Microbacterium paraoxidans HMY-2 with Cd adsorption and alkali production functions and application of microbacterium paraoxidans HMY-2

Abstract

The invention discloses microbacterium paraoxidans HMY-2 with Cd adsorption and alkali production functions and application of the microbacterium paraoxidans HMY-2. The invention discloses a microbacterium paraoxidans HMY-2, which is preserved in China General Microbiological Culture Collection Center on December 30, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.33225. The microbacterium paraoxidans HMY-2 has the advantages that the preservation number is CGMCC No.33225; the highest removal rate of the Cd < 2 + > (50mg / L) can reach 63.14%, and the pH value of the culture medium can be increased to 9.05 from initial 7.0 through metabolism. When the biochar is prepared into a biochar-loaded microbial agent for application, the yield of rice can be increased, and accumulation of heavy metals by rice grains is reduced; the soil property is improved, the pH and Eh value of the soil and the content of available potassium, organic matter and available phosphorus are increased, and a more favorable soil environment is provided for rice growth.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to a strain of Microbacterium paraoxidans ( Microbacterium paraoxydans )HMY-2 and its applications. 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 continue to accumulate in the soil-water-biological system through geological cycles, which not only leads to the degradation of arable land quality, but also harms human health through the amplification effect of the food chain.

[0003] Tropical regions have excellent light and heat conditions, and crops can be planted all year round, making them an important rice production base. Agriculture in tropical regions is highly intensive, with a high multiple cropping index, and the use of chemical fertilizers is 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 parent material of soil in some tropical regions is mainly acidic igneous rocks. Coupled with the high temperature and rainy climate characteristics, the base ions in the soil are easily leached, and the soil is mostly acidic. This will also lead to the activation of heavy metal elements in the soil, increase their effectiveness, promote the absorption and accumulation of crops, and affect the safety of agricultural products. Therefore, it has become an urgent problem to construct a heavy metal barrier planting management technology suitable for the farmland soil-rice system in tropical regions to ensure the safety of agricultural products.

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

[0005] Microbial passivation technology uses the adsorption, complexation and redox properties of microorganisms to heavy metals to change the form and effectiveness of heavy metals in the environment, thereby reducing the harm of heavy metals to the environment and ecosystem. Currently reported heavy metal passivation strains mainly include fungi such as yeast ( Saccharomyces )、Aspergillus( Aspergillus )、Rhizopus spp. Rhizopus arrhizus ); Gram-negative bacteria such as Pseudomonas spp. ( Pseudomonas ), Serratia spp. Serratia ), Escherichia coli ( Escherichia coli ) and Gram-positive bacteria such as Bacillus spp. ( Bacillus ), Staphylococcus spp. Staphylococcus ), Lactobacillus spp. 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: 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.

[0010] 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.

[0011] A bacterial agent prepared by the Microbacterium paraoxydants HMY-2 of the present invention.

[0012] As a preferred embodiment of the present invention, the bacterial agent is the fermentation broth of Microbacterium paraoxydants HMY-2.

[0013] As another preferred embodiment of the present invention, the bacterial agent is a biochar bacterial agent loaded with Microbacterium paraoxidans HMY-2.

[0014] As a further preferred embodiment of the present invention, the biochar agent loaded with Microbacterium paraoxydants HMY-2 is prepared by the following method: the rice husk biochar material passed through a 100-200 mesh sieve and the bacterial suspension are shaken at a volume ratio of 1:1-2 at 120-150 rpm for 20-24 h, and then centrifuged at 3000-4000 rpm for 5-10 min, the supernatant is discarded, and the biochar agent loaded with Microbacterium paraoxydants HMY-2 is washed with sterile saline.

[0015] The Microbacterium paraoxygensis of the present invention ( Microbacterium paraoxydans )Application of HMY-2 in adsorption and fixation of cadmium.

[0016] The Microbacterium paraoxygensis of the present invention ( Microbacterium paraoxydans ) Application of HMY-2 in increasing pH under cadmium stress.

[0017] The invention discloses an application of the bacterial agent in improving plant growth conditions.

[0018] As a preferred embodiment, the present invention provides the use of the bacterial agent in reducing the absorption and accumulation of heavy metal cadmium in plants.

[0019] As a preferred embodiment, the present invention provides the use of the bacterial agent in improving the physical and chemical properties of soil.

[0020] The plant is more preferably rice.

[0021] The third object of the present invention is to provide a method for reducing the absorption and accumulation of heavy metal cadmium by plants, by introducing the bacterial agent into the soil.

[0022] The plant is preferably rice.

[0023] Microbacterium paraoxygensis ( Microbacterium paraoxydans)HMY-2 is used in rice cultivation to passivate soil Cd, reduce its accumulation in rice, improve rice growth and promote soil health. The strain was compounded with biochar to make a bacterial agent, and applied at a 2% addition rate to the soil where two rice varieties (Teyou 9301 and Qi1you 386) were planted. The results showed that the growth and yield of both rice varieties were significantly improved. The root dry weight of Teyou 9301 rice increased by 125.6%, the leaf dry weight increased by 66.0%, the grain dry weight increased by 44.9%, and the number of panicles increased by 36.4%; the root dry weight of Qi1you 386 rice increased by 82.4%, the leaf dry weight increased by 37.1%, the grain dry weight increased by 77.9%, and the thousand-grain weight increased by 6.3%. In addition, the application of the bacterial agent significantly reduced the accumulation of cadmium in rice grains (the Cd in the grains of Teyou 9301 and Qi1you 386 decreased by 77.73% and 84.77%, respectively). At the same time, the application of the inoculant significantly increased soil pH, Eh, available potassium, organic matter and available phosphorus content. In summary, the inoculant of Microbacterium paraoxydense HMY-2 can effectively reduce Cd accumulation in rice grains, promote rice growth, and improve soil physical and chemical properties.

[0024] Beneficial Effects

[0025] The present invention screened out a strain of alkali-producing bacteria that can passivate heavy metals from the soil of Yazhou District, Sanya City, Hainan Province, and sequenced and identified it as Microbacterium paraoxydense ( Microbacterium paraoxydans ) HMY-2, which is sensitive to Cd 2+ The removal rate of (50mg / L) can reach up to 63.14%, and the pH value of the culture medium can be increased from the initial 7.0 to 9.05 through metabolism. The application of biochar-loaded microbial agent can increase rice yield and reduce the accumulation of heavy metals in rice grains; improve soil properties, increase soil pH and Eh value, available potassium, organic matter and available phosphorus content, and provide a more favorable soil environment for rice growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the colony characteristic diagram of Microbacterium paraoxydense on LB solid medium.

[0027] Figure 2 This is the phylogenetic tree of Microbacterium paraoxygenase.

[0028] Figure 3 This is the growth curve of Microbacterium paraoxidans and the changes in culture medium pH under cadmium stress.

[0029] Figure 4 This is the dynamic trend of cadmium removal rate by Microbacterium paraoxidans.

[0030] Figure 5 Changes in cadmium content in rice grains under different treatment conditions. Biomaterial Deposit Information

[0031] HMY-2, classified as Microbacterium paraoxygenase ( 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. DETAILED DESCRIPTION

[0032] The present invention is further described below by way of examples, and the present invention can be better understood according to the following examples. The specific material ratios, process conditions and 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.

[0033] Example 1 Microbacterium paraoxygensis ( Microbacterium paraoxydans ) The culture medium formula is as follows: LB solid medium: peptone 10g, yeast extract 5g, sodium chloride 10g, agar 20g, distilled water 1000mL, pH 7.0-7.2, sterilization at 121℃ for 20min. LB liquid medium: no agar, other conditions are the same as above.

[0034] Strain screening Accurately weigh 10 g of fresh soil sample and dissolve it in a 250 mL Erlenmeyer flask filled with 90 mL of sterile water. Mix well by shaking at 28°C and 180 r / min for 30 min. Take 1 mL of supernatant and dilute with 9 mL of sterile water, and dilute to 10 -8 Take 0.1mL of the diluted solution and inoculate it with Cd 2+ LB solid medium plates (concentrations set to 50, 150, 250, 350, 450, 550 mg / L) were incubated at 30°C for 48 hours. Single colonies with good morphology were selected and scraped with an inoculation loop, and streaked on plates containing heavy metal solid medium for separation and purification, and incubated at 30°C for 2 days. After 5 generations of continuous cultivation, genetically stable heavy metal-resistant strains were obtained and stored in a -80°C refrigerator in the form of a suspension containing 50% glycerol by volume.

[0035] Strain identification A strain with good growth condition was selected on the LB medium plate with Cd concentration of 550 mg / L, such as Figure 1 As shown in the figure, the colonies formed by the strain on the LB plate after culturing for 24 hours are yellow, round, opaque, with neat edges and a colony diameter of 1-3 mm. The strain type is named HMY-2 (see Figure 1 ).

[0036] The strains isolated by the above method were sequenced by Qingke Biotechnology Co., Ltd. Based on the sequencing results of 16S rDNA, the homology was compared with other 16S rDNA sequences in GenBank using Blast software, and a phylogenetic tree was further constructed using MEGA software (see Figure 2 ), the results showed that the bacteria was Microbacterium paraoxygensis ( Microbacterium paraoxydans ). Previous studies have shown that this type of strain has the functions of reducing Cr (VI) to repair contaminated soil / water sources, multi-drug resistance, and removal of chlorobenzene, but there are no reports on heavy metal adsorption and passivation functions. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 30, 2024, with the deposit number CGMCC No. 33225.

[0037] Example 2 Under Cd stress, Microbacterium paraoxydense ( Microbacterium paraoxydans ) and Cd adsorption characteristics 1. Experimental steps The screened HMY-2 strain was inoculated into 100 mL LB liquid medium and cultured at 30°C and 180 r·min⁻¹ until the logarithmic growth phase to prepare the seed solution. A 2% (v / v) inoculum was transferred to a 50 mg / LCd 2+ 20 mL LB medium and Cd-free 2+ The strains were cultured simultaneously in 20 mL LB medium at the same temperature and speed. Destructive sampling was performed at 12 h, 24 h, 48 h, 72 h, 120 h and 168 h of culture, with three replicates for each strain at each time point. Each time, 9 mL of culture solution was centrifuged at 8000 r·min⁻¹ for 10 min to separate the supernatant from the bacteria. The supernatant was filtered through a 0.22 μm sterile filter membrane and stored at 4 °C for later use. ICP-OES was used to determine the heavy metals in the supernatant, and the strain OD and solution pH changes were monitored simultaneously.

[0038] 2. Calculation formula The heavy metal removal rate is calculated as follows: R = (C0-C e ) / C0×100% Where: R is Cd 2+ The removal rate of Cd2+ is 2.34, which is 2.33% of the total Cd2+ solution. 2+ Concentration, mg / L; Ce represents the residual Cd in the supernatant 2+ Concentration, mg / L.

[0039] 3. Experimental results according to Figure 3 As shown in A, HMY-2 showed better growth under Cd stress.600 The value continued to increase and reached a maximum of 1.48 at 72 h. It decreased slightly at 72 h but still maintained at a high level, indicating that HMY-2 can maintain sustained and stable physiological activity under Cd stress.

[0040] In addition, under cadmium stress, HMY-2 significantly increased the pH of the culture medium from an initial 7.0 to a maximum of 9.05 at 72 h, and then decreased slightly to 8.85 ( Figure 3 Therefore, the HMY-2 strain has a significant alkali-producing ability.

[0041] Within 168 hours, as the culture time increased, the removal efficiency of HMY-2 for Cd gradually increased, and the maximum removal efficiency could reach 63.14% ( Figure 4 ). Compared with the removal ability of reported strains under the same conditions (Table 1), the removal effect of HMY-2 on Cd is in the upper middle level and has a good removal effect.

[0042] Table 1 Comparison of heavy metal removal rates by different strains

[0043] Example 3 Microbacterium paraoxygensis ( Microbacterium paraoxydans ) on rice growth and Cd accumulation 1. Preparation of bacterial suspension and biochar-loaded inoculants Bacterial suspension: Transfer the strain cultured to the logarithmic growth phase to a 10 mL centrifuge tube, centrifuge at 12,000 rpm for 5 min, discard the supernatant, add sterile water to resuspend, and then centrifuge again under the same conditions as above. After washing, add a certain volume of sterile water and mix until an OD of 600 =1.0 bacterial solution for later use.

[0044] Biochar-loaded bacterial agent: Rice husk biochar material and bacterial suspension were shaken at 130 rpm for 24 h at a volume ratio of 1:1. After taking out, the mixture was poured into a centrifuge tube and centrifuged at 3000 rpm for 8 min. The supernatant was discarded and the mixture at the bottom of the centrifuge tube was washed 2-3 times with sterile saline. The final mixture was the biochar-loaded bacterial agent.

[0045] 2. Processing group settings Two rice varieties were selected: Teyou 9301 and Qi 1 You 386. Four treatment groups were set up: blank control (CK, without inoculation of bacteria and biochar), inoculation of bacterial suspension (B), addition of 2% biochar (w / w, C), and addition of 2% biochar loaded with bacterial agent (w / w, BC). The colony counts of the bacterial agent and the bacterial suspension were kept consistent, and each treatment was replicated 4 times.

[0046] 3. Potted Plant Arrangement Select an appropriate amount of rice seeds with full grains and uniform size, soak them in 10% sodium hypochlorite for 5 minutes for disinfection, and rinse them repeatedly with distilled water 5-6 times to remove the residual sodium hypochlorite; then soak the rice seeds in 75% ethanol solution for 1 minute, and rinse them repeatedly with distilled water 5-6 times to remove the residual ethanol; finally, soak the rice seeds in distilled water for 48 hours. Transplant the germinated rice seeds into the substrate and culture them for 30 days until the three-leaf one-heart stage.

[0047] Weigh 7 kg of sieved test soil into a bucket with a diameter of 25 cm × 25 cm, mix the different materials thoroughly, add water and let stand for a week, then transplant two rice seedlings of the same growth at the three-leaf and one-heart stage into each pot, one plant in one hole, two holes in one pot, and adopt the same water management and fertilization management during the rice growth period.

[0048] The rice was dried 10 days before harvest, and the rice and rice plants were collected 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, and dried to constant weight at 75°C. Soil samples from the corresponding rhizosphere were collected, air-dried to constant weight in a cool place, and ground to pass 20 and 100 mesh sieves for analysis.

[0049] 4. Experimental results 4.1 Effects of different treatments on rice growth and yield For the rice variety Teyou 9301, the BC treatment had the most significant effect on promoting root growth and leaves, with the root dry weight increased by 125.6% and the leaf dry weight increased by 65.96% compared with the CK group (Table 2). In terms of yield, the B, C and BC treatments significantly increased the grain dry weight compared with CK, but there was no significant difference among the three treatments. Compared with CK, the C and BC treatments could significantly increase the number of rice panicles, and the BC treatment had the best effect.

[0050] Table 2 Agronomic traits of Teyou 9301 under different treatments

[0051] For the rice variety Qi1you386, BC treatment also significantly promoted the increase of root and leaf dry weight, which increased by 82.4% and 37.07% respectively compared with CK. In terms of yield, only BC treatment showed significant differences in grain dry weight and thousand-grain number compared with CK, which increased by 77.9% and 6.3% respectively (Table 3).

[0052] Table 3 Agronomic traits of Xiaqi 1you 386 under different treatments

[0053] In conclusion, for both Teyou 9301 and Qi 1 You 386, BC treatment significantly promoted the growth of rice and increased its yield.

[0054] 4.2 Effects of different treatments on Cd accumulation in rice grains For the rice variety Teyou 9301, the BC treatment significantly reduced the Cd content in rice grains by 77.73% compared with the CK group. There was no significant difference in the accumulation of Cd in rice grains between the B and C treatment groups and the CK group ( Figure 5 For Qi1you386, compared with the CK group, the B, C, and BC treatments significantly reduced the accumulation of Cd in rice grains by 68.99%, 62.37%, and 84.77%, respectively ( Figure 5 In conclusion, BC treatment significantly inhibited the accumulation of Cd in rice, whether it was Teyou 9301 or Qi1you 386.

[0055] 4.3 Effects of different treatments on soil physical and chemical properties For the rice variety Teyou 9301, the soil pH value under BC treatment was significantly increased by 5.7% compared with the CK group, and the soil organic matter was significantly increased by 119.5%. The B, C, and BC treatment groups significantly increased the soil Eh by 124.11mv, 529.61mv, and 550.1mv, respectively. Compared with CK, the C and BC treatments significantly increased the soil available potassium content by 91.16% and 177.87%, respectively; the available phosphorus content also significantly increased by 23.97% and 35.09%, respectively.

[0056] Table 4 Physical and chemical properties of soil of Teyou 9301 potted rice under different treatments

[0057] For the rice variety Qi 1 You 386, the BC treatment significantly increased soil pH and organic matter by 5.8% and 89.0% compared with the CK treatment. The C and BC treatment groups significantly increased soil Eh by 571.91mv and 574.55mv, respectively; the available potassium content and available phosphorus content also increased significantly.

[0058] Table 5 Physical and chemical properties of soil in potted rice field Xiaqi 1you 386 under different treatments

[0059] In general, the addition of bacterial agent B alone had no significant effect on soil properties. The addition of biochar C could increase the soil pH, redox potential, available potassium, organic matter and available phosphorus content. The biochar-loaded bacterial agent BC treatment 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 bacterial agent prepared by the Microbacterium paraoxydants HMY-2 according to claim 1, characterized in that: The bacterial agent is a biochar bacterial agent loaded with oxidative Microbacterium HMY-2, and the biochar bacterial agent loaded with oxidative Microbacterium HMY-2 is prepared by the following method: the rice husk biochar material and the bacterial suspension are shaken at a volume ratio of 1:1-2 at 120-150 rpm for 20-24 hours, and then centrifuged at 3000-4000 rpm for 5-10 minutes, the supernatant liquid is discarded, and the biochar bacterial agent loaded with oxidative Microbacterium HMY-2 is washed with sterile physiological saline to obtain.

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

5. The Microbacterium paraoxygenase according to claim 1 ( Microbacterium paraoxydans ) Application of HMY-2 in increasing pH under cadmium stress.

6. Use of the bacterial agent according to claim 2 or 3 in improving plant growth conditions, reducing plant absorption and accumulation of heavy metal cadmium, and improving soil physical and chemical properties.

7. The use according to claim 6, characterized in that: The plant is rice.

8. A method for reducing the absorption and accumulation of heavy metal cadmium in plants, characterized in that: The bacterial agent according to claim 2 or 3 is inoculated into the soil.

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