CN102021132A - Method for screening and remediation of petroleum-contaminated soil bioremediation agent - Google Patents
Method for screening and remediation of petroleum-contaminated soil bioremediation agent Download PDFInfo
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Abstract
The invention provides a method for the screening and remediation of a petroleum-contaminated soil bioremediation agent, which specifically comprises the following six steps of: collecting a petroleum degradation autochtonous bacterium source and a halophilic bacterium source, screening petroleum degradation autochtonous bacteria, screening halophilic bacteria, screening the petroleum degradation halophilic bacteria, screening a high-efficiency agent for the remediation of petroleum-contaminated soil and selecting optimal conditions for the remediation of the high-efficiency degradation agent by utilizing an orthogonal remediation experiment. The bioremediation agent screened by the screening and remediation method can be applied to the bioremediation of the petroleum-contaminated soil with the salt content of 10 to 20 g/Kg. The method for the screening and remediation of the petroleum-contaminated soil bioremediation agent has high practical value, and can be widely applied in the fields of bioremediation of the petroleum-contaminated soil, beaches, petroleum production wastewater and the like.
Description
Technical field
The invention belongs to oil-polluted soils microorganism recovery technique field, be specifically related to a kind of screening method and restorative procedure of oil-polluted soils biological restoration microbial inoculum.
Background technology
Oil is as " blood of industry ", and its industrial development and application are swift and violent day by day.Yet the especially soil pollution problem of pollution that it brings simultaneously also receives much concern.At present, the most promising oil-polluted soils treatment process of generally acknowledging in the world is a biological restoration.
The research of the bioremediation technology of oil-polluted soils starts from eighties of last century eighties, this technology is to utilize the hydrocarbons in the oil to grow as carbon source and the energy by means of a large amount of microorganisms, by its metabolism, the petroleum pollution degradation in the soil is become carbonic acid gas and water or transforms into other innoxious substance.Microorganism has three aspects to petroleum pollution significant feature process: the one, and the direct absorption of hydrocarbon: some hydro carbons in the oil can directly or indirectly be dissolved in the lipophilic district of cytolemma and enter in the film; The 2nd, the biological degradation of alkane and naphthenic hydrocarbon; The 3rd, the biological degradation of aromatic hydrocarbon, alkene, alkynes.
Microorganism has many influences and restraining factors in repair process, mainly comprise: the degradation capability of microorganism, the physico-chemical property of oil-polluted soils, envrionment conditions etc.Research contents mainly concentrates on three aspects at present: the one, and seed selection is petroleum hydrocarbon degradation bacterium efficiently; The 2nd, probe into the optimal environmental condition that microorganism is repaired; The 3rd, research and develop a series of original positions and ectopic microorganism recovery technique.
The biological restoration of carrying out does not obtain very good effect both at home and abroad, its reason is oil field Contaminated soil not only oleaginousness height but also saltiness also very high (such as the soil salt content 9-20g/Kg of Shengli Oil Field up to the 8-17 of uncontaminated soil doubly), high salinity has strengthened the difficulty that soil organisms is repaired, and makes conventional bacterial classification not reach the good treatment effect.As reference 1: Jiang Changliang, Sun Tieyan, Li Peijun, Deng. the long stockpile formula of oil-polluted soils heterotopic biological recovery technique research [J]. Chinese Journal of Applied Ecology, 2001,12 (2): put down in writing among the 279-282 and adopted composting process that the 4 kinds of dissimilar oil-polluted soils in Liaohe Oil Field are handled, when soil PetroChina Company Limited. hydrocarbon total amount (TPH) is 4.16-7.72g/100g soil, through 53 days operation, the TPH clearance reached 45.2%～56.7%.As reference 2:Fllis B.Harold P.EnvironmentalTechnology, 1992, having put down in writing prefabricated bed that usefulness such as Fllis have filtrate collection and a water circulation system among the 12:447-459 administers this moral GoerTek middle part petroleum-contaminated soil that rubs, the concentration of polycyclic aromatic hydrocarbons is reduced to 324.1mg/kg from 1024.4mg/kg in the soil, and degradation rate is 68%.Reference 3: wangdan long day, Yang Huaijie, Liu Yong, Deng. greasy filth (sand) is handled and comprehensive utilization technique research [J]. Southwest University for Nationalities journal (natural science edition), 2003, selection 2219 well oil sludge and sand such as wangdan long day have been put down in writing among the 29:19-23, adopt the bacterial classification (oil is happy precious) that U.S. microorganism company provides and do not add bacterial classification blank contrast experiment, advanced test in place, test-results show to oleaginousness be the oil sludge and sand of 73660mg/kg add bacterial classification (oil is happy precious) after 60 days the degradation rate of oil-containing be 23.3%.
There are some researches show when salt concn in the soil is 6g/Kg, most of plants, particularly cultivated plant just can not normal growth or can not grow fully, in addition, itself belongs to solid-phase media soil, and these characteristics determine the wherein ununiformity of salinity distribution, and the topsoil of 0-5cm often salinity is the highest, this is not only the most serious owing to the suffered pollution in top layer, and water evaporation quantity wherein is also maximum; Be subjected to the influence of greasy dirt, moisture evaporation, plant absorbing moisture, the soil layer salinity of 5-25cm is also than the height of deep soil.Above-mentioned reason has caused local soil especially to have the salinity that salts out the position to exceed the average salinity of the soil of being measured greatly.
Summary of the invention
Problem at the prior art existence, be the restraining effect of the high salinity in the solution oil-polluted soils to microorganism, common micro-organisms can't be used for the biological treatment of high salinity oil-polluted soils, the invention provides a kind of screening and restorative procedure of oil-polluted soils biological restoration microbial inoculum, screen the biological degradation that the biological restoration microbial inoculum that obtains can be applicable to saltiness 10～20g/Kg oil-polluted soils through this screening and restorative procedure; Screening of oil-polluted soils biological restoration microbial inoculum and restorative procedure that halophilic bacterium is strengthened have very high practical value, can be widely used in biological restoration fields such as being used for oil-polluted soils, links, oil extraction waste water.
A kind of screening and restorative procedure of oil-polluted soils biological restoration microbial inoculum specifically comprise following step:
Step 1: the collection in oil degradation original inhabitants bacterium and halophilic bacterium bacterium source;
Get apart from the oil-polluted soils at oil production waste water in oil field discharge outlet place as oil degradation original inhabitants bacterium bacterium source; Get apart from the oil extraction waste water of oil production waste water in oil field treatment plant water outlet and sludge sewage as halophilic bacterium bacterium source.
Step 2: the screening of oil degradation original inhabitants bacterium;
(1) get soil-like 2g as oil degradation original inhabitants bacterium bacterium source, add and fill in the Erlenmeyer flask (including granulated glass sphere) of 100mL sterilized water, and place shaking culture case room temperature vibration 0.5～2h, oscillation frequency is 200rpm.
(2) from the Erlenmeyer flask of sterilized water, get 5mL solution, add among the 100mL gradient screening and culturing liquid A 37 ℃, the 150rpm shake-flask culture, described gradient screening and culturing liquid A is mass concentration 75% beef-protein medium and 1g/L crude oil.
(3) when gradient screening and culturing liquid A is muddy, from gradient screening and culturing liquid A, get 5mL and be forwarded among the 100mL gradient screening and culturing liquid B 37 ℃, the 150rpm shake-flask culture, described gradient screening and culturing liquid B is that mass concentration is the mixing solutions of 50% beef-protein medium and 2g/L crude oil.
(4) when gradient screening and culturing liquid B is muddy, from gradient screening and culturing liquid B, get 5mL and be forwarded among the 100mL gradient screening and culturing liquid C 37 ℃, the 150rpm shake-flask culture, described gradient screening and culturing liquid C is that mass concentration is the mixing solutions of 25% beef-protein medium and 3g/L crude oil.
(5) when gradient screening and culturing liquid C is muddy, get 5mL and be forwarded in the 100mL minimal medium 37 ℃ from gradient screening and culturing liquid C, the 150rpm shake-flask culture is when minimal medium becomes muddy, obtaining with the oil is the oil degradation original inhabitants bacterium of sole carbon source, and refrigeration.
Described minimal medium composition: contain NH in every 1000mL deionized water
4NO
31g, K
2HPO
43H
2O 1g, KH
2PO
41g, MgSO
47H
2O 0.5g, anhydrous CaCl
20.02g, FeSO
40.01g, MnSO
4H
2O 0.01g, crude oil 4g, pH=7.0,121 ℃ of sterilization 20min.
Step 3: the screening of halophilic bacterium;
Get each 1ml of active sludge and oil extraction waste water, adopt the 100mL beef-protein medium to cultivate, through the gradient acclimation and screening obtain can be under 1-10% salt concn environment well-grown halophilic bacterium composite microbial system, and utilize the halophilic bacterium composite microbial system to prepare cell concentration 10
8～10
9The mixing halophilic bacterium nutrient solution of individual/ml.
Step 4: the screening of oil degradation halophilic bacterium;
With the oil is sole carbon source, is 1.0%～10.0% to be salt gradient with mass concentration, by halophilic bacterium composite microbial system and oil-polluted soils, and the wide oil degradation halophilic bacterium of screening salt tolerant scope.
(1) have a liking for the screening that salinity is the nutrient solution of 1.0% oil degradation halophilic bacterium:
(A) get soil-like 2g, add and fill in the Erlenmeyer flask (including granulated glass sphere) of 100mL sterilized water, and place shaking culture case 200rpm, room temperature vibration 0.5-2h as oil degradation original inhabitants bacterium bacterium source.
(B) get 5mL from the Erlenmeyer flask of sterilized water, add among the 100mL enrichment culture liquid A, every bottle adds mixing halophilic bacterium nutrient solution 5mL, in 37 ℃, and the 150rpm shake-flask culture.Described enrichment culture liquid A is the mixing solutions of mass concentration 75% beef-protein medium, 1g/L crude oil, mass concentration 1.0%NaCl.
(C) when enrichment culture liquid A is muddy, therefrom getting 5mL is forwarded among the 100mL enrichment culture liquid B, 37 ℃, 150rpm shake-flask culture, described enrichment culture liquid B are the mixing solutions that mass concentration 50% beef-protein medium, 2g/L crude oil contain mass concentration 1.0%NaCl.
(D) when enrichment culture liquid B is muddy, therefrom gets 5mL and be forwarded to shake-flask culture among the 100mL enrichment culture liquid C, described enrichment culture liquid C is the mixing solutions that mass concentration 25% beef-protein medium, 3g/L crude oil contain mass concentration 1.0%NaCl.
(E) when enrichment culture liquid C is muddy, therefrom get 5mL and be forwarded to shake-flask culture among the inorganic salt enrichment culture liquid A, described inorganic salt enrichment culture liquid A is minimal medium and 1.0%NaCl, when inorganic salt enrichment culture liquid A became muddy, obtaining with the oil was the nutrient solution that salinity is 1% oil degradation halophilic bacterium of having a liking for of sole carbon source.
(2) from have a liking for the nutrient solution that salinity is 1% oil degradation halophilic bacterium, get 5ml, be forwarded among the inorganic salt enrichment culture liquid B 37 ℃, the 150rpm shake-flask culture when inorganic salt enrichment culture liquid A becomes muddy, obtains having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of X.Described inorganic salt enrichment culture liquid B is that minimal medium contains the NaCl solution that mass concentration is X.
(3) get 5ml from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is X, be forwarded among the inorganic salt enrichment culture liquid C 37 ℃, the 150rpm shake-flask culture when inorganic salt enrichment culture liquid C becomes muddy, obtains having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of Y; Described inorganic salt enrichment culture liquid B is that minimal medium contains the NaCl solution that mass concentration is Y, and 1.0%＜X＜Y＜10.0%.
(4) get 5ml from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is Y, be forwarded among the inorganic salt enrichment culture liquid D 37 ℃, the 150rpm shake-flask culture when inorganic salt enrichment culture liquid D becomes muddy, obtains having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of Z; Described inorganic salt enrichment culture liquid D is that minimal medium contains the NaCl solution that mass concentration is Z, and 1.0%＜X＜Y＜Z＜10.0%.
(5) get 5ml from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is Z, be forwarded among the inorganic salt enrichment culture liquid E 37 ℃, the 150rpm shake-flask culture when inorganic salt enrichment culture liquid E becomes muddy, obtains having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of E; Described inorganic salt enrichment culture liquid E is that to contain mass concentration be 10.0% NaCl solution to minimal medium.
(6) salinity is 1.0% for having a liking for of will obtaining, X, Y, Z and 10.0% oil degradation halophilic bacterium than mixing, obtain oil degradation halophilic bacterium composite fungus agent according to equal volume.
Step 5: the high-effect bacterial screening that oil-polluted soils is repaired;
What (1) obtain in the step 3 has a liking for the dull and stereotyped activation of the corresponding salinity of salinity oil degradation halophilic bacterium composite fungus agent utilization, and with step 2 in the oil degradation original inhabitants bacterium that obtains adopt respectively the beef-protein medium shaking culture to cell concentration all greater than every milliliter 10
9Individual, centrifugal collection thalline prepares two kinds of zymocyte liquids.
(2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into the thin layer of 2cm.
(3) soil is respectively charged in the flowerpot, measures oil-contg and saltiness in the soil, inoculate the zymocyte liquid of oil degradation original inhabitants bacterium and the zymocyte liquid of oil degradation halophilic bacterium composite fungus agent respectively; Throw the bacterium amount for containing 10 in every gram soil
6～10
8Individual bacterium colony, and added once in every 10-15 days.
(4) in soil, added the one time of nutrition material every 10～15 days, abundant mixing, and regulate pH in soil and humidity; Described nutritive substance is sucrose and saltpetre, the amount that at every turn adds is to add 1～10g sucrose in every kilogram of soil, adds 2～16g saltpetre and the water content of regulating soil every day in every kilogram of soil, the humidity that makes soil is 20%～40%, regulates pH=7～8 of soil.
(5) survey oleaginousness in each flowerpot by weighting method, utilize the plate technique method to measure the concentration of thalline, measure and also regulate pH in soil, behind 7 time-of-weeks, measure the degradation rate of soil PetroChina Company Limited. in each flowerpot, obtain the highest high-effect bacterial of degradation rate.
Step 6: utilize quadrature reparation experiment to choose the optimal conditions that high efficiency degradation bacterial agent is repaired:
Utilize quadrature reparation experiment to choose the bacterium amount of throwing, humidity respectively, add the sucrose amount and add the saltpetre amount and throw the influence factor that the bacterium amount is repaired as high-effect bacterial, wherein throw the bacterium amount for adding 10 in every gram soil
6～10
8Individual thalline, humidity is 20%～40%, and adding the sucrose amount is every kilogram of soil 1～10g, and adding the saltpetre amount is every kilogram of soil 2～16g.(1) high-effect bacterial that utilizes screening to obtain carries out quadrature reparation experiment, at first with the dull and stereotyped activation of corresponding salinity, adopts beef-protein medium to be cultured to cell concentration greater than every milliliter 10 again
9Individual, centrifugal collection thalline prepares zymocyte liquid.
(2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into the thin layer of 2cm, regulate pH=7～8 of soil.
(3) soil is respectively charged in the flowerpot, surveys oleaginousness and saltiness in each flowerpot by weighting method.
(4) control in each flowerpot and to throw the bacterium amount and add 10 in every gram soil
6～10
8Individual thalline, humidity is 20%～40%, and adding the sucrose amount is 1～10g in every kilogram of soil, and adding the saltpetre amount is 2～16g in every kilogram of soil; And in soil, added high-effect bacterial, sucrose and saltpetre in per 10～15 days and guarantee thalline and concentrations of nutrient in the soil, humidity is by water spray control sooner or later.
(5) oleaginousness every surveyed each flowerpot in 7 days by weighting method in, utilized the plate technique method to measure the concentration of thalline every 7 days, measured every 3 days and the adjusting pH in soil, behind 7 time-of-weeks, measure the degradation rate of each flowerpot soil PetroChina Company Limited., obtain the highest repairing condition of degradation rate.
The present invention has the following advantages:
1, the screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum disclosed by the invention, the thalline of institute's screening and culturing can well be grown under 6%-10% salt concn environment, and this reparation for the high oil-polluted soils of saltiness provides the bacterium source to guarantee;
2, its best repair condition of the screening of oil-polluted soils biological restoration microbial inoculum disclosed by the invention and restorative procedure is a humidity 40%; Throw bacterium amount 10
8Individual/g soil; To add the saltpetre amount be 16g/kg soil to the best in 2 weeks before reparation, and the middle and later periods promptly repairs and adds the saltpetre amount after the beginning of the 3rd week is 8g/kg soil; Adding the sucrose amount before reparation 3 weeks is 5-10g/kg soil, and the middle and later periods is promptly repaired the 4th week beginning back the best, and to add the sucrose amount be 1-5g/kg soil.Repair 7 week the back be that 16% soil oil removal rate reaches 64.31% for saltiness up to 10g/Kg, oil-contg, repairing effect is higher;
3, the screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum disclosed by the invention; can in the higher oil-polluted soils of saltiness, survive; and can utilize oil to carry out metabolism as carbon source, have certain application prospect at field of environment protection such as oil-polluted soils reparation, oily(waste)waters.
Description of drawings
Fig. 1: the degrade degradation rate variation diagram in time of indigenous bacterium and oil degradation halophilic bacterium of PetroChina Company Limited. of the present invention;
Fig. 2: the degrade screening process figure of indigenous bacterium of the screening of a kind of oil-polluted soils biological restoration microbial inoculum that the present invention proposes and restorative procedure PetroChina Company Limited.;
Fig. 3: the screening process figure of the screening of a kind of oil-polluted soils biological restoration microbial inoculum that the present invention proposes and restorative procedure PetroChina Company Limited. degraded halophilic bacterium.
Embodiment
The present invention is described in detail below in conjunction with drawings and Examples.
The screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum that the present invention proposes specifically comprise following step:
Step 1: the collection in oil degradation original inhabitants bacterium and halophilic bacterium bacterium source;
Get apart from the oil-polluted soils at oil production waste water in oil field discharge outlet place as oil degradation original inhabitants bacterium bacterium source; Get apart from the oil extraction waste water of oil production waste water in oil field treatment plant water outlet and sludge sewage as halophilic bacterium bacterium source.
Step 2: the screening of oil degradation original inhabitants bacterium, as shown in Figure 2;
(1) respectively gets 2g from the soil sample of 9 sampling point collections, add respectively in 9 Erlenmeyer flasks (including granulated glass sphere) that fill the 100mL sterilized water, 9 bottles of soil solution are placed shaking culture case room temperature vibration 0.5-2h, oscillation frequency 200rpm.
(2) from the Erlenmeyer flask of every bottle of sterilized water, get 5mL solution, add among the 100mL gradient screening and culturing liquid A 37 ℃ respectively, the 150rpm shake-flask culture, described gradient screening and culturing liquid A is the mixing solutions of mass concentration 75% beef-protein medium and 1g/L crude oil.
(3) when gradient screening and culturing liquid A is muddy, from gradient screening and culturing liquid A, get 5mL and be forwarded among the 100mL gradient screening and culturing liquid B 37 ℃, the 150rpm shake-flask culture, described gradient screening and culturing liquid B is that mass concentration is the mixing solutions of 50% beef-protein medium and 2g/L crude oil.
(4) when gradient screening and culturing liquid B is muddy, from gradient screening and culturing liquid B, get 5mL and be forwarded among the 100mL gradient screening and culturing liquid C 37 ℃, the 150rpm shake-flask culture, described gradient screening and culturing liquid C is that mass concentration is the mixing solutions of 25% beef-protein medium and 3g/L crude oil.
(5) when gradient screening and culturing liquid C is muddy, from gradient screening and culturing liquid C, get 5mL and be forwarded in the 100mL minimal medium 37 ℃, the 150rpm shake-flask culture, when minimal medium became muddy, obtaining with the oil was the oil degradation original inhabitants bacterium of sole carbon source.
(6) will obtain 9 groups what correspond to 9 place's sampling points is the oil degradation original inhabitants bacterium of sole carbon source with the oil, is numbered B1, B2, B3, B4, B5, B6, B7, B8 and B9 respectively, and in 4 ℃ of refrigerations.
Described minimal medium composition: contain NH in every 1000mL water
4NO
31g, K
2HPO
43H
2O 1g, KH
2PO
41g, MgSO
47H
2O 0.5g, anhydrous CaCl
20.02g, FeSO
40.01g, MnSO
4H
2O 0.01g, crude oil 4g, pH=7.0,121 ℃ of sterilization 20min.
Step 3: the screening of halophilic bacterium
Get each 1ml of active sludge and oil extraction waste water, adopt the 100mL beef-protein medium to cultivate, through the gradient acclimation and screening obtain can be under 1-10% salt concn environment well-grown halophilic bacterium composite microbial system, and utilize the halophilic bacterium composite microbial system to prepare cell concentration 10
8～10
9The mixing halophilic bacterium nutrient solution of individual/ml.
Step 4: the screening of oil degradation halophilic bacterium;
With the oil is sole carbon source, is 1.0%, 2.5%, 5.0%, 7.5%, 10.0% to be salt gradient with mass concentration, by screening oil degradation halophilic bacterium in halophilic bacterium composite microbial system and the oil-polluted soils, as shown in Figure 3.
(1) have a liking for the screening that salinity is the nutrient solution of 1% oil degradation halophilic bacterium:
(A) oil degradation original inhabitants bacterium is respectively got 2g from the soil sample of 9 sampling point collections, adding fills in the Erlenmeyer flask (including granulated glass sphere) of 100mL sterilized water respectively, and 9 bottles of soil solution are placed shaking culture case 200rpm, room temperature vibration 0.5-2h.
(B) every bottle of soil solution is got 5mL, adds respectively among 9 bottles of 100mL enrichment culture liquid A, and adds mixing halophilic bacterium nutrient solution 5mL, 37 ℃, 150rpm shake-flask culture to every bottle.Described enrichment culture liquid A is the mixing solutions of mass concentration 75% beef-protein medium, 1g/L crude oil, mass concentration 1%NaCl.
(C) when enrichment culture liquid A is muddy, get 5mL for every bottle and be forwarded among the 100mL enrichment culture liquid B 37 ℃, 150rpm shake-flask culture, described enrichment culture liquid B are the mixing solutions of mass concentration 50% beef-protein medium, 2g/L crude oil and mass concentration 1%NaCl.
(D) when enrichment culture liquid B is muddy, therefrom get 5mL and be forwarded among the 100mL enrichment culture liquid C 37 ℃, 150rpm shake-flask culture, described enrichment culture liquid C are the mixing solutions of mass concentration 25% beef-protein medium, 3g/L crude oil and mass concentration 1.0%NaCl.
(E) when enrichment culture liquid C is muddy, therefrom get 5mL and be forwarded to shake-flask culture among the inorganic salt enrichment culture liquid A, described inorganic salt enrichment culture liquid A is that minimal medium contains the solution that mass concentration is 1.0%NaCl, when inorganic salt enrichment culture liquid A becomes muddy, obtaining 9 groups is sole carbon source with the oil, has a liking for salinity and be the nutrient solution of 1.0% oil degradation halophilic bacterium.
(2) have a liking for the nutrient solution that salinity is 1.0% oil degradation halophilic bacterium from 9 kinds and respectively get 5ml, be forwarded among the inorganic salt enrichment culture liquid B of saliferous 2.5% 37 ℃ respectively successively, the 150rpm shake-flask culture, when inorganic salt enrichment culture liquid B becomes muddy, obtaining 9 groups is sole carbon source with the oil, has a liking for salinity and be the nutrient solution of 2.5% oil degradation halophilic bacterium.Described inorganic salt enrichment culture liquid B is that minimal medium contains the solution that mass concentration is 2.5%NaCl.
(3) from have a liking for the nutrient solution that salinity is 2.5% oil degradation halophilic bacterium, get 5ml, be forwarded among the inorganic salt enrichment culture liquid C 37 ℃, the 150rpm shake-flask culture when inorganic salt enrichment culture liquid A becomes muddy, obtains having a liking for salinity and is the nutrient solution of 5.0% oil degradation halophilic bacterium.Described inorganic salt enrichment culture liquid C is that to contain mass concentration be 5.0% NaCl solution to minimal medium.
(4) from have a liking for the nutrient solution that salinity is 5.0% oil degradation halophilic bacterium, get 5ml, be forwarded among the inorganic salt enrichment culture liquid D 37 ℃, the 150rpm shake-flask culture when inorganic salt enrichment culture liquid D becomes muddy, obtains having a liking for salinity and is the nutrient solution of 7.5% oil degradation halophilic bacterium; Described inorganic salt enrichment culture liquid D is that to contain mass concentration be 7.5% NaCl solution to minimal medium.
(5) from have a liking for the nutrient solution that salinity is 7.5% oil degradation halophilic bacterium, get 5ml, be forwarded among the inorganic salt enrichment culture liquid E 37 ℃, the 150rpm shake-flask culture when inorganic salt enrichment culture liquid E becomes muddy, obtains having a liking for salinity and is the nutrient solution of 10.0% oil degradation halophilic bacterium; Described inorganic salt enrichment culture liquid E is that to contain mass concentration be 10.0% NaCl solution to minimal medium.
(6) 45 groups of oil degradation halophilic bacterium will having a liking for salinity 1%, 2.5%, 5.0%, 7.5% and 10.0% from five kinds of 9 sampling point correspondences mix according to the oil degradation halophilic bacterium of same sampling point, obtain 9 groups of oil degradation halophilic bacterium corresponding to 9 place's sampling points, numbering is respectively: Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, and 4 ℃ of refrigerations.
Step 5: oil-polluted soils is repaired the screening of high-effect bacterial;
(1) will have a liking for salinity is that 1.0%, 2.5%, 5.0%, 7.5% and 10% oil degradation halophilic bacterium is with having a liking for the dull and stereotyped activation of salinity accordingly, and adopt beef-protein medium at 30 ℃ respectively with oil degradation original inhabitants bacterium, under the 150rpm condition shaking culture to cell concentration all greater than every mL10
9Individual, centrifugal collection thalline prepares two kinds of zymocyte liquids.
(2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into the thin layer of 2cm.
(3) soil is respectively charged in the flowerpot, measures oil-contg and saltiness in the soil.Set wherein that 1 flowerpot does not connect bacterium for the contrast basin, inoculate the zymocyte liquid of oil degradation original inhabitants bacterium in all the other flowerpots respectively and have a liking for the zymocyte liquid that salinity is 1.0%, 2.5%, 5.0%, 7.5% and 10.0% oil degradation halophilic bacterium; Throw the bacterium amount for containing 10 in every gram soil
6～10
8Individual bacterium colony (CFU), and added once in every 10-15 days.In soil, added the one time of nutrition material, fully mixing every 10-15 days.Described nutritive substance is sucrose and saltpetre, and the amount that at every turn adds is to add 1～10g sucrose in every kilogram of soil, adds 2～16g saltpetre in every kilogram of soil; And every day regulate the water content of soil, the humidity that makes soil is 20%～40%, regulates pH=7～8 of soil.Test conditions is as shown in table 1, and the parameter of measuring in the process of the test is as shown in table 2:
Table 1 soil remediation test conditions
| Parameter | Parameter value | Experimental implementation |
| Temperature | Room temperature (20-25 ℃) | |
| |
20％-40％ | Sooner or later spray water every day |
| The pH value | 78 | Measure and regulate |
| Oxygen | Ventilating pit | Sooner or later the oxygenation of digging every day |
| Nutritive substance | Sucrose (1-10g/kg soil), 2-16gKNO 3/ Kg soil | Added in per 15 days |
| Throw the bacterium amount | 10 6-10 8Individual (CFU)/g soil | Added in per 15 days |
(4) oleaginousness every surveyed each flowerpot in 7 days by weighting method in, the concentration of utilizing the plate technique method to survey a hypothallus every 7 days, measured and regulated pH in soil every 3 days, as shown in table 2, behind 7 time-of-weeks, measure the degradation rate of soil PetroChina Company Limited. in each flowerpot, obtain the highest high-effect bacterial of degradation rate.
Table 2 soil remediation location parameter
| Parameter | Measuring method | The sampling interval | |
| | Weighting method | 7 days | |
| Cell concentration | |
7 days |
| The pH | PH meter | 3 days |
The degradation rate of petroleum hydrocarbon is the normal survey index that the oil-polluted soils microorganism is repaired, and has directly reflected the degradation capability of microorganism.Measure the degradation rate of soil in the 19 potted flower basins behind 7 time-of-weeks, as Fig. 1, the oleaginousness of finding the soil of contrast flowerpot has reduced by 17.59%, this mainly is because the nutritive substance that adds has stimulated the growth and the metabolism of indigenous microorganism, impel it to decompose the part petroleum hydrocarbon, in addition, the oxygenizement of the volatilization of short chain petroleum hydrocarbon, air also makes the oleaginousness of soil reduce.The degradation efficiency of oil degradation original inhabitants bacterium all is higher than contrast, and its degradation rate exceeds from 13.99% to 30.3% with respect to the contrast flowerpot, shows the oil degradation original inhabitants bacterium through sieving, taming, and the degradation capability of petroleum hydrocarbon is significantly increased.The degradation efficiency of oil degradation halophilic bacterium Y1-Y9 generally is higher than the oil degradation original inhabitants bacterium B1-B9 of screening, exceed respectively successively: 5.51%, 4.35%, 3.32% ,-1.28%, 2.86%, 2.28% ,-4.88%, 0.26%, 2.25%, show that the oil degradation halophilic bacterium more can adapt to hypersaline environment, brings into play Degradation efficiently simultaneously.Wherein Y1, Y2, Y3 and B2 are the composite fungus agent of degradation capability optimum.
Step 6: utilize quadrature reparation experiment to choose the optimal conditions that high efficiency degradation bacterial agent is repaired:
(1) the high-effect bacterial Y2 that screening is obtained repairs the degradation bacterial agent of experiment, with the dull and stereotyped activation of corresponding salinity 1-10%, adopts beef-protein medium at 30 ℃ then, under the 150rpm condition shaking culture to cell concentration greater than every milliliter 10
9Individual, centrifugal collection thalline prepares zymocyte liquid.
(2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into the thin layer of 2cm, regulate pH=7～8 of soil.
(3) soil is respectively charged in the flowerpot, surveys oleaginousness and saltiness in each flowerpot by weighting method.
(4) set the influence factor of quadrature reparation experiment for throwing bacterium amount, temperature, adding the sucrose amount and add four kinds of factors of saltpetre amount, in soil, add microbial inoculum, sucrose and saltpetre in wherein per 10～15 days and guarantee thalline and concentrations of nutrient in the soil, by watering water management, described throwing bacterium amount factor is to add 10 in every gram soil to humidity for sooner or later
6Individual, 10
7Individual and 10
8Individual three levels, humidity factor is 20%, 30% and 40% 3 level, and is as shown in table 3, and adding sucrose amount factor is every kilogram of soil 1g, 5g and three levels of 10g, and adding saltpetre amount factor is every kilogram of soil 2g, 8g and three levels of 16g.The while controlled temperature is at 20～25 ℃, and is as shown in table 4, and is the logical oxygen of soil by digging, and monitoring and adjusting pH in soil are 7-8.
Table 3 orthogonal experiment level of factor table
Table 4 orthogonal experiment condition
| Experiment condition | Value | Experimental implementation |
| Temperature | Room temperature (20-25 ℃) | Place room temperature environment |
| The pH value | 7-8 | Monitoring is also regulated |
| Oxygen | Oxygenation | If ventilating pit, the oxygenation of digging |
(5) oleaginousness every surveyed each flowerpot in 7 days by weighting method in, as shown in table 5, the concentration of utilizing the plate technique method to survey a hypothallus every 7 days, measured and regulated pH in soil every 3 days, behind 7 time-of-weeks, measure the degradation rate of soil PetroChina Company Limited. in each flowerpot, obtain the combination of the highest level affects factor of degradation rate.
Table 5 orthogonal experiment location parameter
| Parameter | Measuring method | Sample time |
| Oleaginousness | Weighting method | Per 7 days |
| Cell concentration | Colony counting method | Per 7 days |
| The pH value | PH meter | Per 3 days |
(5) quadrature is repaired experimental result
Behind 7 time-of-weeks, measure the degradation rate of soil PetroChina Company Limited. in each flowerpot, obtain degradation rate and show over time, as shown in table 6:
Table 6 degradation rate over time
Repair four kinds of factors of empirical factor level according to quadrature---throw bacterium amount, humidity, add sucrose and add saltpetre and have three kinds of conditions, carry out orthogonal experiment, obtain the result of oil degradation rate under the different factor conditions, as shown in table 7.
The degraded counting rate meter of different level of factor is tested in the reparation of table 7 quadrature
Time Orthogonal experiment results (table 7) is analyzed thus: humidity, throwing bacterium amount are the primary factor that influences repairing effect all the time, and best value is respectively 40% and 10
8Individual/g soil.Saltpetre is as the third-largest influence factor, breed in a large number at reparation initial stage and degradation bacteria, high speed degraded period best dosage be higher than other period, that is: to add sour potassium amount be 16g/kg soil to the nitre the best of repairing preceding 2 weeks, middle and later periods the 3rd week beginning afterwards the best to add the saltpetre amount be 8g/kg soil.Sucrose is a crucial relatively influence factor at the reparation initial stage, plays the acceleration microorganism growth, starts the effect of degraded fast, must add in initial start stage; And add the sucrose amount what are not obvious to the degradation effect influence, consider from economical and practical angle that therefore it is 5～10g/kg soil that preceding 3 all the bests add the sucrose amount, to add the sucrose amount be 1～5g/kg soil to the best after i.e. the 4th week beginning of middle and later periods.
From the orthogonal experiment effect analysis: when Black Liquor with Efficient Bacteria repaired for 7 weeks, in humidity 40%; Throw bacterium amount 10
8Individual/g soil; To add the saltpetre amount be 16g/kg soil to the best in 2 weeks before reparation, and the middle and later periods is a 8g/kg soil; Adding the sucrose amount before reparation 3 weeks is 5～10g/kg soil, adds that oil removal rate reaches 64.31% under the condition that the sucrose amount is 1～5g/kg soil the middle and later periods.
Claims (5)
1. the screening and the restorative procedure of an oil-polluted soils biological restoration microbial inoculum is characterized in that comprising following step:
Step 1: the collection in oil degradation original inhabitants bacterium and halophilic bacterium bacterium source;
Get apart from the oil-polluted soils at oil production waste water in oil field discharge outlet place as oil degradation original inhabitants bacterium bacterium source; Get apart from the oil extraction waste water of oil production waste water in oil field treatment plant water outlet and sludge sewage as halophilic bacterium bacterium source;
Step 2: the screening of oil degradation original inhabitants bacterium;
(1) get soil-like 2g as oil degradation original inhabitants bacterium bacterium source, add and fill the Erlenmeyer flask of 100mL sterilized water, and place shaking culture case room temperature vibration 0.5～2h, oscillation frequency is 200rpm;
(2) get 5mL solution from the Erlenmeyer flask of sterilized water, add shake-flask culture among the 100mL gradient screening and culturing liquid A, described gradient screening and culturing liquid A is the mixing solutions of mass concentration 75% beef-protein medium and 1g/L crude oil;
(3) when gradient screening and culturing liquid A is muddy, get 5mL and be forwarded to shake-flask culture among the 100mL gradient screening and culturing liquid B from gradient screening and culturing liquid A, described gradient screening and culturing liquid B is that mass concentration is the mixing solutions of 50% beef-protein medium and 2g/L crude oil;
(4) when gradient screening and culturing liquid B is muddy, get 5mL and be forwarded to shake-flask culture among the 100mL gradient screening and culturing liquid C from gradient screening and culturing liquid B, described gradient screening and culturing liquid C is that mass concentration is the mixing solutions of 25% beef-protein medium and 3g/L crude oil;
(5) when gradient screening and culturing liquid C is muddy, gets 5mL and be forwarded to shake-flask culture in the 100mL minimal medium from gradient screening and culturing liquid C, when minimal medium became muddy, obtaining with the oil was the oil degradation original inhabitants bacterium of sole carbon source, and refrigeration;
Step 3: the screening of halophilic bacterium;
Get each 1ml of active sludge and oil extraction waste water, adopt the 100mL beef-protein medium to cultivate, obtaining salinity through the gradient acclimation and screening is 1%～20% halophilic bacterium composite microbial system, and utilizes the halophilic bacterium composite microbial system to prepare cell concentration 10
8～10
9The mixing halophilic bacterium nutrient solution of individual/ml;
Step 4: the screening of oil degradation halophilic bacterium;
(1) have a liking for the screening that salinity is the nutrient solution of 1.0% oil degradation halophilic bacterium:
(A) get soil-like 2g, add and fill in the Erlenmeyer flask of 100mL sterilized water, and place the vibration of shaking culture case room temperature as oil degradation original inhabitants bacterium bacterium source;
(B) from the Erlenmeyer flask of sterilized water, get 5mL, add among the 100mL enrichment culture liquid A, and in wherein add mixing halophilic bacterium nutrient solution 5mL shake-flask culture, described enrichment culture liquid A is the mixing solutions of mass concentration 75% beef-protein medium, 1g/L crude oil and 1.0%NaCl;
(C) when enrichment culture liquid A is muddy, therefrom gets 5mL and be forwarded to shake-flask culture among the 100mL enrichment culture liquid B, described enrichment culture liquid B is the mixing solutions of mass concentration 50% beef-protein medium, 2g/L crude oil and 1.0%NaCl;
(D) when enrichment culture liquid B is muddy, therefrom gets 5mL and be forwarded to shake-flask culture among the 100mL enrichment culture liquid C, described enrichment culture liquid C is the mixing solutions of mass concentration 25% beef-protein medium, 3g/L crude oil and 1.0%NaCl;
(E) when enrichment culture liquid C is muddy, therefrom get 5mL and be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid A, described inorganic salt enrichment culture liquid A is that minimal medium and mass concentration are the mixing solutions of 1.0%NaCl, when inorganic salt enrichment culture liquid A became muddy, obtaining with the oil was the nutrient solution that salinity is 1.0% oil degradation halophilic bacterium of having a liking for of sole carbon source;
(2) from have a liking for the nutrient solution that salinity is 1.0% oil degradation halophilic bacterium, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid B, when inorganic salt enrichment culture liquid B becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of X; Described inorganic salt enrichment culture liquid B is that minimal medium contains the NaCl solution that mass concentration is X, and 1.0%＜X＜10.0%;
(3) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is X, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid C, when inorganic salt enrichment culture liquid C becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of Y; Described inorganic salt enrichment culture liquid C is that minimal medium contains the NaCl solution that mass concentration is Y, and 1.0%＜X＜Y＜10.0%;
(4) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is Y, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid D, when inorganic salt enrichment culture liquid D becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of Z; Described inorganic salt enrichment culture liquid D is that minimal medium contains the NaCl solution that mass concentration is Z, and 1.0%＜X＜Y＜Z＜10.0%;
(5) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is Z, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid E, when inorganic salt enrichment culture liquid E becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of E; Described inorganic salt enrichment culture liquid E is that to contain mass concentration be 10.0% NaCl solution to minimal medium;
(6) salinity is 1.0% for having a liking for of will obtaining, X, Y, Z and 10.0% oil degradation halophilic bacterium mix according to the equal volume ratio, obtains oil degradation halophilic bacterium composite fungus agent;
Step 5: the high-effect bacterial screening that oil-polluted soils is repaired;
(1) the dull and stereotyped activation of the corresponding salinity of the oil degradation halophilic bacterium composite fungus agent utilization that obtains in the step 3, and with step 2 in the oil degradation original inhabitants bacterium that obtains adopt respectively the beef-protein medium shaking culture to cell concentration all greater than every milliliter 10
9Individual, centrifugal collection thalline prepares two kinds of zymocyte liquids;
(2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into thin layer;
(3) soil is respectively charged in the flowerpot, measures the oleaginousness and the saltiness of soil in the flowerpot, regulate pH in soil and humidity; In flowerpot, inoculate the zymocyte liquid of oil degradation original inhabitants bacterium and the zymocyte liquid of oil degradation halophilic bacterium composite fungus agent respectively; Throwing bacterium amount is in every gram soil and contains 10
6～10
8Individual bacterium colony, and added once in per 10～15 days;
(4) in flowerpot, added the one time of nutrition material every 10～15 days, abundant mixing, and regulate pH in soil and humidity every day;
(5) survey oleaginousness in each flowerpot by weighting method, utilize the plate technique method to measure the concentration of thalline, measure and also regulate pH in soil, behind 7 time-of-weeks, measure the degradation rate of soil PetroChina Company Limited. in each flowerpot, obtain the highest high-effect bacterial of degradation rate;
Step 6: the reparation of high-effect bacterial;
Utilize the quadrature reparation to test and choose the bacterium amount of throwing, humidity respectively, add sucrose amount and saltpetre amount as the repairing condition of high-effect bacterial, wherein throw the bacterium amount and add 10 in every gram soil
6～10
8Individual thalline, humidity is 20%～40%, and adding the sucrose amount is every kilogram of soil 1～10g, and adding the saltpetre amount is every kilogram of dry ground 2～16g; Obtain oil-polluted soils biological restoration microbial inoculum at last.
2. the screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum according to claim 1 is characterized in that: described minimal medium composition: contain NH in every 1000mL deionized water
4NO
31g, K
2HPO
43H
2O 1g, KH
2PO
41g, MgSO
47H
2O 0.5g, anhydrous CaCl
20.02g, FeSO
40.01g, MnSO
4H
2O 0.01g, crude oil 4g, pH=7.0,121 ℃ of sterilization 20min.
3. the screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum according to claim 1 is characterized in that: the reparation detailed process described in the described step 6 is:
(1) high-effect bacterial that utilizes screening to obtain carries out quadrature reparation experiment, at first with the dull and stereotyped activation of corresponding salinity, adopts beef-protein medium to be cultured to cell concentration greater than every milliliter 10 again
9Individual, centrifugal collection thalline prepares zymocyte liquid;
(2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into the thin layer of 2cm, regulate pH=7～8 of soil;
(3) soil is respectively charged in the flowerpot, by oleaginousness and the saltiness in each flowerpot of gravimetric determination;
(4) control in each flowerpot and to throw the bacterium amount and add 10 in every kilogram of soil
6～10
8Individual thalline, humidity is 20%～40%, and adding the sucrose amount is 1～10g in every kilogram of soil, and adding the saltpetre amount is 2～16g in every kilogram of soil; And in soil, added high-effect bacterial, sucrose and saltpetre in per 10～15 days and guarantee thalline and concentrations of nutrient in the soil, humidity is by water spray control sooner or later;
(5) every 7 days by the oleaginousness in each flowerpot of gravimetric determination, the concentration of utilizing the plate technique method to survey a hypothallus every 7 days was measured and the adjusting pH in soil every 3 days, behind 7 time-of-weeks, measure the degradation rate of each flowerpot soil PetroChina Company Limited., obtain the highest repairing condition of degradation rate.
4. according to the screening and the restorative procedure of claim 1 or 3 described a kind of oil-polluted soils biological restoration microbial inoculums, it is characterized in that: the repairing condition of described oil-polluted soils biological restoration microbial inoculum is: humidity is 40% when using the degraded of oil degradation halophilic bacterium composite fungus agent, and throwing the bacterium amount is 10
8Individual/g soil; The saltpetre dosage in 2 weeks is a 16g/kg soil before reparation, and the saltpetre dosage is a 8g/kg soil after the beginning of the 3rd week; 3 all sucrose dosages are 5～10g/kg soil before reparation, and the sucrose dosage is 1～5g/kg soil after the beginning of the 4th week.
5. the screening and the restorative procedure of a kind of oil-polluted soils biological restoration microbial inoculum according to claim 1 is characterized in that: X=2.5% in the described step 4, Y=5.0%, Z=7.5%.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101475285A (en) * | 2009-01-19 | 2009-07-08 | 辽宁天意实业股份有限公司 | Gas field sewerage and solid pollutant comprehensive processing technological process |
-
2010
- 2010-11-19 CN CN201010552491.2A patent/CN102021132B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101475285A (en) * | 2009-01-19 | 2009-07-08 | 辽宁天意实业股份有限公司 | Gas field sewerage and solid pollutant comprehensive processing technological process |
Non-Patent Citations (3)
| Title |
|---|
| 《Appl Microbiol Biotechnol》 20071231 H. Al-Awadhi等 Alkaliphilic and halophilic hydrocarbon-utilizing bacteria from Kuwaiti coasts of the Arabian Gulf 全文 1-5 第77卷, 2 * |
| 《农业环境科学学报》 20070331 杨玉楠 嗜盐菌强化石油污染土壤生物修复的可行性研究 全文 1-5 第26（增刊）卷, 2 * |
| 《现代农业科学》 20091130 杨若勤 嗜盐菌强化石油污染土壤生物修复过程的初步研究 全文 1-5 第16卷, 第11期 2 * |
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