CN105087538B - A kind of oil degradation microbial inoculum and its preparation method and application - Google Patents
A kind of oil degradation microbial inoculum and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of oil degradation microbial inoculums and its preparation method and application, and its preparation method is：Polyvinyl alcohol and sodium alginate soln are mixed in proportion, add activated carbon mixing, is added in after cooling with 10:1 ratio is with including Chryseobacterium sp：Shank bacterium：Achromobacter：Burkholderia（Each bacterial strain mass ratio is 36：22：18：5）Oil degradation bacterium solution mixed；Obtained mixed solution is instilled into calcium chloride saturation boric acid solution with syringe again, carries out balling-up；After standing, the microballoon in solution is taken out, is washed with the 0.9%NaCl solution after sterilizing, obtains the oil degradation microbial inoculum of microspheres with solid shape.The oil degradation microbial inoculum of the present invention can be used for remedying oil-polluted soils；The form of solid fungicide is also allowed for transporting and stored, and the practice for microorganism remediation oil-polluted soils provides technical support.
Description
Technical field
A kind of mix bacterium agent the present invention relates to oil-polluted soils reparation prepares more particularly to a kind of oil degradation microbial inoculum
And its preparation method and application.
Background technology
Oil is one of main energy of modern society, referred to as " industrial blood ", " gold of black ".Oil quilt
It is widely used in preparing the industrial circles such as natural gas, gasoline, kerosene, volatile oil, lubricating oil, paraffin and pitch.In adding for crude oil
It is easy to cause and spills and leakage accident in work, use and transportational process, oil is caused to enter in the environment such as water body, soil and causes stone
Oily pollution.The complex mixture that oil is made of a variety of hydro carbons, a small amount of non-hydrocarbon compound, it is difficult to be degraded in the environment,
Polycyclic aromatic hydrocarbon in its component is a kind of persistence organic pollutant（POPs）.Petroleum pollution has carcinogenic, teratogenesis, mutagenic
Harm.Processing petroleum wastewater is infected with physical method, chemical method, biological method three classes both at home and abroad.There is processing in physics, chemical method
Of high cost, the halfway problem of pollutant process, repairing method of microorganism is because of the advantages that it is economic, effective, non-secondary pollution quilt
It is widely studied.
The high efficient petroleum degrading bacteria liquid that laboratory is enriched with is applied into actual contaminated site and plays efficient repairing effect
It is a urgent problem to be solved.The effect under different environmental conditions is different to the degradation process of oil pollution for microorganism, with
It causes to influence remediation efficiency；And the bacterium solution used in practice is to use the bacterium solution fermented containing abundant nutriment, is deposited
Limited between when stored, the problem of transport is inconvenient.
The content of the invention
The purpose of the present invention is overcoming deficiency of the prior art, and provide a kind of oil degradation microbial inoculum and preparation method thereof
And purposes.
A kind of preparation method of oil degradation microbial inoculum, is as follows：
1）Polyvinyl alcohol at 80 ~ 100 DEG C is completely dissolved, is mixed in proportion with sodium alginate soln, obtains sea
Mosanom and polyvinyl alcohol solution；
2）Activated carbon mixing is added in into sodium alginate and polyvinyl alcohol solution, it is 5 g/L to make concentration of activated carbon；
3）By step 2）After obtained solution is cooled to 40 DEG C, with oil degradation bacterium solution with 10:1 volume ratio mixing；
4）Under 0 ~ 4 DEG C, stirring condition, with syringe by step 3）The calcium chloride that obtained solution instills concentration 30% is satisfied
And boric acid solution, carry out balling-up；
5）By step 4）Obtained solution is placed in 4 DEG C of refrigerators after standing 26h, the microballoon in solution is taken out, with sterilizing
0.9% NaCl solution washing afterwards, obtains the oil degradation microbial inoculum of microspheres with solid shape.
The step 1）In sodium alginate and polyvinyl alcohol solution in sodium alginate mass percent for 1% ~
2%, the mass percent of polyvinyl alcohol is 8% ~ 10%.
The step 3）In oil degradation bacterium solution be containing Chryseobacterium sp, shank bacterium, achromobacter and primary kirschner
The bacterium solution of bacterium, Chryseobacterium sp：Shank bacterium：Achromobacter：Each bacterial strain mass ratio of Burkholderia is 36：22：18：5, oil degradation bacteria
The OD of liquid600For 1.5, matrix is fermentation medium.
The fermentation medium preparation method is：By 20.0 g/L glucose, 20.0 g/L cornstarch, 0.2 g/L
Potassium dihydrogen phosphate, 5.0 g/L calcium carbonate, 5.0 g/L peptones, 10.0 g/L soybean cake powders and 1000mL deionized waters are mixed
It closes, it is 7.0 to adjust pH value, and the 15min that sterilizes in 120 DEG C.
A diameter of 2 ~ the 5mm of oil degradation microbial inoculum of the microspheres with solid shape.
The oil degradation microbial inoculum that a kind of preparation method using the oil degradation microbial inoculum is prepared.
A kind of method using the oil degradation microbial inoculum remedying oil-polluted soils, specially：To oil pollution soil
Earth carries out turning over pretreatment, by oil degradation microbial inoculum with mass ratio 1:20 dosage is added in oil-polluted soils, mixing；
During entire soil remediation soil moisture content is made to be maintained at 20% ~ 30%.
Sodium alginate is used in the present invention（SA）And polyvinyl alcohol（PVA）Bacterium solution is embedded in inside microballoon as carrier
In net structure, solves the environmental impact factor of microorganism, while the microspheres with solid of balling-up is convenient for storage and transport.This method
As one kind of immobilized microorganism technology, have broad application prospects in microorganism remediation oil pollution practice.
The present invention, as function bacterium solution, adds in activated carbon enhancing adsorptivity, improvement using high efficient petroleum degrading bacteria group in embedding process
Immobilized microorganism technology obtains a kind of preparation method of new and effective oil degradation microbial inoculum.Compared with prior art, it is of the invention
Advantageous effect be：
（1）It is used in the present invention with the high efficient petroleum degrading bacteria group that Chryseobacterium sp etc. is main function bacterium, the microflora degradation
Rate is high, and the synergy between each bacterial strain is good, and environmental suitability is strong；（2）Activated carbon is added in the present invention in embedding process to increase
Strong adsorptivity；（3）The form of globular solids microbial inoculum helps to maintain the activity of microorganism, is readily transported and stores, and is microorganism
The practice of remedying oil-polluted soils provides technical support.
Description of the drawings
Microbial inoculum in Fig. 1 embodiment of the present invention 1 adds in soil petrochina changes of contents after oil-polluted soils；
Microbial inoculum in Fig. 2 embodiment of the present invention 2 adds in soil petrochina changes of contents after oil-polluted soils；
Microbial inoculum in Fig. 3 embodiment of the present invention 3 adds in soil petrochina changes of contents after oil-polluted soils；
Microbial inoculum in Fig. 4 embodiment of the present invention 4 adds in soil petrochina changes of contents after oil-polluted soils.
Specific embodiment
Strain in the present invention is purchased from China General Microbiological culture presevation administrative center（CGMCC）It is micro- with Chinese industrial
Biological inoculum preservation administrative center（CICC）, number be respectively：Chryseobacterium sp（Chryseobacterium, CICC strains volume
Number NO.10999）, achromobacter（Achromobacter, CICC bacterium numberings NO.10400）, Burkholderia
（Burkholderia, CICC bacterium numberings NO.10877）, shank bacterium（Caulobacter, CGMCC bacterium numberings NO.
4.1475）.
A kind of preparation method of oil degradation microbial inoculum provided by the invention, is as follows：
1）Polyvinyl alcohol at 80 ~ 100 DEG C is completely dissolved, is mixed in proportion with sodium alginate soln, obtains sea
Mosanom and polyvinyl alcohol solution；
2）Activated carbon mixing is added in into sodium alginate and polyvinyl alcohol solution, it is 5 g/L to make concentration of activated carbon；
3）By step 2）After obtained solution is cooled to 40 DEG C, with oil degradation bacterium solution with 10:1 volume ratio mixing；
4）Under 0 ~ 4 DEG C, stirring condition, with syringe by step 3）The calcium chloride that obtained solution instills concentration 30% is satisfied
And boric acid solution, carry out balling-up；
5）By step 4）Obtained solution is placed in 4 DEG C of refrigerators after standing 26h, the microballoon in solution is taken out, with sterilizing
0.9% NaCl solution washing afterwards, obtains the oil degradation microbial inoculum of microspheres with solid shape.
The step 1）In sodium alginate and polyvinyl alcohol solution in sodium alginate mass percent for 1% ~
2%, the mass percent of polyvinyl alcohol is 8% ~ 10%.
The step 3）In oil degradation bacterium solution be containing Chryseobacterium sp, shank bacterium, achromobacter and primary kirschner
The bacterium solution of bacterium, Chryseobacterium sp：Shank bacterium：Achromobacter：Each bacterial strain mass ratio of Burkholderia is 36：22：18：5, oil degradation bacteria
The OD of liquid600For 1.5, matrix is fermentation medium.
The fermentation medium preparation method is：By 20.0 g/L glucose, 20.0 g/L cornstarch, 0.2 g/L
Potassium dihydrogen phosphate, 5.0 g/L calcium carbonate, 5.0 g/L peptones, 10.0 g/L soybean cake powders and 1000mL deionized waters are mixed
It closes, it is 7.0 to adjust pH value, and the 15min that sterilizes in 120 DEG C.
A diameter of 2 ~ the 5mm of oil degradation microbial inoculum of the microspheres with solid shape.
The oil degradation microbial inoculum that a kind of preparation method using the oil degradation microbial inoculum is prepared.
A kind of method using the oil degradation microbial inoculum remedying oil-polluted soils, specially：To oil pollution soil
Earth carries out turning over pretreatment, by oil degradation microbial inoculum with mass ratio 1:20 dosage is added in oil-polluted soils, mixing；
During entire soil remediation soil moisture content is made to be maintained at 20% ~ 30%.
Following embodiment can make the technical staff of this professional skill field more fully understand the present invention, but not to appoint
Where formula limitation is of the invention.
Embodiment 1：
A kind of preparation method of oil degradation microbial inoculum in the present embodiment, is as follows：
1）Polyvinyl alcohol at 80 ~ 100 DEG C is completely dissolved, is mixed in proportion with sodium alginate soln, obtains sea
Mosanom and polyvinyl alcohol solution；Sodium alginate（SA）With polyvinyl alcohol（PVA）The quality of sodium alginate in mixed solution
Percentage is 1%, and the mass percent of polyvinyl alcohol is 8%, SA：PVA=1:8；
2）Activated carbon mixing is added in into sodium alginate and polyvinyl alcohol solution, it is 5 g/L to make concentration of activated carbon；
3）By step 2）After obtained solution is cooled to 40 DEG C, with oil degradation bacterium solution with 10:1 volume ratio mixing；Stone
Oil degradation bacterium solution is the bacterium solution containing Chryseobacterium sp, shank bacterium, achromobacter and Burkholderia, and four kinds of bacterial strain mass ratioes are 36：
22：18：5, the OD of bacterium solution600For 1.5, matrix is fermentation medium.Fermentation medium preparation method is：By 20.0 g/L grapes
Sugar, 20.0 g/L cornstarch, 0.2 g/L potassium dihydrogen phosphates, 5.0 g/L calcium carbonate, 5.0 g/L peptones, 10.0 g/L are yellow
Beancake powder and 1000mL deionized waters are mixed, and it is 7.0 to adjust pH value, and the 15min that sterilizes in 120 DEG C.
4）Under 0 ~ 4 DEG C, stirring condition, with 7# syringes by step 3）Obtained solution instills the calcium chloride of concentration 30%
Saturation boric acid solution carries out balling-up, i.e., forms the microsphere containing microorganism during the reaction,；
5）By step 4）Obtained solution is placed in 4 DEG C of refrigerators after standing 26h, the microballoon in solution is taken out, with sterilizing
0.9% NaCl solution washing afterwards, obtains the oil degradation microbial inoculum of microspheres with solid shape, the oil for the microspheres with solid shape being prepared
Degradation bacterial agent diameter is between 2 ~ 5mm.
A kind of oil degradation microbial inoculum is prepared using the preparation method of above-mentioned oil degradation microbial inoculum.
The mass percent of oil-polluted soils petrochina in the present embodiment is 32%.It is repaiied using the oil degradation microbial inoculum
The method of multiple oil-polluted soils, step are:
Step（1）, take the oil degradation microbial inoculum prepared（SA：PVA=1：8）With mass ratio 1:20 dosage is added to
Through in oil-polluted soils that turning over, be placed in it is shady and cool react at room temperature, protect soil moisture content during entire soil remediation
It holds 20% ~ 30%.；
Step（2）, in reaction process, sample quarterlies measures soil petrochina content with weight method；
Step（3）, survey with weight method the variation of oil-polluted soils petrochina content, and examine its degradation effect.With four
3 g of point-score soil sampling, grinding are crossed 2 mm sieves, are added in 50 mL glass centrifuge tubes, chloroform 20 is added into centrifuge tube
Supernatant is put into triangular flask weighed in advance, then chloroform is added in into centrifuge tube by mL, 20 min of ultrasound, centrifugation
20 mL repeat to extract, combining extraction liquid, and heating water bath removes solvent at 70 DEG C, and gravimetric detemination soil petrochina contains
Amount calculates the degradation rate of oil.The oil being initially added is m0, initial triangular flask weight m1, water-bath is evaporated Vee formation bottle weight
m2, then the oil weight in triangular flask system is m=m2-m1, the degradation rate of oil for v=（m0-m）/m0。
In this example, for petroleum hydrocarbon degradation microbial inoculum to the degradation rate of oil-polluted soils up to 3%, microbial inoculum adds in oil-polluted soils
Soil petrochina changes of contents is as shown in Figure 1 afterwards.
Embodiment 2：
In the present embodiment, step 1）Sodium alginate（SA）With polyvinyl alcohol（PVA）The matter of sodium alginate in mixed solution
It is 2% to measure percentage, and the mass percent of polyvinyl alcohol is 8%, SA：PVA=2:8；Remaining step is same as Example 1, is prepared into
To microbial inoculum.
The mass percent of the oil-polluted soils petrochina handled using the microbial inoculum is 32%.In this example, petroleum hydrocarbon degradation
For microbial inoculum to the degradation rate of oil-polluted soils up to 13%, microbial inoculum adds in soil petrochina changes of contents such as Fig. 2 after oil-polluted soils
It is shown.
Embodiment 3：
In the present embodiment, step 1）Sodium alginate（SA）With polyvinyl alcohol（PVA）The matter of sodium alginate in mixed solution
It is 1% to measure percentage, and the mass percent of polyvinyl alcohol is 10%, SA：PVA=1:10；Remaining step is same as Example 1, prepares
Obtain microbial inoculum.
The mass percent of the oil-polluted soils petrochina handled using the microbial inoculum is 32%.In this example, petroleum hydrocarbon degradation
For microbial inoculum to the degradation rate of oil-polluted soils up to 29%, microbial inoculum adds in soil petrochina changes of contents such as Fig. 3 after oil-polluted soils
It is shown.
Embodiment 4：
In the present embodiment, step 1）Sodium alginate（SA）With polyvinyl alcohol（PVA）The matter of sodium alginate in mixed solution
It is 2% to measure percentage, and the mass percent of polyvinyl alcohol is 10%, SA：PVA=2:10；Remaining step is same as Example 1, prepares
Obtain microbial inoculum.
The mass percent of the oil-polluted soils petrochina handled using the microbial inoculum is 32%.In this example, petroleum hydrocarbon degradation
For microbial inoculum to the degradation rate of oil-polluted soils up to 16%, microbial inoculum adds in soil petrochina changes of contents such as Fig. 4 after oil-polluted soils
It is shown.
Claims (6)
1. a kind of preparation method of oil degradation microbial inoculum, which is characterized in that be as follows：
1）Polyvinyl alcohol at 80 ~ 100 DEG C is completely dissolved, is mixed in proportion with sodium alginate soln, obtains alginic acid
Sodium and polyvinyl alcohol solution；
2）Activated carbon mixing is added in into sodium alginate and polyvinyl alcohol solution, it is 5 g/L to make concentration of activated carbon；
3）By step 2）After obtained solution is cooled to 40 DEG C, with oil degradation bacterium solution with 10:1 volume ratio mixing；Described
Oil degradation bacterium solution be the bacterium solution containing Chryseobacterium sp, shank bacterium, achromobacter and Burkholderia, Chryseobacterium sp：Shank bacterium：Nothing
Color bacillus：Each bacterial strain mass ratio of Burkholderia is 36：22：18：5, the OD of oil degradation bacterium solution600For 1.5, matrix is trained for fermentation
Support base；The Chryseobacterium sp is preserved in Chinese industrial Microbiological Culture Collection administrative center, preserving number CICC
NO.10999, the achromobacter are preserved in Chinese industrial Microbiological Culture Collection administrative center, preserving number CICC
NO.10400, the Burkholderia are preserved in Chinese industrial Microbiological Culture Collection administrative center, preserving number CICC
NO.10877, the shank bacterium are preserved in China General Microbiological culture presevation administrative center, and preserving number is CGMCC NO.
4.1475；
4）Under 0 ~ 4 DEG C, stirring condition, with syringe by step 3）Obtained solution instills the calcium chloride saturation boron of concentration 30%
Acid solution carries out balling-up；
5）By step 4）Obtained solution is placed in 4 DEG C of refrigerators after standing 26h, the microballoon in solution is taken out, after sterilizing
0.9% NaCl solution is washed, and obtains the oil degradation microbial inoculum of microspheres with solid shape.
2. a kind of preparation method of oil degradation microbial inoculum as described in claim 1, it is characterised in that：The step 1）In
The mass percent of sodium alginate and sodium alginate in polyvinyl alcohol solution is 1% ~ 2%, the mass percent of polyvinyl alcohol
For 8% ~ 10%.
3. a kind of preparation method of oil degradation microbial inoculum as described in claim 1, it is characterised in that：The fermentation medium
Preparation method is：By 20.0 g/L glucose, 20.0 g/L cornstarch, 0.2 g/L potassium dihydrogen phosphates, 5.0 g/L calcium carbonate,
5.0 g/L peptones, 10.0 g/L soybean cake powders and 1000mL deionized waters are mixed, and it is 7.0 to adjust pH value, and in 120
DEG C sterilizing 15min.
4. a kind of preparation method of oil degradation microbial inoculum according to claim 1, it is characterised in that：The microspheres with solid
A diameter of 2 ~ the 5mm of oil degradation microbial inoculum of shape.
5. a kind of oil degradation microbial inoculum that preparation method using oil degradation microbial inoculum as described in claim 1 is prepared.
A kind of 6. method using oil degradation microbial inoculum remedying oil-polluted soils as claimed in claim 5, which is characterized in that
Oil-polluted soils are carried out to turn over pretreatment, by oil degradation microbial inoculum with mass ratio 1:20 dosage is added to oil pollution
In soil, mixing；During entire soil remediation soil moisture content is made to be maintained at 20% ~ 30%.
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| CN106148226A (en) * | 2016-07-04 | 2016-11-23 | 四川行之智汇知识产权运营有限公司 | A kind of oil degradation micro organism powder |
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| CN110964711B (en) * | 2018-09-28 | 2024-03-22 | 天津大学 | Acid-resistant microbial capsule embedding liquid, microbial capsule, and preparation methods and applications thereof |
| CN110756577A (en) * | 2019-10-30 | 2020-02-07 | 东营金岛环境工程有限公司 | Microbial remediation technology for petroleum-polluted site |
| CN113215009B (en) * | 2020-01-19 | 2022-08-30 | 中国石油天然气集团有限公司 | Composite immobilized microbial agent and preparation method and application thereof |
| CN111718085A (en) * | 2020-06-11 | 2020-09-29 | 中石大蓝天(青岛)石油技术有限公司 | Solid waste treatment method |
| CN111718094A (en) * | 2020-06-11 | 2020-09-29 | 中石大蓝天(青岛)石油技术有限公司 | Treatment method of oil silt |
| CN113399449B (en) * | 2021-06-18 | 2022-09-09 | 西安建筑科技大学 | Environment-friendly reinforcing method for inhibiting dust emission in heavy metal polluted site |
| CN115216425B (en) * | 2022-06-24 | 2023-06-27 | 山东农业大学 | Flavobacterium nepalensis and application thereof in degradation of plastics |
| CN115805232B (en) * | 2022-11-29 | 2023-10-20 | 西南科技大学 | Application method of encapsulated bacteria for efficiently treating hexavalent chromium polluted soil |
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