CN104388312B - Method for screening petroleum degrading bacteria, method for preparing petroleum degrading bacteria microbial inoculum from screened bacteria and application of microbial inoculum - Google Patents
Method for screening petroleum degrading bacteria, method for preparing petroleum degrading bacteria microbial inoculum from screened bacteria and application of microbial inoculum Download PDFInfo
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Abstract
The invention relates to a method for screening petroleum degrading bacteria, wherein an enrichment culture medium, a screening culture medium and an acclimatization culture medium used in the method are culture media taking petroleum as a unique carbon source, and specifically, a solubilization solution of petroleum and polysorbate-80 is added into the culture media. The invention has the advantages that the invention takes the nonionic surfactant polysorbate-80 as the solubilizer, can improve the dissolving effect of petroleum in water and solve the problem that the petroleum is difficult to dissolve in a culture medium; the invention really uses petroleum as the only carbon source, and the culture medium uses petroleum polysorbate-80 solution as the carbon source in the processes of primary screening, domestication, purification and the like of the petroleum degrading bacteria; the screening of the petroleum degrading bacteria in the invention comprises the processes of primary screening, domestication, purification and the like, and the strains are gradually domesticated in a domestication culture medium with gradually rising 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.
Description
Technical Field
The invention relates to the technical field of microbial remediation of petroleum-contaminated soil, in particular to a method for screening petroleum degrading bacteria, a method for preparing a petroleum degrading bacteria microbial inoculum from the screened bacteria and application of the microbial inoculum in petroleum-contaminated soil remediation.
Background
Petroleum is a complex mixture containing many hydrocarbons (normal paraffins, branched paraffins, aromatic hydrocarbons, alicyclic hydrocarbons) and a small amount of other organic compounds (sulfides, nitrides, naphthenic acids, etc.), and is one of the most important energy sources for human beings. The leakage of oil is often accompanied in the process of oil exploitation and transportation, which causes environmental pollution. In particular, Polycyclic Aromatic Hydrocarbons (PAHs) pose a great threat to human health due to their poor bioavailability, long half-life, and mostly teratogenic, carcinogenic, and mutagenic effects. The petroleum industry in China develops rapidly, but serious environmental pollution is caused, and nearly ten million acres of cultivated land are polluted by petroleum hydrocarbon to different degrees. Therefore, repairing petroleum hydrocarbon polluted soil and recovering the ecological function of the soil to ensure the safety of agricultural products are very urgent tasks.
The technology for repairing the petroleum-polluted soil becomes a new research hotspot in the current environmental protection field. In recent years, certain progress has been made at home and abroad in the aspect of treating petroleum pollution by physical methods, chemical methods and biological methods, and the traditional physical methods and chemical methods are expensive, easy to cause secondary pollution and limited in application. The biological method has the advantages of good treatment effect, low cost, small influence on the environment, no secondary pollution, wide application range and the like, and is concerned. The biological method mainly comprises three types of microbial repair, phytoremediation 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 biological method is a good method for repairing petroleum polluted soil so far, and has wide market and development prospect.
In recent years, a technology for repairing petroleum-contaminated soil by using microorganisms is paid attention by researchers, and the key point of the technology is to improve the degradation activity of the microorganisms, wherein one is to enhance the inherent slow microbial degradation process in the nature, and the other is to contact the pollutants with the microorganisms through a bioreactor to strengthen the degradation process and improve the degradation rate. At present, research work in laboratories focuses on the exploration of screening high-efficiency degradation flora, optimizing degradation conditions and degradation means aiming at certain oil, and partial application research is carried out on actual petroleum pollution sites. Screening and oil removing effects of microorganisms for efficiently degrading engine oil of Wangzuoying and the like (Wangzuoying, Wangzui, Guixianlin and the like, industrial microorganisms, 2006,36(1):26-29 and 33.) six microbial strains with degradation capability on high-concentration engine oil and other related petroleum products are screened from soil polluted by petroleum and products at multiple places, and the effects of the six strains on the single and mixed engine oil degradation are researched, and the influence of nutritional ingredients on the engine oil degradation is investigated. Huchangqing and the like (Huchangqing, Bayuting, Xuwei, and the like. screening of filamentous petroleum-degrading fungi and degradation characteristic research thereof [ J ]. Anhui agricultural science 2010,38(4): 1664-. Qiu Qing Hua, etc. (Qiu Qing, Hanipa, Denshao cloud, etc. screening and degradation condition research of petroleum degradation mixed bacteria [ J ] Chinese agronomy report, 2013,29(3): 184) 189.) 68 strains of bacteria, 15 strains of fungi and 6 strains of actinomycetes are obtained from oil contaminated soil, oily wastewater and crude oil through enrichment, domestication and separation; the separated strains are screened through preliminary screening, secondary screening and pairwise mixing antagonism experiments to obtain two groups of petroleum efficient degradation mixed bacteria agents, and the petroleum degradation rate of the two groups of petroleum efficient degradation mixed bacteria agents exceeds 60%. The Yaowanjing (Yaowanjing, screening of efficient oil removal microbial strains and research on enhanced biodegradation of petroleum hydrocarbon by surfactant [ D ]. Chongqing: Chongqing university, 2007.) selects a strain with the degradation capability of more than 60% from soil polluted by petroleum hydrocarbon for a long time through enrichment culture and domestication separation, identifies the strain, performs ultraviolet mutagenesis experiments and optimizes a culture medium, so that the degradation rate of the strain on the petroleum hydrocarbon is obviously improved, and researches on the optimal conditions of microbial degradation of the petroleum hydrocarbon and the influence and mechanism of the surfactant Tween80, SDS, APG and JFC on the petroleum hydrocarbon biodegradation.
A great deal of research work is carried out abroad mainly aiming at the aspects of marine sediment petroleum pollutants, environmental pollution caused by crude oil leakage and the like. The related reports indicate that the oil tanker leakage event of the Alaska prince bay causes large-area pollution, and the JonE.Llidstrom and the like add high-efficiency degrading bacteria to carry out bioremediation, so that the environmental quality is obviously improved, and the event is known as the most successful example of bioremediation. Meanwhile, the environmental protection authorities and important organizations in the United states have vigorously advocated the promotion of bioremediation technology. And R.Margesin (R.Margesin.positional of cold-adapted microorganisms for biological analysis of oil-polarized soil. BioDeterioration & BioDegradation2000(46): 3-10.) is used for screening petroleum degrading strains from the soil polluted by the high mountain and domesticating the petroleum degrading strains. The biological repair technology is regarded as a very effective method for treating large-area polluted areas in Europe. Salanitro finds that most of the soil polluted by petroleum is toxic to organisms, almost no animals can survive at the initial stage of pollution, the yield of crops is only 0-25% of the normal yield, but the soil repaired by a biological method has no influence on animals and plants. S.S.Zinjdard (S.S.Zinjarde, A.A.Pant.Hydrocarbon dispersions from torpidermal Engine environment [ J ]. Marine Pollution Bulletin,2002,44: 118-. K.S.M.Rahman et al (K.S.M.Rahman, J.Thahira-Rahman, P.Lakshmanaplamy, et al.Towards effective crude oil degradation by amino acid bacterial consortium [ J ]. Bioresource Technology,2002,85: 257. 261.) also found that the degradation rate of crude oil by the mixed strain decreased from 78% to 52% when the crude oil concentration increased from l% to 10%.
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. 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. 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. 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. Solid state fermentation has the following disadvantages over liquid state fermentation: limited to microorganisms resistant to low water activity, few options; the fermentation rate is slow, and the period is long; the components of natural raw materials are complex and sometimes change, so that the yield and the quality are influenced; the process parameters are difficult to detect and control; less products, more labor consumption in operation and high strength. The invention researches the production of the petroleum degrading bacteria agent by a liquid state fermentation culture technology, and determines a high-density liquid state submerged fermentation production technology of the petroleum degrading bacteria agent. The patent number CN 103898000A mainly relates to a liquid fermentation method of a petroleum degradation microbial inoculum.
Although the method for screening the petroleum degrading bacteria by using the petroleum hydrocarbon as the only carbon source has been reported, 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 hydrocarbons in the petroleum polluted soil are 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. The patent number CN 102517279A mainly relates to a method for screening a self-transferable broad-host plasmid carrying a petroleum hydrocarbon degradation gene by utilizing triparental mating and butt joint and taking petroleum hydrocarbon as a unique carbon source, and mainly takes petroleum hydrocarbons such as n-octane, benzene, toluene, salicylic acid, catechol and the like as substrates to screen microorganisms; patent No. CN 102533928A mainly relates to a method for screening degradation bacteria using polycyclic aromatic hydrocarbon as a substrate, and single tetracyclic polycyclic aromatic hydrocarbon pyrene or tricyclic polycyclic aromatic hydrocarbon phenanthrene and the like are used as substrates for screening the degradation bacteria.
The viscosity of petroleum is 124.12mm2And/s, the solubility is low, and if the culture medium is prepared by taking petroleum as a carbon source without treatment, the dissolution amount is small, and the petroleum basically sticks to the wall of the container. The patent number CN 102021132A mainly relates to a screening and repairing method of a bioremediation agent for petroleum-polluted soil, which takes crude oil as a substrate. In this experiment1, 2 and 3g of petroleum are added into 100ml of culture medium respectively, under the condition of no solubilizer, the crude oil is hardly dissolved, the concentration of the petroleum in the culture medium is very low, the petroleum is basically hung on the wall of a culture vessel, and the petroleum is added into the culture medium but is not dissolved, so the petroleum plays a little role.
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, the strain culture is only carried out in a small-scale laboratory, and the technical reports of realizing the industrial production of the microbial inoculum by using the petroleum degrading bacteria are less. A patent report (CN 103898000A) of a liquid fermentation method of a petroleum degradation microbial inoculum introduces the liquid fermentation method of the petroleum degradation microbial inoculum, compared with solid fermentation, the fermentation period is shortened by about 60 to 84 hours, but the method improves the thallus yield and improves the oil reduction property of strains by combining oil carbon sources and rapid carbon sources, and soybean oil is used for replacing petroleum in a fermentation culture medium. Because soybean oil is easier to be used as a carbon source by microorganisms and the bioavailability of the soybean oil is far higher than that of petroleum, the method for producing the microbial inoculum without petroleum participation in the fermentation process is not beneficial to ensuring the viability and the degradation efficiency of the microbial inoculum in the application of petroleum-polluted soil treatment engineering.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for screening petroleum degrading bacteria by taking a petroleum polysorbate-80 additive solution as a unique carbon source, inoculating and domesticating the petroleum degrading bacteria in a domestication culture medium with sequentially increased petroleum concentration, taking a strain in the domestication culture medium with the highest petroleum concentration, performing streak culture on an oil-containing plate, selecting a single colony, transferring the single colony to an LB inclined plane until a purified petroleum degrading colony with a single shape is obtained, and preparing a petroleum degrading bacteria agent by using the single colony.
The invention adopts the following technical scheme:
a method for screening petroleum-degrading bacteria uses an enrichment culture medium, a screening culture medium and an acclimatization culture medium which use petroleum as a sole carbon source, and concretely, a solution of petroleum and polysorbate-80 is added into the culture medium.
A method for screening petroleum degrading bacteria comprises the following specific steps:
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
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.
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 single colony from an oil-containing flat plate, transferring the single colony to an LB inclined plane, and culturing for 24h at 30 ℃;
(3) re-inoculating the oil-containing plate from the LB 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.
On the basis of the scheme, the step 3) specifically comprises the following steps:
firstly, a plurality of single strains obtained by primary screening are picked by an inoculating loop and respectively inoculated into 100mL of domesticated culture medium A, and are cultured for a period (the period is 7d when the temperature is 30 ℃ and the rotation speed is 175rpm and the constant temperature is oscillated), then 10mL of culture solution is taken out from the domesticated culture medium A and added into 90mL of domesticated culture medium B, and the culture is carried out for a period. Then, 10mL of the culture medium was taken out of acclimatization medium B and added to 90mL of acclimatization medium C, and the mixture was cultured for one cycle. 10mL of the culture medium was taken out of acclimatization medium C and added to 90mL of acclimatization medium D, and cultured for one cycle. And (3) taking 10mL of culture solution from the acclimatization culture medium D, adding the culture solution into 90mL of acclimatization culture medium E, culturing for a period, and gradually increasing the petroleum concentration gradient from the acclimatization culture medium A to the acclimatization culture medium E.
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, 1000mL of distilled water, 0.1MPa, 121 ℃, and autoclaving for 20 min.
The screening culture medium comprises:
0.025g equivalent petroleum polysorbate-80 solution, 20g agar, 0.5g NaCl (NH4)2SO40.1g，MgSO4·7H2O 0.025g，KNO30.24g，KH2PO40.57g, pH 7.2, 1000mL of distilled water, 0.1MPa, 121 ℃, and 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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 1000mL of distilled water, pH value of 7.2, 0.1MPa,sterilizing with high pressure steam at 121 deg.C 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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 20g of agar, 1000mL of distilled water, 7.2 of pH value, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The LB slant culture medium was:
10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar and 1000mL of distilled water, wherein the pH value is 7.0, the pressure is 0.1MPa, the temperature is 121 ℃, and the sterilization is carried out for 20min by high-pressure steam.
Solution of trace elements: MnSO439.9mg，ZnSO4·7H2O 42.8mg，FeCl2·4H2O15.73mg，CuSO440mg, 1000mL of distilled water.
And (3) strain preservation culture medium: 5g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000mL of distilled water, wherein the pH value is 7.2-7.4, the pressure is 0.1MPa, and the beef extract is sterilized by high-pressure steam at 121 ℃ for 20 min.
A petroleum degrading bacterium agent is prepared from the prepared petroleum degrading bacterium, and the specific preparation method comprises the following steps:
(1) 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；
(2) Mixing the liquid seeds of each single strain and inoculating into a fermentation medium, wherein the ventilation rate is 1:1, the stirring speed is 80rpm, and the total inoculation amount is 10% (V/V);
(3) samples were taken every 2 hours from 4 hours of fermentor culture, and the staining of the samples was observed with a microscope and the density of the cells was measured with a bacteria counter. Until the thallus density is more than 1 x 109cfu, namely obtaining the petroleum degrading bacterial agent.
On the basis of the scheme, the first-stage 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;
wherein: the liquid medium contained per 1000ml of sterile water: adding 0.125g equivalent petroleum-containing polysorbate-80 solution, 3g beef extract, 10g peptone, 5g NaCl, pH 7, and sterilizing at 121 deg.C for 20 min; the solid medium was added with 20g of agar based on the liquid medium.
The fermentation medium is as follows: contains per 1000ml of sterile water: solubilization of petroleum polysorbate-80 with petroleum content of 0.125g equivalentLiquid, glucose 3 g; 25g of corn steep liquor; 3.5g of beef extract; KH (Perkin Elmer)2PO43g；MgSO4·7H2O 0.6g；CaCl20.03g, pH 7, sterilizing at 121 deg.C for 30 min.
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 culture medium in the processes of primary screening, domestication, purification and the like of the petroleum degrading bacteria. 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, purification and the like, and the strains are gradually domesticated in a domestication culture medium with gradually rising 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) After the high-efficiency degradation strain is obtained, the industrial production of the microbial inoculum is realized by adopting a liquid fermentation technology and combining a petroleum carbon source and a high-efficiency carbon source. The invention really realizes the industrial production of the petroleum degrading bacteria agent, thereby meeting the application of the petroleum degrading bacteria agent in the treatment engineering of petroleum-polluted soil. In addition, the method for producing the microbial inoculum with petroleum participating in the whole fermentation process can save the production time and ensure the survival capability and the degradation efficiency of the microbial inoculum in the application of petroleum-polluted soil treatment engineering.
Description of the drawings:
FIG. 1 shows the amount of cell growth and the petroleum degradation rate of each of the domesticated strains.
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 a Critical Micelle Concentration (CMC) in an aqueous solution, the solubility of some water-insoluble or slightly soluble substances in the micellar solution can increase significantly, forming a transparent colloidal solution, which is called solubilization. Polysorbate-80 (polyoxyethylene sorbitan fatty acid ester) is a water-soluble nonionic surfactant, and the nonionic surfactant is not affected by electrolyte and pH value, so that the surfactant has no bactericidal property, and has an HLB (hydrophile-lipophile balance, HLB) value of 15.0, and is suitable for being used as a solubilizer and an oil-in-water (O/W) type emulsion. The polysorbate-80 can improve the solubility of petroleum in a water body, so that the petroleum is dispersed in a solubilizing liquid of the polysorbate-80 more uniformly, and the petroleum pear ester-80 solubilizing liquid is used as a unique carbon source to prepare various microbial culture media. The effects of crude oil concentration and polysorbate-80 concentration on the emulsification effect of the solubilizate solution are shown in tables 1 and 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 polysorbate-80, the crude oil is well emulsified in water when the concentration of the crude oil is less than 3% and the polysorbate-80/crude oil ratio is greater than 2.00; when the polysorbate-80/crude ratio is less than 2.00, 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) and taking back the soil sample, breaking up, removing impurities, uniformly mixing, air-drying, and sealing and storing. Taking 5g of a petroleum-degrading indigenous bacteria source soil sample, adding the sample into a conical flask filled with 100mL of sterile water, and placing the conical flask in a shaking incubator to shake for 2h at room temperature, wherein the shaking frequency is 200 rpm;
(2) 5mL of the supernatant was taken from the flask in sterile water, and 250mL of the enrichment medium was inoculated thereto, followed by shaking culture at 30 ℃ and 175rpm for 48 hours. 0.2mL of the enriched solution is taken to be coated on a plate screening culture medium, and the culture is carried out for 48h at the constant temperature of 30 ℃.
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 medium is A, B, C, D, E, wherein the petroleum concentration gradient is gradually increased, the domestication culture medium is filled in a 250mL triangular flask, and the constant temperature shaking culture at 30 ℃ and 175rpm is carried out for 7d as a period. Meanwhile, an acclimatization culture medium without inoculating the strain is used as a blank control. During acclimatization, firstly, a plurality of single strains obtained by primary screening are selected by an inoculating loop and respectively inoculated into 100mL of fresh acclimatization culture medium A for a period of culture, then 10mL of culture solution is taken out from the acclimatization culture medium A and added into 90mL of fresh acclimatization culture medium B for a period of culture. Then, 10mL of the culture medium was taken out of acclimatization medium B and added to 90mL of fresh acclimatization medium C, and the mixture was cultured for one cycle. 10mL of the culture medium was removed from acclimatization medium C and added to 90mL of fresh acclimatization medium D, and cultured for one cycle. 10mL of the culture medium was removed from acclimatization medium D and added to 90mL of fresh acclimatization medium E, and cultured for one cycle. After acclimatization culture, petroleum degradation of each strain in acclimatization medium E was observed.
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 single colony from an oil-containing flat plate, transferring the single colony to an LB inclined plane, and culturing for 24h at 30 ℃;
(3) re-inoculating the oil-containing plate from the LB 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, 1000mL of distilled water, 0.1MPa, 121 ℃, and autoclaving for 20 min.
The screening culture medium 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), 20g of agar, 0.5g of NaCl, (NH)4)2SO40.1g，MgSO4·7H2O0.025g，KNO30.24g，KH2PO40.57g, pH 7.2, 1000mL of distilled water, 0.1MPa, 121 ℃, and 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，K2HPO4·3H2O1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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，K2HPO4·3H2O1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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，K2HPO4·3H2O1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The domestication culture medium D comprises:
0.100g 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，K2HPO4·3H2O1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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，K2HPO4·3H2O1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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，K2HPO4·3H2O1.31g，MgSO4·7H2O 1.03g，CaCl20.2g, 1mL of trace element solution, 20g of agar, 1000mL of distilled water, 7.2 of pH value, 0.1MPa, 121 ℃, and high-pressure steam sterilization for 20 min.
The LB slant culture medium was:
10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar and 1000mL of distilled water, wherein the pH value is 7.0, the pressure is 0.1MPa, the temperature is 121 ℃, and the sterilization is carried out for 20min by high-pressure steam.
Solution of trace elements: MnSO439.9mg，ZnSO4·7H2O 42.8mg，FeCl2·4H2O 15.73mg，CuSO440mg, 1000mL of distilled water.
And (3) strain preservation culture medium: 5g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000mL of distilled water, wherein the pH value is 7.2-7.4, the pressure is 0.1MPa, and the beef extract is sterilized by high-pressure steam at 121 ℃ for 20 min. 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
The optical density (OD value) of the culture solution is measured by a photoelectric turbidimetry method to indirectly determine the growth amount of the bacteria. Taking a certain amount of culture solution, centrifuging for 20min at 8000 r.min-1, adding equal amount of distilled water to prepare bacterial suspension, diluting by a certain multiple, measuring the OD600 value of the bacterial suspension at 600nm by using a UV-2012C type ultraviolet-visible spectrophotometer as a reference, and simultaneously measuring the petroleum degradation rate of each strain, wherein the result is shown in figure 1.
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
Taking the first-class seeds of the two rings under the aseptic condition, inoculating the seeds into a 500ml triangular flask filled with 200ml of culture medium, and carrying out shake culture. 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 of culture medium into the 50L fermentation tank, and then adding 4L of second-level seed liquid, wherein every 1000ml of sterile water consists of 0.125g equivalent of petroleum-containing polysorbate-80 additive solution (wherein 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, 121 ℃, sterilizing for 30 minutes, the culture conditions are that the stirring rotation speed is 80rpm, the ventilation amount is 1:1, the temperature is 30 ℃, and after culturing for 8 hours, the thallus density is 1.2 × 10 detected by a bacteria counter7cfu, end of culture.
4. Fermenting in a fermentation tank to obtain the microbial inoculum product
400L of medium was added to a 500L fermenter, the medium consisting of sterile water per 1000 ml: 0.125g of petroleumA quantitative polysorbate-80 solution (wherein the mass concentration of petroleum is 2%, the mass concentration of polysorbate-80 is 4%, and the balance is water), and 3g of glucose; 25g of corn steep liquor; 3.5g of beef extract; KH (Perkin Elmer)2PO43g；MgSO40.6g；CaCl20.03g, the pH value is 7, sterilization is carried out for 30min at 121 ℃, the cultured fermentation liquor of the seeding tank is inoculated into a 500L fermentation tank, the culture condition is that the ventilation amount is 1:1, the stirring speed is 80rpm, the temperature is 30 ℃, the fermentation tank is inoculated for 4 hours, staining microscopy and bacterial counting are carried out every 2 hours, the thallus density is detected to be 2.4 × 10 after 10 hours of culture9cfu, and obtaining the petroleum degradation live bacteria preparation.
Claims (4)
1. A method for screening petroleum degrading bacteria is characterized in that an enrichment culture medium, a screening culture medium and an acclimatization culture medium used in the method are culture media taking petroleum as a unique carbon source, and concretely, a solution of petroleum and polysorbate-80 is added into the culture media; the petroleum and polysorbate-80 increasing solution contains 2% of petroleum by mass, 4% of polysorbate-80 by mass and the balance of water.
2. The method for screening petroleum degrading bacteria according to claim 1, which comprises the following steps:
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
Selecting the single strains obtained by the primary screening by using an inoculating loop, and inoculating and domesticating the single strains in a domestication culture medium;
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 single colony from an oil-containing flat plate, transferring the single colony to an LB inclined plane, and culturing for 24h at 30 ℃;
(3) re-inoculating the oil-containing plate from the LB 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.
3. The method for screening petroleum degrading bacteria according to claim 2, wherein the domestication in the step 3) is specifically:
firstly, selecting a plurality of single strains obtained by primary screening by using an inoculating loop, respectively inoculating the strains into 100mL of domestication culture medium A, culturing for a period of 7d under constant temperature oscillation at the temperature of 30 ℃ and the rotating speed of 175rpm, then taking 10mL of culture solution out of the domestication culture medium A, adding the culture solution into 90mL of domestication culture medium B, and culturing for a period; then taking 10mL of culture solution from the domestication culture medium B, adding the culture solution into 90mL of the domestication culture medium C, and culturing for a period; taking 10mL of culture solution from the domestication culture medium C, adding the culture solution into 90mL of the domestication culture medium D, and culturing for a period; and (3) taking 10mL of culture solution from the acclimatization culture medium D, adding the culture solution into 90mL of acclimatization culture medium E, culturing for a period, and gradually increasing the petroleum concentration gradient from the acclimatization culture medium A to the acclimatization culture medium E.
4. The method for screening petroleum-degrading bacteria according to claim 3, wherein:
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 value 7.2, 1000mL of distilled water, 0.1MPa, 121 ℃, and sterilizing for 20min by high-pressure steam;
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 value 7.2, 1000mL of distilled water, 0.1MPa, 121 ℃, and sterilizing for 20min by high-pressure steam;
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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 1000mL of distilled water, 7.2 pH value, 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, 1mL of trace element solution, 20g of agar, 1000mL of distilled water, 7.2 of pH value, 0.1MPa, 121 ℃, and sterilizing for 20min by high-pressure steam;
the LB slant culture medium was:
10g of peptone, 5g of yeast powder, 5g of NaCl, 20g of agar and 1000mL of distilled water, wherein the pH value is 7.0, the pressure is 0.1MPa, the temperature is 121 ℃, and the high-pressure steam sterilization is performed for 20 min;
solution of trace elements: MnSO439.9mg，ZnSO4·7H2O 42.8mg，FeCl2·4H2O 15.73mg，CuSO440mg, 1000mL of distilled water;
and (3) strain preservation culture medium: 5g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000mL of distilled water, wherein the pH value is 7.2-7.4, the pressure is 0.1MPa, and the beef extract is sterilized by high-pressure steam at 121 ℃ for 20 min.
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