CN101717725A - Microbial inoculum for bioremediating coastline polluted by oil spilling and preparation method thereof - Google Patents
Microbial inoculum for bioremediating coastline polluted by oil spilling and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a microbial inoculum for bioremediating a coastline polluted by oil spilling and a preparation method thereof. The microbial inoculum for bioremediating the coastline polluted by oil spilling is made of mixed fermentation liquor and a fixed carrier as raw materials, wherein the mixed fermentation liquor comprises bacillus pumilus CGMCCnO.3384 fermentation liquor, Venice acinetobacter CGMCCnO.3385 fermentation liquor and/or Doxa pseudomonad CGMCCnO.3386 fermentation liquor and/or Ralstonia pickettii CGMCCnO.3387 fermentation liquor. By using the synergic action of different strains on the degradation of various petroleum hydrocarbons, the microbial inoculum improves the degradation speed of petroleum hydrocarbon pollutants and thoroughly degrades various petroleum hydrocarbon pollutants; in addition, a flora formed by a plurality of strains of bacteria has more stable structure and can better adapt to various petroleum-polluted environments. The microbial inoculum rapidly grows on the polluted areas, forms competitive advantages and remarkably increases the quantity of hydrocarbon degraded microbes in the polluted areas, thereby improving the biodegradation rate of crude oil.
Description
Technical field
The invention belongs to oil and petroleum products and pollute bioremediation technology field, shoreline, relate to a kind of microbial inoculum for bioremediating coastline polluted by oil spilling and preparation method particularly.
Background technology
Along with the world growing to oil and goods demand thereof, at sea exploit, transport, load and unload and utilize the oil spill accident in the oil process also day by day to increase.Oil spilling not only causes serious environmental to pollute, and because the genotoxic potential and the biological accumulation effect of petroleum hydrocarbon class pollutant can cause Environment of Alongshore Sea Area quality and biological species diversity index seriously to descend, destroy the function of the ecosystem, also can cause enormous economic loss water industry and tourism.In addition, the polycyclic arene compound in the petroleum hydrocarbon has intensive teratogenesis, carcinogenic and mutagenic effect, may enter human body by food chain, and HUMAN HEALTH is had very big potential hazard.Safety, economic, eco-friendly oil spill treatment process are the research focuses in present environmental pollution improvement field.
Biological restoration be meant utilize biology particularly microorganism come the catalyzed degradation environmental pollutant, reduce or finally eliminate the controlled or spontaneous process of environmental pollution, be the emerging green technology that on the microbiological deterioration basis, grows up.It is little, simple to operate to have investment, is difficult for producing advantages such as secondary pollution, has become the on-the-spot important selection approach of removing petroleum hydrocarbon class pollutant.Late 1980s, the U.S. was in the biological restoration project that Exxon Vadez oil tanker oil is revealed, and removed oil spill in the short period of time, was the beginning of biological restoration successful Application, also started the application beginning of biological restoration in administering pollution of the sea simultaneously.
In oil spill biological restoration process, petroleum hydrocarbon degradation bacterium is the executive of oil degradation, is again core drive wherein.Although having a high potential of indigenous microorganism degradation of contaminant, the domestication time is long, the speed of growth is slow, metabolic activity is low.Biological reinforcedly promptly add contaminant degradation microbial inoculum efficiently, significantly improving the contaminant degradation bacterial classification group and the quantity of contaminated site, thereby improve contaminant degradation efficient, become one of important technical of biological restoration at contaminated site.The microorganism of these inoculations is called as the pioneer microorganism, can enrichment from indigenous microorganism, also can obtain from other zone, and perhaps use genetic engineering bacterium.
Contain multiple complicated ingredients such as various alkane, aromatic hydrocarbons and polycyclic aromatic hydrocarbons in the oil.Studies show that, the petroleum hydrocarbon degradation bacterium of different genera is to the adaptation of different components in the crude oil and utilize the ability difference, a kind of microorganism can only utilize one or more petroleum hydrocarbons, therefore single microorganism can not be finished the degradation process of oil fully, must just may realize the degraded fully of petroleum pollution by the microorganism acting in conjunction of multiple difference in functionality.Make up and mix the petroleum hydrocarbon degradation flora efficiently, antagonistic action between each bacterial strain strengthens between bacterial strain acting synergistically in the reduction mixed bacterial, farthest brings into play the function of every strain bacterium, is the key that improves the biological reinforced efficient of oil spill.
In view of the complicacy of petroleum hydrocarbon contaminant structure and the diversity of kind, select multiple microorganism to form a biosystem, allow wherein each bacterium bring into play its best physiological function, and utilize it to act synergistically degradation of contaminant expeditiously, meet need of practice.People such as Li Peijun disclose a kind of method that adopts the microbial inoculum of the degraded oil that multiple fungi is prepared in 2002, and be applied to the biological restoration of oil-polluted soils, wherein, mixed bacterium is each the single fungi that screens to be cultivated the back equal proportion respectively mix (the publication number: CN 1382758A) that obtains; The king adds peaceful grade and discloses solid microbe of a kind of degraded oil pollutent and petroleum products and preparation method thereof (publication number: CN 101050435A), can be applicable in the bioremediation technology of burst accident emergency processings such as oil-polluted soils and petroleum products leakage, wherein, microbiobacterial agent is to be made by subtilis and Sphingobacterium multivorum mixed fermentation.From domestic and international existing result of study, though the effect of cultivating in biological degradation for mixed bacterium has obtained fully certainly, the part achievement has been applied to reality, and it is still not deep enough that mixed bacterium is cultivated synergistic Study on Mechanism.
Summary of the invention
The purpose of this invention is to provide a kind of bioremediating coastline polluted by oil spilling bacterium.
Second purpose of the present invention provides a kind of microbial inoculum for bioremediating coastline polluted by oil spilling.
The 3rd purpose of the present invention provides a kind of preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling.
Technical scheme of the present invention is summarized as follows:
The bioremediating coastline polluted by oil spilling bacterium, form by following bacterium: bacillus pumilus (Bacillus pumilus), protect a surname in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.3384; Venice acinetobacter calcoaceticus (Acinetobacter venetianus), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.3385 and/or pseudomonas mendocina (Pseudomonas mendocina), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.3386 and/or Pi Shi Rolston bacterium (Ralstonia pickettii), be preserved in Chinese microorganism strain and protect good management committee common micro-organisms center, protecting minus sign is CGMCC No.3387.
A kind of microbial inoculum for bioremediating coastline polluted by oil spilling, ratio in 1.0L～3.0L: 1kg is that raw material is made by mixed fermentation liquid and fixation support, and described mixed fermentation liquid comprises bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and/or pseudomonas mendocina CGMCC No.3386 fermented liquid and/or Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid.
The bacteria concentration of described bacillus pumilus CGMCC No.3384 fermented liquid is 1 * 10
6Individual/ml～1 * 10
10The bacteria concentration of individual/ml, described Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid is 1 * 10
6Individual/ml～1 * 10
10The bacteria concentration of individual/ml, described pseudomonas mendocina CGMCC No.3386 fermented liquid is 1 * 10
6Individual/ml～1 * 10
10The bacteria concentration of individual/ml, described Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid is 1 * 10
6Individual/ml～1 * 10
10Individual/ml, the volume ratio of described bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid, pseudomonas mendocina CGMCC No.3386 fermented liquid and Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid is 1～10: 1～10: 0～4: 0～4.
The volume ratio of described bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid, pseudomonas mendocina CGMCC No.3386 fermented liquid and Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid is 5～8: 2～8: 0.5～3: 0.5～3.
Described fixation support is that diatomite, kaolin, the peat composed of rotten mosses and chitin are at least a.
The preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling comprises the steps:
(1) bacillus pumilus CGMCC No.3384, Venice acinetobacter calcoaceticus CGMCC No.3385 and/or pseudomonas mendocina CGMCC No.3386 and/or Pi Shi Rolston bacterium CGMCC No.3387 are cultivated and fermentor cultivation obtains fermented liquid through slant culture, shake-flask seed respectively;
(2) be 1 * 10 with bacteria concentration
6Individual/ml～1 * 10
10The bacillus pumilus CGMCC No.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
6Individual/ml～1 * 10
10Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and/or the bacteria concentration of individual/ml are 1 * 10
6Individual/ml～1 * 10
10Pseudomonas mendocina CGMCC No.3386 fermented liquid and/or the bacteria concentration of individual/ml are 1 * 10
6Individual/ml～1 * 10
10The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml is 1～10: 1～10 by volume: be mixed into mixed fermentation liquid at 0～4: 0～4;
(3) in 1.0L～3.0L: 1 kilogram ratio mixes described mixed fermentation liquid and fixation support, and lyophilize promptly obtains microbial inoculum for bioremediating coastline polluted by oil spilling.
The volume ratio of the described bacillus pumilus CGMCC of described step (2) No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCCNo.3385 fermented liquid, pseudomonas mendocina CGMCC No.3386 fermented liquid, Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid is 5～8: 2～8: 0.5～3: 0.5～3.
Described fixation support is that diatomite, kaolin, the peat composed of rotten mosses and chitin are at least a.
Advantage of the present invention is:
(1) will carry out composite to several bacterial strains that the petroleum hydrocarbon different components has different degradation properties, utilize the synergy of different strains to the different sorts petroleum hydrocarbon degradation, accelerate the degradation rate of petroleum hydrocarbon contaminant, all kinds of petroleum hydrocarbon contaminants of comparatively thorough degraded, and the flora structure that multi-strain bacteria is formed is more stable, can better adapt to all kinds of oil polluted environments.
(2) absorbing and transforming in the process of alkane, bacterial strain Venice acinetobacter calcoaceticus (Acinetobacter venetianus) of acinetobacter (Acinetobacter sp.) can produce the glycolipid type biological surfactant, this bio-surfactant can play the effect of emulsification solubilising to the petroleum hydrocarbon of contaminated site, helps petroleum hydrocarbon degradation bacterium absorbing hydro carbons.
(3) compare with the indigenous microorganism of contaminated site, microbial inoculum of the present invention can form competitive edge, and significantly increase contaminated site hydrocarbon degradation microbial numbers in the contaminated site growth rapidly, thereby improves the biological degradation rate of crude oil.
Description of drawings
Fig. 1 is for adding the degradation rate of single strain and 60 days crude oil of mix bacterium agent system.
Wherein, TJ-1 represents to add separately the degraded system of TJ-1 bacterium; TJ-2 represents to add separately the degraded system of TJ-1 bacterium; DXH-1 represents to add separately the degraded system of DXH-1 bacterium; DXH-2 represents to add separately the degraded system of DXH-2 bacterium; BXHH-1, BXHH-2, BXHH-3, BXHH-4, BXHH-5 and BXHH-6 represent to add the system of TJ-1, TJ-2, DXH-1 and DXH-2 mixed bacterium, the blending ratio of various bacterium was respectively 4: 4: 1: 1,5: 2: 3: 3,8: 1: 0.5: 0.5,10: 8: 0: 4,1: 10: 4: 0 and 1: 1: 0: 0.
Fig. 2 is for adding the GC-MS figure of 60 days crude oil bubble point oils of TJ-1 single strain system hydrocarbon component.
Fig. 3 is for adding the GC-MS figure of 60 days crude oil bubble point oils of TJ-2 single strain system hydrocarbon component.
Fig. 4 is for adding the GC-MS figure of 60 days crude oil bubble point oils of DXH-1 single strain system hydrocarbon component.
Fig. 5 is for adding the GC-MS figure of 60 days crude oil bubble point oils of DXH-1 single strain system hydrocarbon component.
Fig. 6 is for adding the GC-MS figure that mixed bacterium BXHH-1 thalline is 60 days crude oil bubble point oil hydrocarbon components.
Fig. 7 is for adding the GC-MS figure that mixed bacterium BXHH-2 thalline is 60 days crude oil bubble point oil hydrocarbon components.
Fig. 8 is single strain genomic dna-PCR result.From left to right be followed successively by DXH-1, DXH-2, TJ-1, TJ-2 and mark, Mark is respectively 2000,1000,750,500,250 from top to bottom, 100bp.
Fig. 9 is the bacterial strain TJ-1 phylogenetic tree based on the 16SrDNA sequence homology.
Figure 10 is the bacterial strain TJ-2 phylogenetic tree based on the 16SrDNA sequence homology.
Figure 11 is the phylogenetic tree based on the bacterial strain DHX-1 of 16SrDNA sequence homology.
Figure 12 is the phylogenetic tree based on the bacterial strain DHX-2 of 16SrDNA sequence homology
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
The bacillus pumilus of the bioremediating coastline polluted by oil spilling bacterium that the present invention relates to (Bacillus pumilus) is called for short: TJ-1; Venice acinetobacter calcoaceticus (Acinetobacter venetianus) is called for short: TJ-2; Pseudomonas mendocina (Pseudomonasmendocina) is called for short: DXH-1; Pi Shi Rolston bacterium (Ralstonia pickettii) is called for short: DXH-2.Four strain bacterium all separate in the settling of PORT OF TIANJIN six numbering heads.The living bacteria count of four kinds of bacterium is 10
8Individual/more than the g.
Four kinds of bacterial strains that the present invention relates to are protected a surname on November 3rd, 2009 and are protected good management committee common micro-organisms center in Chinese microorganism strain, and the preserving number of bacillus pumilus (Bacillus pumilus) is CGMCC No.3384; The preserving number of Venice acinetobacter calcoaceticus (Acinetobacter venetianus) is that the preserving number of CGMCC No.3385, pseudomonas mendocina (Pseudomonas mendocina) is that the preserving number of CGMCC No.3386, Pi Shi Rolston bacterium (Ralstoniapickettii) is CGMCC No.3387.And prove that four kinds of bacterial strains all survive.
The bioremediating coastline polluted by oil spilling bacterium is made up of following bacterium: bacillus pumilus CGMCC No.3384, Venice acinetobacter calcoaceticus CGMCC No.3385, pseudomonas mendocina CGMCC No.3386 and Pi Shi Rolston bacterium CGMCCNo.3387.
The bioremediating coastline polluted by oil spilling bacterium is made up of following bacterium: bacillus pumilus CGMCC No.3384, Venice acinetobacter calcoaceticus CGMCC No.3385 and pseudomonas mendocina CGMCC No.3386.
The bioremediating coastline polluted by oil spilling bacterium is made up of following bacterium: bacillus pumilus CGMCC No.3384, Venice acinetobacter calcoaceticus CGMCC No.3385 and Pi Shi Rolston bacterium CGMCC No.3387.
The bioremediating coastline polluted by oil spilling bacterium is made up of following bacterium: bacillus pumilus CGMCC No.3384 and Venice acinetobacter calcoaceticus CGMCC No.3385.
Bacillus pumilus CGMCC No.3384, Venice acinetobacter calcoaceticus CGMCC No.3385 are alkane degradation bacterial, and pseudomonas mendocina CGMCC No.3386 and Pi Shi Rolston bacterium CGMCC No.3387 are the aromatic hydrocarbons degradation bacteria.By a large amount of evidences, bioremediating coastline polluted by oil spilling bacterium of the present invention has synergy to the degraded of petroleum hydrocarbon, can significantly improve the degradation efficiency of petroleum pollution.
Microbial inoculum for bioremediating coastline polluted by oil spilling, ratio in 2.0L: 1kg is that raw material is made by mixed fermentation liquid and diatomite, and mixed fermentation liquid comprises bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCCNo.3385 fermented liquid, pseudomonas mendocina CGMCC No.3386 fermented liquid and Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid.
Microbial inoculum for bioremediating coastline polluted by oil spilling, ratio in 1.0L: 1kg is that raw material is made by mixed fermentation liquid and kaolin, and mixed fermentation liquid comprises bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCCNo.3385 fermented liquid and pseudomonas mendocina CGMCC No.3386 fermented liquid.
Embodiment 7
Microbial inoculum for bioremediating coastline polluted by oil spilling, ratio in 3.0L: 1kg is that raw material is made by the mixed fermentation liquid and the peat composed of rotten mosses, and mixed fermentation liquid comprises bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid.
Embodiment 8
Microbial inoculum for bioremediating coastline polluted by oil spilling, is that 1: 1 peat composed of rotten mosses and chitin are that raw material is made in the ratio of 2.0L: 1kg by mixed fermentation liquid and mass ratio, and mixed fermentation liquid comprises bacillus pumilus CGMCC No.3384 fermented liquid and Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid.
Embodiment 9
The fermentation of bacterium can adopt conventional fermentation process to carry out, and following public method is wherein a kind of, does not limit the present invention.
1, slant culture
With the four kinds of bacterium bacillus pumilus (Bacillus pumilus) that the present invention relates to, preserving number is CGMCC No.3384, is called for short TJ-1; Venice acinetobacter calcoaceticus (Acinetobacter venetianus), preserving number is called for short TJ-2, pseudomonas mendocina (Pseudomonas mendocina) for CGMCC No.3385, protect minus sign and be called for short DXH-1, Pi Shi Rolston bacterium (Ralstonia pickettii) for CGMCC No.3386, preserving number is inoculated into respectively on the 2216E culture medium slant for CGMCC No.3387 is called for short DXH-2, after cultivating 72h under 25 ℃, standby.Wherein, the inclined-plane preparation method is as follows for the agent of 2216E solid culture: take by weighing the 5.0g peptone respectively, 1.0g yeast soaks powder, 0.01g high ferric phosphate, stirring and dissolving is in 1000mL ageing seawater successively, take by weighing the 20g agar powder then, after boiling dissolving, transfer pH 7.6～7.8, behind sterilization 20min under 121 ℃, in ultra-clean work, each specification is to pour the prepared 2216E substratum in the 5mL left and right sides in 180mm * 18mm (length * diameter) test tube into, tilt to place, obtain the 2216E culture medium slant after the cooling.
2, shake-flask seed is cultivated
With the bacterial classification on the above-mentioned inclined-plane, aseptic technique on Bechtop is inoculated into respectively in the 500mL Erlenmeyer flask of the 2216E liquid nutrient medium that fills 200mL, and 25 ℃, 200r/min cultivates 72h, and is standby.Above-mentioned 2216E liquid nutrient medium preparation method is as follows: take by weighing the 5.0g peptone respectively, 1.0g yeast soaks powder, 0.01g high ferric phosphate, stirring and dissolving is in 1000mL ageing seawater successively, divide and install in the 500mL triangular pyramidal bottle, every bottled liquid measure is 200mL, and in 121 ℃ of 20min that sterilize down, every kind of mycetocyte density reaches 5 * 10
7Individual/mL.
3, fermentor cultivation
Above-mentioned seed culture fluid is inserted respectively in the 5L fermentor tank with 5% inoculum size, the sterilized fermention medium of 2.0L wherein is housed.In the fermenting process, control pH is 7.2, and culture temperature is 25 ℃, and rotating speed is 600r/min, and air flow is 2.0L/min, cultivates 60h, and every kind of mycetocyte density reaches 2 * 10
8Individual/mL.Above-mentioned fermention medium preparation method is as follows: take by weighing 60.0g glucose, 140.0g whiteruss, 8.0gKH respectively
2PO
4, 12.0gK
2HPO
4, 20.0g (NH
4)
2SO
4, 40.0gNaCl, 0.4g MgSO
47H
2O, 0.02gCaCl
2, 0.02g Na
2-EDTA adds in the distilled water of 1500mL successively, and after the stirring and dissolving, adds the 10mL trace element solution, is supplemented to 2000mL with distilled water, and pH transfers to 7.5, in 121 ℃ of 20min that sterilize down.Wherein, trace element solution consists of (g/L): ZnSO
47H
2O 0.29; MnSO
4H
2O 0.24; CuSO
47H
2O0.25; (NH
4)
6Mo
7O
244H
2O, 0.17.
Microbial inoculum for bioremediating coastline polluted by oil spilling is that raw material is made in the ratio of 2.0L: 1kg by mixed fermentation liquid and diatomite, and mixed fermentation liquid comprises that volume ratio is 4: 4: 1: 1 bacteria concentration is 1 * 10
8The bacillus pumilus CGMCCNo.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Pseudomonas mendocina CGMCC No.3386 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
8The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml.
Embodiment 11
Microbial inoculum for bioremediating coastline polluted by oil spilling is that raw material is made in the ratio of 2.0L: 1kg by mixed fermentation liquid and diatomite, and mixed fermentation liquid comprises that volume ratio is 5: 2: 3: 3 bacteria concentration is 1 * 10
8The bacillus pumilus CGMCCNo.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Pseudomonas mendocina CGMCC No.3386 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
8The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml.
Embodiment 12
Microbial inoculum for bioremediating coastline polluted by oil spilling is that raw material is made in the ratio of 2.0L: 1kg by mixed fermentation liquid and diatomite, and mixed fermentation liquid comprises that volume ratio is 8: 2: 0.5: 0.5 bacteria concentration is 1 * 10
8The bacillus pumilus CGMCCNo.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Pseudomonas mendocina CGMCC No.3386 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
8The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml.
Embodiment 13
Microbial inoculum for bioremediating coastline polluted by oil spilling is that raw material is made in the ratio of 2.0L: 1kg by mixed fermentation liquid and kaolin, and mixed fermentation liquid comprises that volume ratio is that 1: 10: 4 bacteria concentration is 1 * 10
8The bacillus pumilus CGMCCNo.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
8The pseudomonas mendocina CGMCC No.3386 of individual/ml.
Embodiment 14
Microbial inoculum for bioremediating coastline polluted by oil spilling is that raw material is made in the ratio of 1.0L: 1kg by mixed fermentation liquid and chitin, and mixed fermentation liquid comprises that volume ratio is that 10: 8: 4 bacteria concentration is 1 * 10
6The bacillus pumilus CGMCCNo.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
6Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
8The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml.
Embodiment 15
Microbial inoculum for bioremediating coastline polluted by oil spilling is that raw material is made in the ratio of 3.0L: 1kg by the mixed fermentation liquid and the peat composed of rotten mosses, and mixed fermentation liquid comprises that volume ratio is that 1: 1 bacteria concentration is 1 * 10
10Bacillus pumilus CGMCC No.3384 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
10Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid of individual/ml.
Embodiment 16
The preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling comprises the steps:
(1) bacillus pumilus CGMCC No.3384, Venice acinetobacter calcoaceticus CGMCC No.3385, pseudomonas mendocina CGMCC No.3386 and Pi Shi Rolston bacterium CGMCC No.3387 are cultivated and fermentor cultivation acquisition fermented liquid through slant culture, shake-flask seed respectively;
(2) be 1 * 10 with bacteria concentration
8The bacillus pumilus CGMCC No.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Pseudomonas mendocina CGMCC No.3386 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
8The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml is 4: 4: 1 by volume: 1 mixed evenly becomes mixed fermentation liquid;
(3) in 2.0L: 1 kilogram ratio mixes mixed fermentation liquid and diatomite, and lyophilize promptly obtains microbial inoculum for bioremediating coastline polluted by oil spilling.
Embodiment 17
The preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling comprises the steps:
(1) with embodiment 16;
(2) be 1 * 10 with bacteria concentration
8The bacillus pumilus CGMCC No.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Pseudomonas mendocina CGMCC No.3386 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
8The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml is 5: 2: 3 by volume: 3 mixed evenly becomes mixed fermentation liquid;
(3) in 2.0L: 1 kilogram ratio mixes mixed fermentation liquid and diatomite, and lyophilize promptly obtains microbial inoculum for bioremediating coastline polluted by oil spilling.
Embodiment 18
The preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling comprises the steps:
(1) with embodiment 16;
(2) be 1 * 10 with bacteria concentration
8The bacillus pumilus CGMCC No.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid of individual/ml, bacteria concentration are 1 * 10
8Pseudomonas mendocina CGMCC No.3386 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
8The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml is 8: 2: 0.5 by volume: 0.5 mixed evenly becomes mixed fermentation liquid;
(3) in 2.0L: 1 kilogram ratio mixes described mixed fermentation liquid and diatomite, and lyophilize promptly obtains microbial inoculum for bioremediating coastline polluted by oil spilling.
Embodiment 19
The preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling comprises the steps:
(1) with embodiment 16;
(2) be 1 * 10 with bacteria concentration
6The bacillus pumilus CGMCC No.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
6Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and bacteria concentration be 1 * 10
6The pseudomonas mendocina CGMCC No.3386 fermented liquid of individual/ml is that 1: 10: 4 mixed evenly becomes mixed fermentation liquid by volume;
(3) in 2.0L: 1 kilogram ratio is that 1: 1 the kaolin and the peat composed of rotten mosses mixes with mixed fermentation liquid and mass ratio, and lyophilize promptly obtains microbial inoculum for bioremediating coastline polluted by oil spilling.
The preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling comprises the steps:
(1) with embodiment 16;
(2) be 1 * 10 with bacteria concentration
6The bacillus pumilus CGMCC No.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
6Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
6The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml is that 10: 8: 4 mixed evenly becomes mixed fermentation liquid by volume;
(3) in 1.0L: 1 kilogram ratio is that 1: 1 the kaolin and the peat composed of rotten mosses mixes with mixed fermentation liquid and mass ratio, and lyophilize promptly obtains microbial inoculum for bioremediating coastline polluted by oil spilling.
Embodiment 21
The preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling comprises the steps:
(1) with embodiment 16;
(2) be 1 * 10 with bacteria concentration
10Bacillus pumilus CGMCC No.3384 fermented liquid and the bacteria concentration of individual/ml are 1 * 10
10Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid of individual/ml is 1: 1 by volume: be mixed into mixed fermentation liquid;
(3) in 3.0L: 1 kilogram ratio mixes mixed fermentation liquid and chitin, and lyophilize promptly obtains microbial inoculum for bioremediating coastline polluted by oil spilling.
Embodiment 22
To carry out composite to several bacterial strains that the petroleum hydrocarbon different components has different degradation properties, experimental results show that different strains has synergy can accelerate the degradation rate of petroleum hydrocarbon contaminant to the different sorts petroleum hydrocarbon degradation, all kinds of petroleum hydrocarbon contaminants of comparatively thorough degraded, and the flora structure that multi-strain bacteria is formed is more stable, can better adapt to all kinds of oil polluted environments.(see Fig. 1-Fig. 7)
Experimental results show that, absorbing and transforming in the process of alkane, bacterial strain Venice acinetobacter calcoaceticus (Acinetobacter venetianus) of acinetobacter (Acinetobacter sp.) can produce the glycolipid type biological surfactant, this bio-surfactant can play the effect of emulsification solubilising to the petroleum hydrocarbon of contaminated site, helps petroleum hydrocarbon degradation bacterium absorbing hydro carbons.
Compare with the indigenous microorganism of contaminated site, microbial inoculum of the present invention can form competitive edge, and significantly increase contaminated site hydrocarbon degradation microbial numbers in the contaminated site growth rapidly, thereby improves the biological degradation rate of crude oil.
Strain identification
One, bacterial strain colony characteristics, individual special card and physiological and biochemical property
1. authentication method:
According to " common bacteria system identification handbook " (eastern elegant pearl, 2001) with " the described method of primary outstanding Bacteria Identification handbook (Science Press, 1984) is carried out the analysis of physio biochemical characteristics of bacterial strain.
2. qualification result
Colonial morphology, individual morphology and physiological and biochemical property according to four strain bacterium see Table 1.Can preliminary evaluation TJ-1, TJ-2, DHX-1 and DHX-2 belong to bacillus (Bacillus sp.), acinetobacter calcoaceticus (Acinetobacter sp.), Rhodopseudomonas (Pseudomonas sp.) and Rolston Pseudomonas (Ralstonia sp.).
The colonial morphology of table 1 bacterial strain, individual morphology and physiological and biochemical property
Two, molecular biology identification
1. authentication method and operation:
The petroleum hydrocarbon degradation bacterium single strain that is separated to adopts the 16SrDNA Sequence Identification, and its key step is as follows:
(1) extraction of bacterium single strain DNA
The extraction of strain chromosome DNA: select single bacterium colony, be inoculated into the 2216E slant medium and cultivate 24h for 28 ℃, adopt phenol-chloroform method to extract chromosomal DNA.
(2) structure of 16SrDNA sequential analysis and phylogenetic tree
Use the 16SrDNA universal primer, by Shanghai bio-engineering corporation synthetic primer.Wherein, 16sF be 5 '-AGAGTTTGATCCTGGCTCAG-3 '; 16sR is 5 '-ACGGCTACCTTGTTACGACT-3 '.With the chromosomal DNA that is extracted is that template is carried out pcr amplification.
Utilizing the DNA of Shanghai Bo Ya biotech firm to reclaim test kit reclaims the PCR product band in the agarose gel electrophoresis.The DNA that reclaims is served the Hai Boya order-checking.Sequencing result carries out BLAST on NCBI and Swiss-Prot website analyzes, and utilizes Phylip software package, MEGA 3.1 softwares, uses CLUATALW method and NJ method that nucleotide sequence is carried out the homology evolutionary analysis.
2 qualification results
Based on 16SrDNA each bacterial strain that is separated to is further identified.It is generally acknowledged that the 16SrDNA sequence homology is higher than 97%, can think to belong to a kind of.Fig. 8 represents the pcr amplification result of single strain DNA.
Homology analysis and phylogeny result based on each bacterial strain of 16SrDNA see Fig. 9-Figure 12 respectively.As can be seen, the 16SrDNA sequence homology of TJ-1, TJ-2, DHX-1 and the DHX-2 homology with bacillus pumilus (Bacillus pumilus), Venice acinetobacter calcoaceticus (Acinetobacter venetianus), pseudomonas mendocina (Pseudomonas mendocina) and Pi Shi Rolston bacterium (Ralstonia pickettii) respectively is the highest, near 100%.Can think that this four strains bacterium belongs to bacillus pumilus (B.pumilus), Venice acinetobacter calcoaceticus (A.venetianus), pseudomonas mendocina (P.mendocina) and Pi Shi Rolston bacterium (R.pickettii).
Claims (8)
1. bioremediating coastline polluted by oil spilling bacterium is characterized in that being made up of following bacterium: bacillus pumilus (Bacilluspumilus), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCCNo.3384; Venice acinetobacter calcoaceticus (Acinetobacter venetianus), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.3385 and/or pseudomonas mendocina (Pseudomonasmendocina), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCCNo.3386 and/or Pi Shi Rolston bacterium (Ralstonia pickettii), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.3387.
2. microbial inoculum for bioremediating coastline polluted by oil spilling, it is characterized in that in 1.0L～3.0L: the ratio of 1kg is that raw material is made by mixed fermentation liquid and fixation support, and described mixed fermentation liquid comprises bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and/or pseudomonas mendocina CGMCC No.3386 fermented liquid and/or Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid.
3. microbial inoculum for bioremediating coastline polluted by oil spilling according to claim 2 is characterized in that the bacteria concentration of described bacillus pumilus CGMCC No.3384 fermented liquid is 1 * 10
6Individual/ml～1 * 10
10The bacteria concentration of individual/ml, described Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid is 1 * 10
6Individual/ml～1 * 10
10The bacteria concentration of individual/ml, described pseudomonas mendocina CGMCC No.3386 fermented liquid is 1 * 10
6Individual/ml～1 * 10
10The bacteria concentration of individual/ml, described Pi Shi Rolston bacterium CGMCCNo.3387 fermented liquid is 1 * 10
6Individual/ml～1 * 10
10Individual/ml, the volume ratio of described bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid, pseudomonas mendocina CGMCC No.3386 fermented liquid and Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid is 1～10: 1～10: 0～4: 0～4.
4. microbial inoculum for bioremediating coastline polluted by oil spilling according to claim 3 is characterized in that the volume ratio of described bacillus pumilus CGMCC No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid, pseudomonas mendocina CGMCC No.3386 fermented liquid and Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid is 5～8: 2～8: 0.5～3: 0.5～3.
5. according to claim 2,3 or 4 described microbial inoculum for bioremediating coastline polluted by oil spilling, it is characterized in that described fixation support is that diatomite, kaolin, the peat composed of rotten mosses and chitin are at least a.
6. the preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling is characterized in that comprising the steps:
(1) bacillus pumilus CGMCC No.3384, Venice acinetobacter calcoaceticus CGMCC No.3385 and/or pseudomonas mendocina CGMCC No.3386 and/or Pi Shi Rolston bacterium CGMCC No.3387 are cultivated and fermentor cultivation obtains fermented liquid through slant culture, shake-flask seed respectively;
(2) be 1 * 10 with bacteria concentration
6Individual/ml～1 * 10
10The bacillus pumilus CGMCC No.3384 fermented liquid of individual/ml, bacteria concentration are 1 * 10
6Individual/ml～1 * 10
10Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid and/or the bacteria concentration of individual/ml are 1 * 10
6Individual/ml～1 * 10
10Pseudomonas mendocina CGMCC No.3386 fermented liquid and/or the bacteria concentration of individual/ml are 1 * 10
6Individual/ml～1 * 10
10The Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid of individual/ml is 1～10: 1～10 by volume: be mixed into mixed fermentation liquid at 0～4: 0～4;
(3) in 1.0L～3.0L: 1 kilogram ratio mixes described mixed fermentation liquid and fixation support, and lyophilize promptly obtains microbial inoculum for bioremediating coastline polluted by oil spilling.
7. the preparation method of microbial inoculum for bioremediating coastline polluted by oil spilling according to claim 6 is characterized in that the volume ratio of the described bacillus pumilus CGMCC of described step (2) No.3384 fermented liquid, Venice acinetobacter calcoaceticus CGMCC No.3385 fermented liquid, pseudomonas mendocina CGMCC No.3386 fermented liquid, Pi Shi Rolston bacterium CGMCC No.3387 fermented liquid is 5～8: 2～8: 0.5～3: 0.5～3.
8. according to the preparation method of claim 6 or 7 described microbial inoculum for bioremediating coastline polluted by oil spilling, it is characterized in that described fixation support is that diatomite, kaolin, the peat composed of rotten mosses and chitin are at least a.
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