CN101698537A - In-situ remediation method using peat in underground water polluted by petroleum hydrocarbon as an additive medium - Google Patents
In-situ remediation method using peat in underground water polluted by petroleum hydrocarbon as an additive medium Download PDFInfo
- Publication number
- CN101698537A CN101698537A CN200910217811A CN200910217811A CN101698537A CN 101698537 A CN101698537 A CN 101698537A CN 200910217811 A CN200910217811 A CN 200910217811A CN 200910217811 A CN200910217811 A CN 200910217811A CN 101698537 A CN101698537 A CN 101698537A
- Authority
- CN
- China
- Prior art keywords
- peat
- reaction
- medium
- underground water
- bacteria suspension
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- 239000003415 peat Substances 0.000 title claims abstract description 68
- 239000003209 petroleum derivative Substances 0.000 title claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances data:image/svg+xml;base64,PD94bWwgdmVyc2lvbj0nMS4wJyBlbmNvZGluZz0naXNvLTg4NTktMSc/Pgo8c3ZnIHZlcnNpb249JzEuMScgYmFzZVByb2ZpbGU9J2Z1bGwnCiAgICAgICAgICAgICAgeG1sbnM9J2h0dHA6Ly93d3cudzMub3JnLzIwMDAvc3ZnJwogICAgICAgICAgICAgICAgICAgICAgeG1sbnM6cmRraXQ9J2h0dHA6Ly93d3cucmRraXQub3JnL3htbCcKICAgICAgICAgICAgICAgICAgICAgIHhtbG5zOnhsaW5rPSdodHRwOi8vd3d3LnczLm9yZy8xOTk5L3hsaW5rJwogICAgICAgICAgICAgICAgICB4bWw6c3BhY2U9J3ByZXNlcnZlJwp3aWR0aD0nMzAwcHgnIGhlaWdodD0nMzAwcHgnIHZpZXdCb3g9JzAgMCAzMDAgMzAwJz4KPCEtLSBFTkQgT0YgSEVBREVSIC0tPgo8cmVjdCBzdHlsZT0nb3BhY2l0eToxLjA7ZmlsbDojRkZGRkZGO3N0cm9rZTpub25lJyB3aWR0aD0nMzAwLjAnIGhlaWdodD0nMzAwLjAnIHg9JzAuMCcgeT0nMC4wJz4gPC9yZWN0Pgo8dGV4dCB4PScxMDAuNScgeT0nMTcwLjAnIGNsYXNzPSdhdG9tLTAnIHN0eWxlPSdmb250LXNpemU6NDBweDtmb250LXN0eWxlOm5vcm1hbDtmb250LXdlaWdodDpub3JtYWw7ZmlsbC1vcGFjaXR5OjE7c3Ryb2tlOm5vbmU7Zm9udC1mYW1pbHk6c2Fucy1zZXJpZjt0ZXh0LWFuY2hvcjpzdGFydDtmaWxsOiNFODQyMzUnID5IPC90ZXh0Pgo8dGV4dCB4PScxMjYuMScgeT0nMTg2LjAnIGNsYXNzPSdhdG9tLTAnIHN0eWxlPSdmb250LXNpemU6MjZweDtmb250LXN0eWxlOm5vcm1hbDtmb250LXdlaWdodDpub3JtYWw7ZmlsbC1vcGFjaXR5OjE7c3Ryb2tlOm5vbmU7Zm9udC1mYW1pbHk6c2Fucy1zZXJpZjt0ZXh0LWFuY2hvcjpzdGFydDtmaWxsOiNFODQyMzUnID4yPC90ZXh0Pgo8dGV4dCB4PScxMzguMCcgeT0nMTcwLjAnIGNsYXNzPSdhdG9tLTAnIHN0eWxlPSdmb250LXNpemU6NDBweDtmb250LXN0eWxlOm5vcm1hbDtmb250LXdlaWdodDpub3JtYWw7ZmlsbC1vcGFjaXR5OjE7c3Ryb2tlOm5vbmU7Zm9udC1mYW1pbHk6c2Fucy1zZXJpZjt0ZXh0LWFuY2hvcjpzdGFydDtmaWxsOiNFODQyMzUnID5PPC90ZXh0Pgo8L3N2Zz4K data:image/svg+xml;base64,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 O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 23
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 5
- 238000005067 remediation Methods 0.000 title claims abstract description 5
- 239000000654 additive Substances 0.000 title abstract 3
- 230000000996 additive Effects 0.000 title abstract 3
- 241000894006 Bacteria Species 0.000 claims abstract description 52
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 239000004576 sand Substances 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 238000011049 filling Methods 0.000 claims abstract description 6
- 239000000725 suspension Substances 0.000 claims description 40
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 33
- 230000001580 bacterial Effects 0.000 claims description 21
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound 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[C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 15
- 229910052799 carbon Inorganic materials 0.000 claims description 15
- 238000004321 preservation Methods 0.000 claims description 15
- HNBDQABBWNOTRU-UHFFFAOYSA-N thalline Chemical compound 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CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 230000037250 Clearance Effects 0.000 description 1
- 230000036740 Metabolism Effects 0.000 description 1
- YNPNZTXNASCQKK-UHFFFAOYSA-N Phenanthrene Chemical compound 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- 238000009412 basement excavation Methods 0.000 description 1
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- YNQLUTRBYVCPMQ-UHFFFAOYSA-N ethylbenzene Chemical compound 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- CTQNGGLPUBDAKN-UHFFFAOYSA-N o-xylene Chemical compound 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- 231100000719 pollutant Toxicity 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000005201 scrubbing Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
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Abstract
The invention relates to an in-situ remediation method using peat in underground water polluted by petroleum hydrocarbon as an additive medium, which comprises: domesticating a strain; separating the strain; preprocessing peat; performing amplification culture of functional bacteria; immobilizing the functional bacteria receiving the amplification culture in the wet peat which is heated; uniformly mixing the peat carrying the functional bacteria and coarse sand in a certain volume ratio to prepare a reaction wall supporting medium; and filling the reaction wall supporting medium into a permeable reaction wall. The method has the advantages that: the peat used as a biological reaction wall additive medium can effectively immobilize functional bacteria, are insusceptible to flow away with underground water so as to be lost and can effectively absorb petroleum hydrocarbons, benzenes, toluenes, ethylbenzenes, dimethylbenzenes and naphthalines in the underground water, wherein the immobilization rate of the peat is 87 percent according to measurement in experiment and the absorption effect of the peat on polycyclic aromatic hydrocarbons is particularly obvious; nutrients are effectively fed to the functional bacteria in the reaction wall so as to ensure points in the reaction wall can keep high biomass in an operation period; and the peat used is cheap, widely and readily available and ecologically safe.
Description
Technical field:
The present invention relates to the recovery technique in a kind of groundwater pollution place, especially the Supporting Media and the permeable reactive barrier of the reparation of underground water polluted by petroleum hydrocarbon original position.
Background technology:
In view of the seriousness of underground water polluted by petroleum hydrocarbon, the various countries scholar has extensively carried out the control and the recovery technology research of underground water polluted by petroleum hydrocarbon.Wherein the permeable reactive barrier recovery technique (Permeable Reactive Barrier, PRB) be many developed countries such as present America and Europe emerging be used for the method that original position is removed the underground water pollution components.
Environmental Protection Agency (USEPA) is defined as PRB: pollute pinnule at underground arrangement active material body of wall so that tackle, make and pollute pinnule by behind the reaction medium, its pollutent can be converted into the another kind of form that environment is accepted, thereby realizes that Pollutant levels reach the target of environmental standard.Reaction medium can be removed underground water pollutant by absorption, precipitation, chemical degradation and biological degradation.Aspect the underground water polluted by petroleum hydrocarbon reparation, mainly contain absorption reaction wall and biological respinse wall.
The absorption reaction wall mainly is to add medium such as gac in the PRB body of wall to utilize its physical adsorbability to remove petroleum hydrocarbon contaminated matter in underground water, but adsorption medium usually costs an arm and a leg, and needs periodic replacement, and this has increased the running cost of system greatly.
The biological respinse wall is to add the concrete particle that contains oxygen-releasing compound (OCR) and be total to metabolic substd in the PRB body of wall; OCR is that solid-state superoxide is (as CaO
2, MgO
2Deng), for microorganism provides oxygen source and electron acceptor(EA), make indigenous bacterium produce aerobic biodegradation to petroleum hydrocarbon, the common metabolism that common metabolic substd can be petroleum hydrocarbon provides matrix.But vying each other of may existing between low temperature, oligotrophic and bacterial strain in the actual repair process because in the groundwater environment makes that PRB biological restoration efficient may be lower, simultaneously the avidity of petroleum hydrocarbon original inhabitants degradation bacteria and Supporting Media easily runs off it a little less than along with phreatic flowing, and biological restoration efficient also is subject to external environment in addition influences.
Therefore, how to select a kind of body of wall Supporting Media, make it have relatively cheap, easy acquisition, environmental friendliness, suitable petroleum hydrocarbon contaminant absorption, also can be used as the microbial immobilization carrier and suitable nutrition source is provided, is to solve the reaction wall technology to be applied to the key issue that underground water polluted by petroleum hydrocarbon is repaired bottleneck.
According to above standard, we have done a large amount of tests and test, the interpolation medium of finally selecting peat to repair as underground water polluted by petroleum hydrocarbon reaction in wall.
Peat is the composite soil material that contains different organism resolutions, is a kind of 95% left and right sides porosity and 200m that have
2The porous material of/g specific surface area, and have low price, use convenience, characteristics such as ecological safety, scrubbing power are strong.Contain many biopolymers in the peat, for example: lignin, the soil ulmin of cellulose family and certain mass, these materials contain many active centre and polar functionalities group, be considered to that the non-ionic type organic compound is had high avidity and loading capacity, the biological degradation that the hydrolysate of part component also can be hydrocarbon polymer provides the composite nutrient source, studies show that the humic acid material that contains in the peat has very big loading capacity to petroleum hydrocarbon, and it has low fibre content and high lignin thermolysis material and ash content, make peat can active adsorption over-saturation attitude and solubilised state petroleum hydrocarbon in the underground water, clearance can reach 63-97%.In addition, on the peat medium, not only have higher immobilization rate through originally experimental results show that microorganism, and can keep high biological activity for a long time.External existing peat is applied to example and the experience that the underground water original position is repaired: and peat can be realized by tradition excavation and stemming operation technology when underground repair system is installed.
Summary of the invention:
Purpose of the present invention is exactly at above-mentioned the deficiencies in the prior art, provide a kind of be fit to very much to be used as the underground water polluted by petroleum hydrocarbon reparation with peat as adding the in-situ remediation method that medium is used for underground water polluted by petroleum hydrocarbon.
The objective of the invention is to be achieved through the following technical solutions:
Strain domestication at first, strain separating, pre-treatment peat, next is the function yeast enlarged culturing, the function yeast of enlarged culturing is solidificated on after heat treatment the wet shape peat, again the peat of loading functional bacterium and coarse sand are prepared into reaction body of wall Supporting Media according to certain volume ratio uniform mixing, will react the body of wall Supporting Media then and pack in the permeable reaction wall;
Comprise following order and step:
A, the contaminated site pedotheque is suspended with sterilized water, magnetic force high-speed stirring 30min, use eight layers of filtered through gauze foreign material, rubble then, static 1h, get the 50mL suspension liquid and be connected in the 200mL enrichment medium, 10 ℃ of low temperature, 120rpm cultivated 15 days, get an amount of bacteria suspension then and be connected to again in the fresh enrichment medium, 10 ℃ of low-temperature adaptations; Continuous passage then 5 times, each 9 days, reduce the auxiliary carbon source consumption when going down to posterity gradually at every turn, be sole carbon source until petroleum hydrocarbon;
B, above-mentioned indigenous bacteria suspension is connected in the minimal medium that diesel oil is sole carbon source, continue 10 ℃ of low-temperature adaptations, enrichment, this stage periodic monitor thalline increment and diesel component degraded situation, according to monitoring result, progressively shorten generation time, accelerate the speed that goes down to posterity, till the shortening degradation time is stablized, can not be remained valid to the indigenous flora degradation function;
The separation of c, bacterial classification: the indigenous mixed bacterial suspension that seed selection is arrived, diluting different gradients coats on the inorganic salt solid plate, cultivated 15 days down at 10 ℃, single colony inoculation of growing is rule to the common solid plate is separated to pure bacterial strain, the preservation of inoculation slant preservation substratum; Bacterial classification is through being accredited as: Ye Shi yeast (Yarrowia sp), rhodococcus (Rhodococcus sp.), acinetobacter calcoaceticus (Acinetobacter sp), pseudomonas (Pseudomonas sp), new Sphingobacterium (Novosphingobium sp.);
D, pre-treatment peat, with peat through dry in the shade, broken, sieve after 140 ℃ of thermal treatment 3h, the screening particle diameter is to soak 24 hours with ultrapure water behind the 0.25-1mm, is prepared into wet shape peat;
E, get the pre-waterbearing stratum sand of repairing ground, dry, sieve, it is standby to get particle diameter 0.5-2mm coarse sand;
F, with the isolated five kinds of function yeast enlarged culturing of step c, the seed liquor after activating is seeded in the 300mL minimal medium that contains 400mg/L diesel oil with 10% inoculum size, connect altogether four parallel, 10 ℃, 120rpm shakes cultivation, cultivates 60 hours to logarithmic phase, and the bacterium number is about 10
11Individual/mL, then with the centrifugal 15min of bacteria suspension 8000rpm, with 20mL phosphate buffered saline buffer flushing thalline, again with the centrifugal 15min of bacteria suspension 8000rpm, triplicate, the collection thalline is prepared into bacteria suspension, and (the bacterium number is about 10
10Individual/mL) stand-by;
Wet shape peat is got in the immobilization on peat of g, function yeast, in the Glass Containers of packing into, adds the bacteria suspension of step f preparation, cultivates 48 hours in 10-12 ℃, and five kinds of function yeast of enlarged culturing are immobilized on the peat;
H, the preparation of reaction body of wall Supporting Media are that 1:4 carries out uniform mixing with peat and the coarse sand of immobilization function yeast according to volume ratio, are prepared into and react the body of wall Supporting Media;
The making of i, permeable reactive barrier is packed in the wide groove of 30-35cm being prepared into the layering of reaction body of wall Supporting Media, and every filling one deck all will be paved, and evenly compacting is permeable reactive barrier.
Beneficial effect: peat has higher immobilization rate to microorganism, records the immobilization rate in the experiment and is about 87%; When peat is used as absorption reaction wall interpolation medium, but the petroleum hydrocarbon in the active adsorption underground water is a thing as benzene, toluene, ethylbenzene, dimethylbenzene, naphthalene series substance, phenanthrene, and is wherein particularly evident to the adsorption effect of polycyclic aromatic hydrocarbons; When peat is used as biological respinse wall interpolation medium, effective fixed function bacterium, make it be difficult for liquid food with Groundwater Flow, and can be effectively in the reaction body of wall function yeast supply with nutrition, make that each point can keep higher biomass in the reaction body of wall in the operating period; The low price of the used interpolation medium of the present invention peat, wide material sources obtain and ecological safety easily.
The bacterium classification name:
Classification name: Ye Shi yeast
Latin literary fame: Yarrowia sp
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center
Address: Datun Road, Chaoyang District, Beijing City
Preservation date: on October 12nd, 2009
Deposit number: 3318
Classification name: rhodococcus
Latin literary fame: Rhodococcus sp
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center
Address: Datun Road, Chaoyang District, Beijing City
Preservation date: on October 12nd, 2009
Deposit number: 3322
Classification name: acinetobacter calcoaceticus
Latin literary fame: Acinetobacter sp
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center
Address: Datun Road, Chaoyang District, Beijing City
Preservation date: on October 12nd, 2009
Deposit number: 3323
Classification name: pseudomonas
Latin literary fame: Pseudomonas sp
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center
Address: Datun Road, Chaoyang District, Beijing City
Preservation date: on October 12nd, 2009
Deposit number: 3324
Classification name: new Sphingobacterium
Latin literary fame: Novosphingobium sp.
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center
Address: Datun Road, Chaoyang District, Beijing City
Preservation date: on October 12nd, 2009
Deposit number: 3326
Embodiment:
The present invention is described in further detail below in conjunction with embodiment:
Comprise following order and step: strain domestication, strain separating, the peat pre-treatment, the function yeast enlarged culturing, the function yeast of enlarged culturing is solidificated on after heat treatment the wet shape peat, again the peat of loading functional bacterium and coarse sand are prepared into reaction body of wall Supporting Media according to certain volume ratio uniform mixing, will react the body of wall Supporting Media then and pack in the permeable reaction wall.
Embodiment 1
A, the contaminated site pedotheque is suspended with sterilized water, magnetic force high-speed stirring 30min, use eight layers of filtered through gauze foreign material, rubble then, static 1h, get the 50mL suspension liquid and be connected in the 200mL enrichment medium, 10 ℃ of low temperature, 120rpm cultivated 15 days, get an amount of bacteria suspension then and be connected to again in the fresh enrichment medium, 10 ℃ of low-temperature adaptations; Continuous passage then 5 times, each 9 days, reduce the auxiliary carbon source consumption when going down to posterity gradually at every turn, be sole carbon source until petroleum hydrocarbon;
B, above-mentioned indigenous bacteria suspension is connected in the minimal medium that diesel oil is sole carbon source, continue 10 ℃ of low-temperature adaptations, enrichment, this stage periodic monitor thalline increment and diesel component degraded situation, according to monitoring result, progressively shorten generation time, accelerate the speed that goes down to posterity, till the shortening degradation time is stablized, can not be remained valid to the indigenous flora degradation function;
The separation of c, bacterial classification: the indigenous mixed bacterial suspension that seed selection is arrived, diluting different gradients coats on the inorganic salt solid plate, cultivated 15 days down at 10 ℃, single colony inoculation of growing is rule to the common solid plate is separated to pure bacterial strain, the preservation of inoculation slant preservation substratum; Bacterial classification is through being accredited as: Ye Shi yeast (Yarrowia sp), rhodococcus (Rhodococcus sp.), acinetobacter calcoaceticus (Acinetobacter sp), pseudomonas (Pseudomonas sp), new Sphingobacterium (Novosphingobium sp.);
D, pre-treatment peat, with peat through dry in the shade, broken, sieve after 140 ℃ of thermal treatment 3h, the screening particle diameter is to soak 24 hours with ultrapure water behind the 0.25-1mm, is prepared into wet shape peat;
E, get the pre-waterbearing stratum sand of repairing ground, dry, sieve, get particle diameter 0.5mm coarse sand;
F, with the isolated five kinds of function yeast enlarged culturing of step c, the seed liquor after activating is seeded in the 300mL minimal medium that contains 400mg/L diesel oil with 10% inoculum size, connect altogether four parallel, 10 ℃, 120rpm shakes cultivation, cultivates 60 hours to logarithmic phase, and the bacterium number is about 10
11Individual/mL, then with the centrifugal 15min of bacteria suspension 8000rpm, with 20mL phosphate buffered saline buffer flushing thalline, again with the centrifugal 15min of bacteria suspension 8000rpm, triplicate, the collection thalline is prepared into bacteria suspension, and (the bacterium number is about 10
10Individual/mL);
Wet shape peat slurry is got in the immobilization on peat of g, function yeast, in the Glass Containers of packing into, adds the bacteria suspension of step f preparation, cultivates 48 hours in 10 ℃, and five kinds of function yeast of enlarged culturing are immobilized on the peat;
H, the preparation of reaction body of wall Supporting Media are to carry out uniform mixing at 1: 4 with the peat of immobilization function yeast and coarse sand according to volume ratio, are prepared into and react the body of wall Supporting Media;
The making of i, permeable reactive barrier is packed in the wide groove of 30cm being prepared into the layering of reaction body of wall Supporting Media, and every filling one deck all will be paved, and evenly compacting is permeable reactive barrier.
Embodiment 2
A, the contaminated site pedotheque is suspended with sterilized water, magnetic force high-speed stirring 30min, use eight layers of filtered through gauze foreign material, rubble then, static 1h, get the 50mL suspension liquid and be connected in the 200mL enrichment medium, 10 ℃ of low temperature, 120rpm cultivated 15 days, get an amount of bacteria suspension then and be connected to again in the fresh enrichment medium, 10 ℃ of low-temperature adaptations; Continuous passage then 5 times, each 9 days, reduce the auxiliary carbon source consumption when going down to posterity gradually at every turn, be sole carbon source until petroleum hydrocarbon;
B, above-mentioned indigenous bacteria suspension is connected in the minimal medium that diesel oil is sole carbon source, continue 10 ℃ of low-temperature adaptations, enrichment, this stage periodic monitor thalline increment and diesel component degraded situation, according to monitoring result, progressively shorten generation time, accelerate the speed that goes down to posterity, till the shortening degradation time is stablized, can not be remained valid to the indigenous flora degradation function;
The separation of c, bacterial classification: the indigenous mixed bacterial suspension that seed selection is arrived, diluting different gradients coats on the inorganic salt solid plate, cultivated 15 days down at 10 ℃, single colony inoculation of growing is rule to the common solid plate is separated to pure bacterial strain, the preservation of inoculation slant preservation substratum; Bacterial classification is through being accredited as: Ye Shi yeast (Yarrowia sp), rhodococcus (Rhodococcus sp.), acinetobacter calcoaceticus (Acinetobacter sp), pseudomonas (Pseudomonas sp), new Sphingobacterium (Novosphingobium sp.);
D, pre-treatment peat, with peat through dry in the shade, broken, sieve after 140 ℃ of thermal treatment 3h, the screening particle diameter is to soak 24 hours with ultrapure water behind the 0.25-1mm, is prepared into wet shape peat;
E, get the pre-waterbearing stratum sand of repairing ground, dry, sieve, it is standby to get particle diameter 1mm coarse sand;
F, with the isolated five kinds of function yeast enlarged culturing of step c, the seed liquor after activating is seeded in the 300mL minimal medium that contains 400mg/L diesel oil with 10% inoculum size, connect altogether four parallel, 10 ℃, 120rpm shakes cultivation, cultivates 60 hours to logarithmic phase, and the bacterium number is about 10
11Individual/mL, then with the centrifugal 15min of bacteria suspension 8000rpm, with 20mL phosphate buffered saline buffer flushing thalline, again with the centrifugal 15min of bacteria suspension 8000rpm, triplicate, the collection thalline is prepared into bacteria suspension, and (the bacterium number is about 10
10Individual/mL) stand-by;
Wet shape peat is got in the immobilization on peat of g, function yeast, in the Glass Containers of packing into, adds the bacteria suspension of step f preparation, cultivates 48 hours in 11 ℃, and five kinds of function yeast of enlarged culturing are immobilized on the peat;
H, the preparation of reaction body of wall Supporting Media are to carry out uniform mixing at 1: 4 with the peat of immobilization function yeast and coarse sand according to volume ratio, are prepared into and react the body of wall Supporting Media;
The making of i, permeable reactive barrier is packed in the wide groove of 33cm being prepared into the layering of reaction body of wall Supporting Media, and every filling one deck all will be paved, and evenly compacting is permeable reactive barrier.
Embodiment 3
A, the contaminated site pedotheque is suspended with sterilized water, magnetic force high-speed stirring 30min, use eight layers of filtered through gauze foreign material, rubble then, static 1h, get the 50mL suspension liquid and be connected in the 200mL enrichment medium, 10 ℃ of low temperature, 120rpm cultivated 15 days, get an amount of bacteria suspension then and be connected to again in the fresh enrichment medium, 10 ℃ of low-temperature adaptations; Continuous passage then 5 times, each 9 days, reduce the auxiliary carbon source consumption when going down to posterity gradually at every turn, be sole carbon source until petroleum hydrocarbon;
B, above-mentioned indigenous bacteria suspension is connected in the minimal medium that diesel oil is sole carbon source, continue 10 ℃ of low-temperature adaptations, enrichment, this stage periodic monitor thalline increment and diesel component degraded situation, according to monitoring result, progressively shorten generation time, accelerate the speed that goes down to posterity, till the shortening degradation time is stablized, can not be remained valid to the indigenous flora degradation function;
The separation of c, bacterial classification: the indigenous mixed bacterial suspension that seed selection is arrived, diluting different gradients coats on the inorganic salt solid plate, cultivated 15 days down at 10 ℃, single colony inoculation of growing is rule to the common solid plate is separated to pure bacterial strain, the preservation of inoculation slant preservation substratum; Bacterial classification is through being accredited as: Ye Shi yeast (Yarrowia sp), rhodococcus (Rhodococcus sp.), acinetobacter calcoaceticus (Acinetobacter sp), pseudomonas (Pseudomonas sp), new Sphingobacterium (Novosphingobium sp.);
D, pre-treatment peat, with peat through dry in the shade, broken, sieve after 140 ℃ of thermal treatment 3h, the screening particle diameter is to soak 24 hours with ultrapure water behind the 0.25-1mm, is prepared into wet shape peat;
E, get the pre-waterbearing stratum sand of repairing ground, dry, sieve, it is standby to get particle diameter 2mm coarse sand;
F, with the isolated five kinds of function yeast enlarged culturing of step c, the seed liquor after activating is seeded in the 300mL minimal medium that contains 400mg/L diesel oil with 10% inoculum size, connect altogether four parallel, 10 ℃, 120rpm shakes cultivation, cultivates 60 hours to logarithmic phase, and the bacterium number is about 10
11Individual/mL, then with the centrifugal 15min of bacteria suspension 8000rpm, with 20mL phosphate buffered saline buffer flushing thalline, again with the centrifugal 15min of bacteria suspension 8000rpm, triplicate, the collection thalline is prepared into bacteria suspension, and (the bacterium number is about 10
10Individual/mL) stand-by;
Wet shape peat is got in the immobilization on peat of g, function yeast, in the Glass Containers of packing into, adds the bacteria suspension of step f preparation, cultivates 48 hours in 12 ℃, and five kinds of function yeast of enlarged culturing are immobilized on the peat;
H, the preparation of reaction body of wall Supporting Media are to carry out uniform mixing at 1: 4 with the peat of immobilization function yeast and coarse sand according to volume ratio, are prepared into and react the body of wall Supporting Media;
The making of i, permeable reactive barrier is packed in the wide groove of 35cm being prepared into the layering of reaction body of wall Supporting Media, and every filling one deck all will be paved, and evenly compacting is permeable reactive barrier.
Claims (1)
- One kind with peat as adding the in-situ remediation method that medium is used for underground water polluted by petroleum hydrocarbon, it is characterized in that, pre-treatment peat at first, next is the function yeast enlarged culturing, the function yeast of enlarged culturing is solidificated on after heat treatment the wet shape peat, again the peat of loading functional bacterium and coarse sand are prepared into reaction body of wall Supporting Media according to certain volume ratio uniform mixing, will react the body of wall Supporting Media then and pack in the permeable reaction wall, comprise following order and step:A, the contaminated site pedotheque is suspended with sterilized water, magnetic force high-speed stirring 30min, use eight layers of filtered through gauze foreign material, rubble then, static 1h, get the 50mL suspension liquid and be connected in the 200mL enrichment medium, 10 ℃ of low temperature, 120rpm cultivated 15 days, get an amount of bacteria suspension then and be connected to again in the fresh enrichment medium, 10 ℃ of low-temperature adaptations; Continuous passage then 5 times, each 9 days, reduce the auxiliary carbon source consumption when going down to posterity gradually at every turn, be sole carbon source until petroleum hydrocarbon;B, above-mentioned indigenous bacteria suspension is connected in the minimal medium that diesel oil is sole carbon source, continue 10 ℃ of low-temperature adaptations, enrichment, this stage periodic monitor thalline increment and diesel component degraded situation, according to monitoring result, progressively shorten generation time, accelerate the speed that goes down to posterity, till the shortening degradation time is stablized, can not be remained valid to the indigenous flora degradation function;The separation of c, bacterial classification: the indigenous mixed bacterial suspension that seed selection is arrived, diluting different gradients coats on the inorganic salt solid plate, cultivated 15 days down at 10 ℃, single colony inoculation of growing is rule to the common solid plate is separated to pure bacterial strain, the preservation of inoculation slant preservation substratum; Bacterial classification is through being accredited as: Ye Shi yeast (Yarrowia sp), rhodococcus (Rhodococcus sp.), acinetobacter calcoaceticus (Acinetobacter sp), pseudomonas (Pseudomonas sp), new Sphingobacterium (Novosphingobium sp.);D, pre-treatment peat, with peat through dry in the shade, broken, sieve after 140 ℃ of thermal treatment 3h, the screening particle diameter is to soak 24 hours with ultrapure water behind the 0.25-1mm, is prepared into wet shape peat;E, get the pre-waterbearing stratum sand of repairing ground, dry, sieve, it is standby to get particle diameter 0.5-2mm coarse sand;F, with the isolated five kinds of function yeast enlarged culturing of step c, the seed liquor after activating is seeded in the 300mL minimal medium that contains 400mg/L diesel oil with 10% inoculum size, connect altogether four parallel, 10 ℃, 120rpm shakes cultivation, cultivates 60 hours to logarithmic phase, and the bacterium number is about 10 11Individual/mL, then with the centrifugal 15min of bacteria suspension 8000rpm, with 20mL phosphate buffered saline buffer flushing thalline, again with the centrifugal 15min of bacteria suspension 8000rpm, triplicate, the collection thalline is prepared into bacteria suspension, and (the bacterium number is about 10 10Individual/mL);Wet shape peat is got in the immobilization on peat of g, function yeast, in the Glass Containers of packing into, adds the bacteria suspension of step f preparation, cultivates 48 hours in 10-12 ℃, and five kinds of function yeast of enlarged culturing are immobilized on the peat;H, the preparation of reaction body of wall Supporting Media are to carry out uniform mixing at 1: 4 with the peat of immobilization function yeast and coarse sand according to volume ratio, are prepared into and react the body of wall Supporting Media;The making of i, permeable reactive barrier is packed in the wide groove of 30-35cm being prepared into the layering of reaction body of wall Supporting Media, and every filling one deck all will be paved, and evenly compacting is permeable reactive barrier.
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