Mulberry enterobacter, microbial agent as well as preparation method and application of microbial agent

Abstract

The invention provides mulberry enterobacter, a microbial agent as well as a preparation method and application of the microbial agent. The bacterial strain XJ 10 is classified and named as Enterobacter mori, and is preserved in the Guangdong Microbial Culture Collection Center on February 20, 2025, the preservation number is GDMCC No.65928, and the preservation date is February 20, 2025. The strain XJ 10 can tolerate cadmium-lead combined pollution, has the capabilities of fixing nitrogen, dissolving phosphorus and dissolving potassium, can promote residual cadmium and lead in soil to be converted into effective cadmium and lead, can promote absorption of enrichment plants or hyperaccumulator to cadmium and lead, and can improve the physicochemical properties of the soil at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial remediation applications for soil heavy metal pollution, and specifically relates to an Enterobacter mori, a microbial inoculant, and a preparation method and application thereof. Background Art

[0002] The overall situation of soil heavy metal pollution in China is not optimistic. According to the National Soil Pollution Survey Bulletin in 2014, the total soil over-standard rate reached 16.1%, and the proportion of slightly, moderately, severely, and extremely polluted sites was 11.2%, 2.3%, 1.5%, and 1.1% respectively. The pollution distribution shows that soil pollution in the south is more serious than that in the north, and soil pollution problems are relatively prominent in some regions such as the Yangtze River Delta, the Pearl River Delta, and the old industrial bases in Northeast China. The over-standard rate of cultivated land soil sites is 19.4%, and the over-standard rates of cadmium, mercury, arsenic, copper, lead, chromium, zinc, nickel, etc. at the sites are relatively high. Among them, the over-standard situations of cadmium and lead are relatively prominent. The main sources of pollution are industrial pollution, agricultural pollution, and transportation pollution, such as the emissions from metal smelters, agricultural fertilizers, and vehicle exhausts. Heavy metals in the soil are difficult to degrade, exist and accumulate for a long time, affect soil quality and the ecosystem, threaten the quality safety of agricultural products, and thus endanger human health.

[0003] Phytoremediation is one of the remediation methods considered at home and abroad to be able to remove soil heavy metal pollution, and has advantages such as environmental friendliness, high cost-effectiveness, environmental beautification, and improvement of ecological functions. Microorganisms can change the chemical forms of soil heavy metals through redox reactions and methylation or demethylation, and can also promote the absorption and transportation of heavy metals by plants, and promote the absorption of heavy metals by plants by secreting plant hormones such as auxin and cytokinin. In addition, the cell surface of microorganisms contains various functional groups, such as hydroxyl, amino, carboxyl, etc., and these functional groups can undergo electrostatic adsorption, ion exchange, complexation, etc. with heavy metal ions for biosorption. Moreover, soil microorganisms can dissolve heavy metals through metabolic activities to produce organic acids, amino acids, and other metabolites, and establish a symbiotic relationship with plants to enhance the tolerance to heavy metals. Summary of the Invention

[0004] In view of the above problems, the purpose of the present invention is to provide an Enterobacter mori XJ10, a microbial inoculant, and a preparation method and application thereof. This strain can tolerate cadmium-lead combined pollution, and has the ability of nitrogen fixation, phosphorus solubilization, and potassium solubilization, can promote the conversion of residual cadmium and lead in the soil into available cadmium and lead, promote the absorption of cadmium and lead by enriched plants or hyperaccumulator plants, and at the same time can improve the physical and chemical properties of the soil.

[0005] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0006] The present invention provides Enterobacter mori, strain XJ 10, with a preservation number of GDMCC No. 65928.

[0007] Furthermore, the 16S rDNA sequence of Enterobacter mori XJ 10 consists of 993 base pairs (bp), and its nucleotide sequence is as shown in SEQ ID NO.1. This sequence has been uploaded to the National Center for Biotechnology Information database with an accession number of: PQ451573.1.

[0008] The present invention also provides a microbial inoculum, which contains the above-mentioned Enterobacter mori XJ 10.

[0009] Furthermore, the microbial inoculum also contains a culture medium for culturing Enterobacter mori XJ 10.

[0010] The present invention also provides a preparation method of a microbial inoculum, including: inoculating the above-mentioned Enterobacter mori XJ 10 into an LB medium and culturing it on a shaker, and the obtained bacterial liquid is the microbial inoculum.

[0011] Furthermore, the composition of the LB medium is: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and the pH value is 7.0 - 7.4.

[0012] Preferably, the culture conditions are 28 - 35 °C, 180 - 220 rmp, the pH value is 7.0 - 7.4, and the culture time is 36 - 96 h.

[0013] More preferably, the culture conditions are 30 °C, 180 rmp, and the pH value is 7.2.

[0014] The present invention also provides the application of the above-mentioned Enterobacter mori XJ 10 or the microbial inoculum in the remediation of heavy metal - contaminated soil.

[0015] Furthermore, the heavy metals are cadmium and / or lead.

[0016] Furthermore, the concentration of cadmium is not more than 5 ppm.

[0017] Furthermore, the concentration of lead is not more than 500 ppm.

[0018] The present invention also provides the application of the above-mentioned Enterobacter mori XJ 10 or the microbial inoculum in the preparation of nitrogen - fixing and / or phosphorus - solubilizing and / or potassium - solubilizing fertilizers.

[0019] The beneficial effects of the present invention are as follows:

[0020] The present invention provides an Enterobacter mori XJ 10, a microbial inoculum, and a preparation method and application thereof. Among them, Enterobacter mori XJ 10 and the microbial inoculum can effectively improve the physical and chemical properties of the soil, and the soil organic matter content has increased by 33.1%; and it also has good nitrogen fixation, phosphorus solubilization, and potassium solubilization abilities, and can effectively increase the contents of nitrogen, phosphorus, and potassium in the soil. The contents of total nitrogen, total phosphorus, and total potassium have increased by 25.7%, 32.1%, and 11.0% respectively, while the contents of available nitrogen, phosphorus, and potassium have increased by 67.7%, 26.2%, and 43.2% respectively; at the same time, it can also effectively activate cadmium and lead in the soil, and the available state of cadmium has increased by 11.0%, and the available state of lead has increased by 18.7%; it can convert the difficult-to-use forms of Cd and Pb in heavy metals in the soil into available forms, and the conversion rates of cadmium and lead have increased by 8.06% and 14.0% respectively; it can be used in the field of combined remediation of heavy metal contaminated soil by plants-microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] One or more embodiments are exemplarily illustrated by the pictures in the corresponding drawings. These exemplary illustrations do not constitute a limitation on the embodiments, unless otherwise stated, the figures in the drawings do not constitute a proportional limitation.

[0022] Figure 1 Growth status of Enterobacter mori XJ 10 strain provided by the embodiment of the present invention in LB solid medium and LB liquid medium;

[0023] Figure 2 Detection results of nitrogen fixation, phosphorus solubilization, and potassium solubilization abilities of Enterobacter mori XJ 10 strain provided by the embodiment of the present invention;

[0024] Figure 3 Growth rate curve of Enterobacter mori XJ 10 strain provided by the embodiment of the present invention in LB liquid medium;

[0025] Figure 4 Effect of Enterobacter mori XJ 10 strain provided by the embodiment of the present invention on the available states of Cd and Pb in cadmium-lead contaminated soil;

[0026] Figure 5 Effect of Enterobacter mori XJ 10 strain provided by the embodiment of the present invention on the chemical forms of Cd and Pb in cadmium-lead contaminated soil.

[0027] PRESERVATION DESCRIPTION

[0028] The taxonomic name of the Enterobacter mori strain XJ 10 of the present invention is Enterobacter mori, which is preserved in the Guangdong Provincial Culture Collection Center of Microorganisms. The preservation address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The preservation number is GDMCC No. 65928, and the preservation date is February 20, 2025. Detailed implementation manners

[0029] To more clearly illustrate the present invention, the present invention will be further described in detail below with reference to examples and in conjunction with the accompanying drawings. Those skilled in the art should understand that the specific content described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0030] Examples

[0031] Soil contaminated with Cd-Pb in Nanshan District, Shenzhen City, Guangdong Province was collected as the screening soil. After determination by ICP-OES, the cadmium content was 7.2 mg / kg and the lead content was 524 mg / kg. According to the "Soil Environmental Quality - Risk Control Standards for Soil Pollution of Agricultural Land (Trial)", this pollution reached the risk screening value.

[0032] 1. Media used in the present invention

[0033] Screening medium: LB solid or liquid medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, deionized water or distilled water 1 L. Adding agar makes it LB solid medium, and without agar makes it LB liquid medium. Add 5 ppm of cadmium and 500 ppm of lead to the LB medium for the detection of the tolerance of microorganisms to heavy metals.

[0034] Nitrogen-fixing medium: The nitrogen-fixing ability is detected by Ashby's medium. The specific components are: potassium dihydrogen phosphate 0.2 g / L, magnesium sulfate 0.2 g / L, sodium chloride 0.2 g / L, calcium carbonate 5.0 g / L, mannitol 10.0 g / L, calcium sulfate 0.1 g / L, agar 15.0 g / L, pH value 7.0 ± 0.1 (25 °C).

[0035] Phosphate-solubilizing medium: The phosphate-solubilizing ability is detected by Meng Jina organic phosphorus medium. The specific components are: glucose 10.0 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, egg yolk lecithin 0.2 g / L, calcium carbonate 5.0 g / L, yeast extract powder 0.4 g / L, agar 20.0 g / L, pH value 7.0.

[0036] Potassium-solubilizing medium: The composition of the potassium-solubilizing medium is as follows: sucrose 5.0 g / L, glucose 5.0 g / L, ammonium sulfate 0.5 g / L, yeast powder 0.5 g / L, magnesium sulfate 0.3 g / L, disodium hydrogen phosphate 2.0 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, potassium feldspar 2.0 g / L, agar 15.0 g / L, and the pH is 7.2 ± 0.2 (25 °C).

[0037] 2. Isolation and purification of tolerant strains

[0038] Mix the above-mentioned heavy metal-contaminated soil with sterile water at a ratio of 1:10 for gradient dilution. Shake it at 28 °C and 180 r / min for 30 min, and then let it stand for 20 min to prepare a bacterial suspension. Coat the bacterial suspension on the above-mentioned screened LB solid medium by the spread plate method and culture it at room temperature for 3 - 5 days. Pick the strain with the best growth to perform isolation and purification to obtain the strain Enterobacter mori XJ 10 of the present invention ( Figure 1 , where the left figure shows the growth state of Enterobacter mori XJ 10 strain in LB solid medium, and the right figure shows the growth state of Enterobacter mori XJ 10 strain in LB liquid medium), and the preservation number is GDMCC No.65928.

[0039] 3. Determination of functional tolerant strains

[0040] Inoculate the screened tolerant strain onto nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing media, and observe the nitrogen-fixing, phosphorus-dissolving, and potassium-dissolving abilities of the strain ( Figure 2 , where the left figure shows the detection result of the nitrogen-fixing ability of Enterobacter mori XJ 10 strain, the middle figure shows the detection result of the phosphorus-dissolving ability of Enterobacter mori XJ 10 strain, and the right figure shows the detection result of the potassium-dissolving ability of Enterobacter mori XJ 10 strain). After detection, it is found that the strain can form a nitrogen-fixing circle on the nitrogen-fixing medium, a phosphorus-dissolving circle on the phosphorus-solubilizing medium, and a potassium-dissolving circle on the potassium-solubilizing medium. This indicates that the strain has nitrogen-fixing, phosphorus-dissolving, and potassium-dissolving abilities.

[0041] Detection of growth rate: Inoculate the strain into a conical flask (200 mL) containing LB liquid medium and culture it at room temperature (25 °C). Use an ultraviolet spectrophotometer to detect the OD600 value of the strain every day for a period of 6 days to judge its growth rate ( Figure 3 ).

[0042] 4. Influence of the strain on soil physical and chemical properties

[0043] The strain was inoculated into LB liquid medium and cultured on a shaker to prepare a microbial inoculant. The inoculant was applied to Cd-Pb contaminated soil (Cd: 5 mg / kg; Pb: 500 mg / kg), and after a 2-month soil culture experiment, the physical and chemical properties of the soil were detected.

[0044] The pH value of the soil was detected using a pH meter; the soil organic matter content was detected using the dichromate oxidation-external heating method; the electrical conductivity (EC) was detected using an electrical conductivity detector; the soil cation exchange capacity (CEC) was detected using cobalt(III) hexammine chloride spectrophotometry. The detection results are shown in Table 1.

[0045] Table 1 Effects of Enterobacter mori XJ 10 strain on the physical and chemical properties of Cd-Pb contaminated soil

[0046]

[0047] As can be seen from Table 1, the soil pH decreased by 1.06 units, the soil organic matter increased by 33.3%, the soil electrical conductivity increased by 51.5%, and the soil cation exchange capacity decreased by 18.5%.

[0048] 5. Effects of the strain on soil nitrogen, phosphorus, and potassium

[0049] The total amounts of nitrogen, phosphorus, and potassium in the soil were detected using the Kjeldahl method, acid solution-molybdenum antimony anti-colorimetric method, and flame photometry; the available nitrogen, phosphorus, and potassium in the soil were detected using the alkali diffusion method, sodium bicarbonate extraction-molybdenum antimony anti-colorimetric method, and ammonium acetate extraction-flame photometry. The detection results are shown in Table 2.

[0050] Table 2 Effects of Enterobacter mori XJ 10 strain on the total and available amounts of nitrogen, phosphorus, and potassium in Cd-Pb contaminated soil

[0051]

[0052] As can be seen from Table 2, the contents of total nitrogen, total phosphorus, and total potassium increased by 25.7%, 32.1%, and 11.0% respectively, while the contents of available nitrogen, phosphorus, and potassium increased by 67.7%, 26.2%, and 43.2% respectively.

[0053] 6. Effects of the strain on available Cd / Pb and chemical forms in soil

[0054] The available and chemical forms of Cd / Pb in the soil were detected by DTPA single-step extraction method and BCR sequential extraction method to judge the activation ability of the strain on Cd / Pb in the soil. The detection results are as Figure 4 and Figure 5As shown, the contents of available Cd and Pb increased by 11.0% and 18.7% respectively, while the proportions of residual Cd and Pb in the soil decreased by 5% and 7% respectively. This indicates that the strain has the ability to activate cadmium and lead in the soil, and the increase in the content of available heavy metals in the soil is beneficial to the extraction of heavy metals by (hyper)accumulator plants.

[0055] 7. Molecular identification of the strain

[0056] The 16S rDNA sequence of the tolerance-functional strain is shown in SEQ ID NO.1:

[0057] TACGAATACAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGGGCTTCTCTGGGGGA

[0058] After comparison by the National Center for Biotechnology Information, this tolerance-functional bacterium is Enterobacter mori, a Gram-negative bacterium belonging to the genus Enterobacter of the family Enterobacteriaceae.

[0059] Obviously, the above-mentioned embodiments of the present invention are merely examples for more clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is impossible to enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. An Enterobacter mori, strain XJ 10, with the preservation number of GDMCC No. 65928.

2. The Enterobacter mori according to claim 1, wherein The 16S rDNA sequence of the Enterobacter mori XJ 10 consists of 993 bases, and its nucleotide sequence is as shown in SEQ ID NO.

1.

3. A microbial inoculant, characterized in that, The microbial inoculum contains the Enterobacter mori XJ10 described in claim 1.

4. The microbial inoculum according to claim 3, characterized in that, The microbial inoculum also contains a culture medium for culturing the Enterobacter mori XJ 10.

5. A preparation method of a microbial inoculant, characterized in that, Including: Inoculate the Enterobacter mori XJ10 described in claim 1 into LB medium and culture it on a shaker. The obtained bacterial liquid is the microbial inoculum.

6. The preparation method of the microbial inoculum according to claim 5, wherein, The composition of the LB medium is: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and the pH value is 7.0 - 7.

4.

7. The preparation method of the microbial inoculum according to claim 5, characterized in that, The culture conditions are 28 - 35 °C, 180 - 220 rmp, the pH value is 7.0 - 7.4, and culture for 36 - 96 h; Preferably, the culture conditions are 30 °C, 180 rmp, and the pH value is 7.

2.

8. The application of the Enterobacter mori strain XJ 10 described in claim 1 or the microbial inoculum described in claim 3 in the remediation of heavy metal - contaminated soil.

9. The application according to claim 8, wherein The heavy metals are cadmium and / or lead; Optionally, the concentration of cadmium is not more than 5 ppm; Optionally, the concentration of lead is not more than 500 ppm.

10. The application of the Enterobacter mori XJ 10 described in claim 1 or the microbial inoculum described in claim 3 in the preparation of nitrogen - fixing and / or phosphorus - solubilizing and / or potassium - solubilizing fertilizers.

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