CN114214312A - Charcoal-loaded low-temperature-resistant efficient degradation PAHSMixed bacterium particle and preparation method and application thereof - Google Patents
Charcoal-loaded low-temperature-resistant efficient degradation PAHSMixed bacterium particle and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a low-temperature-resistant efficient degradation PAH (polycyclic aromatic hydrocarbon) with biochar loadingSA mixed bacterium particle and a preparation method and application thereof. The invention takes the mixture of corncobs and pine sawdust with the mass ratio of 1:1 as a carrier to utilize the absorptionThe mixed bacteria agent is fixed by the attaching method. The biological charcoal fertilizer is composed of pseudomonads (Pseudomonas sp.) SDR4 and Mortierella alpina (Mortierella alpina) JDR7 which are screened from freeze-thaw soil according to the volume ratio of 1:1 and biological charcoal. The charcoal immobilized low-temperature degradation resistant PAHs mixed bacteria have good biological activity and can not introduce secondary pollution to soil. Compared with single physical adsorption, chemical oxidation and other methods, the method is more efficient, energy-saving and environment-friendly, and provides a good living environment for microorganisms.
Description
Technical Field
The invention relates to the technical field of microbial immobilization, in particular to a charcoal-loaded low-temperature-resistant efficient-degradation PAHS (polycyclic aromatic hydrocarbons) mixed bacterium particle and a preparation method and application thereof.
The Pseudomonas (Pseudomonas sp.) SDR4 in the invention is already preserved in 19.4.2017 by the common microorganism center of the China general microbiological culture Collection center, the preservation address is west Lu No.1 Hospital No. 3 of the sunward area of Beijing, and the preservation numbers are: GCMCC NO. 14048.
The Mortierella alpina (Mortierella alpine) JDR7 in the invention has been deposited in 5.1.2018 in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms at the location of No. 3 Hospital No.1 of Xilu, Beijing, the sunny region, with the deposition number: GCMCC NO. 15183.
Background
Polycyclic aromatic hydrocarbon compounds (PAHs) are products of incomplete combustion of fossil fuels or pyrolysis of other organic matters, and most PAHs have strong carcinogenicity, distortion and mutagenicity. PAHs are hydrocarbons with benzene rings in the structures, and the physical and chemical properties of the PAHs are influenced by the number of the benzene rings. With the increase of the number of benzene rings, the molecular weight of the polycyclic aromatic hydrocarbon is correspondingly increased, the water solubility and the volatility are gradually reduced, and the lipophilicity is gradually increased, so that the polycyclic aromatic hydrocarbon is more difficult to be degraded by microorganisms in the natural environment while the toxicity is increased. Toxicity weakening and harmless treatment of high-ring-number polycyclic aromatic hydrocarbons often depends on metabolic degradation of specific microorganisms. The degradation of microorganisms is a main way for removing PAHs, and exogenous microorganisms are easily restricted by indigenous microorganism competition and environmental conditions in an in-situ soil environment, so that an ideal repairing effect cannot be obtained.
The microorganism immobilization technology uses an immobilized carrier material to construct a buffer system, and forms a favorable microenvironment to avoid the damage of the repairing microorganisms caused by adverse land conditions and the competition with the indigenous microorganisms of the polluted soil. The immobilized carrier is used as a place for the growth and propagation of microorganisms, and the microorganisms and enzymes secreted by the microorganisms are gathered on the carrier, so that the contact efficiency of the high-efficiency degradation microorganisms and the PAHs is greatly improved. The carrier has great influence on the activity and stability of microorganisms, resulting in different degradation effects. Due to the characteristics of complexity and uniqueness of soil, the requirements on carrier materials for soil remediation are high. Since the carrier to be put into the soil is not recyclable and will become a part of the soil, it is important to select an environmentally friendly carrier. The total straw yield of various crops planted on cultivated land in China is about 7 hundred million tons every year, the crop straws are made into biochar, so that the carbon utilization efficiency is higher, meanwhile, the environmental pollution can be reduced, and the sustainable development of agricultural ecological systems is facilitated.
Biochar provides a good habitat for microorganisms. The carbon source and the nitrogen source which are contained on the surface of the biochar and are partially easy to decompose are beneficial to microbial activity, the number and the activity of soil microorganisms can be improved, gaps of the biochar have great variability, the loose and porous structure and the huge surface area of the biochar can store water and nutrients, the biochar becomes a microenvironment where the microorganisms can live, a hotbed is provided for the growth of microorganisms of a special group, and therefore the circulation of soil nutrient elements is promoted.
Disclosure of Invention
In order to solve the problems, the invention aims to jointly fix the low-temperature-resistant and high-efficiency degradable PAHs fungi and bacteria on charcoal to construct an immobilized low-temperature-resistant fungus-bacteria symbiotic system and provide an optimal technical approach for in-situ remediation of microorganisms in soil polluted by the PAHs under the alternate freeze-thaw condition.
In order to achieve the purpose, the invention adopts the technical scheme that: charcoal-loaded low-temperature-resistant efficient degradation PAHSMixing the mixed bacteria particles, corncobs and pine sawdust to form a carrier, and fixing the mixed bacteria agent by an adsorption method; the mixed microbial inoculum is prepared from Pseudomonas (Pseudomonas sp.) SDR4 with the preservation number of GCMCC NO.14048 and the preservation number of the mixed microbial inoculum is as follows: mortierella alpina (Mortierella alpina) JDR7 of GCMCC No.15183, and the volume ratio is 1: 1.
The above-mentioned low-temperature-resistant efficient degradation PAH with biochar loadingSThe mixed bacteria particles are obtained by washing the corncobs and the pine sawdust with distilled water for 4 times, air-drying for 2 days, drying in an oven at 75 ℃ for 12 hours, and roasting in a muffle furnace at 500 ℃ for 4 hours.
The above-mentioned low-temperature-resistant efficient degradation PAH with biochar loadingSThe volume ratio of the mixed bacteria particle, Pseudomonas (Pseudomonas sp.) SDR4 and Mortierella alpina (Mortierella alpina) JDR7 is 1: 1.
The preparation method of the charcoal-loaded low-temperature-resistant efficient degradation PAHS mixed bacteria particle comprises the following steps:
1) preparation of the microbial inoculum: pseudomonas (Pseudomonas sp.) SDR4 and Mortierella alpina (Mortierella alpina) JDR7 in a volume ratio of 1:1, respectively inoculating into a complete culture medium, and shake-culturing at 15 deg.C and 120r/min for 3d to obtain microbial inoculum;
2) pretreatment of a carrier: washing the corncobs and the pine sawdust with distilled water for 4 times, air-drying for 2 days, drying in a 75 ℃ drying oven for 12 hours, roasting in a muffle furnace for 4 hours at 500 ℃ to obtain 500 ℃ corncob biochar and 500 ℃ pine sawdust biochar, and mixing according to a mass ratio of 1:1 to obtain a biochar mixture;
3) carrier immobilization: sterilizing the biochar mixture at 121 ℃ for 60min, infiltrating the biochar mixture with a proliferation culture solution according to a solid-to-liquid ratio of 1.0ml/g, adding the microbial inoculum prepared in the step 1 according to the inoculum size of 2% after culturing for 12-24h, culturing at constant temperature of 15 ℃, supplementing the proliferation culture solution once every 24h, and continuously supplementing for 3 timesThen, continuing to culture at constant temperature of 15 ℃ for 4-5 days to obtain the PAH loaded with the biochar and capable of resisting low temperature and efficiently degradingSThe mixed bacterium particles CW-SDR4+ JDR 7.
The above-mentioned low-temperature-resistant efficient degradation PAH with biochar loadingSThe preparation method of the mixed bacterium particles comprises the steps of completely culturing 10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar and 1000mL of distilled water, performing pH 7.1-7.2, and sterilizing at 121 ℃ for 20 min.
The above-mentioned low-temperature-resistant efficient degradation PAH with biochar loadingSThe preparation method of the mixed bacterium granule comprises the steps of preparing 4g of sucrose, 3g of yeast extract and KH2PO4 0.5g，(NH4)2HPO4 2g，MgSO4·H20.25g of O, 6.0-6.5 of pH and sterilization at 121 ℃ for 20 min.
The above-mentioned low-temperature-resistant efficient degradation PAH with biochar loadingSThe application of the mixed bacteria particles in degrading polycyclic aromatic hydrocarbon pollutants.
The application and the method are as follows: adding the charcoal-loaded low-temperature-resistant high-efficiency degradation PAH (polycyclic aromatic hydrocarbon) of claim 1 into freeze-thaw soil containing polycyclic aromatic hydrocarbon pollutantsSThe mixed bacteria particles are supplemented with water to ensure that the water content of the soil is 40 percent, and the polycyclic aromatic hydrocarbon pollutants are degraded.
In the application, the polycyclic aromatic hydrocarbon is phenanthrene and pyrene.
The invention has the beneficial effects that: the bacteria used in the invention are low temperature resistant high-efficiency PAHSThe activity of the degrading bacteria can not be inhibited in cold regions, in addition, the used biochar has the characteristics of porosity, high specific surface area and strong adsorbability, provides a plurality of adsorption sites for microorganisms, has low cost and simple preparation process, can not introduce secondary pollution, does not need to be recycled, and in addition, the microenvironment of the biochar immobilized mixed bacteria can effectively shield the malignant competition, phagocytosis and toxicity of indigenous bacteria, bacteriophage and toxic substances to the microorganisms, so that the biochar can play a role in a high efficiency at a moderate temperature in a complex environment.
Drawings
FIG. 1 is a phylogenetic tree for the construction of SDR4 based on the 16SrDNA sequence and the Neighbor-Joining method.
FIG. 2 is a phylogenetic tree for the construction of JDR7 based on the ITS sequence and the Neighbor-Joining method.
FIG. 3 is an electron micrograph of the microbial inoculum.
FIG. 4 is an electron micrograph of a corncob C500 biochar immobilized mixed bacterial agent.
FIG. 5 is an electron micrograph of pine wood chips W500 immobilized mixed microbial inoculum.
FIG. 6 is an electron microscope photograph of the immobilized mixed bacterial agent mixed by corncob C500 biochar and pine sawdust W500 biochar.
FIG. 7 is a graph comparing degradation of phenanthrene (Phe) by five degradation methods.
FIG. 8 is a graph comparing the degradation of pyrene (Pyr) by five degradation methods.
The Pseudomonas (Pseudomonas sp.) SDR4 in the invention is already preserved in 19.4.2017 by the common microorganism center of the China general microbiological culture Collection center, the preservation address is west Lu No.1 Hospital No. 3 of the sunward area of Beijing, and the preservation numbers are: GCMCC NO. 14048.
The Mortierella alpina (Mortierella alpine) JDR7 in the invention has been deposited in 5.1.2018 in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms at the location of No. 3 Hospital No.1 of Xilu, Beijing, the sunny region, with the deposition number: GCMCC NO. 15183.
Detailed Description
The invention is further illustrated by the following examples.
Example 1 selection of Pseudomonas sp (Pseudomonas sp.) SDR4
The screening method of Pseudomonas (Pseudomonas sp.) SDR4 is as follows:
collecting 3 points of soil samples from a sinking irrigation channel, wherein the sampling depth is about 50cm, grinding, sieving by a 2mm sieve, mixing the soil samples, filling into a reagent bag, sealing, storing in a refrigerator at 4 ℃ for later use, and measuring the concentration of PAHs. Simulating the low temperature in winter of the area, freezing the frozen soil sample in a refrigerator at the temperature of-20 ℃ for 20 days, and melting the frozen soil sample in an incubator at the temperature of 15 ℃ for 10 days for later use. Weighing appropriate amount of soil sample, numbering SR (unfrozen soil sample) and SD (frozen soil sample), placing in nutrient medium, naturally enriching and culturing for 4d, and thenInoculating 10ml of the enrichment culture solution into an inorganic salt selective culture medium for selective culture, domesticating by adopting a method of timing quantitative transfer to gradually increase the concentration of a carbon source, and carrying out shake culture for 7d at 15 ℃ and 120 r.min. The initial concentrations of Phe and Pyr in the selection medium were 10, 10 mg.L-1The 2 nd transfer concentration was increased to 20, 20 mg.L-1The 3 rd transfer concentration was increased by 30, 30 mg.L-1Diluting the bacterial sludge suspension with sterile water to obtain 10-2、10-3、10-4、10-5And 10-6A diluted bacterial sludge suspension. Sucking 0.2mL of the strain, respectively coating the strain on a solid nutrient medium plate added with a PAHs film, performing inverted culture at 15 ℃ until obvious colonies visible to the naked eye grow out, continuously scribing the plate for 3 times, selecting a single strain with large colonies and fast growth, inoculating the single strain into a nutrient medium inclined plane sprayed with PAHs, performing inverted culture at 28 ℃,15 ℃ and 4 ℃ for about 10 days, observing the growth condition, and performing primary screening on low-temperature PAHs degrading bacteria. 4 strains of bacteria are primarily screened out and are marked as SDR2-SDR5, the strains obtained by primary screening are inoculated into experimental simulated soil according to the bacterial suspension of 10 percent, and the bacterial concentration is 6 multiplied by 108And (3) supplementing sterile water at any time when the concentration of CFU/mL and Pyr is 30mg/kg to ensure that the soil moisture is 30%, placing the soil in a freeze-thaw circulating box for light-proof culture, freezing for 12h at-5 ℃ and thawing for 12h at 15 ℃, sampling at 0,15,30,45 and 60d respectively, extracting and purifying to perform liquid chromatography analysis, and determining the residual quantity of PAHs in the soil, wherein the result is that the SDR4 has obvious effect.
The strain SDR4 is light yellow, opaque, gram-negative, short rod-shaped, neat in edge, free from spore formation and aerobic on a beef extract peptone plate.
The inorganic salt liquid culture medium comprises K2HPO41.0g,(NH4)2S045g,MgS04.7H200.5 g, pH7.0-7.2, constant volume with distilled water L, sterilizing at 121 ℃ for 25min, and culturing in nutrient medium (CM medium): 10.0g of peptone, 10g of glucose, 5g of yeast powder, 5g of beef extract, 5g of sodium chloride, pH 7.0-7.2, diluting to 1000mL with distilled water, sterilizing at 121 ℃ for 25min, and adding 20g/L of agar powder into a solid culture medium.
Extraction, PCR amplification and sequencing of strain SDR4 gene are completed by Shanghai Biotechnology Limited company. The sequencing results were compared for homology with Blast in GenBank on NCBI website to determine the bacterial species. Genetic distances were calculated using MEGA6 software (molecular evolution Genetics Analysis), phylogenetic trees were constructed using the Neighbor-joining method (Neighbor-joining), and the stability of the numbers was evaluated using Bootstrap Analysis. As can be seen from FIG. 1, the SDR4 strain naturally clusters with the 16S rDNA sequence of Pseudomonas, and the similarity is 99%, so that SDR4 belongs to Pseudomonas.
Example 2 screening of Mortierella alpina (Mortierella alpina) JDR7
The screening method of Mortierella alpina (Mortierella alpina) JDR7 is as follows:
collecting 3 soil samples from polluted areas in the western chicken, sampling to a depth of about 50cm, grinding, sieving with a 2mm sieve, mixing, packaging into reagent bags, sealing, storing in refrigerator at 4 deg.C, and measuring PAHs concentration. Simulating the low temperature in winter of the area, freezing the frozen soil sample in a refrigerator at the temperature of-20 ℃ for 20 days, and melting the frozen soil sample in an incubator at the temperature of 15 ℃ for 10 days for later use. Weighing a proper amount of soil samples, numbering SR (unfrozen soil sample) and SD (frozen soil sample) respectively, placing the soil samples in a nutrient medium for natural enrichment culture for 4d, then taking 10ml of the enrichment culture solution, inoculating the enrichment culture solution in an inorganic salt selective culture medium for selective culture, domesticating by adopting a method of transferring quantitatively at regular time and gradually increasing the carbon source concentration, and carrying out shake cultivation for 7d at 15 ℃ and 120 r.min. The initial concentrations of Phe and Pyr in the selection medium were 10, 10 mg.L-1The 2 nd transfer concentration was increased to 20, 20 mg.L-1The 3 rd transfer concentration was increased by 30, 30 mg.L-1Diluting the bacterial sludge suspension with sterile water to obtain 10-2、10-3、10-4、10-5And 10-6A diluted bacterial sludge suspension. Sucking 0.2mL of the strain, respectively coating the strain on a solid nutrient medium plate added with a PAHs film, performing inverted culture at 15 ℃ until obvious colonies visible to the naked eye grow out, continuously scribing the plate for 3 times, selecting a single strain with large colonies and fast growth, inoculating the single strain into a nutrient medium inclined plane sprayed with PAHs, performing inverted culture at 28 ℃,15 ℃ and 4 ℃ for about 10 days, observing the growth condition, and performing primary screening on low-temperature PAHs degrading bacteria. 4 bacteria were initially screened, as JDR6-JDR8The screened strains are inoculated into experimental simulated soil according to the strain suspension of 10 percent, and the strain concentration is 6 multiplied by 108And (3) supplementing sterile water at any time when the concentration of CFU/mL and Pyr is 30mg/kg to ensure that the soil moisture is 30%, placing the soil in a freeze-thaw circulating box for dark culture, freezing for 12h at-5 ℃ and thawing for 12h at 15 ℃, sampling for 0,15,30,45 and 60d respectively, extracting and purifying to perform liquid chromatography analysis, and determining the residual quantity of PAHs in the soil, wherein the result is that JDR7 has an obvious effect. The strain JDR7 is white on beef extract peptone plate, and has petal-shaped growth at the early stage, developed hypha at the later stage and dense growth.
The extraction, PCR amplification and sequencing of the JDR7 gene are carried out by Shanghai Biotechnology Co. The sequencing results were compared for homology with Blast in GenBank on NCBI website to determine the bacterial species. Genetic distances were calculated using MEGA6 software (molecular evolution Genetics Analysis), phylogenetic trees were constructed using the Neighbor-joining method (Neighbor-joining), and the stability of the numbers was evaluated using Bootstrap Analysis. As can be seen from FIG. 2, the JDR7 strain naturally clustered with the ITS sequences of Mortierella, and the similarity between the ITS sequences was 99%, so that the strain JDR7 belongs to Mortierella.
Example 3 charcoal-loaded low-temperature-resistant efficient degradation PAHS mixed bacteria granule
Culture of Pseudomonas sp (SDR4)
Complete medium: 10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar and 1000mL of distilled water, wherein the pH value is 7.1-7.2, and the sterilization is carried out at 121 ℃ for 20 min.
Taking Pseudomonas (Pseudomonas sp.) SDR4 which is preserved on a slant culture medium and has the preservation number of GCMCC NO.14048, inoculating the Pseudomonas on a complete culture medium according to the inoculation amount of 2%, carrying out shake culture at 15 ℃ and 121r/min for 30-40h until the bacterial density is 6 x 108 CFU.mL < -1 >, and obtaining the Pseudomonas (Pseudomonas sp.) SDR4 bacterial suspension.
Culture of Mortierella alpina (Mortierella alpina) JDR7
Complete medium: 10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar and 1000mL of distilled water, wherein the pH value is 7.1-7.2, and the sterilization is carried out at 121 ℃ for 20 min.
Taking and storing on slant culture mediumMortierella alpina (Mortierella alpina) JDR7 with accession number of GCMCC NO.15183, inoculating the Mortierella alpina JDR 8983 with inoculation amount of 2%, culturing at 15 deg.C for 30-40h at 121r/min by shaking bed until spore density in spore suspension is 6 × 108CFU·mL-1Obtaining the suspension of the fungus of the Mortierella alpina (Mortierella alpina) JDR 7.
(III) pretreatment of the support
Washing the corncobs and the pine cuttings with distilled water for 4 times, air-drying for 2 days, drying in a 75 ℃ drying oven for 12 hours, and roasting in a muffle furnace at 500 ℃ for 4 hours to obtain 500 ℃ corncob biochar C500 and 500 ℃ pine cuttings biochar W500, and mixing according to a mass ratio of 1:1 to form a biochar mixture.
(IV) immobilization of the Carrier
Proliferation culture solution: 4g of cane sugar, 3g of yeast extract and KH2PO4 0.5g，(NH4)2HPO4 2g，MgSO4·H20.25g of O, 6.0-6.5 of pH, and sterilizing for 20min at 121 DEG C
Sterilizing the biochar mixture at 121 ℃ for 60min, infiltrating the biochar mixture with a proliferation culture solution according to a solid-to-liquid ratio of 1.0ml/g, adding the microbial inoculum prepared in the first step according to the inoculum size of 2% after culturing for 12-24h, culturing at constant temperature of 15 ℃, supplementing the proliferation culture solution once every 24h, continuously supplementing for 3 times, and then continuing culturing at constant temperature of 15 ℃ for 4-5d to obtain the biochar-loaded low-temperature-resistant efficient degradation PAHS mixed bacterial particles which are recorded as CW-SDR4+ JDR 7.
Example 4 Low temperature resistant highly effective degradation of PAH by charcoal loadingSApplication of mixed bacterium particles in degradation of PAHs pollutants
Soil for degradation experiments: the surface layer clean soil with the depth of 0-20 cm is adopted in Shenyang ecological test station of Chinese academy of sciences, and the basic physicochemical properties are as follows: the pH was 6.72, the organic carbon was 17.8g/kg, total nitrogen was 1.1g/kg, and total phosphorus was 0.35 g/kg. And (3) sieving the soil by a 1mm sieve, subpackaging the sieved soil into culture bottles, sterilizing 20g of each bottle at the high temperature of 121 ℃ for 60min, and quantitatively adding a soil sample containing Phe and Pyr pollutants into each bottle after cooling.
CW-SDR4+ JDR7 of the present invention was added at 2% inoculum size to soil samples containing Phe, Pyr contaminants and incubated at 15 ℃ to bring the soil moisture to 40% and samples were taken at 0, 7, 14, 21, 28d, respectively.
Comparative example 1
Sterile water (CK) was added at 2% inoculum size to soil samples containing Phe, Pyr contaminants and incubated at 15 ℃ to a soil moisture of 40% and sampled at 0, 7, 14, 21, 28d, respectively.
Comparative example 2
The microbial inoculum (SDR4) and (JDR7) prepared in the steps (I) and (II) of example 3 were added to soil samples containing Phe and Pyr contaminants at an inoculum size of 2%, respectively, and incubated at 15 ℃ to achieve a soil moisture of 40% and sampled at 0, 7, 14, 21 and 28d, respectively.
Comparative example 3
After the 500 ℃ corncob biochar prepared in the step (three) in the example 3 and the microbial inoculum (SDR4) prepared in the step (I) and the step (II) and the microbial inoculum (JDR7) are immobilized by the step (four), the corncob biochar and the microbial inoculum are added into a soil sample containing Phe and Pyr pollutants, and are cultured at 15 ℃ to ensure that the soil moisture is 40 percent and the samples are respectively taken at 0, 7, 14, 21 and 28 d.
Comparative example 4
After the 500 ℃ pine sawdust biochar prepared in the step (three) and the microbial inoculum (SDR4) and JDR7) prepared in the step (one) and the step (two) are immobilized according to the inoculation amount of 2 percent, the immobilized pine sawdust biochar is added into a soil sample containing Phe and Pyr pollutants, the soil sample is cultured at 15 ℃ to ensure that the soil moisture is 40 percent, and the samples are respectively taken at 0, 7, 14, 21 and 28 d.
Weighing 20g of the treated soil sample, sieving the soil sample by a 60-mesh sieve, pouring the soil sample into a 100ml centrifuge tube, adding 30ml of dichloromethane and 15ml of dichloromethane respectively by using a pipette gun, and carrying out ultrasonic treatment for 2 hours each time, wherein the water temperature of an ultrasonic instrument is kept below 30 ℃ during ultrasonic treatment. And (4) putting the ultrasonic centrifuge tube into a centrifuge, and pouring the supernatant into a chicken heart bottle with a corresponding number for rotary evaporation at a speed of 4000r/min after the centrifugation is finished. Absorbing 2mL of n-hexane to dissolve a sample, taking out an upper layer liquid, transferring the upper layer liquid to a silica gel purification column (1g of neutral Al2O3+1g of silica gel +1g of anhydrous sodium chloride), purifying, leaching with 50mL of n-hexane/dichloromethane (volume ratio is 9:1), collecting a filtrate, concentrating to about 1mL, drying by using soft nitrogen, and measuring PAHs (polycyclic aromatic hydrocarbons) by using liquid chromatography at constant volume of 1mL of acetonitrile.
The samples to be detected in the polluted soil are detected by using a fluorescence and ultraviolet detectorMeasuring, wherein the chromatographic condition is that a chromatographic column is ZORBAX Eclipse (4.6m multiplied by 250mm multiplied by 5ug) which is a PAHs analysis special column; the column temperature is 25 ℃; the mobile phase is acetonitrile and water, and the ratio of acetonitrile: 60% of water: 40% flow rate 1.0 mL/min-1The sample introduction amount is 10 mu L, various PAHs are qualitatively determined by chromatographic peak retention time, the PAHs are quantitatively determined by an external standard method, and the method recovery rate is 79.56-92.48 percent.
The results show that: the density and activity of microorganisms can be improved by the mixed biochar discovered by an SEM scanning electron microscope, and compared with the chart shown in fig. 2 and 4, fungi on the mixed biochar are obviously increased, some bacteria and fungi are adsorbed in gaps of the biochar, and some bacteria and fungi are directly adsorbed on the surface of the biochar. Compared with the graph in fig. 3 and 4, the fungi and the bacteria are adsorbed on the biochar, the hyphae of the fungi grow densely and vertically and horizontally on the mixed biochar, and the hyphae can better contact with pollutants along with the continuous extension of the growth outwards.
The charcoal is loaded with low-temperature-resistant high-efficiency degradation PAHs mixed bacteria particles (CW-SDR4+ JDR7) and is put into a soil sample containing Phe and Pyr pollutants, and the effect of repairing the polluted soil of the PAHs in the cold area is obvious. As shown in fig. 2-3, after 28d, the removal rates of Phe and Pyr were 75.28% and 64.87%, respectively, which were improved by 36.82% and 35.03% respectively as compared with the mixed microbial inoculum (SDR4+ JDR7), 6.78% and 6.57% as compared with the corncob biochar alone (C500-SDR4+ JDR7), and 6.93% and 7.59% as compared with the pine wood chip biochar alone (W500-SDR4+ JDR 7). In addition, the biochar has low cost and simple preparation process, does not introduce secondary pollution, and does not need to be recycled.
Claims (9)
1. Charcoal-loaded low-temperature-resistant efficient degradation PAHSThe mixed bacteria particle is characterized in that corncobs and pine sawdust are mixed to be a carrier, and the mixed bacteria agent is fixed by an adsorption method; the mixed microbial inoculum is prepared from Pseudomonas (Pseudomonas sp.) SDR4 with the preservation number of GCMCC NO.14048 and the preservation number of the mixed microbial inoculum is as follows: mortierella alpina (Mortierella alpina) JDR7 of GCMCC NO. 15183.
2. The charcoal-loaded low-temperature-resistant efficient degradation PAHS mixed bacteria particle as claimed in claim 1, wherein: the corncob and pine sawdust are obtained by washing with distilled water for 4 times, air-drying for 2 days, drying in a 75 ℃ oven for 12 hours, and roasting in a muffle furnace for 4 hours at 500 ℃.
3. The charcoal-loaded PAH with low temperature resistance and high efficiency degradation according to claim 1SThe mixed bacterium particle is characterized in that the volume ratio of (Pseudomonas sp.) SDR4 to Mortierella alpina (Mortierella alpina) JDR7 is 1: 1.
4. The PAH of claim 1, which is loaded with biochar and resistant to low temperature and high efficiency degradationSThe preparation method of the mixed bacterium granules is characterized by comprising the following steps:
1) preparation of the microbial inoculum: pseudomonas (Pseudomonas sp.) SDR4 and Mortierella alpina (Mortierella alpina) JDR7 in a volume ratio of 1:1, respectively inoculating into a complete culture medium, and shake-culturing at 15 deg.C and 120r/min for 3d to obtain microbial inoculum;
2) pretreatment of a carrier: washing the corncobs and the pine sawdust with distilled water for 4 times, air-drying for 2 days, drying in a 75 ℃ drying oven for 12 hours, roasting in a muffle furnace for 4 hours at 500 ℃ to obtain 500 ℃ corncob biochar and 500 ℃ pine sawdust biochar, and mixing according to a mass ratio of 1:1 to obtain a biochar mixture;
3) carrier immobilization: sterilizing the biochar mixture at 121 ℃ for 60min, infiltrating the biochar mixture with a proliferation culture solution according to a solid-to-liquid ratio of 1.0ml/g, culturing for 12-24h, adding the microbial inoculum prepared in the step 1 according to the mass percent of 2%, culturing at constant temperature of 15 ℃, supplementing the proliferation culture solution once every 24h, continuously supplementing for 3 times, and then continuously culturing at constant temperature of 15 ℃ for 4-5d to obtain biochar loaded low-temperature-resistant efficient degradation PAHSThe mixed bacterium particles CW-SDR4+ JDR 7.
5. The charcoal-loaded PAH with low temperature resistance and high efficiency degradation according to claim 3SThe preparation method of the mixed bacterium particles is characterized in that the complete culture medium is 10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar and 1000mL of distilled water, the pH value is 7.1-7.2, and the sterilization is carried out for 20min at 121 ℃.
6. The charcoal-loaded PAH with low temperature resistance and high efficiency degradation according to claim 3SThe preparation method of the mixed bacteria granule is characterized in that the proliferation culture solution is 4g of sucrose, 3g of yeast extract and KH2PO4 0.5g，(NH4)2HPO4 2g，MgSO4·H20.25g of O, 6.0-6.5 of pH and sterilization at 121 ℃ for 20 min.
7. The PAH of claim 1, which is loaded with biochar and resistant to low temperature and high efficiency degradationSThe application of the mixed bacteria particles in degrading polycyclic aromatic hydrocarbon pollutants.
8. Use according to claim 7, characterized in that the method is as follows: adding the biochar loaded low-temperature-resistant high-efficiency degradation PAH disclosed in claim 1 into freeze-thaw soil containing PAHs pollutantsSThe mixed bacteria granules are supplemented with water to ensure that the water content of the soil is 40 percent, and PAHs pollutants are degraded.
9. The use of claim 8 wherein the PAHs are phenanthrene and pyrene.
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