阅读说明：本技术 一种生物炭固定化菌剂去除低浓度阿特拉津的方法 (Method for removing low-concentration atrazine by using biochar immobilized microbial agent ) 是由 吉莉 张桂香 李渊 李娜 李雯雯 于 2021-09-17 设计创作，主要内容包括：本发明公开了一种生物炭固定化菌剂去除低浓度阿特拉津的方法,属于生物降解技术领域。本发明通过选取玉米芯,在250℃下缺氧裂解4h,并冷却至室温后再将其取出,研磨过筛后制得生物炭,取生物炭用去离子水制备生物炭母液,取处于对数生长期的菌株,制成OD-(600)＝1的菌悬液,备用；将OD-(600)＝1的菌悬液与生物炭母液混匀振荡制备得到生物炭固定化菌剂,将生物炭固定化菌剂,对土壤中低浓度阿特拉津去除。(The invention discloses a method for removing low-concentration atrazine by a biochar immobilized microbial agent, and belongs to the technical field of biodegradation. The method comprises the steps of selecting corncobs, carrying out anoxic pyrolysis for 4 hours at 250 ℃, cooling to room temperature, taking out the corncobs, grinding and sieving to obtain biochar, preparing biochar mother liquor from the biochar by using deionized water, and preparing OD (origin-destination) from strains in logarithmic growth phase 600 1, preparing bacterial suspension for later use; will OD 600 Uniformly mixing and oscillating the bacterial suspension and the biochar mother liquor to obtain the biochar immobilized microbial inoculum, and removing the low-concentration atrazine in the soil from the biochar immobilized microbial inoculum.)

1. A method for removing low-concentration atrazine by a biochar immobilized microbial agent is characterized by comprising the following steps: the method comprises the following steps:

step 1, taking corncobs for pretreatment, carrying out anoxic cracking under the corncobs, cooling to room temperature, taking out the corncobs, and grinding and sieving to obtain biochar for later use;

step 2, taking the biochar, and fixing the volume with deionized water to prepare biochar mother liquor for later use;

step 3, taking the strain in logarithmic growth phase, and carrying out ultrasonic treatment to prepare OD₆₀₀1, preparing bacterial suspension for later use;

step 4, OD₆₀₀Uniformly mixing and oscillating the bacterial suspension and the biochar mother liquor to obtain a biochar immobilized fungicide;

and 5, removing the low-concentration atrazine in the soil by using the biochar immobilized microbial inoculum.

2. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: the cracking temperature of the anoxic cracking in the step 1 is 250 ℃, and the cracking time is 4 h.

3. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: the corn cob pretreatment specifically comprises the steps of cleaning the corn cobs, drying at 80 ℃ and grinding.

4. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: the sieve mesh sieved in the step 1 is 0.15 mm; and the ultrasonic time in the step 3 is 2 h.

5. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: and 2, taking the biochar in the step 2, and fixing the volume by using deionized water, wherein the volume is specifically that every 500mg of the biochar is fixed to 500mL by using the deionized water.

6. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: the volume ratio of the bacterial suspension to the biochar mother liquor in the step 3 is 1:1, 2:1 or 3: 1.

7. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: the step 4 is carried out under the oscillation condition of 150r/min at 30 ℃ for 24 h.

8. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: the concentration of the low-concentration atrazine is 2 mg/L.

9. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: the inoculation amount conditions are that the pH is 7.63, the temperature is 33 ℃, and the inoculation amount is 10%.

10. The method for removing low-concentration atrazine by using the biochar immobilized microbial agent as claimed in claim 1, wherein the biochar immobilized microbial agent comprises the following steps: the biological degradation efficiency of the biochar immobilized microbial agent on low-concentration atrazine is 88.25%.

Technical Field

The invention belongs to the technical field of biodegradation, and particularly relates to a method for removing low-concentration atrazine by a biochar immobilized microbial agent.

Background

Atrazine is a common triazine benzene herbicide, has high weeding efficiency and low price, is used in a large area for a long time in the world, causes pollution to the environment such as soil, underground water, surface water and the like, and threatens human health. Biodegradation is a sustainable method for removing atrazine from water and soil. At present, researchers have found a plurality of degrading bacteria capable of removing atrazine, however, the biodegradation technology has some defects, such as slow growth of microorganisms caused by toxicity of pollutants, sensitive response of microorganisms to changes of environmental factors, limitation of growth of microorganisms caused by lack of nutrients in a culture medium, and influence on biodegradation efficiency.

Disclosure of Invention

Aiming at the problem of low removal rate of low-concentration atrazine in the prior art, the invention provides a method for removing atrazine by using a biochar immobilized microbial agent.

For atrazine with different initial concentrations, the removal difficulty is different, and the lower the concentration, the greater the removal difficulty.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for removing low-concentration atrazine by a biochar immobilized microbial agent comprises the following steps:

step 1, taking corncobs for pretreatment, carrying out anoxic cracking under the corncobs, cooling to room temperature, taking out the corncobs, and grinding and sieving to obtain biochar for later use;

step 2, taking the biochar, and fixing the volume with deionized water to prepare biochar mother liquor for later use;

step 3, taking the strain in logarithmic growth phase, and carrying out ultrasonic treatment to prepare OD₆₀₀1, preparing bacterial suspension for later use;

step 4, OD₆₀₀Uniformly mixing and oscillating the bacterial suspension and the biochar mother liquor to obtain a biochar immobilized fungicide;

and 5, removing the low-concentration atrazine in the soil by using the biochar immobilized microbial inoculum.

Further, the cracking temperature of the anoxic cracking in the step 1 is 250 ℃, and the cracking time is 4 h.

Further, the corn cob pretreatment is to clean the corn cobs, dry the corn cobs at 80 ℃ and grind the corn cobs.

Further, the sieve mesh sieved in the step 1 is 0.15mm, and the particle size of the biochar is 0.15 mm. The biochar provides a better habitat for the growth and the propagation of microorganisms and is also a better carrier.

Further, the biochar in the step 2 is subjected to volume fixing by deionized water, specifically, every 500mg of biochar is subjected to volume fixing by deionized water to 500 mL.

Further, the volume ratio of the bacterial suspension to the biochar mother liquor in the step 3 is 1:1, 2:1 or 3: 1. The influence of the ratio between the exploration bacteria and the biochar on the atrazine removal effect is also explored, and the later result also shows that the 2:1 effect is the best in three ratios. And the ultrasonic time in the step 3 is 2 h.

Further, the step 4 oscillation condition is 150r/min oscillation at 30 ℃ for 24 h. The bacteria are allowed to grow under appropriate conditions.

Further, the concentration of the low-concentration atrazine is 2 mg/L. The atrazine degraded in high concentration is much but far from the national standard of 0.003mg/L, so the application introduces low-concentration atrazine, and the optimal concentration is determined to be 2mg/L through experiments.

Further, the inoculation amount conditions were that the pH was 7.63, the temperature was 33 ℃, and the inoculation amount was 10%. The optimal condition for degrading atrazine is verified and obtained by a response surface method.

Further, the biodegradation efficiency of the biochar immobilized fungicide on low-concentration atrazine is 88.25%.

Compared with the prior art, the invention has the following advantages:

1. the corncobs are used for preparing biochar at different temperatures so as to prepare the immobilized microbial inoculum.

2. Effectively treats low-concentration atrazine, and the highest removal rate reaches 88.25 percent.

3. The influence of three ratios of the volume ratio of the bacterial suspension to the biochar mother liquor, namely 1:1, 2:1 and 3:1, on the growth of the bacteria and the atrazine degradation is researched, and the result shows that the strain grows the best and has the best effect on the atrazine degradation when the ratio is 2: 1.

4. The influence of three proportions of immobilized microbial agents prepared from 3 types of corncob biochar CC250, CC400 and CC600 prepared at different cracking temperatures on atrazine in the growth season of the strain is researched, and the result shows that (1) the influence effect of three different biochar on the growth of the strain is CC250> CC600> CC 400. (2) When the ratio of the two is 2:1, the biodegradation effect on atrazine is SCC250, SCC400, SCC600 and SCC250 removal effect are the best, and the removal rate can reach 88.25%.

Drawings

FIG. 1 is an SEM image of corncob biochar at different cracking temperatures;

FIG. 2 is an XRD spectrum of corncob biochar at different cracking temperatures;

FIG. 3 shows the free radical signal of the corncob biochar at different cracking temperatures;

FIG. 4 is an electron microscope image of a biochar immobilized microbial agent;

FIG. 5 shows the effect of different ratios of the same charcoal on the growth of the strain;

FIG. 6 shows the effect of different biochar on the growth of the strain at the same ratio;

FIG. 7 is a plot showing strain growth;

FIG. 8 is a graph showing the effect of various types of biochar-immobilized bacteria on atrazine removal.

Detailed Description

Example 1

Example 2

First, experimental instrument and equipment

TABLE 1.1 Experimental instruments

Experimental reagent: atrazine, methanol (chromatographically pure), sodium bicarbonate, calcium chloride, sodium lactate, Vc, L-cysteine hydrochloride, NaOH (analytically pure), HCl (analytically pure), dichloromethane (chromatographically pure), sodium chloride, anhydrous sodium sulfate

II, an experimental method:

2.1 preparation of biochar:

the biochar is prepared by taking corncobs as raw materials. Cleaning corncobs, drying and grinding the corncobs in an oven at 80 ℃, placing the corncobs powder in a crucible, carrying out anoxic cracking in muffle furnaces at 250 ℃, 400 ℃ and 600 ℃ for 4 hours, taking out and grinding the biochar after the biochar is cooled to room temperature in the muffle furnaces, and sieving the biochar by a 0.15mm sieve for later use. The biochar prepared was CC250, CC400 and CC600, respectively.

2.2 characterization of biochar:

and (3) observing the surface structure of the biochar: after 3 different biochar samples are sprayed with gold, the surface morphology and structure of the biochar are observed under a material scanning electron microscope.

And (3) analyzing the biochar elements: the elemental content (C, H, N) was determined by an elemental analyzer (Flash EA 1112) and the elemental O content was calculated by mass balance method with the mass of ash and free moisture subtracted.

Determination of biochar pH and EC: 3 biochar were mixed with water at a ratio of 1:10, respectively, and measured with a pH/EC meter.

Biochar N₂Determination of the BET Surface Area (SA): surface area was measured using an ASAP-2020 surface area Analyzer.

Determination of the biochar crystal structure (XRD): uniformly spreading 3 powdered biochar in a sample tank, compacting, and scanning and measuring on an X-ray diffractometer respectively, wherein the scanning range is 2 theta which is 10-80 degrees.

And (3) testing the biochar persistent free radicals: 3 biochar samples of about 2mg are respectively filled into a quartz capillary tube, and the quartz capillary tube is placed into a resonant cavity of an electron paramagnetic resonance spectrometer to detect an EPR signal.

2.3 preparation of the biochar immobilized microbial inoculum:

respectively weighing 500mg of CC250, CC400 and CC600 biochar, diluting to 500mL with deionized water, and performing ultrasonic treatment for 2h to prepare biochar mother liquor for later use. Collecting strain in logarithmic growth phase, and making into OD₆₀₀Bacterial suspension of 1. Then the OD is measured₆₀₀Adding the bacterial suspension and the biochar mother liquor into an erlenmeyer flask according to the proportion of 1:1, 2:1 and 3:1 respectively, oscillating for 24 hours at the temperature of 30 ℃ and at the speed of 150r/min to enable microorganisms to be adsorbed on the biochar, and adjusting the pH to be 7.63 for later use.

2.4 characterization of the biochar immobilized bacteria agent:

centrifuging the fixed charcoal immobilized microbial inoculum, removing supernatant, repeatedly washing with sterile normal saline for multiple times, removing paraffin and other liquid on the surface, after centrifugation, adding 2.5% glutaraldehyde solution, fixing at 4 deg.C for 24h to maintain the form of the strain on the charcoal structure, drying, and observing the form and loading condition of the strain on the charcoal under a scanning electron microscope.

The influence of the biochar immobilized fungicide on the growth of the strain:

adding the prepared different immobilized microbial agents into an enrichment medium containing atrazine in a proportion of 10 percent to ensure that the concentration of the atrazine in the final solution is 2mg/L and the pH value is 7.63, culturing the atrazine in the solution at the temperature of 33 ℃ under the condition of 150r/min, sampling at certain intervals to measure the bacterial load, and observing the influence of the biochar on the growth of the bacterial strain. Sampling is carried out after shaking up the solution before sampling, and three groups of parallels are arranged. Meanwhile, a blank control is set, wherein an equal amount of bacterial suspension is used as a control group, and the bacterial suspension is required to be adjusted according to the proportion by using sterile physiological saline.

The biochar immobilized fungicide has the following effect of removing atrazine:

adding the prepared immobilized microbial inoculum into an enrichment medium containing atrazine in a proportion of 10% to ensure that the concentration of atrazine in the solution is 2mg/L and the pH is 7.63, culturing the atrazine in the enrichment medium at 33 ℃ and 150r/min, sampling at 2d and 5d respectively, and determining the concentration of atrazine in the solution. Sampling is carried out after shaking up the solution before sampling, three groups of parallels are arranged, and simultaneously the requirements on a blank control group are consistent with the influence of the biochar immobilized fungicide on the growth of the strain.

Third, result and discussion

3.1 characterization of biochar

The basic physicochemical properties and elemental composition of the corncob biochar at different cracking temperatures are shown in table 1.2. As can be seen from table 1.2, the pH values of the corncob biochar are all greater than 7, and are respectively 9.36, 10.19, and 10.00, which increase with the increase of the pyrolysis temperature, because the mineral elements originally combined with the organic matters in the corncob biochar are transformed into carbonate in the cracking process, and these carbonate and other substances are alkaline after being dissolved in water. Similarly, the EC of the corncob biochar increases with the increase of the pyrolysis temperature, and is respectively 1.43, 4.05 and 6.64, which is also related to the accumulation of alkali liquor at high temperature. The ash content of the corncob biochar is relatively stable along with the increase of the pyrolysis temperature, and the ash content of the corncob biochar is almost consistent when the pyrolysis temperature is 400 ℃ and 600 ℃, and is 1.80 and 1.81 respectively. The C content is increased along with the increase of the pyrolysis temperature and respectively reaches 75.76%, 85.65% and 89.88%, however, the H content, the N content and the O content are decreased along with the increase of the pyrolysis temperature and respectively reach from 4.37%, 0.76%, 19.58% to 1.69%, 0.70% and 8.14%, wherein the H content is related to the organic matter component of the plant. The atomic ratios in the corncob biochar prepared at different cracking temperatures have different significances, H/C represents aromaticity, the larger the value is, the lower the aromaticity is, the H/C atomic ratio of CC600 is reduced to 0.23 along with the increase of the cracking temperature, which shows that the original organic components are reduced, and the carbonization degree is enhanced. (O + N)/C represents polarity and hydrophobicity, the smaller the value represents the enhancement of hydrophobicity, the polar group is reduced, and the (O + N)/C value of CC600 is reduced from 0.20 of CC250 to 0.07 with the increase of the cleavage temperature, indicating that the increase of the cleavage temperature leads to the enhancement of hydrophobicity of the biochar and the reduction of the polar group. This is consistent with the results of some other studies on biomass to produce biochar.

TABLE 1.2 basic physicochemical properties and elemental composition of corncob biochar

To further understand the structure and surface morphology characteristics of the corncob biochar, the scanning electron microscope and specific surface area analysis of the materials were performed on 3 different biochar, and the results are shown in fig. 1 and table 1.2. As can be seen from Table 1.2, the specific surface area tends to increase with the increase of the cracking temperature, from 1.71m of CC250²Increase of/g to 295.95m²In the case of the higher pyrolysis temperatures, owing to the aromatic CIncreasing the content of functional groups linking aromatic groups (e.g. -OH, ester C ═ O, aliphatic CH)₂C — O, aromatic C ═ O, and phenol-OH) are destroyed and removed, some fine microporous structures are formed, resulting in an increase in the SA value. FIG. 1 is SEM images of biochar at different cracking temperatures, which better shows the characteristics of the surface pore structure of a sample, wherein (a), (b) and (c) are SEM images of CC250, CC400 and CC600 respectively. As can be seen from FIG. 1, all of the 3 different biochar samples have rich pore structures, and with the increase of the cracking temperature, the pore structures of the biochar are more developed, the surfaces of the CC250 and CC400 biochar samples are rough, the surface of the CC600 biochar sample is smooth and has rich reticular pore structures, and meanwhile, the phenomenon that the micropores are blocked by the debris in the pore structures of the CC250 and CC400 can be seen. The structure of 3 kinds of biological charcoal mainly is netted distribution, arranges evenly, along with the increase of scanning electron microscope multiple, can be clear see the aperture on the biological charcoal lateral wall, and arrange closely between the aperture.

X-ray diffraction analysis (XRD) enables analysis of the internal crystal structure and composition of different biochar. FIG. 2 is XRD patterns of biochar at different cracking temperatures, wherein (a), (b) and (c) respectively represent XRD patterns of CC250, CC400 and CC 600. According to the standard card in Jade software, the main substances existing in 3 different biochar samples are Si and Al₂O₃、SiO₂、Fe₂O₃、CaCO₃And the graphitization degree is enhanced along with the rise of the pyrolysis temperature, the enrichment capacity of inorganic elements is improved, and stable mineral crystals are easier to form. The main characteristic peaks of the CC250 are 28.43 and 40.59 degrees at 2 theta, the main characteristic peaks of the CC400 are 26.82, 28.61 and 40.70 degrees at 2 theta, and the main characteristic peaks of the CC600 are 24.34, 26.65, 28.49, 30.16, 31.43 and 40.71 degrees at 2 theta. It has been shown that the biochar contains Fe₂O₃Part of the crystalline phase is beneficial to the growth of microorganisms, meanwhile, oxide and alkaline salt ions existing in the biochar are not beneficial to the adsorption of atrazine, and the structure of cellulose is damaged due to the increase of cracking temperature.

Research shows that the biochar can generate free radicals in the carbonization process, the generation of the free radicals can stimulate cells of the strain to generate ROS, the oxidation resistance of the cells is reduced, cell death is caused, and the biochar has obvious cytotoxicity. Liao et al found that significant Electron Paramagnetic Resonance (EPR) signals were found in corn stover, rice and wheat straw, and in the main biopolymer components of biomass (cellulose and lignin), indicating that free radicals were generated in the carbonization process of biochar. The PFRs of biochar generally exist in various forms and can recognize the type of free radical according to the g factor of the EPR signal. g >2.0040 is a typical feature of oxygen-centered free radicals, such as semi-rolling free radical anions; g is 2.0030-2.0040, which is a typical characteristic of the combination of oxygen-centered free radicals and carbon-centered free radicals; and when g <2.0030 is a carbon-centered radical, such as an aromatic radical. As shown in fig. 3, which is a free radical signal diagram of the corncob biochar at different cracking temperatures, g factors of BC250, BC400 and BC600 are 2.00249, 2.00254 and 2.00256, respectively, and g values are all less than 2.0030, which indicates that the free radicals contained in the 3 biochar samples are free radicals with carbon as a center, and meanwhile, when the cracking temperature is increased from 250 ℃ to 400 ℃, the EPRs intensity in the biochar samples is also enhanced, and when the cracking temperature is increased from 400 ℃ to 600 ℃, the signal intensity of the EPRs is weakened along with the increase of the cracking temperature, the signal intensity is strongest in the biochar samples prepared by pyrolysis at 400 ℃, and the signal intensity is weakest in the biochar samples prepared by pyrolysis at 600 ℃, which is consistent with the research results of zhangong excess, cuo shu, etc.

3.2 characterization of biochar-immobilized Agents

FIGS. 4(a), (b), (c) and (d) are surface topography observations of biochar-immobilized bacteria agents. As can be seen from FIG. 4(a), the biochar maintains the original natural form during the pyrolysis process, and channels and pores with different sizes are distributed on the surface of the biochar, so that an ideal living site can be provided for microorganisms. Meanwhile, it can be seen that the rod-shaped thalli are loaded on the surface of the biochar and are mainly distributed on the flat part of the biochar surface, because the acting force between the biochar and the microorganisms is weaker, the flat part of the surface is more suitable for the growth of the microorganisms, and the fixing of the microorganisms is facilitated. As can be seen from FIGS. 4(b) and (c), the bacterial strains are present on the charcoal surface in different colony forms, and are partially aggregated as shown in FIGS. 4(b) and 4(d), because the bacterial strains are biofilm-formed on the charcoal or the bacterial cells are aggregated by physical adsorption. The other part is dispersed as shown in fig. 4(a) and 4(c), which is caused by electrostatic attraction between carriers.

3.3 Effect of charcoal-immobilized microbial Agents on growth of the strains

To investigate the effect of different ratios of bacteria liquid/biochar on the growth of the strain, the ratios of 3 different bacteria liquid/biochar were adjusted to 1:1, 2:1 and 3:1, and simultaneously different ratios of bacteria liquid in physiological saline were added to the medium as a blank control, as shown in fig. 5, the effect of different ratios of the same biochar on the growth of the strain.

Studies have shown that different initial bacterial loads can play a critical role in the growth of microorganisms, and that a suitable bacterial load can shorten the time required for the adaptation phase of the microorganism. As can be seen from FIG. 5(a), the growth of the 1:1 bacterial suspension blank is slower, while the growth of 2:1 and 3:1 is the same. The reason is that when the bacterial amount is low, atrazine inhibits the growth of the bacterial strain, so that the bacterial strain is difficult to propagate quickly, the proper increase of the bacterial amount promotes the synergistic effect in the bacterial strain to help the bacterial strain to adapt to the environment better and quickly, and when the bacterial amount reaches a certain value, the excessive increase of the bacterial amount causes the biological capacity in the culture medium to reach the maximum value, the bacterial body competes for utilizing nutrient substances in the culture medium, the growth is limited, and the growth amount of the bacterial strain does not change obviously along with the increase of the bacterial amount. The appropriate amount of bacteria required for different strains varies, depending on the type, nature and type and characteristics of the organism, with more or less utilizing strains requiring a relatively lower amount of bacteria and less utilizing strains requiring a relatively higher amount of bacteria.

As shown in FIGS. 5(b), (c) and (d), the strain was able to rapidly adapt to the environment when the ratio of the bacterial suspension/CC was 2 and 3 for the same biochar, thereby shortening the time required for the adaptation period of the microorganism. However, the bacteria liquid/CC ratio is better than 2, because the adsorption of the charcoal to the bacteria cells gradually reaches a saturation state with the increase of the bacteria liquid concentration, when the bacteria liquid content is higher, the transfer of the charcoal immobilized microorganism material to the substrate is inhibited due to the limited internal space of the charcoal carrier, or the microorganisms have a competitive action due to the narrow space and the lack of nutrient substances, and the bacteria cells immobilized on the charcoal surface cannot obtain sufficient vegetative growth and propagation, thereby reducing the activity. It has also been shown that under the same culture conditions, the more inoculum will result in excessive metabolite production by the immobilized microorganism, and such toxic metabolite will reduce the survival rate of the microorganism and inhibit the growth of the strain. However, in the early stage of the growth of the strain, when the ratio of the bacteria liquid/CC is 3, the growth of the strain is larger than that of the strain with the ratio of 2, mainly because the bacterial amount is relatively large and the growth is relatively fast in the initial stage, but along with the consumption of nutrient substances in the culture medium, the strains have competition effect and also generate excessive metabolites, and the growth of the strain is limited, so that the growth of the strain is better when the ratio of the bacteria liquid/CC is 3 in the early stage.

The addition of biochar has been shown to affect the growth of the strain, Yang et al have studied the effect of different biochar concentrations on the growth of pseudomonas, and the results show that the concentration of pseudomonas cultured with biochar is also significantly increased with the increase of the biochar concentration of BC350-BC700, and is increased with the increase of the biochar concentration, but when the concentration is increased to 500mg/L, the high concentration of biochar prolongs the exponential growth period of bacteria, and the growth of bacteria is inhibited. The results of this study are consistent with the results of this study, and when the concentration of biochar is increased to a certain extent, the excessively high biochar prolongs the exponential growth of the strain and growth is inhibited. However, many studies have found that different types of biochar and immobilized bacteria prepared from strains have different properties and different effects on the growth of the strains, and the effects are related to the types of strains, the types of biochar and the interaction between biochar and pollutants.

The effect of different biochar at the same ratio on strain growth is shown in figure 6. As can be seen from FIG. 6(a), when the ratio of the bacteria liquid to the biochar is 1:1, after 10% of biochar immobilized bacteria agent is added into the culture medium, the growth of the strain is inhibited to different degrees, wherein the degree of inhibition of CC600 is greater, because CC600 has a larger specific surface area and a stronger adsorption capacity to atrazine, the carbon source available for the growth of the strain is insufficient, and the growth of the strain is inhibited. As can be seen from FIGS. 6(b) and (c), when the ratio of bacteria liquid/biochar is 2:1 and 3:1, after 10% biochar immobilized bacteria agent is added into the culture medium, the growth of the strain is promoted to different degrees, because the porous structure of biochar provides a better growth environment for the microorganism, the direct contact between the strain and atrazine is reduced, and the toxic action of atrazine on the strain is relieved.

Research also shows that biochar can be used as exogenous substance input to influence the growth of bacteria, thereby influencing biodegradation. The biological carbon can also release TOC into the solution to directly influence the growth and the reproduction of bacteria, extracellular polymerase of the bacteria is utilized to decompose structurally complex TOC, the microbial concentration is also obviously increased along with the increase of the TOC concentration, meanwhile, the biological carbon also has certain influence on the fatty acid composition of the bacteria, and the higher the TOC content is, the more beneficial the synthesis of the fatty acid is, and the more beneficial the growth of the bacteria is. The microbial cell surface has rich functional groups, and the surface functional groups can form covalent bonds with the biochar surface functional groups, so that bacteria can be immobilized, and the growth of strains is promoted.

A comparison of the effect of different ratios of charcoal on strain growth is shown in FIG. 7. Wherein 7(a) shows the effect of different ratios on the growth of the strain in 3 different biochar, it can be seen from the figure that when the ratio of bacteria liquid/biochar is 1:1, the growth of bacteria in CC250, CC400 and CC600 is obviously lower than 2:1 and 3: 1.7 (b) shows that under the condition that the ratio of bacteria liquid to biochar is 1:1, different biochar influences the growth of the strains, the growth of the strains is inhibited, and the CC600 has the largest inhibiting effect on the strains, and the result is consistent with the research result of the figure 6.

The effect of three different biochar on strain growth was CC250> CC600> CC 400. Research shows that nitrogen (N) is an important material basis for the growth of microorganisms, the C/N value of the biochar plays an important role in the growth and development of the microorganisms, and particularly in soil, when the C/N ratio of organic matters is lower than 25:1, the activity of the microorganisms is stronger, the decomposition of the organic matters is accelerated, and conversely, the activity of the microorganisms is reduced, the decomposition of the organic matters is slowed down, so that the closer the C/N ratio is to 25:1, the better the growth of the microorganisms is. The C/N ratios of the 3 corncob biochar prepared at different cracking temperatures, namely CC250, CC400 and CC600, are respectively 99.68, 115.74 and 128.4, which shows that the C/N ratio of the CC250 is closest to 25:1 and is most suitable for the growth of microorganisms.

From the analysis of physicochemical properties of the biochar, it can be seen that with the increase of the cracking temperature, the H/C of CC250, CC400 and CC600 is respectively reduced from 0.69 to 0.42 and 0.23, which indicates that the higher the aromaticity of the biochar, the less the original organic components, the higher the carbonization degree, the lower the polymerization degree of the carbon, the conversion of aliphatic carbon and aromatic carbon into graphene and other substances, the fixation of N, P and other elements in the organic mechanism of the biochar, the lower the nutrition effect and the lower the availability of microorganisms. The (O + N)/C is respectively reduced from 0.20 to 0.10 and 0.07, which shows that the polar group of the biochar is reduced, the hydrophobicity is enhanced, the adhesion growth effect on microbial cells is reduced, and the growth of microorganisms is not facilitated.

The biochar has volatile organic compounds and persistent free radicals, and the biochar prepared at the medium temperature (400 ℃) has higher polycyclic aromatic hydrocarbons, dioxin and the like than the biochar prepared at the high temperature (400 ℃), and the stable persistent free radicals (comprising semiquinones, phenoxy compounds and phenolic substances) can generate toxicity to microorganisms and induce the microorganisms to generate oxidative stress reaction. As can be seen from EPR (ethylene-propylene rubber) images of corncob biochar at different pyrolysis temperatures, the strength of a free radical signal of CC400 is strongest, and the toxicity to a strain is higher than that of CC600, so that the growth promotion effect of an immobilized microbial inoculum prepared from CC600 on the strain is higher than that of the immobilized microbial inoculum prepared from CC400 on the growth of the strain.

Masiello et al found that biochar can alter microbial intercellular communication by adsorbing signal molecules that many gram-negative bacteria use to regulate gene expression and intraspecies communication. The biochar prepared under the high temperature condition (700 ℃) has higher specific surface area than the biochar prepared under the low temperature condition (300 ℃), adsorbs signal molecules (AHL) and blocks the transmission of signals among cells, thereby influencing the growth of microorganisms. Compared with CC250, CC600 has larger specific surface area and stronger signal molecule adsorption capacity, and can block intercellular signal transmission to a great extent and influence the growth of microorganisms, so that the CC250 has stronger capacity of promoting the growth of strains, and the influence effect of 3 different biochar on the growth of the strains is CC250> CC600> CC 400.

3.4 Effect of biochar-immobilized fungicide on atrazine removal

In order to analyze the atrazine removing capacity of 9 different immobilized bacteria agents, 3 biochar with different proportions and different types and bacteria liquid with different proportions, atrazine with a certain concentration is added into a culture medium, samples are taken after 2d and 5d to determine the atrazine concentration in the solution, and the determined atrazine removing rate is shown in figure 8. Wherein 8(a), (b) and (c) respectively represent the removal effect of the bacteria liquid/biochar immobilized bacteria agents with different types of biochar with the ratios of 1:1, 2:1 and 3:1 on atrazine, JN01 represents the removal effect of bacteria with the same ratio on atrazine, and CC250, CC400 and CC600 respectively represent the removal effect of biochar with the same type and the same ratio on atrazine.

The removal efficiency of the biochar immobilized microbial agents of different types on the atrazine is higher than that of a single bacterial strain and a single biochar on the atrazine. As can be seen from fig. 8(a) (b) (c), the removal rates of atrazine by the single strains 1:1, 2:1, and 3:1 on day 2 were 50.67%, 61.56%, and 64.41%, respectively, and the removal rates of atrazine on day 5 were 55.58%, 69.49%, and 70.52%, respectively, indicating that the removal rate of atrazine gradually increased with the increase of the bacterial load, and the removal rate of atrazine also increased and gradually leveled off with the increase of degradation time. When the ratio of the bacteria liquid is increased from 2:1 to 3:1, the removal rate of the atrazine is gradually stable, and the degradation rate is not obviously improved, because excessive thalli compete for limited atrazine substrate, so that the nutrition of cell thalli is limited, and the removal rate of the atrazine is influenced. Zhao Xinyue et al studied the effect of different inoculum sizes on atrazine removal and the results showed that the atrazine removal showed a significant upward trend when the inoculum size was increased from 1% to 3% and the atrazine removal rate remained essentially unchanged when the inoculum size was increased to 5%. Therefore, when the bacterial quantity reaches a certain degree, the removal rate of the atrazine cannot be improved by increasing the bacterial quantity.

Researches show that different raw materials and process conditions such as pyrolysis temperature, heat transfer rate, retention time and the like in the preparation process influence the performance of the biochar. Therefore, the adsorption effect of the biochar (CC250, CC400 and CC600) prepared at different cracking temperatures on atrazine is studied, and as can be seen from fig. 8(a) (b) (c), the adsorption capacity on atrazine can be improved by increasing the proportion of the biochar mother liquor, wherein the CC250 is increased to 33.22% from 27.64%, the CC400 is increased to 33.61% from 29.91%, and the CC600 is increased to 41.64% from 39.77%. Therefore, the adsorption effect of 3 different biochar on atrazine is CC600> CC400> CC250, which shows that CC250 has poor adsorption effect on atrazine and CC600 has good adsorption effect on atrazine. The biochar prepared from the same raw material has the advantages that the higher the cracking temperature is, the more the microporous structures are, the specific surface area is large, the larger specific surface area and the porous structure of the CC600 can adsorb atrazine through the surface adsorption effect, the pore filling effect and the like, and the relatively strong adsorption capacity is shown.

Wang et al studied the adsorption performance of the biochar produced by peanut shell pyrolysis at 300 ℃, 450 ℃ and 600 ℃ on atrazine, and the results showed that the high temperature biochar (BC600) has higher adsorption capacity when the atrazine concentration is lower, and the contribution of the adsorption capacity to the total adsorption capacity increases with the increase of the pyrolysis temperature, and the high temperature biochar of peanut shell can effectively fix atrazine and prevent the atrazine from migrating to the surface of the earth or underground water. Zheng et al studied the adsorption performance of the biochar prepared from the waste biomass under the condition of 450 ℃ oxygen limitation on atrazine, and found that the adsorption capacity on atrazine is 31% when the particle size of the biochar is 0.24mm, and 44% when the particle size of the biochar is 0.125mm, indicating that the particle size of the biochar has an influence on the adsorption of atrazine. The particle size of the biochar used in the experiment is 0.15mm, and the maximum adsorption capacity of the biochar is 41.64%, which is consistent with the research result.

In the biodegradation process, pollutants can be adsorbed on the surface and the pore structure of the biochar with different concentrations. As can be seen from fig. 8(a), (b) and (c), the biodegradation efficiency of the strain and the biochar immobilized fungicide on atrazine is much higher than the adsorption efficiency of biochar on atrazine, indicating that the atrazine removal effect is mainly biodegradation rather than adsorption. As can be seen from fig. 8(a) and fig. 6(a), when the ratio of the bacterial suspension to the biochar mother liquor is 1:1, the growth of the strain is inhibited to different degrees, but the biodegradation efficiency of the biochar immobilized microbial inoculum to atrazine is still greater than that of the strain to atrazine, which indicates that when the biochar concentration is higher, the adsorption effect of the surface and pore structures of the biochar in the immobilized microbial inoculum to atrazine can influence the carbon source for the growth of the strain, and the toxic effect on the strain is reduced, at this time, the adsorption effect of the biochar can adsorb a large amount of atrazine, so when the ratio of the two is 1:1, the biodegradation efficiency of the immobilized microbial inoculum to atrazine is CC600> CC400> CC250, and the biodegradation efficiency of the 5d to atrazine is 69.76%, 67.41%, and 61.88%, respectively.

As can be seen from fig. 8(b) (c) and 6(b) (c), when the ratio of the two is 2:1 and 3:1, the same regular characteristics are obtained, and the growth promoting effect on the strain is more obvious as the concentration of the biochar is increased, and the biodegradation efficiency of the immobilized microbial agent on atrazine is also more obvious. As can be seen from fig. 8(c), when the ratio of the two is 2:1, the removal efficiency of the biochar-immobilized bacteria agent on atrazine is the best, and the biodegradation effect on atrazine is SCC250> SCC400> SCC 600. Therefore, the biological degradation efficiency of the SCC250 immobilized fungicide on atrazine is the highest, 88.25%, which is obviously higher than that of a single strain (69.49%) and biochar (28.01%).

The immobilized bacteria agent has good removal efficiency, and has two reasons, on one hand, the biochar improves the survival rate of the bacterial strain and can exert stable degradation performance; on the other hand, biochar has nutrients that promote the growth of the strain. Fu et al studied the removal effect of the immobilized microbial inoculum prepared from cinnamon hull, peanut hull biochar and pseudomonas YT-11 on diesel, and the results showed that when 7.5g/L diesel is used as the sole carbon source, the removal rate of 7d on the immobilized microbial inoculum is 69.94% and 64.41%, which is much greater than the removal rate of single bacterium on diesel, and found that the main degradation pathways of the immobilized microbial inoculum include surface adsorption, internal absorption and biodegradation, the surface adsorption is mainly used in the early stage of degradation, and the biodegradation is mainly used in the later stage. Therefore, the strain is fixed by taking the biochar as a carrier, so that the removal effect of a single strain on the atrazine can be effectively improved.

3.5 summary of the above

(1) The basic physicochemical properties of the corncob biochar prepared under the conditions of different cracking temperatures are measured, and the results show that the pH, EC and ash content of 3 different corncob biochar are increased along with the increase of the cracking temperature, the C content is in an increasing trend, the H content, N content and O content are in a decreasing trend, the aromaticity and carbonization degree of the biochar are enhanced, the hydrophobicity is enhanced, the polar groups are reduced, the specific surface area is increased, and the free radical signals are first enhanced and then weakened.

(2) The SEM can observe that rod-like bacteria are supported on the surface of the charcoal, and are mainly distributed on the flat portion of the charcoal surface, and the bacterial strain exists in different colony forms (aggregation-like, dispersion-like) on the charcoal surface.

(3) The growth of the strain can be promoted and the removal effect of the strain on atrazine can be enhanced by properly increasing the concentration of the biochar, and when the concentration of the biochar is too high (1:1), the growth of the strain can be inhibited and the growth period of the strain can be prolonged. When the concentration of atrazine is 2mg/L, under the conditions of pH 7.63, temperature 33 ℃ and 10% of inoculum size, the ratio of the bacterial suspension to the biochar mother liquor is 2:1, the biodegradation efficiency of SCC250 on atrazine is highest and is 88.25%, and the efficiency is obviously higher than that of a single bacterial strain (69.49%) and biochar (28.01%).

Those skilled in the art will appreciate that the invention may be practiced without these specific details. Although illustrative embodiments of the present invention have been described above to facilitate the understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the embodiments, and various changes may be made apparent to those skilled in the art as long as they are within the spirit and scope of the present invention as defined and defined by the appended claims, and all matters of the invention which utilize the inventive concepts are protected.