CN101050435A - Solid microbe agent for degrading petroleum pollution, and petroleum products, and preparation method - Google Patents
Solid microbe agent for degrading petroleum pollution, and petroleum products, and preparation method Download PDFInfo
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- CN101050435A CN101050435A CN 200710013906 CN200710013906A CN101050435A CN 101050435 A CN101050435 A CN 101050435A CN 200710013906 CN200710013906 CN 200710013906 CN 200710013906 A CN200710013906 A CN 200710013906A CN 101050435 A CN101050435 A CN 101050435A
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Abstract
This invention discloses a solid microbial agent for degrading petroleum pollutants and petroleum products. The solid microbial agent is prepared by: mixing primary seed solutions of Bacillus subtilis and Sphingobacterium multivorum at a volume ratio of (0.5-20):1, fermenting to obtain mixed bacterial fermentation solution, and mixing the mixed bacterial fermentation solution and turfy soil at a weight ratio of (0.1-1):1. The active living bacteria number of Bacillus subtilis is 106-1010/mL, and that of Sphingobacterium multivorum is 107-1012/mL. The solid microbial agent has such advantages as high activity in soil, rapid growth and no secondary pollution to environment, and can be used for biological bioremediation of petroleum polluted soil and petroleum product leakage fields.
Description
Technical field
The present invention relates to a kind of solid microbial that is used for petroleum pollution and petroleum products degraded, belong to the bioremediation technology field that oil-polluted soils and petroleum products leak the burst accident emergency processing.
Background technology
Biological restoration is meant and adds some materials (as nutrition, O in contaminate environment
2, microorganism) or improve the ability of these materials, the biotechnology that the natural biological degradation process is quickened.As far back as 1976, the leakage of oil that " Marie Antoinette " number causes in Cape of Good Hope is administered with regard to successfully using this biological restoration means.The biological restoration that Exxon valdez in 1989 carries out in the processing of bay, Alaska oil tanker leakage accident has obtained the extensive approval of society, and from then on bioremediation technology is developed rapidly.
Biological restoration is the biotechnology of a class low consumption, efficient and environmental safety, mainly relies on the natural metabolism process degraded of bacterium, fungi even higher plant and cell free enzyme, removes the pollutent in the environment.Up to now, the microorganism of known energy degraded oil hydro carbons is genus surplus in the of totally 100, kind more than 200, and they belong to bacterium, actinomycetes, mould and algae.The bacterium of degraded oil has some bacterial strain in the genus such as Rhodopseudomonas, acinetobacter, Mycosphaerella, Vibrio.Wherein modal is Rhodopseudomonas, found that it contains the plasmid of multiple degradation of hydrocarbon, and once be used for genetically engineered, to the degraded of short chain alkanes such as ethane, propane, butane or long chain alkane and aromatic hydrocarbons, all there is pseudomonas to participate in, and alkane is degraded fully.Bacterial strain during the mould of common degraded oil has that aspergillus, mould, branch spore are mould etc. and belongs to, white-rot fungi is very high to organic degradation efficiencies such as oil.Often decompose oil and produce yeast protein in the yeast with candiyeast.Find again that in recent years as if cyanobacteria and green alga degradable aromatic hydrocarbons, especially cyanobacteria have the ability of the multiple aromatic hydrocarbons of oxidation.
At present, many scholars study with regard to the microbial metabolism approach of hydrocarbon compound, and they think that the committed step of bacterium and fungus degrading is in the presence of molecular oxygen, the process of the oxidized oxydasis of substrate.Normal alkane changes into carboxylic acid earlier and then carries out deep degraded by β-Yang Hua, forms the short chain fatty acid and the acetyl-CoA of two carbosilane units, emits CO
2, alkane also can transfer ketone earlier to, but is not its main metabolic mode.The alkene of multi-branched mainly changes into dicarboxylic acid and degrades, methyl can influence the carrying out of degraded, the degraded of naphthenic hydrocarbon needs two kinds of oxidasic synergistic oxidations, a kind of oxydase is oxidized to cyclic alcohol with it earlier, then dehydrogenation forms glycol, along with the formation of pyrocatechol, ring disconnects, and pyrocatechol is degraded to the intermediate product of tricarboxylic ring then.Fungi can form the intermediate product with different three-dimensional chemical configurations with degradation by bacteria petroleum hydrocarbon degradation petroleum hydrocarbon compound, bacterium almost always becomes the cis glycol with the petroleum hydrocarbon degradation, fungi then almost is degraded into it trans glycol, many trans glycol are potential carcinogenss, and the cis glycol is nontoxicity then.
Fungi can secrete extracellular enzyme, and these extracellular enzymes can be distributed in the edatope, directly contacts degradation of contaminant with petroleum hydrocarbon.The complicacy of fungal enzyme system and the characteristics that can produce extracellular enzyme make easier complex structure, the pollutent that molecular weight is bigger of utilizing of fungi.People such as Li Pei army in 2002 disclose a kind of preparation method (CN1382758A) of solid fungus to degradate petroleum, and this method uses many fungal strains to be prepared into mix bacterium agent, utilizes the Synergistic degradation effect between fungi that it is acted on the biological restoration of oil-polluted soils.But fungi easily produces deleterious mesostate (as trans glycol) in the process of degradable organic pollutant, soil is caused secondary pollution, and this has just limited its applicability.With the bacterium is that the main microbial preparation of forming exactly can remedy this defective.People such as calendar year 2001 Maruyama Akihiko disclose and have adopted Alcanivorax to belong to the method (JP2001037466) that the fungicide preparation that belongs to Bacillus becomes the heavy oil pollutent in mixed biologic preparation degraded ocean or the relevant environment.Bacterium tends to produce bio-surfactants such as some glycolipids, polysaccharide, protein and lipoprotein in the biodegradation process to organic pollution materials, this class material can the cut oil hydrocarbon, but can strengthen the biology availability of petroleum hydrocarbon to a certain extent, improve the validity of biological restoration.
Summary of the invention
The problem that exists in the biological restoration process at existing fungi or bacterial micro-organism microbial inoculum, the invention provides a kind of solid microbial that is used for petroleum pollution and petroleum products degraded, utilize mutual synergy and the characteristics such as high reactivity in peat composed of rotten mosses carrier of mixt bacteria bacterial strain in the decomposing petroleum hydrocarbon class process, improve degradation effect petroleum pollution.
The present invention also provides the preparation method of this solid microbial, comprises the employed bacterial classification for preparing this microbiobacterial agent, and the cultivation and fermentation method.
A kind of solid microbial that is used for the degraded of petroleum pollution and petroleum products, be by primary seed solution 0.5～2: 1 mixed fermentation by volume of subtilis and Sphingobacterium multivorum mixt bacteria fermented liquid and turfy soil by weight proportion 0.1～1: 1 is mixed and made into.
Solid microbial of the present invention is that wherein the living bacteria count of subtilis is 10 by the mixt bacteria fermented liquid of subtilis and Sphingobacterium multivorum and turfy soil 0.1～1: 1 mixture by weight proportion
6～10
10Individual/ml, the living bacteria count of eating sheath ammonia bacillus is 10 more
7～10
12Individual/ml.
The preparation method of solid microbial of the present invention, with by weight proportion 0.1～1 of the mixt bacteria fermented liquid of subtilis (Bacillus subtillus) and Sphingobacterium multivorum and turfy soil: 1 mixes, wherein, the mixt bacteria preparation of fermentation liquid step of described subtilis (Bacillus subtillus) and Sphingobacterium multivorum is as follows:
1, bacterial classification:
Subtilis (Bacillus subtillus), preserving number: CGMCC No.1950,
Sphingobacterium multivorum (Sphingobacterium multivolum), preserving number: CGMCC No.1951,
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC),
Preservation date: on March 7th, 2007.
2, primary seed solution
Above-mentioned bacterial classification respectively after activating 16～32h on the beef extract-peptone slant medium, is transferred to and shakes bottle activation a 16～32h in liquid LB substratum or the beef-protein medium, as primary seed solution.
3, mixt bacteria fermented liquid
With the primary seed solution subtilises of above-mentioned two bacterium and Sphingobacterium multivorum by volume 0.5～2: 1, total inoculum size 2%～10% is transferred in the fermention medium shaking table and cultivates 16～32h, and rotating speed is 150～250rpm, and culture temperature is 20～30 ℃.In the described liquid fermentation liquid, the living bacteria count of subtilis is 10
6～10
10Individual/ml, the living bacteria count of Sphingobacterium multivorum is 10
6～10
12Individual/ml.
The carbon source that fermention medium in the above-mentioned steps 3 is selected, nitrogenous source are cheap raw material: bean cake powder and W-Gum can also have yeast powder.
Preferably, the fermention medium in the above-mentioned steps 3 is as follows: bean cake powder is 2～6g, and W-Gum is 1～3g, and yeast powder is 1～3g, K
2HPO
40.11g, CaCl
20.01g, MgSO
47H
2O 0.15g, MnSO
40.01g, distilled water 100ml.
The application of solid microbial of the present invention when being used for petroleum pollution and petroleum products contaminated site and carrying out degradation treatment, also can be added tensio-active agent and water, stirs, and is sprinkled upon contaminated site.Add the tensio-active agent of tensio-active agent preferred fatty acid polyoxyethylene ester class.
During above-mentioned application, the degree that the addition of solid fungicide and the addition of tensio-active agent can leak according to the degree or the petroleum products of soil pollution be adjusted.
Technical characterstic of the present invention is that the two strain bacteriums that will have efficient degradation crude oil ability carry out the purebred activation of liquid respectively, in same fermentation condition and same fermention medium, carry out mixed culture by a certain percentage, obtain highly active ferment product, mix with the high-quality turfy soil according to a certain percentage then and mix thoroughly, make solid microbial.Adopt solid microbial of the present invention, carried out on-the-spot pilot experiment in the Liaohe Oil Field, a certain proportion of tensio-active agent of compounding application (as the polyoxyethylene carboxylate class), on-the-spot degradation effect is good.Solid microbial provided by the invention has the advantage that activity is high in soil, growth is rapid, environment is not caused secondary pollution.
Solid microbial and the existing microbiobacterial agent that is used for petroleum pollution and petroleum products degraded of the present invention compares, has following advantage: adopt bacterial isolates with efficient degradation crude oil ability, avoided fungal inoculant in the decomposing petroleum hydrocarbon process, to produce the possibility of toxic metabolite, can not cause secondary pollution to ecological environment of soil, and made full use of identical synergy between bacterium, make the microorganism that adds have very high living bacteria count, thereby more help with physical environment in indigenous bacterium competition nutrition, selected on the other hand solid carrier is to be rich in organic turfy soil, quality is loose, nutritious, microorganism is adsorbed in the peat composed of rotten mosses carrier and can breeds by secondary, and the biological restoration effect of contaminated soil is improved greatly.Solid fungicide preparation process of the present invention is simple to operation, with low cost, has broad application prospects in the biological restoration field in oil-polluted soils and petroleum products leakage accident place.
Embodiment
Two strain bacteriums of the present invention are respectively subtilis (Bacillus subtillus) and Sphingobacterium multivorum (Sphingobacterium multivolum).Wherein a bacillus subtilis separates the earth from China Liaohe Oil Field, another strain Sphingobacterium multivorum separates the earth from Canadian Alberta oil field, the culture presevation of subtilis number is CGMCC No.1950, and the culture presevation of Sphingobacterium multivorum number is CGMCC No.1951.
Embodiment 1: the mixt bacteria preparation of fermentation liquid
The bacterium of adopting is respectively subtilis (Bacillus subtillus) and Sphingobacterium multivorum (Sphingobacterium multivolum).With its respectively through slant culture, shake a bottle activation culture, shake flask fermentation and cultivate and obtain ferment product, the concrete operations step is:
(1) slant medium: adopt ordinary method to prepare the beef extract-peptone solid medium, concrete steps are: extractum carnis 3g, and peptone 5g, NaCl 5g, distilled water 1000ml, pH7.2～7.4, agar 1.5%～2% stirs, in 1.05kg/cm
2, 20～30min sterilizes under 121.3 ℃ of conditions.Sterilization back beveling, on Bechtop, the bacterial strain of aseptic inoculation preservation is to the test tube slant respectively, and bacterial classification is cultivated 16～32h under 25～30 ℃ of conditions, and it is standby to treat that lawn covers with the inclined-plane;
(2) preparation of seed liquor: liquid nutrient medium still is a beef-protein medium, and the liquid nutrient medium branch for preparing is packed in 150～250ml triangular flask, and liquid amount is 20～60ml, with 121.3 ℃ of sterilization 30min.To be cooled to about 40 ℃, picking one ring inclined-plane lawn is inoculated in the triangular flask, places on the shaking table then and cultivates, and adjustings shaking speed is 150～250rpm, cultivates 16～32h under 20～30 ℃ of conditions, cultivation in the fermention medium for the treatment of to pack into after the substratum muddiness.
(3) mixt bacteria preparation of fermentation liquid: the seed liquor of the two strain bacteriums that will activate respectively, the mixed volume ratio of two strain bacterium bacterium liquid is 0.5: 1, inoculum size according to 2%～10% (v/v) inserts in the fermention medium, fermentative medium formula is as follows: bean cake powder is 2～6g, W-Gum is 1～3g, yeast powder is 1～3g, K
2HPO
40.11g, CaCl
20.01g, MgSO
47H
2O 0.15g, MnSO
40.01g, distilled water 100ml.Inoculation is placed on cultivates 22h on the shaking table, temperature is 20～30 ℃, and shaking speed is 150～250rpm.Obtain highly active mixed fermentation liquid under this condition, the living bacteria count of subtilis is 10
7～10
9Individual/ml, the living bacteria count of Sphingobacterium multivorum is 10
8～10
11Individual/ml.
Embodiment 2: the mixt bacteria preparation of fermentation liquid
As described in embodiment 1, different is, changes the ratio of the seed liquor of two strain bacteriums in the step (3), and subtilis and Sphingobacterium multivorum mixed volume ratio are 2.0: 1, obtain highly active mixed fermentation liquid under this condition, the living bacteria count of subtilis is 10
6～10
10Individual/ml, the living bacteria count of Sphingobacterium multivorum is 10
6～10
12Individual/ml.
3: two strain bacteriums of embodiment are tested the degradation capability of crude oil
The two strain bacteriums that activation is good, picking one ring lawn from its inclined-plane respectively, after in the beef extract-peptone liquid nutrient medium, activating 16～32h, make certain density bacteria suspension, be transferred in the crude oil substratum according to 2～10% inoculum size again, place on 20～30 ℃ of shaking tables and cultivate 5～12d, rotating speed is 150～250rpm.With the substratum that does not connect bacterium is blank.Cultivate when finishing, in nutrient solution, add methylene dichloride (DCM), the centrifugal O/w emulsion of breaking, organic phase is placed in the beaker of constant weight in advance with the granular anhydrous sodium sulphate filter dehydration, room temperature nitrogen stripping adopts Residual oil content in the gravimetric determination nutrient solution to constant weight.Formula calculates the biological degradation rate (η %) of petroleum hydrocarbon below adopting:
η(％)＝ω
0-ω
x/ω
0×100
In the formula: ω
0Be Residual oil content in the contrast culture liquid; ω
xBe Residual oil content in the test bacteria culture fluid.
From following table as can be seen, this two strains bacterium degradation capability to crude oil in liquid shaking bottle is all higher, and wherein the degradation capability during two strain bacterium mixed culture is the highest, reaches 51.73% (the 4th row sees the following form).
The determination experiment of each treatment samples degrading crude oil
| Handle sample | Initial oleaginousness (g) | Remaining oleaginousness (g) | Degradation rate (%) |
| Subtilis Sphingobacterium multivorum subtilis+Sphingobacterium multivorum blank | 0.3266 0.3254 0.3211 0.3354 | 0.2159 0.1747 0.1550 0.2922 | 33.89 46.31 51.73 12.88 |
Embodiment 4: the preparation method of solid fungicide, and the concrete operations step is:
(1) prepares the mixt bacteria fermented liquid of subtilis and Sphingobacterium multivorum by the method for embodiment 1.
(2) take by weighing turfy soil 100g, in the 500mL beaker of packing into, the wrapping back is in 121.3 ℃ of sterilization 30min～2h, and the sterilization back is stand-by.
(3) be to mix at 0.5: 1 in proportion with the mixt bacteria fermented liquid of step (1) and the peat composed of rotten mosses of step (2), on Bechtop, fully mix thoroughly, place under the room temperature condition to store.Measure the living bacteria count of bacterium in the made solid fungicide two days later, wherein the living bacteria count of subtilis is 10
7～10
9Individual/ml, the living bacteria count of Sphingobacterium multivorum is 10
8～10
11Individual/ml.
Embodiment 5: the preparation method of solid fungicide
As described in embodiment 4, the blending ratio that different is changes mixt bacteria fermented liquid and the peat composed of rotten mosses is 1: 1 o'clock, measures the living bacteria count of the interior bacterium of made solid fungicide two days later, and wherein the living bacteria count of subtilis is 10
6～10
8Individual/ml, the living bacteria count of Sphingobacterium multivorum is 10
8～10
11Individual/ml.
Embodiment 6: the test of solid fungicide effect
A certain Polluted area carries out the effect of field application test to the solid fungicide that adopts the present invention to prepare in the Liaohe Oil Field.Concrete test method is:
The original position reparation is simulated at the scene, and the heatable adobe sleeping platform that to dig 3 length of sides be the dark 0.5m of 2m (is numbered 1
#, 2
#, 3
#), get oil-containing soil 6m
3, stir, be divided into three parts, 1
#The zone is a blank, adds entry in the soil, 2
#The zone is for adding solid fungicide, tensio-active agent polyoxyethylene carboxylate and the water of about 500 grams, 3
#The zone is a certain amount of active sludge (being mainly indigenous bacterium) and water.These soil of mixing thoroughly are added in the hole of digging, stir soil week about once, the wind reinforcement that works of going forward side by side, the sampling and measuring total petroleum hydrocarbons content tests being treated to three repetitions every three days, to reduce testing error.The mensuration of total petroleum hydrocarbons content adopts conventional weighting method, and calculation formula is: heavy * (the 1-water ratio %) * 1000 of residue weight/aquatic foods soil in oil total hydrocarbon (g/kg)=evaporative flask.After carrying out 2 months by a definite date reparation at the scene.1
#The eventual degradation rate of blank is 40.189%.2
#The degradation rate of solid fungicide is 75.826%, 3
#The degradation rate of active sludge is 45.743%.This shows that the repairing effect optimum of solid peat composed of rotten mosses microbial inoculum has reached more than 70%.
Claims (10)
1, subtilis, culture presevation number is CGMCC No.1950.
2, Sphingobacterium multivorum, culture presevation number is CGMCC No.1951.
3, a kind of solid microbial that is used for the degraded of petroleum pollution and petroleum products, be by primary seed solution 0.5～2: 1 mixed fermentation by volume of the Sphingobacterium multivorum of the subtilis of claim 1 and claim 2 mixt bacteria fermented liquid and turfy soil by weight proportion 0.1～1: 1 is mixed and made into.
4, solid microbial as claimed in claim 3, wherein the living bacteria count of subtilis is 10
6～10
10Individual/ml, the living bacteria count of eating sheath ammonia bacillus is 10 more
7～10
12Individual/ml.
5, a kind of preparation method of solid microbial, with the mixt bacteria fermented liquid of subtilis and Sphingobacterium multivorum and turfy soil by weight proportion 0.1～1: 1 mixes, wherein, the mixt bacteria preparation of fermentation liquid of described subtilis and Sphingobacterium multivorum, step is as follows:
(1) bacterial classification
Subtilis, preserving number: CGMCC No.1950,
Sphingobacterium multivorum, preserving number: CGMCC No.1951,
(2) primary seed solution
Above-mentioned bacterial classification respectively after activating 16～32h on the beef extract-peptone slant medium, is transferred to and shakes bottle activation a 16～32h in liquid LB substratum or the beef-protein medium, as primary seed solution,
(3) mixt bacteria fermented liquid
With the primary seed solution subtilises of above-mentioned two bacterium and Sphingobacterium multivorum by volume 0.5～2: 1, total inoculum size 2%～10% is transferred in the fermention medium shaking table and cultivates 16～32h, and rotating speed is 150～250rpm, and culture temperature is 20～30 ℃.
6, the preparation method of solid microbial as claimed in claim 5 is characterized in that selected carbon source, the nitrogenous source of fermention medium in the described step (3) is bean cake powder and W-Gum.
7, the preparation method of solid microbial as claimed in claim 5 is characterized in that the fermention medium in the described step (3) also has yeast powder.
8, the preparation method of solid microbial as claimed in claim 5, it is characterized in that the fermention medium in the described step (3) is as follows: bean cake powder is 2～6g, and W-Gum is 1～3g, and yeast powder is 1～3g, K
2HPO
40.11g, CaCl
20.01g, MgSO
47H
2O 0.15g, MnSO
40.01g, distilled water 100ml.
9, the application of the described solid microbial of claim 3 is being used for petroleum pollution and the petroleum products contaminated site carries out degradation treatment.
10, the application of solid microbial as claimed in claim 9 wherein also is added with tensio-active agent and water, stirs, and is sprinkled upon contaminated site.
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