CN102021132A - Method for screening and remediation of petroleum-contaminated soil bioremediation agent - Google Patents

Abstract

The invention provides a method for screening and repairing bacterial agents for bioremediation of petroleum-contaminated soil, which specifically includes collecting the source of petroleum-degrading native bacteria and halophilic bacteria, screening of petroleum-degrading native bacteria, screening of halophilic bacteria, and oil-degrading halophilic bacteria. There are six steps: the screening of bacteria, the screening of high-efficiency bacterial agents for oil-contaminated soil remediation, and the use of orthogonal remediation experiments to select the optimal conditions for remediation of highly degradable bacterial agents. The bioremediation bacteria agent screened by the screening and repair method can be applied to the bioremediation of oil-contaminated soil with a salt content of 10 to 20 g/Kg; Its practical value can be widely used in bioremediation fields such as oil-contaminated soil, coastal beaches, and oil production wastewater.

Description

Screening and remediation method of a bioremediation bacterial agent for petroleum-contaminated soil

technical field

The invention belongs to the technical field of microbial remediation of petroleum-contaminated soil, and in particular relates to a screening method and a remediation method of bioremediation bacterial agents for petroleum-contaminated soil.

Background technique

Petroleum, as the "blood of industry", its industrial development and application are increasingly rapid. However, the pollution it brings, especially soil pollution, has also attracted much attention. At present, the most promising oil-contaminated soil treatment method recognized internationally is bioremediation.

The research on the bioremediation technology of petroleum-contaminated soil began in the 1980s. This technology relies on a large number of microorganisms to use hydrocarbons in petroleum as carbon and energy sources to grow, and through their metabolism, the petroleum pollutants in the soil Degraded into carbon dioxide and water or converted into other harmless substances. The main action process of microorganisms on petroleum pollutants has three aspects: one is the direct absorption of hydrocarbons: some hydrocarbons in petroleum can be directly or indirectly dissolved in the lipophilic region of the cell membrane and enter the membrane; the other is the biological absorption of alkanes and naphthenes. The third is the biodegradation of aromatic hydrocarbons, alkenes and alkynes.

Microorganisms have many influences and restrictive factors in the remediation process, mainly including: the degradation ability of microorganisms, the physical and chemical properties of oil-contaminated soil, and environmental conditions. The current research content mainly focuses on three aspects: one is to breed highly efficient petroleum hydrocarbon degrading bacteria; the other is to explore the optimal environmental conditions for microbial remediation; the third is to develop a series of in-situ and ex-situ microbial remediation technologies.

The bioremediation carried out at home and abroad has not achieved very satisfactory results. The reason is that the soil polluted by oilfields not only has high oil content but also high salt content (for example, the soil salt content in Shengli Oilfield is 9-20g/Kg, which is as high as that of unpolluted soil. 8-17 times), high salinity increases the difficulty of soil bioremediation, making conventional strains unable to achieve a good treatment effect. For example, reference 1: Jiang Changliang, Sun Tieyan, Li Peijun, etc. Research on ex-situ bioremediation technology of oil-contaminated soil with long stockpile [J]. Journal of Applied Ecology, 2001, 12(2): 279-282, which recorded the use of compost Four different types of petroleum-contaminated soils in Liaohe Oilfield were treated by the method. When the total petroleum hydrocarbons (TPH) in the soil were 4.16-7.72g/100g soil, after 53 days of operation, the TPH removal rate reached 45.2%-56.7%. . Such as reference 2: Fllis B.Harold P.EnvironmentalTechnology, 1992, 12: 447-459 records that Fllis et al. treat the oily soil in central Stockholm with a prefabricated bed with filtrate collection and water circulation system. The concentration of aromatic hydrocarbons decreased from 1024.4mg/kg to 324.1mg/kg, and the degradation rate was 68%. Reference 3: Wang Danchang, Yang Huaijie, Liu Yong, et al. Research on Oil Sludge (Sand) Treatment and Comprehensive Utilization Technology [J]. Southwest University for Nationalities Journal (Natural Science Edition), 2003, 29:19-23. Well 2219 oil mud sand, using the strains provided by the American Microbiology Company (Youlebao) and the blank comparison experiment without adding strains, entered the field test, and the test results showed that the addition of strains to the oil mud sand with an oil content of 73660mg/kg (Youlebao) The maximum oil degradation rate after 60 days is 23.3%.

Studies have shown that when the salt concentration in the soil is 6g/Kg, most plants, especially cultivated plants, cannot grow normally or cannot grow at all. In addition, the soil itself is a solid medium, which determines the salinity distribution in it. Inhomogeneity, the surface soil of 0-5cm tends to have the highest salinity, which is not only because the surface layer is the most polluted, but also has the largest water evaporation; affected by oil pollution, water evaporation, and plant absorption of water, 5-25cm The soil salinity of the soil is also higher than that of the deeper soil. The above reasons lead to the salinity of local soil, especially the part with salt precipitation, much higher than the measured average soil salinity.

Contents of the invention

In view of the problems existing in the prior art, in order to solve the inhibitory effect of high salinity in oil-contaminated soil on microorganisms, ordinary microorganisms cannot be used for biological treatment of high-salinity oil-contaminated soil, the present invention provides a bioremediation bacterium for oil-contaminated soil The screening and remediation method of the agent, the bioremediation bacterial agent screened by the screening and remediation method can be applied to the biodegradation of oil-contaminated soil with a salt content of 10-20g/Kg; the bioremediation bacteria of oil-contaminated soil strengthened by halophilic bacteria The agent screening and remediation method has high practical value and can be widely used in bioremediation fields such as oil-contaminated soil, coastal beaches, and oil production wastewater.

A method for screening and remediating bacterial agents for bioremediation of oil-contaminated soil, specifically comprising the following steps:

Step 1: Collection of sources of oil-degrading indigenous bacteria and halophilic bacteria;

The oil-contaminated soil at the discharge outlet of oilfield wastewater was used as the source of oil-degrading indigenous bacteria; the oil production wastewater and activated sludge of sewage treatment plant at the outlet of oilfield wastewater treatment plant were used as the source of halophilic bacteria.

Step 2: Screening of indigenous oil-degrading bacteria;

(1) Take 2g of the soil sample as the source of the indigenous oil-degrading bacteria, add it to an Erlenmeyer flask (containing glass beads) filled with 100mL of sterile water, and place it in a shaking incubator for 0.5-2 hours at room temperature. 200rpm.

(2) Get 5mL solution from the Erlenmeyer flask of sterile water, add 37 DEG C in 100mL gradient screening culture fluid A, 150rpm shaking flask culture, described gradient screening culture fluid A is mass concentration 75% beef extract peptone medium and 1g/L crude oil.

(3) When the gradient screening culture solution A is turbid, take 5 mL from the gradient screening culture solution A and transfer it to 100 mL of the gradient screening culture solution B at 37 ° C, 150 rpm shake flask culture, and the gradient screening culture solution B is the mass concentration It is a mixed solution of 50% beef extract peptone medium and 2g/L crude oil.

(4) When the gradient screening culture medium B is turbid, transfer 5mL from the gradient screening culture medium B to 100mL gradient screening culture medium C at 37°C and 150rpm shake flask culture, and the gradient screening culture medium C is the mass concentration It is a mixed solution of 25% beef extract peptone medium and 3g/L crude oil.

(5) When the gradient screening medium C is turbid, take 5 mL from the gradient screening medium C and transfer it to 100 mL of inorganic salt culture medium at 37 ° C, 150 rpm shake flask culture, when the inorganic salt medium becomes turbid, obtain petroleum Oil-degrading indigenous bacteria as the sole carbon source and refrigerated.

The composition of _{the inorganic salt medium: every 1000mL deionized water contains 1g NH4NO3, K2HPO4 · 3H2O1g , KH2PO41g , MgSO4 · 7H2O0.5g} , anhydrous _CaCl2 0.02g, FeSO ₄ 0.01g, MnSO ₄ ·H ₂ O 0.01g, crude oil 4g, pH=7.0, sterilized at 121°C for 20min.

Step 3: screening of halophilic bacteria;

Take 1ml of activated sludge and 1ml of oil production wastewater, and use 100mL of beef extract peptone medium to cultivate. After gradient domestication and screening, a halophilic bacteria complex strain that can grow well in a salt concentration environment of 1-10% is obtained, and the halophilic bacteria is used to compound Bacterial system Prepare a mixed halophilic bacteria culture solution with a bacterial cell concentration of 10 ⁸ -10 ⁹ cells/ml.

Step 4: Screening of petroleum-degrading halophilic bacteria;

Petroleum was used as the only carbon source, with a concentration of 1.0% to 10.0% as the salt gradient, and the oil-degrading halophilic bacteria with a wide range of salt tolerance were screened from the composite strain of halophilic bacteria and oil-contaminated soil.

(1) The screening of the culture fluid of the oil-degrading halophilic bacteria with a halophilic degree of 1.0%:

(A) Take 2 g of soil samples as the source of indigenous oil-degrading bacteria, add them to an Erlenmeyer flask (containing glass beads) filled with 100 mL of sterile water, place in a shaking incubator at 200 rpm, and shake at room temperature for 0.5-2 hours.

(B) Take 5 mL from a Erlenmeyer flask of sterile water, add it to 100 mL of enrichment culture solution A, add 5 mL of mixed halophilic bacteria culture solution to each bottle, and culture in shake flask at 37°C and 150 rpm. The enrichment culture solution A is a mixed solution of 75% beef extract peptone medium, 1 g/L crude oil and 1.0% NaCl in mass concentration.

(C) When the enrichment medium A is turbid, transfer 5mL from it to 100mL enrichment medium B, culture in a shaker flask at 37°C and 150rpm, the enrichment medium B is 50% beef extract peptone Medium, 2g/L crude oil mixed solution containing 1.0% NaCl in mass concentration.

(D) When the enriched culture solution B is turbid, take 5 mL from it and transfer it to 100 mL enriched culture solution C for shaking flask culture. The enriched culture solution C is 25% beef extract peptone medium with a mass concentration of 3 g/ L crude oil contains a mixed solution with a mass concentration of 1.0% NaCl.

(E) When the enriched culture solution C is turbid, take 5 mL from it and transfer it to the inorganic salt-enriched culture solution A for shaking flask culture, and the inorganic salt-enriched culture solution A is an inorganic salt medium and 1.0% NaCl, When the inorganic salt-enriched culture solution A becomes turbid, a culture solution of petroleum-degrading halophilic bacteria with a halophilicity of 1% and petroleum as the sole carbon source is obtained.

(2) Take 5ml from the culture solution of petroleum-degrading halophilic bacteria with a halophilicity of 1%, transfer it to the culture solution B enriched with inorganic salts at 37°C, and cultivate in shake flasks at 150 rpm. When the culture medium A enriched with inorganic salts When it becomes turbid, a culture solution of petroleum-degrading halophilic bacteria having a halophilic degree of X is obtained. The inorganic salt-enriched culture solution B is an inorganic salt medium containing NaCl solution with a mass concentration of X.

(3) Take 5ml from the culture solution of petroleum-degrading halophilic bacteria with a halophilicity of X, transfer it to culture medium C enriched with inorganic salts at 37°C, and cultivate in shake flasks at 150 rpm. When the culture medium C enriched with inorganic salts becomes When it is turbid, a culture solution of petroleum-degrading halophilic bacteria with a halophilicity of Y is obtained; the inorganic salt-enriched culture solution B is an inorganic salt medium containing a NaCl solution with a mass concentration of Y, and 1.0%<X<Y <10.0%.

(4) Take 5ml from the culture solution of the oil-degrading halophilic bacteria with a degree of Y and transfer it to the inorganic salt-enriched culture solution D at 37°C and 150rpm shake flask culture. When the inorganic salt-enriched culture solution D becomes When it is turbid, a culture solution of petroleum-degrading halophilic bacteria with a halophilic degree of Z is obtained; the inorganic salt-enriched culture solution D is an inorganic salt medium containing a NaCl solution with a mass concentration of Z, and 1.0%<X<Y <Z<10.0%.

(5) Take 5ml from the culture solution of petroleum-degrading halophilic bacteria with a halophilic degree of Z, transfer it to the inorganic salt-enriched culture solution E at 37°C, and culture it in a shaking flask at 150rpm. When the inorganic salt-enriched culture solution E becomes When it is turbid, a culture solution of petroleum-degrading halophilic bacteria with a halophilicity of E is obtained; the inorganic salt-enriched culture solution E is an inorganic salt medium containing a NaCl solution with a mass concentration of 10.0%.

(6) Mix the obtained petroleum-degrading halophilic bacteria with a halophilicity of 1.0%, X, Y, Z and 10.0% according to the same volume ratio to obtain a composite bacterial agent for petroleum-degrading halophilic bacteria.

Step 5: Screening of high-efficiency bacterial agents for oil-contaminated soil remediation;

(1) The halophilic oil-degrading halophilic bacteria compound bacteria agent obtained in step 3 was activated by using the corresponding salinity plate, and the oil-degrading native bacteria obtained in step 2 were respectively cultured in beef extract peptone medium until the bacteria were shaken. The concentrations were all greater than 10 ⁹ per milliliter, and the bacterial cells were collected by centrifugation to prepare two kinds of fermentation bacterial liquids.

(2) Take the soil used as the source of the oil-degrading indigenous bacteria, remove impurities, air-dry, break up, pass through a 200-mesh sieve, and mix well, then spread the soil into a thin layer of 2 cm.

(3) Put the soil into flower pots respectively, measure the oil content and salt content in the soil, and inoculate the fermented bacterial liquid of the indigenous oil-degrading bacteria and the fermented bacterial liquid of the oil-degrading halophilic bacteria compound bacterial agent respectively; Each gram of soil contains 10 ⁶ to 10 ⁸ colonies, and it is added every 10-15 days.

(4) Add nutrients to the soil every 10 to 15 days, fully mix, and adjust the pH and humidity of the soil; the nutrients are sucrose and potassium nitrate, and the amount added each time is Add 1-10g of sucrose to one kilogram of soil, add 2-16g of potassium nitrate per kilogram of soil, and adjust the water content of the soil every day so that the humidity of the soil is 20%-40%, and the pH of the soil is adjusted to 7-8.

(5) measure the oil content in each flowerpot by gravimetric method, utilize the plate technology method to measure the concentration of thalline, measure and adjust the pH value of soil, after 7 weeks time, measure the degradation rate of petroleum in the soil in each flowerpot , to obtain the high-efficiency bacterial agent with the highest degradation rate.

Step 6: Use the orthogonal repair experiment to select the optimal conditions for the restoration of highly efficient degradable bacteria agents:

The amount of bacteria, humidity, the amount of sucrose added and the amount of potassium nitrate added were selected as the influencing factors of high-efficiency bacterial agent remediation by using the orthogonal restoration experiment. The amount of bacteria added was 10 ⁶ to 10 ⁸ per gram of soil The humidity is 20%-40%, the dosage of sucrose is 1-10g per kilogram of soil, and the dosage of potassium nitrate is 2-16g per kilogram of soil. (1) Use the high-efficiency bacteria agent obtained by screening to carry out the orthogonal repair experiment, first use the corresponding salinity plate to activate, then use the beef extract peptone medium to cultivate until the bacterial cell concentration is greater than ¹⁰⁹ cells per milliliter, and collect the bacterial cell by centrifugation to prepare Fermentation broth.

(2) Get the soil as the source of the native oil-degrading bacteria, remove impurities, air-dry, break up, pass through a 200-mesh sieve, and mix evenly, spread the soil into a thin layer of 2 cm, and adjust the pH of the soil to 7-8 .

(3) Put the soil into flower pots respectively, and measure the oil content and salt content in each flower pot by gravimetric method.

(4) Control the amount of bacteria in each flowerpot to add 10 ⁶ to 10 ⁸ bacteria per gram of soil, the humidity is 20% to 40%, and the amount of sucrose to be added is 1 to 10g per kilogram of soil. The amount of potassium nitrate added is 2-16g per kilogram of soil; and high-efficiency bacterial agents, sucrose and potassium nitrate are added to the soil every 10-15 days to ensure the content of bacteria and nutrients in the soil, and the humidity is controlled by spraying water in the morning and evening .

(5) Measure the oil content in each flower pot by gravimetric method every 7 days, use the plate technique method to measure the concentration of bacteria every 7 days, measure and adjust the pH value of the soil every 3 days, 7 weeks Finally, the degradation rate of petroleum in the soil of each flowerpot was measured, and the remediation condition with the highest degradation rate was obtained.

The present invention has the following advantages:

1. The present invention discloses a method for screening and repairing bacterial agents for bioremediation of petroleum-contaminated soils. The bacteria that are screened and cultivated can grow well under the environment of 6%-10% salt concentration, which is the oil pollution with high salt content. The remediation of the soil provides a guarantee of the source of bacteria;

2. The screening and restoration method of bioremediation bacterial agent for petroleum-contaminated soil disclosed by the present invention has the optimum restoration conditions of 40% humidity; ¹⁰ bacteria/g soil; the optimal addition of potassium nitrate 2 weeks before restoration The amount of potassium nitrate is 16g/kg soil, and the amount of potassium nitrate is 8g/kg soil after the 3rd week of restoration in the middle and late stages; The best dosage of sucrose after the beginning of the week is 1-5g/kg soil. After 7 weeks of restoration, the oil removal rate of the soil with a salt content as high as 10g/Kg and an oil content of 16% can reach 64.31%, and the repair effect is relatively high;

3. The invention discloses a method for screening and repairing bioremediation bacterial agents for petroleum-contaminated soils, which can survive in petroleum-contaminated soils with high salt content, and can use petroleum as a carbon source for metabolism. , Oily wastewater and other environmental protection fields have certain application prospects.

Description of drawings

Fig. 1: The degradation rate of petroleum-degrading indigenous bacteria and petroleum-degrading halophilic bacteria varies with time in the present invention;

Fig. 2: the screening flowchart of a kind of petroleum-contaminated soil bioremediation bacterium agent proposed by the present invention and the screening process of petroleum-degrading native bacteria in the restoration method;

Fig. 3: A flow chart of the screening of petroleum-degrading halophilic bacteria in the screening of bioremediation bacterial agents for petroleum-contaminated soil and the restoration method proposed by the present invention.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

The screening and restoration method of a kind of bioremediation bacterium of petroleum-contaminated soil that the present invention proposes specifically comprises the following steps:

Step 1: Collection of sources of oil-degrading indigenous bacteria and halophilic bacteria;

The oil-contaminated soil at the discharge outlet of oilfield wastewater was used as the source of oil-degrading indigenous bacteria; the oil production wastewater and activated sludge of sewage treatment plant at the outlet of oilfield wastewater treatment plant were used as the source of halophilic bacteria.

Step 2: Screening of oil-degrading native bacteria, as shown in Figure 2;

(1) Take 2g of the soil samples collected from 9 sampling points, add them to 9 Erlenmeyer flasks (containing glass beads) filled with 100mL sterile water respectively, and place the 9 bottles of soil solution in a shaking incubator at room temperature Oscillate for 0.5-2h, and the oscillation frequency is 200rpm.

(2) Get 5mL solution from the Erlenmeyer flask of every bottle of sterile water, add respectively 37 ℃ in 100mL gradient screening medium A, 150rpm shaking flask culture, described gradient screening medium A is mass concentration 75% beef extract A mixed solution of peptone medium and 1g/L crude oil.

(3) When the gradient screening culture solution A is turbid, take 5 mL from the gradient screening culture solution A and transfer it to 100 mL of the gradient screening culture solution B at 37 ° C, 150 rpm shake flask culture, and the gradient screening culture solution B is the mass concentration It is a mixed solution of 50% beef extract peptone medium and 2g/L crude oil.

(4) When the gradient screening culture medium B is turbid, transfer 5mL from the gradient screening culture medium B to 100mL gradient screening culture medium C at 37°C and 150rpm shake flask culture, and the gradient screening culture medium C is the mass concentration It is a mixed solution of 25% beef extract peptone medium and 3g/L crude oil.

(5) When the gradient screening medium C is turbid, take 5 mL from the gradient screening medium C and transfer it to 100 mL of inorganic salt culture medium at 37 ° C, 150 rpm shake flask culture, when the inorganic salt medium becomes turbid, obtain petroleum Oil-degrading indigenous bacteria as sole carbon source.

(6) The obtained 9 groups corresponding to 9 sampling points of oil-degrading indigenous bacteria with petroleum as the sole carbon source were numbered B1, B2, B3, B4, B5, B6, B7, B8 and B9 respectively, and Refrigerate at 4°C.

The composition of the inorganic salt medium: every 1000mL of water contains 1g of NH ₄ NO ₃ , 1g of K ₂ HPO ₄ 3H ₂ O, 1g of KH ₂ PO ₄ , 0.5g of MgSO ₄ 7H ₂ O, 0.02g of anhydrous CaCl ₂ , FeSO ₄ 0.01g, MnSO ₄ ·H ₂ O 0.01g, crude oil 4g, pH=7.0, sterilized at 121°C for 20min.

Step 3: Screening of halophilic bacteria

Take 1ml of activated sludge and 1ml of oil production wastewater, and use 100mL of beef extract peptone medium to cultivate. After gradient domestication and screening, a halophilic bacteria complex strain that can grow well in a salt concentration environment of 1-10% is obtained, and the halophilic bacteria is used to compound Bacterial system Prepare a mixed halophilic bacteria culture solution with a bacterial cell concentration of 10 ⁸ -10 ⁹ cells/ml.

Step 4: Screening of petroleum-degrading halophilic bacteria;

With petroleum as the only carbon source, with the mass concentration of 1.0%, 2.5%, 5.0%, 7.5%, and 10.0% as the salt gradient, the oil-degrading halophilic bacteria were screened from the halophilic bacteria complex strain and oil-contaminated soil, as shown in the figure 3.

(1) the screening of the culture fluid of the oil-degrading halophilic bacteria with a halophilic degree of 1%:

(A) Take 2g of soil samples collected from 9 sampling points by indigenous oil-degrading bacteria, add them to conical flasks (containing glass beads) filled with 100mL sterile water, and place 9 bottles of soil solution in shaking culture 200rpm in the box, shake at room temperature for 0.5-2h.

(B) Take 5mL of each bottle of soil solution, add it to nine bottles of 100mL enrichment culture solution A, and add 5mL of mixed halophilic bacteria culture solution to each bottle, and shake the flask at 37°C and 150rpm. The enrichment culture solution A is a mixed solution of 75% beef extract peptone medium, 1 g/L crude oil, and 1% NaCl in mass concentration.

(C) When the enrichment culture solution A is turbid, transfer 5 mL from each bottle to 100 mL enrichment culture solution B at 37° C., 150 rpm shake flask culture, and the enrichment culture solution B is a mass concentration of 50% beef extract A mixed solution of peptone medium, 2g/L crude oil and 1% NaCl in mass concentration.

(D) When enrichment medium B is turbid, take 5mL from it and transfer it to 100mL enrichment medium C at 37°C, 150rpm shake flask culture, said enrichment medium C is 25% beef extract peptone culture A mixed solution of base, 3g/L crude oil and 1.0% NaCl in mass concentration.

(E) When the enriched culture solution C is turbid, take 5 mL from it and transfer it to the inorganic salt-enriched culture solution A for shaking flask culture. The inorganic salt-enriched culture solution A is an inorganic salt medium containing a mass concentration of 1.0 %NaCl solution, when the inorganic salt-enriched culture solution A became turbid, 9 groups of oil-degrading halophilic bacteria culture solutions with a halophilic degree of 1.0% were obtained, which used petroleum as the only carbon source.

(2) Take 5ml each from the culture solution of 9 kinds of oil-degrading halophilic bacteria with a halophilicity of 1.0%, and transfer them to the inorganic salt-enriched culture solution B containing 2.5% salt respectively at 37°C and 150rpm shake flask After culturing, when the inorganic salt-enriched culture solution B became turbid, 9 groups of culture solutions of petroleum-degrading halophilic bacteria with a halophilic degree of 2.5% were obtained, which used petroleum as the sole carbon source. The inorganic salt enriched culture solution B is a solution containing 2.5% NaCl in the inorganic salt medium.

(3) Take 5ml from the culture solution of petroleum-degrading halophilic bacteria with a halophilicity of 2.5%, transfer it to the culture solution C enriched with inorganic salts at 37°C, and cultivate in shake flasks at 150 rpm. When the culture solution enriched with inorganic salts A When it became cloudy, a culture solution of petroleum-degrading halophilic bacteria with a halophilicity of 5.0% was obtained. The inorganic salt-enriched culture solution C is an inorganic salt medium containing 5.0% NaCl solution.

(4) Take 5ml from the culture solution of petroleum-degrading halophilic bacteria with a halophilicity of 5.0%, transfer it to the inorganic salt-enriched culture solution D at 37°C, and culture it in a shaking flask at 150 rpm. When the inorganic salt-enriched culture solution D When it becomes turbid, a culture solution of petroleum-degrading halophilic bacteria with a halophilicity of 7.5% is obtained; the inorganic salt-enriched culture solution D is an inorganic salt medium containing a NaCl solution with a mass concentration of 7.5%.

(5) Take 5ml from the culture solution of petroleum-degrading halophilic bacteria with a halophilicity of 7.5%, transfer it to inorganic salt-enriched culture solution E at 37°C, and culture it in a shaking flask at 150 rpm. When the inorganic salt-enriched culture solution E When it becomes turbid, a culture solution of petroleum-degrading halophilic bacteria with a halophilicity of 10.0% is obtained; the inorganic salt-enriched culture solution E is an inorganic salt medium containing a NaCl solution with a mass concentration of 10.0%.

(6) 45 groups of petroleum-degrading halophilic bacteria corresponding to five kinds of halophilic degrees of 1%, 2.5%, 5.0%, 7.5% and 10.0% from 9 sampling points were mixed according to the petroleum-degrading halophilic bacteria at the same sampling point , to obtain 9 groups of oil-degrading halophilic bacteria corresponding to 9 sampling points, numbered respectively: Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, and refrigerated at 4°C.

Step 5: Screening of high-efficiency bacterial agents for oil-contaminated soil remediation;

(1) Activate the oil-degrading halophilic bacteria with a halophilic degree of 1.0%, 2.5%, 5.0%, 7.5% and 10% with the corresponding halophilic degree plate, and use beef extract peptone medium with the native oil-degrading bacteria respectively Under the conditions of 30° C. and 150 rpm, the culture was shaken until the cell concentration was greater than 10 ⁹ cells per mL, and the cells were collected by centrifugation to prepare two fermentation cell liquids.

(2) Take the soil used as the source of the oil-degrading indigenous bacteria, remove impurities, air-dry, break up, pass through a 200-mesh sieve, and mix well, then spread the soil into a thin layer of 2 cm.

(3) Put the soil into flower pots respectively, and measure the oil content and salt content in the soil. One of the flowerpots was set as the control pot without inoculation, and the rest of the flowerpots were inoculated with the fermentation broth of indigenous oil-degrading bacteria and oil with a halophilicity of 1.0%, 2.5%, 5.0%, 7.5% and 10.0%, respectively. Degrading the fermentation broth of halophilic bacteria; the dosage is 10 ⁶ to 10 ⁸ colonies (CFU) per gram of soil, and the dosage is once every 10-15 days. Add nutrients to the soil every 10-15 days and mix well. The nutrient substance is sucrose and potassium nitrate, and the amount added each time is 1-10g sucrose per kilogram of soil, and 2-16g potassium nitrate per kilogram of soil; and the water content of the soil is adjusted every day, so that The humidity of the soil is 20%-40%, and the pH of the soil is adjusted to 7-8. The test conditions are shown in Table 1, and the parameters measured during the test are shown in Table 2:

Table 1 Soil remediation test conditions

parameters Parameter value Experimental operation temperature Room temperature (20-25°C) Humidity 20%-40% Spray water every morning and evening pH value 78 measure and adjust Oxygen Air vent Dig the soil every morning and evening for oxygenation Nutrients Sucrose (1-10g/kg soil), 2-16gKNO ₃ /Kg soil Dosing every 15 days Bacteria dosage 10 ⁶ -10 ⁸ (CFU)/g soil Dosing every 15 days

(4) Measure the oil content in each flowerpot by gravimetric method every 7 days, measure the concentration of the thalline every 7 days by using the plate technique method, measure and adjust the pH value of the soil every 3 days, as shown in the table As shown in 2, after 7 weeks, the degradation rate of petroleum in the soil in each flower pot was measured, and the high-efficiency bacterial agent with the highest degradation rate was obtained.

Table 2 Determination parameters of soil remediation

parameters test methods Sampling interval oil content gravimetric method 7 days Bacteria concentration plate counting method 7 days

pH value pH meter 3 days

The degradation rate of petroleum hydrocarbons is a common indicator for microbial remediation of petroleum-contaminated soils, which directly reflects the degradation ability of microorganisms. After 7 weeks, the degradation rate of the soil in 19 flower pots was measured, as shown in Figure 1, it was found that the oil content of the soil in the control flower pot decreased by 17.59%, which was mainly because the added nutrients stimulated the growth and metabolism of indigenous microorganisms , prompting it to degrade part of the petroleum hydrocarbons. In addition, the volatilization of short-chain petroleum hydrocarbons and the oxidation of air also reduce the oil content of the soil. The degradation efficiency of the indigenous oil-degrading bacteria is higher than that of the control, and its degradation rate is 13.99% to 30.3% higher than that of the control flowerpot, indicating that the sieved and domesticated oil-degrading indigenous bacteria have significantly improved the ability to degrade petroleum hydrocarbons . The degradation efficiency of oil-degrading halophilic bacteria Y1-Y9 is generally higher than that of the screened oil-degrading indigenous bacteria B1-B9, which are higher in turn: 5.51%, 4.35%, 3.32%, -1.28%, 2.86%, 2.28%, -4.88 %, 0.26%, and 2.25%, indicating that the oil-degrading halophilic bacteria are more able to adapt to the high-salt environment, and at the same time exert an efficient degradation effect. Among them, Y1, Y2, Y3 and B2 are the compound bacterial agents with the best degradation ability.

Step 6: Use the orthogonal repair experiment to select the optimal conditions for the restoration of highly efficient degradable bacteria agents:

(1) Use the high-efficiency bacterial agent Y2 obtained through screening to carry out the degrading bacterial agent of the repair experiment, activate it with a plate with a corresponding salinity of 1-10%, and then use beef extract peptone medium to vibrate at 30°C and 150rpm until the bacteria are formed The concentration is greater than 10 ⁹ per milliliter, and the bacteria are collected by centrifugation to prepare a fermentation broth.

(2) Get the soil as the source of the native oil-degrading bacteria, remove impurities, air-dry, break up, pass through a 200-mesh sieve, and mix evenly, spread the soil into a thin layer of 2 cm, and adjust the pH of the soil to 7-8 .

(3) Put the soil into flower pots respectively, and measure the oil content and salt content in each flower pot by gravimetric method.

(4) Set the influencing factors of the orthogonal repair experiment as four factors: the amount of bacteria, temperature, sucrose and potassium nitrate, and the bacteria, sucrose and nitric acid are added to the soil every 10 to 15 days Potassium is used to ensure the content of bacteria and nutrients in the soil, and the humidity is controlled by watering in the morning and evening. The bacterial dosage factor is three levels of 10 ⁶ , 10 ⁷ and 10 ⁸ per gram of soil. Humidity factors are three levels of 20%, 30% and 40%, as shown in Table 3, the factors of the amount of sucrose added are three levels of 1g, 5g and 10g per kilogram of soil, and the factors of the amount of potassium nitrate added are 2g per kilogram of soil , 8g and 16g three levels. At the same time, the temperature is controlled at 20-25°C, as shown in Table 4, and the soil is ventilated with oxygen by turning the soil, and the pH value of the soil is monitored and adjusted to 7-8.

Table 3 Orthogonal experiment factor level table

Table 4 Orthogonal experimental conditions

Experimental conditions value Experimental operation

temperature Room temperature (20-25°C) at room temperature pH value 7-8 monitor and adjust Oxygen Oxygenation Set up ventilation holes, dig soil for oxygenation

(5) measure the oil content in each flowerpot by gravimetric method every 7 days, as shown in table 5, utilize plate technique method to measure the concentration of thalline every 7 days, measure and adjust soil every 3 days After 7 weeks, the degradation rate of petroleum in the soil in each flowerpot was measured, and the combination of factors affecting the level with the highest degradation rate was obtained.

Table 5 Orthogonal Experiment Determination Parameters

parameters test methods sampling time oil content gravimetric method every 7 days Bacteria concentration plate counting method every 7 days pH value pH meter Every 3 days

(5) Orthogonal repair experiment results

After 7 weeks, measure the degradation rate of petroleum in the soil in each flowerpot, obtain the change table of degradation rate with time, as shown in table 6:

Table 6 Degradation rate changes with time

According to the four factors of the level of factors in the orthogonal restoration experiment—the amount of bacteria, the humidity, the addition of sucrose and the addition of potassium nitrate, there are three conditions, and the orthogonal experiment was carried out to obtain the results of the oil degradation rate under different factors, as shown in the table. 7.

Table 7 Degradation rate table of different factor levels in orthogonal repair experiment

According to the results of this orthogonal experiment (Table 7), the humidity and the amount of bacteria are always the primary factors affecting the repair effect, and the optimal values are 40% and 10 ⁸ bacteria/g soil respectively. Potassium nitrate is the third most influential factor. The optimal dosage of potassium nitrate is higher than that in other periods during the initial stage of restoration, the period of large-scale reproduction of degrading bacteria and the period of high-speed degradation, that is, the optimal dosage of potassium nitrate in the first 2 weeks of restoration is 16g/kg Soil, the best dosage of potassium nitrate is 8g/kg soil after the third week in the middle and late stage. Sucrose is a relatively key influencing factor in the early stage of repair, which can accelerate the growth of microorganisms and quickly start the degradation. Considering that the best amount of sucrose added in the first three weeks is 5-10g/kg soil, and the best amount of added sucrose after the beginning of the fourth week is 1-5g/kg soil in the middle and late stages.

From the analysis of the effect of the orthogonal experiment: when the high-efficiency bacteria were repaired for 7 weeks, the humidity was 40%; the amount of bacteria was ¹⁰⁸ /g soil; 8g/kg of soil; 5-10g/kg of sucrose was added 3 weeks before restoration, and 1-5g/kg of sucrose was added in the middle and late stages, and the oil removal rate reached 64.31%.

Claims (5)

1. the screening and the restorative procedure of an oil-polluted soils biological restoration microbial inoculum is characterized in that comprising following step:

Step 1: the collection in oil degradation original inhabitants bacterium and halophilic bacterium bacterium source;

Get apart from the oil-polluted soils at oil production waste water in oil field discharge outlet place as oil degradation original inhabitants bacterium bacterium source; Get apart from the oil extraction waste water of oil production waste water in oil field treatment plant water outlet and sludge sewage as halophilic bacterium bacterium source;

Step 2: the screening of oil degradation original inhabitants bacterium;

(1) get soil-like 2g as oil degradation original inhabitants bacterium bacterium source, add and fill the Erlenmeyer flask of 100mL sterilized water, and place shaking culture case room temperature vibration 0.5～2h, oscillation frequency is 200rpm;

(2) get 5mL solution from the Erlenmeyer flask of sterilized water, add shake-flask culture among the 100mL gradient screening and culturing liquid A, described gradient screening and culturing liquid A is the mixing solutions of mass concentration 75% beef-protein medium and 1g/L crude oil;

(3) when gradient screening and culturing liquid A is muddy, get 5mL and be forwarded to shake-flask culture among the 100mL gradient screening and culturing liquid B from gradient screening and culturing liquid A, described gradient screening and culturing liquid B is that mass concentration is the mixing solutions of 50% beef-protein medium and 2g/L crude oil;

(4) when gradient screening and culturing liquid B is muddy, get 5mL and be forwarded to shake-flask culture among the 100mL gradient screening and culturing liquid C from gradient screening and culturing liquid B, described gradient screening and culturing liquid C is that mass concentration is the mixing solutions of 25% beef-protein medium and 3g/L crude oil;

(5) when gradient screening and culturing liquid C is muddy, gets 5mL and be forwarded to shake-flask culture in the 100mL minimal medium from gradient screening and culturing liquid C, when minimal medium became muddy, obtaining with the oil was the oil degradation original inhabitants bacterium of sole carbon source, and refrigeration;

Step 3: the screening of halophilic bacterium;

Get each 1ml of active sludge and oil extraction waste water, adopt the 100mL beef-protein medium to cultivate, obtaining salinity through the gradient acclimation and screening is 1%～20% halophilic bacterium composite microbial system, and utilizes the halophilic bacterium composite microbial system to prepare cell concentration 10 ⁸～10 ⁹The mixing halophilic bacterium nutrient solution of individual/ml;

Step 4: the screening of oil degradation halophilic bacterium;

(1) have a liking for the screening that salinity is the nutrient solution of 1.0% oil degradation halophilic bacterium:

(A) get soil-like 2g, add and fill in the Erlenmeyer flask of 100mL sterilized water, and place the vibration of shaking culture case room temperature as oil degradation original inhabitants bacterium bacterium source;

(B) from the Erlenmeyer flask of sterilized water, get 5mL, add among the 100mL enrichment culture liquid A, and in wherein add mixing halophilic bacterium nutrient solution 5mL shake-flask culture, described enrichment culture liquid A is the mixing solutions of mass concentration 75% beef-protein medium, 1g/L crude oil and 1.0%NaCl;

(C) when enrichment culture liquid A is muddy, therefrom gets 5mL and be forwarded to shake-flask culture among the 100mL enrichment culture liquid B, described enrichment culture liquid B is the mixing solutions of mass concentration 50% beef-protein medium, 2g/L crude oil and 1.0%NaCl;

(D) when enrichment culture liquid B is muddy, therefrom gets 5mL and be forwarded to shake-flask culture among the 100mL enrichment culture liquid C, described enrichment culture liquid C is the mixing solutions of mass concentration 25% beef-protein medium, 3g/L crude oil and 1.0%NaCl;

(E) when enrichment culture liquid C is muddy, therefrom get 5mL and be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid A, described inorganic salt enrichment culture liquid A is that minimal medium and mass concentration are the mixing solutions of 1.0%NaCl, when inorganic salt enrichment culture liquid A became muddy, obtaining with the oil was the nutrient solution that salinity is 1.0% oil degradation halophilic bacterium of having a liking for of sole carbon source;

(2) from have a liking for the nutrient solution that salinity is 1.0% oil degradation halophilic bacterium, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid B, when inorganic salt enrichment culture liquid B becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of X; Described inorganic salt enrichment culture liquid B is that minimal medium contains the NaCl solution that mass concentration is X, and 1.0%＜X＜10.0%;

(3) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is X, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid C, when inorganic salt enrichment culture liquid C becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of Y; Described inorganic salt enrichment culture liquid C is that minimal medium contains the NaCl solution that mass concentration is Y, and 1.0%＜X＜Y＜10.0%;

(4) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is Y, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid D, when inorganic salt enrichment culture liquid D becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of Z; Described inorganic salt enrichment culture liquid D is that minimal medium contains the NaCl solution that mass concentration is Z, and 1.0%＜X＜Y＜Z＜10.0%;

(5) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is Z, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid E, when inorganic salt enrichment culture liquid E becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of E; Described inorganic salt enrichment culture liquid E is that to contain mass concentration be 10.0% NaCl solution to minimal medium;

(6) salinity is 1.0% for having a liking for of will obtaining, X, Y, Z and 10.0% oil degradation halophilic bacterium mix according to the equal volume ratio, obtains oil degradation halophilic bacterium composite fungus agent;

Step 5: the high-effect bacterial screening that oil-polluted soils is repaired;

(1) the dull and stereotyped activation of the corresponding salinity of the oil degradation halophilic bacterium composite fungus agent utilization that obtains in the step 3, and with step 2 in the oil degradation original inhabitants bacterium that obtains adopt respectively the beef-protein medium shaking culture to cell concentration all greater than every milliliter 10 ⁹Individual, centrifugal collection thalline prepares two kinds of zymocyte liquids;

(2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into thin layer;

(3) soil is respectively charged in the flowerpot, measures the oleaginousness and the saltiness of soil in the flowerpot, regulate pH in soil and humidity; In flowerpot, inoculate the zymocyte liquid of oil degradation original inhabitants bacterium and the zymocyte liquid of oil degradation halophilic bacterium composite fungus agent respectively; Throwing bacterium amount is in every gram soil and contains 10 ⁶～10 ⁸Individual bacterium colony, and added once in per 10～15 days;

(4) in flowerpot, added the one time of nutrition material every 10～15 days, abundant mixing, and regulate pH in soil and humidity every day;

(5) survey oleaginousness in each flowerpot by weighting method, utilize the plate technique method to measure the concentration of thalline, measure and also regulate pH in soil, behind 7 time-of-weeks, measure the degradation rate of soil PetroChina Company Limited. in each flowerpot, obtain the highest high-effect bacterial of degradation rate;

Step 6: the reparation of high-effect bacterial;

Utilize the quadrature reparation to test and choose the bacterium amount of throwing, humidity respectively, add sucrose amount and saltpetre amount as the repairing condition of high-effect bacterial, wherein throw the bacterium amount and add 10 in every gram soil ⁶～10 ⁸Individual thalline, humidity is 20%～40%, and adding the sucrose amount is every kilogram of soil 1～10g, and adding the saltpetre amount is every kilogram of dry ground 2～16g; Obtain oil-polluted soils biological restoration microbial inoculum at last.

2. the screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum according to claim 1 is characterized in that: described minimal medium composition: contain NH in every 1000mL deionized water ₄NO ₃1g, K ₂HPO ₄3H ₂O 1g, KH ₂PO ₄1g, MgSO ₄7H ₂O 0.5g, anhydrous CaCl ₂0.02g, FeSO ₄0.01g, MnSO ₄H ₂O 0.01g, crude oil 4g, pH=7.0,121 ℃ of sterilization 20min.

3. the screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum according to claim 1 is characterized in that: the reparation detailed process described in the described step 6 is:

(1) high-effect bacterial that utilizes screening to obtain carries out quadrature reparation experiment, at first with the dull and stereotyped activation of corresponding salinity, adopts beef-protein medium to be cultured to cell concentration greater than every milliliter 10 again ⁹Individual, centrifugal collection thalline prepares zymocyte liquid;

(2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into the thin layer of 2cm, regulate pH=7～8 of soil;

(3) soil is respectively charged in the flowerpot, by oleaginousness and the saltiness in each flowerpot of gravimetric determination;

(4) control in each flowerpot and to throw the bacterium amount and add 10 in every kilogram of soil ⁶～10 ⁸Individual thalline, humidity is 20%～40%, and adding the sucrose amount is 1～10g in every kilogram of soil, and adding the saltpetre amount is 2～16g in every kilogram of soil; And in soil, added high-effect bacterial, sucrose and saltpetre in per 10～15 days and guarantee thalline and concentrations of nutrient in the soil, humidity is by water spray control sooner or later;

(5) every 7 days by the oleaginousness in each flowerpot of gravimetric determination, the concentration of utilizing the plate technique method to survey a hypothallus every 7 days was measured and the adjusting pH in soil every 3 days, behind 7 time-of-weeks, measure the degradation rate of each flowerpot soil PetroChina Company Limited., obtain the highest repairing condition of degradation rate.

4. according to the screening and the restorative procedure of claim 1 or 3 described a kind of oil-polluted soils biological restoration microbial inoculums, it is characterized in that: the repairing condition of described oil-polluted soils biological restoration microbial inoculum is: humidity is 40% when using the degraded of oil degradation halophilic bacterium composite fungus agent, and throwing the bacterium amount is 10 ⁸Individual/g soil; The saltpetre dosage in 2 weeks is a 16g/kg soil before reparation, and the saltpetre dosage is a 8g/kg soil after the beginning of the 3rd week; 3 all sucrose dosages are 5～10g/kg soil before reparation, and the sucrose dosage is 1～5g/kg soil after the beginning of the 4th week.

5. the screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum according to claim 1 is characterized in that: X=2.5% in the described step 4, Y=5.0%, Z=7.5%.

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