CN101724582A - Immobilized microbial inoculum for remediating PAHs contaminated soil and preparation method thereof - Google Patents
Immobilized microbial inoculum for remediating PAHs contaminated soil and preparation method thereof Download PDFInfo
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Abstract
The invention relates to remediation technology of polycyclic aromatic hydrocarbon (PAHs) contaminated soil, in particular to an immobilized microbial inoculum for remediating the PAHs contaminated soil and a preparation method thereof. The preparation method specifically comprises the following steps of: inoculating a prepared microbial mixed strain liquid into a solid medium, namely a processed microbial inoculum vector; culturing the microbial mixed strain liquid at a constant temperature of between 28 and 30 DEG C in an incubator for 3 to 4 days; and carrying out adsorption immobilization and proliferation to prepare an immobilized mixed microbial preparation. The method specifically adopts crop byproducts as the vector, and has the advantages of high adsorption speed of mixed strains, good proliferation effect, simple operation, no secondary pollution, rich sources of the crop byproducts and low price. The prepared immobilized cell granules are put into the PAHs contaminated soil in a percentage of 2 to 4 percent, which can effectively remove PAHs contaminants in the soil. The degradation rate of the PAHs processed by 70d can reach 74 percent when the concentration of the PAHs in the contaminated soil is 84mg/kg.
Description
Technical field
The present invention relates to the recovery technique of polycyclic aromatic hydrocarbons (PAHs) contaminated soil, specifically a kind of immobilized microbial inoculum of repairing the PAHs contaminated soil and preparation method thereof.
Background technology
Conventional environment remediation technology mainly refers to: chemical repairing method, physics repairing method, restoration of the ecosystem method and biological restoration method, both often are classified as unified technology the back, generally take the original position mode on the engineering.Bioremediation technology is as the emerging technology of less energy-consumption, non-secondary pollution, be suitable for the soil remediation of the slight or intermediate pollution of big area, have advantages such as expense is low, easy to operate, environmentally safe, and can improve the soil, increase fertility of land, therefore have and use widely and development prospect.
China has carried out big quantity research from the nineties in 20th century at the oil-polluted soils bioremediation technology, has all obtained significant achievement in research from theoretical and application.But still have a large amount of polluted farmland soils to need to repair at present, be badly in need of carrying out the biological restoration research of farmland pollution soil.
Shen is comforted feed ditch and is established in nineteen sixty, 70 kilometers of total lengths, start from the Fushun second branch factory of oil, admit the trade effluent and the sanitary sewage of the discharging of Fushun City enterprise of tens of family on the way, these discharge waters enter Shenyang in Dongling District deep-well town, tiltedly wear Dongling District, the village enters Shujiatun District in White Tower fort town llama peace, imports Taizihe River at last.Because life-time service feed ditch sewage irrigates, the feed ditch upstream area in deep-well town, caused the rice seedling speed of growth slowly, phenomenon such as mashed root, grain be flat, the rice of output has dense oil flavor and aromatics smell.When secular dirty filling caused severe contamination for soil and farm crop, the underground water of this region and surface water quality also be subjected to seriously influencing.This area has a lot of wells to be subjected in various degree pollution, and shallow ground water has dense peculiar smell and greasy dirt.Investigation according to Liaoning Province's quarantine station relevant department shows, the chronic disease among the crowd of this area and the sickness rate of malignant disease obviously increase, and the farm crop in dirty irrigated area (paddy rice, vegetables etc.) are sold in Shenyang City and the Fushun City basically, exist potential to threaten to city resident healthy.From the eighties, along with the reinforcement of people to sewage irrigation harm understanding and government's attention, dirty irrigated area has progressively begun clear water and has irrigated the transformation and the shunting engineering construction of removing contamination, and whole dirty irrigated area has realized substantially that in 2000 the clear water in farmland is irrigated in the district.But because area, Liaoxi-Shenyang shortage of water resources, particularly dry year are particularly serious, the dirty phenomenon of irritating in indivedual areas still exists.In order to administer and repair the dirty for many years PAHs contaminated soil that causes of irritating, we have researched and developed microbial immobilized recovery technique IMR (Immobilized MicrobeRemediation).
Immobilization technology is meant the method for utilizing chemistry or physics, with free cell (microorganism) or enzyme, in the area of space that is positioned to limit, and makes it keep active a kind of technology for a long time.The key of microbial immobilized technology is suitable solid support material of screening and the immobilization technology of determining optimization.Utilize the IMR technology to repair the more existing successful examples of polluted-water, but repairing polluted soil yet there are no report.Can the core of PAHs contaminated soil recovery technique be the Degradation of efficient degrading bacteria to PAHs, but be crucial with the degradation capability of degradation bacteria efficiently bringing into play incisively and vividly.Immobilization technology is applied in the reparation of PAHs contaminated soil, can make microorganism keep active for a long time, and it is high-effect that it also can be played consistently in complex environment.
Summary of the invention
The immobilized microbial inoculum preparation method of the reparation PAHs contaminated soil that to the purpose of this invention is to provide good, the widely used crop by-product of a kind of repairing effect be carrier.
To achieve these goals, technical scheme of the present invention is:
Repair the immobilized microbial inoculum of PAHs contaminated soil: by counting 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 1-3: 2-4: 1-3 by weight, the bacterial classification of Gordon Salmonella, joint bacterium, Alcaligenes, pale bacillus, pseudomonas, bacillus and mycobacterium, surplus is a solid medium.
Solid medium by weight percentage, the soybean cake powder of the rice chaff of 55-65%, the corn cob of 11-16%, 1.0-2.0%, the K of 0.06-0.1%
2HPO
4, the wheat bran of 20-25%, the MgSO of 0.025-0.05%
4.7H
2The sucrose of O, 0.8-1.2%, surplus are moisture; PH7.0-7.5.Described bacterial classification needs to concentrate cultivation in the enrichment culture that contains polycyclic aromatic hydrocarbons, and it is: Gordon Salmonella, joint bacterium, Alcaligenes, pale bacillus, pseudomonas, bacillus and mycobacterium are counted in the enrichment medium that contains the PAHs pollutent of bacterial classification inoculation at 30-100ml of 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 1-3: 2-4: 1-3 by weight, then throw in the PAHs contaminated soil of enrichment medium weight percent 10-15%, 28～30 ℃ of temperature, cultivate a week in 120～130rpm/min constant temperature shaking table, stand-by; The bacterium liquid of above-mentioned cultivation by weight for the inoculum size of 10-15% is inoculated in the minimal medium, 28～30 ℃ of temperature, is cultivated 5-7d in 120～130rpm/min constant temperature shaking table, and the microorganism mixed bacteria liquid that obtains is primary seed solution.
Repair the preparation method of the immobilized microbial inoculum of PAHs contaminated soil, enrichment, cultivate and domestication: at first with Gordon Salmonella, the joint bacterium, Alcaligenes, pale bacillus, pseudomonas, the bacterial classification that bacillus and mycobacterium are counted 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 1-3: 2-4: 1-3 by weight is cultured to logarithmic phase in containing the enrichment medium of polycyclic aromatic hydrocarbons, with the bacterial classification of logarithmic phase in crop by-product as solid increment substratum 28～30 ℃ of temperature, under adsorb, fix and propagation 3-4d, breed 2-3 time, promptly get and be immobilized microbial inoculum.
Described bacterial classification needs to concentrate cultivation in the enrichment culture that contains polycyclic aromatic hydrocarbons, and it is: Gordon Salmonella, joint bacterium, Alcaligenes, pale bacillus, pseudomonas, bacillus and mycobacterium are counted in the enrichment medium that contains the PAHs pollutent of bacterial classification inoculation at 30-100ml of 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 1-3: 2-4: 1-3 by weight, then throw in the PAHs contaminated soil of enrichment medium weight percent 10-15%, 28～30 ℃ of temperature, cultivate a week in 120～130rpm/min constant temperature shaking table, stand-by; The bacterium liquid of above-mentioned cultivation is inoculated in the minimal medium for the inoculum size of 10-15% by weight, 28～30 ℃ of temperature, cultivate 5-7d in 120～130rpm/min constant temperature shaking table, the microorganism mixed bacteria liquid that obtains is primary seed solution logarithmic phase bacterium liquid.
Described enrichment medium is counted by weight, 0.1-0.2% glucose, 0.01-0.5%NaCl, 0.05-0.1% peptone, 0.1-1.0%NH
4NO
3, 0.05-0.1% yeast extract paste, 0.1-0.5%K
2HPO
4, 0.01-0.05%MgSO
4.7H
2O, surplus is a sterilized water; PH7.0-7.5; Simultaneously add PAHs in enrichment medium, making its total amount concentration is 100-200mg/L.Described minimal medium is counted by weight: 0.01-0.5%NaCl, 0.1-1.0%NH
4NO
3, 0.5-0.1%K
2HPO
4, 0.01-0.05%MgSO
4.7H
2O, surplus are sterilized water; PH7.0-7.5.
During described spawn culture with logarithmic phase, be that the inoculum size of 10-15% is added to and adsorbs fixedly 3-4d in the solid medium according to weight percent, keep water content 55%-65% the bacterial classification of logarithmic phase, for propagation once; Be solid spawn then, in the same way, breed 2-3 time again, i.e. being fixed microbial inoculum with propagation product once.
Described solid medium by weight percentage, the soybean cake powder of the rice chaff of 55-65%, the corn cob of 11-16%, 1.0-2.0%, the K of 0.06-0.1%
2HPO
4, the wheat bran of 20-25%, the MgSO of 0.025-0.05%
4.7H
2The sucrose of O, 0.8-1.2%, surplus are moisture; PH7.0-7.5.Described solid increment substratum is mixed by the multiple kinds of crops byproduct, various materials is crushed to particle size diameter at 0.1-0.5cm, and uses the Ca (OH) of 1.2-2% before use earlier
2Water logging bubble sterilization 24h.
Principle: be used to repair the PAHs contaminated soil with multiple kinds of crops byproduct mixture as solid support material, adopt the absorption fixing means; The mixing microorganisms of cultivating the efficient degradation PAHs that tames is carried out immobilization, the preparation mix bacterium agent; The immobilized microbial inoculum of preparation is carried out the original position reparation to the PAHs contaminated soil.
The present invention has following advantage:
1. the present invention selects multiple kinds of crops byproduct mixture as solid support material, it is more suitable that solid support material is crushed to diameter 0.1-0.5cm scope, its crop by-product porosity is fit to the absorption of microorganism, can fix complete microorganism cells, avoid the stability of artificial destruction biological enzyme activity and biochemical reaction; Rate of propagation is fast, effective.
The present invention be chosen in for many years in the aged PAHs contaminated soil cultivate, domestication superior microorganism mixed bacterium, can keep active after the immobilization for a long time; The microenvironment of immobilized cell particle helps strengthening the degradation capability of these bacterial classifications, and it is high-effect that it is played consistently under complicated environmental condition.
3. the crop by-product mixture is the immobilized microbial inoculum of carrier, can directly add according to pollution level, and is simple to operate, do not produce secondary pollution.Such material source is abundant, low price.Reduce investment outlay, simplify flow process, safe and reliable in the practice, expense is cheap economically.This class material is added to the permeability that can increase soil in the soil, through corrupt for a long time, also can increase the fertility of soil.
4. the present invention adopts the microorganism reparation, is specially crop by-product carrier absorption fixation of microbe, and is fast to the mixed bacterium rate of adsorption, cultivation effect good.The immobilized microbial inoculum of its preparation is rendered in the PAHs contaminated soil according to 2%-4%, can make that the PAHs of difficult degradation is effectively removed in the contaminated soil, when the PAHs of high-molecular weight, difficult degradation in contaminated soil total amount concentration is 84mg/kg, through the processing of 70d, the degradation rate of PAHs total amount can reach 74%.
Description of drawings
Fig. 1 repairs PAHs contaminated soil design sketch for immobilized microorganism microbial inoculum of the present invention.
Fig. 2 repairs PAHs contaminated soil design sketch for immobilized microorganism microbial inoculum of the present invention.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1
Crop by-product is the method for carrier fixation of microbe, and concrete steps are: prepare seed liquor earlier, handle carrier simultaneously; Hybrid bacterial strain adsorbs fixing in solid medium and propagation then.Concrete steps are as follows:
1) preparation of bacterium liquid:
With bacterial classification Gordon Salmonella (Gordona sp.), joint bacterium (Arthrobaccer sp.), Alcaligenes (Alealigenes sp.), pale bacillus (Ochrobactrum), pseudomonas (Psedomonassp.), bacillus (Bacillus sp.), mycobacterium (Mycobacterium sp.), (all purchase in Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences microbial strains storehouse, it is that opening anyly can be bought per capita; The bacterial classification that is adopted simultaneously all can bought on the market) above-mentioned bacterial classification is counted by weight, 1: 1: 1: 1: 2: 3: 2 ratio was seeded in the enrichment medium that contains the PAHs pollutent of 90ml, then throw in the dirty irrigated area of 10g (village, back, north, Fushun City's Triratna village topsoil soils) aged PAHs contaminated soil, in 30 ℃, shaking culture 7d in the 120rpm/min constant temperature shaking table, stand-by.
The enrichment medium of the described PAHs of containing pollutent is: 2.5ml PAHs solution is joined in the aseptic 500ml triangular flask, make that polycyclic aromatic hydrocarbons total amount concentration is 200mg/L in the enrichment medium; After treating that solvent evaporates is intact, the aseptic enrichment medium of the 90ml that branch is installed is poured in the triangular flask again.Described substratum is by weight percentage: glucose 0.1%, peptone 0.1%, yeast extract paste 0.1%, NaCl0.5%, NH
4NO
30.1%, K
2HPO
40.5%, MgSO
47H
2O 0.05%, pH7.2.Sterilising conditions is: 0.1Mpa moist heat sterilization 30min.
Then above-mentioned kind of liquid is transferred in the 150ml triangular flask of 30ml minimal medium, makes that PAHs total amount concentration is 200mg/L in the substratum by 10% inoculum size.In 28 ℃, carry out the domestication second time under the 120rpm/min condition, cultivate, cultivate 7d.Promptly as the microorganism mixed bacteria liquid, making kind of liquid is logarithmic phase, i.e. primary seed solution with this triplicate.
Described minimal medium is by weight percentage: NaCl 0.01%, NH
4NO
30.5%, K
2HPO
40.5%, MgSO
47H
2O 0.05%, pH7.2.Sterilising conditions is: 0.1Mpa moist heat sterilization 30min.
2) immobilization of hybrid bacterial strain
Above-mentioned primary seed solution is joined through in the pretreated solid medium by 10% inoculum size, and thorough mixing, be that the 80ml primary seed solution inserts in the 800g solid medium, keep water content 60%, absorption, fixing multiplication culture 3d under 28 ℃ of conditions, promptly propagation once is secondary seed, presses above-mentioned propagation mode, breed secondary again, be immobilized microbial inoculum.
Solid support material (from ecological station, ten li rivers of Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences), i.e. solid culture based formulas: rice chaff 55%, corn cob 16%, sucrose 1.0%, wheat bran 25%, soybean cake powder 1.0%, KH
2PO
40.06%, MgSO
4.7H
2O 0.026%, (NH
4)
2HPO
40.2%, 1.8%CaO.Add water behind the mixing, moisture content 55%, placement is spent the night.Regulate pH7.5 next day.Be sub-packed in the high-temperature resistance plastice bag every bag of 800g.0.1mpa sterilization 1h, placement is spent the night, and carries out the sterilization second time again under similarity condition.
The said fixing microbial inoculum is acted on effect is in the polycyclic aromatic hydrocarbon pollution of artificial preparation: in the polycyclic aromatic hydrocarbon pollution of artificial preparation (the PAHs total amount is 83.72mg/kg), the said fixing microbial inoculum of adding 2%, keeping soil moisture content is static placement under 30%, 28 ℃ of condition.Do contrast (CK) test with same method, promptly only add sterile carrier in the contaminated soil.The residual quantity of PAHs in the period sampling measuring soil, experimental result is seen Fig. 1.
When PAHs total amount concentration was 83.72mg/kg in the soil, through the processing of 70d, concentration was reduced to 21.35mg/kg, and degradation rate reaches 74.50%, exceeds 6.98 percentage points than CK.The suitable survival environment condition of experimental result explanation regulating and controlling microbial adds solid medium indigenous microorganism of the present invention and also can bring into play better action.Experimental result illustrates that microbial immobilized microbial inoculum is gratifying for repairing PAHs contaminated soil effect.
Embodiment 2 difference from Example 1 are:
1) preparation of bacterium liquid:
Bacterial classification is Gordon Salmonella (Gordona sp.), joint bacterium (Arthrobaccer sp.), Alcaligenes (Alealigenes sp.), pale bacillus (Ochrobactrum), pseudomonas (Psedomonassp.), bacillus (Bacillus sp.), mycobacterium (Mycobacterium sp.) is counted above-mentioned bacterial classification by weight, 0.5: 1.5: 0.5: 1.5: 3: 4: 3 ratio was seeded in the enrichment medium that contains the PAHs pollutent of 100ml, then throw in the PAHs contaminated soil of the dirty Irrigation District Ageing of 15g, in 28 ℃, shaking culture 5d in the 130rpm/min constant temperature shaking table, stand-by.
The enrichment medium of the described PAHs of containing pollutent is: 1.25ml PAHs solution is joined in the aseptic 500ml triangular flask, make that polycyclic aromatic hydrocarbons total amount concentration is 100mg/L in the enrichment medium; After treating that solvent evaporates is intact, the aseptic enrichment medium of the 100ml that branch is installed is poured in the triangular flask again.Described enrichment medium is by weight percentage: glucose 0.2%, peptone 0.06%, yeast extract paste 0.06%, NaCl 0.01%, NH
4NO
31.0%, K
2HPO
40.1%, MgSO
47H
2O 0.01%, pH7.5.Sterilising conditions is: 0.1Mpa moist heat sterilization 30min.
Then above-mentioned kind of liquid is transferred in the 150ml triangular flask of 30ml minimal medium, makes that PAHs total amount concentration is 100mg/L in the substratum by 15% inoculum size.In 30 ℃, carry out the domestication second time under the 130rpm/min condition, cultivate, cultivate 5d.Promptly as the microorganism mixed bacteria liquid, making kind of liquid is logarithmic phase, i.e. primary seed solution with this triplicate.
Described minimal medium is by weight percentage: NaCl 0.5%, NH
4NO
30.1%, K
2HPO
40.1%, MgSO
47H
2O 0.01%, pH7.5.Sterilising conditions is: 0.1Mpa moist heat sterilization 30min.
2) immobilization of hybrid bacterial strain
Above-mentioned primary seed solution is joined through in the pretreated solid medium by 15% inoculum size, and thorough mixing, be that the 80ml primary seed solution inserts in the 800g solid medium, keep water content 65%, absorption, fixing multiplication culture 4d under 30 ℃ of conditions, promptly propagation once is secondary seed, presses above-mentioned propagation mode, breed secondary again, be immobilized microbial inoculum.
Solid support material (from ecological station, the Chinese Academy of Sciences ten li rivers of ecological institute), solid culture based formulas: rice chaff 65%, corn cob 11%, sucrose 0.8%, wheat bran 20%, soybean cake powder 1.2%, KH
2PO
40.06%, MgSO
4.7H
2O 0.026%, (NH
4)
2HPO
40.2%, 1.4%CaO.Add water behind the mixing, moisture content 60%, placement is spent the night.Regulate pH7.5 next day.Be sub-packed in the high-temperature resistance plastice bag every bag of 800g.0.1mpa sterilization 1h, placement is spent the night, and carries out the sterilization second time again under similarity condition.
The said fixing microbial inoculum is acted on effect is in the polycyclic aromatic hydrocarbon pollution of artificial preparation: in the polycyclic aromatic hydrocarbon pollution of artificial preparation (the PAHs total amount is 83.72mg/kg), the immobilization mix bacterium agent of adding 2%, keeping soil moisture content is static placement under 30%, 28 ℃ of condition.Do contrast (CK) test with same method, promptly only add sterile carrier in the contaminated soil.The residual quantity of PAHs in the period sampling measuring soil, experimental result is seen Fig. 2.
When PAHs total amount concentration was 84.23mg/kg in the soil, through the processing of 70d, concentration was reduced to 22.01mg/kg, and degradation rate reaches 73.87%.In the same time, the CK degradation rate reaches 67.52%.Experimental result illustrates that solid medium of the present invention also has better effect to indigenous microorganism.The microbial immobilized microbial inoculum of the present invention's preparation is gratifying for repairing PAHs contaminated soil effect.
Embodiment 3 difference from Example 1 are:
Repair the preparation method of the immobilized microbial inoculum of PAHs contaminated soil, enrichment, cultivation and domestication: at first Gordon Salmonella, joint bacterium, Alcaligenes, pale bacillus, pseudomonas, bacillus and mycobacterium are counted 0.5: 0.5: 0.5 by weight: 0.5: 2: 3: 2 bacterial classification inoculation was in the enrichment medium that contains the PAHs pollutent of 50ml, then throw in the PAHs contaminated soil of enrichment medium weight percent 12%, in 29 ℃, shaking culture 6d in the 125rpm/min constant temperature shaking table, stand-by.
The enrichment medium of the described PAHs of containing pollutent is: PAHs solution is added in the enrichment medium, and making its polycyclic aromatic hydrocarbons total amount concentration is 150mg/L.Described enrichment medium is by weight percentage: glucose 0.15%, peptone 0.08%, yeast extract paste 0.08%, NaCl 0.1%, NH
4NO
30.8%, K
2HPO
40.3%, MgSO
47H
2O 0.03%, pH7.2.
Then above-mentioned kind of liquid is transferred in the 150ml triangular flask of 30ml minimal medium, makes that PAHs total amount concentration is 150mg/L in the substratum by 15% inoculum size.In 29 ℃, carry out the domestication second time under the 125rpm/min condition, cultivate, cultivate 6d.Promptly as the microorganism mixed bacteria liquid, making kind of liquid is logarithmic phase, i.e. primary seed solution with this triplicate.
Described minimal medium is by weight percentage: NaCl 0.1%, NH
4NO
30.5%, K
2HPO
40.3%, MgSO
47H
2O 0.04%, pH7.2.
2) immobilization of hybrid bacterial strain
Above-mentioned primary seed solution is joined through in the pretreated solid medium by 13% inoculum size, and thorough mixing, be that the 80ml primary seed solution inserts in the 800g solid medium, keep water content 55%, absorption, fixing multiplication culture 4d under 29 ℃ of conditions, promptly propagation once is secondary seed, presses above-mentioned propagation mode, breed secondary again, be immobilized microbial inoculum.
Solid support material (from ecological station, the Chinese Academy of Sciences ten li rivers of ecological institute), solid culture based formulas: rice chaff 60%, corn cob 13%, sucrose 1%, wheat bran 23%, soybean cake powder 1.5%, KH
2PO
40.08%, MgSO
4.7H
2O0.04%.
Above-mentioned detailed description specifies at embodiments of the invention, and this embodiment is not in order to limit claim of the present invention.
Claims (10)
1. immobilized microbial inoculum of repairing the PAHs contaminated soil, it is characterized in that: by counting 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 1-3: 2-4: 1-3 by weight, the bacterial classification of Gordon Salmonella, joint bacterium, Alcaligenes, pale bacillus, pseudomonas, bacillus and mycobacterium, surplus is a solid medium.
2. by the immobilized microbial inoculum of the described reparation of claim 1 PAHs contaminated soil, it is characterized in that: described solid medium by weight percentage, the soybean cake powder of the rice chaff of 55-65%, the corn cob of 11-16%, 1.0-2.0%, the K of 0.06-0.1%
2HPO
4, the wheat bran of 20-25%, the MgSO of 0.025-0.05%
4.7H
2The sucrose of O, 0.8-1.2%, surplus are moisture; PH7.0-7.5.
3. by the described immobilized microbial inoculum of repairing the PAHs contaminated soil of claim 1, it is characterized in that: described bacterial classification needs to concentrate cultivation in the enrichment culture that contains polycyclic aromatic hydrocarbons, and it is: with Gordon Salmonella, the joint bacterium, Alcaligenes, pale bacillus, pseudomonas, bacillus and mycobacterium are counted the bacterial classification inoculation of 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 1-3: 2-4: 1-3 by weight in the enrichment medium that contains the PAHs pollutent of 30-100ml, then throw in the PAHs contaminated soil of enrichment medium weight percent 10-15%, 28～30 ℃ of temperature, cultivate a week in 120～130rpm/min constant temperature shaking table, stand-by;
The bacterium liquid of above-mentioned cultivation by weight for the inoculum size of 10-15% is inoculated in the minimal medium, 28～30 ℃ of temperature, is cultivated 5-7d in 120～130rpm/min constant temperature shaking table, and the microorganism mixed bacteria liquid that obtains is primary seed solution.
4. preparation method by the immobilized microbial inoculum of the described reparation of claim 1 PAHs contaminated soil, enrichment, cultivate and domestication, it is characterized in that: at first with Gordon Salmonella, the joint bacterium, Alcaligenes, pale bacillus, pseudomonas, the bacterial classification that bacillus and mycobacterium are counted 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 1-3: 2-4: 1-3 by weight is cultured to logarithmic phase in containing the enrichment medium of polycyclic aromatic hydrocarbons, with the bacterial classification of logarithmic phase in crop by-product as solid increment substratum 28～30 ℃ of temperature, under adsorb, fix and propagation 3-4d, breed 2-3 time, promptly get and be immobilized microbial inoculum.
5. by the described preparation method who repairs the immobilized microbial inoculum of PAHs contaminated soil of claim 4, it is characterized in that: described bacterial classification needs to concentrate cultivation in the enrichment culture that contains polycyclic aromatic hydrocarbons, and it is: with Gordon Salmonella, the joint bacterium, Alcaligenes, pale bacillus, pseudomonas, bacillus and mycobacterium are counted the bacterial classification inoculation of 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 1-3: 2-4: 1-3 by weight in the enrichment medium that contains the PAHs pollutent of 30-100ml, then throw in the PAHs contaminated soil of enrichment medium weight percent 10-15%, 28～30 ℃ of temperature, cultivate a week in 120～130rpm/min constant temperature shaking table, stand-by;
The bacterium liquid of above-mentioned cultivation is inoculated in the minimal medium for the inoculum size of 10-15% by weight, 28～30 ℃ of temperature, cultivate 5-7d in 120～130rpm/min constant temperature shaking table, the microorganism mixed bacteria liquid that obtains is primary seed solution logarithmic phase bacterium liquid.
6. by the described preparation method who repairs the immobilized microbial inoculum of PAHs contaminated soil of claim 4, it is characterized in that: described enrichment medium is counted by weight, 0.1-0.2% glucose, 0.01-0.5%NaCl, 0.05-0.1% peptone, 0.1-1.0%NH
4NO
3, 0.05-0.1% yeast extract paste, 0.1-0.5%K
2HPO
4, 0.01-0.05%MgSO
4.7H
2O, surplus is a sterilized water; PH7.0-7.5; Simultaneously add PAHs in enrichment medium, making its total amount concentration is 100-200mg/L.
7. by the described preparation method who repairs the immobilized microbial inoculum of PAHs contaminated soil of claim 4, it is characterized in that: described minimal medium is counted by weight: 0.01-0.5%NaCl, 0.1-1.0%NH
4NO
3, 0.5-0.1%K
2HPO
4, 0.01-0.05%MgSO
4.7H
2O, surplus are sterilized water; PH7.0-7.5.
8. by the described preparation method who repairs the immobilized microbial inoculum of PAHs contaminated soil of claim 4, it is characterized in that: during described spawn culture with logarithmic phase, is that the inoculum size of 10-15% is added to and adsorbs fixedly 3-4d in the solid medium with the bacterial classification of logarithmic phase according to weight percent, keep water content 55%-65%, for breeding once; Be solid spawn then, in the same way, breed 2-3 time again, i.e. being fixed microbial inoculum with propagation product once.
9. by the described preparation method who repairs the immobilized microbial inoculum of PAHs contaminated soil of claim 8, it is characterized in that: described solid medium by weight percentage, the soybean cake powder of the rice chaff of 55-65%, the corn cob of 11-16%, 1.0-2.0%, the K of 0.06-0.1%
2HPO
4, the wheat bran of 20-25%, the MgSO of 0.025-0.05%
4.7H
2The sucrose of O, 0.8-1.2%, surplus are moisture; PH7.0-7.5.
10. by the described preparation method who repairs the immobilized microbial inoculum of PAHs contaminated soil of claim 9, it is characterized in that: described solid increment substratum is mixed by the multiple kinds of crops byproduct, various materials are crushed to particle size diameter at 0.1-0.5cm, and use the Ca (OH) of 1.2-2% before use earlier
2Water logging bubble sterilization 24h.
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| CN102583772A (en) * | 2011-01-14 | 2012-07-18 | Bm有限公司 | Microbial preparation comprising mixed microorganisms (bm-s-1), and a biological treatment method for rivers and lakes and a sludge autodigestion process using the same |
| CN102583771A (en) * | 2011-01-14 | 2012-07-18 | Bm有限公司 | Biological treatment method and waste-water treatment agent for refractory wastewater |
| CN102776135A (en) * | 2011-05-12 | 2012-11-14 | 中国科学院沈阳应用生态研究所 | Mixed inoculant for degrading polycyclic aromatic hydrocarbon pollutants |
| CN103920706A (en) * | 2014-04-16 | 2014-07-16 | 内蒙古科技大学 | Method for repairing rare earth polluted soil by immobilized mixed fungicide |
| CN104028550A (en) * | 2014-07-10 | 2014-09-10 | 内蒙古科技大学 | Method for repairing rare earth pollution by using coupling between suaeda glauca and immobilized microbial agent |
| TWI473765B (en) * | 2011-08-09 | 2015-02-21 | Omya Int Ag | Surface-treated calcium carbonate for binding and bioremediating hydrocarbon-containing compositions |
| CN104475444A (en) * | 2014-11-04 | 2015-04-01 | 上海交通大学 | Preparation, application and usage method for biocarbon-immobilized composite pollution-degrading bacterium particle |
| CN104646411A (en) * | 2013-11-20 | 2015-05-27 | 沈阳工业大学 | Carbofuran contaminated soil repair method of immobilized bacillus |
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| WO2015191582A1 (en) * | 2014-06-09 | 2015-12-17 | BiOWiSH Technologies, Inc. | Microbial compositions for hydrocarbon remediation and methods of use thereof |
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| CN106670226A (en) * | 2016-12-09 | 2017-05-17 | 天长市天龙泵阀成套设备厂 | Biological remediation method for organic pollution of soil |
| CN108114976A (en) * | 2017-12-19 | 2018-06-05 | 轻工业环境保护研究所 | A kind of method of repairing polluted soil |
| CN108504591A (en) * | 2018-03-23 | 2018-09-07 | 辽宁大学 | The composite bacteria agent of polycyclic aromatic hydrocarbon pollutant and its application in a kind of degradation freeze-thaw soils |
| CN108559721A (en) * | 2018-05-15 | 2018-09-21 | 北京师范大学 | It is a kind of purification air complex micro organism fungicide and its application |
| CN108624530A (en) * | 2018-05-15 | 2018-10-09 | 中国环境科学研究院 | The immobilized microbial inoculum and preparation method of a kind of in-situ immobilization contaminated sediment remediation and application |
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| CN117243100A (en) * | 2023-10-17 | 2023-12-19 | 东北农业大学 | Irrigation mode optimization method for promoting degradation of polycyclic aromatic hydrocarbon in paddy field bottom mud of rice-crab co-cropping-microorganism coupling system |
| WO2024051268A1 (en) * | 2022-09-05 | 2024-03-14 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Polycyclic aromatic hydrocarbons contaminated soil remediation agent based on mechanism of plant-microbe combined action and preparation method therefor and use thereof |
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2008
- 2008-10-29 CN CN 200810228376 patent/CN101724582A/en active Pending
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| CN102583771A (en) * | 2011-01-14 | 2012-07-18 | Bm有限公司 | Biological treatment method and waste-water treatment agent for refractory wastewater |
| CN102583772B (en) * | 2011-01-14 | 2014-07-09 | Bm有限公司 | Microbial preparation comprising mixed microorganisms (bm-s-1), and a biological treatment method for rivers and lakes and a sludge autodigestion process using the same |
| CN102583771B (en) * | 2011-01-14 | 2014-07-09 | Bm有限公司 | Biological treatment method and waste-water treatment agent for refractory wastewater |
| CN102583772A (en) * | 2011-01-14 | 2012-07-18 | Bm有限公司 | Microbial preparation comprising mixed microorganisms (bm-s-1), and a biological treatment method for rivers and lakes and a sludge autodigestion process using the same |
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| CN108504591A (en) * | 2018-03-23 | 2018-09-07 | 辽宁大学 | The composite bacteria agent of polycyclic aromatic hydrocarbon pollutant and its application in a kind of degradation freeze-thaw soils |
| CN108504591B (en) * | 2018-03-23 | 2021-06-25 | 辽宁大学 | Complex microbial inoculant for degrading polycyclic aromatic hydrocarbon pollutants in freeze-thaw soil and application thereof |
| CN108559721A (en) * | 2018-05-15 | 2018-09-21 | 北京师范大学 | It is a kind of purification air complex micro organism fungicide and its application |
| CN108624530A (en) * | 2018-05-15 | 2018-10-09 | 中国环境科学研究院 | The immobilized microbial inoculum and preparation method of a kind of in-situ immobilization contaminated sediment remediation and application |
| CN113458140A (en) * | 2021-06-02 | 2021-10-01 | 广东新泓环境咨询有限公司 | Polycyclic aromatic hydrocarbon soil pollution remediation method and application |
| CN114602967A (en) * | 2022-03-04 | 2022-06-10 | 南京农业大学 | Method for removing USEPA PAHs in soil by using functional indigenous flora solid microbial inoculum |
| CN114602967B (en) * | 2022-03-04 | 2022-12-23 | 南京农业大学 | Method for removing USEPA PAHs in soil by using functional indigenous flora solid microbial inoculum |
| WO2024051268A1 (en) * | 2022-09-05 | 2024-03-14 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Polycyclic aromatic hydrocarbons contaminated soil remediation agent based on mechanism of plant-microbe combined action and preparation method therefor and use thereof |
| CN117243100A (en) * | 2023-10-17 | 2023-12-19 | 东北农业大学 | Irrigation mode optimization method for promoting degradation of polycyclic aromatic hydrocarbon in paddy field bottom mud of rice-crab co-cropping-microorganism coupling system |
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