CN110038894B - Microbial remediation method for petroleum hydrocarbon contaminated soil - Google Patents
Microbial remediation method for petroleum hydrocarbon contaminated soil Download PDFInfo
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Abstract
The invention discloses a microbial remediation method for petroleum hydrocarbon contaminated soil, which belongs to the technical field of remediation of contaminated soil, and particularly relates to a microbial remediation method for petroleum hydrocarbon contaminated soil.
Description
Technical Field
The invention belongs to the technical field of remediation of contaminated soil, and particularly relates to a microbial remediation method of petroleum hydrocarbon contaminated soil.
Background
With the development of science and technology, the usage amount of the motor vehicles is continuously increased, and gas stations of the motor vehicles are visible anywhere on roads in urban areas or towns. For safety reasons, the gasoline and diesel storage tanks and the oil delivery pipes in the gasoline station are usually buried underground. Along with the increase of burying time, the phenomenon of damaged seepage can appear in oil storage tank and defeated oil pipe, and then leads to its peripheral soil to receive certain pollution.
At present, the method for treating the oil sludge at home and abroad comprises the following steps: solvent extraction, hot-wash, solidification, pyrolysis, biological (in situ, ex situ). The solvent extraction method and the hot washing method have large energy consumption and high cost, the solidification method is easy to cause secondary pollution, and the hot connection method can be used for recovering oil-containing sludge of crude oil. The microbial technology is widely accepted by experts at home and abroad due to unique advantages, is an extension of the traditional biological treatment method, and has the advantages of large treatment area, no secondary pollution, low cost, various and flexible forms, no damage to soil structures and the like.
Numerous studies have shown that the biodegradability of petroleum hydrocarbons varies depending on the type and size of the hydrocarbon molecules they contain. Petroleum hydrocarbons of medium chain length (C10-C24) are most susceptible to degradation; short chain petroleum hydrocarbons are generally toxic to many microorganisms, making their degradation difficult for microorganisms. Diesel and gasoline are the main pollutants in contaminated soil near petrol stations. Wherein the diesel oil is a complex hydrocarbon (C10-C22) mixture, the main components of the gasoline are C5-C12 aliphatic hydrocarbon and naphthenic hydrocarbon, and when the soil contains the diesel oil and the gasoline simultaneously, microorganisms are difficult to propagate in the soil well, so that the repairing effect of the microorganisms on the petroleum hydrocarbon polluted soil is poor.
Disclosure of Invention
Aiming at the defects in the prior art, the first object of the invention is to provide a microbial remediation method for petroleum hydrocarbon contaminated soil, wherein microorganisms can multiply in a large amount in the contaminated soil containing diesel oil and gasoline, and the method has a good soil remediation effect.
In order to achieve the first object, the invention provides the following technical scheme: a microbial remediation method of petroleum hydrocarbon contaminated soil comprises the following steps: firstly, collecting and mixing polluted soil at different depths near a gas station to obtain a sample, and after the sample is dried in the air, crushing, removing impurities, sieving by a 20-mesh sieve, and mixing to obtain soil powder; spreading 1g of the soil powder obtained in the step I on a separation culture medium which respectively takes diesel oil and gasoline as carbon sources, and placing the separation culture medium in an incubator at the temperature of 30-36 ℃ for culturing for 96 hours to obtain diesel oil degradation bacterial colonies and gasoline degradation bacterial colonies; respectively picking diesel oil degrading bacterial colonies and gasoline degrading bacterial colonies on a culture medium in the step II by strain amplification, inoculating the picked diesel oil degrading bacterial colonies into a liquid culture medium taking diesel oil as a carbon source, simultaneously inoculating the picked gasoline degrading bacterial colonies into a liquid culture medium taking gasoline as a carbon source, then placing the two liquid culture mediums on a shaking table, and culturing for 7d at the temperature of 30-36 ℃ and at the speed of 150r/min to obtain a diesel oil degrading strain culture solution and a gasoline degrading strain culture solution with the viable count of 1.5 multiplied by 109-2.0 multiplied by 109/L; compounding strains, namely compounding the diesel oil degrading strain culture solution and the gasoline degrading strain culture solution obtained by the step three according to the weight ratio of 1:1 to obtain a composite strain culture solution; fifthly, restoring and collecting the polluted soil near the gas station by using the strain, putting the composite strain culture solution obtained in the step (iv) into the polluted soil, putting 1L of strain solution into every 1m of 3 soil, turning the soil until the strain culture solution is uniformly dispersed, stacking the strain culture solution into a pile, keeping the water content of the soil at 20-30% at room temperature, and periodically stirring and ventilating.
By adopting the technical scheme, the invention collects the soil near the gas station to be repaired, respectively screens out strains capable of decomposing diesel oil and gasoline by using a separation culture medium, increases the strain amount by using a liquid culture medium, and finally directly puts the prepared strain culture solution into the soil near the corresponding gas station. Therefore, strains screened from the soil needing to be repaired can be well adapted to the environment of the soil and propagate in large quantities, so that diesel oil and gasoline can be quickly and effectively decomposed, carbon dioxide and water which are pollution-free to the environment are correspondingly generated, and a good soil repairing effect is achieved.
Further, in the step (II), the components of the separation culture medium comprise, by weight, 0.5-1 part of beef extract, 0.5-1 part of peptone, 3-5 parts of NaCl, 20-25 parts of agar, 0.01-0.03 part of antifungal agent, 1-2 parts of diesel oil or gasoline, 2-5 parts of biosurfactant and 1000 parts of distilled water.
By adopting the technical scheme, the beef extract and the peptone in the separation culture medium are used as nutrient components of the strains, the antifungal agent can inhibit fungi and actinomycetes existing in soil, NaCl is used for adjusting the osmotic pressure of strain propagation, and agar can promote the separation culture medium to be in a solid state, so that the separation of the strains is facilitated; the biosurfactant promotes diesel oil and gasoline to be better dispersed in distilled water. Therefore, the strain capable of decomposing diesel oil or gasoline firstly decomposes beef extract and peptone to carry out certain amount of reproduction; after the strain is propagated to a certain amount, the nutrients of the beef extract and the peptone are not enough to meet the requirement of the strain for propagation, and the strain which is difficult to decompose diesel oil or gasoline is apoptotic due to lack of nutrient substances, so that the strain which can decompose diesel oil or gasoline is screened out, and the strain has good repairing effect and environmental friendliness on the soil polluted by diesel oil and gasoline.
Further, in the second step, the antifungal agent is a mixture of cycloheximide and nystatin.
Further, in the second step, the weight ratio of the cycloheximide to the nystatin is 1: 1.
By adopting the technical scheme, the cycloheximide is an antibiotic generated by fermenting actinomycetes, has an inhibiting effect on pathogenic bacteria such as yeast, army, protozoa and the like, and has no obvious inhibiting effect on bacteria; nystatin is a polyene antifungal drug that can bind to sterols on fungal cell membranes, causing a change in the permeability of the cell membrane, which results in loss of important cellular contents and thus an antifungal effect. When cycloheximide and nystatin are used simultaneously and the weight ratio is 1:1, the stability of nystatin can be increased, the screened strains can quickly and effectively decompose diesel oil and gasoline in soil, and the specificity is good.
Further, in the second step, the biosurfactant is sophorolipid.
By adopting the technical scheme, the sophorolipid is a glycolipid biosurfactant, can better promote diesel oil and gasoline to be better dispersed in distilled water, and is convenient for strains to uniformly distribute and grow. The microbial inoculum has the characteristics of no toxicity, 100 percent biodegradability, high temperature resistance, high salt resistance, environmental friendliness and the like, so the microbial inoculum has small damage to strains, and can be used as a nutrient component of the strains to a certain extent for the growth of the strains. Compared with other biosurfactants, the strain correspondingly screened has better degradation effect.
Further, in the second step, the preparation method of the separation medium comprises: weighing beef extract, peptone, NaCl, agar, biosurfactant, diesel oil, gasoline and distilled water according to the set weight parts, placing in a conical flask, stirring until all components are uniformly dispersed, sterilizing at 121 ℃ for 20min, cooling to 50-60 ℃, adding an antifungal agent, uniformly mixing, uniformly subpackaging in a culture dish, and cooling to solidify to obtain the beef extract.
By adopting the technical scheme, the separation culture medium is subjected to high-temperature sterilization treatment after uniformly dispersing the components in the preparation process, so that the conditions of agglomeration, precipitation and the like of the components are reduced, the original microorganisms in the culture medium can be eliminated by the sterilization treatment, the competition phenomenon caused by survival among the microorganisms is reduced, and the separation culture medium is favorable for better strain screening. The antifungal agent is added after sterilization, so that the decomposition of the antifungal agent caused by heating and pressurization is reduced, and good antifungal performance is ensured.
Further, in the third step, the liquid culture medium comprises, by weight, 1-3 parts of peptone, 3-5 parts of yeast extract, 1-2 parts of glucose, 1-2 parts of diesel oil or gasoline, 2-5 parts of a biosurfactant and 1000 parts of distilled water.
Further, in the third step, the bioactive agent is sophorolipid.
By adopting the technical scheme, the peptone, the yeast extract and the glucose provide sufficient nutrients for strain expansion culture, the strains are favorably and rapidly propagated, and the environmental adaptability of the strains is also ensured on the basis that diesel oil and gasoline provide carbon sources required by the growth of the strains, so that the amplified strains can be better suitable for the corresponding soil needing to be repaired. The liquid culture medium can be used for quickly amplifying the required strains and has the characteristics of simple components and excellent amplification effect.
Further, in the third step, the preparation method of the liquid medium comprises: weighing the components of the liquid culture medium according to the set weight parts, placing the components in a conical flask, stirring until the components are uniformly dispersed, sterilizing at 121 ℃ for 20min, and cooling to obtain the liquid culture medium.
By adopting the technical scheme, the liquid culture medium is uniformly dispersed and then subjected to high-temperature sterilization treatment in the preparation process, so that the conditions of agglomeration, precipitation and the like of the components are reduced, the original microorganisms in the culture medium can be eliminated through the sterilization treatment, the competition phenomenon caused by survival among the microorganisms is reduced, and the liquid culture medium is favorable for better strain amplification.
In conclusion, the invention has the following beneficial effects: 1. according to the method, the soil to be repaired is collected, the required repairing strain is screened from the soil, the strain is expanded and cultured, and the strain is put in, so that diesel oil and gasoline can be rapidly and effectively decomposed, carbon dioxide and water which are pollution-free to the environment are correspondingly generated, and the method has good soil repairing effect and environmental friendliness; 2. the invention selects the self-made separation culture medium and liquid culture medium, the strains which are correspondingly deleted and amplified can quickly and effectively degrade diesel oil and gasoline in soil, and the invention has the characteristics of simple components and good effect.
Drawings
FIG. 1 is a general operational process diagram of microbial remediation of petroleum hydrocarbon contaminated soil.
FIG. 2 is a diagram of the detailed operation of the microorganism for remediating petroleum hydrocarbon contaminated soil.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
1. Materials and apparatus the reagents of the invention are all commercially available in chemical purity.
1M NaOH solution: is prepared by dissolving 4g of NaOH in 100mL of distilled water.
1M HCl solution: is prepared by dissolving 8.77mL of concentrated hydrochloric acid with the mass percent of 38% in 100mL of distilled water.
An incubator: available from Shanghai Hotan scientific instruments Inc. under model number 250A.
Shaking table: available from Shanghai Hotan scientific instruments Inc. under the model number HT-211 series.
An autoclave: purchased from Xuzhou Huaibo instruments, Inc. under the model LDZH-200 KBS.
An aseptic operation table: purchased from Shanghai Hotan scientific instruments Inc. under the model number SW-CJ-2D (2G).
2. Example 2.1 preparation of isolation Medium a, weighing: diesel degradation colony culture medium: 0.5-1 part of beef extract, 0.5-1 part of peptone, 3-5 parts of NaCl, 20-25 parts of agar, 0.01-0.03 part of antifungal agent, 1-2 parts of diesel oil, 2-5 parts of biosurfactant and 1000 parts of distilled water.
Gasoline degradation colony culture medium: 0.5-1 part of beef extract, 0.5-1 part of peptone, 3-5 parts of NaCl, 20-25 parts of agar, 0.01-0.03 part of antifungal agent, 1-2 parts of gasoline, 2-5 parts of biosurfactant and 1000 parts of distilled water.
b. Dissolving: pouring distilled water into a conical flask, sequentially adding beef extract, peptone, NaCl and agar, fully mixing, adding a biosurfactant, mixing, adding diesel oil or gasoline, and stirring until the components are uniformly dispersed to respectively obtain a diesel oil mixed solution and a gasoline mixed solution.
c. And (3) sterilization: sealing the conical bottle with cotton plug and kraft paper, placing into autoclave, and sterilizing at 121 deg.C for 20 min.
d. Subpackaging and storing: taking out the sterilized conical flask together with the mixed solution, placing into an aseptic operation table, turning on an ultraviolet lamp, cooling the mixed solution to 50-60 ℃, turning off the ultraviolet lamp and turning on a ventilation program, sterilizing an antifungal agent packaging bottle with alcohol, bringing the sterilized antifungal agent packaging bottle into the aseptic operation table together with an operator, adding the antifungal agent into the diesel mixed solution or the gasoline mixed solution, uniformly subpackaging the mixture into an empty culture dish, cooling to solidify to obtain a diesel separation culture medium and a gasoline separation culture medium, sealing and packaging by a preservative film, and storing at 4 ℃.
2.2, preparation of liquid medium a, weighing:
diesel oil degrading strain culture solution: 1-3 parts of peptone, 3-5 parts of yeast extract, 1-2 parts of glucose, 1-2 parts of diesel oil, 2-5 parts of biosurfactant and 1000 parts of distilled water.
Gasoline degradation strain culture solution: 1-3 parts of peptone, 3-5 parts of yeast extract, 1-2 parts of glucose, 1-2 parts of gasoline, 2-5 parts of biosurfactant and 1000 parts of distilled water.
b. Dissolving: pouring distilled water into a conical flask, sequentially adding peptone, yeast extract and glucose, mixing, adding biosurfactant, mixing, adding diesel oil or gasoline, and stirring until the components are uniformly dispersed to obtain a diesel oil liquid culture medium and a gasoline liquid culture medium.
c. And (3) sterilization: sealing the conical bottle with cotton plug and kraft paper, placing into autoclave, and sterilizing at 121 deg.C for 20 min.
d. And (4) cooling and storing: taking out the sterilized conical flask filled with diesel oil liquid culture medium or gasoline liquid culture medium, cooling to room temperature, and storing at 4 deg.C.
2.3 a method for microbial remediation of petroleum hydrocarbon contaminated soil, see figures 1 and 2, comprising the steps of: firstly, collecting and mixing polluted soil at positions 10cm, 50cm, 1m, 2m and 5m near a gas station to obtain a sample, air-drying the sample, crushing the sample, removing impurities, sieving the sample with a 20-mesh sieve, and mixing the sample uniformly to obtain soil powder; secondly, screening and selecting 3g of soil powder obtained in the first step by a strain sieve, paving the soil powder on a separation culture medium which respectively takes diesel oil and gasoline as carbon sources, and culturing the separation culture medium in an incubator at the temperature of 30-36 ℃ for 96 hours to obtain diesel oil degradation bacterial colonies and gasoline degradation bacterial colonies; respectively picking a diesel oil degrading bacterial colony and a gasoline degrading bacterial colony on the culture medium in the second step, inoculating the picked diesel oil degrading bacterial colony into a liquid culture medium taking diesel oil as a carbon source, simultaneously inoculating the picked gasoline degrading bacterial colony into a liquid culture medium taking gasoline as a carbon source, then placing the two liquid culture mediums on a shaking table, and culturing for 7 days at the temperature of 30-36 ℃ and at the speed of 150r/min to obtain a diesel oil degrading strain culture solution and a gasoline degrading strain culture solution with the viable count of 1.5 multiplied by 109-2.0 multiplied by 109/L; compounding strains, namely compounding the diesel oil degrading strain culture solution and the gasoline degrading strain culture solution obtained by the step three according to the weight ratio of 1:1 to obtain a composite strain culture solution; fifthly, restoring and collecting the polluted soil near the gas station by using the strain, putting the composite strain culture solution obtained in the step (iv) into the polluted soil, putting 1L of strain solution into every 1m of 3 soil, turning the soil until the strain culture solution is uniformly dispersed, stacking the strain culture solution into a pile, keeping the water content of the soil at 20-30% at room temperature, and periodically stirring and ventilating.
2.4, examples 1-5 the components and amounts of the separation medium, the components and amounts of the liquid medium, and the parameters of the microbial remediation process were adjusted based on the procedure of 2.1-2.3, as shown in Table one below.
TABLE parameter adjustment for examples 1-5
2.5 comparative example
Comparative example 1: application publication No. CN106734181A discloses a microbial remediation method for petroleum-contaminated soil.
Comparative example 2: application publication No. CN101091957B discloses a microbial remediation method for petroleum-contaminated soil.
3. The invention collects the polluted soil near the gas stations in 5 different areas and with different pollution degrees as samples, and the samples are used as 5 parallel experiments to verify the repairing effect of the invention.
The above 5 samples were treated according to the remediation methods of examples 1 to 6 and comparative examples 1 and 2, respectively, and the diesel oil reduction and the gasoline reduction of the contaminated soil were measured on days 7, 15, 30 and 45 of remediation, respectively. The results of the measurements are shown in Table II below.
TABLE two repair results for examples 1-6 and comparative examples 1 and 2
Referring to Table two, comparing the remediation results of examples 1-6 with the remediation results of comparative examples 1 and 2, respectively, it can be seen that the present invention degrades > 80% diesel and > 75% gasoline at day 45 of soil remediation, whereas comparative examples 1 and 2 degrade only > 60% diesel and > 45% gasoline at day 45 of soil remediation. Therefore, the method can quickly and effectively repair the soil polluted by diesel oil and gasoline, and has the characteristics of short repair period, excellent repair effect and environmental friendliness.
Comparing the repair results of examples 1-3 with the repair results of examples 4-6, respectively, it can be seen that examples 1-3 degrade > 80% diesel and > 75% gasoline on day 30, and examples 4-6 require 45 days to achieve this degradation. Thus, example 1 to example 3 are preferred examples.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (7)
1. A microbial remediation method of petroleum hydrocarbon contaminated soil is characterized by comprising the following steps:
firstly, soil collection
Collecting contaminated soil at different depths near a gas station, uniformly mixing to obtain a sample, air-drying, crushing, removing impurities, sieving with a 20-mesh sieve, and uniformly mixing to obtain soil powder;
② screening strains
Spreading 3g of the soil powder obtained in the first step on a separation culture medium which respectively takes diesel oil and gasoline as carbon sources, and placing the separation culture medium in an incubator at the temperature of 30-36 ℃ for culturing for 96 hours to obtain diesel oil degradation bacterial colonies and gasoline degradation bacterial colonies;
③ amplification of strains
Respectively picking diesel oil degrading bacterial colony and gasoline degrading bacterial colony on the culture medium in the second step, inoculating the picked diesel oil degrading bacterial colony into liquid culture medium with diesel oil as carbon source, simultaneously inoculating the picked gasoline degrading bacterial colony into liquid culture medium with gasoline as carbon source, placing the two liquid culture mediums on a shaking table, and culturing at 30-36 ℃ and 150r/min for 7d to obtain the viable count of 1.5 multiplied by 109-2.0×109Diesel oil degrading strain culture solution and gasoline degrading strain culture solution in each liter;
fourthly, compounding of strains
Compounding the diesel oil degrading strain culture solution and the gasoline degrading strain culture solution obtained by the step three according to the weight ratio of 1:1 to obtain a composite strain culture solution;
fifthly, strain repair
Collecting contaminated soil near a gas station, putting the composite strain culture solution obtained in the step (IV) into the contaminated soil, putting 1L of strain solution into every 1m of 3 soil, turning the soil until the strain culture solution is uniformly dispersed, piling the strain culture solution into a pile, keeping the water content of the soil at 20-30% at room temperature, and periodically stirring and ventilating;
in the second step, the components of the separation culture medium comprise, by weight, 0.5-1 part of beef extract, 0.5-1 part of peptone, 3-5 parts of NaCl, 20-25 parts of agar, 0.01-0.03 part of antifungal agent, 1-2 parts of diesel oil or gasoline, 2-5 parts of biosurfactant and 1000 parts of distilled water;
the biosurfactant is sophorolipid.
2. The method of claim 1, wherein the antifungal agent is a mixture of cycloheximide and nystatin.
3. The microbial remediation method of petroleum hydrocarbon contaminated soil as claimed in claim 2, wherein in step (ii), the weight ratio of cycloheximide to nystatin is 1: 1.
4. The microbial remediation method of petroleum hydrocarbon contaminated soil as claimed in claim 1, wherein in step (II), the preparation method of the separation medium is as follows: weighing beef extract, peptone, NaCl, agar, biosurfactant, diesel oil, gasoline and distilled water according to the set weight parts, placing in a conical flask, stirring until all components are uniformly dispersed, sterilizing at 121 ℃ for 20min, cooling to 50-60 ℃, adding an antifungal agent, uniformly mixing, uniformly subpackaging in a culture dish, and cooling to solidify to obtain the beef extract.
5. The microbial remediation method of petroleum hydrocarbon contaminated soil as claimed in claim 1, wherein in step (c), the liquid medium comprises, by weight, 1-3 parts of peptone, 3-5 parts of yeast extract, 1-2 parts of glucose, 1-2 parts of diesel oil or gasoline, 2-5 parts of biosurfactant, and 1000 parts of distilled water.
6. A method as recited in claim 5, wherein said bioactive agent is sophorolipid in step (iii).
7. The microbial remediation method of claim 5, wherein the liquid medium is prepared by: weighing the components of the liquid culture medium according to the set weight parts, placing the components in a conical flask, stirring until the components are uniformly dispersed, sterilizing at 121 ℃ for 20min, and cooling to obtain the liquid culture medium.
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