Bacillus halophilous HMF09 and application thereof

Abstract

The invention relates to halophilous bacillus (Halophilous sp.) HMF09, and the preservation number of the halophilous bacillus is CGMCC (China General Microbiological Culture Collection Center) No.30475. The strain has high saline-alkaline tolerance and efficient cadmium ion immobilization capability, and can be applied to reduce the ion activity of heavy metal cadmium in soil, reduce stress of saline-alkaline on crops and promote crop growth.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a halobacillus strain HMF09 and an application thereof. Background Art

[0002] Heavy metal contaminated soil refers to the phenomenon that heavy metals are introduced into the soil due to human activities, causing the heavy metal content in the soil to be significantly higher than the original content and causing the deterioration of the ecological environment. The heavy metal cadmium (Cd) has strong chemical activity, high mobility, persistent toxicity and is easily enriched in the food chain. It can affect the normal physiological activities of crop enzyme systems, cell and organelle structures, metabolism, etc., and poses a potential threat to human health.

[0003] Saline-alkali soil is a general term for saline soil and alkaline soil. Saline soil mainly refers to salinized soil with high chloride or sulfate content. The soil is alkaline, but the pH is not necessarily very high. Alkaline soil refers to soil containing carbonate or heavy phosphate, with a high pH and alkaline soil. Saline-alkali soil has low organic matter content, low soil fertility, poor physical and chemical properties, and many anions and cations that are harmful to crops, making it difficult for crops to grow.

[0004] The soil in some parts of my country is simultaneously polluted by heavy metals and has salinity problems, which has a dual impact on plants and greatly affects their growth and development.

[0005] Solving the problem of soil heavy metal pollution and salinization is urgent. Studies have shown that some functional microorganisms can form precipitation with heavy metals by producing substances such as carbonates, hydrogen sulfide and phosphates, thereby reducing the mobility and bioavailability of heavy metals in the soil, and are therefore widely used in the remediation of heavy metal-contaminated soil. For cadmium pollution in soil heavy metal pollution, microbial adsorption and degradation is a low-cost, environmentally friendly and efficient method for treating cadmium-contaminated soil.

[0006] However, there are currently few strains with high salt-alkali tolerance and efficient cadmium fixation. Therefore, screening strains and microbial products that can promote plant growth and reduce the activity of heavy metal ions in the soil, that is, reduce the content of effective cadmium in the soil, and reduce the stress of salt and alkali on crops is of great significance for the sustainable agricultural development and efficient utilization of resources in saline-alkali land. Summary of the invention

[0007] The invention aims to provide a halophilic bacillus HMF09 with high salt-alkali tolerance and high-efficiency cadmium ion fixation ability. The strain can reduce the ion activity of soil heavy metal cadmium, reduce the stress of salt and alkali on crops, and promote crop growth.

[0008] The present invention adopts the following technical solution: A halophilic Bacillus ( Halobacillus sp.) HMF09, deposited on April 30, 2024 in the China General Microbiological Culture Collection Center, Beijing, China, with the deposit number CGMCC No.30475.

[0009] Furthermore, the halobacillus sp. HMF09 is resistant to salt and alkali.

[0010] Furthermore, the halobacillus sp. HMF09 can tolerate the heavy metal cadmium.

[0011] Furthermore, the halophilic Bacillus HMF09 fixes or adsorbs heavy metal cadmium.

[0012] Furthermore, the halobacillus sp. HMF09 can promote plant growth.

[0013] An application of the above-mentioned halobacillus HMF09 in treating soil contaminated by cadmium.

[0014] An application of the above-mentioned halophilic bacillus HMF09 in treating saline-alkali soil.

[0015] An application of the above-mentioned halobacillus HMF09 in treating saline-alkali soil contaminated by cadmium.

[0016] An application of the above-mentioned halobacillus HMF09 in promoting crop growth in a soil environment contaminated by cadmium.

[0017] A microbial agent comprising the above-mentioned halophilic Bacillus HMF09.

[0018] The beneficial effects of the present invention are as follows: the halophilic Bacillus HMF09 strain of the present invention has good salt-alkali resistance and the ability to reduce the content of heavy metal cadmium, and can grow normally in a high-salt-alkali culture medium and a cadmium-containing environment. At the same time, the halophilic Bacillus HMF09 strain of the present invention can effectively alleviate the inhibition of the plant's nutritional growth process under salt-alkali stress, improve the plant's salt-alkali resistance, and promote plant growth.

[0019] The present invention proposes new methods and ideas for the utilization of saline-alkali land and the prevention and treatment of cadmium metal-contaminated soil, and obtains excellent bacterial strain resources with better salt-alkali resistance and vitality promotion and can be used for the prevention and treatment of heavy metal-contaminated soil for production selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of Halobacillus sp. HMF09.

[0021] Figure 2 This is the cell morphology of Halobacillus sp. HMF09.

[0022] Figure 3This is the phylogenetic tree of Halobacillus sp. HMF09 constructed based on 16S rDNA. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the embodiments and drawings. The protection scope of the present invention is not limited to the embodiments, and any changes made by those skilled in the art within the scope defined by the claims also fall within the protection scope of the present invention.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] Example 1 Screening of strain HMF09 (1) Isolation of strains Soil samples were collected from a wheat experimental field (saline-alkali soil) in Emin County, Tacheng District, Xinjiang. 5 g of soil sample was added to a conical flask containing 100 mL of sterile water and shaken at 180 r / min for 30 min to fully disperse the microorganisms in the test soil sample in the sterile water to obtain a soil mixture. The soil mixture was diluted in a gradient with a concentration gradient of 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Apply the soil mixture, take 100 μL of the bacterial suspension and apply it to LB solid medium containing 10% sodium chloride for the isolation of salt-tolerant microorganisms. Invert the plate and place it in a 30°C constant temperature incubator for 48 to 96 hours.

[0026] (2) Purification of strains According to the differences in colony morphology, color and size, a single microbial colony was picked and purified several times until a monoclonal strain was obtained. The purified strain was stored in a −80°C refrigerator with 30% glycerol. According to the colony morphology, 6 salt-tolerant strains resistant to 10% sodium chloride were isolated and purified, and they were numbered HH1, HH2, HH3, HH4, HH5, and HH6 in the order of purification.

[0027] (3) Discovery and preliminary screening of cadmium-resistant strains Prepare PDA medium (200 g potato, 20 g glucose, 1000 mL ultrapure water). Inoculate the strains numbered HH1, HH2, HH3, HH4, HH5, and HH6 onto solid plate medium with a cadmium chloride concentration of 25 mmol / L for screening and culture. Set 3 replicates for each concentration, culture at a constant temperature of 28°C for 4-5 days, and observe and record the characteristics of single colonies.

[0028] (4) Re-screening of cadmium-resistant strains Under sterile conditions, individual colonies on PDA solid culture medium with a cadmium chloride concentration of 25 mmol / L were picked up with an inoculation needle and streaked onto PDA solid culture medium plates with cadmium chloride concentration gradients of 10 mmol / L, 25 mmol / L and 50 mmol / L. A strain with good growth and full colonies was obtained on the plate with a concentration of 25 mmol / L. After three purifications, the strain was numbered HH4.

[0029] (5) Test on the tolerance of HH4 strain to cadmium-containing medium 1) Preparation of cadmium-containing culture medium LB solid cadmium-containing medium: 0.0816 g cadmium chloride, 5 g yeast extract, 10 g peptone, 10 g NaCl, 20 g agar, add distilled water to make up to 1 L, sterilize at 121°C for 30 min, prepare a culture medium with a cadmium ion concentration of 50 mg / L, and set aside.

[0030] 2) Experimental treatment After appropriately diluting the seed solution of the pure strain HH4 on a clean bench, take 200 μL and spread it evenly on the LB solid cadmium-containing plate with a spreading rod. After it is completely dried in the shade, seal the plate with a sealing film and place it in a 35°C constant temperature shaker for culture. After 72 hours, measure the bacterial concentration in the liquid culture medium.

[0031] 3) Result analysis As shown in Table 1, after 72 hours of culture, the bacterial concentration of strain HH4 increased from OD 600 =0.039 increased to OD 600 =1.676, which proves that strain HH4 can grow normally in an environment with a cadmium content of 50 mg / L. It not only has strong salt tolerance, but also can survive well in a cadmium-contaminated environment. Strain HH4 was named HMF09.

[0032] Table 1 Test results of cadmium resistance of strain HH4 .

[0033] Example 2 Identification of strain HMF09 (1) Morphological characteristics The colonies are round and orange-yellow in color, with smooth and neat edges and no protrusions. Figure 1 As shown. The bacteria are rod-shaped, have spores, and are Gram-positive. Figure 2 shown.

[0034] (2) Physiological and biochemical characteristics The physiological and biochemical characteristics of strain HMF09 were identified, and the results are shown in Table 2.

[0035] Table 2 Physiological and biochemical characteristics of strain HMF09 .

[0036] (3) Molecular biological characteristics The genomic DNA of strain HMF09 was extracted and used as a template to perform PCR amplification using bacterial 16S rDNA universal primers. The amplified product was recovered and sequenced to obtain a DNA sequence containing 1500 bp (as shown in SEQ ID No. 1). The sequencing results were entered into the GeneBank database for BLAST comparison analysis. The 16S rDNA sequence was compared with the NCBI database, and it was found that the consistency between HMF09 and Halobacillus halophilus reached 99%. The results of constructing the phylogenetic tree are shown in the following figure. Figure 3 According to the strain morphology, sequencing analysis and physiological and biochemical test results, HMF09 was identified as Halobacillus sp. Halobacillus sp .).

[0037] The halophilic Bacillus HMF09 was deposited in the China General Microbiological Culture Collection Center on April 30, 2024, with the address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No.30475.

[0038] Example 3 Determination of salt and alkali resistance of Halobacillus sp. HMF09 (1) Determination of salt tolerance The activated strain HMF09 was selected and inoculated into 100 mL of LB liquid culture medium with NaCl concentrations of 10 g / L, 50 g / L, 100 g / L, 150 g / L, and 200 g / L, and cultured in an incubator at 35 °C for 3 days. The growth of the bacteria was observed, and the bacterial concentration of the liquid in the culture medium was measured. The results are shown in Table 3.

[0039] Table 3 Salt tolerance test results of strain HMF09 .

[0040] Strain HMF09 still has good activity when the salt content is 150g / L, and the strain can tolerate high-salt environment.

[0041] (2) Determination of alkali resistance The activated strains were selected and inoculated into 100 mL of LB liquid culture medium with pH values ​​of 7, 8, 9, 10, and 11, and cultured in an incubator at 35°C for 3 days. The growth of the bacteria was observed, and the concentration of the bacteria in the liquid culture medium was measured. The results are shown in Table 4.

[0042] Table 4 Alkali resistance test results of strains .

[0043] Strain HMF09 still had good activity at pH 10, indicating that the strain could tolerate alkaline environment.

[0044] Example 4 Test on the curing effect of strain HMF09 on cadmium ions (1) Preparation of cadmium-containing culture medium LB liquid cadmium-containing medium: 15 mg cadmium chloride (99.99%), 5 g yeast extract, 10 g peptone, 10 g NaCl, add sterile water to 1 L, and sterilize at 121°C for 15 min.

[0045] (2) Preparation of HMF09 bacterial suspension The strain stored at -80°C was streaked on LB solid medium for activation, and two colonies were picked with an inoculation loop and inoculated into a triangular flask containing 50 mL LB liquid medium. The culture was cultured at 35°C for 48 h to obtain the fermentation broth of HMF09. The fermentation broth was centrifuged at high speed (10000 r / min), the clear liquid was poured out to obtain bacterial mud, and then diluted with an appropriate amount of sterile water to a viable bacterial count of 1×10 8 cfu / mL to obtain HMF09 bacterial suspension.

[0046] Preparation method of LB liquid culture medium: 5 g yeast extract, 10 g peptone, 10 g NaCl, 1000 mL water, pH 7.2, sterilization at 121°C for 30 min.

[0047] (3) Experimental treatment Take the bacterial suspension of HMF09 respectively, add it into three Erlenmeyer flasks containing 50mL / 250mL LB liquid cadmium culture medium at an inoculum volume of 10% (volume ratio), and culture at 30℃ and 180r / min for 48h, 72h, and 96h respectively to obtain the treatment liquid of strain HMF09. The three treatment liquids were centrifuged at high speed (10000r / min), and the supernatant was filtered with a 0.22 filter membrane to determine the cadmium ion content. The experiment was repeated 3 times, and the percentage of cadmium ion fixation was calculated using the LB liquid cadmium culture medium as the control.

[0048] (4) Results analysis The results are shown in Table 5. The cadmium ion concentration of the control group was 9.20 mg / L, while the cadmium ion concentrations in the solution were 8.42 mg / L, 5.28 mg / L and 2.78 mg / L after treatment with the strain HMF09 for 48 h, 72 h and 96 h, respectively. After 96 h, the cadmium ion content in the solution decreased by 69.78%, proving that the strain HMF09 has a significant effect on the solidification of cadmium ions.

[0049] Table 5 Effect of strain HMF09 on the fixation of cadmium ions in liquid cadmium-containing culture medium .

[0050] Example 5 Experiment on the solidification of heavy metal cadmium ions in soil by strain HMF09 (1) Preparation of bacterial suspension: The preparation method is the same as that in Example 4, and the number of viable bacteria is 1×10 8 cfu / mL.

[0051] (2) First, Cd 2+ The soil with a content of 50 mg / kg was dried at 80℃ for 5 hours, placed at room temperature, and then the bacterial suspension was added to the sterilized soil (20g soil plus 10ml bacterial suspension), mixed well and placed at room temperature for 3 days. After taking out some soil and air-drying it, the DTPA extraction method (GB / T23739-2009) was used to determine the effective cadmium content in the soil. The treatment without bacterial suspension was used as the control group, and each treatment was repeated three times.

[0052] From the results in Table 6, it can be seen that under the action of the strain HMF09, the cadmium ion content in the treatment group decreased by 29.12% compared with the control group, indicating that the halophilic Bacillus HMF09 has a significant solidification effect on heavy metal cadmium ions in the soil.

[0053] Table 6 Effect of strain HMF09 on the fixation of heavy metal cadmium in soil (unit: mg / kg) .

[0054] Example 6 Experiment on the growth-promoting effect of strain HMF09 on crops 1. Wheat germination test (1) Seed disinfection: Select wheat seeds of uniform size and full grains, soak them in 70% alcohol for 1 min, then soak them in 3% NaClO for 10 min, then rinse them 4 times with sterile water and dry them.

[0055] (2) Preparation of HMF09 bacterial suspension: The preparation method is the same as that in Example 4, and the number of viable bacteria is 1×10 8 cfu / mL.

[0056] (3) Germination test: First, use a viable bacterial count of 1×10 8cfu / mL bacterial suspension was used to soak wheat seeds (T1~T4), placed in a dark place for 12 h, and drained. The control group soaked seeds (CK1~CK4) with sterile water; then NaCl solutions with concentrations of 0mmol / L, 100mmol / L, 150mmol / L and 200mmol / L were prepared respectively, and sterile filter papers sterilized in advance were soaked, and placed in culture dishes. Each concentration corresponded to 2 culture dishes, which were treatment and control respectively; finally, the soaked seeds were placed on the soaked filter paper, 50 seeds per dish, and each treatment was repeated 3 times. The culture dishes were placed flat in the incubator and cultured at 26℃. The germination of wheat seeds was counted every day, and the results are shown in Table 7.

[0057] Table 7 Statistical results of the wheat germination promotion experiment with Halobacillus sp. HMF09 .

[0058] It can be seen from Table 7 that under the same concentration of salt stress, the germination rate of wheat seeds treated with HMF09 bacterial suspension was higher than that treated with sterile water; when the concentration of NaCl solution was in the range of 0-200 mmol / L, the germination rate increased with the increase of salt concentration, which further indicated that strain HMF09 could improve the salt stress tolerance of wheat seeds and increase its germination rate.

[0059] (II) Potted experiment on promoting wheat growth by strain HMF09 Wheat seeds with consistent germination potential were selected from CK1 and transplanted into pots (18 cm high, 16 cm in diameter) filled with agronomic soil. The experimental soil was a mixture of nutrient soil, vermiculite and perlite in a ratio of 3:1:1.

[0060] The pot experiment was divided into 2 groups and 4 treatments, as shown in Table 8. In the first group (T5, CK5), 270 mL of deionized water was applied after the agronomic soil was filled in the flower pots, as the salt-free stress group. T5 took 2 mL of bacterial suspension and applied it to the roots of the seedlings every 7 days, and CK5 was poured with the same amount of water as the control; in the second group (T6, CK6), 270 mL of saline with a NaCl concentration of 100 mmol / L was applied after the agronomic soil was filled in the flower pots, as the salt stress group. T6 took 2 mL of bacterial suspension and applied it to the roots of the seedlings every 7 days, and CK6 was poured with the same amount of water as the control. 10 wheat seedlings were transferred into each pot, and 3 pots were sown for each treatment, and cultured in a light incubator (26℃, light 16h / d). 100 mL of water was applied every 2 days, and the growth of the seedlings was observed regularly. Plant height and root length were measured after 40 days.

[0061] Table 8 Statistical results of the experiment on promoting wheat growth by Halobacillus sp. HMF09 .

[0062] The above results show that under salt stress and non-salt stress conditions, the plant height and root length of wheat in the treatment group were significantly higher than those in the control group, and under salt stress, the plant height and root length increased more than the control, further indicating that Halobacillus halophilus HMF09 can promote the growth of wheat.

[0063] Example 7 Preparation of Halobacillus sp. HMF09 powder (1) Preparation of LB liquid culture medium: same as in Example 4.

[0064] (2) Activation of strains: Pick a colony of Halobacillus subtilis HMF09, inoculate it into a test tube containing 10 mL of LB liquid culture medium, and culture it at a constant temperature of 160 rpm and 35°C for 24 h for activation.

[0065] (3) Preparation of seed solution: Take 5 mL of the activated bacterial solution and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of LB liquid culture medium. Incubate the culture at 160 rpm and 35°C for 24 h to obtain the seed solution.

[0066] (4) Preparation of fermentation broth: 180 mL of the prepared seed solution was inoculated into a 6 L small fermentation tank containing 3.6 L of LB liquid medium, and cultured at 160 rpm and 35 °C for 48 h to obtain the fermentation broth of Halobacillus sp. HMF09. The effective viable count was 3.31 × 10 9 cfu / mL.

[0067] (5) Preparation of bacterial agent: The fermentation liquid and soluble starch were mixed evenly at a mass ratio of 10:1, and sprayed in a freeze dryer to obtain the powder of Halobacillus sp. HMF09. The effective viable bacterial count was 3.15×10 10 cfu / g.

[0068] Example 8 Potted Planting Test of Halophilous Bacillus HMF09 on Cadmium-Containing Saline-Alkaline Soil Preparation of cadmium-containing saline-alkali soil: The soil taken from Huanghua area of ​​Cangzhou (salt content of 12.2 g / kg, pH of 8.12) was prepared into saline-alkali soil with a cadmium ion content of 20 mg / kg using cadmium chloride reagent, sterilized at 128°C for 30 minutes, and placed for 1 day for use.

[0069] Seed treatment: Select evenly plump corn seeds, soak them in 50ppm sodium hypochlorite solution for 1 hour, then wash the seeds 3 times with deionized water, and germinate them at 25℃ until they turn white.

[0070] Preparation of HMF09 bacterial suspension: Take 1 g of the HMF09 powdered bacterial agent prepared in Example 7, add it into 1 L of deionized water, stir and dissolve it for later use.

[0071] The pot experiment was conducted with two treatments, the experimental group and the control group, with 10 pots in each treatment. Each pot (18 cm high, 16 cm in diameter) was filled with 1.5 kg of saline-alkali soil containing cadmium, and one corn seed was planted at a sowing depth of about 5 cm. After sowing, 270 mL of bacterial suspension was applied to each pot in the experimental group until the soil was completely moistened without seeping from the bottom of the pot, and 270 mL of deionized water was applied to the control group. The pots were moved into the tissue culture room for culture at 25 °C, and deionized water was applied once every 4 days, 100 mL each time, to keep the soil moist. After the corn grew two true leaves, 90 mL of the prepared bacterial suspension was applied to each pot in the experimental group, and 90 mL of deionized water was applied to the control group. After 20 days of continuous culture, the plant height, stem diameter, taproot length, and cadmium ion content in the aboveground and roots of the corn plants were measured to investigate the effects of the halobacillus HMF09 bacterial agent on corn biomass and cadmium ion content in corn plants.

[0072] Method for detecting cadmium ion content in corn plants: Take 100g of samples from the aboveground part and root of corn plants, wash them with clean water, then rinse them with EDTA solution and deionized water three times respectively, wipe the water on the surface of the plants with a towel, and dry them in an oven at 105°C to constant weight. Ash the aboveground samples and root samples in a muffle furnace, and determine the cadmium content of the samples after ashing by atomic absorption spectrophotometry, and then calculate the cadmium content of the aboveground part and root of corn plants.

[0073] As shown in Table 9, under the condition of cadmium ion content of 20 mg / kg in saline-alkali soil, the height, stem diameter and taproot length of corn plants irrigated with the bacillus halophilus HMF09 inoculant were significantly better than those of the control group, indicating that the strain HMF09 inoculant can effectively promote corn growth and inhibit the toxic effects of cadmium ions in the soil. At the same time, the cadmium content in the aboveground and roots of corn plants treated with the HMF09 inoculant was significantly reduced by 20.76% and 31.44%, respectively, indicating that the HMF09 inoculant has a good fixation effect on heavy metal cadmium in the soil, reduces the absorption of cadmium ions by the roots and their transport to the aboveground parts, and greatly reduces the content of harmful cadmium ions in corn plants.

[0074] The experimental results show that the application of HMF09 microbial agent is conducive to the safe cultivation and development of farmland soil contaminated by heavy metal cadmium.

[0075] Table 9 Effects of strain HMF09 on corn plant biomass and cadmium ion content .

[0076] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A halophilic Bacillus ( Halobacillus sp .) HMF09, characterized in that, The deposit number is CGMCC No.30475.

2. The halobacillus sp. HMF09 according to claim 1, characterized in that It is resistant to salt and alkali.

3. The halobacillus sp. HMF09 according to claim 1, characterized in that It is resistant to the heavy metal cadmium.

4. The halobacillus sp. HMF09 according to claim 1, characterized in that Can fix or adsorb heavy metal cadmium.

5. The halobacillus sp. HMF09 according to claim 1, characterized in that It promotes plant growth.

6. Use of the halobacillus sp. HMF09 as claimed in claim 1 in treating soil contaminated by cadmium.

7. Use of the halophilic Bacillus HMF09 as claimed in claim 1 in treating saline-alkali soil.

8. Use of the halobacillus sp. HMF09 as claimed in claim 1 in treating saline-alkali soil contaminated by cadmium.

9. Use of the halobacillus sp. HMF09 as claimed in claim 1 in promoting crop growth in a soil environment contaminated by cadmium.

10. A microbial agent comprising the halophilic Bacillus HMF09 according to claim 1.

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