CN102199430B - Immobilized microsphere for remedying calcium carbonate-containing petroleum polluted soil as well as preparation method and application - Google Patents
Immobilized microsphere for remedying calcium carbonate-containing petroleum polluted soil as well as preparation method and application Download PDFInfo
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Abstract
The invention relates to an immobilized microsphere for remedying calcium carbonate-containing petroleum polluted soil as well as a preparation method and application. The immobilized microsphere particles are spherical; air gaps are formed inside the immobilized microsphere particles; the diameter of the microspher is 2.5-3.0 mm; the immobilized microsphere particles comprise sodium alginate as an embedded material, solid calcium carbonate as a pore former and a sustained release agent as well as attapulgite as an adsorbent for adsorbing petroleum hydrocarbon degrading bacteria and nutritive elements. The preparation method of the immobilized microsphere comprises the steps of: with the sodium alginate as the embedded material, the attapulgite as the adsorbent and the calcium carbonate as the pore former and the sustained release agent, embedding degrading bacteria and nutrient components capable of degrading the petroleum hydrocarbon; carrying out high-concentration bacterial liquid suspension, immobilized microsphere formation and calcium carbonate removal, reproducing and re-culturing. The immobilized microsphere has the advantages of high nutrition, protecting performance, slow release performance, activity and stability, favorable mass transfer performance, convenience for use, and the like.
Description
Technical field
The present invention relates to a kind of immobilized microspheres for remediation of petroleum contaminated soil, its preparation method and application thereof, belong to microbial preparation field.
Background technology
Microorganism recovery technique is acknowledged as the most vital soil cleaning technique, having obtained extensive and long-range development aspect oil-polluted soils reparation, has obtained many breakthroughs and progress.Generally speaking, for microbial remedy technology for contaminated soil by petroleum, its core is the Degradation of efficient degrading bacteria to oil, and this is particularly important in repairing at the scene.But in practical application, soil physical chemistry environment more complicated due to petroleum pollution, adding the external source bacterium adding is free bacterium, make the composition of pollutent in degradation process trend towards more complicated, the toxicity of some secondary metabolites and middle degraded product is larger, and they are difficult to be degraded by microorganisms.The deficiency of free microorganism repair just displays gradually, as in unit volume effectively degradation bacteria concentration low, with indigenous bacterium competition in weak tendency, toxin immunity infringement ability etc.The introducing of immobilization technology can well overcome these drawbacks, not only not only can solve the weak problem of free external source bacterium competitive power, strengthens the free resistance to environmental impact ability of external source bacterium, and can greatly improve the repairing effect of pollutent.
At present, existing microbial preparation in the world, mainly take bacterium liquid and dry powder as main, and in its use procedure, be unfavorable for storage and transport, affected by soil ecosystem and cause the activity inducement phase long, there is larger error in remediation efficiency and laboratory level, therefore the strong microbiobacterial agent of active high, the resistance to Environment impact of exploitation seems particularly important.Immobilized microorganism technique (immobilized microorganism) is the biotechnology that the enzyme immobilization technology of 20 century 70s in biochemical industry grows up.It is to utilize the means of physics or chemistry that free cell or enzyme are positioned or are defined in specific area of space, makes it keep catalytic activity repeatedly used a kind of technology.Not only can increase substantially reaction microorganism concn, keep efficient bacterial classification, and can strengthen the resistance to Environment impact of microorganism.
In fact, at field of environment protection, immobilization technology is applied in the application of water treatment aspect relatively extensively and has obtained many phasic results.Take petroleum polluting water body reparation as example, Bao Mutai, the employing sodium alginates such as Gong Yuanjiao are done fixedly degradation bacteria processing petroleum pollution water sample of carrier, Zhang Hui, Li Peijun etc. adopt fixedly microbacterium processing oil pollution surface water of polyvinyl alcohol, and result all shows that immobilized microspheres has good degradation property to crude oil and is significantly higher than free bacterium.But aspect soil pollution reparation, immobilization technology is in the stage at the early-stage, development also sticks to laboratory level, as, the people such as Wang Xin ，Li Pei army adopt polyvinyl alcohol and sodium alginate phenanthrene and benzo [a] pyrene that fixedly foreign bacteria is degraded in soil, and result shows, it is feasible that immobilization introduction bacterium is applied to soil organic contamination reparation, and specific ionization bacterium has greater advantage.The nonuniformity of edatope adds that the complicacy of oil composition has determined that immobilization technology will overcome many problems and challenge in oil-polluted soils reparation field, as the not returnability of immobilization particle, the life-span of carrier, mass-transfer performance, degrade start time, preservation condition etc.
Summary of the invention
For the deficiency of existing microbial remedy technology for contaminated soil by petroleum and the problem of corresponding microorganism preparation technology of preparing slower development, the object of the present invention is to provide a kind of immobilized microspheres that can be used for oil-polluted soils reparation and preparation method thereof.The advantages such as this immobilized microspheres has nutritional type, protectiveness, slow-releasing, Activity and stabill is high, mass transfer performances good and easy to use.Preparation method is usingd sodium alginate as embedded material, attapulgite is as sorbent material, calcium carbonate is as pore former and sustained release dosage, degradation bacteria and the nutritive ingredient of embedding degradable petroleum hydrocarbon, through high-concentration bacterial liquid suspend, immobilization balling-up and the preparation process of cultivating again except calcium carbonate, propagation, being fixed microballoon.
The present invention relates to a kind of immobilized microspheres for remediation of petroleum contaminated soil, it is characterized in that: particle is spherical shape, there is space inside, its microsphere diameter is 2.5～3.0mm, described immobilized microspheres particle comprises sodium alginate as embedded material, solid carbonic acid calcium is as pore former and sustained release dosage, and attapulgite is as sorbent material, and its absorption has petroleum hydrocarbon degradation bacterium and nutritive element.
Wherein, the mass ratio of the sodium alginate in described immobilized microspheres, attapulgite and calcium carbonate is 1-6: 0.5-3: 1-10, and preferred proportion is 2.5-3.5: 0.5-1.5: 3-7, most preferably 3: 1: 5.
The preparation method who the present invention relates to a kind of immobilized microspheres for remediation of petroleum contaminated soil, is characterized in that, the method comprises the following steps:
(1) cultivate and prepare the bacteria suspension of petroleum hydrocarbon degradation bacterium;
(2) put into deionized water after sodium alginate is mixed with attapulgite, high-temperature heat sterilization, keeps 40-60 ℃, solid carbonic acid calcium is dropped in sodium alginate-attapulgite mixture, mix, continue to keep 40-60 ℃, obtain sodium alginate-attapulgite-calcium carbonate gel;
(3) step (1) gained bacteria suspension is joined in step (2) gained coagulant liquid, mix;
When embedding Paracoccus sp. bacterium, in mixture, the concentration of petroleum hydrocarbon degradation bacterium is preferably 0.01-0.04g/ml, most preferably 0.02g/ml; The concentration of sodium alginate is preferably 0.01-0.06g/ml, further preferred 0.025-0.035g/ml, most preferably 0.03g/ml; The concentration of attapulgite is preferably 0.005-0.03g/ml, further preferred 0.005-0.015g/ml, most preferably 0.01g/ml; The concentration of solid carbonic acid calcium is preferably 0.01-0.1g/ml, further preferred 0.03-0.07g/ml; 0.05g/ml most preferably;
(4) by step (3) gained mixture, be added dropwise in calcium chloride solution, Quick uniform mixes, and obtains spherical granules; When embedding Paracoccus sp. bacterium, wherein the concentration of calcium chloride solution is preferably 0.02-0.08g/ml, further preferred 0.02-0.04g/ml, most preferably 0.02g/ml;
(5) after step (4) spherical granules balling-up 5-30min, collect spherical granules, be placed in dilute hydrochloric acid and react, until without obvious Bubble formation, clean;
(6) step (5) gained particle is placed in to calcium chloride solution, soaks, clean;
(7) step (6) gained transfer of granules is entered in proliferated culture medium, shaking table is cultivated, and cleans, and prepares immobilized microspheres.
Wherein, when described step (4) is mixed with calcium chloride, the concentration ratio of petroleum hydrocarbon degradation bacterium, sodium alginate, attapulgite, calcium carbonate and calcium chloride can be preferably 1-4: 1-6: 0.5-3: 1-10: 2-8, further preferred 1-4: 2.5-3.5: 0.5-1.5: 3-7: 2-4, most preferably 2: 3: 1: 5: 2.
Wherein, the solvent of the described bacteria suspension of described step (1) is proliferated culture medium, and its composition is: glucose 20g/L, yeast extract paste 3g/L, peptone 2g/L, NH
4nO
31g/L, MgSO
47H
2o 0.2g/L, KCl 0.2g/L, the wherein pH=7.8 of this proliferated culture medium.
Wherein, described step (1) is cultivated and is prepared the step of the bacteria suspension of petroleum hydrocarbon degradation bacterium and is:
A preparation LB liquid nutrient medium, high-temperature sterilization, is cooled to room temperature, obtains enrichment medium;
Preparation proliferated culture medium, high-temperature sterilization, cooling preservation;
B is in aseptic operating platform, and the inoculum size by 5% in steps A gained enrichment medium is inoculated petroleum hydrocarbon degradation bacterium, then, in 25 ℃ of-30 ℃ of constant-temperature tables vibrations, cultivates, and collects bacterium liquid, the centrifugal wet thallus that obtains;
C mixes wet thallus with proliferated culture medium.
Wherein, described step (4) utilizes constant flow pump to drip described mixture to described calcium chloride solution.
The invention still further relates to the immobilized microspheres for remediation of petroleum contaminated soil calciferous that above method prepares.
The low temperature resistant petroleum hydrocarbon degradation bacterium of described immobilized microspheres embedding can be Paracoccus sp. or Halomonas sp..
The invention still further relates to described immobilized microspheres for the application of decomposing petroleum hydrocarbon, be preferred for the application at degraded in soil petroleum hydrocarbon.
Accompanying drawing explanation
Fig. 1-a is the no concave-convex rod soil of control group one preparation of the present invention and the form picture of the immobilized microspheres particle that calcium carbonate adds.
Fig. 1-b is the form picture that has the immobilized microspheres particle that attapulgite adds without calcium carbonate of control group of the present invention two preparations.
Fig. 1-c is the mode of appearance picture of the immobilized microspheres particle of the embodiment of the present invention 4 preparations.
Fig. 1-d is the scanning electron microscope (SEM) photograph of the immobilized microspheres granule interior of the embodiment of the present invention 4 preparations.
Fig. 1-e is the scanning electron microscope (SEM) photograph of the immobilized microspheres embedding bacterium of the embodiment of the present invention 4 preparations.
Fig. 2 is the active comparison of the immobilized microspheres succinodehydrogenase prepared of the present invention and control group, wherein X-coordinate is successively corresponding to free bacterium, microorganism in the immobilized microspheres of control group one, microorganism in the immobilized microspheres of control group two, in the immobilized microspheres of embodiment 4, the succinodehydrogenase enzyme of microorganism is lived.
Fig. 3 is the comparison of the immobilized microspheres prepared of the present invention and control group to the degradation property of high concentration petroleum pollution soil, wherein X-coordinate is successively corresponding to blank sample, free bacterium, the immobilized microspheres of control group one, the immobilized microspheres of control group two, the degradation property of the immobilized microspheres of embodiment 4.
The preservation information of bacterial strain of the present invention
Bacterial strain Paracoccus sp., this bacterial strain is in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms common micro-organisms center April 8 in 2010; Postcode: 100101) preservation, deposit number is CGMCC No.3718, the secondary coccus (Paracoccus sp.) of taxonomy called after.
Bacterial strain Halomonas sp., this bacterial strain is in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms common micro-organisms center April 8 in 2010; Postcode: 100101) preservation, is numbered CGMCC No.3717, taxonomy called after salt sporangium (Halomonas sp.).
embodiment
The petroleum hydrocarbon degradation bacterium of immobilized microspheres institute embedding of repairing for oil-polluted soils microorganism of the present invention, can make a general reference all bacterial classifications that can decomposing petroleum hydrocarbon, related Paracoccus sp. and Halomonas sp. bacterial strain etc. in the patent application that is 201010239621.7 such as applicant's application number, following examples only be take Paracoccus sp. as example.
These two kinds of degradation bacteria are near the Dagang Oilfield of Tianjin oil-polluted soils, the low temperature resistant petroleum hydrocarbon degradation bacterium obtaining through artificial enrichment culture, separation and purification.The patent application that is 201010239621.7 by application number quotes in full.
The concrete preparation process of two kinds of petroleum hydrocarbon degradation bacteriums that can embedding in the embodiment of the present invention is:
(1) domestication step
Petroleum pollution soil sample is utilized to physiological saline solution, in access crude oil inorganic salt oily(waste)water liquid nutrient medium, at 10 ℃ of shaking tables, cultivate 10-20 days, after get quantitative culture liquid and continue to cultivate 10-20 days, obtain bacterium liquid, in above-mentioned culturing process, add common 5-10ml sterilizing crude oil in batches;
(2) purification procedures
Gained bacterium liquid in step (1) is carried out to gradient dilution, be applied on crude oil inorganic salt oily(waste)water solid medium, cultivate 3-5 days, streak culture;
(3) domestication, streak culture repeatedly
Domestication, streak culture repeatedly, until obtain the pure bacterium of many strains.
The concrete preparation process of the immobilized microspheres of repairing for oil-polluted soils microorganism of the present invention is as follows:
(1) preparation of bacteria suspension:
The preparation of A substratum:
Enrichment medium: for inoculating petroleum hydrocarbon degradation bacterium, by microbiology ordinary method preparation LB liquid nutrient medium, utilize ordinary method to carry out high-temperature sterilization processing, can be at 121 ℃ high-temperature sterilization 30min, be cooled to room temperature, obtain enrichment medium.
Proliferated culture medium: for providing nutrition to petroleum hydrocarbon degradation bacterium, comprise glucose 20g/L, yeast extract paste 3g/L, peptone 2g/L, NH
4nO
31g/L MgSO
47H
2o 0.2g/L, KCl 0.2g/L, pH=7.8, utilizes ordinary method to carry out high-temperature sterilization processing, can be at 115 ℃ high-temperature sterilization 20min, cooling standby.
B enrichment culture:
From the slant medium of preservation petroleum hydrocarbon degradation bacterium, use aseptic water washing bacterium liquid to common LB liquid nutrient medium, shaking table obtains seed liquor after cultivating 12h; In aseptic operating platform, in enrichment medium, by the inoculum size of 5% (v/v), inoculate in the seed liquor of low temperature resistant petroleum hydrocarbon degradation bacterium; After, be placed in constant-temperature table, regulate shaking speed 150rpm, cultivate at 25 ℃-30 ℃, preferably 24-60h; Collect bacterium liquid, the centrifugal 10min of 4000rpm, collecting precipitation, obtains wet thallus, can be placed in afterwards 4 ℃ of preservations.
The preparation of C bacteria suspension:
By wet thallus dilution, solvent can adopt sterilized water, and preferably the employing aforesaid proliferated culture medium of material that can supplement the nutrients, mixes wet thallus with proliferated culture medium, can save backup in 4 ℃ afterwards.
(2) preparation of sodium alginate-attapulgite-calcium carbonate gel liquid:
Sodium alginate mixes with attapulgite, throws in into deionized water, utilizes conventional high-temperature heat sterilization to process, can be at 121 ℃ high-temperature heat sterilization 30min, be then placed in 40-60 ℃ of water-bath and be incubated.Then, by mixing in the mixture of analytically pure solid carbonic acid calcium input sodium alginate-attapulgite, better with vortex instrument mixed effect, continue water bath heat preservation.
(3) bacteria suspension adds:
In step (2) gained coagulant liquid, add step (1) gained bacteria suspension, better with vortex instrument mixed effect.
If the wet thallus concentration in mixture is too small, the activity of the immobilized microspheres finally making is inadequate, if excessive, can stop up the hole of immobilized microspheres, and can cause thalline waste; The concentration of sodium alginate is less than finite concentration, does not become ball, but while surpassing finite concentration, the immobilized microspheres of preparation has serious conditions of streaking, and in operating process, coagulant liquid is too sticky, and preparation is got up more difficult; If the concentration of attapulgite is too low, do not have the effect of improving immobilized microspheres performance, but when its concentration surpasses to a certain degree, in the sodium alginate-attapulgite-calcium carbonate of preparation-bacterium mixture, have more precipitation, immobilized microspheres composition is difficult to homogeneous; If the concentration of solid carbonic acid calcium is too low, cannot pore-forming, do not have the effect of improving microsphere particle hole, when the excessive concentration of calcium carbonate, preparation process hydrochloric acid used can increase and the corresponding prolongation of pore-forming time meeting, make thalline poisoned by hydrochloric acid larger, and the physical strength of the immobilized microspheres particle of final preparation also can weaken, frangible.
It should be noted that, the bacterium of embedding is different, and its preferred material concentration of institute and ratio also can be different.When embedding Paracoccus sp. bacterium, in mixture, the concentration of petroleum hydrocarbon degradation bacterium is preferably 0.01-0.04g/ml, most preferably 0.02g/ml; The concentration of sodium alginate is preferably 0.01-0.06g/ml, further preferred 0.025-0.035g/ml, most preferably 0.03g/ml; The concentration of attapulgite is preferably 0.005-0.03g/ml, further preferred 0.005-0.015g/ml, most preferably 0.01g/ml; The concentration of solid carbonic acid calcium is preferably 0.01-0.1g/ml, further preferred 0.03-0.07g/ml; 0.05g/ml most preferably;
(4) immobilization balling-up:
By step (3) gained mixture, splash in calcium chloride solution, drop and calcium chloride Contact are balling-up, obtain spherical granules.
Wherein, the concentration of calcium chloride solution is too low, cannot balling-up, but the excessive concentration of calcium chloride solution, its calcium ion can produce and poison thalline, affect the activity of bacterium.When embedding Paracoccus sp. bacterium, wherein the concentration of calcium chloride solution is preferably 0.02-0.08g/ml, further preferred 0.02-0.04g/ml, most preferably 0.02g/ml.
Wherein, when described step (4) mixture mixes with calcium chloride, in order to make reactant ratio suitable, and reaction is carried out smoothly, preferably the concentration ratio of petroleum hydrocarbon degradation bacterium, sodium alginate, attapulgite, calcium carbonate and calcium chloride is 1-4: 1-6: 0.5-3: 1-10: 2-8, preferred 1-4: 2.5-3.5: 0.5-1.5: 3-7: 2-4, more preferably 2: 3: 1: 5: 2.Preferably utilize constant flow pump to carry out dropwise operation, the object of doing is like this that the balling-up speed of reaction and quality are improved.In operation, magnetic stirring apparatus is set in calcium chloride solution, to guarantee the contact area of mixture liquid and calcium chloride.
(5) except calcium carbonate:
After balling-up 5-30min, rely on visual inspection judgement herein, during as spherical shape, with tweezers, remove to prick become bead, as sensation high resilience, balling-up hardness is moderate, illustrates that institute's balling-up reaches requirement.Generally, the time is too short, and balling-up hardness is inadequate; Overlong time, balling-up hardness can meet the demands, but can extend except reaction times of calcium carbonate, means thalline damage greatlyr, therefore when just forming whippy bead, can start next-step operation.And then collect spherical granules, be placed in dilute hydrochloric acid and react with remove portion calcium carbonate pore-forming.Dilute hydrochloric acid can be 0.2M HCI.If the volume of dilute hydrochloric acid is too many, larger to the murder by poisoning of bacterium, therefore can choose according to the grain amount obtaining, generally choose the dilute hydrochloric acid of 1～1.2 times of volume of particle volume.Wherein, when without obvious Bubble formation, can finish this reaction, clean gained particle, can utilize sterilized water or physiological saline to carry out.
(6) immobilization strengthening:
Step (5) gained particle is placed in to calcium chloride solution and soaks, general 12-24h, then cleans, and cleaning process is available physiological saline or sterilized water all.If the amount of described calcium chloride solution is too many, too large to the toxic action of bacterium, can choose according to the amount of gained particle, generally choose the calcium chloride solution of 1/2～1 times of volume of particle volume.
(7) propagation is cultivated again:
By step (6) gained particle, be transferred in proliferated culture medium, the effect of proliferated culture medium is herein to promote to be adsorbed on the bacterial strain regrowth on microballoon, can select conventional any proliferated culture medium, the present invention, for convenient, has selected the described proliferated culture medium of step (1).And the amount of proliferated culture medium used, if very little, additional nutritive substance is not enough, can, according to how many selections of prepared immobilization particle, generally use immobilization microorganism particles volume 2-5 multiplication culture base unit weight doubly to cultivate again.Then, in 25 ℃, to be not more than the rotating speed shaking table of 100rpm, cultivate, preferably shaking table is cultivated 36-60h at a slow speed,, then clean several times, preparing immobilized microspheres, this immobilized microspheres can save backup at 4 ℃ afterwards.Above cleaning step object is to remove calcium chloride solution or the proliferated culture medium impurity adsorbing on immobilized microspheres, and all available sterilized water and physiological saline clean.
Foregoing has been described immobilized microspheres of the present invention and preparation method thereof.Immobilized microspheres particle prepared by the present invention is compared with existing microbial preparation, and its obvious feature is, absorption method and entrapping method are organically combined, and not only can guarantee the density of thalline but also can guarantee the picked-up of nutritive substance; And calcium carbonate is as the introducing of pore-creating agent and sustained release dosage, not only can guarantees the mass transfer performances of particle but also can strengthen its slow-releasing; In embedding process, added the required nutritive substance of microorganism growth, can temporarily obtain nutrient supply after making immobilized microbial inoculum render to contaminated soil environment, resistance to Environment impact strengthens.In addition, microbial inoculum preparation process simple economy, the embedded material adopting (sodium alginate is polysaccharose substance, and attapulgite is mineral dust) is natural materials, and with low cost, good biocompatibility and biodegradability are good, environmental friendliness and safety.The present invention has broad application prospects in the biological restoration process of oil-polluted soils.
The following examples will further be set forth the present invention, but should not be understood to limitation of the scope of the invention.
In following examples, the situation of key instrument used and equipment is as shown in the table:
The reagent that following examples adopt except bacterial strain, all come from commercially available, the source that following table is main agents:
| Reagent name | Source or producer |
| Attapulgite | Xuyi, Jiangxi |
| Sodium alginate | Chemical Reagent Co., Ltd., Sinopharm Group |
| Calcium chloride | Chemical Reagent Co., Ltd., Sinopharm Group |
| MTT | AMRESCO |
| Dimethyl sulfoxide (DMSO) | Xilong Chemical Co., Ltd |
| Normal hexane | Chemical Reagent Co., Ltd., Sinopharm Group |
| Acetone | Chemical Reagent Co., Ltd., Sinopharm Group |
| Calcium carbonate | Chemical Reagent Co., Ltd., Sinopharm Group |
| Hydrochloric acid | Modern east, Beijing fine chemicals company limited |
The bacterial strain Paracoccus sp. using in embodiments of the invention and the Preparation Example of Halomonas sp.:
Separated and the domestication of Paracoccus sp. and Halomonas sp.
Near the polluted soil Dagang Oilfield oil well of collection in worksite Tianjin, take crude oil as sole carbon source, adopts crude oil inorganic salt oily(waste)water liquid culture and domestication oil degradation bacteria.The preparation of crude oil inorganic salt oily(waste)water substratum, dilutes constant volume by inorganic salt liquid substratum mother liquor through oil field waste, adds crude oil and forms.
Inorganic salt (10 *) liquid nutrient medium mother liquor (ultrapure water solution): NaCl 50g/L, K
2hPO
410g/L, NH
4h
2pO
410g/L, (NH
4)
2sO
410g/L, MgSO
47H
2o 2g/L, KNO
330g/L;
Inorganic salt liquid substratum: inorganic salt liquid substratum mother liquor (10 *) 100ml, with ultrapure water, be settled to 1000ml, utilize 0.2M sodium hydroxide or 0.1M hydrochloric acid to regulate pH=5.0;
Inorganic salt oily(waste)water liquid nutrient medium: inorganic salt liquid substratum (10 *) 100ml, add oil field waste 700ml, then be settled to 1000ml with ultrapure water, utilize 0.2M sodium hydroxide or 0.1M hydrochloric acid to regulate pH=5.0.
Crude oil inorganic salt oily(waste)water liquid nutrient medium: inorganic salt oily(waste)water substratum 1000ml, 8g sterilizing crude oil.
Crude oil inorganic salt oily(waste)water solid culture based component is: inorganic salt oily(waste)water liquid nutrient medium 1000ml, add agar 15g, and solid medium surface uniform is coated with 20 μ l sterilizing crude oil.
Domestication step: the petroleum pollution soil sample of the about 1g of physiological saline solution, access is equipped with in the 250mL Erlenmeyer flask of 100mL " crude oil inorganic salt oily(waste)water liquid " substratum, and every kind of liquid nutrient medium is done two repetitions.10 ℃, under 150r/min condition, be placed in shaking table and cultivate 10d (wherein, every 5d adds 1mL sterilizing crude oil); Then get a certain amount of above-mentioned nutrient solution access and be equipped with in the 250mL triangular flask of the corresponding substratum of 100mL, under equal temperature condition, continue to cultivate 10d (adding 1mL sterilizing crude oil every 5d).
Isolation and purification step: " crude oil inorganic salt oily(waste)water liquid culture " the bacterium liquid after domestication carries out gradient dilution (10
-1～10
-8), coating 50ul, to corresponding solid domestication substratum, cultivates 3 days.The bacterium colony that obtains different amts after cultivation is dull and stereotyped, chooses even, the distinguishable flat board of concentration, with the bacterium colony that transfering loop picks on flat board, rules to crude oil inorganic salt oily(waste)water solid medium.
Repeatedly tame through 25 generations, streak culture in crude oil inorganic salt oily(waste)water solid medium, obtain the pure bacterium of many strains, pure bacterium is merely able to verify the ability of its degraded oil through being seeded to inorganic salt oily(waste)water liquid nutrient medium.Finally obtained two strain high efficient petroleum degrading bacterias, a kind of is Paracoccus sp., and this bacterium is accredited as paracoccus through 16SrDNA, and another kind is Halomonas sp., and this Pseudomonas is in salt monospore Pseudomonas.
The embodiment for preparing immobilized microspheres:
Embodiment 1
(1) bacteria suspension preparation:
The preparation of A substratum: (substratum of following examples all adopts the substratum of embodiment 1 preparation, so the step of following examples (1) A omission, starts to describe from step (1) B)
Enrichment medium: press several milliliters of LB liquid nutrient mediums of microbiology ordinary method preparation: Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 5g/L, pH=7.0.
Above-mentioned enrichment medium is divided and installed in several 1000ml triangular flasks, and every bottle is cultivated base unit weight is volume 4/5, inserts in high-temperature sterilization unit, in 121 ℃ of sterilizing 30min, is cooled to room temperature.
Proliferated culture medium (according to the preparation of biology ordinary method): glucose 20g/L, yeast extract paste 3g/L, peptone 2g/L, NH
4nO
31g/L, MgSO
47H
2o 0.2g/L, KCl 0.2g/L, pH=7.8.115 ℃ of high-temperature sterilization 20min, cooling standby.
B enrichment culture:
Enrichment culture: in aseptic operating platform, inoculate Paracoccus sp. bacterial strain by the inoculum size of 5% (v/v) in enrichment medium; After, be placed in constant-temperature shaking incubator, regulate shaking speed 150rpm, cultivate 48h at 25 ℃; Collect bacterium liquid, the centrifugal 10min of 4000rpm, obtains wet thallus, is placed in 4 ℃ of preservations.
The preparation of C bacteria suspension:
1g wet thallus mixes with 10ml proliferated culture medium, makes bacteria suspension, and 4 ℃ save backup.
(2) preparation of sodium alginate-attapulgite-calcium carbonate gel liquid:
2.5g sodium alginate is mixed with 0.5g attapulgite, throw in the deionized water into 90ml, high-temperature heat sterilization 30min at 121 ℃, is then placed in 40 ℃ of water-baths and is incubated.Now, the analytically pure solid carbonic acid calcium of 3g is dropped in the mixture of sodium alginate-attapulgite, vortex instrument mixes, and continues water bath heat preservation.
(3) bacteria suspension adds:
In step (2) gained 90ml coagulant liquid, add 10ml step (1) gained bacteria suspension, utilize vortex instrument to mix.Wherein, the concentration of wet thallus is 0.01g/ml, and the concentration of sodium alginate is 0.025g/ml, and the concentration of attapulgite is 0.005g/ml, and the concentration of calcium carbonate is 0.03g/ml.
(4) immobilization balling-up:
Constant flow pump, by step (3) gained 100ml mixture, splashes in the calcium chloride solution that 100ml concentration is 0.02g/ml, and drop and calcium chloride Contact are balling-up.Magnetic stirring apparatus is installed in calcium chloride solution, to guarantee the contact area of liquid and calcium chloride.
(5) except calcium carbonate:
After balling-up 5min, collect spherical granules 50ml, be placed in 50ml0.2M HCI, reaction is pore-forming with remove portion calcium carbonate.Wherein, floating and rise when particle, during without obvious Bubble formation, can finish this reaction, with sterile water wash.
(6) immobilization strengthening:
50ml step (5) gained particle is placed in to 25ml calcium chloride solution, soaks 24h, utilize sterile water wash.
(7) propagation is cultivated again:
By 50ml step (6) gained particle, be transferred in the made proliferated culture medium of 100ml step (1), in 25 ℃ of rotating speed shaking tables with 100rpm, cultivate at a slow speed 36h, physiological saline cleans three times, prepares immobilized microspheres.
Embodiment 2
(1) bacteria suspension preparation:
B enrichment culture:
Enrichment culture: in aseptic operating platform, inoculate Paracoccus sp. bacterial strain by 5% inoculum size in enrichment medium; After, be placed in constant-temperature shaking incubator, regulate shaking speed 150rpm, cultivate 72h at 25 ℃; Collect bacterium liquid, the centrifugal 10min of 4000rpm, obtains wet thallus, is placed in 4 ℃ of preservations.
The preparation of C bacteria suspension:
4g wet thallus mixes with 10ml proliferated culture medium, makes bacteria suspension, and 4 ℃ save backup.
(2) preparation of sodium alginate-attapulgite-calcium carbonate gel liquid:
3.5g sodium alginate is mixed with 1.5g attapulgite, throw in the deionized water into 90ml, high-temperature heat sterilization 30min at 121 ℃, is then placed in 60 ℃ of water-baths and is incubated.Now, the analytically pure solid carbonic acid calcium of 7g is dropped in the mixture of sodium alginate-attapulgite, vortex instrument mixes, and continues water bath heat preservation.
(3) bacteria suspension adds:
In step (2) gained 90ml coagulant liquid, add 10ml step (1) gained bacteria suspension, utilize vortex instrument to mix.Wherein, the wet thallus concentration in mixture is 0.4g/ml, and the concentration of sodium alginate is 0.035g/ml, and the concentration of attapulgite is 0.015g/m, and the concentration of calcium carbonate is 0.07g/ml.
(4) immobilization balling-up:
Constant flow pump, by step (3) gained 100ml mixture, splashes in the calcium chloride solution that 100ml concentration is 0.04g/ml, and drop and calcium chloride Contact are balling-up.Magnetic stirring apparatus is installed in calcium chloride solution, to guarantee the contact area of liquid and calcium chloride.
(5) except calcium carbonate:
After balling-up 5min, collect spherical granules 90ml, be placed in 90ml0.2M HCI, reaction is pore-forming with remove portion calcium carbonate.Wherein, floating and rise when particle, during without obvious Bubble formation, can finish this reaction, with sterile water wash.
(6) immobilization strengthening:
90ml step (5) gained particle is placed in to 45ml calcium chloride solution, soaks 18h, utilize sterile water wash.
(7) propagation is cultivated again:
By 90ml step (6) gained particle, be transferred in the made proliferated culture medium of 300ml step (1), in the rotating speed shaking table of 25 ℃ of 100rpm, cultivate at a slow speed 60h, physiological saline cleans three times, prepares immobilized microspheres.
Embodiment 3
(1) bacteria suspension preparation:
B enrichment culture:
Enrichment culture: in aseptic operating platform, inoculate Paracoccus sp. bacterial strain by 5% inoculum size in enrichment medium; After, be placed in constant-temperature shaking incubator, regulate shaking speed 150rpm, cultivate 60h at 25 ℃; Collect bacterium liquid, the centrifugal 10min of 4000rpm, obtains wet thallus, is placed in 4 ℃ of preservations.
The preparation of C bacteria suspension:
3g wet thallus mixes with 10ml proliferated culture medium, makes bacteria suspension, and 4 ℃ save backup.
(2) preparation of sodium alginate-attapulgite-calcium carbonate gel liquid:
3g sodium alginate is mixed with 1g attapulgite, throw in the deionized water into 90ml, high-temperature heat sterilization 30min at 121 ℃, is then placed in 60 ℃ of water-baths and is incubated.Now, the analytically pure solid carbonic acid calcium of 5g is dropped in the mixture of sodium alginate-attapulgite, vortex instrument mixes, and continues water bath heat preservation.
(3) bacteria suspension adds:
In step (2) gained 90ml coagulant liquid, add 10ml step (1) gained bacteria suspension, utilize vortex instrument to mix.Wherein, the wet thallus concentration in mixture is 0.3g/ml, and the concentration of sodium alginate is 0.03g/ml, and the concentration of attapulgite is 0.01g/ml, and the concentration of calcium carbonate is 0.05g/ml.
(4) immobilization balling-up:
Constant flow pump, by step (3) gained 100ml mixture, splashes in the calcium chloride solution that 100ml concentration is 0.03g/ml, and drop and calcium chloride Contact are balling-up.Magnetic stirring apparatus is installed in calcium chloride solution, to guarantee the contact area of liquid and calcium chloride.
(5) except calcium carbonate:
After balling-up 8min, collect spherical granules 60ml, be placed in 60ml0.2M HCI, reaction is pore-forming with remove portion calcium carbonate.Wherein, floating and rise when particle, during without obvious Bubble formation, can finish this reaction, with sterile water wash.
(6) immobilization strengthening:
60ml step (5) gained particle is placed in to 30ml calcium chloride solution, soaks 12h, utilize sterile water wash.
(7) propagation is cultivated again:
By 60ml step (6) gained particle, be transferred in the made proliferated culture medium of 300ml step (1), in the rotating speed shaking table of 25 ℃ of 100rpm, cultivate at a slow speed 48h, physiological saline cleans three times, prepares immobilized microspheres.
Embodiment 4
Laboratory is by experiment of single factor and orthogonal experiment, the final optimal proportion of each moiety of being fixed microballoon, and its preparation process is as follows:
(1) bacteria suspension preparation:
B enrichment culture:
Enrichment culture: in aseptic operating platform, inoculate Paracoccus sp. bacterial strain by 5% inoculum size in enrichment medium; After, be placed in constant-temperature shaking incubator, regulate shaking speed 150rpm, cultivate 48h at 25 ℃; Collect bacterium liquid, the centrifugal 10min of 4000rpm, obtains wet thallus, is placed in 4 ℃ of preservations.
The preparation of C bacteria suspension:
2g wet thallus mixes with 10ml proliferated culture medium, makes bacteria suspension, and 4 ℃ save backup.
(2) preparation of sodium alginate-attapulgite-calcium carbonate gel liquid:
3.0g sodium alginate is mixed with 1.0g attapulgite, throw in the deionized water into 90ml, high-temperature heat sterilization 30min at 121 ℃, is then placed in 60 ℃ of water-baths and is incubated.Now, the analytically pure solid carbonic acid calcium of 5g is dropped in the mixture of sodium alginate-attapulgite, vortex instrument mixes, and continues water bath heat preservation.
(3) bacteria suspension adds:
In step (2) gained 90ml coagulant liquid, add 10ml step (1) gained bacteria suspension, utilize vortex instrument to mix.Wherein, the wet thallus concentration in mixture is 0.02g/ml, and the concentration of sodium alginate is 0.03g/ml, and the concentration of attapulgite is 0.01g/ml, and the concentration of calcium carbonate is 0.05g/ml.
(4) immobilization balling-up:
Constant flow pump, by step (3) gained 100ml mixture, splashes in the calcium chloride solution that 100ml concentration is 0.02g/ml, and drop and calcium chloride Contact are balling-up.Calcium chloride solution is placed on magnetic stirring apparatus, to guarantee the contact area of liquid and calcium chloride.
(5) except calcium carbonate:
After balling-up 10min, collect spherical granules 50ml, be placed in 50ml0.2M HCI, reaction is pore-forming with remove portion calcium carbonate.Wherein, floating and rise when particle, during without obvious Bubble formation, can finish this reaction, with sterile water wash.
(6) immobilization strengthening:
50ml step (5) gained particle is placed in to 25ml calcium chloride solution, soaks 24h, utilize sterile water wash.
(7) propagation is cultivated again:
By 50ml step (6) gained particle, be transferred in the made proliferated culture medium of 100ml step (1), in 25 ℃ of rotating speed shaking tables with 100rpm, cultivate at a slow speed 60h, physiological saline cleans three times, prepares immobilized microspheres.
In order to investigate, in the present embodiment, adding the immobilized microspheres of attapulgite and calcium carbonate compares with traditional immobilized microbial inoculum, its degradation property of its advantage, spy arranges two groups of comparative examples tests, be that control group one does not add attapulgite and calcium carbonate, add with control group two immobilized microspheres that attapulgite does not add calcium carbonate, its preparation process is as follows:
Control group one
Except following steps, control group one is identical with the step of embodiment 4.Step (2) is the preparation of sodium alginate gel liquid, and concrete steps are:
3.0g sodium alginate is thrown in to the deionized water into 90ml, and high-temperature heat sterilization 30min at 121 ℃, is then placed in 60 ℃ of water-baths and is incubated.
Control group two
Except following steps, control group two is identical with the step of embodiment 4.
Step (2) is the preparation of sodium alginate-attapulgite coagulant liquid, and concrete steps are:
3.0g sodium alginate and 1.0g attapulgite are mixed, throw in the deionized water into 90ml, high-temperature heat sterilization 30min at 121 ℃, is then placed in 60 ℃ of water-baths and is incubated.
Embodiment 5-10 discusses in immobilized microspheres preparation process, the impact of each constituent concentration on test-results.
Embodiment 5
Except following explanation, the step of the present embodiment is consistent with embodiment 4.
During step (2) preparation sodium alginate-attapulgite-calcium carbonate gel, the sodium alginate adding is 6g, the attapulgite adding is 1g, the calcium carbonate adding is 5g, therefore each constituent concentration in step (4) mixture is: sodium alginate 0.06g/ml, attapulgite 0.01g/ml, calcium carbonate 0.05g/ml.
The concentration of the calcium chloride solution of step (4) is 0.02g/ml.
Embodiment 6
Except following explanation, the step of the present embodiment is consistent with embodiment 4.
During step (2) preparation sodium alginate-attapulgite-calcium carbonate gel, the sodium alginate adding is 1g, the attapulgite adding is 1g, the calcium carbonate adding is 5g, therefore each constituent concentration in step (4) mixture is: sodium alginate 0.01g/ml, attapulgite 0.01g/ml, calcium carbonate 0.05g/ml.
The concentration of the calcium chloride solution of step (4) is 0.02g/ml.
Sodium alginate concentration in embodiment 5 is too high, the gel of preparation is partially sticky, there is certain conditions of streaking in the immobilized microorganism microbial inoculum particle of final preparation, affected the quality of balling-up, pass through test of many times, when sodium alginate concentration is during higher than 0.035g/ml, become bead all to have certain conditions of streaking.And the sodium alginate concentration of embodiment 6 is lower, balling ratio is low, and by test of many times, when sodium alginate concentration is during lower than 0.025g/ml, balling ratio is all not ideal enough.And under the constant condition of other condition, when the concentration of the sodium alginate in step (4) mixture is 0.025-0.035g/ml, prepared product balling ratio is high, there is no conditions of streaking, gel viscosity is moderate, and preparation is easier to.
Embodiment 7
Except following explanation, the step of the present embodiment is consistent with embodiment 4.
During step (2) preparation sodium alginate-attapulgite-calcium carbonate gel, the sodium alginate adding is 3g, the attapulgite adding is 3g, the calcium carbonate adding is 5g, therefore each constituent concentration in step (4) mixture is: sodium alginate 0.03g/ml, attapulgite 0.03g/ml, calcium carbonate 0.05g/ml.
The concentration of the calcium chloride solution of step (4) is 0.02g/ml.
The concentration of the attapulgite of the present embodiment is higher, middle more attapulgite precipitation, the final immobilized microspheres composition heterogeneity of occurring in the sodium alginate-attapulgite-calcium carbonate of preparation-bacterium mixture.Actual when attapulgite concentration surpasses 0.015g/ml, all easily produce this situation.By test of many times, under the constant condition of other condition, the concentration of the attapulgite in step (4) mixture is 0.005-0.015g/ml, and prepared product is not precipitation, relatively homogeneous almost.
Embodiment 8
Except following explanation, the step of the present embodiment is consistent with embodiment 4.
During step (2) preparation sodium alginate-attapulgite-calcium carbonate gel, the sodium alginate adding is 3g, the attapulgite adding is 1g, the calcium carbonate adding is 10g, therefore each constituent concentration in step (4) mixture is: sodium alginate 0.03g/ml, attapulgite 0.01g/ml, calcium carbonate 0.1g/ml.
The concentration of the calcium chloride solution of step (4) is 0.02g/ml.
The amount of step (5) hydrochloric acid soln of pore-forming for removing calcium carbonate is 100ml.
Embodiment 9
Except following explanation, the step of the present embodiment is consistent with embodiment 4.
During step (2) preparation sodium alginate-attapulgite-calcium carbonate gel, the sodium alginate adding is 3g, the attapulgite adding is 1g, the calcium carbonate adding is 1g, therefore each constituent concentration in step (4) mixture is: sodium alginate 0.03g/ml, attapulgite 0.01g/ml, calcium carbonate 0.01g/ml.
The concentration of the calcium chloride solution of step (4) is 0.02g/ml.
The amount of step (5) hydrochloric acid soln of pore-forming for removing calcium carbonate is 10ml.
The concentration of the calcium carbonate of embodiment 8 is higher, the physical strength of the immobilized microspheres particle of preparation weakens, fragile, and hydrochloric acid used can increase, and pore-forming time lengthening, mean that thalline is poisoned by hydrochloric acid stronger, surveying enzyme work is 0.024, and the enzyme value alive of preparing particle far below embodiment 4, passes through test of many times, when calcium carbonate concentration surpasses 0.07g/ml, institute embedding bacterium is all had to certain influence.The concentration of the calcium carbonate of embodiment 9 is lower, and prepared immobilized microspheres pore-forming is not enough, and by test of many times, when calcium carbonate concentration is during lower than 0.03g/ml, pore-forming all has deficiency.By test of many times, when the concentration range of the calcium carbonate in the mixture of step (4) is 0.03-0.07g/ml, the immobilized microspheres physical strength making is moderate, and can not affect the activity of thalline.
Except following explanation, the step of the present embodiment is consistent with embodiment 4.
During step (2) preparation sodium alginate-attapulgite-calcium carbonate gel, the sodium alginate adding is 3g, and the attapulgite adding is 1g, the calcium carbonate adding is 5g, therefore each constituent concentration of gel is: sodium alginate 0.03g/ml, attapulgite 0.01g/ml, calcium carbonate 0.05g/ml.
The concentration of the calcium chloride solution of step (4) is 0.08g/ml.
The calcium chloride solution concentration of the present embodiment is higher, corresponding calcium ion concn is higher, because calcium ion itself is toxic to thalline, larger to microbial activity damage in immobilized microspheres preparation process, surveying enzyme work is 0.026, and the enzyme value alive of preparing particle far below embodiment 4, passes through test of many times, when calcium chloride concentration is during higher than 0.04g/ml, the microbial activity of institute's embedding is all had to certain influence.By test of many times, when the concentration of calcium chloride solution is 0.02-0.04g/ml, microbial activity is not had to damage substantially, and can form more uniform bead.
Test result analysis:
(1) micro-structure diagram
Immobilized microspheres particle is processed through two fixing, the ethanol series solution gradient dehydrations of washed with de-ionized water, glutaraldehyde-osmic acid, critical point drying and metal spraying.(operating all according to routine operation above) finally uses the immobilized microspheres of the S-4800 of Hitachi awkward silence at a meeting emission scan electron microscope observation control group one, control group two, embodiment 4.
Apparent structure Fig. 1-the c and native apparent structure Fig. 1-a, the control group two adding without calcium carbonate of control group one no concave-convex rod that after comparative example's 4 interpolation calcium carbonate, remove the immobilized microspheres particle in hole have attapulgite without the immobilized microspheres particle apparent structure Fig. 1-b of calcium carbonate interpolation, can find out add attapulgite add again calcium carbonate product surface have many folds and a space, from its structure, can indirectly reflect that granule interior is polynuclear plane, more be conducive to microorganism existence.And the inner scanning Electronic Speculum Fig. 1-d of observation embodiment 4 immobilized microspheres can find out that there is space the inside of immobilized microspheres, be conducive to the growth of thalline, be conducive to transmission and the transportation of energy and material.Scanning electron microscope (SEM) photograph 1-e by embodiment 4 immobilized microspheres embedding bacterium can find out again, and thalline is distribution situation on sodium alginate-attapulgite-calcium carbonate complex carrier, rod-short be the thalline of embedding, be evenly attached on carrier.As can be seen here, the interpolation of attapulgite and calcium carbonate makes the form of immobilized microspheres that noticeable change occur, and is more conducive to transmission and the transportation of energy and material.
(2) activity of immobilized microspheres
The free bacterium that is 1ml immobilized microspheres and its isodose by volume carries out the comparison that succinodehydrogenase enzyme is lived, to characterize the activity of immobilized microspheres.Wherein, reaction conditions is, 1ml immobilized microspheres particle is dissolved in 9ml0.2M Trisodium Citrate, gets 30ul lysate, dilutes the MTT that adds 300ul0.5% after 100 times, is placed in the 2h that develops the color at 30 ℃; After reaction finishes, 10000rpm/min is centrifugal, then adds 4ml dmso solution precipitated product; Under 525nm, survey absorbancy.Absorbance is higher, shows that the succinic dehydrogenase activity of microorganism is higher, also shows that the quantity of microorganism is more (seeing Fig. 2).As shown in Figure 2, free bacterium is after fixing, and by multiplication culture, its quantity has obtained phenomenal growth, illustrates that free bacterium can grow in granule interior; And the microbial inoculum particle of interpolation attapulgite and calcium carbonate, its enzyme work will illustrate that the introducing of attapulgite and calcium carbonate has improved the pore texture of granule interior higher than any one control group, is more conducive to thalli growth.
(3) degradation property of immobilized microspheres
The application criterion of immobilized microspheres is its degradation capability to petroleum hydrocarbon, and therefore, the present invention determines its character by measuring the degradation capability of petroleum hydrocarbon.
The test conditions design of immobilized microspheres degraded soil Petroleum Hydrocarbon: high concentration petroleum pollution soil and soil ratio are 2: 1,2.5%, 25 ℃ of condition of immobilized microspheres inoculum size, 150rmp shaking table is cultivated 15d, three revision tests.Measure soil total petroleum hydrocarbon and measure by ordinary method, in order to guarantee accurate testing degree, adopt quick solvent extration, with reference to instrument, use standard method of test.Measuring method is: 10g soil sample is mixed and packed abstraction pool into 8g diatomite, using normal hexane: acetone=1: 1 as extraction agent, receiving flask is collected extraction liquid, and nitrogen blows to constant weight, weight difference value obtains TPH (total petroleum hydrocarbon amount in soil), calculates the content η=G of soil Petroleum Hydrocarbon
always/ G
soil, and then the degradation rate that obtains petroleum hydrocarbon
Wherein, solvent extraction instrument working procedure is fast: Heating temperature 170 degree, and preheating 100s, pressure 10Mpa, static extracting time 720s, nitrogen blows 60s, twice of re-extract.Wherein, free bacterium is set and compares, the free dosage of bacterium is identical with the initial embedding amount of immobilized microbial inoculum, and degradation condition is also identical.The degradation rate of petroleum hydrocarbon is higher shows that the degradation property of microbiobacterial agent is better, the results are shown in Figure 3.Wherein, the degradation rate of the immobilized microspheres of embodiment 4 is the highest, up to 50%, higher than free bacterium also higher than the immobilized microspheres of control group one and control group two.In experimentation, As time goes on, the residual continuous stripping of calcium carbonate in immobilized microspheres, but the volume of particle does not change, and after calcium carbonate stripping, particle hole obviously will strengthen, and illustrates that the mass-transfer performance of immobilized microspheres is strengthened.As can be seen here, under the same terms, add attapulgite and calcium carbonate, the petroleum hydrocarbon degradation rate of immobilized microspheres is significantly improved.
(4) the resistance to Environment impact of immobilized microspheres
In order to investigate the resistance to Environment impact of immobilized microspheres, under same Degrading experiment condition, temperature setting is set to 10 ℃, the petroleum hydrocarbon degradation rate of embodiment 4 still reaches 42%; And change soil ratio is set to 1: 2, its petroleum hydrocarbon degradation rate still reaches 45%; And change temperature is set to 20 ℃, its petroleum hydrocarbon degradation rate still can reach 44.9%.Show, the resistance to Environment impact of immobilized microspheres that adds attapulgite and calcium carbonate is strong, can keep efficient degradation property.
(5) introducing of constant flow pump
Immobilized microspheres medical needle tubing or the drop-burettes of adopting are prepared in laboratory traditionally, and its rate of addition is slower, and 5-15ml/min is difficult to meet the demand that fixedly prepared by microballoon in enormous quantities.The preparation of immobilized microspheres of the present invention, has introduced constant flow pump, can realize the possibility of scale operation, according to the difference of constant flow pump model, can meet different turnout, can reach 2000ml/min and higher drop rate.The for example reaction of embodiment 4, if without constant flow pump, it (is mainly time for adding that step (4) is prepared the time that volume is the immobilization microorganism particles of 100ml, because liquid Contact is balling-up, the time of balling-up can ignore) be 15min, if and adopt constant flow pump, its preparation time can reach 1/10th of 15min, 1 minute half.
Claims (17)
1. an immobilized microspheres for remediation of petroleum contaminated soil calciferous, it is characterized in that: particle is spherical shape, there is space inside, its microsphere diameter is 2.5～3.0mm, described immobilized microspheres particle comprises sodium alginate as embedded material, solid carbonic acid calcium is as pore former and sustained release dosage, and attapulgite is as sorbent material, and its absorption has petroleum hydrocarbon degradation bacterium and nutritive element; The mass ratio of described sodium alginate, attapulgite and calcium carbonate is 1-6: 0.5-3: 1-10.
2. immobilized microspheres as claimed in claim 1, is characterized in that, the mass ratio of sodium alginate, attapulgite and calcium carbonate is 2.5-3.5: 0.5-1.5: 3-7.
3. immobilized microspheres as claimed in claim 2, is characterized in that, the mass ratio of sodium alginate, attapulgite and calcium carbonate is 3: 1: 5.
4. a preparation method for immobilized microspheres for remediation of petroleum contaminated soil calciferous, is characterized in that, the method comprises the following steps:
(1) cultivate and prepare the bacteria suspension of petroleum hydrocarbon degradation bacterium;
A preparation LB liquid nutrient medium, high-temperature sterilization, is cooled to room temperature, obtains enrichment medium; Preparation proliferated culture medium, high-temperature sterilization, cooling preservation;
B is in aseptic operating platform, and the inoculum size by 5% in steps A gained enrichment medium is inoculated petroleum hydrocarbon degradation bacterium, then, in 25 ℃ of-30 ℃ of constant-temperature tables vibrations, cultivates, and collects bacterium liquid, the centrifugal wet thallus that obtains;
C mixes wet thallus with proliferated culture medium;
The solvent of described bacteria suspension is proliferated culture medium, and its composition is: glucose 20g/L, yeast extract paste 3g/L, peptone 2g/L, NH
4nO
31g/L, MgSO
47H
2o0.2g/L, KCl0.2g/L, the wherein pH=7.8 of this proliferated culture medium;
(2) put into deionized water after sodium alginate is mixed with attapulgite, high-temperature heat sterilization, keeps 40-60 ℃, solid carbonic acid calcium is dropped in sodium alginate-attapulgite mixture, mix, continue to keep 40-60 ℃, obtain sodium alginate-attapulgite-calcium carbonate gel;
(3) step (1) gained bacteria suspension is joined in step (2) gained coagulant liquid, mix; In mixture, the concentration of petroleum hydrocarbon degradation bacterium is 0.01-0.04g/ml; The concentration of sodium alginate is 0.01-0.06g/ml; The concentration of attapulgite is 0.005-0.03g/ml; The concentration of solid carbonic acid calcium is 0.01-0.1g/ml;
(4) by step (3) gained mixture, be added dropwise in calcium chloride solution, Quick uniform mixes, and obtains spherical granules; Wherein the concentration of calcium chloride solution is 0.02-0.08g/ml;
(5) after step (4) spherical granules balling-up 5-30min, collect spherical granules, be placed in dilute hydrochloric acid and react, until without obvious Bubble formation, clean;
(6) step (5) gained particle is placed in to calcium chloride solution, soaks, clean;
(7) step (6) gained transfer of granules is entered in proliferated culture medium, shaking table is cultivated, and cleans, and prepares immobilized microspheres.
5. the preparation method of immobilized microspheres as claimed in claim 4, is characterized in that, in the mixture of described step (3), the concentration of sodium alginate is 0.025-0.035g/ml; The concentration of attapulgite is 0.005-0.015g/ml; The concentration of solid carbonic acid calcium is 0.03-0.07g/ml.
6. the preparation method of immobilized microspheres as claimed in claim 5, is characterized in that, the concentration of described petroleum hydrocarbon degradation bacterium is 0.02g/ml; The concentration of sodium alginate is 0.03g/ml; The concentration of attapulgite is 0.01g/ml; The concentration of solid carbonic acid calcium is 0.05g/ml.
7. the preparation method of immobilized microspheres as claimed in claim 4, is characterized in that, in described step (4), the concentration of calcium chloride solution used is 0.02-0.04g/ml.
8. the preparation method of immobilized microspheres as claimed in claim 7, is characterized in that, the concentration of described calcium chloride solution is 0.02g/ml.
9. the preparation method of immobilized microspheres as claimed in claim 4, it is characterized in that, when described step (4) is mixed with calcium chloride, the concentration ratio=1-4 of petroleum hydrocarbon degradation bacterium, sodium alginate, attapulgite, calcium carbonate and calcium chloride: 1-6: 0.5-3: 1-10: 2-8.
10. the preparation method of immobilized microspheres as claimed in claim 9, is characterized in that, the concentration ratio of described petroleum hydrocarbon degradation bacterium, sodium alginate, attapulgite, calcium carbonate and calcium chloride is 1-4: 2.5-3.5: 0.5-1.5: 3-7: 2-4.
The preparation method of 11. immobilized microspheres as claimed in claim 10, is characterized in that, the concentration ratio of described petroleum hydrocarbon degradation bacterium, sodium alginate, attapulgite, calcium carbonate and calcium chloride is 2: 3: 1: 5: 2.
The preparation method of 12. immobilized microspheres as claimed in claim 4, is characterized in that, described step (4) utilizes constant flow pump to drip described mixture to described calcium chloride solution.
13. 1 kinds of immobilized microspheres for remediation of petroleum contaminated soil products calciferous, the preparation method by the immobilized microspheres described in claim 4-12 any one prepares.
14. immobilized microspheres as described in claim 1 or 2 or 13, is characterized in that, the petroleum hydrocarbon degradation bacterium of described immobilized microspheres embedding is Paracoccus sp.D17 or Halomonas sp.D24.
The preparation method of 15. immobilized microspheres as claimed in claim 4, is characterized in that, the petroleum hydrocarbon degradation bacterium of described immobilized microspheres embedding is Paracoccus sp.D17 or Halomonas sp.D24.
Described in 16. claims 1 or 13, immobilized microspheres is for the application of decomposing petroleum hydrocarbon.
Described in 17. claims 1 or 13, immobilized microspheres is for the application at degraded in soil petroleum hydrocarbon.
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| CN102886379A (en) * | 2012-09-28 | 2013-01-23 | 天津莱特化工有限公司 | Method for biologically and intensively repairing petroleum contaminated soil |
| CN103923907B (en) * | 2014-04-29 | 2016-09-14 | 金陵科技学院 | New application of nano-structure calcium carbonate hollow microspheres |
| CN105032919B (en) * | 2015-07-30 | 2017-07-04 | 青岛理工大学 | A kind of biological renovation method at oil pollution sandy beach |
| CN106748092A (en) * | 2016-12-12 | 2017-05-31 | 广州市水之道生态环境修复有限公司 | A kind of microorganism growth regulator with composite construction of lean soil restoration of the ecosystem |
| CN106754546A (en) * | 2017-01-18 | 2017-05-31 | 汕头大学 | One plant of Halomonas bacterial strain and the immobilized microbial inoculum containing it of dissolving dinoflagellate |
| CN106591279A (en) * | 2017-02-08 | 2017-04-26 | 光合强化（北京）生物科技有限公司 | Modified attapulgite used for immobilizing polycyclic aromatic hydrocarbon degrading bacteria, and preparation method thereof |
| CN107523502B (en) * | 2017-10-20 | 2020-04-24 | 刘泽洋 | Method for preparing soluble and releasable particles applied to microbial fermentation culture solution |
| CN107936973B (en) * | 2017-11-05 | 2020-07-24 | 江西环保股份有限公司 | Heavy metal contaminated soil CaCO3Preparation method of/C repairing agent |
| CN107674869A (en) * | 2017-11-06 | 2018-02-09 | 中国科学院寒区旱区环境与工程研究所 | A kind of preparation, measure and the experimental method of attapulgite immobilization oil degradation microbial inoculum |
| CN109054846B (en) * | 2018-06-20 | 2021-01-22 | 上海工程技术大学 | Preparation method of soil conditioner based on layer-by-layer self-assembled microspheres |
| 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 |
| CN111234831B (en) * | 2020-01-15 | 2021-09-10 | 河南科技学院 | Application of sodium alginate and calcium chloride in preparation of cadmium embedding agent, cadmium embedding method and application |
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| CN102199430A (en) | 2011-09-28 |
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