Chitosan modified charcoal immobilized complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of immobilized microorganisms and microbial environment restoration, and particularly relates to a chitosan modified charcoal immobilized complex microbial inoculant as well as a preparation method and application thereof. The chitosan modified biochar immobilized complex microbial inoculant provided by the invention comprises complex bacteria, biochar and chitosan, the complex bacteria are fixedly attached to the surface of the biochar and the interiors of pores, and the chitosan forms a layer of uniform membrane structure on the surface of the biochar. The chitosan modified biochar immobilized complex microbial inoculant not only improves the viable count and stability of immobilized microorganisms, but also can significantly improve the degradation rate of pollutants, has good acid-base tolerance and salt tolerance, can be suitable for pollutant degradation and environment restoration or improvement in various acid-base environments and salt environments, and has wide application prospects. The method has good environmental adaptability and good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical fields of immobilized microorganism technology and microbial environmental restoration technology, and specifically relates to a chitosan-modified biochar immobilized composite bacterial agent and a preparation method and application thereof. Background Art

[0002] At present, the application of immobilized microbial technology in the field of contaminated soil remediation, especially the research on the remediation of oil-contaminated soil, has attracted widespread attention from environmental protection workers. The core of microbial remediation technology for oil-contaminated soil lies in the degradation of pollutants by microorganisms, and how to efficiently exert this degradation effect is the key to successfully achieving bioremediation of oil-contaminated soil. Directly adding free microorganisms to contaminated soil often makes it difficult for microorganisms to adapt to the complex soil environment, and also forms a competitive relationship with indigenous microorganisms, making it difficult to ensure the activity of microorganisms and greatly reducing their degradation ability. Immobilized microbial technology fixes free microorganisms on specific carriers, making them highly dense and active, and able to proliferate rapidly and in large quantities under suitable conditions.

[0003] Biochar has become one of the commonly used carriers for immobilized microorganism technology due to its advantages such as large specific surface area, well-developed pores, and high organic matter content. However, when biochar is directly used to immobilize microorganisms, there are problems such as low microbial biological activity, low loading capacity, and weak microbial attachment, which seriously limit the application effect and stability of biochar in immobilized microbial carriers. Therefore, the modification of biochar has become a research hotspot in this field.

[0004] Chitosan is a natural polymer that is non-toxic and non-polluting. It has good biocompatibility, biodegradability and biological activity. It is widely used in bioengineering, medicine, chemical industry, food, environmental protection and other fields. Its environmental protection and eco-friendliness also make its application research have broad prospects and significance. Chitosan is used to modify biochar to solve the defects of activated carbon carriers in order to obtain microbial carriers with better performance. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention proposes a chitosan-modified biochar immobilized composite bacterial agent, which not only increases the viable count and stability of immobilized microorganisms, but also significantly improves the degradation rate of pollutants. At the same time, it has good acid-base tolerance and salt tolerance, and can be used for pollutant degradation and environmental restoration or improvement in various acid-base environments and salt environments, and has good environmental adaptability.

[0006] The technical solution of the present invention is a method for preparing a chitosan-modified biochar immobilized composite bacterial agent, the steps comprising:

[0007] (1) mixing the composite bacterial solution, the protective agent and the biochar, and storing the mixture at 2-8° C. to prepare a biochar-immobilized composite bacterial agent;

[0008] (2) Add the biochar-immobilized composite bacterial agent to the chitosan acetic acid solution and mix well, then add alkali to adjust the pH to 7, and freeze-dry to obtain the chitosan-modified biochar-immobilized composite bacterial agent.

[0009] In step (1), before the composite bacterial solution is mixed with the protective agent and the biochar, it is first centrifuged at a centrifugal speed of 4000-5000 r / min, preferably 4000 r / min.

[0010] In step (1), the composite bacterial solution is OD 600 =TB1 bacterial solution with OD of 1.0-1.2 600 =1.0-1.2 TX3 bacterial solution is mixed in a volume ratio of 0.5-2:1. Preferably, the volume ratio of TB1 bacterial solution to TX3 bacterial solution is 2:1, and the OD of TB1 bacterial solution to TX3 bacterial solution is 600 =1.0.

[0011] The preparation of the TB1 bacterial solution includes: inoculating TB1 into a culture medium, culturing at 37°C and 150 rpm, centrifuging, and resuspending with sterile saline to an OD of 600 = 1.0. The constant temperature shaking culture time is 10-14 hours to ensure that the strain is in the best active state. Before resuspending with sterile saline, wash it with sterile saline 2-3 times.

[0012] The preparation of the TX3 bacterial solution includes: inoculating TX3 into a culture medium, culturing at 37°C and 150 rpm, centrifuging, and resuspending with sterile saline to an OD of 600 =1.0. The constant temperature shaking culture time is 10-14 hours, preferably 12 hours, to ensure that the strain is in the best active state. Before resuspending with sterile saline, wash with sterile saline 2-3 times.

[0013] The culture medium comprises 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, and 1 L of distilled water.

[0014] In step (1), the dosage ratio of biochar to composite bacterial solution is 0.5-3 g:500 mL, preferably 1 g:500 mL; the volume ratio of protective agent to composite bacterial solution is 0.5-2:150, preferably 1:150; the protective agent is glycerol, preferably 50% (v / v) glycerol.

[0015] In step (1), the particle size of the biochar is 0.15-0.425 mm, and the preparation of the biochar includes: firing the biomass raw material at a carbonization temperature of 500°C-600°C. The fired product is then ground and passed through a 40-mesh-100-mesh nylon sieve to prepare biochar with a particle size of 0.15-0.425 mm. This particle size range of biochar helps microorganisms to attach better, while retaining the excellent properties of biochar, providing an ideal carrier for subsequent immobilization.

[0016] In step (1), the culture medium is stored at 2-8°C overnight, preferably at 4°C overnight, to ensure the stability of the bacterial solution and the activity of the strain.

[0017] By precisely controlling the above-mentioned culture conditions in step (1) and using protective agents, the activity and stability of the composite bacterial community are greatly improved.

[0018] In step (2), the mass ratio of the biochar-immobilized composite bacterial agent to chitosan is 0.5-1.5:1, preferably 0.75:1; the concentration of chitosan in the chitosan acetic acid solution is 0.03-0.05 g / mL, preferably 0.04 g / mL; the chitosan has a deacetylation degree of >90% and a viscosity of <100 mPa·s, and the acetic acid concentration is 5% (v / v).

[0019] The preparation of the chitosan acetic acid solution comprises: slowly dissolving chitosan in an acetic acid solution. The concentration of chitosan in the chitosan acetic acid solution is 0.03-0.05 g / mL, preferably 0.04 g / mL; the chitosan is chitosan with a deacetylation degree greater than 90% and a viscosity less than 100 mPa·s, and the concentration of the acetic acid solution is 5% (v / v).

[0020] In step (2), the base is an inorganic base and / or an organic base, and the inorganic base includes but is not limited to carbonates, bicarbonates, hydroxides, phosphates, hydrogen phosphates, etc., such as sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, sodium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, etc., preferably sodium hydroxide or potassium hydroxide, more preferably 10% (w / v, g / mL) sodium hydroxide solution or potassium hydroxide solution. The organic base includes but is not limited to ether sodium, ether potassium, sodium ethoxide, potassium ethoxide, sodium acetate, potassium acetate, sodium acetamide, etc.

[0021] In step (2), alkali is slowly added to adjust the pH to 7, thereby inducing chitosan to form a film on the surface of the biochar.

[0022] The present invention also provides a chitosan-modified biochar immobilized composite bacterial agent prepared by the above-mentioned preparation method of the chitosan-modified biochar immobilized composite bacterial agent, which comprises composite bacteria, biochar and chitosan. The composite bacteria are fixedly attached to the surface and pores of the biochar, and the chitosan forms a uniform film structure on the surface of the biochar.

[0023] The composite bacteria include TB1 and TX3, and the OD 600 =TB1 bacterial solution with OD of 1.0-1.2 600 =1.0-1.2 TX3 bacterial solution is mixed in a volume ratio of 0.5-2:1. Preferably, the volume ratio of TB1 bacterial solution to TX3 bacterial solution is 2:1, and the OD of TB1 bacterial solution to TX3 bacterial solution is 600 =1.0. Preferably, the TB1 bacterial solution and the TX3 bacterial solution are mixed and then centrifuged at a speed of 4000-5000 r / min, preferably 4000 r / min.

[0024] The chitosan-modified biochar-immobilized composite bacterial agent prepared by the preparation method of the chitosan-modified biochar-immobilized composite bacterial agent of the present invention has a fixed bacterial amount of more than 10 11 CFU / g, after 70 days of storage, the solid bacteria count is still>10 10 CFU / g, significantly improving the number of live bacteria and stability of immobilized microorganisms; the degradation rate of benzo[a]pyrene is as high as 67.01%, and the degradation rate of diesel is as high as 73.67%, significantly improving the degradation rate of pollutants with combined pollution of polycyclic aromatic hydrocarbons and petroleum hydrocarbons; it has degradation activity in the range of pH 4-10 and salt concentration of 4-40g / L, and maintains better degradation activity in the neutral to weakly alkaline environment of pH 6-9 and salt concentration range of 8-25g / L, showing good environmental adaptability.

[0025] The chitosan-modified biochar-immobilized composite bacterial agent provided by the present invention is used to prepare products for use in polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons. The product has degradation activity on polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, and has repair activity and / or improvement activity on the pollution caused by polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons. The product can be applied to environmental systems such as soil and water bodies.

[0026] The polycyclic aromatic hydrocarbons refer to aromatic compounds containing two or more benzene rings, which are divided into non-condensed ring type and condensed ring type, such as biphenyl, bipolyphenyl, naphthalene, anthracene, phenanthrene, benzo[a]pyrene, etc., preferably benzo[a]pyrene.

[0027] The petroleum hydrocarbons are organic compounds composed of carbon and hydrogen, mainly including alkanes, olefins, aromatic hydrocarbons and cycloalkanes, etc., alkanes such as methane and ethane, olefins such as ethylene and propylene, aromatic hydrocarbons such as benzene, cycloalkanes such as cyclohexane, etc.

[0028] Pollution caused by PAHs and / or petroleum hydrocarbons includes but is not limited to pollution caused by oil, gasoline, diesel, kerosene, paint, coatings, plastics, rubber, ink, lubricants, asphalt, petroleum coke, liquefied gas, pesticides, fertilizers, detergents, fibers, etc.

[0029] The above-mentioned repair product of the present invention can be in liquid, solid, or semi-solid state, and can be prepared into a desired state as required.

[0030] The present invention also provides a product for use in polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, wherein the product contains the chitosan-modified biochar immobilized composite bacterial agent provided by the present invention. The product has degradation activity for polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, and has repair activity and / or improvement activity for the pollution caused by polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, and can be applied to environmental systems such as soil and water bodies.

[0031] Compared with the prior art, the advantages of the present invention are:

[0032] (1) The present invention uses strain TB1 and strain TX3 for immobilization. Compared with the degradation of benzo[a]pyrene and diesel by a single strain, the degradation rate of strain TB1 and strain TX3 after combination is significantly improved. By utilizing the solubility, bioaffinity and structural characteristics of chitosan, a biofilm is formed on the surface of the biochar immobilized composite bacterial agent, providing a more suitable living microenvironment for the immobilized composite bacterial agent and enhancing the attachment and growth of microorganisms on the biochar surface. The experimental results show that the amount of immobilized microorganisms on biochar prepared by the adsorption method is less than 10 8 CFU / g, the immobilized bacteria amount of the chitosan modified biochar immobilized composite bacterial agent prepared by the present invention is more than 10 11 CFU / g, effectively improving the ability of microorganisms to degrade pollutants. After 70 days of storage, the fixed bacteria amount of chitosan modified biochar immobilized composite bacteria agent was still >10 10 CFU / g.

[0033] (2) By comparing the degradation of composite pollution by free composite bacteria and chitosan-modified biochar-immobilized composite bacteria under different pH and salt concentration environments, the present invention found that the pH tolerance and salt tolerance of the chitosan-modified biochar-immobilized composite bacteria were significantly better than those of the free composite bacteria, and its degradation rate of diesel and benzo[a]pyrene composite pollution was significantly higher than that of the free composite bacteria.

[0034] The chitosan-modified biochar-immobilized composite bacterial agent of the present invention exhibits degradation activity in the pH range of 4-10 and the salt concentration range of 4-40 g / L, and the degradation activity is significantly enhanced in a neutral to weakly alkaline environment of pH 6-9 and the salt concentration range of 8-25 g / L, showing good environmental adaptability, and providing an efficient and feasible solution for actual contaminated soil remediation.

[0035] (3) The present invention applies chitosan-modified biochar-immobilized composite bacterial agent to the polycyclic aromatic hydrocarbon-petroleum hydrocarbon composite contaminated soil remediation experiment. The results show that when 5% chitosan-modified biochar-immobilized composite bacterial agent is added to the composite contaminated soil with a benzo[a]pyrene concentration of 10.74 mg / kg and a diesel concentration of 11.93 g / kg, the degradation rate of benzo[a]pyrene is as high as 67.01% and the degradation rate of diesel is as high as 73.67% after 70 days of remediation. This fully proves the high efficiency and feasibility of the chitosan-modified biochar-immobilized composite bacterial agent in the actual contaminated soil remediation.

[0036] (4) The present invention proposes the use of chitosan-modified biochar immobilized composite bacterial agent, which can not only increase the number of live bacteria and stability of immobilized microorganisms, but also significantly improve the degradation rate of pollutants. Chitosan, by virtue of its solubility, bioaffinity and structural characteristics, creates a more suitable living microenvironment for microorganisms, allowing microorganisms to more firmly attach to the surface of biochar, greatly increasing the number of live bacteria of immobilized microorganisms, and thus improving their ability to degrade pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a physical picture of the chitosan-modified biochar immobilized composite bacterial agent.

[0038] Figure 2 This is a scanning electron micrograph of the composite bacterial agent immobilized on chitosan-modified biochar.

[0039] Figure 3 The effect of different pH values ​​on the degradation of PAHs-PE by chitosan-modified biochar immobilized composite bacterial agent and free TB1:TX3=2:1. Green: diesel; Orange: benzo[a]pyrene.

[0040] Figure 4 Effects of different salt concentrations on the degradation of PAHs-PE by chitosan-modified biochar immobilized composite bacterial agent and free TB1:TX3=2:1. Green: diesel; Orange: benzo[a]pyrene.

[0041] Figure 5 This is a diagram showing the effect of chitosan-modified biochar-immobilized composite bacterial agent on degrading soil benzo[a]pyrene.

[0042] Figure 6 This is a diagram showing the effect of chitosan-modified biochar-immobilized composite bacterial agent on degrading diesel in soil. DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] Example 1: Preparation of composite bacteria

[0045] Selection of strains: Bacillus cereus TB1 (CGMCC No. 30496, China National Microbiological Culture Collection Center) and Acinetobacter sp. TX3 (CGMCC No. 30200, China National Microbiological Culture Collection Center) with the ability to degrade polycyclic aromatic hydrocarbons and petroleum hydrocarbons were selected. Strains TB1 and TX3 were isolated from soil that had been contaminated by petroleum for a long time in the laboratory. After multiple rounds of screening and identification, it was determined that they have the ability to degrade polycyclic aromatic hydrocarbons and petroleum hydrocarbons. Among them, strain TB1 has the ability to produce lipopeptide surfactants. The biological surface activity can enhance the solubility of polycyclic aromatic hydrocarbons and petroleum hydrocarbons, promote the contact between microorganisms and pollutants, and help promote the degradation of polycyclic aromatic hydrocarbons and petroleum hydrocarbons by the composite bacterial agent.

[0046] Culture medium: The culture medium for strain growth consists of 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, and 1 L distilled water.

[0047] Strain culture conditions: strains TB1 and TX3 were inoculated into corresponding culture media, respectively, and cultured in an air bath with constant temperature shaking at 37°C and 150 r / min.

[0048] Preparation of composite bacterial flora: After culturing the activated strains TB1 and TX3 for 12 h, centrifuge at 4000 r / min and resuspend in sterile saline to an OD of 600 =1.0, take 1000mL of strain TB1 bacterial suspension and 500mL of strain TX3 bacterial suspension, mix them and centrifuge at 4000r / min to prepare wet cells of composite bacteria.

[0049] Example 2: Preparation of chitosan-modified biochar immobilized composite bacterial agent

[0050] Preparation of chitosan solution: Accurately weigh 4.0g of chitosan with a deacetylation degree of >90% and a viscosity of <100mPa·s, and slowly add it into 100mL of 5% (v / v) acetic acid solution. Under mild conditions, chitosan gradually fully blends with the acetic acid solution until it is completely dissolved, forming a uniform and stable chitosan solution, which lays the foundation for subsequent immobilization operations.

[0051] Pretreatment of biochar: The biochar used in this experiment is derived from corn stalks, which are fired at a precise carbonization temperature of 500°C and have unique physical and chemical properties. After screening, biochar particles with a particle size range of 0.150-0.425mm were obtained. Subsequently, the wet bacteria prepared in proportion were taken and 10mL of 50% (v / v) glycerol was added as a freeze-drying protective agent. The wet bacteria, protective agent mixture and 3.0g of the above biochar were fully mixed and stored overnight at a low temperature of 4°C. Through this process, the biochar successfully achieved the preliminary fixation of the composite bacteria, and a biochar-immobilized composite bacterial agent was prepared.

[0052] Immobilization of composite bacterial agent: Add 3.0g of biochar-immobilized composite bacterial community to the previously prepared chitosan solution, and use magnetic stirring to allow the two to fully contact and react in the solution for 1 hour. Subsequently, slowly add 10% (w / v, g / mL) NaOH solution, and accurately adjust the pH value of the solution to 7.0. Under this pH condition, chitosan undergoes a series of physical and chemical changes to form a uniform membrane structure on the surface of biochar. After the reaction is completed, the solution is filtered, and the resulting filter residue is transferred to a freeze dryer for freeze drying. The freeze-dried product is a chitosan-modified biochar-immobilized composite bacterial community, which is ground and can pass through a 10-mesh nylon sieve. The finished product is in the form of Figure 1 As shown. Scanning electron microscopy observation of chitosan modified biochar immobilization composite bacterial agent, as shown Figure 2 As shown in the figure, the strains are densely distributed on the film formed by chitosan, and the red circles are bacteria fixed on the biochar structure. Due to the effect of chitosan, the added bacterial agent is tightly and firmly attached to the surface of biochar and inside the pores. At the same time, chitosan can also provide a certain protection for microorganisms, so that the microbial flora of the chitosan-modified biochar immobilized composite bacterial agent can survive and function more stably in a complex environment and degrade organic pollutants more effectively.

[0053] Example 3: Comparison of the degradation ability of benzo[a]pyrene and diesel combined pollution

[0054] Free single strain TB1 bacterial solution (OD 600 =1.0, 5% v / v), free single strain TX3 bacterial solution (OD 600=1.0, 5% v / v), free TB1 and TX3 composite bacterial liquid (5% v / v), biochar immobilized composite bacterial agent prepared by adsorption method (i.e., biochar immobilized composite bacterial agent prepared in the "pretreatment of biochar" operation in Example 2, 5% w / v), and chitosan modified biochar immobilized composite bacterial agent (5% w / v) were added to a composite contaminated culture medium (pH=7.0, salt concentration 12 g / L) containing 10 mg / L benzo[a]pyrene and 10 g / L diesel, and cultured at 37°C, 150 r / min in an air bath constant temperature shaking culture for 7 days. After 7 days, the degradation rate of benzo[a]pyrene was determined by high performance liquid chromatography, and the degradation rate of diesel was determined by weight method. Among them, the free TB1 and TX3 composite bacterial liquid were set into three comparison groups according to the volume ratio of TB1 bacterial liquid to TX3 bacterial liquid of 1:1, 2:1, and 1:2, and the OD of TB1 bacterial liquid was 0.04477 W / L. 600 =1.0, TX3 bacterial solution OD 600 =1.0. The results are shown in Table 1:

[0055] Table 1

[0056]

[0057]

[0058] Example 4: Determination of viable counts by plate method

[0059] 1.0 g chitosan-modified biochar immobilized composite bacterial agent was added to 100 mL sterile water and magnetically stirred for 12 h. 100 uL of the clear solution was taken and spread on a NA medium plate after continuous dilution at a certain multiple. The plate was then placed in a constant temperature incubator at 37 ° C for incubation. The bacterial plate count method was used to determine the number of bacteria. The experiment was repeated three times. The experimental determination of the number of viable bacteria in the chitosan-modified biochar immobilized composite bacterial agent was > 10 11 CFU / g. After 70 days of storage at 4℃, the number of viable bacteria in the chitosan-modified biochar immobilized composite bacterial agent was still >10 10 CFU / g. The fixed bacteria amount of the composite bacterial agent immobilized by chitosan modified biochar was significantly higher than that of the biochar immobilized bacterial agent prepared by adsorption method <10 8 CFU / g, and its solid bacteria content is stable and can be stored for a long time.

[0060] Example 5: Effect of pH on the degradation of polycyclic aromatic hydrocarbons-petroleum hydrocarbons by chitosan-modified biochar immobilized composite bacterial agent

[0061] The composite contaminated culture medium was prepared, and the concentrations of benzo[a]pyrene and diesel were 10 mg / L and 10 g / L, respectively. Free composite bacterial solution (5% v / v) and chitosan-modified biochar-immobilized composite bacterial agent (5% w / v) were added to the composite contaminated culture medium. The OD of TB1 in the free composite bacterial solution was 600=1.0, TX3 bacterial solution OD 600 =1.0, the volume ratio of TB1 bacterial solution to TX3 bacterial solution was 2:1. Different pH gradients were set, namely 4, 5, 6, 7, 8, 9, and 10, and three replicates were set for each pH value. A blank control group CK with inactivated composite bacteria was set. The composite contaminated culture medium with chitosan-modified biochar immobilized composite bacteria was placed in a constant temperature shaking culture at 37°C and 150r / min in an air bath for 7 days. After 7 days, the degradation rate of benzo[a]pyrene was determined by high performance liquid chromatography, and the degradation rate of diesel was determined by weight method.

[0062] Benzo[a]pyrene was determined by high performance liquid chromatography: after 7 days of degradation, 0.6g of sodium chloride was added to each 20ml of culture medium, and an equal amount of dichloromethane was added after dissolution. Ultrasonic extraction was performed for 30 minutes. After the end of ultrasound, the liquid was separated, the supernatant was collected, and the extraction was repeated twice. After the extraction, the supernatant was collected and added with anhydrous sodium sulfate until there was no flowing anhydrous sodium sulfate, and it was placed for 30 minutes to dry, and then purified with a silica gel column. Weigh the mass m1 of the dry ground-mouth eggplant-shaped bottle, collect all the extracts and transfer them to the ground-mouth eggplant-shaped bottle, and concentrate them under reduced pressure in a 60℃ rotary evaporator constant temperature water bath. After the dichloromethane was completely evaporated and cooled, weigh the mass m2 of the ground-mouth eggplant-shaped bottle. Add 5mL of acetonitrile to fully dissolve the substance in the eggplant-shaped bottle, take 1mL and pass it through a 0.22um hydrophobic polytetrafluoroethylene filter membrane syringe filter into a 1.5mL brown sample bottle, and send it to a high performance liquid chromatograph to determine the retention time and peak area, and use the standard curve to determine the concentration of benzo[a]pyrene.

[0063] The HPLC detection conditions are as follows: reverse phase column (AQ-C18, 5um, 4.6×250mm), column temperature 30°C; mobile phase acetonitrile: water = 8:2, isocratic elution; UV detection wavelength 254nm, volume flow rate 1.0mL / min, injection volume 10uL.

[0064] The formula for determining diesel degradation rate by weight method is:

[0065] m CK2 : The mass of the CK extract in the control group after rotary concentration and drying in the ground eggplant-shaped bottle;

[0066] m CK1 : The quality of the CK dry ground eggplant-shaped bottle in the control group;

[0067] m2: the mass of the extract in the treatment group after rotary concentration and drying in the ground-mouth eggplant-shaped bottle;

[0068] m1: The mass of dry ground-mouth eggplant-shaped bottles in the treatment group.

[0069] like Figure 3As shown in the figure, the degradation effect of free composite bacterial liquid on benzo[a]pyrene and diesel is significantly lower than that of chitosan modified biochar immobilized composite bacterial agent. Chitosan modified biochar immobilized composite bacterial agent maintains good degradation efficiency in the range of pH 4-10, with the degradation rate of benzo[a]pyrene not less than 26%, and the degradation rate of diesel not less than 30%, and has good tolerance to pH. Especially at pH 6-8, the degradation effect of benzo[a]pyrene and diesel is the best, the diesel degradation rate can reach more than 60%, the highest is 65%, and the benzo[a]pyrene degradation rate can reach more than 59%, the highest is 66%. In the acidic environment of pH 4 and the alkaline environment of pH 10, the degradation rate is relatively low. This shows that the chitosan modified biochar immobilized composite bacterial agent has good degradation performance in the neutral to weakly alkaline environment of pH 6-9. When the pH value is too high or too low, the activity of the strain is inhibited and the degradation efficiency is reduced. This is because pH affects the growth and metabolism of bacteria, as well as the activity of enzymes. Under acidic conditions, excessive hydrogen ion concentration will destroy the stability of cell membranes and affect the transport and metabolism of substances; under alkaline conditions, excessive hydroxide ion concentration will affect the conformation and activity of enzymes, thereby reducing degradation efficiency.

[0070] Example 6: Effect of salt concentration on degradation of polycyclic aromatic hydrocarbons-petroleum hydrocarbons by chitosan-modified biochar immobilized composite bacterial agent

[0071] The composite contaminated culture medium was prepared, and the concentrations of benzo[a]pyrene and diesel were 10 mg / L and 10 g / L, respectively. Free composite bacterial solution (5% v / v) and chitosan-modified biochar-immobilized composite bacterial agent (5% w / v) were added to the composite contaminated culture medium. The OD of TB1 in the free composite bacterial solution was 600 =1.0, TX3 bacterial solution OD 600 =1.0, the volume ratio of TB1 bacterial solution to TX3 bacterial solution was 2:1. Different salt concentration gradients were set, namely 4g / L, 8g / L, 12g / L, 16g / L, 20g / L, 25g / L, 30g / L, and 40g / L. Three replicates were set for each salt concentration value, and a blank control group CK with inactivated composite bacteria was set. The composite contaminated culture medium with chitosan-modified biochar immobilized composite bacteria was placed in a constant temperature shaking culture at 37°C and 150r / min in an air bath for 7 days. The degradation rate of benzo[a]pyrene was determined by high performance liquid chromatography, and the degradation rate of diesel was determined by weight method.

[0072] like Figure 4As shown in the figure, the degradation effect of free composite bacterial liquid on benzo[a]pyrene and diesel is significantly lower than that of chitosan modified biochar immobilized composite bacterial agent. Under a certain range of salt concentration (4-40g / L), especially in the salt concentration range of 8-25g / L, the degradation rate of chitosan modified biochar immobilized composite bacterial agent on benzo[a]pyrene and diesel can be maintained between 40% and 70%, and the degradation rate of benzo[a]pyrene can reach up to 61.89%, and the degradation rate of diesel can reach up to 61.14%. Although the degradation rate is low at a higher salt concentration of 40g / L, it shows good degradation performance in the medium salt concentration range. Therefore, it can be considered that the chitosan modified biochar immobilized composite bacterial agent has a certain salt concentration tolerance and can maintain a relatively stable degradation rate.

[0073] Example 7: Application of chitosan-modified biochar immobilized composite bacterial agent in contaminated soil remediation

[0074] Clean soil was collected, and stones and plant and animal residues were screened out and naturally air-dried before grinding through a 10-mesh nylon sieve for later use. Benzo[a]pyrene and diesel were dissolved in dichloromethane and fully mixed with the air-dried and sieved clean soil. The soil was placed in a fume hood and aged for 7 days after the dichloromethane was completely volatilized. The concentrations of benzo[a]pyrene and diesel in the soil were measured. A simulated contaminated soil containing benzo[a]pyrene-diesel was prepared, and the concentrations of polycyclic aromatic hydrocarbons and petroleum hydrocarbons were 10.74 mg / kg and 11.93 g / kg, respectively.

[0075] Chitosan modified biochar immobilized composite bacteria agent was added to the contaminated soil at a ratio of 5% (w / w), and three repeated experimental groups were set up, and a blank control group (CK) was set up. Watering was carried out every 24 hours to maintain the soil moisture content at 60%. Different time points were set during the entire cultivation period, and sampling was carried out at 0d, 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d, and 70d. After taking 100g of soil sample and air-drying it naturally, the air-dried soil sample was ground and weighed to take 10.0g, and 50mL of dichloromethane extract was added. After sealing, it was shaken vigorously to make the soil and the extract fully mixed and then ultrasonically extracted for 30min, and the extraction was repeated 3 times. After centrifugation at 4000r / min for 5min, the upper organic phase was filtered through a 500-mesh filter and dried over anhydrous sodium sulfate. After obtaining the extract, the degradation rate of benzo[a]pyrene was determined by high performance liquid chromatography, and the degradation rate of diesel was determined by weight.

[0076] By analyzing the degradation experimental soil samples at different time points, the degradation rate of benzo[a]pyrene was as follows: Figure 5 As shown in Figure 2, the degradation rate of diesel is Figure 6As shown. The degradation rate of benzo[a]pyrene by the chitosan-modified biochar-immobilized composite bacterial agent was low at 8.51% within 7 days. The degradation rate of diesel in 7 days could reach 30.10%. With the extension of time, the degradation efficiency of benzo[a]pyrene and diesel gradually increased. At 70 days, the degradation rate of benzo[a]pyrene by the chitosan-modified biochar-immobilized composite bacterial agent reached 67.01%, and the degradation rate of diesel reached 73.67%. The chitosan-modified biochar-immobilized composite bacterial agent had a good degradation effect on benzo[a]pyrene and diesel. During the entire experimental period, the degradation effect of the bacterial agent on diesel was more significant than that on benzo[a]pyrene. The degradation rates of benzo[a]pyrene and diesel in the control group (CK) were low throughout the experiment, indicating that the chitosan-modified biochar-immobilized composite bacterial agent played a key role in the degradation process.

[0077] The present invention has been introduced in detail above, and the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a chitosan-modified biochar immobilized composite bacterial agent, characterized in that the steps include: (1) mixing the composite bacterial solution, the protective agent and the biochar, and storing the mixture at 2-8° C. to prepare a biochar-immobilized composite bacterial agent; (2) Add the biochar-immobilized composite bacterial agent to the chitosan acetic acid solution and mix well, then add alkali to adjust the pH to 7, and freeze-dry to obtain the chitosan-modified biochar-immobilized composite bacterial agent.

2. The preparation method according to claim 1, characterized in that: In step (1), the composite bacterial solution is centrifuged before being mixed with the protective agent and the biochar.

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), the dosage ratio of biochar to composite bacterial solution is 0.5-3 g:500 mL, the volume ratio of protective agent to composite bacterial solution is 0.5-2:150, and the composite bacterial solution is OD 600 =TB1 bacterial solution with OD of 1.0-1.2 600 =1.0-1.2 TX3 bacterial solution is mixed according to a volume ratio of 0.5-2:

1.

4. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of the biochar-immobilized composite bacterial agent to chitosan is 0.5-1.5:1, and the concentration of chitosan in the chitosan acetic acid solution is 0.03-0.05 g / mL.

5. A chitosan-modified biochar immobilized composite bacterial agent prepared by the preparation method according to any one of claims 1 to 4, characterized in that: It comprises composite bacteria, biochar and chitosan. The composite bacteria are fixedly attached to the surface and pores of the biochar, and the chitosan forms a uniform film structure on the surface of the biochar.

6. The chitosan-modified biochar immobilized composite bacterial agent according to claim 5, characterized in that: The composite bacteria include TB1 and TX3.

7. The chitosan-modified biochar-immobilized composite bacterial agent prepared by the preparation method of any one of claims 1 to 4 or the chitosan-modified biochar-immobilized composite bacterial agent described in claim 5 or 6 is used to prepare products for use in polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons.

8. The use according to claim 7, characterized in that: The product is applicable to soil or water, has degradation activity on polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, and / or has restoration activity and / or improvement activity on pollution caused by polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons.

9. A product for use in polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, characterized in that: The invention comprises a chitosan-modified biochar-immobilized composite bacterial agent prepared by the preparation method of any one of claims 1 to 4 or a chitosan-modified biochar-immobilized composite bacterial agent according to claim 5 or 6.

10. The product according to claim 9, characterized in that The product is applicable to soil or water, has degradation activity on polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons, and / or has restoration activity and / or improvement activity on pollution caused by polycyclic aromatic hydrocarbons and / or petroleum hydrocarbons.

Images