CN117645875B - Microbial remediation organic pollution soil conditioner - Google Patents
Microbial remediation organic pollution soil conditioner Download PDFInfo
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Abstract
The invention relates to a microbial remediation organic contaminated soil conditioner, which is beneficial to microorganisms, and can be used for remarkably improving the physical and chemical properties of organic contaminated soil and improving the water-retaining property, air permeability and nutrition condition of the soil so as to promote the healthy growth of plants. Therefore, the microbial remediation organic contaminated soil conditioner can effectively improve the organic contaminated soil environment and promote plant growth through the unique microbial combination and the application of the compound HK-018, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a microbial remediation organic contaminated soil conditioner.
Background
Microbial agents play a vital role in the improvement of modern agricultural soils and lightly organically contaminated soils, and they contain active microorganisms such as bacteria, fungi and actinomycetes that are capable of improving soil quality, promoting crop growth and maintaining ecological balance. These microbial agents act primarily by promoting plant growth and nitrogen fixation. They produce hormones such as indoleacetic acid (IAA) and Gibberellin (GA) required for plant growth to promote plant root development and overall growth, and to improve crop yield and quality. Meanwhile, nitrogen-fixing bacteria such as rhizobium and cyanobacteria can convert nitrogen in the atmosphere into nitrogen compounds which can be directly absorbed by plants, which helps to reduce fertilizer use and reduce environmental pollution.
In addition, the microbial agent increases the soil fertility by decomposing organic substances (including pollutants) in the soil, and participates in the circulation of mineral substances in the soil, thereby improving the availability and availability of nutrients. They can also improve the physical structure of the soil, enhance the porosity and air permeability of the soil, and contribute to the growth and development of root systems. In the aspect of biological control, the microbial agent can effectively control plant diseases by producing antibiotics or other inhibiting substances, and reduce the dependence on chemical pesticides. With technological development and importance on environmental protection, the application of microbial agents is expanding continuously, researchers are aiming at developing more effective and environment-friendly microbial agent products and searching microbial strains suitable for different soils and crops so as to improve the application effect of the microbial agents in agricultural production.
However, in practical applications, the use effect of microbial agents is affected by many factors, and some of the main problems faced by them cannot be ignored. First, microbial agents are extremely demanding in terms of their environment, requiring specific temperatures, humidity and pH values for their growth and development. These microbial agents are typically cultured and propagated in a controlled laboratory environment and then applied in a real-world environment. However, the complexity of a real-world environment far exceeds laboratory conditions.
For example, too high or too low an air temperature may have an effect on microbial activity, making it unable to grow and reproduce normally. Under high temperature conditions, the cells may be subjected to heat stress, causing denaturation of cellular proteins, which in turn affects the normal function of the cells. In contrast, under low temperature conditions, the growth and proliferation of the cells may be inhibited because the metabolic activity in the cells is slowed down in the low temperature environment, resulting in slow growth of the cells. In addition, the requirements of the microbial agents for humidity and pH are quite specific. Too dry or humid conditions, too acidic or too basic conditions, may lead to an inability of the microorganism to survive properly.
In addition to environmental conditions, the state of the soil also has a significant impact on the survival and action of microbial agents. The microbial agent needs to grow and reproduce in the soil, so that the quality of the soil, the content of organic matters, the content of nutrient elements such as nitrogen, phosphorus, potassium and the like can directly influence the activity of the microbial agent. The tightly textured soil may restrict the flow of air and moisture, affecting the diffusion and growth of microbial agents. Soil lacking organic matter may not provide enough nutrients to support the growth of microorganisms. The lack of main nutrient elements such as nitrogen, phosphorus, potassium and the like can limit the growth and the reproduction of the microbial agent.
In summary, while microbial agents have advantages in restoring soil pollution, improving soil structure, increasing soil biodiversity, and increasing soil long-term fertility, they have relatively poor fertility stability due to their high sensitivity to environmental conditions and soil conditions. Under different living environments and soil conditions, the microbial inoculum may be greatly reduced in bacterial load, thereby affecting fertility performance. In some cases, its fertility may even be inferior to chemical fertilizers. Therefore, for this situation, future research and development is required to find and cultivate microbial agents which are more stable, more adaptive and higher in fertility, so that the requirements of environmental protection and high yield of crops can be met.
Disclosure of Invention
In order to solve or partially solve the problems existing in the related art, the application of the invention provides a microbial remediation organic contaminated soil conditioner.
The invention provides a microbial remediation organic contaminated soil conditioner, which comprises the following components: bacillus subtilis, bacillus coagulans, trichoderma harzianum and Compound HK-018, which is selected from the group consisting of compounds represented by formula (I)
（I）。
Further, the bacterial amount of bacillus subtilis, bacillus coagulans and trichoderma harzianum is 1:1:1.
Further, the weight ratio of the compound HK-018 in the microbial remediation organic contaminated soil conditioner is 10-20%.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
The beneficial technical effects of the invention are as follows:
the compound 3-amino-1-carboxyl-2-methoxycarbonyl propionic acid plays a key role in microbial agents, and functional groups such as amino, carboxyl, methoxycarbonyl and the like in the molecular structure of the compound have various positive interactions with soil microorganisms. These interactions not only promote the physiological state and survival environment of the microorganism, but also improve the overall function of the microbial agent.
Specifically, the interaction with bacillus subtilis (Bacillus subtilis) significantly improves the production capacity of antibiotics and effectively inhibits the growth of pathogenic microorganisms. The action of the bacillus coagulans and the bacillus coagulans (Bacillus coagulans) strengthens the process of decomposing organic matters in soil, releases more nutrient substances absorbable by plants and improves the soil fertility. In addition, interaction with trichoderma harzianum (Trichoderma harzianum) enhances the ability of fungi to break down organic matter and antibiotic production, thereby improving soil structure and controlling pathogenic microorganisms.
Another advantage of such compounds is their decomposition process. Functional groups such as amino, carboxyl and methoxycarbonyl are easy to hydrolyze and biodegrade in natural environment, and are converted into harmless substances such as ammonia, water, carbon dioxide and the like. The process does not produce toxic or harmful byproducts and has no negative effect on the soil or water ecosystem. Thus, the use of the compound not only increases the fertility and biodiversity of the soil, but also helps to maintain the health and balance of the soil ecosystem.
Overall, the structural characteristics of 3-amino-1-carboxyl-2-methoxycarbonyl propionic acid make the 3-amino-1-carboxyl-2-methoxycarbonyl propionic acid have remarkable advantages in improving soil quality and plant growth environment, and provide important support for sustainable agriculture and soil management.
Drawings
FIG. 1 is a graph showing the comparison of rice growth in each group at the eighth week of the test example of the present invention;
wherein, from left to right in proper order is: blank, control, 0.1mg/ml treatment, 0.5mg/ml treatment, 1mg/ml treatment.
Detailed Description
Alternative embodiments of the present application will be described in more detail below with reference to the accompanying drawings. While the drawings illustrate alternative embodiments of the present application, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.
The invention provides a microbial remediation organic contaminated soil conditioner, which comprises the following components: bacillus subtilis, bacillus coagulans, trichoderma harzianum and Compound HK-018, which is selected from the group consisting of compounds represented by formula (I)
（I）。
In microbial agents, the structural characteristics of 3-amino-1-carboxyl-2-methoxycarbonyl propionic acid determine the interaction mode between the microbial agents and strains.
Interaction of 3-amino-1-carboxy-2-methoxycarbonylpropionic acid with Bacillus subtilis
Bacillus subtilis (Bacillus subtilis) inhibits the growth of pathogenic microorganisms in microbial agents, mainly by producing antibiotics. Amino and carboxyl in the compound are used as polar functional groups, and form hydrogen bonds with amino acid residues on the cell wall of bacillus subtilis, so that the physiological state and the antibiotic production capacity of the compound are improved. Meanwhile, methoxycarbonyl in the compound interacts with biomolecules in bacterial cells to further regulate the bioactivity.
Interaction of 3-amino-1-carboxy-2-methoxycarbonylpropionic acid with Bacillus coagulans
The main function of bacillus coagulans (Bacillus coagulans) in microbial agents is to decompose organic substances in soil and release nutrients for plant absorption. Amino and carboxyl groups in the compound react with amino acid residues on the cell wall of bacillus coagulans, so that the metabolic activity of the bacillus coagulans is improved. In addition, methoxycarbonyl groups of the compounds enhance the process of decomposing organic materials by bacillus coagulans.
Interaction of 3-amino-1-carboxy-2-methoxycarbonylpropionic acid with Trichoderma harzianum
Trichoderma harzianum (Trichoderma harzianum) is a soil fungus whose main function is to break down organic matter in the soil and to inhibit the growth of pathogenic microorganisms by producing antibiotics and enzymes. Amino and carboxyl groups in the compound form hydrogen bonds with amino acid residues on the cell wall of trichoderma harzianum, thereby changing the physiological state of fungi and the inhibition capability of the fungi on pathogenic microorganisms. Methoxycarbonyl groups in the compounds further enhance the ability of fungi to decompose organic substances.
In general, the structural features of 3-amino-1-carboxy-2-methoxycarbonyl propionic acid, such as amino, carboxy and methoxycarbonyl, enable complex interactions with species in a particular microbial agent. These interactions not only alter the physiological state of the species, but also improve their living environment and the overall function of the microbial agent. This interaction promotes improvement of soil quality and improvement of plant growth environment, by increasing fertility of soil and improving soil structure, while improving disease resistance of plants.
In addition, functional groups such as amino, carboxyl and methoxycarbonyl groups contained in the molecular structure of 3-amino-1-carboxyl-2-methoxycarbonylpropionic acid are liable to be hydrolyzed and biodegraded in natural environments. Amino and carboxyl groups can be rapidly converted into ammonia, water and carbon dioxide, while methoxycarbonyl groups can be decomposed into water and carbon dioxide. These decomposition products are all harmless substances common in nature and are easily utilized by soil microorganisms. In the decomposition process of the compound, toxic or harmful byproducts are not generated, so that the soil or water ecological system is not negatively influenced. The breakdown of the compounds enhances soil fertility and biodiversity while maintaining the health and balance of the soil ecosystem.
In one embodiment of the application, the bacterial amount of bacillus subtilis, bacillus coagulans and trichoderma harzianum is 1:1:1.
In one embodiment of the application, the weight ratio of the compound HK-018 in the microbial remediation organic contaminated soil conditioner is 10-20%.
For clarity, the following examples are provided in detail.
Example 1
Step one: amino and carboxyl protection
10 g of 3-aminopropionic acid are dissolved in 100 ml of dichloromethane.
15 g of Boc anhydride was added and stirred at room temperature for 2 hours to protect the amino group.
The addition of 20 g of propionic anhydride was continued and stirred for another 4 hours to protect the carboxyl group.
After the completion of the reaction, the organic layer was washed with water.
The organic layer was separated using diethyl ether.
The organic layer was dried and concentrated to give the double protected product.
Step two: methoxycarbonyl group introduction
The double protected product was dissolved in a mixture of 15 ml formic acid and 30 ml methanol.
Heating to reflux temperature and continued for 4 hours.
After completion, the mixture was diluted with water.
The product was extracted with diethyl ether.
The organic layer was dried and concentrated to give an intermediate after methoxycarbonyl incorporation.
Step three: removing protecting groups
The intermediate product was dissolved in water.
20 ml of 1M hydrochloric acid was added thereto and stirred at room temperature for 2 hours.
The product was extracted with diethyl ether.
The organic layer was dried and concentrated to give the final product after removal of the protecting groups. (i.e., compound HK-018,3-amino-1-carboxy-2-methoxycarbonylpropionic acid).
1H NMR (300 MHz, DMSO-d6) δ 11.99 (1H, br s, COOH), 3.69 (3H, s, CH3), 2.92-3.10 (4H, m, CH2), 2.60 (1H, m, CH), 1.50 (2H, br s, NH2)。13C NMR (100 MHz, DMSO-d6) δ 177.3 (C, 1-carboxyl), 173.0 (C, 1-carboxyl), 52.2 (CH3, aliphatic), 41.5 (CH2, aliphatic), 41.1 (CH, aliphatic), 34.9 (CH2, aliphatic)。
Test example 1
Culturing strains: first, bacillus subtilis, bacillus coagulans and trichoderma harzianum were cultured in LB medium, MRS medium, PDA medium, respectively, at an inoculum size of 0.1%. The inoculated culture medium was placed in a constant temperature and humidity box at 30℃and 70% humidity for 48 hours.
Compound treatment: 3-amino-1-carboxy-2-methoxycarbonylpropionic acid was dissolved in sterile water to prepare a mother liquor of 100 mg/ml. The corresponding volumes were removed from the 100mg/ml mother liquor and sterile water was added to prepare working solutions with final concentrations of 0.1mg/ml, 0.5mg/ml and 1 mg/ml. And adding the working solution into the culture medium of the experimental group, and uniformly mixing. No compound was added to the control group.
Inoculating strains: the cultured strain was added again to the culture medium of the control group and the experimental group at an inoculum size of 0.1%, and cultured again at 30℃and a humidity of 70% for 48 hours.
Colony count: the growth of the strain under the different treatments was determined using plate counting. Specifically, 1ml of the bacterial liquid is taken and serially diluted to 10 -6 0.1ml of the diluted solution was spread on an agar medium, and after incubation at 30℃for 24 hours, the solution was counted by a microscope.
Soil treatment and planting: each group was sterilized by ultraviolet light to obtain 500g of sterilized soil. The blank group is not added with the microbial inoculum, the control group is added with the microbial inoculum which is not treated by the compound according to the soil proportion of 5 percent and mixed, the experimental group is added with the microbial inoculum which is treated by the compound according to the soil proportion of 5 percent and mixed, and 5 rice seeds are sown in each soil. After sowing, the soil was cultured at 25℃and 70% humidity.
And (3) measuring growth parameters: from the day of planting, the growth of rice was recorded weekly. Comprises measuring plant height and measuring fresh weight and dry weight of rice by an electronic balance. Determination of dry weight the rice samples were dried to constant weight at 105 ℃. The observation was continued for 8 weeks and 3 replicates were recorded.
Soil pollution measurement: soil samples of each group were taken after 8 weeks and tested under the following conditions:
experimental data and analysis:
colony count results (Unit: CFU/mL)
Bacillus subtilis
Control group: 1.2 X 10 8
Treatment group 0.1 mg/ml: 1.5 X 10 8
Treatment group 0.5 mg/ml: 1.5 X 10 8
Treatment group 1 mg/ml: 2.0 X 10 8
Bacillus coagulans
Control group: 2.5 X 10 8
Treatment group 0.1 mg/ml: 3.0 X 10 8
Treatment group 0.5 mg/ml: 3.1 X 10 8
Treatment group 1 mg/ml: 3.8 X 10 8
Trichoderma harzianum
Control group: 3.0 X 10 7
Treatment group 0.1 mg/ml: 3.5 X 10 7
Treatment group 0.5 mg/ml: 4.0 X 10 7
Treatment group 1 mg/ml: 4.2 X 10 7
Conclusion: after each strain is treated by the compound 3-amino-1-carboxyl-2-methoxycarbonyl propionic acid, the colony count is increased, and the colony count is correspondingly increased along with the increase of the treatment dosage. These results indicate that the compound helps to promote the growth and reproductive activity of these species.
Results of measurement of growth parameters (growth at week eight see FIG. 1):
conclusion:
with the increase of the concentration of the compound, the degradation effect of the pollutant is more obvious, and the compound promotes the degradation of petroleum hydrocarbon and Polycyclic Aromatic Hydrocarbon (PAHs) in soil through the interaction between the compound and different microorganisms.
Plant height increases: in the 1mg/ml treatment group, a significant increase in plant height indicated that the compound had a positive effect on plant growth. This is due to the soil improvement effect, since the improvement of soil structure and nutrient status contributes to better development of the root system, thereby promoting growth of plants.
Fresh weight and dry weight increase: an increase in fresh and dry weight also indicates an increase in the overall biomass of the plant due to the improvement in soil nutrition and the improvement in soil environment. The compound improves the water retention and air permeability of soil, so that plants can absorb water and nutrition more effectively.
Differential effect of different concentrations: the differences in growth parameters between the treatment groups further supported the effect of the compounds on soil improvement. With increasing compound concentration, the improvement of plant growth is more remarkable, which reflects the gradual improvement of soil environmental quality.
Overall, the experimental results show that the addition of the compound significantly promotes the growth of plants, due to the improving effect of the compound on the soil quality. The increase in plant height, fresh weight and dry weight is a direct result of the improvement in physical and chemical properties of the soil and the improvement in nutritional status. Therefore, it is considered that the compound has a remarkable soil improvement effect, and this effect is enhanced with an increase in treatment concentration.
The foregoing description of the embodiments of the present application is illustrative, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
Claims (1)
1. The microbial remediation organic contaminated soil conditioner is characterized by comprising the following components: bacillus subtilis, bacillus coagulans, trichoderma harzianum and Compound HK-018, which is selected from the group consisting of compounds represented by formula (I)
（I）；
The bacterial amount of the bacillus subtilis, the bacillus coagulans and the trichoderma harzianum is 1:1:1;
the weight ratio of the compound HK-018 in the microbial remediation organic contaminated soil conditioner is 10-20%.
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