CN108220277B - Single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum and preparation method and application thereof - Google Patents
Single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum and preparation method and application thereof Download PDFInfo
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
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Abstract
The invention discloses a single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum and a preparation method and application thereof. The method is characterized in that pseudomonas SDR4 and mortierella alpina JDR7 obtained by screening in freeze-thaw soil are mixed according to the volume of 1:1 and corncob serving as a carrier. The single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum has good biological activity, low cost and simple preparation process, does not introduce secondary pollution, does not need to be recycled, and can effectively shield malignant competition, phagocytosis and toxicity of indigenous bacteria, bacteriophage and toxic substances to microorganisms, so that the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum can stably exert high efficiency in a complex environment. The invention has obvious effect of repairing the freeze-thaw soil polluted by the polycyclic aromatic hydrocarbon in cold areas.
Description
Technical Field
The invention relates to a microorganism immobilization technology, in particular to a method for degrading polycyclic aromatic hydrocarbon in freeze-thaw soil in cold regions by using corncobs as carriers and fixing low-temperature resistant mixed bacteria by an adsorption method.
The Pseudomonas (Pseudomonas sp.) SDR4 is preserved in the general microorganism center of China general microbiological culture Collection center (CGMCC) in 2017, 4 and 19 months, and the preservation number is CGMCC NO. 14048.
The Mortierella alpina (Mortierella alpina) JDR7 in the invention is preserved in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms in 2018, 1 month and 5 days, and the preservation number is CGMCC NO. 15183.
Background
Polycyclic Aromatic Hydrocarbons (PAHs) are organic compounds formed by fusing 2 or more than 2 benzene rings, and 16 common PAHs are listed as pollution-controlling substances by the United States Environmental Protection Agency (USEPA). PAHs have long-term residue, are easy to generate biological enrichment and biological amplification phenomena and are difficult to degrade in the environment; has the characteristics of two halves, namely longer half-life period and semi-volatility; three high, high stability, high accumulation and high toxicity. Due to the high octanol-water distribution coefficient and poor water solubility of the compound, the compound is often adsorbed on soil particles. Therefore, the soil becomes a main carrier of PAHs, and the PAHs enter the human body through a food chain by migrating through a soil-plant system, thereby affecting the health of the human body. Since the higher the number of benzene rings, the greater the lipid solubility, the lower the water solubility and the lower the biodegradability, fewer microorganisms are able to degrade high molecular weight PAHs.
Microbial degradation is the major pathway for removal of PAHs. Research shows that free microorganisms added into a repair field can cause great difference between an application result and an experimental result due to malignant competition of indigenous bacteria or difficulty in adapting to the environment, so that the free microorganisms cannot be well applied to in-situ contaminated soil repair. The research shows that the degradation of the strains in the microbial remediation of PAHs contaminated soil is often better than that of a single strain in the intra-seed combination or inter-seed combination degradation, but the key point is to exert the degradation capability of the high-efficiency degrading strain in a dripping way. The microorganism grows on the surface of the carrier, so that the contact chance of the microorganism and the PAHs in an adsorption state can be increased, and the degradation effect of the microorganism on the PAHs is promoted. The carrier material in soil remediation is not only an adhesive for microorganisms, but also a good buffer system for the microenvironment provided by the carrier, so that the damage of adverse conditions of soil can be shielded, and most importantly, the carrier can provide sufficient nutrition for exogenous microorganisms, and the density and activity of soil microorganisms are improved. Therefore, in the large-scale popularization and application of the microbial immobilization technology to repair the freeze-thaw soil polluted by PAHs in northern cold areas, the screening of the low-temperature-resistant efficient PAHs degrading bacteria and the selection of the immobilized carrier are the key points, and the selection of the immobilization method is the next step.
Disclosure of Invention
In order to solve the problems, the invention aims to screen low-temperature-resistant efficient PAHs degrading bacteria, jointly fix fungi and bacteria on a biomass degradable carrier material corncob, construct an immobilized low-temperature fungi-bacteria symbiotic system and provide an optimal technical approach for in-situ remediation of PAHs polluted soil microorganisms under the alternate freeze-thaw condition.
In order to achieve the purpose, the invention adopts the technical scheme that: a single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum is prepared by fixing the mixed microbial inoculum by adopting an adsorption method on a carrier prepared by mixing corncobs, wheat bran and bean cakes; the mixed microbial inoculum is prepared by mixing Pseudomonas (Pseudomonas sp.) SDR4 with the preservation number of CGMCC NO.14048 and Mortierella alpina (Mortierella alpina) JDR7 with the preservation number of CGMCC NO.15183 according to the volume ratio of 1: 1.
The single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum comprises the following components in parts by weight: wheat bran: bean cake 1: (0.05-0.08): (0.05-0.10).
The preparation method of the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed bacterial agent comprises the following steps:
1) preparing a mixed microbial inoculum: inoculating Pseudomonas (Pseudomonas sp.) SDR4 with the preservation number of CGMCC NO.14048 and Mortierella alpina (Mortierella alpina) JDR7 with the preservation number of CGMCC NO.15183 into a complete culture medium according to the volume ratio of 1:1, and performing shake mixed culture at 15 ℃ and 120r/min for 3d to prepare a mixed microbial inoculum;
2) pretreatment of a carrier: crushing corncobs, adding the crushed corncobs into quicklime water solution, soaking and uniformly stirring, covering a film and sealing for 24 hours; filtering to obtain corncobs, uniformly mixing the treated corncobs with wheat bran and bean cakes, adjusting the water content to be 40-50% and the pH value to be 6.5-7.0 to obtain a pretreated carrier;
3) adsorption and fixation: sterilizing the pretreated carrier at 121 ℃ for 60min, infiltrating the pretreated carrier with a proliferation culture solution according to a solid-to-liquid ratio of 1.0ml/g, and culturing for 12-24 h; adding the mixed microbial inoculum prepared in the step 1) according to the inoculum size of 10%, culturing at constant temperature of 15 ℃, supplementing the proliferation culture solution once every 24h, continuously supplementing for 3 times, and culturing at constant temperature of 15 ℃ for 4-5 days to obtain the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum.
According to the preparation method of the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum, the complete culture medium comprises 5g of beef extract, 10g of peptone, 10g of glucose, 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 at 121 ℃ for 20 min.
In the preparation method of the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum, the multiplication 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.
The single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed bacterial agent is applied to degradation of polycyclic aromatic hydrocarbon pollutants in freeze-thaw soil. The method comprises the following steps: and adding the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum into freeze-thaw soil containing polycyclic aromatic hydrocarbon pollutants, and adding or not adding water to ensure that the water content of the soil is 20-30%.
The polycyclic aromatic hydrocarbon is an organic compound formed by fusing 2 or more than 2 benzene rings together. Preferably, the polycyclic aromatic hydrocarbon is phenanthrene, pyrene and benzo [ a ] pyrene.
The screening method of the Pseudomonas (Pseudomonas sp.) SDR4 provided by the invention comprises the following steps:
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) respectively, placing in nutrient medium for natural enrichment culture for 4d, inoculating 10mL of the enrichment culture solution in inorganic salt selective medium for selective culture, performing acclimation by adopting a method of periodically and quantitatively switching to gradually increase carbon source concentration, and performing acclimation at 15 ℃ for 120 r.min－1And (7) carrying out shake culture for 7 d. Initial concentrations of Phe, Pyr and BaP in the selection medium were 10, 10 and 5 mg.L－1The 2 nd transfer concentration was increased to 20, 20 and 10 mg.L－1The 3 rd transfer concentrations were increased to 30, 30 and 15 mg.L－1. Diluting 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 suspension, respectively coating on solid nutrient medium plates added with PAHs membranes, performing inverted culture at 15 ℃ until visible obvious bacterial colonies grow out, continuously scribing the plates and rotating the plates for 3 times, picking out large bacterial colonies and growing more than the bacterial coloniesFast single bacterial strain is inoculated into the slant of nutrient medium sprayed with PAHs, and is respectively cultured for about 10 days at 28, 15 and 4 ℃ in an inverted way, the growth condition is observed, and the low-temperature PAHs degrading bacteria are preliminarily screened. 4 strains of bacteria are primarily screened out and are marked as SDR2-SDR5, the strains obtained by primary screening are suspended into experimental simulated soil according to the bacterial suspension of 10 percent, and the bacterial concentration is 6 multiplied by 108CFU/mL, the concentration of Pyr and BaP are respectively 30mg/kg and 15 mg/kg. And supplementing sterile water at any time to ensure that the soil moisture is 30%, placing the soil in a freeze-thaw circulating box for dark culture, freezing for 12h at the temperature of-5 ℃, unfreezing for 12h at the temperature of 15 ℃, sampling for 0, 15, 30, 45 and 60d respectively, extracting, purifying, performing liquid chromatography, and determining the residual quantity of PAHs in the soil, wherein the result is that the SDR4 has a remarkable 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.
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 MEGA 5.0 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 strain SDR4 naturally clusters with the 16S rDNA sequence of Pseudomonas, and has a similarity of 99%, so that the strain SDR4 belongs to Pseudomonas sp.
The inorganic salt liquid culture medium comprises: k2HPO41.0g，(NH4)2SO4 5g，MgSO4﹒7H20.5g of O, 7.0-7.2 of pH, 1L of distilled water for constant volume, and sterilizing at 121 ℃ for 25min, wherein the nutrient medium (CM medium) is as follows: 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.
(II) screening of Mortierella alpina (Mortierella alpina) JDR7 provided by the invention
3 points from the contaminated area of the chicken WestAnd (3) sampling soil samples with the sampling depth of about 50cm, grinding, sieving by a 2mm sieve, mixing the soil samples, filling the mixture 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) respectively, placing in nutrient medium for natural enrichment culture for 4d, inoculating 10mL of the enrichment culture solution in inorganic salt selective medium for selective culture, performing acclimation by adopting a method of periodically and quantitatively switching to gradually increase carbon source concentration, and performing acclimation at 15 ℃ for 120 r.min－1And (7) carrying out shake culture for 7 d. Initial concentrations of Phe, Pyr and BaP in the selection medium were 10, 10 and 5 mg.L－1The 2 nd transfer concentration was increased to 20, 20 and 10 mg.L－1The 3 rd transfer concentrations were increased to 30, 30 and 15 mg.L－1. Diluting 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. Primarily screening 3 strains of fungi, recording as JDR6-JDR8, and placing the strains obtained by primary screening into experimental simulated soil according to 10% of strain suspension, wherein the strain concentration is 6 multiplied by 108CFU/mL, the concentration of Pyr and BaP are respectively 30mg/kg and 15 mg/kg. And (2) supplementing sterile water at any time to ensure that the soil moisture is 30%, placing the soil in a freeze-thaw circulating box for dark culture, freezing for 12h at the temperature of-5 ℃, unfreezing for 12h at the temperature of 15 ℃, sampling for 0, 15, 30, 45 and 60d respectively, extracting, purifying, performing liquid chromatography, and determining the residual quantity of PAHs in the soil, wherein the JDR7 effect is obvious.
The strain JDR7 is white on a beef extract peptone plate, petal-shaped in the early growth stage and developed hypha in the later growth stage, grows densely, is tubular under a scanning electron microscope, and is an industrial oil-producing aerobic fungus with good food safety evaluation record.
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 MEGA 5.0 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.
The invention has the following advantages: in the invention, the applied bacteria are low temperature resistant high-efficiency PAHs degrading bacteria, the activity of the bacteria can not be inhibited in cold regions, in addition, the used corncobs have loose surface shapes, uniform internal tissues, developed pores, low cost and simple preparation process, and secondary pollution can not be introduced without recovery. In addition, the microenvironment of the corn cob immobilized mixed bacteria can effectively shield malignant competition, phagocytosis and poison of indigenous bacteria, bacteriophage and toxic substances to microorganisms, so that the corn cob immobilized mixed bacteria can stably exert high efficiency in a complex environment. The carrier corncob can improve the concentration of the microorganism observed by SEM (scanning electron microscope), and the removal rate of Phe, Pyr and BaP is 58.49%, 45.91% and 37.07% respectively after 60 days.
Drawings
Pseudomonas (Pseudomonas sp.) SDR4, deposit name: china general microbiological culture Collection center (CGMCC), abbreviated as: CGMCC, storage unit address: west road No.1, north chen, chaoyang district, beijing, zip code: 100101. the preservation date is 2017, 4 and 19 months, and the preservation number is CGMCC NO. 14048.
Mortierella alpina (Mortierella alpine) JDR7, deposit name: china general microbiological culture Collection center (CGMCC), abbreviated as: CGMCC, storage unit address: west road No.1, north chen, chaoyang district, beijing, zip code: 100101. the preservation date is 2018, 1 month and 5 days, and the preservation number is CGMCC NO. 15183.
FIG. 1 is a phylogenetic tree for the construction of SDR4 based on the 16S rDNA sequence and the Neighbor-Joining method.
FIG. 2 is a phylogenetic tree for the construction of JDR7 based on the ITS sequences and the Neighbor-Joining method.
FIG. 3 is a photograph of a scanning electron microscope of X2000 of the mixed immobilized cob.
FIG. 4 is a graph comparing the degradation of phenanthrene (Phe) by three degradation methods.
FIG. 5 is a graph comparing the degradation of pyrene (Pyr) by three degradation methods.
FIG. 6 is a graph showing the comparison of the degradation of benzo [ a ] pyrene (Bap) by three degradation methods.
Detailed Description
Embodiment of the method for degrading polycyclic aromatic hydrocarbon pollutants in freeze-thaw soil
Culture of Pseudomonas sp (SDR 4)
Complete medium: 5g of beef extract, 10g of peptone, 10g of glucose, 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 beef extract is sterilized at 121 ℃ for 20 min.
Inoculating Pseudomonas (Pseudomonas sp.) SDR4 with the preservation number of CGMCC NO.14048, which is preserved on a slant culture medium, into a complete culture medium according to the inoculation amount of 5 percent, and carrying out shake culture at 15 ℃ and 120r/min for 30-40 h until the bacterial density is 6 multiplied by 108CFU·mL－1Obtaining the bacterial suspension of the Pseudomonas (Pseudomonas sp.) SDR 4.
Culture of Mortierella alpina (Mortierella alpina) JDR7
Complete medium: 5g of beef extract, 10g of peptone, 10g of glucose, 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 beef extract is sterilized at 121 ℃ for 20 min.
Collecting Mortierella alpina (Mortierella alpina) JDR7 with preservation number of CGMCC NO.15183, inoculating 5% of the culture medium into complete culture medium, shake culturing at 15 deg.C and 120r/min for 30-40 hr until spore density in spore suspension is 6 × 108CFU·mL－1And obtaining the fungus suspension of the Mortierella alpina (Mortierella alpina) JDR 7.
(III) Mixed bacterial agent
Complete medium: 5g of beef extract, 10g of peptone, 10g of glucose, 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 beef extract is sterilized at 121 ℃ for 20 min.
According to the volume ratio of 1:1, taking Pseudomonas (Pseudomonas sp.) SDR4 bacterial suspension and Mortierella alpina (Mortierella alpina) JDR7 fungal bacterial suspension, respectively inoculating the bacterial suspensions into a complete culture medium according to the inoculation amount of 10%, and carrying out shake mixed culture at 15 ℃ for 3d by 120r/min to obtain the mixed microbial inoculum.
(IV) pretreatment of the support
Crushing fresh and dried corncobs to the particle size of 1.5-2 cm, and adding the crushed corncobs to 0.1 mol.L-1Soaking and stirring quicklime water solution uniformly, covering with a film and sealing for 24 h; filtering to obtain corncobs, uniformly mixing the treated corncobs with wheat bran and bean cakes, adjusting the water content to be 45% and the pH value to be 6.5-7.0 to obtain a pretreated carrier; according to weight ratio, corncob: wheat bran: bean cake 1: (0.05-0.08): (0.05-0.10).
(V) adsorption fixation
The proliferation culture solution is: 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 sterilization at 121 ℃ for 20 min.
Sterilizing the pretreated carrier prepared in the step (four) at 121 ℃ for 60min, infiltrating the pretreated carrier with a proliferation culture solution according to a solid-to-liquid ratio of 1.0ml/g, and culturing for 12-24 h; and (3) adding the mixed microbial inoculum prepared in the step (three) according to the inoculation amount of 10%, culturing at a constant temperature of 15 ℃, supplementing a proliferation culture solution every 24 hours, continuously supplementing for 3 times, then continuing culturing at the constant temperature of 15 ℃ for 4-5 days, naturally fixing part of microorganisms on a carrier along with the growth of the mixed microbial inoculum during the culture process, washing the cells of the immobilized mixed microbial inoculum with deionized water for several times after the culture is finished, and preparing the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum which is marked as Y-SDR4+ JDR 7.
(VI) degradation
Soil for degradation experiments: the surface layer clean soil is 0-20 cm collected from Shenyang ecological test station of Chinese academy of sciences, the soil is meadow brown soil, and the basic physicochemical properties are as follows: the pH was 6.72, the organic carbon was 17.8g/kg, the total nitrogen was 1.1g/kg, the total phosphorus was 0.35g/kg, and the exchange capacity (CEC) was 45.04 mg/kg. And (3) sieving the soil by using a 2mm sieve, subpackaging the sieved soil into culture bottles, sterilizing each bottle for 60min at the high temperature of 121 ℃, cooling, quantitatively adding acetone solutions of Phe, Pyr and Bap into each bottle, uniformly mixing the acetone solutions to ensure that the initial concentrations of Phe, Pyr and Bap are 120mg/L, 120mg/L and 60mg/L respectively, and standing overnight to obtain a soil sample containing Phe, Pyr and Bap pollutants.
The invention comprises the following steps: adding the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed bacterial agent (Y-SDR4+ JDR7) into a soil sample containing Phe, Pyr and Bap pollutants according to the inoculation amount of 2%, placing the soil sample into a freeze-thaw circulating box to be cultured in a dark place, freezing the soil sample for 12 hours at the temperature of-5 ℃, unfreezing the soil sample for 12 hours at the temperature of 15 ℃, replenishing sterile water at any time to enable the soil moisture to be 20%, and sampling the soil sample for 0, 15, 30, 45 and 60 days respectively.
Comparative example 1: and (2) directly adding the mixed microbial inoculum (SDR4+ JDR7) prepared in the step (three) into a soil sample containing Phe, Pyr and Bap pollutants, putting the soil sample into a freeze-thaw circulating box to be cultured in the dark, freezing the soil sample for 12 hours at the temperature of-5 ℃, unfreezing the soil sample for 12 hours at the temperature of 15 ℃, supplementing sterile water at any time to ensure that the soil moisture is 20 percent, and sampling the soil sample at 0, 15, 30, 45 and 60 days respectively.
Comparative example 2: adding sterile water (CK) into a soil sample containing Phe, Pyr and Bap pollutants according to the inoculation amount of 2%, placing the soil sample in a freeze-thaw circulating box for dark culture, freezing for 12h at-5 ℃ and unfreezing for 12h at 15 ℃, supplementing sterile water at any time to ensure that the soil moisture is 20%, and sampling at 0, 15, 30, 45 and 60 days respectively.
(seventh) result
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 into a pipette gun for ultrasonic treatment for 2 times, and performing ultrasonic treatment for 2 hours each time, wherein the water temperature in an ultrasonic instrument is kept below 30 ℃ during ultrasonic treatment. And putting the ultrasonic centrifuge tube into a centrifuge, centrifuging for 10min at the rotating speed of 4000r/min, and pouring the supernate into a heart-shaped bottle with a corresponding number for rotary evaporation after the centrifugation is finished. Absorbing 2mL of n-hexane to dissolve a sample, transferring the sample to a purification column (1g of neutral A12O3+1g of silica gel +1g of anhydrous sodium sulfate) for purification, leaching with 30mL of n-hexane and dichloromethane (volume ratio is 1:1), collecting filtrate, concentrating to about 1mL, drying by using soft nitrogen, and measuring PAHs (polycyclic aromatic hydrocarbons) by using 1mL of acetonitrile at constant volume and liquid chromatography.
Detecting samples to be detected of the polluted soil by using a fluorescence and ultraviolet detector, wherein the chromatographic conditions are as follows: the chromatographic column is a special column for polycyclic aromatic hydrocarbon analysis ZORBAX eclipsePAH (4.6m is multiplied by 250mm is multiplied by 5 um); the column temperature is 25 ℃; the mobile phase is chromatographic pure acetonitrile and water, wherein the acetonitrile is 60 percent to 40 percent of water, and the flow rate is 1.000 mL/min; the sample amount is 10 mu L, various PAHs are qualitatively determined by chromatographic peak retention time, and quantitatively determined by an external standard method, and the recovery rate of the method is 79.56-92.48 percent.
Corncobs are natural organic matters widely existing in the environment, mainly comprise cellulose (32-36%), hemicellulose (35-40%) and lignin, are loose in surface shape, uniform in internal tissue and developed in pores. As shown in FIG. 3, SEM scanning electron microscope shows that the corn cob can improve the density and activity of microorganisms, the filamentous fungus JDR7 grows densely and is criss-cross netted, some bacteria are adsorbed on the surface of the corn cob and attached to the surface of fungal hyphae and move along with the growth of the hyphae, so that the corn cob can better contact with pollutants and play a role in bacteria-fungus synergy.
As shown in figures 2-4, the corn cob immobilized mixed bacteria are put into the soil polluted by PAHs, and the remediation effect on the freeze-thaw soil polluted by polycyclic aromatic hydrocarbons in cold regions is obvious. After 60 days, the removal rates of Phe, Pyr and BaP are 58.49%, 45.91% and 37.07% respectively, which are improved by 23.20%, 8.76% and 9.66% respectively compared with those of a free bacterium SDR4+ JDR 7. In addition, the corncob has wide sources, low price and good air permeability in northern areas, has certain biodegradability, is decomposed and converted into organic matters to become important components of soil, and cannot cause secondary pollution to the soil.
Claims (2)
1. The preparation method of the single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum is characterized by comprising the following steps: pseudomonas with preservation number of CGMCC NO.14048 ()Pseudomonas sp.) Mortierella alpina (with preservation number of CGMCC number 15183) and SDR4Mortierella alpine ) JDR7 was inoculated into complete medium at a volume ratio of 1:1And carrying out shake bed mixed culture at 15 ℃ for 3d at 120r/min to obtain a mixed microbial inoculum, and preparing the immobilized mixed microbial inoculum by using corncobs as materials and adopting an adsorption immobilization method.
2. The single-carrier low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed microbial inoculum prepared by the method of claim 1 is applied to degradation of polycyclic aromatic hydrocarbon pollutants in freeze-thaw soil.
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| CN110152622B (en) * | 2019-06-12 | 2021-10-22 | 辽宁大学 | Biomass adsorbent for adsorbing PAHs in water and preparation method and application thereof |
| CN111117993B (en) * | 2020-01-15 | 2023-04-07 | 辽宁大学 | Biomass immobilized fungus-bacterium mixed particle for degrading PAHs in soil as well as preparation method and application thereof |
| CN114214312A (en) * | 2022-01-05 | 2022-03-22 | 辽宁大学 | Charcoal-loaded low-temperature-resistant efficient degradation PAHSMixed bacterium particle and preparation method and application thereof |
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