Microbacterium paraoxydans HMY-2 with Cd adsorption and alkali production functions and its applications

Abstract

The present invention discloses Microbacterium paraoxydans HMY-2 with both Cd adsorption and alkali production functions and its application. Microbacterium paraoxydans ( Microbacterium paraoxydans ) HMY-2 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 30, 2024, with the deposit number CGMCC No. 33225. It has an adsorption effect on Cd

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Microbacterium paraoxydans Microbacterium paraoxydans HMY-2 with the ability to passivate heavy metal cadmium and produce alkali and its application. Background Art

[0002] Heavy metal pollution has become a global environmental problem, and its ecological risks are particularly prominent in the process of industrialization and urbanization. Heavy metals such as lead, cadmium, mercury, chromium, and arsenic are continuously enriched in the soil-water-biological system through geological cycles, which not only leads to the degradation of arable land quality but also endangers human health through the food chain amplification effect.

[0003] The tropical region has superior light and heat conditions and can grow crops throughout the year, making it an important rice production base. Agriculture in the tropical region is highly intensive, with a high multiple cropping index and a fertilizer application rate much higher than the average level in subtropical and temperate regions. This planting pattern can lead to environmental problems such as soil acidification, reduced organic matter, nutrient imbalance, and continuous accumulation of heavy metals. In addition, the soil parent material in some tropical regions is mainly acidic igneous rock, and coupled with the characteristics of high temperature and abundant rainfall, the base ions in the soil are easily leached, and the soil is mostly acidic, which will also lead to the activation of heavy metal elements in the soil, increased availability, and promotion of the absorption and accumulation of heavy metals by crops, affecting the safety of agricultural products. Therefore, it has become an urgent problem to construct a heavy metal control planting management technology suitable for the farmland soil-rice system in the tropical region to ensure the safety of agricultural products.

[0004] At present, the main methods for controlling heavy metals in farmland soil are agronomic regulation techniques (such as lime adjustment, optimized fertilization, crop variety screening, foliar barrier, etc.), soil improvement techniques (passivators and conditioners), and biological techniques (microorganisms and plants). Among them, the microbial passivation technique has become a research hotspot for heavy metal pollution treatment due to its environmental friendliness, low cost, and easy operation.

[0005] The microbial passivation technique uses the adsorption, complexation, redox and other characteristics of microorganisms to change the forms and availability of heavy metals in the environment, thereby reducing the harm of heavy metals to the environment and ecological systems. The reported heavy metal passivating strains mainly include fungi such as the genus Saccharomyces Saccharomyces ), the genus Aspergillus Aspergillus ), the genus Rhizopus Rhizopus arrhizus ); Gram-negative bacteria such as the genus Pseudomonas Pseudomonas ), the genus Serratia Serratia ), Escherichia coli Escherichia coli ), etc., and Gram-positive bacteria such as the genus Bacillus Bacillus ), the genus Staphylococcus Staphylococcus ), the genus Lactobacillus Lactobacillus) etc. However, in practical application, the added heavy metal passivation strains are subject to competition from indigenous microorganisms, resulting in colonization difficulties and unstable effects. Therefore, screening indigenous passivation strains and using carrier fixation methods to increase the colonization ability of strains and ensure the function of strains have become the preferred means.

[0006] At present, natural polymer materials such as sodium alginate and chitosan are commonly used as microbial carriers, but they are expensive and have poor stability. In contrast, biochar is low in cost, and its porous structure and high specific surface area can provide a place for microorganisms to attach and live, enhance their resistance and survival rate, and promote their functions. It can also adsorb heavy metals through surface functional groups (-COOH, -OH, etc.), thereby improving the passivation and remediation effect. In addition, the alkaline characteristics of biochar (pH=8-10) can also help increase soil pH, making it suitable for use in acidic soils.

[0007] Therefore, this technology targets the characteristics of soil acidification and Cd pollution in tropical areas. First, Cd passivation strains are screened, while taking into account the ability to produce alkali, and Cd passivation is promoted from two aspects: direct adsorption and alkaline metabolites to adjust soil pH, and indirectly changing the heavy metal occurrence form. Then, biochar is loaded to form a microbial-biochar composite microbial passivation technology, which is applied to rice cultivation to achieve the purpose of blocking and controlling soil heavy metals, reducing accumulation in rice grains, and adjusting soil pH, increasing soil organic matter, and other soil improvement effects, ultimately ensuring the sustainable use of soil. Summary of the invention

[0008] The present invention aims to provide a strain of Microbacterium paraoxidans having the ability to adsorb Cd and produce alkali ( Microbacterium paraoxydans )HMY-2, to solve the problems raised in the above background technology.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] A strain of Microbacterium paraoxidans with the ability to adsorb Cd and produce alkali ( Microbacterium paraoxydans ) HMY-2 was deposited at the General Microbiology Center of China Microbiological Culture Collection Committee on December 30, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33225.

[0011] The colony characteristics and bacterial morphology of the Microbacterium paraoxydense are as follows: after the strain is cultured on the LB plate for 24 hours, the colonies formed are yellow in color, round, opaque, with neat edges, and the colony diameter is 1-3 mm.

[0012] A bacterial agent prepared by the Microbacterium paraoxydants HMY-2 of the present invention.

[0013] As a preference of the present invention, the microbial agent is the fermentation broth of Microbacterium paraoxydans HMY-2.

[0014] As another preference of the present invention, the microbial agent is a biochar microbial agent loaded with Microbacterium paraoxydans HMY-2.

[0015] As a further preference of the present invention, the biochar microbial agent loaded with Microbacterium paraoxydans HMY-2 is prepared by the following method: The rice husk biochar material passing through a 100-200 mesh sieve and the bacterial suspension are oscillated at a volume ratio of 1:1-2 and 120-150 rpm for 20-24 h, and then centrifuged at 3000-4000 rpm for 5-10 min. The supernatant liquid is discarded, and the biochar microbial agent loaded with Microbacterium paraoxydans HMY-2 is obtained by washing with sterile normal saline.

[0016] The application of the Microbacterium paraoxydans ( Microbacterium paraoxydans ) HMY-2 of the present invention in adsorbing and fixing cadmium.

[0017] The application of the Microbacterium paraoxydans ( Microbacterium paraoxydans ) HMY-2 of the present invention in increasing pH under cadmium stress.

[0018] The application of the microbial agent of the present invention in improving the growth condition of plants.

[0019] As a preference, the present invention provides the application of the microbial agent in reducing the absorption and accumulation of heavy metal cadmium by plants.

[0020] As a preference, the present invention provides the application of the microbial agent in improving the physical and chemical properties of soil.

[0021] The plant is further preferably rice.

[0022] The third object of the present invention provides a method for reducing the absorption and accumulation of heavy metal cadmium by plants, which is to introduce the microbial agent into the soil.

[0023] The plant is preferably rice.

[0024] Microbacterium paraoxydans ( Microbacterium paraoxydans)HMY-2 is applied to rice cultivation to passivate Cd in the soil, reduce its accumulation in rice, improve the growth condition of rice and promote soil health. The strain is compounded with biochar to prepare a microbial agent, which is applied to the soil for growing two kinds of rice (Teyou 9301 and Qi 1 You 386) at an addition amount of 2%. The results show that the growth and yield of both kinds of rice are significantly improved. For Teyou 9301 rice, the dry weight of roots increases by 125.6%, the dry weight of leaves increases by 66.0%, the dry weight of grains increases by 44.9%, and the number of panicles increases by 36.4%; for Qi 1 You 386 rice, the dry weight of roots increases by 82.4%, the dry weight of leaves increases by 37.1%, the dry weight of grains increases by 77.9%, and the 1000-grain weight increases by 6.3%. In addition, the application of the microbial agent significantly reduces the accumulation of cadmium in rice grains (the Cd in grains of Teyou 9301 and Qi 1 You 386 is reduced by 77.73% and 84.77% respectively). At the same time, the application of the microbial agent significantly increases the soil pH, Eh, available potassium, organic matter and available phosphorus contents. To sum up, the Microbacterium paraoxydans HMY-2 microbial agent can effectively reduce the Cd accumulation in rice grains, promote the growth of rice and improve the physical and chemical properties of the soil.

[0025] Beneficial effects

[0026] A strain of alkali-producing bacteria capable of passivating heavy metals was screened from the soil in Yazhou District, Sanya City, Hainan Province in the present invention, and its sequencing identification was Microbacterium paraoxydans ( Microbacterium paraoxydans )HMY-2, and its highest removal rate for Cd 2+ (50 mg / L) can reach 63.14%, and the pH value of the culture medium can be increased from the initial 7.0 to 9.05 through metabolic action. Preparing it into a biochar-loaded microbial agent for application can increase the rice yield, reduce the accumulation of heavy metals in rice grains; improve the soil properties, increase the soil pH and Eh values, available potassium, organic matter and available phosphorus contents, and provide a more favorable soil environment for the growth of rice. Description of the drawings

[0027] Figure 1 It is a colony characteristic diagram of Microbacterium paraoxydans on LB solid medium.

[0028] Figure 2 It is a phylogenetic tree diagram of Microbacterium paraoxydans.

[0029] Figure 3 It is the growth curve of Microbacterium paraoxydans and the change of the pH value of the culture medium under cadmium stress.

[0030] Figure 4 It is the dynamic change trend of the cadmium removal rate of Microbacterium paraoxydans.

[0031] Figure 5 It is the change of cadmium content in rice grains under different treatment conditions.

[0032] Biological material preservation information

[0033] HMY-2, classified as Microbacterium paraoxydans ( Microbacterium paraoxydans ) HMY-2, was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on December 30, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33225. Detailed implementation manners

[0034] The present invention will be further described below through examples. According to the following examples, the present invention can be better understood. The specific material ratios, process conditions and their results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0035] Example 1 Isolation, purification and identification of Microbacterium paraoxydans ( Microbacterium paraoxydans )

[0036] The culture medium formula is as follows:

[0037] LB solid medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, 1000 mL of distilled water, pH 7.0 - 7.2, sterilized at 121 °C for 20 min. LB liquid medium: without agar, other conditions are the same as above.

[0038] Strain screening

[0039] Accurately weigh 10 g of fresh soil sample, dissolve it in a 250 mL Erlenmeyer flask containing 90 mL of sterile water, and shake and mix well at 28 °C and 180 r / min on a shaker for 30 min. Take 1 mL of the supernatant and dilute it with 9 mL of sterile water, and dilute it successively to 10 -8 . Take 0.1 mL of the diluted solution and inoculate it onto an LB solid medium plate containing Cd 2+ (concentrations set at 50, 150, 250, 350, 450, 550 mg / L), and incubate it upside down at 30 °C for 48 h. Select and scrape off well-shaped single colonies with an inoculation loop, and streak and purify them on a heavy metal-containing solid medium plate, and incubate it upside down at 30 °C for 2 d. Continuously culture for 5 generations to obtain a genetically stable heavy metal-tolerant strain, and store it in a -80 °C refrigerator in the form of a suspension containing 50% (v / v) glycerol.

[0040] Strain identification

[0041] A strain with good growth condition was screened out on an LB medium plate with a Cd concentration of 550 mg / L, such as Figure 1As shown, the colonies formed after culturing the strain on an LB plate for 24 h were yellow, round, opaque, with neat edges, and the colony diameter was 1-3 mm. The strain type was named HMY-2 (see Figure 1 ).

[0042] The strain isolated and screened by the above method was sequenced by Qingke Biotechnology Co., Ltd. According to the sequencing results of 16S rDNA, the homology comparison with other 16S rDNA sequences was carried out in GenBank using Blast software, and a phylogenetic tree was further constructed using MEGA software (see Figure 2 ). The results showed that the bacterium was Microbacterium paraoxydans ( Microbacterium paraoxydans ). Previous studies have shown that this type of strain has functions such as reducing Cr(VI) to repair contaminated soil / water sources, multidrug resistance, and removing chlorobenzene, but there are no relevant reports on the function of heavy metal adsorption and passivation. At present, this strain has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on December 30, 2024, with the deposit number CGMCC No. 33225.

[0043] Example 2 Growth and Cd adsorption characteristics of Microbacterium paraoxydans ( Microbacterium paraoxydans )

[0044] 1. Experimental steps

[0045] The obtained HMY-2 strain was inoculated into 100 mL of LB liquid medium and cultured with shaking at 30 °C and 180 r·min⁻¹ until the logarithmic growth phase to prepare a seed solution. A 2% (v / v) inoculum was transferred to 20 mL of LB medium containing 50 mg / L Cd 2+ and 20 mL of LB medium without Cd 2+ , and cultured simultaneously under the same temperature and rotation speed conditions. Destructive sampling was carried out at 12 h, 24 h, 48 h, 72 h, 120 h, and 168 h of culture, and three parallels were set for each strain at each time point. Each time, 9 mL of the culture solution was centrifuged at 8000 r·min⁻¹ for 10 min to separate the supernatant and the cells. The supernatant was filtered through a 0.22 μm sterile filter membrane and stored at 4 °C for later use. The heavy metals in the supernatant were determined by ICP-OES, and the OD of the strain and the pH change of the solution were monitored simultaneously.

[0046] 2. Calculation formula

[0047] The calculation formula for the heavy metal removal rate is as follows: R=(C0 - C e ) / C0×100%

[0048] In the formula: R is Cd 2+Removal rate; C0 represents the initial Cd concentration in the solution, mg / L; Ce represents the residual Cd concentration in the supernatant, mg / L. 2+ Concentration, mg / L; Ce represents the residual Cd concentration in the supernatant, mg / L. 2+ Concentration, mg / L.

[0049] 3. Experimental results

[0050] According to Figure 3 A in, under Cd stress, HMY-2 showed good growth conditions. The OD value continued to increase and reached the maximum of 1.48 at 72 h. There was a slight decrease at 72 h, but it still remained at a relatively high level, indicating that under Cd stress, HMY-2 could maintain continuous and stable physiological activity. 600 Value continued to rise and reached the maximum of 1.48 at 72 h. There was a slight decrease at 72 h, but it still remained at a relatively high level, indicating that under Cd stress, HMY-2 could maintain continuous and stable physiological activity.

[0051] In addition, under cadmium stress, HMY-2 could significantly increase the pH value of the medium, from the initial 7.0 to the maximum of 9.05 at 72 h, and then slightly decreased to 8.85 (B in Figure 3 ). Therefore, the HMY-2 strain had significant alkali-producing ability.

[0052] Within 168 h, with the increase of culture time, the removal rate of Cd by HMY-2 gradually increased, and the highest removal rate could reach 63.14% ( Figure 4 ). Compared with the removal ability of the reported strains under the same conditions (Table 1), the removal effect of HMY-2 on Cd was in the upper-middle level and had a good removal effect.

[0053] Table 1 Comparison of heavy metal removal rates by different strains

[0054]

[0055] Example 3 Control effect of Microbacterium paraoxydans ( Microbacterium paraoxydans ) on rice growth and Cd accumulation

[0056] 1. Preparation of bacterial suspension and biochar-loaded bacterial agent

[0057] Bacterial suspension: Transfer the strain cultured to the logarithmic growth phase into a 10 mL centrifuge tube, centrifuge at 12000 rpm for 5 min, discard the supernatant, resuspend with sterile water, and then centrifuge again under the same conditions as above. After washing, add a certain volume of sterile water and mix until a bacterial solution with OD 600 = 1.0 is obtained and reserved.

[0058] Biochar-loaded inoculant: The rice husk biochar material and the bacterial suspension were oscillated for 24 h at a volume ratio of 1:1 and 130 rpm. After taking out, the mixture was poured into a centrifuge tube and centrifuged at 3000 rpm for 8 min. The supernatant liquid was discarded, and the mixture at the bottom of the centrifuge tube was washed 2-3 times with sterile normal saline. The finally obtained mixture was the biochar-loaded inoculant.

[0059] 2. Treatment group settings

[0060] Two types of rice were selected: Teyou 9301 and Qi 1 You 386; Four treatment groups were set: blank control (CK, without inoculation and biochar), inoculated with bacterial suspension (B), added 2% biochar (w / w, C), added 2% biochar-loaded inoculant (w / w, BC), ensuring that the colony counts of the inoculant and the bacterial suspension were the same, and each treatment had 4 replicates.

[0061] 3. Pot arrangement

[0062] An appropriate amount of plump and uniform-sized rice seeds were selected, soaked in 10% sodium hypochlorite for 5 min for disinfection, and rinsed repeatedly with distilled water 5-6 times to remove the residual sodium hypochlorite; Then the rice seeds were soaked in 75% ethanol solution for 1 min and rinsed repeatedly with distilled water 5-6 times to remove the residual ethanol; Finally, the rice seeds were soaked in distilled water for 48 h. The germinated rice seeds were transplanted into the substrate and cultured for 30 days until the three-leaf and one-heart stage.

[0063] Weigh 7 kg of sieved test soil into a water bucket with a diameter of 25 cm × 25 cm, fully mix different materials, add water and let it stand for one week, then transplant two rice seedlings with the same growth at the three-leaf and one-heart stage into each pot, one plant per hole, two holes per pot, and the same water management and fertilization management were adopted during the rice growth period.

[0064] Ten days before rice harvest, the water was drained, and the rice grains and rice plants were collected respectively after maturity. The collected samples were washed with deionized water to remove sediment and impurities, placed in an oven at 105°C for 30 min for blanching, and dried to a constant weight at 75°C. The corresponding rhizosphere soil samples were collected, air-dried to a constant weight in a cool place, and ground through 20- and 100-mesh sieves for analysis.

[0065] 4. Experimental results

[0066] 4.1 Effects of different treatments on rice growth and yield

[0067] For the rice variety Teyou 9301, the BC treatment had the most significant promoting effect on root growth and leaves. Its root dry weight increased by 125.6% compared to the CK group, and the leaf dry weight increased by 65.96% compared to CK (Table 2). In terms of yield, the B, C, and BC treatments all significantly increased the grain dry weight compared to CK, but there was no significant difference among the three treatments. Compared to CK, the C and BC treatments could significantly increase the panicle number of rice, and the BC treatment had the best effect.

[0068] Table 2 Agronomic traits of Teyou 9301 under different treatments

[0069]

[0070] For the rice variety Qi 1 You 386, the BC treatment also significantly promoted the increase in root and leaf dry weights, which were increased by 82.4% and 37.07% respectively compared to CK. In terms of yield, compared to CK, only the BC treatment showed significant differences in grain dry weight and 1000-grain weight, which were increased by 77.9% and 6.3% respectively (Table 3).

[0071] Table 3 Agronomic traits of Qi 1 You 386 under different treatments

[0072]

[0073] In summary, whether it is Teyou 9301 or Qi 1 You 386, the BC treatment significantly promoted the growth of rice and increased the yield.

[0074] 4.2 Effects of different treatments on Cd accumulation in rice grains

[0075] For the rice variety Teyou 9301, compared with the CK group, the BC treatment significantly reduced the Cd content in rice grains by 77.73%. There was no significant difference in Cd accumulation in grains among the B, C treatment groups and the CK group (A in Figure 5 ). For the Qi 1 You 386 variety, compared with the CK group, the B, C, and BC treatments all significantly reduced the Cd accumulation in rice grains, by 68.99%, 62.37%, and 84.77% respectively (B in Figure 5 ). In summary, whether it is Teyou 9301 or Qi 1 You 386, the BC treatment significantly inhibited the Cd accumulation in rice.

[0076] 4.3 Effects of different treatments on soil physical and chemical properties

[0077] For the rice variety Teyou 9301, under the BC treatment, the soil pH value increased significantly by 5.7% compared to the CK group, and the soil organic matter increased significantly by 119.5%. The Eh values of the soil in the B, C, and BC treatment groups increased significantly, by 124.11 mv, 529.61 mv, and 550.1 mv respectively. Compared with CK, the C and BC treatments significantly increased the available potassium content in the soil, by 91.16% and 177.87% respectively; the available phosphorus content also increased significantly by 23.97% and 35.09% respectively.

[0078] Table 4 Physical and Chemical Properties of the Soil of Pot-grown Rice of Teyou 9301 under Different Treatments

[0079]

[0080] For the rice variety Qi 1 You 386, compared with the CK treatment, the BC treatment significantly increased the soil pH value and organic matter, by 5.8% and 89.0% respectively. The Eh values of the soil in the C and BC treatment groups increased significantly, by 571.91 mv and 574.55 mv respectively; the available potassium content and the available phosphorus content also increased significantly.

[0081] Table 5 Physical and Chemical Properties of the Soil of Pot-grown Rice of Qi 1 You 386 under Different Treatments

[0082]

[0083] Generally speaking, the impact of the treatment with only the inoculant B on the soil properties was not significant. The treatment with the biochar C could increase the soil pH value, redox potential, available potassium, organic matter, and available phosphorus content. The treatment with the biochar loaded with the inoculant BC had the best effect, providing a more favorable soil environment for rice growth.

Claims

1. A strain of Microbacterium paraoxidans with the ability to adsorb Cd and produce alkali ( Microbacterium paraoxydans ) HMY-2 was deposited at the General Microbiology Center of China Microorganism Culture Collection Committee on December 30, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.33225.

2. The bacterial agent prepared by the Microbacterium paraoxydants HMY-2 according to claim 1, characterized in that: The bacterial agent is OD 600 =1.0 bacterial suspension of Microbacterium paraoxydense HMY-2.

3. A biochar-loaded bacterial agent, characterized in that: The biochar-loaded bacterial agent is prepared by the following method: the rice husk biochar material and the bacterial agent prepared from the Microbacterium paraoxydense HMY-2 according to claim 2 are mixed at a volume ratio of 1:1-2, shaken at 120-150 rpm for 20-24 h, then centrifuged at 3000-4000 rpm for 5-10 min, the supernatant liquid is discarded, and the biochar-loaded bacterial agent is washed with sterile saline to obtain the biochar-loaded bacterial agent.

4. The Microbacterium paraoxygenase according to claim 1 ( Microbacterium paraoxydans )Application of HMY-2 in adsorption and fixation of cadmium.

5. Use of the biochar-loaded bacterial agent according to claim 3 in improving soil pH under cadmium stress.

6. The use of the biochar-loaded bacterial agent according to claim 3 in improving plant growth conditions, reducing plant absorption and accumulation of heavy metal cadmium, and improving soil physical and chemical properties, wherein the plant is rice, and the improvement of soil physical and chemical properties is to increase the pH and Eh value, available potassium, organic matter and available phosphorus content of the soil.

7. A method for reducing the absorption and accumulation of heavy metal cadmium in plants, characterized in that: The biochar-loaded bacterial agent according to claim 3 is inoculated into the soil, and the plant is rice.

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