Bioremediation agent as well as preparation method and application thereof

Abstract

The invention discloses a bioremediation agent as well as a preparation method and application thereof. The bioremediation agent comprises hydrothermal carbon and pichia pastoris loaded on the hydrothermal carbon. The bioremediation agent disclosed by the invention can be used for treating pollutants with relatively high accumulation amount, for example, the degradation of quinclorac by microorganisms is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural technology, in particular to a biological remediation agent and a preparation method and application thereof. BACKGROUND

[0002] The widely used high-stability rice field herbicide quinclorac is prone to cause soil accumulation and residue under intensive planting, affecting the production stability of the crop rotation system and the ecological safety of the farmland. Although the microbial remediation technology has the advantages of high efficiency and environmental friendliness, the specific degrading bacteria still have problems such as low survival rate and easy loss of function after entering the soil. The pollutants may have a stress effect on the microorganisms, affecting their growth and metabolism, and the complexity of the soil environment, such as pH change, nutrient deficiency, competition with indigenous bacteria, etc. may also limit the survival and function of the degrading bacteria. Therefore, how to enhance the adaptability of microorganisms in contaminated soil and improve their degradation efficiency of pollutants has become a problem to be solved in the field of pollution bioremediation.

[0003] The immobilized microorganism technology provides a protective barrier for microorganisms by wrapping or adsorbing microorganisms with carrier materials, thereby reducing the stress of the external environment on the microorganisms. At the same time, the carrier material can also supply appropriate nutrients, thereby improving the colonization rate and biomass of the microorganisms. In addition, the immobilized strains can reduce the compensation of the probiotic function during the degradation of pollutants, thereby improving the degradation efficiency of pollutants. Therefore, the immobilized microorganisms usually have the advantages of high biomass, strong stability, low loss rate, and fast reaction speed. Biochar is widely used for immobilizing microorganisms due to its rich pore structure and surface functional groups, which can improve the activity of microorganisms and the degradation efficiency of pollutants. At present, the research on the combination of carbon-based materials and microorganisms for remediation mainly focuses on the synergistic degradation of bacteria-biochar. The research on hydrothermal carbon is relatively new, and its preparation process and characteristics are different from those of biochar. There are few studies on this new type of carbon material.

[0004] In recent years, fungi have gradually attracted attention in the degradation of organic pollutants. Among them, yeasts are considered to have high potential for in-situ application in soil due to their rich co-metabolic enzyme system, fast growth rate, and strong ecological occupation ability compared with specific degrading bacteria which are prone to lose functional genes. However, current research mainly focuses on the degradation system of bacteria-immobilized carriers, and the synergistic effect of fungi and immobilized carriers is relatively less studied. SUMMARY

[0005] The present application aims to solve at least one of the above technical problems in the prior art. To this end, the purpose of the present application is to provide a biological remediation agent and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0007] In a first aspect, the present invention provides a bioremediation agent comprising hydrothermal carbon and Pichia pastoris loaded on the hydrothermal carbon.

[0008] In this invention, hydrothermal carbon is used to load Pichia pastoris via pore-adsorption. On one hand, the hydrothermal carbon provides an environmental barrier for Pichia pastoris, isolating it from adverse environmental influences and allowing it to survive and function better. On the other hand, the hydrothermal carbon provides a carbon source for Pichia pastoris, promoting its growth. The two form a hydrothermal carbon-Pichia pastoris synergistic treatment system, which, as a bioremediation agent, can degrade harmful substances.

[0009] In some embodiments of the present invention, the loading of Pichia pastoris is 1.0 × 10⁻⁶. 8 ~4.0×10 10 CFU / g hydrothermal carbon, such as 5.0×10 8 ~3.0×10 10 CFU / g hydrothermal carbon, 1.0×10 9 ~3.0×10 10 CFU / g hydrothermal carbon, 2.825×10 9 ~2.825×10 10 CFU / g hydrothermal carbon, 2.825×10 9 ~2.26×10 10 CFU / g hydrothermal carbon, 2.825×10 9 ~1.41×10 10 CFU / g hydrothermal carbon, etc.

[0010] In some embodiments of the present invention, the average particle size of the hydrothermal carbon is 50-300 μm, such as 100-250 μm, 150 μm, 180 μm, 200 μm, 220 μm, or 250 μm.

[0011] In some embodiments of the present invention, the average specific surface area of ​​the hydrothermal carbon is 10–50 m². 2 / g, such as 12~40m 2 / g. The specific surface area of ​​the micropores in the hydrothermal carbon is 0.01–4.0 m². 2 / g. In this invention, the specific surface area of ​​the hydrothermal carbon was measured using the Brunol-Emmett-Teller (BET) method, specifically using an automated surface and porosity analyzer (Micromeritics ASAP 2460, USA) at 77.3 K with nitrogen static adsorption technology.

[0012] In some embodiments of the present invention, the pore volume of the hydrothermal carbon is 0.05–0.3 cm³. 3 / g; the pore volume of the micropores in the hydrothermal carbon is 2.0 × 10⁻⁶.-4 ~15×10 -4 cm 3 / g.

[0013] In some embodiments of the present application, the average pore size of the hydrothermal carbon is 18-30 nm; wherein the mesopore adsorption diameter (measured during nitrogen adsorption process) of the hydrothermal carbon is 18-26 nm; and the mesopore desorption diameter (measured during nitrogen desorption process) of the hydrothermal carbon is 15-26 nm.

[0014] In some embodiments of the present application, the C / N ratio of the hydrothermal carbon is 15-25.

[0015] In some embodiments of the present application, the hydrothermal carbon comprises at least one of feces-based hydrothermal carbon and straw-based hydrothermal carbon.

[0016] In some embodiments of the present application, the hydrothermal carbon comprises at least one of pig manure hydrothermal carbon, cow manure hydrothermal carbon and straw hydrothermal carbon.

[0017] In some embodiments of the present application, the preparation method of the hydrothermal carbon comprises subjecting feces or straw to a hydrothermal reaction with water to obtain the hydrothermal carbon.

[0018] In some embodiments of the present application, the temperature of the hydrothermal reaction is 200-240℃. In the present application, the above hydrothermal reaction can effectively hydrolyze cellulose and hemicellulose in straw and feces, while avoiding excessive aromatic condensation, thereby improving the pore structure and nutrient release capacity. A low temperature, such as <200℃, is difficult to degrade lignocellulose, and a high temperature, such as >240℃, reduces the bioavailability of dissolved organic matter (DOM). A temperature of 200-240℃, such as 220℃, balances the carbon fixation capacity and biocompatibility.

[0019] In some embodiments of the present application, the time of the hydrothermal reaction is 8-24 h; such as 10-20 h, 12-18 h, 15-24 h, 16 h, 18 h, 21 h, 23 h, etc. In the present application, appropriately prolonging the hydrothermal time can promote the structural evolution of the hydrothermal carbon, enhance the stability, reduce the inhibition of DOM on crops, and reduce the risk of leaching of harmful substances.

[0020] In some embodiments of the present application, the preparation method of the hydrothermal carbon can be a conventional technique in the art, or can be the preparation method described in Environmental Technology & Innovation Volume 38, May 2025, 104118. The entire contents of this document are incorporated into the specification of the present application.

[0021] In some embodiments of the present application, the bioremediation agent further comprises a culture medium; the culture medium comprises potato culture medium, inorganic culture medium.

[0022] In some embodiments of the present application, the culture medium comprises a carbon source; the carbon source comprises at least one of sucrose, trehalose, glucose, mannitol, starch, maltose, fructose, glycerol, and methanol; the mass concentration of the carbon source is 0.1-10%.

[0023] In some embodiments of the present application, the culture medium further comprises an ion balancing agent, such as NaCl; the mass concentration of the ion balancing agent is 0.01-8%, such as 0.02-5%.

[0024] In some embodiments of the present application, the pH of the culture medium is 3-10, such as 3-9, etc.

[0025] In a second aspect of the present application, a preparation method of the bioremediation agent is provided, comprising the following steps: culturing Pichia pastoris in a culture medium containing hot carbon to obtain the bioremediation agent.

[0026] In a third aspect of the present application, a method for degrading pollutants is provided, comprising the following steps: adding the bioremediation agent to the pollutants for degradation.

[0027] In some embodiments of the present application, the pollutants comprise at least one of quinclorac, organochlorine pesticide, organophosphorus pesticide, polycyclic aromatic hydrocarbon, phenol, o-xylene, abamectin, and heavy metal ion.

[0028] In some embodiments of the present application, the degradation is performed at 15-45℃, such as 20-40℃.

[0029] In some embodiments of the present application, the degradation is performed at a pH of 3-10, such as 3-9, etc.

[0030] In some embodiments of the present application, the pollutants comprise at least one of pollutants on crops, pollutants in water bodies, and pollutants in soil.

[0031] In some embodiments of the present application, the mass-volume ratio of the pollutants to the bioremediation agent is (1-100) mg / L:(300-1500) mg / L.

[0032] The present application has the following beneficial effects:

[0033] The bioremediation agent of the present application can significantly improve the degradation of quinclorac by microorganisms, and the degradation improvement rate is as high as 10.4-44.9%.

[0034] The bioremediation agent of the present application can treat pollutants with high accumulation amount, for example, the concentration of quinclorac that can be treated can reach about 100 mg / L.

[0035] The preparation method of the bioremediation agent of the present application is simple and convenient for popularization. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The influence of different hydrothermal carbons (left) and DOMs (right) in Example 1 of the present application on the biomass of Pichia pastoris.

[0037] Figure 2 The effect of the bioremediation agent with different Pichia pastoris inoculation amounts in Example 2 of the present application on the degradation of quinclorac.

[0038] Figure 3 The effect of the bioremediation agent on the degradation of quinclorac at different temperatures in Example 3 of the present application.

[0039] Figure 4 The effect of the bioremediation agent on the degradation of quinclorac at different pH values in Example 3 of the present application.

[0040] Figure 5 The effect of the bioremediation agent on the degradation of quinclorac at different NaCl concentrations in Example 4 of the present application.

[0041] Figure 6 The effect of the bioremediation agent on the degradation of quinclorac at different carbon sources in Example 4 of the present application.

[0042] Figure 7 The effect of the bioremediation agent on the degradation of quinclorac at different concentrations of carbon sources in Example 5 of the present application.

[0043] Figure 8 The zero-order kinetic model of the bioremediation agent degrading quinclorac in Example 6 of the present application.

[0044] Figure 9 The first-order kinetic model of the bioremediation agent degrading quinclorac in Example 6 of the present application.

[0045] Figure 10 The second-order kinetic model of the bioremediation agent degrading quinclorac in Example 6 of the present application.

[0046] Figure 11 The isothermal degradation curve of the bioremediation agent degrading quinclorac in Example 7 of the present application.

[0047] Figure 12These are TEM images of yeast cells during the degradation of dichloroquinoline acid by the bioremediation agent in Example 7 of the present invention; wherein, (a) and (c) are transmission electron micrographs of Pichia pastoris at 6d and 10d, respectively; and (b) and (d) are transmission electron micrographs of the bioremediation agent at 6d and 10d, respectively.

[0048] Figure 13 The images show the growth of tomatoes under different treatment groups under dichloroquinoline acid stress in Example 8 of this invention; where (a) to (c) correspond to the tomato plant, leaf, and root system diagrams under 0.1 mg / L dichloroquinoline acid, respectively; and (d) to (f) correspond to the tomato plant, leaf, and root system diagrams under 0.5 mg / L dichloroquinoline acid, respectively.

[0049] Figure 14 These are the tomato growth indicators under different treatment groups under 0.1 mg / L quinclorac stress in Example 8 of this invention; where (a) is the fresh weight of the aboveground parts of the tomato; (b) is the tomato plant height; (c) is the chlorophyll content of the tomato; (d) is the stem diameter of the tomato; (e) is the root length of the tomato; and (f) is the root weight of the tomato.

[0050] Figure 15 These are the tomato growth indicators under different treatment groups under 0.5 mg / L quinclorac stress in Example 8 of this invention; where (a) is the fresh weight of the aboveground parts of the tomato; (b) is the tomato plant height; (c) is the chlorophyll content of the tomato; (d) is the stem diameter of the tomato; (e) is the root length of the tomato; and (f) is the root weight of the tomato.

[0051] Figure 16 The values ​​represent the CAT(a), POD(b), and SOD(c) activities in plant roots treated with 0.1 mg / L quinclorac acid in Example 8 of this invention.

[0052] Figure 17 The values ​​represent the CAT(a), POD(b), and SOD(c) activities in plant roots treated with 0.5 mg / L quinclorac acid in Example 8 of this invention. Detailed Implementation

[0053] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0054] Raw material source:

[0055] Rice straw was collected from experimental fields of South China Agricultural University, pig manure came from an ecological farm in Zhaoqing City, Guangdong Province, and cow manure came from a commercial farm in Guangzhou City, Guangdong Province.

[0056] Pichia sp. G130 was isolated from earthworm manure and preserved in the National Agricultural and Environmental Microbial Germplasm Repository of Guangdong Academy of Agricultural Sciences (No. GDAAS20130) and the Guangdong Microbial Culture Collection Center (No. GDMCC No. 65197). The single colony of Pichia sp. was white to yellowish on solid medium, round, with neat edges, smooth or slightly raised surface, and grew well in potato dextrose agar (PDA) medium. The optimal growth conditions were pH 5.0-6.0 and temperature 28-30℃.

[0057] In the following examples, the preparation method of hydrothermal carbon is as follows:

[0058] 30 g of straw, pig manure and cow manure were weighed, respectively, and added to 250 mL of deionized water. After ultrasonic dispersion, they were stirred uniformly. The mixture was transferred to a hydrothermal reactor and hydrothermal reaction was carried out at 220℃. The reaction time of straw and cow manure hydrothermal carbon was set to 10, 12, 15 and 18 h, and the reaction time of pig manure hydrothermal carbon was set to 15, 18, 21 and 24 h. The hydrothermal carbon product was recovered by vacuum filtration on a 0.22 μm filter membrane and washed repeatedly with deionized water until the filtrate was clear. The washed hydrothermal carbon was freeze-dried at-40℃. The dried hydrothermal carbon was ground to powder and transferred to a sealed bag and stored in a-20℃ refrigerator to ensure the stability of DOM. The three kinds of hydrothermal carbon materials were named as HS (hydrochar of straw), HSM (hydrochar of swine manure) and HCM (hydrochar of cattle manure), respectively. The samples with different hydrothermal times were named with “-” plus the hydrothermal time (h) for distinction, such as straw hydrothermal carbon with different hydrothermal times were HS-10, HS-12, HS-15 and HS-18, respectively. Correspondingly, pig manure and cow manure hydrothermal carbon were named as HSM-15-HSM-24 and HCM-10-HCM-18, respectively.

[0059] Before preparing the bioremediation agent, the hydrothermal carbon raw material was dried at 60℃, crushed and passed through a 60 mesh screen to ensure uniform particle size, with particle size ≤250 μm.

[0060] Preparation of culture medium and solution:

[0061] Potato Dextrose Broth (PDB, 1 L): 200 g peeled potato was cut into small pieces and boiled for 20 min. After boiling, the filtrate was collected by filtering through eight layers of gauze. 20 g glucose was added to the filtrate, and the volume was made up to 1 L with deionized water. The pH was adjusted to 6.0 with 1 M NaOH and 1 M HCl. The prepared medium was sterilized by autoclaving at 121 °C for 30 min. If a solid PDA medium is needed, 2% V / V agar powder was added to the PDB medium and sterilized.

[0062] Mineral Medium Salt (MSM, 1 L): (NH4)2SO4 1.5 g, K2HPO4 1.5 g, MgSO4·7H2O 0.2 g, NaCl 0.5 g, fructose 5 g, trace elements 1 mL (trace element formula: ZnSO4·7H2O 22 g, H3BO3 11.4 g, MnCl2·4H2O 5.06 g, CoCl2·6H2O 1.61 g, (NH4)6Mo7O 24 ·4H2O 1.1 g, CaCl2 0.5 g, CuSO4·5H2O 0.5 g), iron element 0.25 mL (iron solution formula: EDTA-Na2 5 g, FeSO4·7H2O 5 g), dissolved with deionized water, and the pH was adjusted to 5.0 with 1 M NaOH and 1 M HCl. After autoclaving at 115 °C for 30 min, it was ready for use.

[0063] 10×PBS buffer (1 L): NaCl 80 g, KCl 2 g, Na2HPO4 14.4 g, KH2PO4 2.4 g, dissolved with deionized water, and the pH was adjusted to 6.8 with 1 M NaOH and 1 M HCl. When used, it needs to be diluted to 1×PBS buffer.

[0064] Yeast suspension: 20 h activated yeast was inoculated into PDB medium and cultured at 30 °C and 160 rpm for 20 h to the logarithmic growth phase. An appropriate amount of the bacterial solution was centrifuged at 4000 rpm for 5 min, and the collected bacterial cells were washed three times with high-purity water to remove residual medium. The bacterial cells were resuspended in PBS buffer, and the concentration of the bacterial suspension was adjusted to OD 600 1.0 using a spectrophotometer. The prepared yeast suspension was ready for use.

[0065] Example 1

[0066] In this example, a bioremediation agent was prepared, and the specific process was as follows:

[0067] The yeast suspension was inoculated into 20 mL PDB medium at a 5% V / V inoculation amount, and 0.01 g of hydrothermal carbon prepared from different raw materials and different hydrothermal times or 0.5 mL of hydrothermal carbon DOM (with a DOC content equal to that of the hydrothermal carbon) was added, respectively. The culture was incubated at 30°C in a 180 rpm shaker for 120 h. After the incubation, the bacterial cells were centrifuged at 4000 rpm for 5 min, and the supernatant was discarded to prepare the bioremediation agent.

[0068] The mass of the dried bacterial cells was recorded to evaluate the effect of the hydrothermal carbon on the growth of the yeast.

[0069] Figure 1 The effects of the hydrothermal carbon prepared from different raw materials and different hydrothermal times and its dissolved organic matter (DOM) on the biomass of Pichia sp. G130 yeast were demonstrated. Overall, the yeast could be well loaded and grown in the different hydrothermal carbon and its derived DOM treatment groups, and the difference in the yeast biomass and the change under different hydrothermal times were small. The yeast biomass was significantly positively correlated with the DOC content, CDOM content and F max content of component C3, indicating that for the yeast, the DOC, especially the CDOM and C3 (humic acid substances) in the FDOM, can serve as a potential carbon source to provide part of the energy and nutritional support for the growth of the yeast.

[0070] Example 2

[0071] In this example, a bioremediation agent was prepared and used to degrade quinclorac. The specific process was as follows:

[0072] The yeast suspension was inoculated into 20 mL MSM medium (pH = 5) containing 0.05% NaCl and 0.5% fructose at inoculation amounts of 1%, 2%, 5%, 8% and 10% V / V, respectively, and 0.01 g of hydrothermal carbon material (HS-18, HSM-24 and HCM-18) was added to form a gradient of carbon-bacteria compound ratio. The yeast was pre-cultured in a constant temperature shaker at 30°C and 180 rpm for 2 h to better bind the yeast to the hydrothermal carbon material, and a bioremediation agent system was prepared.

[0073] Quinclorac stock solution was added to the bioremediation agent system to make the concentration of quinclorac in the reaction system reach 25 mg / L, and the reaction was carried out at 30°C and 180 rpm for 120 h. After the incubation, the sample was centrifuged at 13000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm nylon filter and transferred to a liquid chromatography vial. The residual amount of quinclorac in the supernatant was determined, and the degradation rate of quinclorac was calculated according to the following formula:

[0074]

[0075] In the formula, D 二氯喹啉酸— Degradation rate of quincloracic acid, %; C0— Concentration of quincloracic acid in blank control group, mg / L; C t —Concentration of quinclorac acid after incubation, mg / L.

[0076] The blank control group was 0.5% fructose MSM medium (pH=5) without dichloroquinoline. The treatment control groups were anhydrous thermal carbon reaction system with an equal proportion of yeast suspension added alone and yeast-free reaction system with an equal mass of hydrothermal carbon added alone. All treatment groups were set up in triplicate.

[0077] The results are as follows Figure 2 As shown. From Figure 2 It can be seen that bioremediation agents with different yeast inoculum sizes can effectively degrade quinclorac acid. When the yeast inoculum size is 5% (1.41 × 10⁻⁶), the degradation effect is significantly reduced. 10 The degradation rate was best improved when the inoculum concentration was CFU / g hydrothermal carbon. The degradation rates of the HS-18, HSM-24, and HCM-18 bioremediation agents were increased by 9.5%, 8.0%, and 25.5% respectively compared to the yeast-only treatment group. A 1% inoculum (2.825 × 10⁻⁶) was used. 9 The degradation rates of the bioremediation agents HS-18, HSM-24, and HCM-18 (CFU / g hydrothermal carbon) were increased by 16.8%, 13.6%, and 22.3% respectively compared to yeast, with the highest degradation rate improvement being second only to 5% of the inoculum.

[0078] Taking into account that yeast can fully exert its degradation function and avoid excessive competition for carbon sources and nutrients, thus affecting the overall degradation efficiency, a 1% inoculum amount can maximize the degradation function of yeast in the bioremediation agent.

[0079] Example 3

[0080] This embodiment prepares a bioremediation agent and degrades dichloroquinoline acid. The specific process is as follows:

[0081] Weigh 0.01 g of hydrothermal carbon material into a 50 mL centrifuge tube, add 5% v / v yeast suspension, and use MSM medium at pH 5 as the reaction system. After pre-culturing for 2 h, adjust the contaminant concentration of the system to 25 mg / L dichloroquinoline acid, and react at 20℃, 25℃, 30℃, 35℃, and 40℃ for 120 h at 180 rpm.

[0082] The pH of MSM medium was adjusted to 3, 4, 5, 6, 7, 8, and 9 using 0.1M HCl and 0.1M NaOH, respectively. 0.01g of hydrothermal carbon material was weighed into a 50mL centrifuge tube, and 5% v / v yeast suspension was added. After pre-culturing for 2h, the contaminant concentration of the system was adjusted to 25mg / L dichloroquinoline acid. The mixture was then placed at 30℃ and reacted at 180rpm for 120h.

[0083] MSM medium without quinclorac as blank control group, anhydrous hydrochar reaction system with the same proportion of yeast suspension and anhydrous hydrochar reaction system with the same proportion of yeast suspension as treatment control group, all treatment groups were set in triplicate. After the culture, the sample was centrifuged at 13000 rpm for 3 min, the supernatant was filtered through a 0.22 μm nylon filter and transferred to a liquid chromatography vial. The residual amount of quinclorac in the supernatant was determined by the method of Example 2, and the degradation rate of quinclorac was calculated by the formula of Example 2. The results are shown in Table 3. Figure 3 、 Figure 4

[0084] As can be seen from Table 3, the yeast can better degrade quinclorac at 20-40℃. Between 20-40℃, the degradation rate showed a rising trend on the curve, and reached the highest value at 40℃. Compared with the yeast treatment group, the degradation rates of HS, HSM and HCM bioremediation agents for quinclorac were increased by 26.6%, 19.8% and 33.3%, respectively, followed by 30℃, which was increased by 11.8%-28.2%. Considering that the optimum growth temperature of yeast is 28-35℃, this temperature range is suitable for bioremediation agents to degrade quinclorac. Figure 3 As can be seen from Table 4, acidic conditions can better meet the degradation of quinclorac by bioremediation agents. Overall, the degradation rate of quinclorac showed a trend of first increasing and then decreasing with the change of pH, and the degradation rate was higher in the range of pH 3-pH 6, and the degradation effect of the combined treatment of different hydrochar materials in this range was different. The degradation rates of HS-18, HSM-24 and HCM-18 bioremediation agents at pH 3 were increased by 22.1%, 25.2% and 40.6% compared with the yeast group, respectively, and at pH 6, they were increased by 19.3%, 10.4% and 40.2% compared with the yeast treatment group, respectively.

[0085] Figure 4 Considering that the suitable growth pH of yeast is 3-6, and too low pH may affect the normal growth of soil pH, microorganisms and plants. Therefore, considering the degradation effect and application potential, pH 6 shows better degradation effect of quinclorac.

[0086] Example 4

[0087] In this example, a bioremediation agent was prepared and used to degrade quinclorac, and the specific process was as follows:

[0088] In this example, a bioremediation agent was prepared and used to degrade quinclorac, and the specific process was as follows:

[0089] ​​MSM medium was prepared with NaCl mass concentration of 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 5% respectively, 0.01 g of hydrothermal carbon material (HS-18, HSM-24, HCM-18) was weighed in a 50 mL centrifuge tube, 5% V / V yeast suspension was added, and after 2 h of pre-culture, the pollutant concentration of the system was adjusted to 25 mg / L quinclorac, and then it was placed at 30°C under 180 rpm for 120 h.

[0090] MSM medium was prepared with glucose, fructose, glycerol as carbon source at 0.5% V / V (containing 0.05wt% NaCl), 0.01 g of hydrothermal carbon material was weighed in a 50 mL centrifuge tube, 5% V / V yeast suspension was added, and after 2 h of pre-culture, the pollutant concentration of the system was adjusted to 25 mg / L quinclorac, and then it was placed at 30°C under 180 rpm for 120 h.

[0091] MSM medium without quinclorac was used as a blank control group, and a hydrothermal carbon reaction system with the same proportion of yeast suspension and a yeast-free reaction system with the same mass of hydrothermal carbon were used as treatment control groups. All treatment groups were set in triplicate. After the culture was completed, the sample was centrifuged at 13000 rpm for 3 min, the supernatant was filtered through a 0.22 μm nylon filter, and then transferred to a liquid chromatography vial. The residual amount of quinclorac in the supernatant was determined by the method of Example 2 for the NaCl concentration experiment and the carbon source experiment, and the degradation rate of quinclorac was calculated according to the formula of Example 2. The results are shown in Figure 5 、 Figure 6

[0092] As can be seen from Figure 5 , the bioremediation agent can well degrade quinclorac under different NaCl concentrations. An appropriate amount of NaCl can maintain the osmotic pressure balance of yeast cells and promote their stable growth. When the NaCl concentration is 0.5%, the degradation of the bioremediation agent of HS-18 and HSM-24 groups reaches the maximum value, which is increased by 34.6% and 27.0% respectively compared with the yeast treatment group, while the degradation rate of the bioremediation agent of HCM-18 group reaches the maximum value at 0.1% NaCl concentration, which is increased by 44.9% compared with the yeast treatment group. In addition, when the NaCl concentration is 0.05%, the degradation rate is increased by 18.3% to 30.5%. Considering the growth requirements of yeast, the mass concentration of NaCl is 0.05%, which can provide appropriate ionic strength to support normal yeast metabolism.

[0093] As can be seen from Figure 6 ​It can be seen that fructose, glucose, glycerol and other do not need additional enzymolysis, can be absorbed by yeast and rapidly metabolized, promote its growth and enzyme synthesis, which can provide better carbon source for bioremediation agent, thereby improving the degradation rate of quinclorac. Among them, fructose significantly improves the degradation rate of quinclorac, and the bioremediation agents of HS-18, HSM-24 and HCM-18 groups reach the highest degradation rate, which is increased by 24.2%, 18.3% and 32.8% respectively compared with the yeast treatment group. The degradation rate of each treatment group of glucose is increased by 24.9% to 49.6% compared with the yeast treatment group; the bioremediation agents of HS-18 and HCM-18 groups in the glycerol treatment group are increased by 4.8% and 33.6% respectively compared with the yeast treatment group, and the bioremediation agent of HSM-24 group is decreased by 4.8%.

[0094] Example 5

[0095] In this embodiment, a bioremediation agent is prepared and used to degrade quinclorac, and the specific process is as follows:

[0096] 0.01 g of hydrothermal carbon material was weighed into a 50 mL centrifuge tube, 5% V / V yeast suspension was added, and MSM medium with fructose concentration of 0.1%, 0.2%, 0.5%, 1%, 2%, 5% and 10% was used as the reaction system. After 2h of pre-culture, the system pollutant concentration was adjusted to 25mg / L quinclorac, and the reaction was carried out at 30℃ and 180rpm for 120h.

[0097] The MSM medium without quinclorac was used as a blank control group, and the reaction system without hydrothermal carbon and the same proportion of yeast suspension and the reaction system without yeast and the same amount of hydrothermal carbon were used as treatment control groups. After the culture was completed, the sample was centrifuged at 13000rpm for 3min, the supernatant was filtered through a 0.22μm nylon filter, and then transferred to a liquid chromatography vial. Three parallel groups were set for each treatment group. The residual amount of quinclorac in the supernatant was determined by the method of Example 2, and the degradation rate of quinclorac was calculated according to the formula of Example 2. The results are shown in Figure 7 .

[0098] From Figure 7 It can be seen that different carbon source concentrations can promote the degradation of bioremediation agent to quinclorac. With the increase of fructose concentration, the degradation rate of bioremediation agent also shows an upward trend. Among them, the degradation rates of bioremediation agents of HS-18 and HCM-18 groups reach the peak value when the fructose concentration is 10%, which is increased by 5.0% and 8.1% respectively compared with the yeast treatment group, and the degradation rate of bioremediation agent of HSM-24 group is the highest when the fructose concentration is 2%, which is increased by 3.1% compared with the yeast treatment group.

[0099] Example 6

[0100] This embodiment uses a bioremediation agent to degrade dichloroquinoline acid. The specific process is as follows:

[0101] The reaction system was prepared using MSM medium (pH=6) containing 0.05% NaCl and 2% fructose, with a reaction temperature of 30℃ and a carbon-to-bacterial ratio of 1%. 0.01 g of hydrothermal carbon material was weighed into a 50 mL centrifuge tube, and 1% v / v yeast suspension was added. After pre-culturing for 2 h, the contaminant concentrations were adjusted to 25 mg / L and 100 mg / L dichloroquinoline acid. The mixture was then incubated at 30℃ and 180 rpm for 120 h, and samples were taken on days 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0102] MSM medium without quinclorac acid was used as a blank control group. Anhydrous thermal carbon reaction system with an equal proportion of yeast suspension added alone and yeast-free reaction system with an equal mass of hydrothermal carbon added alone were used as treatment control groups. All treatment groups were set up in triplicate. After cultivation, samples were centrifuged at 13000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm nylon filter and transferred to a liquid chromatography vial. The residual amount of quinclorac acid in the supernatant was determined using the method of Example 2, and the degradation rate of quinclorac acid was calculated using the same formula as in Example 2.

[0103] The time-series degradation behavior of dichloroquinoline acid by hydrothermal carbon and yeast was fitted using zero-order kinetics, first-order kinetics, and second-order kinetics, respectively, and the kinetic equations are as follows:

[0104] C t =C0-k0t

[0105]

[0106] In the formula, C t —Concentration of dichloroquinoline acid at time t, mg / L; C0—Initial concentration of dichloroquinoline acid, mg / L; k0—Zero-order reaction rate constant, min -1 k1—First-order reaction rate constant, min -1 k2—Second-order reaction rate constant, min -1 .

[0107] Table 1 Kinetic parameters of 100 mg / L dichloroquinoline acid degradation by yeast using different raw materials, hydrothermal carbon, and yeast

[0108]

[0109] Based on the fitting results of the 100 mg / L dichloroquinoline acid level ( Figure 8 to Figure 10 (Table 1) shows that the 100 mg / L treatment group showed better degradation kinetic fitting compared to the 25 mg / L treatment group. R0 in zero-order, pseudo-first-order, and pseudo-second-order kinetic models of yeast2 The R values ​​for the HS-18 group of bioremediation agents in the zero-order, pseudo-first-order, and pseudo-second-order kinetic models were 0.4924, 0.4915, and 0.9906, respectively. 2 The R values ​​for the HSM-24 group of bioremediation agents in the zero-order, pseudo-first-order, and pseudo-second-order kinetic models were 0.9475, 0.9794, and 0.9976, respectively. 2 The R values ​​for the HCM-18 group of bioremediation agents in the zero-order, pseudo-first-order, and pseudo-second-order kinetic models were 0.8614, 0.7443, and 0.9795, respectively. 2 The values ​​were 0.9913, 0.9640, and 0.9249, respectively. This indicates that the zero-order, pseudo-first-order, and pseudo-second-order kinetic models can all effectively simulate the degradation of dichloroquinoline acid by the HS-18 and HCM-18 group of bioremediation agents (R0). 2 >0.9). Based on the correlation coefficient analysis of reaction orders for different treatments, the pseudo-second-order kinetic curves showed good linear fit in the yeast group, the HS-18 group of bioremediation agents, and the HSM-24 group of bioremediation agents (R0.9). 2 The >0.97 (mean squared ratio) may be due to the strong adsorption or binding between yeast and dichloroquinoline acid during the degradation process of these treatments. Therefore, pseudo-second-order kinetics are more suitable for simulating the degradation behavior of 100 mg / L dichloroquinoline acid in these three groups. The degradation process of the HCM-18 group of bioremediation agents is more in line with the zero-order kinetic model.

[0110] The rate constant K value reflects the ideal degradation rate of dichloroquinoline acid (DCA) for different treatment groups. The results showed that the HCM-18+G130 treatment group had the highest K value (0.842), which was 1.684 times that of the yeast-only treatment group, indicating that the addition of HCM-18 significantly improved the degradation rate of DCA. This may be because the larger average pore size and specific surface area of ​​HCM facilitate yeast attachment and enhance its metabolic activity. HS and HCM have higher DOM content, which may serve as an additional carbon source, further promoting the degradation of DCA. In addition, DOM can enhance the activity of degrading bacteria, promote the secretion of extracellular polymers, and facilitate biofilm formation. Overall, the HCM-18 and HS-18 groups of bioremediation agents showed superior degradation effects.

[0111] Example 7

[0112] This embodiment uses a bioremediation agent to degrade dichloroquinoline acid. The specific process is as follows:

[0113] Take 0.01 g of hydrothermal carbon material in a 50 mL centrifuge tube, add 1% V / V yeast suspension, and use MSM medium containing 0.05% NaCl, 2% fructose (pH=6) as the reaction system. After 2h of pre-culture, adjust the pollutant concentration of the system to 0, 1, 5, 25, 50, 100, 200 mg / L quinclorac. Place in a 30°C temperature with 180 rpm for shaking reaction, and take samples at 5 days and 10 days respectively to explore the degradation effect of bioremediation agent under different initial quinclorac concentrations.

[0114] Use MSM medium without quinclorac as a blank control group, and use a hydrothermal carbon reaction system with the same proportion of yeast suspension and a yeast-free reaction system with the same mass of hydrothermal carbon as treatment control groups. All treatment groups are set in triplicate. After the end of the culture, centrifuge the sample at 13000 rpm for 3 min, take the supernatant, filter it through a 0.22 μm nylon filter, and then transfer it to a liquid chromatography vial. The residual amount of quinclorac in the supernatant is determined by the method of Example 2, and the degradation rate of quinclorac is calculated by the formula of Example 2. The results are shown in Figure 11 .

[0115] From Figure 11 it can be seen that the degradation rate of the bioremediation agent group is always higher than that of the yeast treatment group alone, which indicates that hydrothermal carbon can alleviate the toxic effects of quinclorac on yeast. However, different initial quinclorac concentrations have an impact on the degradation effect of bioremediation agent. At 5 days, each treatment group showed similar degradation rates when the quinclorac concentration was below 50 mg / L. When the quinclorac concentration exceeded 50 mg / L, the degradation rate of each treatment group decreased. At 10 days, when the quinclorac concentration was below 25 mg / L, the degradation rate of the yeast treatment group did not change significantly compared to the 5th day, indicating that the yeast may have lost its activity. In contrast, the degradation rate of the bioremediation agent group increased compared to the 5th day, indicating that the hydrothermal carbon may have provided additional nutrients to the yeast, allowing it to continue to degrade quinclorac. When the quinclorac concentration was higher than 50 mg / L, the degradation rate of each treatment group was higher than that of the 5th day. In particular, under the condition of 100 mg / L high-concentration quinclorac, the bioremediation agent of HS-18 and HCM-24 groups increased by 16.5 times and 1.6 times respectively compared to the corresponding treatment group at the 5th day. This indicates that under high-concentration quinclorac stress, the degradation activity of yeast is inhibited, but the addition of hydrothermal carbon improves the degradation capacity of yeast, thereby improving the overall degradation efficiency.

[0116] Overall, HCM-18+G130 had the best degradation effect on both short-term and prolonged degradation time, and still showed the alleviation of yeast toxicity effect under the concentration of 100 mg / L quinclorac. On the one hand, it may be due to the similar pore size of HCM-18 to the size of yeast, which is beneficial for yeast to avoid quinclorac stress in the hydrothermal carbon pore, so as to retain part of the metabolic activity to improve the degradation efficiency. At the same time, the organic acids in the DOM derived from hydrothermal carbon can be used as energy for microorganisms, helping microorganisms to better cope with stress conditions during the degradation process.

[0117] Figure 12 The TEM images of yeast cells during the degradation of quinclorac by HCM-18 group bioremediation agent are shown when the concentration of quinclorac is 100 mg / L. At the 6th day, the yeast cells appear vacuolization phenomenon (Fig. 6(a)), which indicates that high concentration of quinclorac has a stronger impact on yeast. Figure 12 As can be seen from Fig. 6(c), the connection between yeast and hydrothermal carbon is not tight, and the number of particulate matter around the yeast cells increases, which may be related to hydrothermal carbon particles or quinclorac degradation products. At the 10th day, the yeast cells show more significant vacuolization phenomenon (Fig. 6(b)). Figure 12 As can be seen from Fig. 6(c), the connection between yeast and hydrothermal carbon is not tight, and the number of particulate matter around the yeast cells increases, which may be related to hydrothermal carbon particles or quinclorac degradation products. At the 10th day, the yeast cells show more significant vacuolization phenomenon (Fig. 6(b)). Figure 12 As can be seen from Fig. 6(c), the connection between yeast and hydrothermal carbon is not tight, and the number of particulate matter around the yeast cells increases, which may be related to hydrothermal carbon particles or quinclorac degradation products. At the 10th day, the yeast cells show more significant vacuolization phenomenon (Fig. 6(b)). Figure 12 As can be seen from Fig. 6(c), the connection between yeast and hydrothermal carbon is not tight, and the number of particulate matter around the yeast cells increases, which may be related to hydrothermal carbon particles or quinclorac degradation products. At the 10th day, the yeast cells show more significant vacuolization phenomenon (Fig. 6(b)).

[0118] Example 8

[0119] This example studies the phytotoxicity alleviation effect of bioremediation agent on tomato growth under different quinclorac stress, and the specific process is as follows:

[0120] Two stress levels of 0.1 mg / kg and 0.5 mg / kg quinclorac were set, and at each stress level, no repair group, only hydrothermal carbon group, only Pichia G130 group, and biological remediation agent group were set. In addition, a group of uncontaminated soil without adding drugs was set as the positive control for tomato growth. The effects of hydrothermal carbon-yeast combined treatment on tomato growth and the effect of relieving tomato quinclorac phytotoxicity were studied. Before seedling, the tomato seeds were first disinfected with 1% sodium hypochlorite solution for 10 min, then washed thoroughly with sterile water for 6-8 times, then a suitable amount of peat soil was laid in the seedling pot, and the tomato seeds were buried so that they were located 1 cm below the soil surface. The seedling pots were placed in the plant culture room, and the culture conditions were set as temperature 25°C, humidity 40%, light duration 16h / day, and watering every day. The volume of the plastic flowerpot used was 350cm 3 .

[0121] For the drug-containing soil used in this example, the preparation method of uncontaminated soil with drugs was used to ensure that the stress response of crops came from quinclorac phytotoxicity as much as possible. 200 mg / L quinclorac stock solution was prepared 3 days before transplanting, and quinclorac treatment solution was prepared by adding high-purity water to the soil of the negative control group and the treatment group. The positive control group soil without adding drugs was added with the corresponding amount of high-purity water, and stirring was performed to make the water and quinclorac evenly distributed. After standing overnight to allow the methanol to completely volatilize, the positive control soil with consistent water content and the test soil with quinclorac concentrations of 0.1 mg / kg and 0.5 mg / kg were obtained. One day before transplanting, HCM-18, yeast suspension, and the synergistic culture liquid pretreated for 2h were added to the corresponding treatment group soil, respectively. The addition amount of the treatment group was calculated according to the optimal carbon-bacteria ratio, wherein the yeast addition amount was set to 1×10 -8 CFU / g, and the HCM-18 addition amount was 0.67%, and the balance was performed overnight after adding the treatment. Healthy plants with a height of 10 cm were selected and transferred to the flowerpots that had completed the above pretreatment and placed in the plant culture room for culture, and 6 parallels were set for each treatment group. The culture conditions were set as temperature 25°C, humidity 40%, light duration 16h / day, watering every morning during the culture process, and complete nutrient solution was poured every 5 days. To ensure sufficient colonization of yeast, 1×10 -8 CFU / g of yeast suspension was added again on the 7th day after transplanting. After 18 days of culture, the plants and treatment soil were harvested, the leaf chlorophyll content was determined using a chlorophyll meter, the tomato plant height and fresh weight were measured, the roots were washed and the rhizosphere scanner was used to measure the root length, and the plant root weight was measured.

[0122] To evaluate the physiological response of tomato plants to quinclorac stress and the alleviating effect of bioremediation agent treatment on stress, 0.1 g of tomato roots was taken to determine the enzyme activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the roots of the plants. The determination method is as follows:

[0123] (1) SOD activity determination

[0124] By taking 0.1-0.5 g of fresh plant sample, adding 5 mL of pre-cooled phosphate buffer, grinding into homogenate, and centrifuging at 4000 rpm for 10 min, the supernatant was taken as the crude enzyme solution. In a 10 mL centrifuge tube, 1.5 mL of phosphate buffer, 0.3 mL of Met solution, 0.3 mL of NBT solution, 0.3 mL of EDTA-Na2 solution, 0.3 mL of riboflavin solution, 0.05 mL of enzyme solution, and 0.25 mL of distilled water were added, mixed, and then placed under 4000Lx sunlight for 20 minutes. Another two 10 mL centrifuge tubes were prepared, and buffer was used instead of enzyme solution. The reagents were added according to the above steps, and one tube was placed in sunlight for reaction, and the other tube was placed in the dark. After the reaction was completed, the blank control tube without light was used to set zero, and the absorbance of each tube was measured. The 50% inhibition of NBT reduction was taken as one enzyme activity unit.

[0125] (2) CAT activity determination

[0126] Two small test tubes were taken as sample and control tubes, respectively. In the control tube, 1.5 mL of buffer solution, 0.1 mL of enzyme extract, 0.2 mL of distilled water, 1.0 mL of sulfuric acid, and 0.2 mL of hydrogen peroxide solution were added in sequence; in the sample tube, 1.5 mL of buffer solution, 0.1 mL of enzyme extract, 1.2 mL of distilled water, and then 0.2 mL of hydrogen peroxide solution were added in sequence, and 1 mL of sulfuric acid was added immediately after timing for 4 minutes to terminate the reaction. At 240 nm, distilled water was used to set zero, and the absorbance of the sample and control tubes was measured, respectively. The milligrams of H2O2 generated per gram per minute was taken as one enzyme activity unit.

[0127] (3) POD activity determination

[0128] Weigh 0.1–0.2 g of sample, grind with 1 mL of 0.5 mol / L phosphate buffer (pH 5.5), homogenize under ice bath conditions, transfer to a test tube, rinse the mortar with 4 mL of buffer solution, transfer to the test tube, mix well, and incubate at 5°C for 10 minutes. Centrifuge at room temperature and store the supernatant for later use. Using 0.05 mol / L phosphate buffer (pH 5.5) as a blank reference for zeroing, take 1 mL of the supernatant enzyme solution, add 2 mL of 0.05 mol / L phosphate buffer (pH 5.5), add 1 mL of 0.05 mol / L guaiacol, and finally add 1 mL of 2% hydrogen peroxide. Shake immediately, pour quickly into a cuvette, and measure the absorbance of the solution at a wavelength of 470 nm. Record the absorbance every 30 seconds for a total of 4 minutes. (The last sentence appears to be incomplete and requires further context.) 470 A change of 0.01 nm in absorbance is defined as one unit of enzyme activity.

[0129] Figure 13 It showed 0.1 mg / L ( Figure 13 (a)~(c)) and 0.5mg / L ( Figure 13 The growth of tomatoes under dichloroquinoline stress (d) to (f). Figure 13 (a) shows that tomato growth was significantly inhibited under 0.1 mg / L quinclorac acid (NCK, control group), resulting in stunted plants and yellowing leaves. The HCM-18, G130, and HCM-18+G130 groups all improved tomato growth, producing taller plants and greener leaves, indicating that these two individual treatments, as well as the bioremediation agent treatment, all had a certain alleviating effect on quinclorac acid stress. (Observation of leaf morphology and color changes...) Figure 13 As shown in (b) of the diagram, the HCM-18+G130 group exhibited better leaf morphology and color, demonstrating a certain protective effect. From the root growth status ( Figure 13 In (c) it can be seen that the NCK group has significantly shorter roots and fewer root hairs, indicating that quinclorac stress inhibits root growth. The HCM-18 and HCM-18+G130 groups have longer and more developed roots, indicating that they alleviate the toxic effects of quinclorac on the roots.

[0130] Figure 13 In the middle (d) group, it can be observed that the tomato plants in the NCK group were severely damaged, with wilted leaves and stunted growth, indicating that 0.5 mg / L quinclorac acid caused strong toxicity to tomato growth. The growth of plants in the HCM-18 and G130 groups improved somewhat, but they were still shorter than those treated with 0.1 mg / L. The HCM-18+G130 group performed better; compared to the NCK group, the plants were slightly taller and the leaves were slightly greener, indicating that the bioremediation agent treatment still had a certain mitigating effect on high concentrations of quinclorac acid. Changes in leaf morphology and color (…) Figure 13As shown in (e), the leaves in the NCK group were smaller than those in the 0.1 mg / L dichloroquinoline acid treatment group. The leaves in the HCM-18 and G130 groups were darker in color, but showed slight yellowing at the edges, indicating that the stress was partially alleviated but still somewhat affected. The leaves in the HCM-18+G130 group were relatively greener and had better morphology; although some yellowing remained, it was a significant improvement compared to the NCK group. Observation of root growth showed... Figure 13 In the middle (f) group, the NCK group had short roots and fewer branches, indicating that high concentrations of quinclorac acid affected lateral root growth. The roots of the HCM-18 group were shorter and somewhat inhibited compared to the NCK group, indicating that HCM-18 failed to alleviate the damage to tomato roots caused by high concentrations of quinclorac acid. The G130 group and the HCM-18+G130 group had relatively well-developed root systems, showing significant improvement compared to the NCK group.

[0131] Figure 14 The data shows the various growth indicators of tomatoes corresponding to the reaction system at 0.1 mg / L quinclorac acid, from... Figure 14 (a) and Figure 14 As shown in (b), there were no significant differences in aboveground part weight and plant height among the treatments. The chlorophyll content of each treatment was also observed. Figure 14 As shown in (c), the chlorophyll content of the NCK group was significantly higher than that of the PCK group, and the chlorophyll content of the HCM-18, G130 and HCM-18+G130 groups was also significantly higher than that of the NCK group, indicating that the above treatments can increase the chlorophyll content. Figure 14 Figure (d) shows the stem diameter of plants under each treatment. It can be observed that compared to the PCK group, the stem diameter of the NCK group was significantly reduced, and the stem diameter further decreased after HCM-18 treatment. The G130 and HCM-18+G130 groups partially alleviated the trend of stem diameter decline, increasing it by 0.6% and 7.4% respectively compared to NCK. Figure 14 As shown in (e), the PCK group had the longest root length. The root lengths of the HCM and HCM-18+G130 groups were both longer than those of the NCK group. There was no significant difference in root length among the treatment groups, and all were shorter than the PCK group. This indicates that the HCM and bioremediation agent groups can alleviate the stress of quinclorac acid on tomato roots and promote their growth. Figure 14As shown in (f), the root fresh weight of the HCM-18+G130 treatment group was higher than that of the PCK and NCK groups, but the root length was lower than that of the PCK, indicating that the root length was shorter but the root system was developed. This may be due to the fact that the HCM and the bioremediation agent group are rich in organic matter and nutrients, which may promote the thickening of the root system and the development of lateral roots. In summary, quinclorac treatment (NCK group) has a certain negative impact on tomato growth, especially the reduction of chlorophyll content and stem diameter. HCM-18, G130 and HCM-18+G130 may alleviate the impact of quinclorac to some extent, showing that the chlorophyll content is restored, the stem diameter is increased, etc. This may be due to the fact that HCM is rich in pore structure, high organic carbon, nitrogen content and humus content, which maintains the soil structure and nutrients, provides a suitable survival environment for yeast to promote plant growth. In addition, the antioxidant enzymes produced during yeast metabolism can enhance the antioxidant capacity of plants, improve the physiological state of plants after phytotoxicity, restore normal metabolic processes such as photosynthesis and respiration, and help plants allocate more resources to the synthesis of chlorophyll, thereby increasing chlorophyll content and stem diameter.

[0132] At the 0.5 mg / L quinclorac level, Figure 15 In (a), Figure 15 In (b), and Figure 15 As shown in (d), there was no significant difference between the quinclorac treatment group (NCK) and each of the addition groups (HCM-18, G130, HCM-18+G130), and the results were significantly lower than those of the PCK group, indicating that 0.5 mg / L quinclorac treatment inhibited the growth of the aboveground part and the development of the stem diameter, and each treatment failed to significantly restore it. Observing the chlorophyll content of the plant (c) showed that the chlorophyll content of the NCK group and each treatment group was higher, and the high concentration of quinclorac stimulated the synthesis of chlorophyll. This may be due to the fact that quinclorac may affect the biosynthesis pathway of chlorophyll, such as promoting the synthesis of delta-amino levulinic acid (ALA), thereby increasing the chlorophyll content. On the other hand, it may inhibit the chlorophyll degradation pathway, such as reducing the activity of chlorophyllase, thereby prolonging the life of chlorophyll. The chlorophyll content of the yeast group and the HCM+G130 group was slightly higher than that of NCK, indicating that yeast and its combined action with HCM helped to alleviate the impact of quinclorac on the plant photosynthetic system, promote the accumulation of chlorophyll, and improve the plant's ability to adapt to the stress environment. There was no significant change in root length among the treatment groups, among which the root length of the HCM-18 group showed greater variability (e). By comparing the root fresh weight (f), it was found that the root fresh weight of the HCM-18+G130 treatment group was higher than that of the PCK and NCK groups, but the root length was lower than that of the PCK, indicating that the root length was shorter but the root system was developed. This may be due to the fact that the HCM and the bioremediation agent group are rich in organic matter and nutrients, which may promote the thickening of the root system and the development of lateral roots. In summary, quinclorac treatment (NCK group) has a certain negative impact on tomato growth, especially the reduction of chlorophyll content and stem diameter. HCM-18, G130 and HCM-18+G130 may alleviate the impact of quinclorac to some extent, showing that the chlorophyll content is restored, the stem diameter is increased, etc. This may be due to the fact that HCM is rich in pore structure, high organic carbon, nitrogen content and humus content, which maintains the soil structure and nutrients, provides a suitable survival environment for yeast to promote plant growth. In addition, the antioxidant enzymes produced during yeast metabolism can enhance the antioxidant capacity of plants, improve the physiological state of plants after phytotoxicity, restore normal metabolic processes such as photosynthesis and respiration, and help plants allocate more resources to the synthesis of chlorophyll, thereby increasing chlorophyll content and stem diameter. Figure 15 Figure 15 Figure 15 ​​It can be found that HCM-18+G130 group showed a certain recovery trend in root fresh weight, indicating that the combined treatment might have a certain promoting effect on root growth, and the root length of HCM-18+G130 group was shorter and the root system was heavier, indicating that the effect of HCM-18+G130 group on the root system under 0.5 mg / L quinclorac stress was consistent with that of 0.1 mg / L quinclorac. In summary, the negative effect of 0.5 mg / L quinclorac treatment on tomato growth was more significant than that of 0.1 mg / L, especially the decrease in shoot fresh weight and stem diameter. HCM-18, G130 and HCM-18+G130 treatments failed to significantly improve growth inhibition, but HCM-18 might have a certain promoting effect on root length, and HCM-18+G130 had a certain recovery effect on plant height, chlorophyll content and root fresh weight.

[0133] Under external environmental stress, a large number of superoxide free radicals O2 - · will be produced in the plant body - · may accelerate the aging of plant leaves, thereby reducing the antioxidant defense ability of plants, while root catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) can effectively reduce the number of O2 - · and improve the stress resistance of plants. Figure 16 The activities of CAT, POD and SOD in the roots of plants in each treatment group under 0.1 mg / L quinclorac were shown. The results showed that there was no significant difference in CAT activity among the treatment groups, which remained at 350-400 mg / (g·min), indicating that the quinclorac treatment at this concentration had little effect on the accumulation of H2O2. Compared with the normal level of POD activity in the PCK group, the POD activity in the NCK group decreased significantly, indicating that the accumulation of O2 - · caused by quinclorac treatment inhibited the activity of POD. HCM-18 and G130 treatments significantly reduced the activity of POD to about 500 U / (g·min), while the activity of POD in the HCM-18+G130 treatment group was effectively improved to close to the normal level of the PCK group, which was significantly higher than that of the other three treatments. This may be due to the combined effect of the two, which to some extent affected the balance of oxidative stress and more effectively regulated the ROS level, making the POD system partially recover. The SOD activity in the NCK group was the highest, which was significantly higher than that in the PCK group, indicating that quinclorac induced oxidative stress, and the plant enhanced the activity of SOD to scavenge O2 -SOD activity was slightly lower than NCK group, but still higher than G130 and HCM-18+G130 treatment groups, which was not significant, indicating that the mitigation of oxidative stress by hydrothermal carbon was limited. SOD activity of G130 and HCM-18+G130 treatment groups was further reduced, close to PCK group, indicating that these treatments might be more effective in reducing oxidative stress, reducing the demand for SOD in plants.

[0134] Overall, 0.1 mg / L quinclorac induced oxidative stress, up-regulated SOD activity, and down-regulated POD activity, which might be due to the direct inhibition of quinclorac. HCM-18+G130 treatment effectively restored part of the POD activity and adjusted SOD activity to close to PCK level, indicating that the combined action of hydrothermal carbon and yeast might regulate the oxidative stress response to some extent and improve the stress resistance of plants.

[0135] Figure 17 The antioxidant enzyme activity in the roots of plants treated with 0.5 mg / L quinclorac was shown. The results showed that the CAT activity of the NCK group was up-regulated, indicating that quinclorac induced oxidative stress. The CAT activity of the HCM-18 treatment group was comparable to that of the NCK group, while the CAT activity of the G130 and HCM-18+G130 treatment groups was significantly reduced, indicating that the effect of yeast might alleviate oxidative stress and reduce the dependence of plants on CAT. The POD activity of the NCK group was slightly higher than that of the PCK group, indicating that quinclorac might affect the activity of POD. The POD activity in the HCM-18+G130 treatment group was the lowest. This indicates that HCM-18+G130 might reduce the demand for POD activity in plants by reducing ROS production, or hydrothermal carbon might affect the antioxidant regulation ability of yeast to some extent. SOD activity showed significant differences among different treatments. Higher SOD activity was required to scavenge O2 - · produced in normal metabolic processes. Compared with PCK, the SOD activity of each treatment group was significantly reduced, indicating that high concentration of quinclorac might exacerbate plant damage or change the type of ROS from O2 - · to H2O2. The SOD activity of the HCM-18 and HCM-18+G130 treatment groups was slightly lower than that of the NCK group, while the SOD activity of the G130 treatment group was the lowest, indicating that the above treatments might remove part of the generated O2 - ·.

[0136] In general, 0.5 mg / L quinclorac induced more severe oxidative stress in tomato seedlings, enhanced CAT activity in tomato roots, and significantly reduced SOD activity, indicating that quinclorac might promote ROS accumulation mainly in the form of H2O2. The CAT and SOD activities of the G130 and HCM-18+G130 treatment groups were significantly lower than those of the NCK group, indicating that the ROS level was reduced, and the tomato did not need to rely on high levels of CAT and SOD activity to alleviate stress damage, reducing the overall burden on the tomato antioxidant system.

[0137] The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A bioremediation agent, characterized by: The Pichia pastoris is loaded on the hydrothermal carbon.

2. The bio-repair agent according to claim 1, characterized in that: The Pichia pastoris has a loading of 1.0 x 10 8 ~ 4.0 x 10 10 CFU / g hydrochar.

3. The bio-repair agent according to claim 1, characterized in that: The average specific surface area of the hydrothermal carbon is 10 to 50 m 2 / g; and / or the specific surface area of the micropores in the hydrothermal carbon is 0.01 to 4.0 m 2 / g.

4. The bio-repair agent of claim 1, wherein: The hydrothermal carbon has a pore volume of 0.05 to 0.3 cm 3 / g; and / or, the hydrothermal carbon has a pore volume of micropores of 2.0 x 10 -4 -15 x 10 -4 cm 3 / g.

5. The bio-repair agent according to claim 1, characterized in that: The hydrothermal carbon comprises at least one of fecal-based hydrothermal carbon and straw-based hydrothermal carbon.

6. The bio-repair agent of claim 1, wherein: The bioremediation agent further comprises a culture medium; the culture medium comprises a carbon source and / or an ion balancing agent.

7. The biological repair agent of claim 6, wherein: The culture medium satisfies at least one of the following conditions: (I) the mass concentration of the carbon source is 0.1-10%; (II) the mass concentration of the ion balancing agent is 0.01-8%; (III) the pH of the culture medium is 3-10.

8. A method for producing the bio-remediating agent according to any one of claims 1 to 7, characterized by: The method comprises the following steps: culturing Pichia pastoris in a culture medium containing hydrothermal carbon to obtain the bioremediation agent.

9. A method of degrading a pollutant, characterized by: The method comprises the following steps: The bioremediation agent of any one of claims 1-7 is added to the pollutants for degradation.

10. The method of degrading pollutants of claim 8, wherein: The pollutants comprise at least one of quinclorac, organochlorine pesticide, organophosphorus pesticide, polycyclic aromatic hydrocarbon, phenol, o-xylene, abamectin and heavy metal ion.

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