance as the abscissa and the phosphorus concentration (mg / L) as the ordinate, plot the standard curve as Figure 4 shown. Inoculate the seed liquid into the phosphorus-solubilizing liquid medium at an inoculation amount of 1% (v / v). At the same time, set up a control group without inoculation, with three replicates in each group. Shake and culture at 35 °C and 180 rpm / min for 7 days. Take the fermented bacterial liquid every 24 h and centrifuge it at 10,000 rpm / min for 10 min. Take the supernatant as the test solution. Pipette 0.5 mL of the supernatant, dilute it with water to 12.5 mL, add 2 mL of potassium persulfate solution, stopper tightly, and place it in an autoclave for digestion at 121 °C for 30 min. After the autoclaving is completed, take it out and cool, add 0.5 mL of ascorbic acid solution and mix well. After 30 s, add 1 mL of molybdate solution, mix well thoroughly, let it stand at room temperature for 15 min, and measure its absorbance at a wavelength of 700 nm. The results are shown in Table 1 and Figure 5 as shown. As the number of culture days increases, the content of soluble phosphorus in the supernatant of the bacterial liquid also increases. On the 5th day of shake culture, the content of soluble phosphorus in the supernatant of the bacterial liquid is 400.07 ± 12.06 mg / L. Calculate the phosphorus solubilization rate according to the phosphorus content in the supernatant. It can be seen that the phosphorus solubilization ability of Pseudomonas fluorescens MPEB0009802 in this application is relatively strong.

[0046] Table 1

[0047]

[0048] Example 3

[0049] Qualitative and quantitative analysis of the ammonia nitrogen removal ability of strain MPEB0009802

[0050] 3.1 Qualitative analysis of the ammonia nitrogen removal ability of strain MPEB0009802

[0051] The ammonia nitrogen degradation ability of Pseudomonas fluorescens MPEB0009802 was preliminarily evaluated using ammonia nitrogen solid medium: 10 μL of the overnight cultured MPEB0009802 bacterial solution was pipetted onto the surface of the ammonia nitrogen solid medium and incubated in an incubator at 35 °C for 7 days to observe whether the strain could grow normally. The colony morphology of MPEB0009802 in the ammonia nitrogen solid medium was as Figure 6 shown, and it could be seen that the strain could grow normally in the ammonia nitrogen solid medium.

[0052] 3.2 Quantitative analysis of the ammonia nitrogen removal ability of strain MPEB0009802

[0053] The ammonia nitrogen degradation ability of Pseudomonas fluorescens MPEB0009802 was quantitatively analyzed by the Nessler's reagent spectrophotometry method. Preparation of the potassium sodium tartrate solution: Weigh 50 g of potassium sodium tartrate and dissolve it in 100 mL of water, heat and boil to expel ammonia, and make up the volume to 100 mL after cooling sufficiently. Plotting of the ammonia nitrogen standard curve: Accurately pipette 0.0 mL, 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, 2.0 mL of the 10 mg / L ammonia nitrogen standard working solution (prepared freshly before use) into 25 mL colorimetric tubes respectively, add water to 10 mL, and the ammonia nitrogen concentrations are 0.00, 0.4, 0.8, 1.2, 1.6, 2.0 mg / L standard solutions. Add 200 μL of the potassium sodium tartrate solution, mix well by shaking, and then add 300 μL of the Nessler's reagent and mix well by shaking. Let it stand at room temperature for 10 min and measure the absorbance at a wavelength of 420 nm. Plot the standard curve with the absorbance as the abscissa and the ammonia nitrogen concentration (mg / L) as the ordinate as Figure 7 shown. The seed liquid was transferred to the ammonia nitrogen liquid medium at an inoculation amount of 1% (v / v). At the same time, a control group without inoculation was set up, with three replicates in each group. Incubate with shaking at 35 °C and 180 rpm / min for 7 days. Take the fermented bacterial liquid every 24 h, centrifuge at 10000 rpm / min for 10 min, and take the supernatant as the test solution. Pipette 0.2 mL of the supernatant, dilute it with water to 10 mL, add 200 μL of the potassium sodium tartrate solution, mix well by shaking, and then add 300 μL of the Nessler's reagent and mix well by shaking. Let it stand at room temperature for 10 min and measure the absorbance at a wavelength of 420 nm. The results are shown in Table 2 and Figure 8As shown, with the increase in the number of culture days, the ammonia nitrogen content in the supernatant of the bacterial solution decreased accordingly. On the 2nd day of shaking culture, the ammonia nitrogen content in the supernatant of the bacterial solution decreased from 250 mg / L to 95.73 ± 8.69 mg / L. Calculating the ammonia nitrogen degradation rate based on the ammonia nitrogen content in the supernatant, it can be seen that the ammonia nitrogen degradation ability of Pseudomonas fluorescens MPEB0009802 of the present application is relatively strong.

[0054] Table 2

[0055]

[0056] Example 4

[0057] Qualitative analysis of siderophore production by strain MPEB0009802

[0058] The ability of Pseudomonas fluorescens MPEB0009802 to produce siderophores was preliminarily evaluated using a CAS detection medium: 10 μL of the overnight culture of MPEB0009802 bacterial solution was spotted on the surface of an iron-free solid medium and incubated in an incubator at 35°C for 24 h. Approximately 10 mL of CAS solid medium (cooled to about 40°C) was added by the plate covering method, and it was observed whether a yellow or orange-yellow halo was produced around the strain within 12 h. The colony morphology of the siderophore-producing strain MPEB0009802 was as Figure 9 shown, and a yellow or orange-yellow halo was produced around the strain.

[0059] Example 5

[0060] Qualitative analysis of lipase production by strain MPEB0009802

[0061] The ability of Pseudomonas fluorescens MPEB0009802 to produce lipase was preliminarily evaluated using a lipase screening medium: 10 μL of the overnight culture of MPEB0009802 bacterial solution was spotted on the surface of the lipase screening medium and incubated in an incubator at 35°C for 24 h. According to the fact that lipase can decompose Tween 80 and the product can combine with Ca 2+ to form a white precipitate ring around the colony, and thus the strains capable of producing lipase were preliminarily screened. The colony morphology of the lipase-producing strain MPEB0009802 was as Figure 10 shown, and a white precipitate ring was formed around the colony.

[0062] Those skilled in the art should understand that the above embodiments are only exemplary embodiments, and various changes, substitutions, and alterations can be made without departing from the spirit and scope of the present invention.

Claims

1. A Pseudomonas fluorescens strain MPEB0009802, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), deposit address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, deposit number: GDMCC No: 65112.

2. A preparation comprising the Pseudomonas fluorescens as claimed in claim 1.

3. Use of the Pseudomonas fluorescens according to claim 1 or the preparation according to claim 2 in sewage treatment.

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