CN104450597A - Preparation method of petroleum degrading bacteria solid inoculant and method for restoring petroleum-polluted soil by using prepared solid inoculant - Google Patents
Preparation method of petroleum degrading bacteria solid inoculant and method for restoring petroleum-polluted soil by using prepared solid inoculant Download PDFInfo
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- CN104450597A CN104450597A CN201410756751.6A CN201410756751A CN104450597A CN 104450597 A CN104450597 A CN 104450597A CN 201410756751 A CN201410756751 A CN 201410756751A CN 104450597 A CN104450597 A CN 104450597A
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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
Abstract
The invention relates to a preparation method of a petroleum degrading bacteria solid inoculant. The preparation method comprises the following steps: A, screening and domestication of the petroleum degrading bacteria; B, preparation of a seed culture solution; C, fermentation of the solid inoculant; and D, drying, pulverization, measurement and packaging of composted products. The petroleum degrading bacteria solid inoculant has the beneficial effect of improving the petroleum dissolution effect by taking a nonionic surfactantpolysorbate-80 (tween 80) as a solubilizer. The preparation method has the advantages that solid fermentation raw materials are easy to get and the process is relatively simple; and the solid inoculant contains a large amount of carbon and nutrient elements so as to provide more proper matrix for the growth of the bacteria, has strong affinity for the microorganisms, has immobilization efficiency, is capable of improving the competitiveness and degrading efficiency of the added microorganisms and indigenous microorganisms, and is convenient to transport and operate agriculturally and suitable for the large-scale in-situ biological remediation of the petroleum-polluted soil.
Description
Technical field
The present invention relates to microbial remedy technology for contaminated soil by petroleum field, particularly a kind of method of preparation method of oil degradation bacteria solid fungicide and the remedying oil-polluted soil of solid fungicide prepared therefrom.
Background technology
Along with developing rapidly of economy, the edatope problem that the mankind cause is day by day serious.Soil, as the non-renewable resource of the mankind, is the basis that the mankind depend on for existence.The hazardous and noxious substances of industrial or agricultural discharge grows with each passing day, and shows a rising trend to the organic contamination that soil causes.Wherein, the oil discharged in the exploitation of oil, transport, the process such as processing and accumulating is particularly serious to the harm of edatope.The mixture that oil is made up of number of chemical material, these chemistries are different, mainly comprise aromatic hydrocarbon, stable hydrocarbon, resene, pitch etc.Petroleum hydrocarbon excessive in soil may excess residual and accumulation in plant materials, by the inrichment of food chain, the physical and mental health of the mankind is suffered damage.In addition, in oil, some pollutant component can penetrate into soil shallow ground water with precipitation, and polluted underground water water quality, is finally detrimental to health.Wherein to harm larger be polycyclic aromatic hydrocarbons (PAHs) class material, polycyclic aromatic hydrocarbons has the effects such as teratogenecity, carinogenicity and mutagenicity, and lower boiling lubrication oils and oil fuel can cause the chemical pneumonitis of human body, dermatitis, anaesthetize and the harm such as to suffocate.
At present, China's oil contaminated soil remediation method comprises three kinds: physical restoration, chemical restoration and biological restoration.Compared with physics, chemical restoration, biological renovation method has the advantages such as low cost, non-secondary pollution and process effect be better.Biological process mainly comprises microorganism remediation, phytoremediation and plant-microorganism combine d bioremediation three kinds.Microorganism remediation technology utilizes the indigenous microorganism in soil or supplements the high-effective microorganism through domestication to contaminate environment, and under the envrionment conditions optimized, accelerate decomposition pollutent, repairs contaminated soil.Phytoremediation take sun power as power, utilize a green technology (Yi Xiaojun of the organic pollutants such as plant soil restoration PetroChina Company Limited., Dang Zhi, stone forest, Deng. the phytoremediation [J] of organic pollutant contaminated soil. agriculture environmental protection, 2002, 21 (5): 477-479), there is processing cost low, biomass is large, the advantage such as to beautify the environment, become the one preferred technique (Song Yufang that people remove pollutent, permitted China, Ren Liping, Deng. the biological restoration research [J] of two kind of plant Soil Under Conditions mineral oil in fluid and polycyclic aromatic hydrocarbons (PAHs). Chinese Journal of Applied Ecology, 2001, 12 (1): 108-112.).Plant-microorganism combined remediation technology, as an emerging technology, has played the advantage of phytoremediation technology, compensate for simple microorganism remediation simultaneously and has the deficiency that biomass is little, soil removability is weak, processing costs is high.Plant-microorganism combined remediation technology processes a kind of reasonable method of oil-polluted soils up to now, and it has broad mass market and development prospect.
Microorganism remediation technology just has research in early days, treatment in situ, in-situ treatment and bio-reactor three kinds of microorganism remediation modes are derived, such technology has been widely used in the reparation (Shen Tiemeng of oil-polluted soils, yellow Guoqiang, Li Ling, Deng. oil-polluted soils bio-ventilation is repaired and intensifying technology [J]. Techniques and Equipment for Environmental Pollution Control, 2002,3 (7): 67-69).Li Peijun utilizes biological composting method to repair dissimilar oil-polluted soils, after 60d, oil total hydrocarbon clearance is 38.37% ~ 56.74%, relatively low (the Li Peijun of processing costs, Guo Shuhai, Sun Tiebu. dissimilar oil contaminated soil bioremediation technology research [J]. Chinese Journal of Applied Ecology, 2002,13 (11): 1455-1458.).Domestic research in phytoremediation is still in the starting stage, research in plant and microbial association reparation rare report especially, and the plant resources of China's abundant provides sufficient material for research work, therefore, the advantage of this respect should be made full use of, probe into plant and microorganism to the combine d bioremediation effect of oil-polluted soils.
Plant-microbial association repairing effect depends primarily on plant-microflora to the absorption of organic pollutant and metabolic capacity, and organic contaminants in soil is purified by plant and the rhizospheric microorganism system of coexisting thereof.Plant-microbial association system has good Degradation to organic pollutant.Its ultimate principle is: 1) conversion of plant root exudation to bacterium has hormesis; 2) plant for microorganism provide existence place and the aerobic transformation in transferable oxygen Shi Gen district can normally carry out, this is also mechanism (the Anderson T.A. that plant promotes root-zone microorganism mineralization, Guthrie E.A., Walton B.T..Bioremediation in therhizophere, plant roots and associated microbes clean contaminatedsoil [J] .Environ.Sci.Technol., 1993 (27): 2630-2636.).
There is the industrial fermentation processes producing yeast thalline 19th century, and have employed the technology of ventilation and feed supplement; There is the production of microbial single-cell albumen in 20 century 70s.Fermentation is that microorganism grows and forms the process of meta-bolites in artificial culture environment, is also the common method that microbiobacterial agent is produced.Current microorganism live bacteria preparation series products mainly contains solid state fermentation and liquid state fermentation two kinds of modes.Solid state fermentation is that microorganism is not having or substantially do not having the fermentation mode on the solid state substrate of free-water, and gas, liquid, solid three-phase in solid state substrate is also deposited, and namely contains water and water-insoluble substance in porous solid state substrate.The spread of microorganism remediation oil-polluted soils technology just needs a large amount of microbiobacterial agents, and the domestic research to oil degradation microbial inoculum has only been carried out laboratory and cultivated on a small scale, does not realize suitability for industrialized production.
Now have been reported the method about taking petroleum hydrocarbon as sole carbon source screening oil degradation bacteria, but be for carbon source is screened mostly with single petroleum hydrocarbon or several petroleum hydrocarbon and intermediate product thereof, its culture environment and the miscellaneous practical situation of oil-polluted soils Petroleum Hydrocarbon differ greatly, the viability of bacterial classification in actual oil-polluted soils of therefore screening under this condition, degradation efficiency are not ideal enough, and the actual effect used it in the harnessing project of oil-polluted soils can not get ensureing.
The triage techniques major part of existing oil degradation bacteria is based on laboratory study, with certain single-component petroleum hydrocarbon for substrate carries out bacterial screening, again with the degradation effect of this petroleum hydrocarbon for substrate evaluation institute strain screening, it is also only the spawn culture of laboratory bench scale.
Summary of the invention
For the defect existed in prior art, the object of this invention is to provide a kind of method of preparation method of oil degradation bacteria solid fungicide and the remedying oil-polluted soil of solid fungicide prepared therefrom.
The present invention adopts following technical scheme:
A preparation method for oil degradation bacteria solid fungicide, concrete steps are as follows:
The screening of A, oil degradation bacteria, domestication
1) collection in bacterium source
Get refinery throughout the year by the soil of petroleum pollution as oil degradation original inhabitants bacterium bacterium source.
2) primary dcreening operation of oil degradation original inhabitants bacterium
(1) be taken as the pedotheque into oil degradation original inhabitants bacterium bacterium source, add and fill in the Erlenmeyer flask of sterilized water, be placed in shaking culture case shaken at room temperature 0.5 ~ 2h, oscillation frequency is 200rpm;
(2) from the Erlenmeyer flask of step (1), supernatant liquor is got, access enrichment medium, shaking culture 48h on temperature 30 DEG C, rotating speed 175rpm shaking table.Getting the pregnant solution after cultivation is applied on screening culture medium flat board, 30 DEG C of constant temperature culture 48h.
3) tame
The single bacterial strain of many strains obtained with the above-mentioned primary dcreening operation of transfering loop picking carries out inoculation domestication in domestication substratum, concrete steps are: be first inoculated into 100mL respectively with the single bacterial strain of many strains that transfering loop picking primary dcreening operation obtains and tame in culture medium A, cultivate one-period (cultivating 7d under temperature 30 DEG C, rotating speed 175rpm constant temperature oscillation is one-period), then from domestication culture medium A, taking out 10mL nutrient solution joins in the domestication substratum B of 90mL, cultivates one-period.From domestication substratum B, take out 10mL nutrient solution again join in 90mL domestication culture medium C, cultivate one-period.From domestication culture medium C, take out 10mL nutrient solution join in 90mL domestication substratum D, cultivate one-period.From domestication substratum D, take out 10mL nutrient solution joins in the domestication substratum E of 90mL, cultivates one-period, and from domestication culture medium A to domestication substratum E, petroleum concentration gradient raises step by step.
4) purifying of bacterial classification
(1) get the nutrient solution after domestication, at the flat lining out of oil-containing, cultivate 7d for 30 DEG C;
(2) from picking list bacterium colony switching LB inclined-plane oil-containing flat board, 24h is cultivated for 30 DEG C;
(3) oil-containing flat board is again inoculated by LB inclined-plane;
(4) above-mentioned steps is repeated, until obtain the single purifying bacterium colony of form;
(5) colony inoculation after purifying, in culture presevation medium slant, after 30 DEG C of cultivation 24h, is stored in 4 DEG C of refrigerators, for subsequent use.
B, prepare seed culture fluid
The oil degradation bacterial strain obtained by purifying is individually seeded in seed culture medium, after first order seed, secondary seed, seeding tank progressively enlarged culturing, as liquid seeds; The cell density of liquid seeds reaches 1*10
7cfu; Wherein: first order seed adopts solid medium slant culture, and culture condition is 30 DEG C, cultivates 24 hours; Secondary seed is that liquid culture medium shaking table is cultivated, and culture condition is 30 DEG C, rotating speed 175rpm, cultivates 10-12 hour; Seed tank culture condition is liquid nutrient medium stir culture, and culture condition is 30 DEG C of constant temperature, rotating speed 80rpm, air flow 1:1.
The fermentation of C, solid fungicide
By the waste biomass material after dry air, pulverize or roll over the particulate state being broken into 1-5mm; Be contained in resistant to elevated temperatures plastics bag by biological particles and other batchings, 110 DEG C of sterilizings 1 hour are stand-by;
Seed culture fluid in seeding tank is sprayed onto windrow surface according to the solid-to-liquid ratio of weight ratio 10:1, by ferment-seeded nutrient solution and solid fermentation substratum Homogeneous phase mixing; Postvaccinal material is put on solid fermentation bed and carries out multiplication culture 3-5 days under 28-30 DEG C of condition, then obtained microbe immobilizing material;
By the primary seed solution in step B in proportion 1:10 (v/w) access in the fixation support handled well, keep moisture 50-55%, adsorb under 28-30 DEG C of condition, fixing, increment 3-5 days; Rise in value 3 times as stated above, obtain immobilized microbial inoculum.
D, composting product drying, efflorescence, metering, packaging
Immobilized microbial inoculum is dry under temperature is 40 ~ 60 DEG C of conditions, be 6 ~ 10% to water content, microbial inoculum is pulverized, cross 5mm sieve.
On the basis of such scheme, the solubilization liquid all containing oil and Tween-80 in the substratum used in steps A and B.
On the basis of such scheme, the solubilization liquid PetroChina Company Limited. mass concentration of described oil and Tween-80 is 2%, and Tween-80 mass concentration is 4%, remaining as water.
On the basis of such scheme, described enrichment medium is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, NaCl 0.5g, (NH
4)
2sO
40.1g, MgSO
47H
2o 0.025g, KNO
30.24g, KH
2pO
40.57g, pH value 7.2, distilled water 1000mL, 0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described screening culture medium is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, agar 20g, NaCl0.5g, (NH
4)
2sO
40.1g, MgSO
47H
2o 0.025g, KNO
30.24g, KH
2pO
40.57g, pH value 7.2, distilled water 1000mL, 0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication culture medium A is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum B is:
Containing the oil Tween-80 solubilization liquid of oil 0.050g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication culture medium C is:
Containing the oil Tween-80 solubilization liquid of oil 0.075g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum D is:
Containing the oil Tween-80 solubilization liquid of oil 0.100g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum E is:
Containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described oil-containing flat board substratum is:
Containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, agar 20g, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
LB inclined-plane substratum is:
Peptone 10g, yeast powder 5g, NaCl 5g, agar 20g, distilled water 1000mL, pH value 7.0,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Trace element solution: MnSO
439.9mg, ZnSO
47H
2o 42.8mg, FeCl
24H
2o15.73mg, CuSO
440mg, distilled water 1000mL.
Culture presevation substratum: extractum carnis 5g, peptone 10g, NaCl5g, agar 20g, distilled water 1000mL, pH value 7.2 ~ 7.4,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Oil degradation bacteria solid fungicide prepared by aforesaid method is for remedying oil-polluted soil.Concrete method is: add prepared oil degradation bacteria solid fungicide in oil-polluted soils according to 10-100 kg/acre of soil, auxiliary applying chemical fertilizer and fertilizer simultaneously, the 0-30cm that turns over shows soil mixing, the one simultaneously in maize planting, soya bean and clover three kinds of crops.
The invention has the beneficial effects as follows:
(1) the present invention is using nonionogenic tenside Tween-80 (POLYSORBATE 80) as solubilizing agent, first prepare oil Tween-80 solubilization liquid, again a certain amount of oil Tween-80 solubilization liquid is joined in substratum, the solute effect of oil can be improved, solve oil and dissolve difficult problem in the medium.
(2) really accomplish to take oil as sole carbon source, in the primary dcreening operation of oil degradation bacteria, domestication, purifying, the process such as the preparation of seed liquor and the fermentation of solid fungicide substratum all with oil Tween-80 solubilization liquid for carbon source.The petroleum concentration gradient of taming in bacterial strain domestication process in substratum raises gradually, bacterial strain is tamed gradually and adapts to oil-containing substratum, the viability of bacterial classification in actual soil of therefore screening under this condition, degradation efficiency are improved, and the actual effect used it in oil-polluted soils harnessing project is protected.
(3) screening of patent PetroChina Company Limited. of the present invention degradation bacteria comprises the processes such as primary dcreening operation, domestication, multiple sieve and purifying, in the domestication substratum that bacterial strain raises gradually in petroleum concentration gradient, tamed gradually, adaptation take oil as the growth and breeding of single carbon source, improves it to the tolerance of oil and degradation efficiency thereof.
(4) patent of the present invention solid fermentation raw materials used is easy to get, technique is comparatively simple, and solid fungicide contains a large amount of carbon element and nutritive element, and for bacterial growth provides more suitably matrix, strong to microorganism avidity, immobilization efficiency is high.Improve add competitive power and the degradation efficiency of microorganism and indigenous microorganism.Solid fungicide is convenient to transport and agricultural operation, is applicable to the extensive biology in situ reparation of oil-polluted soils.
(5) the present invention strengthens remedying oil-polluted soils by alfalfa and the combined action of petroleum efficient degradation mix bacterium agent.The method investment is less, and quantities is little, and technical requirements is lower, does not cause secondary pollution as a kind of green in-situ reparation, simultaneously can not spoiled soil ecotope, also contributes to improving degradation problem under Soil degradation and productivity.
Accompanying drawing illustrates:
Fig. 1 is thalli growth amount and the petroleum degradation rate of each bacterial strain after domestication;
Fig. 2 is the total flora number of different times in soil;
Fig. 3 is the clearance of different times total petroleum hydrocarbon in soil.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail.
Embodiment 1
The screening of high efficient petroleum degrading bacteria bacterial strain
1, the collection in oil degradation original inhabitants bacterium bacterium source
To get near Shandong Province's Shengli Oil Field refinery storage tank throughout the year by the soil of petroleum pollution as oil degradation original inhabitants bacterium bacterium source.The method of sampling: get with little spade the pedotheque that thickness is 10cm, mix after multi-point sampling, it is for subsequent use that soil sampling 3kg takes back laboratory.
2, oil Tween-80 solubilization liquid preparation
Tensio-active agent reaches micelle-forming concentration (critical micelleconcentration in aqueous, CMC) after, some water-insolubles or the solubleness of microsolubility material in micellar solution can significantly increase, form transparent colloidal solution, this effect is called solubilising (solubilization).Tween-80 (polyoxylene sorbitan fatty acid fat) is water soluble nonionic surfactant, because nonionogenic tenside does not affect by ionogen and pH value, not having microbe killing properties can, and its HLB value (hydrophile-lipophile balance value, hydrophile-lipophile balance, HLB) be 15.0 be applicable to doing solubilizing agent, be applicable to making oil-in-water (O/W) type emulsion.Tween-80 can improve the solubleness of oil in water body, make it disperse in oil Tween-80 solubilization liquid evenly, the present invention with oil pear ester-80 solubilization liquid for sole carbon source prepares various microbiological culture media.Crude oil concentration and Tween-80 concentration on the impact of solubilization liquid emulsifying effectiveness as shown in table 1, table 2.
Table 1 crude oil concentration is on the impact of solubilizing agent emulsifying effectiveness
Table 2 Tween-80 concentration is on the impact of solubilizing agent emulsifying effectiveness
As can be seen from table 1 and table 2, in oil Tween-80 solubilization liquid preparation process, when crude oil concentration is less than 3%, and when Tween-80/crude oil ratio is greater than 2, crude oil emulsification in water is better; When Tween-80/crude oil ratio is less than 2, crude oil not emulsification in water, only forms spherical oil droplet.When crude oil concentration equals 3%, when Tween-80/crude oil ratio is less than 1.67, crude oil not emulsification in water, only forms spherical oil droplet; When Tween-80/crude oil ratio is greater than 2.00, crude oil emulsification in water is better, but stops the layering of stirring profit serious.When crude oil concentration is greater than 3%, crude oil not emulsification in water, only forms spherical oil droplet.
3, the screening of oil degradation original inhabitants bacterium;
(1) after pedotheque is fetched, through broken loose, removal of impurities, mixing, air-dry after sealed storage.Get oil degradation original inhabitants bacterium bacterium source pedotheque 5g, add the Erlenmeyer flask filling 100mL sterilized water, and be placed in shaking culture case shaken at room temperature 2h, oscillation frequency is 200rpm;
(2) from the Erlenmeyer flask of sterilized water, 5mL supernatant liquor is got, access 250mL enrichment medium, temperature 30 DEG C, rotating speed 175rpm shaking table shaking culture 48h.Get 0.2mL pregnant solution and be applied to plate screening substratum, 30 DEG C of constant temperature culture 48h.
4, tame
The single bacterial strain of many strains obtained with the above-mentioned primary dcreening operation of transfering loop picking carries out inoculation domestication in domestication substratum.
Domestication substratum is respectively A, B, C, D, E, and petroleum concentration gradient wherein raises gradually, and the triangular flask splendid attire of domestication substratum 250mL, temperature 30 DEG C, rotating speed 175rpm constant-temperature shaking culture 7d are one-period.Simultaneously not access the domestication substratum of bacterial strain as blank.During domestication, first be inoculated in the fresh domestication culture medium A of 100mL respectively with the single bacterial strain of many strains that the above-mentioned primary dcreening operation of transfering loop picking obtains, cultivate one-period, then from domestication culture medium A, take out 10mL nutrient solution and join in the fresh domestication substratum B of 90mL, cultivate one-period.From domestication substratum B, taking out 10mL nutrient solution again joins in the fresh domestication culture medium C of 90mL, cultivates one-period.From domestication culture medium C, take out 10mL nutrient solution joins in the fresh domestication substratum D of 90mL, cultivates one-period.From domestication substratum D, take out 10mL nutrient solution joins in the fresh domestication substratum E of 90mL, cultivates one-period.In 5 cycles so repeatedly, after domestication is cultivated, observe the oil degradation situation of each bacterial strain in domestication substratum E.
5, the purifying of bacterial classification
(1) get the domestication nutrient solution in last cycle of ring, at the flat lining out of oil-containing, cultivate 7d for 30 DEG C;
(2) from picking list bacterium colony switching LB inclined-plane oil-containing flat board, 24h is cultivated for 30 DEG C;
(3) oil-containing flat board is again inoculated by LB inclined-plane;
(4) above-mentioned steps is repeated, until obtain the single purifying bacterium colony of form;
(5) colony inoculation after purifying, in culture presevation medium slant, after 30 DEG C of cultivation 24h, is stored in 4 DEG C of refrigerators, for subsequent use.
Used medium is prepared:
Described enrichment medium is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), NaCl 0.5g, (NH
4)
2sO
40.1g, MgSO
47H
2o 0.025g, KNO
30.24g, KH
2pO
40.57g, pH value 7.2, distilled water 1000mL, 0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described screening culture medium is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), agar 20g, NaCl0.5g, (NH
4)
2sO
40.1g, MgSO
47H
2o 0.025g, KNO
30.24g, KH
2pO
40.57g, pH value 7.2, distilled water 1000mL, 0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication culture medium A is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum B is:
Containing the oil Tween-80 solubilization liquid of oil 0.050g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication culture medium C is:
Containing the oil Tween-80 solubilization liquid of oil 0.075g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum D is:
Containing the oil Tween-80 solubilization liquid of oil 0.100g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum E is:
Containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described oil-containing flat board substratum is:
Containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, agar 20g, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
LB inclined-plane substratum is:
Peptone 10g, yeast powder 5g, NaCl 5g, agar 20g, distilled water 1000mL, pH value 7.0,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Trace element solution: MnSO
439.9mg, ZnSO
47H
2o 42.8mg, FeCl
24H
2o15.73mg, CuSO
440mg, distilled water 1000mL.
Culture presevation substratum: extractum carnis 5g, peptone 10g, NaCl5g, agar 20g, distilled water 1000mL, pH value 7.2 ~ 7.4,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
5, the cultivation of oil degradation bacteria, screening and identification of morphology result
The method raised by enrichment culture and petroleum concentration gradient carries out taming, screening, and repeatedly to rule separation through flat board, purifying primary dcreening operation obtains 7 single bacterial strains, as shown in table 3.
Table 3 bacterium colony and thalli morphology observations
By at 30 DEG C, after on the constant-temperature table of 175rpm, 5d is cultivated in shaking table concussion, observe growth characteristics in domestication substratum E of these 7 single bacterial strains and hybrid bacterial strain 1 and hybrid bacterial strain 2 and measure thalli growth amount and the petroleum degradation rate thereof of these 7 single bacterial strains and 2 parallel mixing bacterial strains, wherein hybrid bacterial strain 1 is the Homogeneous phase mixing of 7 kinds of single bacterial strains, and hybrid bacterial strain 2 is strain X-2 and X-6 Homogeneous phase mixing.The growth characteristics of each bacterial strain is as shown in table 4,
The growth characteristics of each bacterial strain of table 4
Comprehensively analyze as can be seen from table 4 and Fig. 1, the emulsification degree of oil is more complete, and the thalli growth amount of bacterial strain is larger, and the petroleum degradation rate of bacterial strain is higher.Thalli growth amount becomes positive correlation with petroleum degradation rate.The petroleum emulsification degree of X-2 and X-6 bacterial strain is comparatively complete, and thalli growth amount is comparatively large, and petroleum degradation rate is higher, is therefore the dominant strain of degraded oil.Test-results also shows, different strains is different to the degradation effect of oil, and hybrid bacterial strain 2 falls oily better effects if than hybrid bacterial strain 1, therefore with hybrid bacterial strain 2 for bacterial classification prepares oil degradation microbial inoculum.
Embodiment 2
Oil degradation microbial inoculum liquid fermenting is produced
1, first order seed is cultivated
Under aseptic condition, the oil degradation bacteria hybrid bacterial strain 2 of Example 1 purifying is seeded in test tube slant substratum.Substratum forms every 1000ml sterilized water: containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), extractum carnis 3g, peptone 10g, NaCl 5g, agar powder 20g, pH value is 7,121 DEG C, sterilizing 20 minutes.Culture condition is: 30 DEG C, cultivates 24 hours, as first order seed.
2, secondary seed is cultivated
Get two ring first order seed accesses under aseptic condition to be equipped with in the 500ml triangular flask of 200ml substratum.Shake flask medium forms every 1000ml sterilized water: containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), extractum carnis 3g, peptone 10g, NaCl 5g, pH value is 7,121 DEG C, sterilizing 20 minutes.Culture condition is 30 DEG C, rotating speed 80rpm, cultivates 9 hours, as secondary seed.
3, seed tank culture
By volume secondary seed accesses in 50L fermentor tank by the inoculum size of 10%.In 50L fermentor tank, add 40L substratum and access secondary seed solution 4L.Substratum forms every 1000ml sterilized water: containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, (wherein oil mass concentration is 2%, and Tween-80 mass concentration is 4%, remaining as water.), extractum carnis 3g, peptone 10g, NaCl 5g, pH value is 7,121 DEG C, sterilizing 30 minutes.Culture condition is mixing speed 80rpm, air flow 1:1, temperature 30 DEG C, cultivates after 8 hours, and detecting cell density through counting apparatus for bacteria is 1.2 × 10
7cfu, cultivates and terminates.
4, solid fungicide fermentation increment
By the waste biomass material after dry air, pulverize or roll over the particulate state being broken into 2mm; Be contained in resistant to elevated temperatures plastics bag by biological particles and other batchings, 110 DEG C of sterilizings 1 hour are stand-by.Nutrient solution prepared by step (3) is sprayed onto windrow surface according to the solid-to-liquid ratio of weight ratio 10:1, Homogeneous phase mixing; Postvaccinal material is put on solid fermentation bed and under 30 DEG C of conditions, carries out multiplication culture 3 days, then obtained microbe immobilizing material.Solid fermentation culture medium prescription comprises the raw material of following weight percent: wheat bran 30 ~ 35%, maize straw powder 25 ~ 45%, Pericarppium arachidis hypogaeae powder 20 ~ 25%, glucose 0.5 ~ 1%, ammonium sulfate 3 ~ 5%, K
2hPO
40.3 ~ 0.5%, MgSO
47H
2o 0.1 ~ 0.3%, water material weight ratio is 1:1 ~ 4:1; Control water ratio and be about 50-55%, regulate pH 7.2, by above-mentioned primary seed solution in proportion 1:10 (v/w) access in the fixation support handled well, keep moisture 50-55%, adsorb under 30 DEG C of conditions, fixing, rise in value 3 days; Rise in value 3 times as stated above, obtain immobilization mix bacterium agent.
5, composting product drying, efflorescence, metering, packaging
After solid fermentation process terminates, heap body drying temperature is 45 DEG C, is 6%, is pulverized by windrow to heap body water content, and cross 5mm sieve, for subsequent use after detecting, its living bacteria count is 800,000,000/g, and moisture content is 4%, pH is 7.0; Utilize packaging facilities to carry out measuring, package, namely make oil degradation solid composite fungus agent.
Embodiment 3
Oil degradation solid composite fungus agent and plant combined remedying oil-polluted soils
1, Experimental Base is chosen and is processed
This test is chosen Dongying City oil recovery factory and is carried soil near oil machine, and this soil is located in be carried near oil machine, petroleum-polluted, and oil total hydrocarbon content is 4g/Kg, and its physiochemical properties of soil is as shown in table 5.The present invention takes alfalfa-microbial association remedying oil-polluted soils, planting density is approximately 8000 plants/acre, add prepared oil degradation bacteria solid fungicide in oil-polluted soils according to 10-100 kg/acre of soil, the 0-30cm that turns over shows soil mixing.When Alfalfa Growing, with film, soil is covered, keep moisture, and apply Controlled Release Fertilizer, guarantee the nutrient needed for plant normal growth.In the seedling stage of growth, Drip irrigation bag of mating formation respectively, adopts the mode of drip irrigation, waters it.
Table 5 soil sample basis character
2, collecting soil sample
Pedotheque for soil sample collector get the soil of underground about 20cm.This test has sampling four times altogether, is respectively planting season, seedling stage, mature stage and harvesting time.After pedotheque is fetched, after air-dry, broken loose, removal of impurities, mixing, cross 60 mesh sieve, then sealed storage.
3, the separation of bacterium in soil, counting
(1) prepare liquid soil: take soil sample 10g, put into the Erlenmeyer flask containing 90mL sterilized water, shaking table jolting 30min, makes soil sample fully mix with water, is disperseed by bacterium.
(2) prepare blank plate: sterilized substratum heat fused waited to be chilled to 55 ~ 60 DEG C, mix and be down flat plate respectively afterwards, each substratum falls three flat boards, and indicates the title of substratum, soil sample numbering and experiment date with marking pen.
(3) be coated with: the planar bottom surface of each substratum is write weaker concn with marking pen respectively, then draws each 0.1mL of respective concentration soil dilution liquid respectively with Sterile pipette, drip carefully in corresponding planar surface middle position.With sterile glass be coated with rod by bacteria suspension along concentric(al) circles direction lightly to external expansion, make it to be evenly distributed, left at room temperature 10min, makes bacterium immersion enter substratum.
(4) cultivate: agar plate is overturn, to make at the bottom of ware upward, cultivate at being inverted in incubator 37 DEG C.
(5) count: choose clearly bacterium colony marking pen and at dull and stereotyped reverse side, bacterium colony is counted.
4, the mensuration of total petroleum hydrocarbon
The selection of wavelength: compound concentration is 100mg/L petroleum oil, in the enterprising line scanning of ultraviolet spectrophotometer, the wavelength obtaining maximum absorption band place crude oil is 228nm.
The drafting of typical curve: get clean 50mI colorimetric cylinder 6, add oil-containing 0.2 successively, 0.5,1.0,1.2, the oil standard solution of 1.5,2.0mg, is diluted to scale marks with sherwood oil, after shaking up, take sherwood oil as reference liquid, survey its absorbancy (A) at wavelength 228nm place, draw the typical curve of crude oil standardized solution.
Soil sample is analyzed: accurately take dry sample 10.00g and be placed in 50mL colorimetric cylinder, add 20mL trichloromethane, colorimetric cylinder is put into ultrasonic sound appratus water-bath supersound extraction 15min, static filtration.Extracting solution is collected in the clean beaker of 50mL, extracts again once with 20mL trichloromethane.Merge extracted twice liquid, be evaporated to dry in (65 ± 5) DEG C water bath with thermostatic control, then use petroleum ether dissolution residue, constant volume in 50mL colorimetric cylinder, survey its absorbancy in 228nm place, then gone out the content of oil in sample by typical curve Equation for Calculating.
5, soil remediation interpretation of result
Through alfalfa vegetative period of about 100 days, sample four times respectively in planting season, seedling stage, mature stage and harvesting time, as shown in Figure 2, in its soil, the content of total petroleum hydrocarbon as shown in Figure 3 for the change of its Soil Microorganism total amount.
Claims (7)
1. a preparation method for oil degradation bacteria solid fungicide, is characterized in that: concrete steps are as follows:
The screening of A, oil degradation bacteria, domestication
1) collection in bacterium source
Get refinery throughout the year by the soil of petroleum pollution as oil degradation original inhabitants bacterium bacterium source.
2) primary dcreening operation of oil degradation original inhabitants bacterium
(1) be taken as the pedotheque into oil degradation original inhabitants bacterium bacterium source, add and fill in the Erlenmeyer flask of sterilized water, be placed in shaking culture case shaken at room temperature 0.5 ~ 2h, oscillation frequency is 200rpm;
(2) from the Erlenmeyer flask of step (1), supernatant liquor is got, access enrichment medium, shaking culture 48h on temperature 30 DEG C, rotating speed 175rpm shaking table.Getting the pregnant solution after cultivation is applied on screening culture medium flat board, 30 DEG C of constant temperature culture 48h.
3) tame
The single bacterial strain of many strains obtained with the above-mentioned primary dcreening operation of transfering loop picking carries out inoculation domestication in domestication substratum, concrete steps are: be first inoculated into 100mL respectively with the single bacterial strain of many strains that transfering loop picking primary dcreening operation obtains and tame in culture medium A, cultivate one-period (cultivating 7d under temperature 30 DEG C, rotating speed 175rpm constant temperature oscillation is one-period), then from domestication culture medium A, taking out 10mL nutrient solution joins in the domestication substratum B of 90mL, cultivates one-period.From domestication substratum B, take out 10mL nutrient solution again join in 90mL domestication culture medium C, cultivate one-period.From domestication culture medium C, take out 10mL nutrient solution join in 90mL domestication substratum D, cultivate one-period.From domestication substratum D, take out 10mL nutrient solution joins in the domestication substratum E of 90mL, cultivates one-period, and from domestication culture medium A to domestication substratum E, petroleum concentration gradient raises step by step.
4) purifying of bacterial classification
(1) get the nutrient solution after domestication, at the flat lining out of oil-containing, cultivate 7d for 30 DEG C;
(2) from picking list bacterium colony switching LB inclined-plane oil-containing flat board, 24h is cultivated for 30 DEG C;
(3) oil-containing flat board is again inoculated by LB inclined-plane;
(4) above-mentioned steps is repeated, until obtain the single purifying bacterium colony of form;
(5) colony inoculation after purifying, in culture presevation medium slant, after 30 DEG C of cultivation 24h, is stored in 4 DEG C of refrigerators, for subsequent use.
B, prepare seed culture fluid
The oil degradation bacterial strain obtained by purifying is individually seeded in seed culture medium, after first order seed, secondary seed, seeding tank progressively enlarged culturing, as liquid seeds; The cell density of liquid seeds reaches 1*10
7cfu; Wherein: first order seed adopts solid medium slant culture, and culture condition is 30 DEG C, cultivates 24 hours; Secondary seed is that liquid culture medium shaking table is cultivated, and culture condition is 30 DEG C, rotating speed 175rpm, cultivates 10-12 hour; Seed tank culture condition is liquid nutrient medium stir culture, and culture condition is 30 DEG C of constant temperature, rotating speed 80rpm, air flow 1:1.
The fermentation of C, solid fungicide
By the waste biomass material after dry air, pulverize or roll over the particulate state being broken into 1-5mm; Be contained in resistant to elevated temperatures plastics bag by biological particles and other batchings, 110 DEG C of sterilizings 1 hour are stand-by;
Seed culture fluid in seeding tank is sprayed onto windrow surface according to the solid-to-liquid ratio of weight ratio 10:1, by ferment-seeded nutrient solution and solid fermentation substratum Homogeneous phase mixing; Postvaccinal material is put on solid fermentation bed and carries out multiplication culture 3-5 days under 28-30 DEG C of condition, then obtained microbe immobilizing material;
By the primary seed solution in step B in proportion 1:10 (v/w) access in the fixation support handled well, keep moisture 50-55%, adsorb under 28-30 DEG C of condition, fixing, increment 3-5 days; Rise in value 3 times as stated above, obtain immobilized microbial inoculum.
D, composting product drying, efflorescence, metering, packaging
Immobilized microbial inoculum is dry under temperature is 40 ~ 60 DEG C of conditions, be 6 ~ 10% to water content, microbial inoculum is pulverized, cross 5mm sieve.
2. the preparation method of a kind of oil degradation bacteria solid fungicide according to claim 1, is characterized in that: the solubilization liquid all containing oil and Tween-80 in the substratum used in steps A and B.
3. the preparation method of a kind of oil degradation bacteria solid fungicide according to claim 2, is characterized in that: the solubilization liquid PetroChina Company Limited. mass concentration of described oil and Tween-80 is 2%, and Tween-80 mass concentration is 4%, remaining as water.
4. the preparation method of a kind of oil degradation bacteria solid fungicide according to any one of claim 1-3, is characterized in that:
Described enrichment medium is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, NaCl 0.5g, (NH
4)
2sO
40.1g, MgSO
47H
2o 0.025g, KNO
30.24g, KH
2pO
40.57g, pH value 7.2, distilled water 1000mL, 0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described screening culture medium is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, agar 20g, NaCl0.5g, (NH
4)
2sO
40.1g, MgSO
47H
2o 0.025g, KNO
30.24g, KH
2pO
40.57g, pH value 7.2, distilled water 1000mL, 0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication culture medium A is:
Containing the oil Tween-80 solubilization liquid of oil 0.025g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum B is:
Containing the oil Tween-80 solubilization liquid of oil 0.050g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication culture medium C is:
Containing the oil Tween-80 solubilization liquid of oil 0.075g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum D is:
Containing the oil Tween-80 solubilization liquid of oil 0.100g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described domestication substratum E is:
Containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Described oil-containing flat board substratum is:
Containing the oil Tween-80 solubilization liquid of oil 0.125g equivalent, NaCl 10g, NH
4nO
31g, KH
2pO
40.5g, K
2hPO
43H
2o 1.31g, MgSO
47H
2o 1.03g, CaCl
20.2g, trace element solution 1mL, agar 20g, distilled water 1000mL, pH value 7.2,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
LB inclined-plane substratum is:
Peptone 10g, yeast powder 5g, NaCl 5g, agar 20g, distilled water 1000mL, pH value 7.0,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
Trace element solution: MnSO
439.9mg, ZnSO
47H
2o 42.8mg, FeCl
24H
2o15.73mg, CuSO
440mg, distilled water 1000mL.
Culture presevation substratum: extractum carnis 5g, peptone 10g, NaCl5g, agar 20g, distilled water 1000mL, pH value 7.2 ~ 7.4,0.1MPa, 121 DEG C, high pressure steam sterilization 20min.
5. the oil degradation bacteria solid fungicide prepared of any one of claim 1-4 is for remedying oil-polluted soil.
6. oil degradation bacteria solid fungicide according to claim 5 is used for remedying oil-polluted soil, it is characterized in that: concrete method is: prepared oil degradation bacteria solid fungicide is added in oil-polluted soils according to 10-100 kg/acre of soil, auxiliary applying chemical fertilizer and fertilizer simultaneously, the 0-30cm that turns over shows soil mixing, the one simultaneously in maize planting, soya bean and clover three kinds of crops.
7. oil degradation bacteria solid fungicide according to claim 6 is used for remedying oil-polluted soil, it is characterized in that: described clover is alfalfa.
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| CN114602967B (en) * | 2022-03-04 | 2022-12-23 | 南京农业大学 | Method for removing USEPA PAHs in soil by using functional indigenous flora solid microbial inoculum |
| CN115305201A (en) * | 2022-09-16 | 2022-11-08 | 中南大学 | Microbial agent for efficiently treating oily sludge and preparation method thereof |
| CN115305201B (en) * | 2022-09-16 | 2023-09-22 | 中南大学 | Microbial agent for efficiently treating oily sludge and preparation method thereof |
| CN115488141A (en) * | 2022-10-24 | 2022-12-20 | 常州大学 | Deep degradation method of oil sludge scum microbial agent in crude oil exploitation and refining processes |
| CN115488141B (en) * | 2022-10-24 | 2023-10-27 | 常州大学 | Deep degradation method for sludge scum microbial agent in crude oil exploitation and refining process |
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