CN111088246A - Biochar immobilized naphthalene degrading microbial inoculum and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a preparation method of a biochar immobilized naphthalene degrading microbial inoculum, which comprises the following steps: weighing biomass raw materials, mixing with water, heating for reaction, cooling at room temperature after the reaction is finished, carrying out solid-liquid separation, cleaning a solid product, and carrying out freeze drying and grinding to obtain biochar; mixing the naphthalene degrading strain and the biochar in an LB liquid culture medium, and placing the mixture in a constant-temperature shaking table for shaking culture to obtain uniform mixed bacterial liquid; and mixing the mixed bacterial liquid with a sodium alginate solution, obtaining the sodium alginate mixed bacterial liquid after magnetic stirring is uniform, dropwise adding the sodium alginate mixed bacterial liquid into a calcium chloride solution, standing, placing into a refrigerator for crosslinking reaction, and obtaining the biochar immobilized naphthalene degrading microbial inoculum after the reaction is finished. The biochar immobilized naphthalene degrading microbial inoculum prepared by the invention has an obvious effect of repairing polycyclic aromatic hydrocarbon polluted soil, is mild in reaction, low in cost, free of secondary pollution and wide in engineering application prospect.
Description
Technical Field
The invention relates to a charcoal immobilized naphthalene degrading microbial inoculum and a preparation method and application thereof, belonging to the technical field of microbial remediation of polluted soil.
Background
Naphthalene is a polycyclic aromatic hydrocarbon substance with a 2-ring structure, low toxicity and high efficiency, and is widely applied to the production of phthalic anhydride, dye intermediates, rubber additives, pesticides and the like. In 10 months in 2017, the international cancer research institution of the world health organization lists naphthalene in a 2B category carcinogen list, and then in 12 months in 2017, China lists the naphthalene in a first group of priority control chemical directory. At present, the remediation technology for polycyclic aromatic hydrocarbon-polluted soil mainly comprises heat treatment, chemical oxidation, chemical leaching and the like, and although the methods have obvious effect on removing polycyclic aromatic hydrocarbon, the environmental problems such as secondary pollution and the like are easily caused. In contrast, the microbial remediation technology has the advantages of economy, high efficiency, environmental friendliness and the like, and is an important process for effectively converting and removing polycyclic aromatic hydrocarbon substances in the polluted soil. Therefore, obtaining the functional microorganism with the high-efficiency degradation capability of the polycyclic aromatic hydrocarbon is the precondition and the key for realizing the success of the microbial remediation of the polluted soil.
In the early work, the inventor obtains a naphthalene high-efficiency degrading strain from petroleum hydrocarbon contaminated soil through screening, and the strain is identified as Paenibacillus napus (Paenibacillus napus) through 16S rDNA sequencing and homology ratio peer-to-peer analysis, and the GenBank accession number is SRR 10341461. Physical and chemical properties and degradation characteristics are analyzed, and the strain can grow and reproduce by taking naphthalene as a unique carbon source, has good metabolic capability on naphthalene, and can achieve a naphthalene degradation removal rate of 83.6 percent within 5 days under the condition of a culture medium. Considering the problems that free strains are easy to run off in practical application and are difficult to compete with soil indigenous microorganisms, the research and development of a proper immobilization technology is a key for ensuring that functional microorganisms can efficiently degrade pollutants. After the functional strains with good degradation capability obtained by screening are mixed with the immobilized carrier, the functional strains can be attached and grown in the immobilized carrier and on the surface of the immobilized carrier, and in practical application, on one hand, the density of the degrading strains in the polluted soil can be enriched, the adverse effect of the external environment and soil indigenous microorganisms on the functional strains can be weakened to a certain extent, and the degradation and removal effect of the functional strains on target pollutants is enhanced; on the other hand, the immobilized carrier can promote the directional migration of the pollutants to a certain extent, strengthen the interface contact between the functional strains and the pollutants, and improve the degradation and removal effect of the pollutants.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a naphthalene degrading bacteria immobilization method and is used for combined remediation of polycyclic aromatic hydrocarbon contaminated soil.
In order to solve the technical problems, the invention provides a preparation method of a biochar immobilized naphthalene degrading microbial inoculum, which is characterized by comprising the following steps:
step 1): weighing a biomass raw material, mixing the biomass raw material with water in a polytetrafluoroethylene lining tank, putting the mixture into a stainless steel reaction kettle, heating for reaction, cooling at room temperature after the reaction is finished, carrying out solid-liquid separation, cleaning a solid product by using distilled water and absolute ethyl alcohol, and carrying out freeze drying and grinding to obtain biochar;
step 2): mixing the naphthalene degrading strain subjected to freezing preservation and the biochar obtained in the step 1) in an LB liquid culture medium, and placing the mixture in a constant-temperature shaking table for shaking culture to obtain uniform mixed bacterial liquid;
step 3): mixing the mixed bacterial liquid obtained in the step 2) with a sodium alginate solution, obtaining the sodium alginate mixed bacterial liquid after magnetic stirring is uniform, dropwise adding the sodium alginate mixed bacterial liquid into a calcium chloride solution, standing, placing in a refrigerator at 4 ℃ for crosslinking reaction, and obtaining the biochar immobilized naphthalene degrading microbial inoculum after the reaction is finished.
Preferably, the biomass raw material in the step 1) is lignin, cellulose or hemicellulose.
Preferably, the mass ratio of the biomass raw material to the water in the step 1) is 1 (10-30).
Preferably, the heating temperature in the step 1) is 220-280 ℃, and the heating reaction time is 2-4 h.
Preferably, the freeze-drying time in the step 1) is 12-24 h.
Preferably, the naphthalene degrading strain in the step 2) is Paenibacillus napus, the GenBank accession number is SRR10341461, and naphthalene can be used as a unique carbon source for growth and propagation.
Preferably, the mass ratio of the naphthalene degrading strain, the biochar and the LB liquid culture medium in the step 2) is 1 (20-50) to 250.
Preferably, the temperature of the constant temperature shaking table in the step 2) is controlled to be 37 ℃, the oscillation rate is 100-150 r/min, and the oscillation time is 12-24 h.
Preferably, the volume ratio of the mixed bacterial liquid to the sodium alginate solution in the step 3) is 1:1, the mass concentration of the sodium alginate solution is 2% -4%, the mass concentration of the calcium chloride solution is 2% -5%, and the volume ratio of the mixed bacterial liquid to the calcium chloride is 1: 2-4.
Preferably, the crosslinking reaction time in the step 3) is 12-24 h.
The invention also provides the charcoal immobilized naphthalene degrading microbial inoculum prepared by the method.
The invention also provides application of the biochar immobilized naphthalene degrading microbial inoculum in remediation of soil polluted by polycyclic aromatic hydrocarbons.
Preferably, the application comprises: fully mixing the biochar immobilized naphthalene degrading microbial inoculum with the polycyclic aromatic hydrocarbon polluted soil, simultaneously adding an inorganic salt solution, uniformly stirring, and sampling every 12 hours to determine the concentration of naphthalene residue in the polluted soil.
More preferably, in the application, the mass ratio of the biochar immobilized naphthalene degrading microbial inoculum to the soil polluted by the polycyclic aromatic hydrocarbon is 2-3: 1000, and the mass ratio of the inorganic salt solution to the soil polluted by the polycyclic aromatic hydrocarbon is 8-15: 100.
Further, the content of the components of the inorganic salt solution is 1g/L Na2HPO4、0.75g/L KH2PO4、0.5g/LNH4Cl、0.1g/L MgSO4·7H2O、0.01g/L CaCl2、0.5g/L NaNO3、0.03g/L FeSO4·7H2O and 0.005L/L trace element solution with pH value of 7.0-7.2, wherein the content of the trace element solution is 5g/L CuCl2·2H2O、1g/L ZnCl2、0.5g/L CoCl2·6H2O and 0.06g/L H3BO3。
Compared with the prior art, the invention has the following beneficial effects:
(1) the method of the invention fixes the naphthalene degrading strain in the charcoal carrier, on one hand, the charcoal can provide stable growth environment for the naphthalene degrading strain, and on the other hand, the environmental concentration of the naphthalene degrading strain can be improved. In addition, the biochar has a certain specific surface area, and can directionally adsorb polycyclic aromatic hydrocarbon substances in the polluted soil on the surface, enhance the interface contact and microbial reaction of the biochar and a naphthalene degradation strain, and realize the combined remediation of the polluted soil. The biochar is a good soil repairing and improving material, does not produce harmful influence on soil microorganisms, and has the advantages of being green, sustainable and the like.
(2) The method utilizes the biochar immobilized naphthalene degrading microbial inoculum and adopts sodium alginate for embedding, so that the method can be applied to remediation of the soil polluted by the polycyclic aromatic hydrocarbon, can obtain a better remediation effect, has the beneficial effects of mild reaction, low cost, no secondary pollution and the like compared with remediation methods such as chemical oxidation, heat treatment and the like, and has a wide engineering application prospect.
Drawings
FIG. 1 is a scanning electron microscope of the MCC280, a biochar material prepared in example 1 of the invention;
FIG. 2 is a diagram of an immobilized naphthalene degrading microbial inoculum of a biochar material MCC280 prepared in example 1 of the invention;
FIG. 3 is a scanning electron microscope of the MCC220 of the biochar material prepared in example 2 of the invention;
FIG. 4 is a diagram of the immobilized naphthalene degrading microbial inoculum of the biochar material MCC220 prepared in example 2 of the invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
The reagents used in the present invention: microcrystalline cellulose, disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium chloride, magnesium sulfate heptahydrate, calcium chloride, sodium nitrate, ferrous sulfate heptahydrate, copper chloride dihydrate, zinc chloride, cobalt chloride hexahydrate and boric acid are analytically pure and purchased from national pharmaceutical group chemical reagent (Shanghai) Co., Ltd; dichloromethane, n-hexane, and acetonitrile were all chromatographically pure and purchased from sigma aldrich trade ltd.
The naphthalene degrading strain is Paenibacillus naphthaphilus (Paenibacillus naphthalins) obtained by screening from petroleum hydrocarbon polluted soil by the inventor, and the GenBank accession number is SRR 10341461.
The detection method of the naphthalene degradation removal rate in the soil comprises the following steps: the determination is carried out according to the national standard of high performance liquid chromatography for determination of polycyclic aromatic hydrocarbons of soil and sediments (HJ 784-2016).
Example 1
The embodiment provides a preparation method of a biochar immobilized naphthalene degrading microbial inoculum, which comprises the following specific steps:
step 1: weighing 2g of microcrystalline cellulose, mixing the microcrystalline cellulose with water in a polytetrafluoroethylene lining tank, putting the mixture into a stainless steel reaction kettle, heating the mixture to 280 ℃ for reaction for 2 hours, cooling the mixture at room temperature after the reaction is finished, carrying out solid-liquid separation at the rotating speed of 12000r/min, repeatedly cleaning a solid product by using distilled water and absolute ethyl alcohol, carrying out freeze drying for 12 hours, and grinding to obtain the biochar named MCC280 (shown in figure 1);
step 2: mixing 0.1g of the naphthalene degradation strain subjected to freezing preservation and 2g of MCC280 biochar in 100mL of LB liquid medium, placing the mixture in a constant-temperature shaking table for shake culture for 12 hours at the temperature of 37 ℃ and the shaking rate of 150r/min to obtain uniform mixed bacterial liquid;
and step 3: mixing the mixed bacterial liquid with a sodium alginate solution according to a volume ratio of 1:1, wherein the mass concentration of the sodium alginate solution is 4%, magnetically stirring uniformly to obtain the sodium alginate mixed bacterial liquid, dropwise adding the sodium alginate mixed bacterial liquid into a calcium chloride solution with the mass concentration of 2% by using a 1mL needle tube, wherein the volume ratio of the sodium alginate mixed bacterial liquid to the calcium chloride solution is 1:2, standing, placing in a 4 ℃ refrigerator for a crosslinking reaction for 12 hours, and obtaining a biological carbon-280 MCC (see figure 2) degradation microbial agent after the reaction is finished.
Fully mixing the biological carbon MCC280 immobilized naphthalene degrading microbial inoculum (3g) and polycyclic aromatic hydrocarbon polluted soil (1kg) according to the mass ratio of 3:1000, wherein the mass concentration of naphthalene in the polluted soil is 50mg/L, adding an inorganic salt solution (150mL), uniformly stirring, and then placing in a constant-temperature shaking table at 37 ℃ for shaking culture for 5 days at the shaking speed of 150 r/min. Through determination, the naphthalene degradation removal rate in the polluted soil can reach 90.3%.
Example 2
The embodiment provides a preparation method of a biochar immobilized naphthalene degrading microbial inoculum, which comprises the following specific steps:
step 1: weighing 5g of microcrystalline cellulose, mixing the microcrystalline cellulose with water in a polytetrafluoroethylene lining tank, putting the mixture into a stainless steel reaction kettle, heating the mixture to 220 ℃ for reaction for 2 hours, cooling the mixture at room temperature after the reaction is finished, carrying out solid-liquid separation at the rotating speed of 12000r/min, repeatedly cleaning a solid product by using distilled water and absolute ethyl alcohol, carrying out freeze drying for 12 hours, and grinding to obtain the biochar named MCC220 (shown in figure 3);
step 2: mixing 0.1g of the naphthalene degradation strain subjected to freezing preservation and 5g of MCC220 biochar in 100mL of LB liquid medium, placing the mixture in a constant-temperature shaking table for shake culture for 12 hours at the temperature of 37 ℃ and the shaking rate of 150r/min to obtain uniform mixed bacterial liquid;
and step 3: and mixing the mixed bacterial liquid with a sodium alginate solution according to a volume ratio of 1:1, wherein the mass concentration of the sodium alginate solution is 2%, magnetically stirring uniformly to obtain the sodium alginate mixed bacterial liquid, dropwise adding the sodium alginate mixed bacterial liquid into a calcium chloride solution with the mass concentration of 5% by using a 1mL needle tube, wherein the volume ratio of the sodium alginate mixed bacterial liquid to the calcium chloride solution is 1:4, standing, placing in a refrigerator at 4 ℃ for crosslinking reaction for 24 hours, and obtaining the biological carbon MCC220 immobilized naphthalene degrading microbial inoculum (shown in figure 4) after the reaction is finished.
Fully mixing the biochar-immobilized MCC220 immobilized naphthalene degrading microbial inoculum (2g) and polycyclic aromatic hydrocarbon polluted soil (1kg) according to the mass ratio of 2:1000, wherein the mass concentration of naphthalene in the polluted soil is 50mg/L, adding an inorganic salt solution (80mL), uniformly stirring, and then placing in a constant-temperature shaking table at 37 ℃ for shaking culture for 5 days at the shaking speed of 150 r/min. Through determination, the naphthalene degradation removal rate in the polluted soil can reach 80.9%.
Claims (14)
1. A preparation method of a biochar immobilized naphthalene degrading microbial inoculum is characterized by comprising the following steps:
step 1): weighing a biomass raw material, mixing the biomass raw material with water in a polytetrafluoroethylene lining tank, putting the mixture into a stainless steel reaction kettle, heating for reaction, cooling at room temperature after the reaction is finished, carrying out solid-liquid separation, cleaning a solid product by using distilled water and absolute ethyl alcohol, and carrying out freeze drying and grinding to obtain biochar;
step 2): mixing the naphthalene degrading strain subjected to freezing preservation and the biochar obtained in the step 1) in an LB liquid culture medium, and placing the mixture in a constant-temperature shaking table for shaking culture to obtain uniform mixed bacterial liquid;
step 3): mixing the mixed bacterial liquid obtained in the step 2) with a sodium alginate solution, obtaining the sodium alginate mixed bacterial liquid after magnetic stirring is uniform, dropwise adding the sodium alginate mixed bacterial liquid into a calcium chloride solution, standing, placing in a refrigerator at 4 ℃ for crosslinking reaction, and obtaining the biochar immobilized naphthalene degrading microbial inoculum after the reaction is finished.
2. The method for preparing the biochar-immobilized naphthalene-degrading bacterial agent according to claim 1, wherein the biomass raw material in the step 1) is lignin, cellulose or hemicellulose.
3. The preparation method of the biochar-immobilized naphthalene degrading microbial inoculum according to claim 1, wherein the mass ratio of the biomass raw material to water in the step 1) is 1 (10-30).
4. The method for preparing the biochar-immobilized naphthalene degrading microbial inoculum according to claim 1, wherein the heating temperature in the step 1) is 220-280 ℃, and the heating reaction time is 2-4 h; the freeze drying time is 12-24 h.
5. The method for preparing the charcoal-immobilized naphthalene degrading bacterial agent of claim 1, wherein the naphthalene degrading bacterial strain in step 2) is Paenibacillus napus, GenBank accession number is SRR10341461, and naphthalene can be used as a unique carbon source for growth and propagation.
6. The preparation method of the biochar-immobilized naphthalene degrading microbial inoculum according to claim 1, wherein the mass ratio of the naphthalene degrading strain, the biochar and the LB liquid culture medium in the step 2) is 1 (20-50): 250.
7. The method for preparing the biochar-immobilized naphthalene degrading microbial inoculum according to claim 1, wherein the temperature of the constant-temperature shaking table in the step 2) is controlled to be 37 ℃, the oscillation rate is 100-150 r/min, and the oscillation time is 12-24 h.
8. The method for preparing the biochar-immobilized naphthalene degrading bacterial agent according to claim 1, wherein the volume ratio of the mixed bacterial liquid to the sodium alginate solution in the step 3) is 1:1, the mass concentration of the sodium alginate solution is 2% -4%, the mass concentration of the calcium chloride solution is 2% -5%, and the volume ratio of the sodium alginate mixed bacterial liquid to the calcium chloride is 1 (2-4).
9. The preparation method of the biochar-immobilized naphthalene degrading microbial inoculum according to claim 1, wherein the crosslinking reaction time in the step 3) is 12-24 h.
10. A biochar-immobilized naphthalene-degrading bacterial agent prepared by the method of any one of claims 1 to 9.
11. The application of the biochar-immobilized naphthalene degrading microbial inoculum of claim 10 in repairing polycyclic aromatic hydrocarbon-polluted soil.
12. The application of the biochar-immobilized naphthalene-degrading bacterial agent in remediation of soil polluted by polycyclic aromatic hydrocarbons as claimed in claim 11, wherein the application comprises: fully mixing the biochar immobilized naphthalene degrading microbial inoculum with the polycyclic aromatic hydrocarbon polluted soil, simultaneously adding an inorganic salt solution, uniformly stirring, and sampling every 12 hours to determine the concentration of naphthalene residue in the polluted soil.
13. The application of the biochar-immobilized naphthalene degrading microbial inoculum in repairing polycyclic aromatic hydrocarbon-polluted soil according to claim 12, wherein the mass ratio of the biochar-immobilized naphthalene degrading microbial inoculum to the polycyclic aromatic hydrocarbon-polluted soil is 2-3: 1000, and the mass ratio of an inorganic salt solution to the polycyclic aromatic hydrocarbon-polluted soil is 8-15: 100.
14. The application of the biochar-immobilized naphthalene degrading microbial inoculum in repairing polycyclic aromatic hydrocarbon polluted soil according to claim 12, wherein the content of the inorganic salt solution is 1g/L Na2HPO4、0.75g/L KH2PO4、0.5g/L NH4Cl、0.1g/L MgSO4·7H2O、0.01g/L CaCl2、0.5g/L NaNO3、0.03g/L FeSO4·7H2O and 0.005L/L trace element solution with pH value of 7.0-7.2, wherein the content of the trace element solution is 5g/L CuCl2·2H2O、1g/LZnCl2、0.5g/L CoCl2·6H2O and 0.06g/L H3BO3。
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| CN109212187A (en) * | 2018-09-06 | 2019-01-15 | 东华大学 | It is a kind of detect polycyclic aromatic hydrocarbon class enzyme linked immunological kit and its application |
| CN109212187B (en) * | 2018-09-06 | 2022-01-11 | 东华大学 | ELISA-like kit for detecting polycyclic aromatic hydrocarbons and application thereof |
| CN111662728A (en) * | 2020-07-29 | 2020-09-15 | 杨凌职业技术学院 | Walnut branch biochar-based fertilizer soil remediation agent and preparation method and application thereof |
| CN111662728B (en) * | 2020-07-29 | 2021-12-21 | 杨凌职业技术学院 | Walnut branch biochar-based fertilizer soil remediation agent and preparation method and application thereof |
| CN111909925A (en) * | 2020-08-13 | 2020-11-10 | 青岛农业大学 | Method for removing soil 2,4, 6-trichlorophenol by porous biomass charcoal immobilized degrading strain |
| CN112795557A (en) * | 2020-12-30 | 2021-05-14 | 天津开发区坤禾生物技术有限公司 | Nutrition-rich immobilized freeze-dried microbial agent and preparation method and application thereof |
| CN114058507A (en) * | 2021-10-21 | 2022-02-18 | 东莞理工学院 | Carbon-coupled compound microbial inoculum and preparation method and application thereof |
| CN114029340A (en) * | 2021-11-09 | 2022-02-11 | 中国科学院南京土壤研究所 | Application of biological PRB of biological carbon coupled microorganism in restoration of polycyclic aromatic hydrocarbon polluted site |
| CN115055515A (en) * | 2022-08-02 | 2022-09-16 | 贵州省生物研究所 | Microbial remediation method for micro-plastics in garbage-polluted soil |
| CN115318812A (en) * | 2022-08-25 | 2022-11-11 | 广东工业大学 | Method for repairing benzo (a) pyrene polluted soil |
| CN116371905A (en) * | 2023-03-20 | 2023-07-04 | 南京农业大学 | Method for removing PAEs in environment by utilizing functional indigenous flora solid microbial inoculum |
| CN116371905B (en) * | 2023-03-20 | 2023-10-13 | 南京农业大学 | Method for removing PAEs in environment by utilizing functional indigenous flora solid microbial inoculum |
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