CN104140962A - Immobilization method for petroleum degrading bacterial group - Google Patents
Immobilization method for petroleum degrading bacterial group Download PDFInfo
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- CN104140962A CN104140962A CN201310523187.9A CN201310523187A CN104140962A CN 104140962 A CN104140962 A CN 104140962A CN 201310523187 A CN201310523187 A CN 201310523187A CN 104140962 A CN104140962 A CN 104140962A
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Abstract
Belonging to the field of microorganism immobilization, the invention discloses an immobilization method for a petroleum degrading bacterial group. Specifically, the method comprises the steps of: (1) liquid fermentation of the petroleum degrading bacterial group; and (2) immobilization of the petroleum degrading bacterial group on a diatomite/activated carbon composite carrier. The immobilization method for a petroleum degrading bacterial group provided by the invention has the advantages of low cost, benefit for large-scale application and high oil removing efficiency. The immobilized degrading bacterial group has a strong synergistic effect and large degradation capacity, and is suitable for biodegradation of various oily solid wastes.
Description
Technical field:
The present invention relates to a kind of process for fixation of oil degradation flora, belong to microorganism fixed network.
Background technology:
In recent years, demand oil and goods thereof day by day being increased along with countries in the world, the a large amount of oil-containing solid waste things that produce in Exploration of Oil And Gas, production drilling process, because of it, contain the pollutents such as petroleum hydrocarbon, heavy metal and organism, be put into national danger wastes, if not treated just directly discharge, will cause serious harm to ecotope.Therefore, oil-containing solid waste is polluted has become the great environmental problem that people must face.In the solid waste treatment technique of oil-containing, the advantages such as biologic treating technique is safe because having, economic, efficient, non-secondary pollution, become the important means of on-the-spot oil-containing solid waste thing safe handling and disposal, hole oil and the oil sheet that particularly to mechanism, cannot remove, when chemical agent is used in restriction again simultaneously, use biologic treating technique can show larger superiority.
Oil is a kind of multiple hydro carbons (normal alkane, branched paraffin, aromatic hydrocarbons and alicyclic hydrocarbon) and complex mixture of other organism (sulfide, nitride and naphthenic) on a small quantity of containing.A typical oil sample contains different hydrocarbon compounds and can reach 200～300 kinds.Different microorganisms has different degradation capabilities to petroleum component, and single oil degradation bacteria is difficult to oil thoroughly to remove from environment, and therefore, the complicacy of oil composition has determined that its degraded needs the common participation of multiple-microorganism.The structure of oil degradation flora can make full use of symbiosis and synergy between each degradation bacteria, realizes the synchronous degraded of petroleum component, greatly improves degradation rate and the degree of petroleum pollution, effectively shortens the degradation cycle of petroleum pollution.
Microbial immobilized technology is by adopting the method for physics or chemistry that free microorganism is fixed in restriceted envelope region, retaining its intrinsic activity, and the modern biological project technique that can be repeated and use continuously.Immobilization technology about oil degradation list bacterium has been reported, and 200910255830.8 disclose a kind of method for immobilizing oil degradation bacteria that reed earth's surface coring is carrier of take; 201210006059.2 disclose immobilizing oil degradation bacteria method and application thereof, and the immobilization list bacterium of these two kinds of methods, due to the limitation of single bacterium degradation bacteria to petroleum hydrocarbon degradation kind, can not be realized the synchronous degraded of petroleum component, causes degradation cycle longer.
Summary of the invention:
Main purpose of the present invention is to process in order to overcome existing biological process the existing defect that oil-containing solid waste thing speed is not high, the cycle is long, proposes a kind of process for fixation of oil degradation flora.
Technical scheme of the present invention is as follows:
A process for fixation for oil degradation flora, comprises the following steps:
(1) oil degradation flora liquid fermenting;
(2) immobilization of oil degradation flora on diatomite/activated carbon composite carrier.
Particularly, described oil degradation flora comprises three kinds of single bacterium, is respectively pseudomonas Pseudomonus sp., bacillus sp. and micrococci Micrococcus sp., according to mass ratio 1:1:1, mixes.
Particularly, the described liquid fermenting of step (1) comprises following process: to being equipped with in the fermentor tank of fermention medium of high-temperature sterilization, according to the inoculum size inoculation oil degradation flora liquid seeds of 5-10%, in 30-37 ℃, 100-120r/min blowing air is cultivated 12-60h; By the inoculum size of 5-10%, proceed to the cultivation of transferring in another fermentor tank that fresh fermention medium is housed, after switching number of times is greater than 5, obtain oil degradation bacteria group fermented liquid.
Particularly, described oil degradation flora liquid seeds preparation comprises following process: get respectively described single bacterium, even Deng mass mixing, total inoculum size according to 5-10% is inoculated in fermention medium, in 30-37 ℃, 100-120r/min, blowing air cultivate 12-36h to substratum PetroChina Company Limited. degradation flora total quantity be 10
8more than CFU/mL, obtain oil degradation flora liquid seeds.
Particularly, the component that described fermention medium comprises following weight fraction: 8-10% peptone, 3-5% beef extract, 8-10%NaCl, the pH of described substratum is neutral, liquid fermentation medium needs sterilizing to use.
Particularly, the immobilization of the described oil degradation flora of step (2) comprises following process: the diatomite/activated carbon composite carrier equal-volume by the oil degradation bacteria group fermented liquid in step (1) after with sterilizing mixes, obtain thalline-carrier mixed solution, then mixed solution is splashed into CaCl
2in solution, continuously stirring 12-24h, room temperature dries up, standby.
Particularly, described diatomite/activated carbon composite carrier, use procedure, the mass ratio that gac and diatomite mix is 1:1.
Beneficial effect of the present invention:
(1) immobilization material using is gac and diatomite, and wide material sources are effective to the immobilization role of microorganism species, the life-span is long, cost is low, is conducive to commercialization, industrialization.
(2) the described method of invention can be immobilized onto several degradation bacteria on carrier simultaneously, and synergy is strong, can realize the synchronous degraded of oil different components, effectively improves oil degradation speed, is applicable to the biological degradation of all kinds of oil-containing solid waste things.
Embodiment:
Embodiment 1:
(1) oil degradation flora liquid fermenting
Degradation flora liquid seeds preparation: get respectively the mass mixings such as pseudomonas, genus bacillus and micrococci even, total inoculum size according to 5% is inoculated in 500mL taper triangle shaking flask, the liquid amount of every bottle of fermention medium is 300mL, 30 ℃, 100r/min blowing air is cultivated 12h, to substratum PetroChina Company Limited. degradation flora total quantity be 10
8more than CFU/mL, obtain oil degradation flora liquid seeds.
Wherein, the component that fermention medium comprises following weight fraction: 8% peptone, 3% beef extract, 8%NaCl, the pH of described substratum is neutral, sterilizing is used.
Flora liquid fermenting: to being equipped with in the fermentor tank of fermention medium of high-temperature sterilization, according to 5% inoculum size inoculation degradation flora liquid seeds, 30 ℃, 100r/min blowing air is cultivated 12h; Inoculum size by 5% proceeds to the cultivation of transferring in another fermentor tank that fresh fermention medium is housed, after switching 6 times, by being fixed of oil degradation bacteria group fermented liquid.
(2) immobilization of oil degradation flora on diatomite/activated carbon composite carrier:
Diatomite/gac fixation support the equal-volume of the oil degradation bacteria group fermented liquid that step (1) is obtained after with sterilizing mixes, and obtains thalline-carrier mixed solution, and mixed solution is splashed into CaCl gently
2in solution, after continuously stirring 12h, room temperature dries up, standby.
Wherein, stone diatomite/activated carbon composite carrier, use procedure, the mass ratio that gac and diatomite mix is 1:1.
Embodiment 2
Degradation flora liquid seeds preparation: get respectively the mass mixings such as pseudomonas, genus bacillus and micrococci even, total inoculum size according to 10% is inoculated in 500mL taper triangle shaking flask, the liquid amount of every bottle of fermention medium is 300mL, 37 ℃, 120r/min blowing air is cultivated 36h, to substratum PetroChina Company Limited. degradation flora total quantity be 10
8more than CFU/mL, obtain oil degradation flora liquid seeds.
Wherein, the component that fermention medium comprises following weight fraction: 10% peptone, 5% beef extract, 10%NaCl, the pH of described substratum is neutral, sterilizing is used.
Flora liquid fermenting: to being equipped with in the fermentor tank of fermention medium of high-temperature sterilization, according to 10% inoculum size inoculation degradation flora liquid seeds, 37 ℃, 120r/min blowing air is cultivated 48h; Inoculum size by 10% proceeds to the cultivation of transferring in another fermentor tank that fresh fermention medium is housed, after switching 6 times, by being fixed of oil degradation bacteria group fermented liquid.
(2) immobilization of oil degradation flora on diatomite/activated carbon composite carrier:
Diatomite/gac fixation support the equal-volume of the oil degradation bacteria group fermented liquid that step (1) is obtained after with sterilizing mixes, and obtains thalline-carrier mixed solution, and mixed solution is splashed into CaCl gently
2in solution, after continuously stirring 24h, room temperature dries up, standby.
Wherein, stone diatomite/activated carbon composite carrier, use procedure, the mass ratio that gac and diatomite mix is 1:1.
Embodiment 3
Degradation flora liquid seeds preparation: get respectively the mass mixings such as pseudomonas, genus bacillus and micrococci even, total inoculum size according to 8% is inoculated in 500mL taper triangle shaking flask, the liquid amount of every bottle of fermention medium is 300mL, 35 ℃, 110r/min blowing air is cultivated 24h, to substratum PetroChina Company Limited. degradation flora total quantity be 10
8more than CFU/mL, obtain oil degradation flora liquid seeds.
Wherein, the component that liquid fermentation medium comprises following weight fraction: 9% peptone, 4% beef extract, 9%NaCl, the pH of described substratum is neutral, sterilizing is used.
Flora liquid fermenting: to being equipped with in the fermentor tank of fermention medium of high-temperature sterilization, according to 8% inoculum size inoculation degradation flora liquid seeds, 35 ℃, 120r/min blowing air is cultivated 24h; Inoculum size by 8% proceeds to the cultivation of transferring in another fermentor tank that fresh fermention medium is housed, after switching 6 times, by being fixed of oil degradation bacteria group fermented liquid.
(2) immobilization of oil degradation flora on diatomite/activated carbon composite carrier:
Diatomite/gac fixation support the equal-volume of the oil degradation bacteria group fermented liquid that step (1) is obtained after with sterilizing mixes, and obtains thalline-carrier mixed solution, and mixed solution is splashed into CaCl gently
2in solution, after continuously stirring 24h, room temperature dries up, standby.
Wherein, stone diatomite/activated carbon composite carrier, use procedure, the mass ratio that gac and diatomite mix is 1:1.
The analyzing of applying effects of immobilization degradation flora in the degraded of oil base drilling cuttings Petroleum Hydrocarbon
Application example 1:
Oil degradation flora: by embodiment 1 immobilization
Oil base drilling cuttings: initial oil-contg 30000mg/kg
In application, first in oil base drilling cuttings, add nutritive salt, make the C:N:P=100:10:1(mass ratio in oil base drilling cuttings), adjust solid waste thing upper strata leach liquor pH is 7.0-8.0 simultaneously, and the applied amount that is 0.05% according to weight ratio by immobilization degradation flora adds in oil base drilling cuttings, adds successively subsequently electron acceptor(EA), tensio-active agent, raising agent, and water content is controlled at 20%～30%, mixes.Every 7d turns over once, after 15 days, measures oil base drilling cuttings Petroleum Hydrocarbon content, and petroleum hydrocarbon content is down to 10350mg/kg by 30000mg/kg, and oil removing rate reaches 65.6%.
Application example 2:
Oil degradation flora: by embodiment 2 immobilizations
Oil base drilling cuttings: initial oil-contg 85000mg/kg
In application, first in oil base drilling cuttings, add nutritive salt, make the C:N:P=100:10:1(mass ratio in oil base drilling cuttings), adjust solid waste thing upper strata leach liquor pH is 7.0-8.0 simultaneously, and the applied amount that is 0.05% according to weight ratio by immobilization degradation flora adds in oil base drilling cuttings, adds successively subsequently electron acceptor(EA), tensio-active agent, raising agent, and water content is controlled at 20%～30%, mixes.Every 7d turns over once, after 30 days, measures oil base drilling cuttings Petroleum Hydrocarbon content, and petroleum hydrocarbon content is down to 20100mg/kg by 85000mg/kg, and oil removing rate reaches 76.4%.
Application example result shows, immobilized bacterium group energy is enough carries out efficient degradation to oil base drilling cuttings Petroleum Hydrocarbon pollutent, degrade after 30 days, oil-contg is 2%, reaches HJ607-2011 < < waste mineral oil and recycles drilling cuttings safety dumping standard in Pollution control technology standard > >.
Claims (7)
1. a process for fixation for oil degradation flora, is characterized in that, comprises the following steps:
(1) oil degradation flora liquid fermenting;
(2) immobilization of oil degradation flora on diatomite/activated carbon composite carrier;
Wherein, the described oil degradation flora of step (1) comprises pseudomonas Pseudomonus sp., bacillus sp. and micrococci Micrococcus sp..
2. the process for fixation of oil degradation flora according to claim 1, is characterized in that: pseudomonas Pseudomonus sp., bacillus sp. and micrococci Micrococcus sp., mix according to mass ratio 1:1:1.
3. the process for fixation of oil degradation flora according to claim 1 and 2, it is characterized in that: described oil degradation flora liquid fermenting comprises following process: to being equipped with in the fermentor tank of fermention medium of high-temperature sterilization, according to the inoculum size inoculation oil degradation flora liquid seeds of 5-10%, in 30-37 ℃, 100-120r/min blowing air is cultivated 12-60h; By the inoculum size of 5-10%, proceed to the cultivation of transferring in another fermentor tank that fresh fermention medium is housed, after switching number of times is greater than 5, obtain oil degradation bacteria group fermented liquid.
4. the process for fixation of oil degradation flora according to claim 3, it is characterized in that, described oil degradation flora liquid seeds preparation comprises following process: get oil degradation flora, total inoculum size according to 5-10% is inoculated in fermention medium, in 30-37 ℃, 100-120r/min, blowing air cultivate 12-36h to substratum PetroChina Company Limited. degradation flora total quantity be 10
8more than CFU/mL, obtain oil degradation flora liquid seeds.
5. the process for fixation of oil degradation flora according to claim 1 and 2, is characterized in that, the component that described fermention medium comprises following weight fraction: 8-10% peptone, and 3-5% beef extract, 8-10%NaCl, the pH of described substratum is neutral.
6. the process for fixation of oil degradation flora according to claim 1 and 2, it is characterized in that, the immobilization of described oil degradation flora comprises following process: the diatomite/activated carbon composite carrier equal-volume by oil degradation bacteria group fermented liquid after with sterilizing mixes, obtain thalline-carrier mixed solution, then mixed solution is splashed into CaCl
2in solution, continuously stirring 12-24h, room temperature dries up, standby.
7. the process for fixation of oil degradation flora according to claim 1 and 2, is characterized in that, described stone diatomite/activated carbon composite carrier, and use procedure, the mass ratio that gac and diatomite mix is 1:1.
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| CN106587358A (en) * | 2016-12-16 | 2017-04-26 | 河北大学 | Oil leakage degradation method for lakes, channels and ships |
| CN106636059A (en) * | 2017-01-09 | 2017-05-10 | 河海大学 | Preparation method of immobilized petroleum degradation enzyme preparation |
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