CN101760455B - Method for immobilizing petroleum degradation bacteria by taking surface root hair of reed as carrier - Google Patents
Method for immobilizing petroleum degradation bacteria by taking surface root hair of reed as carrier Download PDFInfo
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- CN101760455B CN101760455B CN2009102558308A CN200910255830A CN101760455B CN 101760455 B CN101760455 B CN 101760455B CN 2009102558308 A CN2009102558308 A CN 2009102558308A CN 200910255830 A CN200910255830 A CN 200910255830A CN 101760455 B CN101760455 B CN 101760455B
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- degradation bacteria
- oil degradation
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- petroleum
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Abstract
The invention relates to a method for immobilizing petroleum degradation bacteria by taking surface root hairs of reeds as vectors, which intensifies the decontamination capacity of a natural wetland for a petroleum polluted water body. The method is characterized by comprising the following steps of: firstly, screening out purified petroleum degradation bacteria strains by soil polluted by petroleum; then inoculating the purified petroleum degradation bacteria strains to a liquid enriched medium so as to culture petroleum degradation bacteria turbid liquid; finally adding sodium alginate to the prepared petroleum degradation bacteria turbid liquid, mixing, and then uniformly spraying to the root hairs of the reeds so as to achieve immobilization 5 hours later, i.e. immobilizing the petroleum degradation bacteria on the surface root hairs of the wetland reeds. The invention has the advantages of extensive material sources, simple process, low cost and good purifying effect, can achieve very good degradation effect even if the petroleum degradation bacteria turbid liquid with lower concentration is used and can be popularized and used on a large area of a reed wetland.
Description
Technical field
The invention belongs to and strengthen the clean dirty functional area of natural wetland, specially referring to reed face of land coring is the method for immobilizing oil degradation bacteria of carrier.
Background technology
The pollution that all is subjected in various degree over half of current China surface water body badly influences people's production and life.The method of purifying contaminated surface water body is a lot, mainly is divided into physical cleaning method, chemical purification and biological clarification.Wherein the physical cleaning method is to adopt method physics, machinery that the polluted surface water body is manually purified, and this class methods technology is simple, but expense is high and cure the symptoms, not the disease.Chemical purification mainly is to point in the water body that pollutes to add chemical agent, by medicament and pollution substance generation chemical reaction, pollutent in the water body is removed, this class methods investment is big, in timeliness and big waters or the mobile big place of water body certain limitation is arranged also, also easily cause secondary pollution.Biological clarification is to utilize waterplant to the absorption of pollution substance and the Degradation of adsorption function and microorganism, and adopt the artificial measures to create the environment that more helps waterplant and microorganism growth and breeding, cultivate strong waterplant of a large amount of detergent powers and microorganism, to improve a kind of purifying method to oxidation operation degradation efficiency in the polluted-water, this class methods reduced investment, good purification, can also promote simultaneously recovering naturally of aquatic ecosystem, frequency of utilization is at most a wetland purification at present.
Use the wetland purification polluted-water and adopt artificial swamp mostly, waste time and energy.China's natural wetland area is big, it is wide to distribute, can purifying water body but also can use manpower and material resources sparingly as rationally being used not only.But because technical problem, the application of natural wetland purifying contaminated surface water body is also fewer.In view of natural wetland clean dirty mainly be the adsorption that utilizes wetland soil, the sorption of wetland plant and the Degradation of plant rhizosphere microbe thereof, under field conditions (factors), the clean dirty ability of wetland is very limited, especially at specific pollutant.And by prolonging the hydraulic detention time of sewage in wetland, in the hope of improving the decontamination effect improving of natural wetland, can cause pollutent in soil, to be accumulated because of the long residence time again, thereby influence the throughput of wetland.
Summary of the invention
The purpose of this invention is to provide a kind of is the method for immobilizing oil degradation bacteria of carrier with the exposed coring in the reed face of land, to strengthen the clean dirty ability of natural wetland to the petroleum pollution water body, overcomes the deficiency of prior art.
The present invention is a carrier with the exposed coring in the wetland reed face of land of petroleum pollution, from the oil-polluted soils of on-the-spot wetland collection, carry out the separation of oil degradation bacteria, and laboratory domestication and cultivation, obtain salt tolerant type oil degradation bacteria, be fixed in root of phragmites communis palpus carrier surface again, concrete grammar comprises the steps:
1, the screening of oil degradation bacteria
(1) with the soil 10g of petroleum pollution, put into the 250mL Erlenmeyer flask, with 100mL physiological saline abundant mixing, treat that microorganism all is discharged into physiological saline in the soil after, leave standstill and treat solid-liquid separation.Getting its supernatant liquor 1mL adds in the liquid enrichment medium, at 30 ℃, 120rmin
-1Condition under in the shaking culture case, cultivated 7～10 days.After treating the nutrient solution muddiness, therefrom pipette 1mL liquid again and add in the new liquid enrichment medium, culture condition is the same, repeats to operate more than at least three times equally; From above-mentioned liquid enrichment medium, draw 0.2mL bacterium liquid and evenly coat on the solid screening culture medium flat board, 30 ℃ of constant temperature culture, until growing bacterium colony; Select wherein bigger single bacterium colony, the line switching is gone in the new solid screening culture medium, cultivates.After waiting to grow new bacterium colony, repeat aforesaid operations again, until the oil degradation bacterial classification that obtains purifying.
2, preparation oil degradation bacteria suspension liquid
Oil degradation bacteria with above-mentioned purifying, be inoculated in the liquid enrichment medium with a transfering loop picking part, under 120r/min, 30 ℃ of conditions, cultivated 5 days, treat to carry out the coated plate counting after the bacterium liquid muddiness, determine that the oil degradation bacteria suspension liquid concentration for preparing is 2.85 * 10
6～5.7 * 10
6Individual/mL.
The composition of above-mentioned liquid enrichment medium comprises: diesel oil 1g/L, NaNO
33.0g/L, K
2HPO
40.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O 0.5g/L, KCl 0.5g/L, FeSO
47H
2O 0.001g/L, CaCl
20.002g/L, pH 7.0-7.2.
3, the immobilization of oil degradation bacteria
(1) pre-treatment of coring: the face of land coring of the reed that will gather at wetland, soak with sterilized water and to clean in 12 hours, dry after 5 hours with soaked in absolute ethyl alcohol again.
(2) immobilization: taking by weighing the 0.7g root of phragmites communis must evenly be laid in the culture dish; Get concentration 2.85 * 10
6～5.7 * 10
6Oil degradation bacteria suspension liquid 50mL in individual/mL scope adds sodium alginate 0.1g again, and being sprayed onto root of phragmites communis after the mixing equably must reach immobilization after last 5 hour.
The present invention has that material source is extensive, technology is simple, and expense is low, good purification, and the big area that can be used for actual reed wetland such as promotes the use of at advantage.
Embodiment
The present invention generally is subjected to the practical situation of petroleum pollution according to Liaohe River estuarine wetland, the coring exposed with the reed wetland face of land is carrier, from the oil-polluted soils of on-the-spot wetland collection, carry out the separation of oil degradation bacteria, and laboratory domestication and cultivation, obtain salt tolerant type oil degradation bacteria, be fixed in root of phragmites communis palpus carrier surface again, concrete steps are as follows:
1, the screening of oil degradation bacteria
(1) with the soil 10g of petroleum pollution, put into the 250mL Erlenmeyer flask, with 100mL physiological saline abundant mixing, treat that microorganism all is discharged into physiological saline in the soil after, leave standstill and treat solid-liquid separation.
(2) in the supernatant liquor of step (1), pipette 1mL, add in the liquid enrichment medium 30 ℃, 120rmin
-1Condition under in the shaking culture case, cultivated 7～10 days.After treating the nutrient solution muddiness, therefrom pipette 1mL liquid again, add in the new liquid enrichment medium, culture condition is the same, repetitive operation 5 times.
(3) concentrate absorption 0.2mL bacterium liquid evenly to coat on the solid screening culture medium flat board from above-mentioned liquid enrichment culture after 5 times are cultivated, 30 ℃ of constant temperature culture are until growing bacterium colony; Select wherein bigger single bacterium colony, the line switching is gone in the new solid screening culture medium, cultivates.After waiting to grow new bacterium colony, repeat aforesaid operations again, until the oil degradation bacterial classification that obtains purifying.
2, preparation oil degradation bacteria suspension liquid
With the oil degradation bacteria of above-mentioned purifying, be inoculated in the liquid enrichment medium with a transfering loop picking part, 120r/min, 30 ℃ cultivated five days.Treat to carry out the coated plate counting after the bacterium liquid muddiness, determine that the oil degradation bacteria suspension liquid concentration for preparing is 5.7 * 10
6Individual/mL.
The composition of aforesaid liquid enrichment medium comprises: diesel oil 1g/L, NaNO
33.0g/L, K
2HPO
40.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O 0.5g/L, KCl 0.5g/L, FeSO
47H
2O 0.001g/L, CaCl
20.002g/L, pH7.0-7.2.
3, the immobilization of oil degradation bacteria
(1) pre-treatment of coring: will gather the face of land coring of reed at river mouth, Liaohe River reed wetland, and soak with sterilized water and cleaned in 12 hours, and dry after 5 hours with soaked in absolute ethyl alcohol again.
(2) immobilization: according to the actual (real) thickness of the estuarine wetland reed face of land, Liaohe River coring, getting an amount of root of phragmites communis must evenly be laid in the culture dish; The oil degradation bacteria suspension liquid of getting step 2 adds sodium alginate, sprays equably after the mixing or drips to and carry out immobilization on the root of phragmites communis palpus.
Embodiment 1
Getting the oil degradation bacteria concentration is 5.70 * 10
6The suspension liquid 50mL of individual/mL, add the 0.1g sodium alginate, drip to equably after mixing on the root of phragmites communis palpus, immobilization immersed the immobilizing oil degradation bacteria carrier in the micro-polluted water of simulating the petroleum pollution of river mouth, the Liaohe River (100mL seawater+300mL tap water+1g diesel oil) after 5 hours.After under 16 ℃ of conditions of room temperature 20 days, the immobilizing oil degradation bacteria carrier is 60% to the decontamination effect improving of oil micro-polluted water.
Embodiment 2
Getting the oil degradation bacteria concentration is 3.82 * 10
6Individual/mL suspension liquid 50mL, add the 0.1g sodium alginate, drip to equably after mixing on the root of phragmites communis palpus, immobilization immersed the immobilizing oil degradation bacteria carrier in the micro-polluted water of simulating the petroleum pollution of river mouth, the Liaohe River (100mL seawater+300mL tap water+1g diesel oil) after 5 hours.After under 16 ℃ of conditions of room temperature 20 days, the immobilizing oil degradation bacteria carrier is 59% to the decontamination effect improving of micro-polluted water.
Embodiment 3
Getting the oil degradation bacteria concentration is 2.85 * 10
6Individual/mL suspension liquid 50mL, add the 0.1g sodium alginate, drip to equably after mixing on the root of phragmites communis palpus, immobilization immersed the immobilizing oil degradation bacteria carrier in the micro-polluted water of simulating the petroleum pollution of river mouth, the Liaohe River (100mL seawater+300mL tap water+1g diesel oil) after 5 hours.After under 16 ℃ of conditions of room temperature 20 days, the immobilizing oil degradation bacteria carrier is 58% to the decontamination effect improving of micro-polluted water.
As seen, even at 2.85 * 10 of lower concentration
6Under individual/mL and the lower temperature, oil degradation bacteria suspension liquid of the present invention still has stronger detergent power and effect to petroleum waste water, and this helps promoting the use of in the reed wetland big area greatly.
Claims (3)
1. one kind is the method for immobilizing oil degradation bacteria of carrier with reed face of land coring, it is characterized in that at first filtering out oil degradation bacteria, prepare the oil degradation bacteria suspension liquid then, at last with immobilizing oil degradation bacteria on the coring of the wetland reed face of land, wherein the process for fixation of oil degradation bacteria is as follows:
(1) pre-treatment of coring: with the face of land coring of reed, soaked 12 hours with sterilized water earlier, dry after 5 hours with soaked in absolute ethyl alcohol again;
(2) immobilization: taking by weighing the 0.7g root of phragmites communis must evenly be laid in the culture dish; Get concentration 2.85 * 10
6～5.7 * 10
6Oil degradation bacteria suspension liquid 50mL in individual/mL scope adds sodium alginate 0.1g again, and being sprayed onto root of phragmites communis after the mixing equably must reach immobilization after last 5 hour.
2. method for immobilizing oil degradation bacteria as claimed in claim 1 is characterized in that the screening method of above-mentioned oil degradation bacteria is as follows:
(1) with the soil 10g of petroleum pollution, put into the 250mL Erlenmeyer flask, with 100mL physiological saline abundant mixing, treat that microorganism all is discharged into physiological saline in the soil after, leave standstill and treat solid-liquid separation; Getting its supernatant liquor 1mL adds in the liquid enrichment medium, at 30 ℃, 120rmin
-1Condition under in the shaking culture case, cultivated 7～10 days; After treating the nutrient solution muddiness, therefrom pipette 1mL again and add in the identical liquid enrichment medium of another composition, continue under the same conditions to cultivate, repetitive operation is more than at least 3 times;
(2) therefrom draw 0.2mL bacterium liquid again and evenly coat on the solid screening culture medium flat board, 30 ℃ of constant temperature culture, until growing bacterium colony; Select wherein bigger single bacterium colony, line switching goes in the new solid screening culture medium to cultivate, wait to grow new bacterium colony after, repeat the line switching and cultivate operation again, until the oil degradation bacterial classification that obtains purifying, the composition of above-mentioned liquid enrichment medium comprises: diesel oil 1g/L, NaNO
33.0g/L, K
2HPO
40.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O0.5g/L, KCl 0.5g/L, FeSO
47H
2O 0.001g/L, CaCl
20.002g/L, pH 7.0-7.2.
3. method for immobilizing oil degradation bacteria as claimed in claim 1 is characterized in that the method for above-mentioned preparation oil degradation bacteria suspension liquid is as follows:
Oil degradation bacteria with above-mentioned purifying, be inoculated in the liquid enrichment medium with a transfering loop picking part, under 120r/min, 30 ℃ of conditions, cultivated 5 days, treat to carry out the coated plate counting after the bacterium liquid muddiness, determine that the oil degradation bacteria suspension liquid concentration for preparing is 2.85 * 10
6～5.7 * 10
6Individual/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102558308A CN101760455B (en) | 2009-12-31 | 2009-12-31 | Method for immobilizing petroleum degradation bacteria by taking surface root hair of reed as carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102558308A CN101760455B (en) | 2009-12-31 | 2009-12-31 | Method for immobilizing petroleum degradation bacteria by taking surface root hair of reed as carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101760455A CN101760455A (en) | 2010-06-30 |
| CN101760455B true CN101760455B (en) | 2011-11-16 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979338A (en) * | 2010-10-28 | 2011-02-23 | 中国海洋大学 | Method for purifying surface water body of reed wetland by using oil degradation bacterium immobilized carrier |
| CN102657986B (en) * | 2012-04-25 | 2014-05-14 | 中国科学院武汉植物园 | Method for removing suspended particulate matters from urban air by using soil algae |
| CN106636059A (en) * | 2017-01-09 | 2017-05-10 | 河海大学 | Preparation method of immobilized petroleum degradation enzyme preparation |
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Granted publication date: 20111116 Termination date: 20121231 |