CN110484449B - Carbendazim degrading bacterium protective agent and preparation method and application thereof - Google Patents

Carbendazim degrading bacterium protective agent and preparation method and application thereof Download PDF

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CN110484449B
CN110484449B CN201910697819.0A CN201910697819A CN110484449B CN 110484449 B CN110484449 B CN 110484449B CN 201910697819 A CN201910697819 A CN 201910697819A CN 110484449 B CN110484449 B CN 110484449B
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黄星
卢家森
杨涵
祁鹏
何健
洪青
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Nanjing Agricultural University
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Abstract

The invention discloses a protective agent for carbendazim degrading bacteria, a preparation method and application thereof. The protective agent can effectively solve the problems of low viable count, short preservation period, easy bacterial contamination and the like of the microbial agent; the method has the advantages of effectively maintaining the performance of the microbial inoculum, improving the survival rate of effective bacteria, prolonging the preservation life of the microbial inoculum, ensuring that the microbial inoculum can be preserved at normal temperature, being not easy to be infected by bacteria, having low cost and convenient use, effectively reducing the transportation and preservation costs, and further ensuring the practical application of the pesticide degradation microbial inoculum in pesticide degradation.

Description

Protective agent for carbendazim degrading bacteria and preparation method and application thereof
Technical Field
The invention belongs to the field of microbial degradation, relates to protection of a microbial agent for pesticide degradation, and particularly relates to a protective agent for carbendazim degrading bacteria, and a preparation method and application thereof.
Background
Due to the random use of pesticides and the lack of reasonable scientific supervision and use management, pesticide residues cause environmental degradation, seriously damage the balance of an ecological system and cause structural damage and functional loss of the ecological system. Pesticide pollution is non-point source pollution, and the physical and chemical remediation method has certain defects. The microorganism has inherent characteristics, so that the microorganism plays an important role in pesticide degradation. The method for remedying the pollution caused by pesticide residues by using microorganisms is known to be a method which is cheap, safe, effective and free from secondary pollution. At present, a large number of degradable microorganisms are screened and separated aiming at various pesticide pollutants at home and abroad, and various microbial degradation preparations and matched products are developed and applied to biological in-situ remediation, however, the preservation of the biological agent is always a difficult problem, the application potential of the biological agent is directly influenced, and the problems of low viable count, short preservation period, easy bacterial contamination and the like generally exist in the liquid microbial agent in the market at present. The preservation by drying and the like can prolong the preservation period, but the preservation cost is too high and the equipment technology is complex. Therefore, the research on the protective agent suitable for prolonging the preservation time of the microbial inoculum is very important for prolonging the preservation time of the microbial inoculum at normal temperature.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a protective agent for carbendazim degrading bacteria, which can effectively solve the problems of low viable count, short preservation period, easy bacterial contamination and the like of a microbial agent; the performance of the microbial inoculum can be effectively maintained, the survival rate of effective bacteria is improved, the preservation life of the microbial inoculum is prolonged, the microbial inoculum can be preserved at normal temperature, and the microbial inoculum is not easy to contaminate.
The invention also provides a preparation method and application of the carbendazim degrading bacteria protective agent.
The technical scheme is as follows: in order to achieve the purpose, the protective agent for carbendazim degrading bacteria consists of a stabilizing agent, a nutritional agent, a preservative and water, wherein the stabilizing agent is carboxymethyl cellulose, the nutritional agent is sodium citrate and calcium chloride, and the preservative is sodium benzoate.
Wherein the addition concentration of the carboxymethyl cellulose in the protective agent is 10-30 g/L.
The addition concentration of the sodium citrate in the protective agent is 10-30 g/L, and the addition concentration of the calcium chloride in the protective agent is 10-30 g/mL.
Wherein the adding concentration of the sodium benzoate in the protective agent is 50 g/L.
The optimal protective agent of the invention comprises the following components: the adding concentration of the sodium citrate in the protective agent is 30g/L, the adding concentration of the calcium chloride in the protective agent is 20g/L, the adding concentration of the carboxymethyl cellulose in the protective agent is 30g/L, and the adding concentration of the sodium benzoate in the protective agent is 50 g/L. The concentration of the carboxymethyl cellulose in the bacterial liquid fermentation liquid is 3g/L, the concentration of the sodium citrate is 3g/L, the adding concentration of the calcium chloride is 2g/L, and the concentration of the sodium benzoate is 5 g/L.
The preparation method of the protective agent for carbendazim degrading bacteria is characterized in that carboxymethyl cellulose, sodium citrate, calcium chloride, sodium benzoate and water are mixed in proportion to prepare the protective agent.
The protective agent for carbendazim degrading bacteria is applied to protecting carbendazim degrading bacteria.
Wherein the carbendazim degrading microbial inoculum is liquid thallus.
Preferably, the protective agent of the carbendazim degrading bacteria is added into the carbendazim degrading bacteria agent, so that the concentration of carboxymethyl cellulose in the bacteria agent is 1-3 g/L, the concentration of sodium citrate in the bacteria agent is 1-3 g/L, the addition concentration of calcium chloride in the bacteria agent is 1-3 g/L, and the concentration of sodium benzoate in the bacteria agent is 5 g/L.
Preferably, the carbendazim-degrading bactericide is protected to effectively maintain the performance of the bactericide, improve the survival rate of effective bacteria and prolong the preservation life of the bactericide.
Preferably, the carbendazim-protecting degrading microbial inoculum is an application of the carbendazim-protecting degrading microbial inoculum in degrading residual carbendazim pesticides in soil.
Inorganic salt medium (MM) in the present invention: NH (NH) 4 NO 3 1.0g/L，KH 2 PO 4 0.5g/L，K 2 HPO 4 1.5g/L，NaCl 1.0g/L，MgSO 4 ·7H 2 O 0.2g/L，pH 7.0。
LB culture medium: 5.0g/L yeast extract, 10.0g/L peptone, 10.0g/L NaCl, H 2 O1L, pH 7.0, solid medium added with 1.5% -2.0% agar.
djl-6 fermentation Medium: 8g/L of glucose, 0.8g/L of ammonium sulfate, 0.2g/L of yeast extract, 0.1g/L of sodium chloride, 0.5g/L of calcium carbonate, 1.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate and pH 7.0-7.2.
The carbendazim (purity 99.8%) original drug is provided by Jiangsu New Yiyi farm chemical factory. Carbendazim-degrading strain Rhodococcus guinshi sp. nov. DJL-6(DJL-6), (Rhodococcus guinshi sp. nov., a carbazazim-mapping bacterium, International journal of systematic and evalutional microbiology, Vol.12, 2007).
The liquid microbial inoculum is easy to be polluted in the storage process, so that the number of the mixed strains is increased, and the number of the target strains is reduced. The preservative is used as an additive substance in the microbial inoculum, and can effectively reduce the pollution of mixed bacteria. According to the invention, three preservatives with bacteriostatic and mildewproof effects, namely sodium benzoate, calcium propionate and potassium sorbate, are added to calculate the mixed bacteria rate of the djl-6 strain, so that the preservative with good bacteriostatic effect and relatively high viable bacteria number is screened out, and then the viable bacteria number and the mixed bacteria number of the preservative are detected by adding the preservatives with different concentrations, so that the adding concentration of the preservative in the liquid fermentation liquor is determined. The research shows that the degrading bacteria all use sodium benzoate as the bacteriostatic agent with the best effect. Can maintain a higher viable count and does not produce mixed bacteria pollution.
The liquid microbial inoculum has poor stability and rapid decrease of viable count in the preservation process. The addition of the protective agent is an important means for increasing the number of effective viable bacteria of the microbial product and prolonging the preservation period of the microbial product. The addition of a protectant aids in the dispersion and survival of the microorganisms. The invention shows that the microbial inoculum protective agent screened by the orthogonal test can effectively improve the survival rate of bacteria.
Has the beneficial effects that: compared with the prior art, the invention has the following advantages:
the stabilizer component of the carbendazim degrading microbial inoculum protective agent disclosed by the invention is carboxymethyl cellulose, so that the stability of the internal environment of the microbial inoculum can be maintained, and the survival rate of a bacterial strain is further improved. The nutrient components are sodium citrate and calcium chloride, which can reduce the influence of environmental change on the degradation performance of the microbial agent. The preservative is sodium benzoate to prevent microbial agent from being polluted by other bacteria, and the protective agent of the microbial agent is added into the microbial agent according to a certain concentration of effective components and is stored at room temperature.
The protective agent can preserve the carbendazim pesticide degradation microbial agent at normal temperature, and above all, effectively improves the degradation effect of the microbial agent under the condition of normal-temperature preservation, and the preservation method has low cost and convenient use, effectively reduces the transportation and preservation cost, and further ensures the practical application of the carbendazim pesticide degradation microbial agent in pesticide degradation.
The protective agent of the invention adopts sodium benzoate as a preservative, has low cost and can effectively prevent the pollution of mixed bacteria. The protective agent disclosed by the invention adopts sodium citrate and calcium chloride as nutritional agents, can effectively maintain the performance of the microbial inoculum, prolongs the storage life of the microbial inoculum, can meet the requirement of the microbial inoculum on pesticide residue degradation, and has practical value.
The carbendazim degrading bacteria protective agent can effectively improve the survival rate of bacteria, and the number of live bacteria in the fermentation liquor of the carbendazim degrading bacteria djl-6 which is preserved for 30 days by adding the protective agent can be improved by 52.03 percent; the liquid microbial inoculum (fermentation liquor) of the strain djl-6 which is added with the protective agent and stored for 30 days can degrade 93.65% of carbendazim with the concentration of 50mg/L at most in 3 days; the liquid microbial inoculum (fermentation liquor) of the bacterial strain djl-6 which is added with the protective agent and is preserved for 45 days can degrade the carbendazim with the concentration of 5mg/kg dry soil to 95.23 percent within 10 days.
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FIG. 1 is a graph of the degradation of carbendazim by strain djl-6;
FIG. 2 is a graph showing the effect of the strain djl-6 on the degradation experiment of carbendazim by using different combinations of protective agents.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1
The protective agent of carbendazim degrading bacteria comprises the following components:
10g of carboxymethyl cellulose, 20g of sodium citrate, 20g of calcium chloride and 50g of sodium benzoate are weighed, mixed uniformly and then the volume is determined to 1000mL by using sterile water.
Example 2
The protective agent of carbendazim degrading bacteria comprises the following components:
weighing 10g of carboxymethyl cellulose, 30g of sodium citrate, 30g of calcium chloride and 50g of sodium benzoate, uniformly mixing, and then using sterile water to fix the volume to 1000 mL.
Example 3
The protective agent of carbendazim degrading bacteria comprises the following components:
weighing 20g of carboxymethyl cellulose, 30g of sodium citrate, 10g of calcium chloride and 50g of sodium benzoate, uniformly mixing, and then using sterile water to fix the volume to 1000 mL.
Example 4
The protective agent of carbendazim degrading bacteria comprises the following components:
30g of carboxymethyl cellulose, 30g of sodium citrate, 20g of calcium chloride and 50g of sodium benzoate are weighed, mixed uniformly and then the volume is determined to 1000mL by using sterile water.
Example 5
The protective agent of carbendazim degrading bacteria comprises the following components:
weighing 10g of carboxymethyl cellulose, 10g of sodium citrate, 10g of calcium chloride and 50g of sodium benzoate, uniformly mixing, and then using sterile water to fix the volume to 1000 mL.
Example 6
The protective agent of carbendazim degrading bacteria comprises the following components:
weighing 20g of carboxymethyl cellulose, 30g of sodium citrate, 30g of calcium chloride and 50g of sodium benzoate, uniformly mixing, and then using sterile water to fix the volume to 1000 mL.
Example 7
Preservation effect of pesticide carbendazim residue degrading bacterium agent
Preparation of fermentation broth
A single colony of strain djl-6 was picked in liquid LB and cultured on a shaker at 30 ℃ at 160rpm to logarithmic growth phase. Inoculating into a 2L triangular flask containing 1000mL fermentation medium according to the inoculation amount of 1% by volume, and culturing to the late logarithmic growth phase.
The protective agent prepared in the embodiment 1 to 4 is added into a bacterial liquid (fermentation liquid) to be stored at normal temperature (for example, the protective agent is added into the bacterial liquid fermentation liquid in the embodiment 1, the concentration of sodium citrate in the fermentation liquid is 3g/L, the concentration of calcium chloride is 2g/L, the concentration of carboxymethyl cellulose is 3g/L, and the concentration of sodium benzoate is 5g/L), sampling is carried out at 30d, colony counting is carried out by adopting a dilution coating plate method, and the same fermentation liquid which is stored for 30 days is not added with the protective agent in a comparative example. The results are shown in Table 1.
Example 8
Carbendazim degradation effect in inorganic salt culture medium
The protective agent prepared in the examples 1 to 4 is added into a bacterial liquid (fermentation liquid) for storage at normal temperature, the bacterial liquid is taken out after 30d, the bacterial liquid is inoculated into 20ml of a liquid inorganic salt culture medium with the carbendazim concentration of 50mg/kg according to the inoculation amount of 3 percent of the volume ratio, the culture medium is placed in a shaking table for 3d of culture at 30 ℃ and 150rpm, then sampling is carried out, the content of the carbendazim is measured by using HPLC, the degradation rate is calculated, the protective agent is not added in a comparative example, the same fermentation liquid is stored for 30 days, and the result is shown in Table 2.
Example 9
Degradation effect of carbendazim residue in soil
The protective agent prepared in the examples 1 to 4 is added into a bacterial liquid (fermentation liquid) to be stored at normal temperature, the mixture is taken out at 45d, 10ml of the mixture is taken to be inoculated into 100g of soil with the final concentration of carbendazim of 5mg/kg dry soil, a sample is taken at 10d, the content of the carbendazim is measured by using HPLC, the degradation rate is calculated, the protective agent is not added into the same fermentation liquid stored for 45 days in a comparative example, and the result is shown in Table 3.
Determination of carbendazim content
Adding dichloromethane with the same volume into a sample to be detected, sufficiently shaking and extracting, removing upper-layer waste liquid, adding a proper amount of anhydrous sodium sulfate into a lower-layer organic phase, putting 1.5mL into a centrifuge tube, placing the centrifuge tube in a ventilation kitchen for drying, adding 300 mu L of methanol (chromatographic purity) for dissolving after complete volatilization, filtering by an organic phase filter (the aperture is 0.22 mu m), and determining the content of carbendazim in the sample by HPLC.
High performance liquid chromatography conditions: column, Kromasil 100-5C18(4.6 mm. times.250 mm); mobile phase, 60% methanol + 20% water; flow rate, 1 mL/min; detection wavelengths, 240nm and 280 nm; sample size, 20 μ L.
Extraction and determination of residual carbendazim in soil
Adding 5g of the differently processed soil samples to be detected into a clean 50mL centrifuge tube, adding 10mL of methanol (analytically pure) into the centrifuge tube, oscillating on a vortex instrument to ensure that the soil samples are completely soaked by the methanol, and then oscillating and extracting for 1h in a shaking table at 30 ℃ and 160rpm to ensure that the carbendazim can be completely dissolved in the methanol. Taking out, centrifuging at 6000rpm for 10min, transferring the supernatant into a new clean centrifuge tube, shaking the lower layer precipitate with vortex instrument, and repeating the above steps. Mixing the supernatants, air drying in a fume hood, adding 1mL of methanol for dissolving, filtering with 0.22 μm organic phase filter membrane, refrigerating for testing, and measuring carbendazim content in the sample by HPLC under the same conditions.
TABLE 1 colony count results (concentration of each component in bacterial solution in g/L)
Figure BDA0002148665020000051
TABLE 2 degradation rate of carbendazim in inorganic salt medium (each component is calculated as g/L in bacteria solution)
Figure BDA0002148665020000061
TABLE 3 degradation rate of residual carbendazim in soil (each component is calculated as g/L in bacterial liquid)
Figure BDA0002148665020000062
As can be seen from tables 1-3, the protective agent of the microbial inoculum consisting of sodium benzoate, carboxymethyl cellulose, sodium citrate and calcium chloride is added into the microbial inoculum of the carbendazim degrading bacteria, and compared with the comparative example without the protective agent, the protective agent of the invention effectively improves the effective viable count and the degradation rate of the microbial inoculum under the condition of normal-temperature storage.
Example 10
Preparation of seed liquid and measurement of cell growth
Selecting a single bacterial colony of the strain djl-6, culturing in a liquid LB test tube at 160rpm and 30 ℃ to logarithmic phase, taking out the bacterial colony with the volume ratio of 1%, inoculating the bacterial colony into a triangular flask containing 100mL of fermentation medium, culturing at 160rpm and 30 ℃ to logarithmic phase, then placing the flask in a centrifuge, centrifuging at 6000rpm for 10min, collecting the thallus, adding sterile water for resuspending, repeating the step of washing the thallus three times, and finally using the sterile water for resuspending until the OD600 is about 0.8 to serve as seed liquid.
The amount of cell growth was measured by a spectrophotometer, and the amount of growth of the strain was expressed by absorbance at a wavelength of 600 nm.
Degrading carbendazim by using a strain djl-6:
the seed solution of the strain djl-6 was added to 100mL of an inorganic salt medium containing 50mg/L of carbendazim at an inoculum size of 3% by volume, cultured at 30 ℃ and 150rpm, sampled every 8 hours, and the concentration of carbendazim in the sample was determined by liquid chromatography.
The concentration of carbendazim is 50 mg.L -1 The inorganic salt liquid culture medium is inoculated with seed liquid of a strain djl-6 by an inoculum size of 3 percent in volume ratio, is subjected to shaking culture in a constant temperature shaking table at 30 ℃ and 160rpm, samples are taken every 8 hours, and the concentration of the carbendazim is measured, as shown in a degradation curve of figure 1, the strain djl-6 can be degraded by 50 mg.L within 3 days -1 Carbendazim of (1).
Example 11
Preparation of fermentation broth
A single colony of strain djl-6 was picked in liquid LB and cultured on a shaker at 30 ℃ at 160rpm to logarithmic growth phase. Inoculating into a 2L triangular flask containing 1000mL fermentation medium according to the inoculation amount of 1% by volume, and culturing to the late logarithmic growth phase. The number of colonies was counted by dilution plating.
Preliminary screening of bacterial agent bacteriostatic agent
After the strain djl-6 is fermented, 300mL of fermentation liquor is respectively taken and added into a 500mL triangular flask, calcium propionate, sodium benzoate and potassium sorbate are added into the flask, the addition amount is 5g/L of the fermentation liquor, meanwhile, a reagent-free control group is arranged, equal-volume sterile water is added into the flask, and three treatment groups are arranged in parallel. Sealing with sealing film, and storing at normal temperature in dark place. After standing for 3d, sampling, counting the degrading bacteria djl-6 and the mixed bacteria by adopting a dilution coating plate method, and calculating the mixed bacteria rate.
Adding 5g/L of potassium sorbate, sodium benzoate and calcium propionate into the fermentation liquor of the strain djl-6, respectively, standing at normal temperature in the dark for 3 days, diluting, coating, counting, and calculating the rate of infectious microbes. As can be seen from Table 4, after different bacteriostatic agents with effective use concentrations of 5g/L are added into the fermentation broth and stored for 3 days, the total bacteria number in each treatment is smaller than that of a control group without the bacteriostatic agent, the mixed bacteria number is lower than that of the control group, the mixed bacteria rate in the control group without the bacteriostatic agent reaches 3.88%, and in the treatment with the different bacteriostatic agents, the sodium benzoate treatment effect is the best, and the mixed bacteria pollution does not occur, so the sodium benzoate is selected as the bacteriostatic agent of the fermentation broth of the degrading bacteria djl-6 to carry out the subsequent bacteriostatic agent re-screening test.
TABLE 4 influence of different preservatives on the microbial contamination rate of djl-6 fermentation broth
Figure BDA0002148665020000071
Antiseptic rescreening
After the strain djl-6 is fermented, 300mL of fermentation liquor is respectively taken and added into 500mL of triangular flasks, sodium benzoate with different concentrations is respectively added as a preservative for research, the addition amounts are respectively 1, 3, 5, 7 and 10g/L of the fermentation liquor, a control group without any sodium benzoate is simultaneously arranged, sterile water with the same volume is added into the control group, and three treatment sets are arranged in parallel. Sealing with sealing film, storing at normal temperature in dark place for 3d, sampling, counting the degrading bacteria djl-6 and the mixed bacteria by dilution coating plate method, and calculating the mixed bacteria rate.
According to the primary screening result of the preservative, sodium benzoate is selected as the preservative to carry out subsequent experiments. Adding sodium benzoate with different concentrations into the fermentation liquor of the strain djl-6 respectively to make the final concentrations of the sodium benzoate respectively 1, 3, 5, 7 and 10g/L of the fermentation liquor, placing the fermentation liquor at normal temperature and storing in dark for 3d, sampling, diluting and coating to calculate the colony count, and calculating the rate of mixed bacteria. As can be seen from Table 5, when the effective concentration of the added sodium benzoate exceeds 5g/L, no contamination by other bacteria occurs. And when the concentration is 5g/L, the viable count is higher compared with other treatments without contamination. Therefore, for the strain djl-6, if sodium benzoate is selected as the preservative, sodium benzoate with a concentration of 5g/L can be selected for research.
TABLE 5 influence of sodium benzoate with different concentrations on the microbial contamination rate of djl-6 fermentation broth
Figure BDA0002148665020000081
Example 12
Screening of microbial inoculum protector
Screening for Single factor protective Agents
Single bacterial colonies of the strain djl-6 are respectively picked up in liquid LB, placed in a shaker at 30 ℃ and cultured at 160rpm until the logarithmic phase, and then inoculated into a triangle containing 500mL of liquid fermentation medium by the inoculum size of 1% in volume ratio for continuous culture until the logarithmic phase. Then the mixture is put into a 50mL sterile screw centrifugal tube sterilized at high temperature under the aseptic condition, sodium benzoate is added into the centrifugal tube to ensure that the concentration of the sodium benzoate is 5g/L, and then calcium chloride, magnesium chloride, dextrin, fulvic acid, carboxymethyl cellulose, sodium acetate and sodium citrate which are preliminarily screened are respectively added into the centrifugal tube as protective aids, so that the effective use concentrations of the aids in the test tube are respectively 1g/L and 2g/L, a control group without the aids is arranged at the same time, sterile water with the same volume is added into the control group, and three treatment devices are arranged in parallel. Then, samples were taken at 30d and the number of viable cells was counted by dilution plating.
Screening of optimal substance concentration of degrading bacteria auxiliary agent
According to the experimental result of the single-factor assistant, three types of screened protective agents are divided into three factors of inorganic salt assistant, organic matter assistant and stabilizing agent, two different substances are taken from each factor as two levels, and three-factor two-level L4 (2) 3 ) The optimal substance combination of the degrading bacteria agent auxiliary agent is determined by orthogonal experiments.
Screening for optimal use concentration of degrading bacteria auxiliary agent substance combination
According to the results of orthogonal experiments on substance combinations, three auxiliary agents are selected as three factors, three different effective use concentrations are selected from each factor as three levels, and three-factor three-level L9 (3) 3 ) The optimal concentration combination of the degrading bacteria agent auxiliary agent is determined by orthogonal experiments.
Screening of test strain protective agents:
research on influence of inorganic salt addition on preservation effect of degradation bacterium agent
Each inorganic salt is set to have two different concentrations, and fermentation liquor without the addition of the inorganic salt auxiliary agent is used as a reference, and each concentration is set to have three repetitions.
As shown in Table 6, MgCl was added to the fermentation broth of the strain djl-6 2 、CaCl 2 The survival rate of the strain djl-6 in the fermentation liquor can be obviously improved under two effective use concentrations of 1g/L and 2g/L, and 2g/L CaCl is added 2 The survival rate can be improved by 12.79 percent after the treatment, and 0.20 percent of MgCl is added 2 The survival rate is improved by 19.02 percent
TABLE 6 effective use concentrations of organic salts and their effect on the survival enhancement rate of djl-6
Figure BDA0002148665020000091
Research on influence of organic matter addition on preservation effect of degradation bacterium agent
Each organic matter is set with two different concentrations, and the fermentation liquid without organic matter assistant is used as the reference, and each concentration is set with three repetitions. The survival rate is shown in the following table.
As can be seen from Table 7, for the fermentation broth of the strain djl-6, the added sodium acetate and sodium citrate can significantly improve the survival rate of the strain djl-6 in the fermentation broth under two effective use concentrations of 1g/L and 2g/L, the survival rate can be improved by 19.34% after 2g/L of sodium acetate is added, and the survival rate is improved by 26.23% after 2g/L of sodium citrate is added.
TABLE 7 effective use concentrations of organic species and their effect on djl-6 survival enhancement rates
Figure BDA0002148665020000092
Research on influence of added stabilizer on preservation effect of degradation bacterium agent
Each stabilizer is set with two different concentrations, fermentation liquor without the stabilizer is used as a reference for research and screening, and each concentration is set with three repetitions. The survival rate is shown in the following table.
As can be seen from Table 8, for the fermentation broth of the strain djl-6, the survival rate of the strain djl-6 in the fermentation broth can be remarkably improved under two effective use concentrations of 1g/L and 2g/L by adding dextrin and carboxymethyl cellulose, the survival rate can be improved by 20.79% after 0.20% of dextrin is added, and the survival rate is improved by 23.68% after 0.20% of sodium carboxymethyl cellulose is added
TABLE 8 effective use concentrations of stabilizers and their effect on the survival enhancement rate of djl-6
Figure BDA0002148665020000093
Figure BDA0002148665020000101
Orthogonal screening for protective Agents
Screening of optimum substance combinations for built-up protectants
From the results of the one-factor experiment, a three-factor two-level orthogonal table L4 (2) was designed 3 ) To determine the optimal combination of substances, and to classify and design several substances with improving effect on the survival rate of djl-6 into three factors according to organic matter (A), stabilizer (B) and inorganic salt (C), wherein each factor takes two substances as two levels. The factors and levels, test design and results are shown in the table below.
From orthogonal experiments table 9, it can be seen that the primary and secondary sequences affecting the increased survival rate of strain djl-6 are: a. the>C>B, organic matter has the greatest effect on the survival rate of the strain djl-6, inorganic salt has the next least effect, and stabilizer has the least effect. The most preferred combination is A 1 B 2 C 2 I.e. sodium citrate, carboxymethylcellulose, CaCl 2 Compared to the other combinations of substances screened in this study, the effect on the survival rate of djl-6 was greatest. Therefore, sodium citrate, carboxymethyl cellulose, CaCl are selected 2 The compound is used as a compounding aid of djl-6 liquid for further research.
TABLE 9 orthogonal test table for combination of degrading bacteria djl-6
Figure BDA0002148665020000102
Screening of optimum use concentration of compound protective agent
According to the results of the compound protective agent combination orthogonal test, a three-factor three-level orthogonal table L9 (3) is designed 3 ) To determine djl-6 the combination of concentrations of adjuvant substances, the sodium citrate (A), carboxymethyl cellulose (B) and MgCl are screened 2 (C) Three factors were designed, and three concentrations were used for each factor as three levels (wherein the sodium benzoate concentration was added to the test at 5 g/L). The factors and level settings, test design and results are shown in table 10 below.
TABLE 10 Combined orthogonal test Table for concentration of degrading bacteria djl-6
Figure BDA0002148665020000103
Figure BDA0002148665020000111
From the table, the primary and secondary levels between the factors are: a. the>B>C, the optimal level is A 3 B 3 C 2 Therefore, the optimal use concentration of the compound protective agent of the degrading bacteria djl-6 screened and researched by the test is as follows: 3g/L sodium citrate +3g/L carboxymethylcellulose +2g/LCaCl 2 。
Example 13
Degradation experiment of preserved microbial inoculum on pesticide in inorganic salt culture medium
The optimum combination experiment 9 of the concentration orthogonal experiment of the strain djl-6, and the combination of experiment 4, experiment 7 and experiment 8 were verified. And (3) inoculating the bacterium solution after the combination preservation for 30 days into 20mL of inorganic salt liquid culture medium with the carbendazim concentration of 50mg/L according to the inoculation ratio of 3% by volume, measuring the concentration of the carbendazim in the bacterium solution after 3 days by using HPLC, and calculating the degradation rate. As shown in FIG. 2, the highest degradation rate of the composition is the orthogonal composition 8 (i.e., the formulation of example 3), which achieves a degradation rate of 93.65%, while the degradation rates of the remaining compositions are significantly improved compared with CK (without any protective agent), which achieves a degradation rate of only 67.74% under the same conditions.
Degradation experiment of preserved microbial inoculum on pesticides in soil
The optimum combination experiment 9 of the concentration orthogonal experiment of the strain djl-6, and the combination of experiment 4, experiment 7 and experiment 8 were verified. And taking the bacteria liquid after 45 days of combined preservation, taking 10ml of the bacteria liquid, inoculating the bacteria liquid into 100g of soil with the final concentration of the carbendazim of 5mg/kg dry soil, measuring the concentration of the carbendazim in the soil after 10 days by using HPLC, and calculating the degradation rate. The highest degradation rate of the composition is the orthogonal combination 9 (namely the formula of the example 4), the degradation rate of the orthogonal combination 9 reaches 95.23%, the degradation rate of the other combinations is obviously improved compared with that of CK (without any protective agent), and the degradation rate of CK (without any protective agent microbial inoculum) can only reach 48.82% under the same condition.
The liquid microbial inoculum is easily polluted in the storage process, so that the number of mixed strains is increased, and the number of target strains is reduced. The preservative is used as an additive substance in the microbial inoculum, and can effectively reduce the pollution of mixed bacteria. According to the research, three preservatives, namely sodium benzoate, calcium propionate and potassium sorbate, with bacteriostatic and mildewproof effects are added, the mixed bacteria rate of the strain djl-6 is calculated, bacteriostatic agents with good bacteriostatic effects and relatively high viable bacteria number are screened out, then the viable bacteria number and the mixed bacteria number of the strain are detected by adding the preservatives with different concentrations, and the adding concentration of the preservative in the liquid fermentation liquor is determined. The research shows that the degrading bacteria all use sodium benzoate as the bacteriostatic agent with the best effect. Can maintain a higher viable count and does not produce mixed bacteria pollution.
The liquid microbial inoculum has poor stability and rapid decrease of viable count in the preservation process. The addition of the protective agent is an important means for increasing the number of effective viable bacteria of the microbial product and prolonging the storage life of the microbial product. The addition of a protectant aids in the dispersion and survival of the microorganisms. The research shows that the microbial inoculum auxiliary agent screened by the orthogonal test can effectively improve the survival rate of bacteria, wherein the viable count in the fermentation liquor of djl-6 can be improved by 52.03%. The liquid microbial inoculum of the strain djl-6 which is added with the protective agent and stored for 30 days can degrade 93.65% of carbendazim with the concentration of 50mg/L at most in 3 days; the liquid microbial inoculum of the bacterial strain djl-6 which is added with the protective agent and stored for 45 days can degrade the carbendazim with the concentration of 5mg/kg dry soil to 95.23 percent in 10 days.

Claims (5)

1. The application of a protective agent for carbendazim degrading bacteria in protecting carbendazim degrading bacteria is characterized in that the protective agent for carbendazim degrading bacteria is composed of a stabilizing agent, a nutrient, a preservative and water, wherein the stabilizing agent is carboxymethyl cellulose, the nutrient is sodium citrate and calcium chloride, and the preservative is sodium benzoate; the addition concentration of the carboxymethyl cellulose in the protective agent is 10-30 g/L, the addition concentration of the sodium citrate in the protective agent is 10-30 g/L, the addition concentration of the calcium chloride in the protective agent is 10-30 g/L, and the addition concentration of the sodium benzoate in the protective agent is 50 g/L; the carbendazim degrading bacteria areRhodococcus qingshengii sp.nov.djl-6。
2. The use as claimed in claim 1, wherein the carbendazim-degrading bacteria protectant is prepared by mixing carboxymethylcellulose, sodium citrate, calcium chloride, sodium benzoate and water in proportion.
3. The use of claim 1, wherein the carbendazim-degrading bacterial agent is a liquid bacterial body.
4. The use of claim 1, wherein the carbendazim-degrading bacterial agent is protected to effectively maintain the performance of the bacterial agent, improve the survival rate of effective bacteria and prolong the preservation life of the bacterial agent.
5. The use as claimed in claim 1, wherein the carbendazim-protecting degrading microbial inoculum is a carbendazim pesticide which is used for degrading soil residues by using the carbendazim-protecting degrading microbial inoculum.
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