Application of Amycolatopsis kukumannii
By using the Tucumanamic acid bacteria KC 316 fermentation broth as a bacterial agent, the problem of insufficient plant growth and heavy metal enrichment capabilities under heavy metal pollution environment was solved, and the efficient growth and heavy metal enrichment of peacock grass in the polluted environment was achieved, thereby avoiding secondary pollution in the environment.
Patent Information
- Application Number
- CN202510256817.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to effectively promote plant growth and improve the plant's ability to enrich heavy metals under heavy metals polluted environments. Traditional methods have problems of high costs and secondary environmental pollution.
The fermentation broth of Amycolatopsis tucumanensis KC 316 was used as a bacteria agent to promote the growth of peacock grass in a heavy metal-contaminated environment by changing the nutrients and enzyme activities required for plant growth.
It significantly improves the growth performance and heavy metal enrichment ability of peacock grass in heavy metal-polluted environments, including increased soluble protein, POD and SOD activities, as well as the content of chlorophyll and soluble sugars, thereby improving the physiological indicators and heavy metal enrichment efficiency of plants.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and relates to the application of a strain of Amycota tucumanensis, and specifically relates to the application of Amycota tucumanensis in the preparation of a bacterial agent for promoting plant growth in a heavy metal polluted environment and improving the heavy metal enrichment capacity of plants. Background Art
[0002] In recent years, the rapid advancement of industrialization has brought about significant economic development, but it has also caused serious problems of heavy metal pollution in the soil, especially in the soil of tailings fields, ore piles and other areas, because they are rich in heavy metal elements such as lead, zinc, cadmium, etc. If not handled properly, it is very easy to cause long-term pollution to the surrounding environment. These heavy metal elements not only destroy the ecological balance of the soil, but also accumulate through the food chain, posing a potential threat to human health. Studies have shown that Cd is one of the heavy metals that is most easily absorbed and transported by plants. Even at lower concentrations, it can cause many harms to plants, such as replacing the sulfhydryl groups on the functional groups of proteins to activate or inhibit enzyme activity, interfere with ion metabolism, promote the formation and accumulation of free radicals, and destroy the structure of biofilms, affecting the main metabolic processes, not only inhibiting plant growth, but even causing death in severe cases. If the human body frequently consumes foods with excessive Cd content, it is easy to induce gene mutations.
[0003] In view of this situation, traditional physical and chemical methods such as soil leaching and chemical stabilization are widely used. Although these methods can reduce the toxicity of heavy metals to a certain extent, they are often accompanied by high treatment costs, soil structure damage and secondary pollution to the environment, and their comprehensive benefits are not ideal. Traditional soil remediation methods include physical remediation, chemical remediation and biological remediation, among which biological remediation has attracted widespread attention due to its advantages of low cost and small impact on the environment. Among them, the use of specific plants to remediate heavy metal contaminated soil has attracted much attention due to its green, economical and sustainable characteristics. In the field of microbial engineering, the use of microorganisms and their metabolites to promote plant growth and repair soil environment has become an important research direction. Microorganisms can promote plant growth by producing plant hormones, dissolving minerals, fixing atmospheric nitrogen and other mechanisms, and can also repair polluted soil environments through processes such as adsorption, degradation and transformation. In the field of plant nutrition, plant growth and development are inseparable from the supply of nutrients. Nutrients include macroelements (such as nitrogen, phosphorus, potassium) and trace elements (such as iron, zinc, copper), which are essential for plant physiological metabolism and growth and development. In addition, enzyme activity and other factors (such as antioxidant capacity) also play an important role in the growth and stress resistance of plants. For example, cosmos, as a highly adaptable herbaceous plant of the Asteraceae family and the genus Tagetes, has the ability to tolerate barrenness, drought and enrich heavy metals, making it one of the preferred plants in the field of bioremediation. However, despite the above advantages of cosmos, it still faces challenges in practical applications. On the one hand, its growth volume is limited, making it difficult to meet the needs of large-scale restoration; on the other hand, although cosmos has a high enrichment of heavy metals, it is restricted by various factors, and its restoration efficiency still needs to be improved. Therefore, finding a microbial strain that can promote the growth of cosmos and improve its heavy metal enrichment capacity is of great significance for achieving efficient bioremediation.
[0004] Amycolatopsis tucumanensis KC136 is reported to be useful for the prevention and treatment of nematode diseases, and also has the effect of promoting plant growth. After searching, there is no related report in the prior art that this strain has the ability to promote plant growth and improve the ability of plant heavy metal accumulation under heavy metal pollution environment conditions. Summary of the invention
[0005] Based on the above technical problems, the present invention intends to provide an application of Amycolatopsis tucumánensis to promote plant growth and improve the heavy metal accumulation capacity of plants under heavy metal pollution conditions.
[0006] To achieve the above object, the present invention provides the following technical solutions:
[0007] The present invention protects the use of Amycolatopsis tucumanensis in the preparation of a bacterial agent for promoting plant growth in a heavy metal polluted environment and improving the heavy metal enrichment ability of plants. The classification name of the Amycolatopsis tucumanensis is (Amycolatopsis tucumanensis) KC 316, which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 2, 2022, and its deposit number is CGMCC No.26012.
[0008] Furthermore, the bacterial agent is the fermentation broth of Amycolax tucumiensis.
[0009] Furthermore, the fermentation liquid of Amycolax tucumiensis is used in the preparation of a bacterial agent for promoting plant growth in a high-cadmium and high-lead environment.
[0010] Furthermore, the fermentation liquid of Amycolax tucumiensis is used in the preparation of a bacterial agent that promotes plant enrichment of heavy metals in soil.
[0011] Furthermore, the fermentation liquid of Amycolax tucumiensis is used in the preparation of a microbial agent for improving plant physiological indicators.
[0012] The present invention also protects the preparation method of the Tucuman Amycolatopsis fermentation liquid as follows:
[0013] Activation of S1 strain
[0014] Take the preserved KC 316 strain, streak it onto the modified ISP 2 medium, place the plate in a constant temperature incubator, set the temperature to 35-37°C, and culture for 8-10 days, during which the growth of the strain is regularly observed;
[0015] Preparation of S2 seed solution
[0016] The activated single colony in S1 was inoculated into the seed culture medium and cultured under shaking conditions of 28°C and 200 rpm for 4-5 days to obtain the seed solution;
[0017] S3 Fermentation
[0018] Take 5 mL of the seed solution in S2, add it into a container containing fermentation medium, and culture it at 25-35° C. and 130-200 rpm / min for 3-10 days under shaking fermentation to obtain fermentation liquid, wherein the fermentation medium is ISP 2 improved medium.
[0019] Preferably, the preparation process of the ISP 2 improved medium is as follows: add 5g glucose, 5g soy peptone, 5g yeast extract in sequence, then add a small amount of trace salt and a small amount of microorganisms, dissolve them in water, adjust the pH of the solution to 7.5, and finally add water to 1L, and sterilize at 121°C for 15 minutes.
[0020] Preferably, the preparation method of the seed culture medium is: dissolve 5g yeast extract, 10g malt extract and 4g glucose in water, adjust the pH value of the solution to 7.0-7.2, dilute to 1L with water, and then sterilize at 121°C for 30min.
[0021] Preferably, the fermentation liquid obtained in claim 6 is diluted 1000 times and then used for root irrigation at a dosage of 30-40 mL per pot of plants.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] The present invention protects the use of Amycolatopsis tucumanensis KC 316 fermentation liquid in the preparation of a microbial agent that promotes plant growth in a heavy metal polluted environment and improves the heavy metal enrichment capacity of plants under polluted environmental conditions. Experiments have shown that the Amycolatopsis tucumanensis KC 316 fermentation liquid provided by the present invention can promote the growth of cosmos under polluted conditions by changing the nutrients and enzyme activities required for the growth of cosmos. Among them, the soluble protein content increased significantly by 5.62% compared with the control group; the POD activity increased significantly by 15.00% compared with the control group; the SOD activity increased significantly by 2.29% compared with the control group; the proline content increased significantly by 15.04% compared with the control group; the MDA content decreased significantly by 5.93% compared with the control group; the soluble sugar content increased significantly by 20.78% compared with the control group; the chlorophyll content increased significantly by 59.74% compared with the control group, thereby promoting the germination rate of cosmos seeds in a high cadmium environment by 20.00%; The plant height of cosmos in high cadmium and high lead soil during flowering period increased by 22.68% and the root length increased by 22.20%. It can also advance the flowering time of cosmos by 2 days, and the number of flower buds increased by 21.43% compared with the control group. The fresh weight of roots after treatment increased by 54.46%, stems by 86.43%, leaves by 52.84%, and the whole plant by 60.38% compared with the control group. The dry weight of roots increased by 35.29%, stems by 85.71%, leaves by 53.03%, and the whole plant by 72.28% compared with the control group. The experiment also found that the enrichment of three heavy metals in cosmos can be promoted after treatment with KC316 fermentation liquid. The contents and enrichment coefficients of the three heavy metals in the leaves and roots were greater than those in the control group; the lead enrichment increased by 56.25%, and the extraction rate increased by 0.72%; the zinc enrichment increased by 145.78%, and the extraction rate increased by 1.83%; the cadmium enrichment increased by 98.78%, and the extraction rate increased by 18.71%; the soil pH decreased by 0.73 pH units compared with the control group; the total nitrogen, organic matter, and organic carbon contents increased slightly compared with the control group, increasing by 3.36%, 5.25%, and 5.25%, respectively; the heavy metal Cd, Pb, and Zn contents decreased compared with the control group, decreasing by 34.22%, 40.94%, and 32.53%, respectively. It is proved that the fermentation liquid of Amycolatopsis tucumanensis KC 316 can promote the growth of cosmos and improve the ability of cosmos to enrich heavy metals in the soil, so that the problem of heavy metal contaminated soil is further solved, and no secondary pollution is caused to the environment, which is safer for humans and animals. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 For different Cd 2+Effect of KC316 fermentation liquid on root length and plant height of cosmos at different concentrations;
[0025] Figure 2 This is the effect of KC 316 fermentation liquid on the germination rate of cosmos seeds;
[0026] Figure 3 This is the effect of KC316 fermentation liquid on the root length and plant height of cosmos;
[0027] Figure 4 This is the effect of KC316 fermentation liquid on the fresh weight of different parts of Marigold;
[0028] Figure 5 This is the effect of KC316 fermentation liquid on nutrients, enzyme activity and other factors of Marigold;
[0029] Figure 6 This is a schematic diagram of the plant growth comparison between the control group and the KC 316 fermentation liquid treatment group; Figure 6 A is the control group; Figure 6 B is the KC 316-treated group. DETAILED DESCRIPTION
[0030] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0031] The experimental methods in the following examples are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. DMSO is the full name of dimethyl sulfoxide. Unless otherwise specified, the quantitative tests in the following examples are repeated three times, and the results are averaged.
[0032] The classification name of Amycolatopsis tucumanensis used in the present invention is (Amycolatopsis tucumanensis) KC 316, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 2, 2022, and its deposit number is CGMCC No.26012.
[0033] Example 1 Preparation of Fermentation Broth of Amycota tucumiensis KC316
[0034] The preparation method of the fermentation broth of Amycolax tucumiensis KC316 is as follows:
[0035] Activation of S1 strain: Take the KC 316 strain preserved in a glycerol tube, streak it onto the ISP 2 improved medium, and culture it for 3-5 days; the preparation process of the ISP 2 improved medium is as follows: add 5g glucose, 5g soy peptone, 5g yeast extract in sequence, and then add a small amount of trace salt and a small amount of microorganisms, dissolve them in water, adjust the pH of the solution to 7.5, and finally add water to 1L, and sterilize at 121°C for 15 minutes.
[0036] Preparation of S2 seed solution: inoculate the activated single colony in S1 into the seed culture medium, and culture it under shaking at 28°C and 200rpm for 5 days; the preparation method of the seed culture medium is as follows: dissolve 5g yeast extract, 10g malt extract and 4g glucose in water, adjust the pH value of the solution to 7.0-7.2, dilute to 1L with water, and then sterilize at 121°C for 30min;
[0037] S3: Take 5 mL of the seed solution in S2, add it into a container containing fermentation medium, and culture it at 25-35°C and 130-200 rpm / min for 3-10 days to obtain fermentation liquid, wherein the fermentation medium is ISP 2 improved medium.
[0038] Example 2 Promoting the growth of cosmospermum oleifera using the fermentation liquid of Amycota tucumiensis KC316
[0039] This example provides the effect of the fermentation liquid of Amycolatopsis tucumanensis KC 316 on the growth-promoting ability of Marigold in a high-cadmium and high-lead environment and the effect on improving the ability of Marigold to enrich heavy metals in the soil.
[0040] 1. Petri dish growth promotion test: Sterilized and dried filter paper was spread in a petri dish, and then the cosmos seeds were placed in it. Ten seeds were placed in one petri dish, and each group was repeated 3 times. Then 10 mL of cadmium chloride solution of different concentrations was sucked by a pipette and added to different petri dishes to soak the filter paper. The treatment group was set as heavy metal Cd 2+ The concentrations were 0.6, 3, 6, 10, and 30 mg / L. Finally, 10 μL of a 1000-fold dilution of the fermentation broth of Amycolatopsis tucumanensis KC 316 obtained in Example 1 was added to each culture dish, and 10 μL of a 1000-fold diluted blank culture medium was added as a control group.
[0041] Place the culture dish containing the seeds in a constant temperature and light incubator, set the culture conditions to 28°C, 50% humidity, and 12-hour light intensity 0-100% cycle. Observe every 8 hours to ensure sufficient water, and record the root length and bud length of the cosmos on time every day. The results are as follows: Figure 1 shown.
[0042] from Figure 1 The results showed that the root length of the KC316 treated group of cosmos increased by 25.80%, 33.78%, 31.56%, and 32.77% respectively compared with the control group under the same concentration of cadmium chloride at 0.6, 3, 6, and 10 mg / kg; the plant height increased by 20.34%, 23.74%, 21.98%, and 22.34% respectively compared with the control group. It can be seen that the KC316 strain has a strong anti-inflammatory effect under different Cd concentrations of 0.6, 3, 6, and 10 mg / kg. 2+ The root growth and plant height of cosmos were promoted at all concentrations, but the 2+ The concentration of 3 mg / kg had the greatest growth-promoting effect.
[0043] 2. Seed germination rate test: Control group A is 3 mg / L Cd 2+ Solution, treatment group B was added with 3 mg / L Cd 2+ The KC316 fermentation liquid was cultured in a light incubator with three replicates per group. The germination rate of cosmos was recorded from the third day. The results are as follows Figure 2 shown.
[0044] From the above experimental results, it can be seen that the seed germination rate of the KC 316 treatment group increased compared with the control group, and the germination rate increased the most on the third day, reaching 20%.
[0045] 3. Pot experiment: The effect of fermentation liquid of Amycolatopsis tucumanensis KC 316 on the growth promotion of cosmos in flowering period under high cadmium (30 mg / kg) and high lead (3305 mg / kg) environment was determined by outdoor pot experiment:
[0046] 1) Root length and plant height: Control group A was irrigated with 30 mL of blank culture medium, and treatment group B was irrigated with 30 mL of 1000-fold diluted KC 316 fermentation solution. Each group was repeated six times. The root length and plant height of cosmos were observed and recorded. The results are as follows: Figure 3 shown.
[0047] From the above experimental results, it can be seen that on the 63rd day, the fermentation liquid of Amycolatopsis tucumiensis KC 316 promoted the increase of plant height of Marigold 22.68% and root length of 22.20%.
[0048] 2) Fresh weight of different tissue parts: When the culture reached 63 days, the fresh weight of the plants, roots, stems and leaves of the cosmos were weighed. Figure 4 shown.
[0049] The results showed that after KC316 treatment, the fresh weight of the cosmos in the treatment group was significantly higher than that in the control group. The fresh weight of the root increased by 54.46%, the stem increased by 86.43%, the leaf increased by 52.84%, and the whole plant increased by 60.38%.
[0050] 4. Effect of KC 316 on the ability of cosmos to enrich heavy metals in soil
[0051] This example provides the effects of Amycolatopsis tucumán KC 316 and the fermentation broth of the fermentation product on the ability of cosmos to enrich heavy metals in soil.
[0052] Bioaccumulation factor (BCF) = heavy metal content in plants / heavy metal content in soil;
[0053] The amount of heavy metals accumulated by cosmos = aboveground biomass * heavy metal content;
[0054] Extraction rate of heavy metals by cosmos = extraction amount of cosmos / (heavy metal content in soil*soil quality)
[0055] The statistical results are shown in Tables 1-3.
[0056] Table 1 Heavy metal contents and enrichment coefficients of cosmos after treatment with fermentation broth of strain KC316
[0057]
[0058] Note: Different letters in the table indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).
[0059] As shown in Table 1, KC316 treatment can promote the enrichment of three heavy metals in cosmos. The contents and enrichment coefficients of the three heavy metals in leaves and roots were greater than those in the control group; the contents and enrichment coefficients of Zn and Cd heavy metals in the stems increased compared with the control group, while the content and enrichment coefficient of Pb did not increase.
[0060] Table 2 Effect of KC316 fermentation broth on the extraction amount and extraction rate of heavy metals from cosmos (fresh weight)
[0061]
[0062] The results in Table 2 show that in the KC316 treatment group, the lead enrichment increased by 56.25%, and the extraction rate increased by 0.72%; the zinc enrichment increased by 145.78%, and the extraction rate increased by 1.83%; the cadmium enrichment increased by 98.78%, and the extraction rate increased by 18.71%.
[0063] Table 3 Physical and chemical properties and heavy metal content of soil in mining area
[0064]
[0065] Note: Different letters in the table indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).
[0066] The results in Table 3 show that after KC316 treatment, the soil pH decreased by 0.73 pH units compared with the control group, the total nitrogen, organic matter and organic carbon contents increased by 3.36%, 5.25% and 5.25% respectively compared with the control group, and the contents of heavy metals Cd, Pb and Zn were significantly reduced by 34.22%, 40.94% and 32.53% respectively compared with the control group.
[0067] Example 3 Determination of the ability of KC316 to improve nutrients and other factors in cosmos
[0068] The KC316 strain treatment group and the control group were treated with cosmos ( Figure 6 )The chlorophyll, soluble sugar, soluble protein, malondialdehyde (MDA), proline content and superoxide dismutase (SOD) and peroxidase (POD) activities of the leaves were determined.
[0069] Effects of strain KC316 on chlorophyll, soluble sugar, soluble protein, proline, and the activities of POD and SOD in cosmos plants Figure 5 As shown. The results showed that the soluble sugar content of cosmos in the KC316 treatment group increased significantly by 20.78% compared with the control group; the chlorophyll content increased extremely significantly by 59.74%; the soluble protein content increased significantly by 5.62%; the POD activity increased extremely significantly by 15.00%; the SOD activity increased significantly by 2.29%; and the proline content increased extremely significantly by 15.04%. In summary, the experiment proves that the fermentation liquid of the Tucuman pseudomycota KC 316 provided by the present invention can promote the growth of cosmos, improve the ability of cosmos to enrich heavy metals in the soil, and improve the physiological indicators of plants in a high cadmium and high lead environment, so that the problem of heavy metal contaminated soil is further solved, and no secondary pollution is caused to the environment, which is safer for humans and animals, and has important application value for ecological restoration and environmental protection.
Claims
1. The use of Amycota tucumana in the preparation of a bacterial agent for promoting plant growth in a heavy metal pollution environment and improving the heavy metal enrichment capacity of plants, characterized in that: The classification name of the Tucuman pseudomycotic acid bacteria is (Amycolatopsis tucumanensis) KC 316, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 2, 2022, and its deposit number is CGMCC No.26012.
2. The use according to claim 1, characterized in that: The bacterial agent is the fermentation liquid of Amycolax tucumiensis.
3. The use according to claim 2, characterized in that: The application of the Amycolatopsis tucumanicum fermentation liquid in the preparation of a bacterial agent for promoting plant growth in a high-cadmium and high-lead environment.
4. The use according to claim 2, characterized in that: The application of the Tucumán Amycolatopsis fermentation liquid in the preparation of a bacterial agent that promotes plant enrichment of heavy metals in soil.
5. The use according to claim 3, characterized in that: The application of the Amycolatopsis tucumanicum fermentation liquid in the preparation of a bacterial agent for improving the physiological indicators of plants in a high-cadmium and high-lead environment.
6. The use according to claim 3, characterized in that: The preparation method of the Tucuman Amycolatopsis fermentation broth is as follows: Activation of S1 strain Take the preserved KC 316 strain, streak it onto the modified ISP 2 medium, place the plate in a constant temperature incubator, set the temperature to 35-37°C, and culture for 8-10 days, during which the growth of the strain is regularly observed; Preparation of S2 seed solution The activated single colony in S1 was inoculated into the seed culture medium and cultured under shaking conditions of 28°C and 200 rpm for 4-5 days to obtain the seed solution; S3 Fermentation Take 5 mL of the seed solution in S2, add it into a container containing fermentation medium, and culture it at 25-35° C. and 130-200 rpm / min for 3-10 days to obtain fermentation liquid, wherein the fermentation medium is ISP 2 improved medium.
7. The use according to claim 6, characterized in that: The preparation process of the ISP 2 improved medium is as follows: 5g of glucose, 5g of soy peptone, and 5g of yeast extract are added in sequence, and then a small amount of trace salt and a small amount of microorganisms are added to dissolve them in water, the pH of the solution is adjusted to 7.5, and finally water is added to make up to 1L, and sterilized at 121°C for 15 minutes.
8. The use according to claim 6, characterized in that: The preparation method of the seed culture medium is as follows: 5g yeast extract, 10g malt extract and 4g glucose are dissolved in water, the pH value of the solution is adjusted to 7.0-7.2, the volume is fixed to 1L with water, and then sterilized at 121°C for 30min.
9. The use according to claim 6, characterized in that: The fermentation liquid obtained in claim 6 is diluted 1000 times, and then 30-40 mL of the liquid is used for root irrigation of each potted plant.