CN104450597B - Preparation method of petroleum degrading bacteria solid microbial inoculum and method for repairing petroleum-polluted soil by using solid microbial inoculum prepared by preparation method - Google Patents
Preparation method of petroleum degrading bacteria solid microbial inoculum and method for repairing petroleum-polluted soil by using solid microbial inoculum prepared by preparation method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
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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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Abstract
The invention relates to a preparation method of a solid microbial inoculum of petroleum degrading bacteria, which comprises the following specific steps: A. screening and domesticating petroleum degrading bacteria, B, preparing a seed culture solution, C, fermenting a solid microbial inoculum, D, drying a piled product, pulverizing, metering and packaging. The invention has the beneficial effects that the non-ionic surfactant polysorbate-80 (Tween 80) is used as a solubilizer, so that the dissolving effect of petroleum can be improved. The solid fermentation raw materials used in the invention are easy to obtain, the process is simple, the solid microbial inoculum contains a large amount of carbon and nutrient elements, provides a more appropriate substrate for bacterial growth, and has strong affinity to microorganisms and high immobilization efficiency. Improving the competitiveness and degradation efficiency of the added microorganisms and indigenous microorganisms. The solid microbial inoculum is convenient for transportation and agricultural operation, and is suitable for large-scale in-situ bioremediation of petroleum-polluted soil.
Description
Technical Field
The invention relates to the technical field of microbial remediation of petroleum-contaminated soil, in particular to a preparation method of a solid microbial inoculum of petroleum degrading bacteria and a method for remediating petroleum-contaminated soil by using the prepared solid microbial inoculum.
Background
With the rapid development of economy, the soil environment problem caused by human beings is becoming more serious. Soil is used as a non-renewable resource for human beings, and is the basis on which human beings rely for survival. The emission of toxic and harmful substances from industry and agriculture is increasing day by day, and the organic pollution to soil is on a growing trend. Among them, the oil discharged during the development, transportation, processing, storage and transportation of oil has serious harm to the soil environment. Petroleum is a mixture of many chemical substances, which are chemically different and mainly include aromatic hydrocarbons, saturated hydrocarbons, resins, asphalt, etc. Excessive petroleum hydrocarbons in the soil may be excessively remained and accumulated in the plant body, and the physical and mental health of human beings is damaged through the enrichment of food chain. In addition, some pollutant components in the petroleum permeate into the ground water of the shallow soil layer along with precipitation, so that the water quality of the ground water is polluted, and the human health is finally harmed. The Polycyclic Aromatic Hydrocarbons (PAHs) are harmful to human bodies, the polycyclic aromatic hydrocarbons have the effects of teratogenicity, carcinogenicity, mutagenicity and the like, and the lubricating oils and fuel oils with low boiling points can cause the harm of chemical pneumonia, dermatitis, anesthesia, asphyxia and the like to human bodies.
At present, the method for restoring the petroleum polluted soil in China comprises three methods: physical repair methods, chemical repair methods, and biological repair methods. Compared with physical and chemical repairing methods, the biological repairing method has the advantages of low cost, no secondary pollution, good treatment effect and the like. The biological method mainly comprises three methods of microbial repair, plant repair and plant-microbial combined repair. The microbial repairing technology is to utilize indigenous microbes in soil or supplement domesticated efficient microbes to polluted environment, and to accelerate the decomposition of pollutants under optimized environmental conditions to repair polluted soil. The plant restoration is a green technology (Yixian Yun, Danzhi, Shilin, and the like. the plant restoration of the organic pollutant polluted soil [ J ]. the agricultural environmental protection, 2002,21(5): 477-. As a new technology, the plant-microorganism combined remediation technology plays the advantages of the plant remediation technology and simultaneously makes up the defects of small biomass, weak decontamination capability and high treatment cost of simple microorganism remediation. The plant-microorganism combined remediation technology is a good method for treating petroleum-polluted soil so far, and has wide market and development prospect.
The microbial remediation technology has been studied in the early days, and three microbial remediation modes, namely in-situ treatment, in-situ treatment and a bioreactor, have been derived, and the technology is widely applied to the remediation of petroleum-polluted soil (Shen iron Meng, Huang Guo, Li Ling, and the like, the biological ventilation remediation of the petroleum-polluted soil and the enhancement technology thereof [ J ], the environmental pollution treatment technology and equipment, 2002,3(7): 67-69). The Lespedek utilizes a biological composting method to restore the soil polluted by different types of petroleum, the total hydrocarbon removal rate of the petroleum after 60 days is 38.37-56.74%, and the treatment cost is relatively low (Lespedek, Guohui, Sunpu. research on the biological restoration technology of the soil polluted by different types of crude oil [ J ]. applied ecology newspaper, 2002,13(11): 1455-. The research on plant restoration in China is still in the initial stage, the research on the combined restoration of plants and microorganisms is rarely reported, and the abundant plant resources in China provide sufficient materials for research work, so that the advantages in the aspect are fully utilized to explore the combined restoration effect of the plants and the microorganisms on the petroleum-polluted soil.
The combined restoration effect of the plants and the microorganisms mainly depends on the absorption and metabolism capability of the plant-microorganism system on the organic pollutants, and the organic pollutants in the soil are purified by the plant and rhizosphere microorganism coexistence system. The plant-microorganism combined system has good degradation effect on organic pollutants. The basic principle is as follows: 1) the plant root zone secretion has stimulation effect on the transformation of bacteria; 2) plants provide a locus for microorganisms and can transfer oxygen to enable normal aerobic transformation of the root zone, which is also the mechanism by which plants promote mineralization of microorganisms in the root zone (Anderson T.A., Guthrie E.A., Walton B.T., Bioremediation in the rhizosphere, plant roots and associated microorganisms bound soil [ J ]. environ.Sci.Technol.,1993(27): 2630) 2636.).
The industrial fermentation process for producing yeast has appeared in the 19 th century, and the technology of aeration and feeding is adopted; the production of microbial single-cell proteins occurred in the 70's of the 20 th century. Fermentation is the process by which microorganisms grow and form metabolites in an artificial culture environment and is also a common method of microbial agent production. At present, the products of the microorganism live bacteria preparation mainly have two modes of solid fermentation and liquid fermentation. Solid state fermentation is the fermentation of microorganisms on a solid substrate with no or substantially no free water, wherein three phases of gas, liquid and solid coexist, i.e. the porous solid substrate contains water and water-insoluble substances. The large-area popularization of the technology for remedying the petroleum-polluted soil by the microorganisms needs a large amount of microbial agents, and the domestic research on the petroleum degrading microbial agents only develops the small-scale culture in laboratories and does not realize the industrial production.
The method for screening petroleum degrading bacteria by using petroleum hydrocarbon as a unique carbon source has been reported, but most of the methods are used for screening by using single petroleum hydrocarbon or a plurality of petroleum hydrocarbons and intermediate products thereof as the carbon source, and the culture environment of the method is far from the actual situation that the petroleum hydrocarbon in the petroleum polluted soil is various, so the survival capacity and the degradation efficiency of the strains screened under the condition in the actual petroleum polluted soil are not ideal, and the actual effect of the strains in the treatment engineering of the petroleum polluted soil cannot be guaranteed.
Most of the existing screening technologies for petroleum degrading bacteria are mainly based on laboratory research, strain screening is carried out by taking a certain single-component petroleum hydrocarbon as a substrate, then the degradation effect of the screened strain is evaluated by taking the petroleum hydrocarbon as the substrate, and the screening technology is only strain culture in a small test scale in a laboratory.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of a solid microbial inoculum of petroleum degrading bacteria and a method for repairing petroleum-polluted soil by using the solid microbial inoculum prepared by the preparation method.
The invention adopts the following technical scheme:
a preparation method of a solid microbial inoculum of petroleum degrading bacteria comprises the following specific steps:
A. screening and domesticating petroleum degrading bacteria
1) Collection of bacterial sources
Soil polluted by petroleum throughout the year in an oil refinery is taken as a source of the indigenous bacteria for petroleum degradation.
2) Primary screen for oil degradation indigenous bacteria
(1) Taking a soil sample as a petroleum degrading indigenous bacteria source, adding the soil sample into a conical flask filled with sterile water, and placing the conical flask in a shaking incubator to shake for 0.5-2 h at room temperature, wherein the shaking frequency is 200 rpm;
(2) and (3) taking the supernatant from the conical flask in the step (1), inoculating the supernatant into an enrichment medium, and performing shake culture on a shaking table at the temperature of 30 ℃ and the rotating speed of 175rpm for 48 h. Coating the cultured enrichment solution on a screening culture medium plate, and culturing at constant temperature of 30 ℃ for 48 h.
3) Domestication
Inoculating the primarily screened single strains with an inoculating loop, and inoculating the primarily screened single strains into a domestication culture medium A of 100m L, culturing for a period (culturing for 7D at a constant temperature of 30 ℃ and a rotation speed of 175rpm is a period), then taking out a 10m L culture solution from the domestication culture medium A and adding the culture solution into a domestication culture medium B of 90m L, culturing for a period, taking out a 10m L culture solution from the domestication culture medium B and adding the culture solution into a 90m L domestication culture medium C, culturing for a period, taking out a 10m L culture solution from the domestication culture medium C and adding the culture solution into a 90m L domestication culture medium D, culturing for a period, taking out a 10m L culture solution from the domestication culture medium D and adding the culture medium E into the 90m L domestication culture medium E, and culturing for a period, wherein the oil concentration is gradually increased.
4) Purification of bacterial species
(1) Streaking the domesticated culture solution on an oil-containing plate, and culturing at 30 deg.C for 7 d;
(2) picking a single colony from an oil-containing flat plate, transferring the single colony to an L B inclined plane, and culturing for 24h at 30 ℃;
(3) re-inoculating the oil-containing plate from the L B slant;
(4) repeating the steps until a purified colony with a single form is obtained;
(5) the purified bacterial colony is inoculated on the slant of a strain preservation culture medium, cultured for 24 hours at 30 ℃ and stored in a refrigerator at 4 ℃ for later use.
B. Preparation of seed culture solution
Respectively and independently inoculating the petroleum degrading strains obtained by purification into a seed culture medium, and gradually performing amplification culture on the strains through a first-stage seed, a second-stage seed and a seed tank to obtain liquid seeds; the thallus density of the liquid seed reaches 1 x 107cfu; wherein: the first-level seeds are cultured by adopting a solid culture medium slant under the culture condition of 30 ℃ for 24 hours; shake culture with liquid culture medium as secondary seedCulturing at 30 deg.C and 175rpm for 10-12 hr; the seeding tank culture condition is liquid culture medium stirring culture, the culture condition is constant temperature of 30 ℃, the rotating speed is 80rpm, and the ventilation volume is 1: 1.
C. Fermentation of solid microbial inoculum
Crushing or grinding the waste biomass raw material dried by air into particles of 1-5 mm; the biomass particles and other ingredients are put into a high-temperature resistant plastic bag and sterilized for 1 hour at 110 ℃ for standby;
spraying a seed culture solution in a seed tank to the surface of the compost according to a solid-liquid ratio of 10:1 by weight, and uniformly mixing a fermentation seed culture solution with a solid fermentation culture medium; putting the inoculated material on a solid fermentation bed, and carrying out enrichment culture at the temperature of 28-30 ℃ for 3-5 days to obtain a microorganism immobilization material;
b, inoculating the primary seed solution in the step B into the treated immobilized carrier according to the proportion of 1:10(v/w), keeping the water content at 50-55%, and adsorbing, fixing and increasing the value for 3-5 days at the temperature of 28-30 ℃; adding value for 3 times according to the above method to obtain immobilized bacteria agent.
D. Drying, pulverizing, metering, and packaging the piled product
Drying the immobilized microbial inoculum at the temperature of 40-60 ℃ until the water content is 6-10%, crushing the microbial inoculum, and sieving by a 5mm sieve.
On the basis of the above protocol, the media used in steps a and B both contained spiked solutions of petroleum and polysorbate-80.
On the basis of the scheme, the petroleum mass concentration in the petroleum and polysorbate-80 increasing solution is 2%, the polysorbate-80 mass concentration is 4%, and the balance is water.
On the basis of the scheme, the enrichment medium is as follows:
0.025g equivalent petroleum polysorbate-80 solubilization solution, 0.5g NaCl, (NH)4)2SO40.1g，MgSO4·7H2O 0.025g，KNO30.24g，KH2PO40.57g, pH 7.2, distilled water 1000m L, 0.1MPa, 121 ℃, autoclaving for 20 min.
The screening culture medium comprises:
0.025g equivalent petroleum-containing polysorbate-80 solution, 20g agar, 0.5g NaCl, (NH)4)2SO40.1g，MgSO4·7H2O 0.025g，KNO30.24g，KH2PO40.57g, pH 7.2, distilled water 1000m L, 0.1MPa, 121 ℃, autoclaving for 20 min.
The domestication culture medium A comprises:
0.025g equivalent petroleum polysorbate-80 solution, 10g NaCl, NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium B comprises:
0.050g equivalent petroleum-containing polysorbate-80 solubilization solution, 10g NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium C comprises:
0.075g equivalent of petroleum-containing petroleum polysorbate-80 solubilization solution, 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium D comprises:
0.100g equivalent petroleum-containing polysorbate-80 solubilization solution, 10g NaCl, NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium E comprises:
0.125g equivalent petroleum-containing polysorbate-80 solubilization solution, 10g NaCl, NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The culture medium for the oil-containing flat plate comprises:
0.125g equivalent petroleum-containing polysorbate-80 solubilization solution, 10g NaCl, NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, agar 20g, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
L B the culture medium for the slant was:
10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar, 1000m of distilled water L, pH value of 7.0, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
Solution of trace elements: MnSO439.9mg，ZnSO4·7H2O 42.8mg，FeCl2·4H2O 15.73mg，CuSO440mg of distilled water (1000 m L).
The strain preservation culture medium comprises 5g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000m of distilled water L, the pH value is 7.2-7.4, the pressure is 0.1MPa, and the culture medium is sterilized by high-pressure steam for 20min at 121 ℃.
The solid microbial inoculum of the petroleum degrading bacteria prepared by the method is used for repairing petroleum-polluted soil. The specific method comprises the following steps: the prepared solid microbial inoculum of the petroleum degrading bacteria is added into the petroleum polluted soil according to 10-100 kg/mu of land, meanwhile, a chemical fertilizer and an organic fertilizer are applied in an auxiliary manner, 0-30cm of surface soil is ploughed and mixed evenly, and one of three crops of corn, soybean and alfalfa is planted at the same time.
The invention has the beneficial effects that:
(1) according to the invention, the non-ionic surfactant polysorbate-80 (Tween 80) is used as a solubilizer, the petroleum polysorbate-80 solubilization solution is prepared firstly, and then a certain amount of petroleum polysorbate-80 solubilization solution is added into the culture medium, so that the dissolving effect of petroleum can be improved, and the problem that the petroleum is difficult to dissolve in the culture medium is solved.
(2) The method really uses petroleum as a unique carbon source, and uses petroleum polysorbate-80 solution as the carbon source in the processes of primary screening, domestication and purification of petroleum degrading bacteria, preparation of seed solution, fermentation of solid microbial inoculum and the like. In the acclimation process of the strains, the petroleum concentration gradient in the acclimation culture medium is gradually increased, and the strains are gradually acclimated to adapt to the oil-containing culture medium, so that the survival capability and the degradation efficiency of the strains screened under the condition in the actual soil are improved, and the actual effect of the strains in the treatment engineering of the petroleum-polluted soil is guaranteed.
(3) The screening of the petroleum degrading bacteria in the invention comprises the processes of primary screening, domestication, secondary screening, purification and the like, and strains are gradually domesticated in a domestication culture medium with gradually-increased petroleum concentration gradient, so that the strain is suitable for growth and propagation by taking petroleum as a single carbon source, and the tolerance of the strain to the petroleum and the degradation efficiency of the strain are improved.
(4) The solid fermentation raw materials used in the invention are easy to obtain, the process is simple, the solid microbial inoculum contains a large amount of carbon and nutrient elements, provides a more appropriate substrate for bacterial growth, and has strong affinity to microorganisms and high immobilization efficiency. Improving the competitiveness and degradation efficiency of the added microorganisms and indigenous microorganisms. The solid microbial inoculum is convenient for transportation and agricultural operation, and is suitable for large-scale in-situ bioremediation of petroleum-polluted soil.
(5) The method is used for strengthening and repairing the oil-polluted soil by combining the alfalfa and the oil efficient degradation mixed fungicide. The method has the advantages of less investment, small engineering quantity and lower technical requirement, does not cause secondary pollution when used as a green in-situ remediation, does not damage the ecological environment of the soil, and is also beneficial to improving the problems of soil degradation, productivity reduction and the like.
Description of the drawings:
FIG. 1 shows the amount of growth of the cells and the petroleum degradation rate of each of the domesticated strains;
FIG. 2 shows the total number of colonies in the soil at different periods;
FIG. 3 is a graph of total petroleum hydrocarbon removal in soil at different times.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example 1
Screening of high-efficiency petroleum degrading bacterial strain
1. Collection of indigenous bacteria sources for petroleum degradation
Soil polluted by petroleum around an oil storage tank of a petroleum refinery in the Shengli oil field of Shandong province all the year round is taken as a source of the indigenous bacteria for petroleum degradation. The sampling method comprises the following steps: a soil sample with the thickness of 10cm is taken by a small shovel, the soil sample is uniformly mixed after multi-point sampling, and 3kg of the soil sample is taken and is brought back to a laboratory for later use.
2. Preparation of petroleum polysorbate-80 solubilization liquid
After the surfactant reaches Critical Micelle Concentration (CMC) in an aqueous solution, the solubility of some water-insoluble or slightly-soluble substances in the micelle solution can be obviously increased to form a transparent colloidal solution, and the effect is called solubilization (polyoxyethylene sorbitan fatty acid ester), namely, polysorbate-80 (polyoxyethylene sorbitan fatty acid ester) is a water-soluble nonionic surfactant which is not influenced by electrolyte and pH value and has no bactericidal performance, and the H L B value (hydrophile-lipophile balance value, H L B) is 15.0, so that the polysorbate-80 is suitable for being used as a solubilizer and an oil-in-water (O/W) type emulsion, the solubility of petroleum in the petroleum polysorbate-80 can be improved, so that the petroleum is uniformly dispersed in the petroleum polysorbate-80 solution, various microbial culture mediums are prepared by using the polysorbate-80 solution as a unique carbon source, and the effect of the polysorbate-80 on the solubilization of the crude oil is shown in a water solubilization solution table 2.
TABLE 1 Effect of crude oil concentration on the emulsification Effect of solubilizers
TABLE 2 influence of polysorbate-80 concentration on the emulsifying Effect of the solubilizers
As can be seen from tables 1 and 2, in the preparation of the solubilization solution of petroleum polysorbate-80, the crude oil is well emulsified in water when the crude oil concentration is less than 3% and the polysorbate-80/crude oil ratio is greater than 2; when the polysorbate-80/crude ratio is less than 2, the crude does not emulsify in water, but only forms spherical oil droplets. When the concentration of the crude oil is equal to 3%, the crude oil is not emulsified in water and only spherical oil drops are formed when the ratio of polysorbate-80 to the crude oil is less than 1.67; when the ratio of polysorbate-80 to crude oil is greater than 2.00, the crude oil is well emulsified in water, but the oil and water are seriously separated when stirring is stopped. When the concentration of the crude oil is more than 3%, the crude oil is not emulsified in water and only spherical oil drops are formed.
3. Screening the petroleum degrading indigenous bacteria;
(1) taking 5g of a soil sample of the indigenous bacteria source for petroleum degradation, adding the soil sample into a conical flask containing 100m L sterile water, placing the conical flask in a shaking incubator, and shaking at room temperature for 2h and 200 rpm;
(2) taking 5m L supernatant from a conical flask of sterile water, inoculating 250m L enrichment medium, performing shaking culture at 30 ℃ and 175rpm on a shaking table for 48h, taking 0.2m L enrichment solution, coating the enrichment solution on a flat plate screening medium, and performing constant temperature culture at 30 ℃ for 48 h.
4. Domestication
And (4) selecting the primarily screened multiple single strains by using an inoculating loop, and inoculating and domesticating the single strains in a domestication culture medium.
The domestication culture media are A, B, C, D, E respectively, the petroleum concentration gradient of the domestication culture media gradually rises, the domestication culture media are filled in a triangular flask with the thickness of 250m L, the temperature is 30 ℃, the rotating speed is 175rpm, the domestication culture media without the inoculated strains are used as a blank control, when in domestication, a plurality of single strains obtained by primary screening are firstly picked by an inoculating loop and respectively inoculated into a domestication culture medium A with the thickness of 100m L fresh, the single strains are cultured for a period, then 10m L culture solution is taken out from the domestication culture medium A and added into a domestication culture medium B with the thickness of 90m L fresh, the single strains are cultured for a period, then 10m L culture solution is taken out from the domestication culture medium B and added into a domestication culture medium C with the thickness of 90m L fresh domestication culture medium C, the single period is cultured, 10m L culture solution is taken out from the domestication culture medium C and added into a domestication culture medium D with the thickness of 90m L fresh domestication culture medium D, the single period is taken out from the domestication culture medium D and added into a domestication culture medium C of 90m L fresh domestication culture medium D, the single strain is repeatedly observed in the same.
5. Purification of bacterial species
(1) Taking the domestication culture solution of the last cycle, streaking on an oil-containing plate, and culturing at 30 deg.C for 7 d;
(2) picking a single colony from an oil-containing flat plate, transferring the single colony to an L B inclined plane, and culturing for 24h at 30 ℃;
(3) re-inoculating the oil-containing plate from the L B slant;
(4) repeating the steps until a purified colony with a single form is obtained;
(5) the purified bacterial colony is inoculated on the slant of a strain preservation culture medium, cultured for 24 hours at 30 ℃ and stored in a refrigerator at 4 ℃ for later use.
Preparing a used culture medium:
the enrichment medium is as follows:
0.025g equivalent petroleum-containing polysorbate-80 solubilization solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), 0.5g of NaCl (NH)4)2SO40.1g，MgSO4·7H2O 0.025g，KNO30.24g，KH2PO40.57g, pH 7.2, distilled water 1000m L, 0.1MPa, 121 ℃, autoclaving for 20 min.
The screening culture medium comprises:
0.025g equivalent petroleum-containing polysorbate-80 solution (wherein the mass concentration of the petroleum is 2 percent, polysorbate-80 mass concentration of 4%, the remainder being water. ) Agar 20g, NaCl 0.5g, (NH)4)2SO40.1g，MgSO4·7H2O0.025g，KNO30.24g，KH2PO40.57g, pH 7.2, distilled water 1000m L, 0.1MPa, 121 ℃, autoclaving for 20 min.
The domestication culture medium A comprises:
0.025g equivalent petroleum-containing polysorbate-80 solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium B comprises:
0.050g equivalent petroleum-containing polysorbate-80 solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium C comprises:
0.075g equivalent of petroleum-containing petroleum polysorbate-80 solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium D comprises:
polysorbate with 0.100g equivalent of petroleumEster-80 solubilization solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water.), NaCl 10g, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium E comprises:
0.125g equivalent petroleum-containing polysorbate-80 solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The culture medium for the oil-containing flat plate comprises:
0.125g equivalent petroleum-containing polysorbate-80 solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, agar 20g, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
L B the culture medium for the slant was:
10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar, 1000m of distilled water L, pH value of 7.0, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
Solution of trace elements: MnSO439.9mg，ZnSO4·7H2O 42.8mg，FeCl2·4H2O 15.73mg，CuSO440mg of distilled water (1000 m L).
The strain preservation culture medium comprises 5g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000m of distilled water L, the pH value is 7.2-7.4, the pressure is 0.1MPa, and the culture medium is sterilized by high-pressure steam for 20min at 121 ℃.
5. Results of culturing, screening and morphological identification of petroleum degrading bacteria
Domestication and screening are carried out by enrichment culture and petroleum concentration gradient increasing methods, and 7 single strains are obtained by repeated streaking separation and purification primary screening of a flat plate, as shown in table 3.
TABLE 3 results of observing the morphology of colonies and cells
After shaking culture for 5d on a constant-temperature shaking table at 30 ℃ and 175rpm, observing the growth characteristics of the 7 single strains and the mixed strains 1 and 2 in the domestication culture medium E and measuring the thallus growth amounts and the petroleum degradation rates of the 7 single strains and the 2 parallel mixed strains, wherein the mixed strain 1 is the uniform mixing of the 7 single strains, and the mixed strain 2 is the uniform mixing of the strains X-2 and X-6. The growth characteristics of each strain are shown in table 4,
TABLE 4 growth characteristics of the respective strains
From the comprehensive analysis in Table 4 and FIG. 1, it can be seen that the more complete the emulsification of petroleum, the greater the amount of cell growth of the strain, and the higher the petroleum degradation rate of the strain. The growth amount of the thalli and the petroleum degradation rate form a positive correlation. The X-2 and X-6 strains have complete petroleum emulsification degree, large thallus growth amount and high petroleum degradation rate, so the strain is an advantageous strain for degrading petroleum. The test result also shows that different strains have different petroleum degradation effects, and the mixed strain 2 has better petroleum degradation effect than the mixed strain 1, so the mixed strain 2 is used as a strain to prepare the petroleum degradation microbial inoculum.
Example 2
Liquid fermentation production of petroleum degradation microbial inoculum
1. First order seed culture
The petroleum-degrading bacteria mixed strain 2 purified in example 1 was aseptically inoculated into a test tube slant medium. Medium composition per 1000ml sterile water: 0.125g equivalent petroleum-containing polysorbate-80 solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar powder, the pH value is 7, the temperature is 121 ℃, and the sterilization is carried out for 20 minutes. The culture conditions were: cultured at 30 ℃ for 24 hours to obtain primary seeds.
2. Second stage seed culture
The two rings of the first-order seeds are taken under aseptic conditions and inoculated into a 500ml triangular flask filled with 200ml of culture medium. Shake flask medium composition per 1000ml sterile water: 0.125g equivalent of petroleum-containing polysorbate-80 solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), 3g of beef extract, 10g of peptone and 5g of NaCl, the pH value is 7, and the beef extract is sterilized at 121 ℃ for 20 minutes. The culture conditions were 30 ℃ and 80rpm, and the secondary seeds were obtained after 9 hours of culture.
3. Seeding tank culture
Inoculating the second-level seeds into a 50L fermentation tank according to the inoculation amount of 10% by volume, adding 40L culture medium into a 50L fermentation tank, and adding 4L second-level seed liquid, wherein every 1000ml of sterile water consists of 0.125g equivalent of petroleum-containing petroleum polysorbate-80 additive solution (the mass concentration of the petroleum is 2%, the mass concentration of the polysorbate-80 is 4%, and the balance is water), 3g of beef extract, 10g of peptone, 5g of NaCl, the pH value is 7, the temperature is 121 ℃, the sterilization is carried out for 30 minutes, the culture condition is that the stirring rotation speed is 80rpm, the ventilation amount is 1:1, the temperature is 30 ℃, and after 8 hours of culture, the thallus density is 1.2 × 10 detected by a bacteria counter7cfu, end of culture.
4. Solid microbial inoculum fermentation increment
Crushing or grinding the waste biomass raw material dried by air into particles of 2 mm; the biomass particles and other ingredients are put into a high-temperature resistant plastic bag and sterilized for 1 hour at 110 ℃ for standby. Spraying the culture solution prepared in the step (3) onto the surface of the stockpile according to the solid-liquid ratio of 10:1 by weight, and uniformly mixing; placing the inoculated material on a solid fermentation bed, and performing proliferation culture at 30 ℃ for 3 daysPreparing the microorganism immobilized material. The formula of the solid fermentation culture medium comprises the following raw materials in percentage by weight: 30-35% of wheat bran, 25-45% of corn straw powder, 20-25% of peanut shell powder, 0.5-1% of glucose, 3-5% of ammonium sulfate and K2HPO40.3～0.5％、MgSO4·7H20.1-0.3% of O, and the weight ratio of water to material is 1: 1-4: 1; controlling the water content to be about 50-55%, adjusting the pH value to be 7.2, inoculating the primary seed liquid into the treated immobilized carrier according to the proportion of 1:10(v/w), keeping the water content to be 50-55%, adsorbing and fixing at the temperature of 30 ℃, and increasing the value for 3 days; adding value for 3 times according to the method to obtain the immobilized mixed microbial inoculum.
5. Drying, pulverizing, metering, and packaging the piled product
After the solid fermentation process is finished, the drying temperature of the pile is 45 ℃ until the water content of the pile is 6%, the pile is crushed, and is sieved by a 5mm sieve for standby after detection, the effective viable count is 8 hundred million/g, the water content is 4%, and the pH is 7.0; and (3) metering and packaging by using packaging equipment to prepare the petroleum degradation solid complex microbial inoculum.
Example 3
Combined remediation of petroleum-polluted soil by using petroleum-degrading solid complex microbial inoculum and plants
1. Experimental base selection and processing
The test selects the land near the oil extractor of a certain oil extraction plant in Dongying city, the land is near the oil extractor and is polluted by petroleum, the total hydrocarbon content of the petroleum is 4g/Kg, and the basic physicochemical properties of the soil are shown in Table 5. The method adopts alfalfa-microorganism combined remediation of the petroleum-polluted soil, the planting density is about 8000 plants/mu, the prepared petroleum degrading bacteria solid microbial inoculum is added into the petroleum-polluted soil according to the land of 10-100 kg/mu, and the surface soil of 0-30cm is ploughed and mixed evenly. When the alfalfa is planted, the soil is covered by a film, the moisture is kept, and the controlled release fertilizer is applied to ensure the nutrients required by the normal growth of plants. In the growing seedling stage, the drip irrigation bags are respectively paved and irrigated in a drip irrigation mode.
TABLE 5 soil sample basic Properties
2. Soil sample collection
The soil sample is about 20cm underground soil taken by a soil sampler. The test takes four samples in total, namely a planting period, a seedling period, a strong year period and a harvesting period. Taking back the soil sample, air-drying, breaking, removing impurities, uniformly mixing, sieving with a 60-mesh sieve, and sealing for storage.
3. Separation and counting of bacteria in soil
(1) Preparing soil solution, weighing 10g of soil sample, placing into a triangular flask containing 90m L sterile water, shaking for 30min by a shaking table to fully mix the soil sample with the water, and dispersing the bacteria.
(2) Preparing a blank plate: heating and melting the sterilized culture mediums, cooling to 55-60 ℃, uniformly mixing, pouring the culture mediums into plates, pouring three plates into each culture medium, and marking the name, soil sample number and experimental date of the culture medium by using a marker.
(3) Coating, namely writing the diluted concentration on the bottom surface of each plate of each culture medium by using a marker pen, then respectively sucking the soil diluent with the corresponding concentration by using a sterile pipette with the concentration of 0.1m L respectively, carefully dripping the soil diluent at the central position of the surface of the corresponding plate, slightly expanding the bacterial suspension outwards along the concentric circle direction by using a sterile glass coating rod to ensure that the bacterial suspension is uniformly distributed, and standing for 10min at room temperature to ensure that the bacterial liquid is immersed in the culture medium.
(4) Culturing: the agar plate was inverted with the dish bottom facing up and placed upside down in the incubator at 37 ℃ for culturing.
(5) Counting: the colonies which are clear are selected and counted on the reverse side of the plate by using a marker pen.
4. Determination of total petroleum hydrocarbons
The wavelength is selected by preparing a petroleum solution with the concentration of 100 mg/L, and scanning on an ultraviolet spectrophotometer to obtain the crude oil with the wavelength of 228nm at the maximum absorption peak.
Drawing a standard curve: taking 6 clean 50mI colorimetric tubes, sequentially adding oil standard solutions containing 0.2, 0.5, 1.0, 1.2, 1.5 and 2.0mg of oil, diluting the solutions to scale marks by using petroleum ether, shaking the solutions uniformly, and measuring the absorbance (A) of the solutions at a wavelength of 228nm by using the petroleum ether as a reference solution to obtain a standard curve of the crude oil standard solution.
Soil sample analysis, namely accurately weighing 10.00g of a dry sample, placing the dry sample into a 50m L colorimetric tube, adding 20m L trichloromethane, placing the colorimetric tube into an ultrasonic device water bath for ultrasonic extraction for 15min, standing and filtering, collecting an extracting solution in a 50m L clean beaker, extracting the extracting solution once again by using 20m L trichloromethane, combining the two extracting solutions, evaporating the extracting solutions to dryness in a (65 +/-5) DEG C constant-temperature water bath, dissolving residues by using petroleum ether, fixing the volume in a 50m L colorimetric tube, measuring the absorbance at 228nm, and calculating the content of oil in the sample by using a standard curve equation.
5. Analysis of soil remediation results
After the growing period of about 100 days of alfalfa, four times of sampling are respectively carried out in the planting period, the seedling period, the strong year period and the harvesting period, the total amount of microorganisms in soil is changed as shown in figure 2, and the content of total petroleum hydrocarbon in soil is changed as shown in figure 3.
Claims (5)
1. A preparation method of a solid microbial inoculum of petroleum degrading bacteria is characterized by comprising the following steps: the method comprises the following specific steps:
A. screening and domesticating petroleum degrading bacteria
1) Collection of bacterial sources
Taking the soil polluted by petroleum throughout the year by an oil refinery as a petroleum degradation indigenous bacteria source;
2) primary screen for oil degradation indigenous bacteria
(1) Taking a soil sample as a petroleum degrading indigenous bacteria source, adding the soil sample into a conical flask filled with sterile water, and placing the conical flask in a shaking incubator to shake for 0.5-2 h at room temperature, wherein the shaking frequency is 200 rpm;
(2) taking the supernatant from the conical flask in the step (1), inoculating the supernatant into an enrichment medium, and carrying out shake culture on a shaking table at the temperature of 30 ℃ and the rotating speed of 175rpm for 48 h; coating the cultured enrichment solution on a screening culture medium flat plate, and culturing at constant temperature of 30 ℃ for 48 h;
3) domestication
Inoculating and domesticating a plurality of single strains obtained by primary screening by using an inoculating loop in a domestication culture medium, and specifically comprises the steps of inoculating the plurality of single strains obtained by primary screening by using the inoculating loop into a 100m L domestication culture medium A respectively, culturing for a period, then taking a 10m L culture solution from the domestication culture medium A, adding the 10m L culture solution into a 90m L domestication culture medium B, culturing for a period, taking a 10m L culture solution from the domestication culture medium B, adding the 10m L culture solution into a 90m L domestication culture medium C, culturing for a period, taking a 10m L culture solution from the domestication culture medium C, adding the 10m L culture solution into a 90m L domestication culture medium D, culturing for a period, adding the 10m L culture solution into the 90m L domestication culture medium E from the domestication culture medium D, culturing from the domestication culture medium A to the domestication culture medium E, and gradually increasing the petroleum concentration gradient;
the one cycle is as follows: culturing at 30 deg.C and rotation speed of 175rpm under constant temperature shaking for 7 d;
4) purification of bacterial species
(1) Streaking the domesticated culture solution on an oil-containing plate, and culturing at 30 deg.C for 7 d;
(2) picking a single colony from an oil-containing flat plate, transferring the single colony to an L B inclined plane, and culturing for 24h at 30 ℃;
(3) re-inoculating the oil-containing plate from the L B slant;
(4) repeating the steps until a purified colony with a single form is obtained;
(5) inoculating the purified bacterial colony on a strain preservation culture medium slant, culturing at 30 ℃ for 24h, and storing in a refrigerator at 4 ℃ for later use;
B. preparation of seed culture solution
Respectively and independently inoculating the petroleum degrading strains obtained by purification into a seed culture medium, and gradually performing amplification culture on the strains through a first-stage seed, a second-stage seed and a seed tank to obtain liquid seeds; the thallus density of the liquid seed reaches 1 x 107cfu; wherein: the first-level seeds are cultured by adopting a solid culture medium slant under the culture condition of 30 ℃ for 24 hours; the secondary seeds are cultured in a liquid culture medium for 10-12 hours by shaking table under the conditions of 30 ℃ and 175rpm of rotation speed; the seeding tank culture condition is liquid culture medium stirring culture, the culture condition is constant temperature of 30 ℃, the rotating speed is 80rpm, and the ventilation volume is 1: 1;
C. fermentation of solid microbial inoculum
Crushing or grinding the waste biomass raw material dried by air into particles of 1-5 mm; the biomass particles and other ingredients are put into a high-temperature resistant plastic bag and sterilized for 1 hour at 110 ℃ for standby;
spraying a seed culture solution in a seed tank to the surface of the compost according to a solid-liquid ratio of 10:1 by weight, and uniformly mixing a fermentation seed culture solution with a solid fermentation culture medium; putting the inoculated material on a solid fermentation bed, and carrying out enrichment culture at the temperature of 28-30 ℃ for 3-5 days to obtain a microorganism immobilization material;
b, inoculating the primary seed solution in the step B into the treated immobilized carrier according to the proportion of 1:10(v/w), keeping the water content at 50-55%, and adsorbing, fixing and proliferating for 3-5 days at the temperature of 28-30 ℃; proliferating for 3 times to obtain immobilized bacteria agent;
D. drying, pulverizing, metering, and packaging the piled product
Drying the immobilized microbial inoculum at the temperature of 40-60 ℃ until the water content is 6-10%, crushing the microbial inoculum, and sieving by a 5mm sieve; the culture medium used in the steps A and B contains solubilization liquid of petroleum and polysorbate-80, the mass concentration of the petroleum in the solubilization liquid of the petroleum and polysorbate-80 is 2%, the mass concentration of the polysorbate-80 is 4%, and the balance is water.
2. The method for preparing the solid microbial inoculum of the petroleum degrading bacteria according to claim 1, which is characterized in that:
the enrichment medium is as follows:
solubilization solution containing petroleum 0.025g equivalent of petroleum and polysorbate-80, NaCl 0.5g, (NH)4)2SO40.1g，MgSO4·7H2O 0.025g，KNO30.24g，KH2PO40.57g, pH 7.2, distilled water 1000m L, 0.1MPa, 121 deg.C, and high pressure steam sterilizing for 20 min;
the screening culture medium comprises:
solubilization solution containing petroleum 0.025g equivalent of petroleum and polysorbate-80, agar 20g, NaCl 0.5g, (NH)4)2SO40.1g，MgSO4·7H2O 0.025g，KNO30.24g，KH2PO40.57g, pH 7.2, distilled water 1000m L, 0.1MPa, 1Sterilizing with high pressure steam at 21 deg.C for 20 min;
the domestication culture medium A comprises:
the additive solution containing 0.025g equivalent of petroleum and polysorbate-80, 10g of NaCl and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, high pressure steam sterilization for 20 min;
the domestication culture medium B comprises:
0.050g equivalent petroleum-containing additive solution of petroleum and polysorbate-80, 10g NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, high pressure steam sterilization for 20 min;
the domestication culture medium C comprises:
oil and polysorbate-80 solubilization solution containing 0.075g equivalent of oil, 10g of NaCl, NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, high pressure steam sterilization for 20 min;
the domestication culture medium D comprises:
oil and polysorbate-80 solution containing 0.100g equivalent of oil, 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, high pressure steam sterilization for 20 min;
the domestication culture medium E comprises:
0.125g equivalent petroleum-containing additive solution of petroleum and polysorbate-80, 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, high pressure steam sterilization for 20 min;
the culture medium for the oil-containing flat plate comprises:
0.125g equivalent petroleum-containing additive solution of petroleum and polysorbate-80, 10g of NaCl, and NH4NO31g，KH2PO40.5g，K2HPO4·3H2O 1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, trace element solution 1m L, agar 20g, distilled water 1000m L, pH 7.2, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min;
l B the culture medium for the slant was:
10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar, 1000m of distilled water L, pH value of 7.0, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min;
solution of trace elements: MnSO439.9mg，ZnSO4·7H2O 42.8mg，FeCl2·4H2O 15.73mg，CuSO440mg, 1000m L distilled water;
the strain preservation culture medium comprises 5g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000m of distilled water L, wherein the pH value is 7.2-7.4, the pressure is 0.1MPa, and the culture medium is sterilized by high-pressure steam for 20min at 121 ℃.
3. The solid microbial inoculum of petroleum degrading bacteria prepared by the method of claim 1 or 2 is used for repairing petroleum-polluted soil.
4. The solid microbial inoculum of petroleum degrading bacteria according to claim 3 for remediating petroleum-contaminated soil, which is characterized in that: the specific method comprises the following steps: the prepared solid microbial inoculum of the petroleum degrading bacteria is added into the petroleum polluted soil according to 10-100 kg/mu of land, meanwhile, a chemical fertilizer and an organic fertilizer are applied in an auxiliary manner, 0-30cm of surface soil is ploughed and mixed evenly, and one of three crops of corn, soybean and alfalfa is planted at the same time.
5. The solid microbial inoculum of petroleum degrading bacteria of claim 4 for remediating petroleum-contaminated soil, which is characterized in that: the alfalfa is alfalfa.
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