Arthrobacter sp. And application thereof
By screening and isolating highly efficient nitrogen-fixing A. pseudo-Archid pseudo-Archid XWN1 and applying it to mine soil, the problem of lack of nitrogen in the mine soil was solved, the improvement of soil nitrogen fertilizer and the promotion of plant growth was achieved, and the ecological restoration effect was significant.
Patent Information
- Application Number
- CN202411905251.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-03-28
AI Technical Summary
The soil degradation and pollution of harmful substances caused by coal mining, especially in the lack of soil nitrogen in mine pollution sites, which is difficult to support the normal operation of microbial ecosystems and vegetation ecosystems.
A strain of Pseudarthrobacter sp. XWN1 was screened and isolated. This strain had high nitrogen fixing ability and applied it to mining soil by preparing microbial preparations, providing nitrogen elements to the soil through nitrogen fixation.
The pseudoarbicum XWN1 can effectively fix nitrogen, produce a large amount of ammonia nitrogen fertilizer, significantly improve the nitrogen content of the soil, promote plant growth and soil fertility, and have good application prospects in the restoration of mine pollution sites.
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Figure CN119709514A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, relates to a microbial nitrogen fixation technology and the application of a microbial fertilizer in production, and specifically relates to a strain of Pseudoarthrobacter and an application thereof. Background Art
[0002] Coal mines are an important energy source, but long-term coal mining will cause serious damage to the surrounding environment. On the one hand, coal mining destroys the integrity of the land, leading to soil degradation, reduced fertility and other problems. On the other hand, harmful substances such as heavy metals enter the environment, causing secondary pollution to the surrounding environment. Therefore, ecological restoration of mining sites such as mining areas and spoil dumps is an urgent problem to be solved. Among them, the poor soil on the site has become one of the main problems of ecological restoration. Long-term ecological damage has led to a lack of nutrients in the soil on the site, especially nitrogen, which is the main nutrient element for biological growth. Poor soil can hardly support the normal operation of the underlying microbial ecosystem and the upper vegetation ecosystem.
[0003] Microbial remediation is the use of the metabolism of microorganisms to repair sites. Microbial remediation has received widespread attention due to its wide range of metabolic pathways, strong environmental adaptability, and vigorous growth and reproduction. Nitrogen-fixing bacteria are a type of microorganism that can convert N2 in the air into nitrogen-containing compounds, and can continuously supply the obtained nitrogen elements for plant growth. Nitrogen-fixing microbial fertilizers are fertilizers made using the nitrogen-fixing ability of nitrogen-fixing bacteria. Compared with traditional nitrogen fertilizers, nitrogen-fixing microbial fertilizers have the characteristics of simple operation, high vegetation absorption efficiency, and environmental friendliness. Screening out nitrogen-fixing strains with efficient nitrogen-fixing ability and preparing them into microbial fertilizers for use has excellent application prospects in agricultural production and contaminated site remediation. Summary of the invention
[0004] In order to solve the above problems, the purpose of the present invention is to provide a strain of Pseudoarthrobacter, which can effectively fix nitrogen and provide nitrogen elements for the nutrient-poor soil in mines.
[0005] Another object of the present invention is to provide a method for isolating Pseudoarthrobacter.
[0006] The third object of the present invention is to provide a method for amplifying Pseudomonas aeruginosa
[0007] The fourth object of the present invention is to provide a microbial preparation.
[0008] A fifth object of the present invention is to provide the use of the above-mentioned Arthrobacter spp.
[0009] In order to achieve the above-mentioned purpose, the present invention provides a strain of Pseudarthrobacter sp., which is scientifically named as Pseudarthrobacter sp. XWN1, and the preservation unit is General Microbiology Center of China Microbiological Culture Collection Administration; the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is November 26, 2024, and the preservation number is CGMCC No. 32802.
[0010] The present invention also provides a method for isolating the above-mentioned Pseudoarthrobacter, comprising the following steps:
[0011] 1) Inoculate the soil sample into sterile water at a ratio of 1:9, shake thoroughly for 30 minutes to allow the microorganisms in the soil to fully enter the sterile water, and collect the bacterial solution;
[0012] 2) Dilute the bacterial solution to sterile water at a volume ratio of 1:10 to 10 -1 Repeat the dilution process until 10 -6 , 10 -3 Up to 10 -6 The three concentrations of dilutions were spread on the separation medium plate, and inverted in a 30°C incubator to culture until obvious single colonies grew on the separation medium plate;
[0013] 3) The single colony was streaked and purified, and the purification was repeated three times to obtain a single strain of microorganism.
[0014] Furthermore, the preparation method of the separation culture medium plate is as follows: 10g of mannitol, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.1g of calcium sulfate, and 5g of calcium carbonate are added to 1L of water, 15g of agar powder is added, the pH is adjusted to 7, and the separation medium plate is poured into a sterile culture dish while hot after high temperature and high pressure sterilization at 121°C for 20min, and then cooled to obtain a separation culture medium plate.
[0015] The present invention also provides a method for amplifying the above-mentioned Pseudoarthrobacter, comprising the following steps: inoculating Pseudoarthrobacter into an amplification culture medium at a 10% inoculation ratio, setting the culture conditions to 30° C., 180 rpm, and a culture time of 5 days.
[0016] Furthermore, the preparation method of the expansion medium is as follows: add 10g of mannitol, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.1g of calcium sulfate, and 5g of calcium carbonate into 1L of water, adjust the pH to 7, sterilize at 121°C for 20min, and cool for use.
[0017] The invention also provides a microbial preparation containing the above-mentioned Pseudoarthrobacter.
[0018] The present invention also provides the use of the above-mentioned Pseudoarthrobacter in microbial nitrogen fixation.
[0019] The present invention also provides application of the above-mentioned Pseudoarthrobacter in the restoration of mine contaminated sites.
[0020] Furthermore, the Pseudoarthrobacter was inoculated into an amplification medium and cultured at 180 rpm and 30° C. for 3 days to prepare a bacterial solution; then 1 L of the bacterial solution was added to 15 kg of soil, and 0.5 L of the bacterial solution was supplemented every 2 days for a total of 3 times.
[0021] The beneficial effects of the present invention are:
[0022] The present invention provides a strain of Pseudoarthrobacter XWN1, which can effectively fix nitrogen, produce a large amount of ammonia nitrogen fertilizer, provide a large amount of nutrients for crop production, and has good application prospects in the restoration of contaminated sites and the like. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a diagram of the growth status of the Pseudoarthrobacter XWN1 provided by the present invention on LB culture medium.
[0024] Figure 2 The present invention provides a phylogenetic tree of Pseudoarthrobacter XWN1 based on 16S rDNA.
[0025] Figure 3 The invention provides a method for producing NH4 in amplification medium by using the pseudoarthrobacter XWN1 + Situation statistics chart.
[0026] Figure 4 This is a statistical graph of the growth of Pseudomonas aeruginosa XWN1 under different pH conditions provided by the present invention.
[0027] Figure 5 This is a statistical graph of the growth of Pseudomonas aeruginosa XWN1 provided by the present invention under different sodium chloride concentration conditions.
[0028] Figure 6 This is a statistical graph of the growth of Pseudomonas aeruginosa XWN1 under different temperature conditions provided by the present invention. DETAILED DESCRIPTION
[0029] The embodiments of the present invention will be described in detail and comprehensively below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.
[0030] Example 1 Isolation and identification of strains
[0031] (1) Preparation of bacterial suspension:
[0032] Soil samples collected from the spoil dump area of Shaanxi Xiwan open-pit coal mine were inoculated into sterile water at a ratio of 1:9, and fully shaken for 30 minutes with a rotary oscillator to allow the microorganisms in the soil to fully enter the sterile water, and the bacterial solution was collected.
[0033] (2) Culture medium used for separation and screening:
[0034] Liquid separation medium: add 10g mannitol, 0.2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 0.1g calcium sulfate, and 5g calcium carbonate into 1L water, adjust the pH to 7, sterilize at 121℃ and high temperature and high pressure for 20min, and cool for use.
[0035] Separation medium plate: add 10g of mannitol, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.1g of calcium sulfate, and 5g of calcium carbonate to 1L of water, add 15g of agar powder, adjust the pH to 7, sterilize at 121℃ and high temperature and high pressure for 20min, then pour into a sterile culture dish while hot, and obtain a separation medium plate after cooling.
[0036] (3) Microbial isolation and screening:
[0037] Pipette 100 μL of bacterial solution and add it to 900 μL of sterile water. Shake well and record as 10. -1 Repeat the dilution process until 10 -6 , select 10 -3 Up to 10 -6 Three dilution gradients were applied on the separation medium plate. The culture plate was inverted and cultured in a 30°C incubator. After a single colony was clearly grown on the culture plate, a well-growing microbial colony was selected for streaking purification. After repeating the purification three times, a pure single strain of microorganisms with nitrogen-fixing effect was obtained. After the above separation and purification process, a strain with nitrogen-fixing effect was obtained. The bacteria grew in the form of round colonies on the nitrogen-free separation medium plate, with a milky white color and neat and smooth edges.
[0038] The 16S rDNA of the bacterium was amplified by PCR and then sequenced to obtain the sequence shown in Seq ID No.1. The sequence was compared with the NCBI database by Blast. The comparison results showed that the homology with Pseudarthrobacter equi strain IMMIB L-1606 was the highest, reaching 99.42%. The strain was named Pseudarthrobacter sp. XWN1, abbreviated as Pseudarthrobacter XWN1.
[0039] The above-mentioned Pseudoarthrobacter XWN1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 26, 2024, with the deposit number: CGMCC No.32802.
[0040] Example 2: Bacteria fix N2 to produce NH4 + Capacity test
[0041] Amplification medium: add 10g mannitol, 0.2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 0.1g calcium sulfate, and 5g calcium carbonate into 1L water, adjust the pH to 7, sterilize at 121℃ and high temperature and high pressure for 20min, and cool for use.
[0042] Inoculate 20 mL of amplification medium with 10% inoculation ratio of Pseudoarthrobacter XWN1 seed solution, set the culture conditions to 30°C, 180 rpm, culture time for 5 days, and take samples for testing at 24-hour intervals. The results are as follows: Figure 3 shown.
[0043] from Figure 3 It can be seen that after inoculation of Pseudomonas aeruginosa XWN1, the ammonia nitrogen content in the culture system increased significantly. The content was 8.02 mg / L on the first day, 15.73 mg / L on the second day, and significantly increased to 28.98 mg / L on the third day. On the fourth day of culture, the ammonia nitrogen content in the culture medium reached a maximum of 33.90 mg / L. Subsequently, the content decreased to 23.22 mg / L. The reason may be that the carbon source and other nutrients in the culture medium were exhausted, resulting in reduced microbial activity. According to the above experimental results, Pseudomonas aeruginosa XWN1 has good nitrogen fixation ability, and the NH4 + It can provide a nitrogen source for plant growth on the site.
[0044] Example 3 Adaptability of strains to different growth conditions
[0045] Liquid LB medium: add 10 g of sodium chloride, 10 g of tryptone, and 5 g of yeast extract to 1 L of water, adjust the pH to 7, and sterilize at 121°C for 20 min.
[0046] The pH of the liquid LB medium was adjusted by hydrochloric acid and sodium hydroxide, and the pH gradient was set to 5, 6, 7, 8, and 9. Pseudomonas aeruginosa XWN1 was inoculated at a 1% inoculation ratio, and its OD was detected after 24 hours of culture. 600 The statistical results are as follows Figure 4 shown.
[0047] from Figure 4 It can be seen that the growth state of Pseudomonas aeruginosa XWN1 was best under pH 7 and 8 conditions, and the OD 600The values were all 6.24, and the OD 600 was 3.45, but the growth was inhibited in the pH 10 environment, OD 600 The results showed that the bacteria had good adaptability to weak alkaline environment. 600 The value of 5.51 indicates good adaptability, but the growth of Pseudomonas aeruginosa XWN1 is significantly inhibited as the acidity increases. In summary, Pseudomonas aeruginosa XWN1 has the potential for application in alkaline environments.
[0048] The salt concentration of the liquid LB medium was adjusted by sodium chloride, and the salt concentration gradient was set to 2, 4, 6, and 8. Pseudomonas aeruginosa XWN1 was inoculated at a 1% inoculation ratio, and its OD was detected after culturing for 24 hours. 600 The statistical results are as follows Figure 5 shown.
[0049] from Figure 5 It can be seen that salt concentration has an inhibitory effect on the growth of Pseudomonas aeruginosa XWN1, but Pseudomonas aeruginosa XWN1 has a good tolerance to a salt concentration of 4%. Under 2% and 4% conditions, the OD600 of Pseudomonas aeruginosa XWN1 after 24 hours of growth was 5.66 and 5.20, which was only slightly lower than 6.25 in the control group, indicating that Pseudomonas aeruginosa XWN1 has an excellent tolerance to a salt concentration of 4%.
[0050] The LB culture medium was placed in different temperature environments, with a temperature gradient of 20, 25, 30, 35, and 40°C, and inoculated with Pseudomonas aeruginosa XWN1 at a 1% inoculation ratio. The culture was carried out for 3 days, and the OD was detected at 24-hour intervals. 600 The statistical results are as follows Figure 6 shown.
[0051] from Figure 6 It can be seen that the adaptability of Pseudomonas aeruginosa XWN1 under low temperature conditions is higher than that under high temperature conditions. It can hardly grow under 40°C conditions, but the effect on growth is not obvious under 20°C conditions. After 24 hours of cultivation, the OD600 can still reach 3.52. This result shows that Pseudomonas aeruginosa XWN1 has good tolerance to low temperature.
[0052] Example 4
[0053] Select alfalfa seeds with full and uniform grains and soak them in sterile water for 2 hours. Add 1.5 kg of mining soil to the flower pot after high temperature and high pressure sterilization. The soil used was collected from an open-pit coal mine dump in Yulin City, Shaanxi Province. Sow the soaked seeds evenly in the flower pot, cover the surface with a thin layer of soil, and add water to moisten the soil. Cultivate for 3 days.
[0054] Inoculate the strain into the amplification medium and culture at 180rpm and 30℃ for 3 days. After the alfalfa seeds germinate, add 100mL of microbial culture solution. Add 100mL of blank culture medium to the control group. Add 50mL of bacterial solution every 2 days for a total of 3 times. Add 250mL of bacterial solution to the experimental group and 250mL of culture medium to the control group.
[0055] The seedlings were cultured in a light room at a temperature of 25°C, a humidity of 35%, and a light intensity of 16 h. After 60 days of culture, the seedling height and root length were measured, and the root dry weight was measured after drying in an oven at 65°C to constant weight. The soil was tested for organic matter, total nitrogen, and available nitrogen content, and the results are shown in Table 1.
[0056] Table 1 XWN1 bacteria promote the growth of alfalfa
[0057]
[0058] As can be seen from Table 1, the addition of Pseudoarthrobacter XWN1 significantly improved the growth of alfalfa and the nitrogen content in the soil. Compared with the control group, the plant height of alfalfa increased by 21%, the root length increased by 2%, and the dry weight increased by 30%. The soil fertility was also greatly improved, with the organic matter content increased by 72%, the total nitrogen content increased by 50%, and the available nitrogen content increased by 172%. After potted experiments, Pseudoarthrobacter XWN1 exerted excellent plant growth promotion and soil fertility effects in the soil of the mine dump, which well reflects the application potential of Pseudoarthrobacter XWN1 in the ecological restoration of coal mining areas.
[0059] It can be seen from the above examples that the Pseudomonas aeruginosa XWN1 provided by the present invention has good tolerance to weakly alkaline environments, 4% salt concentration and low temperature conditions, and has great potential for application in complex mining environments. It can adapt to complex ecological environments, and combined with its ability to absorb nitrogen to produce ammonia nitrogen fertilizer, it can play a role in mine restoration.
[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A strain of Pseudoarthrobacter, characterized in that The scientific name of this Pseudarthrobacter sp. is: Pseudarthrobacter sp. XWN1, and the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is: November 26, 2024, and the preservation number is: CGMCC No.32802.
2. A method for isolating Pseudoarthrobacter as claimed in claim 1, characterized in that: The steps include: 1) Inoculate the soil sample into sterile water at a ratio of 1:9, shake thoroughly for 30 minutes to allow the microorganisms in the soil to fully enter the sterile water, and collect the bacterial solution; 2) Dilute the bacterial solution to sterile water at a volume ratio of 1:10 to 10 -1 Repeat the dilution process until 10 -6 , 10 -3 Up to 10 -6 The three concentrations of dilutions were spread on the separation medium plate, and inverted in a 30°C incubator to culture until obvious single colonies grew on the separation medium plate; 3) The single colony was streaked and purified, and the purification was repeated three times to obtain a single strain of microorganism.
3. The separation method according to claim 2, characterized in that The preparation method of the separation culture medium plate is as follows: 10g of mannitol, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.1g of calcium sulfate, and 5g of calcium carbonate are added to 1L of water, 15g of agar powder is added, the pH is adjusted to 7, and the mixture is sterilized at 121°C and high temperature and high pressure for 20min, then poured into a sterile culture dish while hot, and cooled to obtain a separation culture medium plate.
4. A method for amplifying Pseudoarthrobacter as claimed in claim 1, characterized in that: The method comprises the following steps: inoculating pseudoarthrobacter into the amplification culture medium at a 10% inoculation ratio, setting the culture conditions at 30° C. and 180 rpm, and the culture time is 5 days.
5. The amplification method according to claim 4, characterized in that The preparation method of the expansion medium is as follows: add 10g of mannitol, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.1g of calcium sulfate, and 5g of calcium carbonate into 1L of water, adjust the pH to 7, sterilize at 121°C and high temperature and high pressure for 20min, and cool for use.
6. A microbial preparation, characterized in that: Contains the Pseudoarthrobacter as claimed in claim 1.
7. Use of the Pseudoarthrobacter as claimed in claim 1 or the microbial preparation as claimed in claim 6 in microbial nitrogen fixation.
8. The use according to claim 7, characterized in that The Pseudoarthrobacter was inoculated into the amplification medium and cultured at 180 rpm and 30° C. for 3 days to prepare a bacterial solution; then 1 L of the bacterial solution was added to 15 kg of soil, and 0.5 L of the bacterial solution was supplemented every 2 days for a total of 3 times.
9. Use of the Pseudoarthrobacter as claimed in claim 1 or the microbial preparation as claimed in claim 6 in the remediation of mine contaminated sites.
10. The use according to claim 7, characterized in that The Pseudoarthrobacter was inoculated into the amplification medium and cultured at 180 rpm and 30° C. for 3 days to prepare a bacterial solution; then 1 L of the bacterial solution was added to 15 kg of soil, and 0.5 L of the bacterial solution was supplemented every 2 days for a total of 3 times.