CN103045579B - Microbial remediation curing adsorbing bacterial preparation applicable to marine environment petroleum pollution as well as preparation method and application of same - Google Patents

Microbial remediation curing adsorbing bacterial preparation applicable to marine environment petroleum pollution as well as preparation method and application of same Download PDF

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CN103045579B
CN103045579B CN201210549044.0A CN201210549044A CN103045579B CN 103045579 B CN103045579 B CN 103045579B CN 201210549044 A CN201210549044 A CN 201210549044A CN 103045579 B CN103045579 B CN 103045579B
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microbial inoculum
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bacterium
bacterial
marine
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CN103045579A (en
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马挺
李国强
梁凤来
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Nankai Univ
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Abstract

The invention provides a microbial remediation curing adsorbing bacterial preparation applicable to marine environment petroleum pollution as well as a preparation method and an application of the microbial remediation curing adsorbing bacterial preparation. The preparation process of the curing adsorbing bacterial preparation comprises the following steps: selecting microbial strains suitable for degrading crude oil in a polluted area; enlarging cultivation step by step so as to obtain a fermenting bacteria liquid with bacteria concentration of 108-1010 bacteria/mL; and then adding 100-1000 g/L porous medium (zeolite or ceramsite) to the fermenting bacteria liquid; further adding 0.1-2 g/L flocculating agent to the fermenting bacteria liquid; sufficiently mixing the fermenting bacteria liquid, the porous medium and the flocculating agent; standing an obtained mixture for 0.5-24 h so as to prepare the curing adsorbing bacterial preparation with bacterial adsorbing rate of not less than 90%. The curing adsorbing bacterial preparation can be applied to biological remediations of marine environment petroleum pollutions such as marine shoal muds or marine bottom sediments; highly efficient petroleum hydrocarbon degrading bacteria and nutritions can be carried in the marine bottom sediments; and the retention times of the highly efficient petroleum hydrocarbon degrading bacteria and the nutritions in the shoals can be prolonged; the survival rate of target bacteria in a target location is obviously increased; and the application range of a remediation bacterial preparation is enlarged.

Description

A kind of microorganism that is applicable to ocean environment petroleum pollution is repaired and solidifies absorption microbial inoculum and its preparation method and application
Technical field
The invention belongs to microbial biotechnology and Environmental Biotechnology field, specifically, relate to a kind of microorganism that is applicable to Marine shoal and thalassogenic sedimentation substance environment petroleum pollution reparation and solidify absorption bacterial preparation process, and the application in Marine shoal and marine bottom sediment petroleum pollution biological restoration.
Background technology
Along with taking place frequently of offshore oil development and oil tanker accident, offshore oil pollution is day by day serious, has directly affected the development of sea farming and fish industry.Marine oil overflow not only directly pollutes harm, and because removing completely, some irreducible oils are difficult to degraded under field conditions (factors), and poisonous, objectionable constituent have accumulation, thereby marine ecology is caused to long-term infringement.Within 2005, national monitoring result shows, the ecological monitored space of Bohai Offshore, particularly HUANGHE ESTUARY and Laizhou Wan, petroleum pollution is still more serious, and marine organisms kind and quantity reduce, spawning ground serious degradation, cause the factor of this phenomenon a lot, wherein the aggravation of hydrocarbon contamination can not be ignored.Therefore,, when greatly developing offshore oil exploitation and transportation, must see that petroleum pollution event is very urgent by the technical study of petroleum pollution.Especially the oil spill accident occurring in Dalian on July 16th, 2010, and after the oil spill accident occurring at Penglai, shandong Province afterwards, the fact that ocean year increases, carries out the great attention that the improvement that reduces or eliminates petroleum pollution in ocean is subject to government day by day.
After oil spill accident occurs, through physical method (as machinery recovery etc.), can remove part surface oil spilling, remaining oil spilling, under the effect of wind, wave, stream, can drift to beach, and seabed is sunk in the meeting that density is larger.In oil, the aromatic component of difficult degradation (carcinogenic substance) prolonged stay, in beach soil and thalassogenic sedimentation substance environment, causes huge persistence infringement to Marine ecosystems and HUMAN HEALTH.
Microbial treatment technology is the efficient remedying oil-polluted new technology of generally acknowledging in the world, refers to and utilizes microorganism to carry out catalyzed degradation environmental pollutant, reduces or finally eliminate the controlled or spontaneous process of environmental pollution.Microorganism can adapt to the ecotope of various complexity, its breeding metabolic capacity is extremely strong, various toxic substances in energy fast degradation oil, and have cheap, natural environmental protection, secondary pollution few, to many advantages such as person poultry harmless, become the on-the-spot important selection approach that oil spill is repaired of removing.The oil spilling that adopts this technical finesse physical method to remove is the optimal path of Revegetation of Eco-environment, has been subject to national governments, scientist and entrepreneur's great attention.Development and popularization microbial treatment technology are applicable to the national conditions of China, and to protection China marine eco-environment, the sustainable development that maintains oceanic resources has great scientific meaning and using value.
In the process of processing in petroleum pollution in ocean biological restoration, often can use the alkane degradation bacterial strain of low temperature salt tolerant, they absorb hydro carbons in the mode of oneself, then participate in self metabolism, finally generate small molecular organic acid or gas.Research for bio-augmentation method has two development trends: the one, and for the ecosystem of microbiological deterioration scarce capacity, add inoculating microbe to do supplementary; The 2nd, the crude oil type that can not degrade for indigenous microorganism or important oil composition, the degrading microorganism of selection or transformation premium properties." nature and acceleration biological treating research project " that United States Government subsidizes tries hard to by modern molecular biology means, on the basis of illustrating pollutent metabolic mechanism, develop suitable molecular regulation means, to reduce nutrition addition and the biological accumulation amount of degradation bacteria in biological restoration process; Also there is scholar to using Pseudomonas aeruginosa as acceptor, the various plasmids that pseudomonas putida etc. is carried proceed to wherein, formed " super have a liking for oily engineering bacteria " with multiple plasmid, for marine oil overflow, processed, its high tens times of energy force rate wild mushroom removing greasy dirt arrives hundred times.Domestic also have scholar to utilize mixed bacterium synergy to strengthen the degradation effect of petroleum hydrocarbon, king adds the peaceful people of grade and discloses solid microbe mix bacterium agent of a kind of degraded oil pollutent and petroleum products and preparation method thereof (publication number: CN101050435A), can be applicable in the bioremediation technology of the burst accident emergency processings such as oil-polluted soils and petroleum products leakage, wherein, microbiobacterial agent is to be made by subtilis and the mixed fermentation of many food Sphingol single-cells.The people such as Zhu Shengfeng disclose microbial inoculum for bioremediating coastline polluted by oil spilling and preparation method's (publication number: CN101717725A), utilize the microbiobacterial agents such as the logical bacterium at least one fixation support embedding bacillus pumilus such as diatomite, kaolin, the peat composed of rotten mosses and chitin, Venice acinetobacter calcoaceticus, pseudomonas mendocina and Pi Shi Rolls, to improve oil spill shoreline biological degradation effect.These inventions can improve the biological restoration efficiency of petroleum pollution, also can be applied in repair district, shallow sea, but because the density of microbial inoculum is less, can not be applied to the restoring area of marine bottom sediment, have therefore limited its range of application.
Accelerate the development of domestic bioremediation technology; the needs that meet China's marine environmental protection development; strengthen the research of low temperature salt tolerant alkane degradation bacterium, microbiological deterioration mechanism, slow-release nutrient and the hydrophobic fixation support of Geng Duo, be conducive to development and the industrial applications of marine oil spill bioremediation technology.The biological restoration of Marine shoal petroleum pollution has good development prospect.
Summary of the invention
The present invention seeks to solve existing repairing method of microorganism is that the remediation microbial inoculum using exists microbial inoculum density less, can not be applied to the restoring area of marine bottom sediment etc., therefore limited the problem of microbial inoculum range of application, provide a kind of microorganism that is applicable to the ocean environment petroleum pollutions such as Marine shoal and marine bottom sediment to repair and solidify absorption bacterial preparation process, and the application in the biological restoration of the ocean environment petroleum pollutions such as Marine shoal and marine bottom sediment.
The microorganism that is applicable to Marine shoal and the petroleum pollution of thalassogenic sedimentation substance environment provided by the invention is repaired and solidifies absorption microbial inoculum, be dense by bacterium be 10 8~ 10 10in the microorganism zymocyte liquid of the applicable degraded target oil product of individual/mL, add 100 ~ 1000g/L porous medium and 1 ~ 2g/L flocculation agent, be fully mixed.
Described microorganism comprises: rhodococcus (Rhodococcus erythoropolis T7-3, be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, its preserving number is CGMCC No.6104, hereinafter to be referred as T7-3), or burkholderia (Burkholderia mallei ATCC23344, buy in U.S.'s typical case's culture center, its article No. is ATCC23344, hereinafter to be referred as ATCC23344), or acinetobacter calcoaceticus (Acinetobacter haemolyticus ATCC19194, buy in U.S.'s typical case's culture center, its article No. is ATCC19194, hereinafter to be referred as ATCC19194), or genus bacillus (Bacillus licheniformis ATCC14580, buy in U.S.'s typical case's culture center, its article No. is ATCC14580, hereinafter to be referred as ATCC14580), or pseudomonas (Pseudomonas aeruginosa PA01, buy in U.S.'s typical case's culture center, its article No. is ATCC15692, hereinafter to be referred as ATCC15692), or the equal proportion mixture (hereinafter to be referred as mixed bacterium) of these bacterium.
Described porous medium is zeolite or haydite.
The present invention provides a kind of microorganism that is applicable to Marine shoal and the petroleum pollution of thalassogenic sedimentation substance environment to repair curing absorption bacterial preparation process simultaneously, and detailed process is:
1st, choose the microbial strains (concrete microorganism as previously mentioned) that is applicable to pollution degradation region crude oil;
2nd, through slant culture, shake-flask seed fermentation and fermentor tank cascade amplification, obtaining bacterium dense is 10 8~ 10 10the zymocyte liquid of individual/mL;
3rd, in the zymocyte liquid of the 2nd step, add 100 ~ 1000g/L porous medium, fully mix; Described porous medium is zeolite or haydite;
4th, in the mixture of the 3rd step, add flocculation agent 1 ~ 2g/L, fully mix, mix rear standing 0.5 ~ 24h, make the curing absorption microbial inoculum of thalline adsorption rate >=90%.
The present invention also provides the above to solidify the application of absorption microbial inoculum in the biological restoration of Marine shoal or the petroleum pollution of thalassogenic sedimentation substance environment.Concrete application process is, by 1 ~ 2kg/m 2repaired area adds described curing absorption microbial inoculum, by 0.2 ~ 1kg/m 2repaired area adds nutritive salt fertilizer.Described nutritive salt fertilizer is sulfate of ammoniac or Sodium phosphate dibasic.
The present invention is solidified absorption microbial inoculum and is all conducive to hold for a long time bacterium under lab simulation, beach and seabed field condition, and decomposing petroleum hydrocarbon, therefore can be used for the biological restoration field of the on-the-spot petroleum pollution in beach and seabed.
Advantage of the present invention and positively effect:
The invention provides a kind of method of utilizing zeolite or haydite absorption petroleum hydrocarbon high efficiency degradation bacterial agent and nutrition agent thereof, petroleum hydrocarbon efficient degrading bacteria and nutrition thereof can be carried in marine bottom sediment, and can extend petroleum hydrocarbon efficient degrading bacteria and nutrition agent thereof in the residence time of beach, obviously improve object bacteria in the survival rate of objective, expand the range of application of remediation microbial inoculum, therefore there is Marine shoal and marine bottom sediment petroleum pollution biological restoration ability.
Accompanying drawing explanation
Fig. 1 is the degradation rate temporal evolution graphic representation of alkane degradation bacterium to crude oil.
Fig. 2 is the residual microorganism effect comparison diagram of supernatant liquor before and after solid material absorption (A is that the fermented liquid supernatant 100 μ l of not absorption are coated with dull and stereotyped effect, and B is that the fermented liquid supernatant 100 μ l after absorption are coated with dull and stereotyped effect).
Fig. 3 is fermented liquid outside drawing before and after solid material absorption (A is the fermented liquid after absorption, and B is the fermented liquid not adsorbing).
Fig. 4 is that before and after solid material absorption, scanning electron microscope (SEM) is schemed (A is that the material porous medium before absorption is inner, and B is the material porous medium inside after adsorbing).
Fig. 5 is total plate count change curve in simulating lab test settling.
Fig. 6 is alkane degradation bacterium sum change curve in simulating lab test settling.
Fig. 7 is the petroleum hydrocarbon degradation graphic representation of simulating lab test.
Fig. 8 is total petroleum hydrocarbon change curve in beach field experiment settling.
Fig. 9 is total count change curve in beach field experiment settling.
Figure 10 is the change curve of alkane degradation bacterium in beach field experiment settling.
Figure 11 is 60 days degradation rate histograms of total petroleum hydrocarbon in beach test in place settling.
Embodiment
Embodiment 1, rhodococcus T7-3 solidify the preparation method of absorption microbial inoculum
1, the screening of degraded bacterial classification
To pollute region, collect crude oil (the present embodiment be take extra large two station commingled crudes temporarily as example, hereinafter to be referred as crude oil), the mass volume ratio with 2% joins minimal medium (KH 2pO 43.48g/L, Na 2hPO 41.5g/L, MgSO 40.70g/L, (NH 4) 2sO 44g/L, NaCl20g/L, yeast powder 0.05g/L, pH7.0 ~ 7.2) in, forming crude oil minimal medium, 121 ℃ of sterilizing 20min, screen the bacterial classification good to oil degradation performance, and culture condition is 25 ℃, 150r/min, inoculum size 2%, incubation time is 7d.
The present embodiment is selected rhodococcus (Rhodococcus erythoropolis) T7-3 from 30 kinds of bacterial strains to be selected, (on May 11st, 2012 be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; its address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica; its preserving number is CGMCC No.6104, suggestion Classification And Nomenclature is that rhodococcus erythropolis (Rhodococcus erythoropolis) is hereinafter to be referred as T7-3).Be seeded to the crude oil minimal medium with sterilizing, measured the oil degradation rate of bacterial classification in degradation process, wherein: the rear crude content of crude content-degraded before oil degradation rate %=(degraded) former oil concentration * 100% before/degraded, crude content is measured and measured with reference to GB GB17378.4-2007 method.The results are shown in Figure shown in 1.As can be seen from the figure, T7-3 is 75% to the degradation rate of crude oil.
2, the enlarged culturing of bacterial classification
Strain expanded culture method of the present invention is pure through dull and stereotyped minute, and slant culture, shake-flask seed are cultivated and four steps of fermentor cultivation, and final somatic cells density reaches 2 * 10 8individual/more than mL.That select below is a kind of in microorganism described in summary of the invention, does not limit the present invention.
1), flat board divides pure
By t bacteria 7-3, with on disinfection inoculation ring picking list colony inoculation to 1 solid medium flat board, line repeatedly, cultivates after 72h at 25 ℃, observes and has or not miscellaneous bacteria, to verify the purity of object bacteria.
Wherein, No. 1 the dull and stereotyped preparation method of solid medium is as follows: take respectively 5.0g peptone, 1.0g yeast powder, stirring and dissolving, in 1000mL ageing seawater, then takes 18g agar powder successively, boils after dissolving, adjust pH7.6 ~ 7.8, at 121 ℃, after sterilizing 20min, be down to 50 ℃, with every 20mL, pour and be laid in culture dish (d=10cm) bottom into, obtain solid medium flat board after cooling.
2), slant culture
To on above-mentioned flat board, verify that correct single bacterium colony, with disinfection inoculation ring picking one ring, is inoculated in No. 1 culture medium slant, line repeatedly, cultivates after 72h at 25 ℃, standby.
Wherein, No. 1 culture medium slant preparation method is as follows: take respectively 5.0g peptone, 1.0g yeast powder, stirring and dissolving, in 1000mL ageing seawater, then takes 18g agar powder successively, boil after dissolving, adjust pH7.6 ~ 7.8, at 121 ℃, after sterilizing 20min, be down to 50 ℃, with every 5mL, be sub-packed in 180mm * 18mm(length * diameter) in test tube, 10 ° of-20 ° of inclination room temperatures are placed, and obtain culture medium slant after cooling.
3), shake-flask seed is cultivated
By the bacterial classification on above-mentioned inclined-plane, on Bechtop, aseptic technique is inoculated in the 500mL Erlenmeyer flask of No. 1 liquid nutrient medium that 200mL is housed, and 25 ℃, 200r/min cultivates cell density and just reached 5 * 10 8individual/mL stops cultivating, standby.
Wherein No. 1 liquid nutrient medium preparation method is as follows: take respectively 5.0g peptone, and 1.0g yeast powder, stirring and dissolving is in 1000mL ageing seawater successively, and every 200mL divides and installs in 500mL Erlenmeyer flask, sterilizing 20min at 121 ℃.
4), fermentor cultivation
Above-mentioned seed liquor nutrient solution being housed, 40 ~ 60% liquid measures is amplified to fermentation culture in the fermentor tank of sterilization fermentation substratum respectively step by step with the inoculum size of 3-5%.In fermenting process, controlling pH is 7.2, and culture temperature is 25 ℃, and rotating speed is 600r/min, and air flow is 1.0 ~ 2.0L/min, cultivates 60h and reaches 2 * 10 to somatic cells density 8individual/more than mL.
The preparation method of above-mentioned fermention medium is as follows: take respectively 30.0g glucose, 80.0g whiteruss, 4.0g potassium primary phosphate, 6.0g dipotassium hydrogen phosphate, 10.0g ammonium sulfate, 25.0g sodium-chlor, 0.2g magnesium sulfate, 0.01g calcium chloride, 0.005g vitamin H and add to successively in the distilled water of 1000mL, after stirring and dissolving, adjust pH to 7.2, in 115 ℃ of sterilizing 15min.
3, solidify the absorption of absorption microbial inoculum
Following public be t bacteria 7-3 and solid material with the preparation process of several different ratioss, do not limit the present invention.
T7-3 seed liquor and zeolite (or haydite) are carried out to adsorption experiment with the listed ratio of table 1, wherein after 1-9 group reinforcing body adsorption medium, stir 2min static 1.5h again, system after absorption is got supernatant liquor 100 μ l dilutions and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in initial seed liquid dense (cfu2), calculate biomass (cfu3) and adsorption rate (%) (see figure 2) of zeolite adsorption, wherein the method for calculation of adsorption rate are as follows: cfu3=cfu2-cfu1, adsorption rate %=cfu3/cfu2 * 100%.The microbial inoculum that the 3rd group of experiment of take forms is for No. 3 example, and before zeolite adsorption, T7-3 cell concentration is 9 * 10 8cFU/mL, after absorption, cell concentration is 1.9 * 10 8cFU/mL, adsorption rate reaches 78.9%, average every g zeolite adsorption 1.8 * 10 9cFU thalline.
The adsorption of table 1 porous medium to T7-3 somatic cells
Group Bacterium number Bacterium liquid ml Zeolite g Haydite g Potassium aluminium sulfate g Adsorption rate % Bacterium material ratio
1 T7-3 25 2.5 / / 35.3 10∶1
2 T7-3 25 7.5 / / 59.1 10∶3
3 T7-3 25 12.5 / / 78.9 2∶1
4 T7-3 25 15 / / 76.7 5∶3
5 T7-3 25 17.5 / / 84.5 10∶7
6 T7-3 25 20 / / 88.7 5∶4
7 T7-3 25 25 / / 91.2 1∶1
8 T7-3 25 / 15 / 92.2 5∶3
9 T7-3 25 / 20 / 85.5 5∶4
As can be seen from Table 1, the 25ml bacterium liquid of take is example, material add-on 2.5g, and during mass volume ratio 100g/L, corresponding bacterium material is than being 10:1; Material add-on 12.5g, during mass volume ratio 500g/L, corresponding bacterium material is than being 2:1, by that analogy.Visible material add-on porous medium in 100 ~ 1000g/L all has certain adsorption to somatic cells.More than adding material 500g/L, thalline adsorption rate just can reach more than 75%, and before and after solid material absorption, fermented liquid outside drawing as shown in Figure 3, illustrates that the solid materials such as zeolite or haydite are higher to thalline adsorption efficiency, can be applied to the preparation of solid fungicide.Solid microbial sample after blank zeolite and absorption is carried out to scanning electron microscope (SEM) to be detected, as shown in Figure 4, can obviously see the thalline being adsorbed on zeolite, and cell density is larger, illustrate use zeolite to carry out thalline absorption as carrier to prepare solid microbial be feasible.
Embodiment 2, burkholderia ATCC23344 solidify the preparation method of absorption microbial inoculum
1, the screening of degraded bacterial classification
To pollute region, collect crude oil (the present embodiment be take extra large two station commingled crudes temporarily as example, hereinafter to be referred as crude oil), the mass volume ratio with 2% joins minimal medium (KH 2pO 43.48g/L, Na 2hPO 41.5g/L, MgSO 40.70g/L, (NH 4) 2sO 44g/L, NaCl20g/L, yeast powder 0.05g/L, pH7.0 ~ 7.2) in, screening the bacterial classification good to oil degradation performance, culture condition is 25 ℃, 150r/min, inoculum size 2%, incubation time is 7d.
The present embodiment is selected burkholderia (Burkholderia malleiATCC23344 from 30 kinds of bacterial strains to be selected, buy in U.S.'s typical case's culture center, its article No. is ATCC23344, hereinafter to be referred as ATCC23344), be seeded to the crude oil minimal medium of bacterium, measured the oil degradation rate of bacterial classification in degradation process, wherein: the rear crude content of crude content-degraded before oil degradation rate %=(degraded) former oil concentration * 100% before/degraded, crude content is measured and measured with reference to GB GB17378.4-2007 method.The results are shown in Figure shown in 1.As can be seen from the figure, ATCC23344 is 68% to the degradation rate of crude oil.
2, the enlarged culturing of bacterial classification
Second step with embodiment 1.
3, solidify the absorption of absorption microbial inoculum
Following public be bacterial classification ATCC23344 and solid material with the preparation process of several different ratioss, do not limit the present invention.
ATCC23344 seed liquor and solid absorption medium zeolite (or haydite) are carried out to adsorption experiment with the listed ratio of table 2, after reinforcing body adsorption medium, stir 2min static 1.5h again, system after absorption is got supernatant liquor 100 μ l dilutions and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in initial seed liquid dense (cfu2), calculate biomass (cfu3) and the adsorption rate (%) of zeolite adsorption, wherein the method for calculation of adsorption rate are as follows: cfu3=cfu2-cfu1, adsorption rate %=cfu3/cfu2 * 100%.The microbial inoculum that the 14th group of experiment of take forms is example No. 14, and adsorption rate reaches 86.7%.
The adsorption of table 2 porous medium to ATCC23344 somatic cells
Group Bacterium number Bacterium liquid ml Zeolite g Haydite g Potassium aluminium sulfate g Adsorption rate % Bacterium material ratio
11 ATCC19194 25 2.5 / / 76.8 10∶1
12 ATCC15692 25 7.5 / / 79.5 10∶3
13 ATCC23344 25 12.5 / / 83.2 2∶1
14 ATCC23344 25 15 / / 86.7 5∶3
15 ATCC23344 25 17.5 / / 85.9 10∶7
16 ATCC23344 25 20 / / 89.7 5∶4
17 ATCC23344 25 25 / / 92.2 1∶1
18 ATCC23344 25 / 15 / 93.2 5∶3
19 ATCC23344 25 / 20 / 95.5 5∶4
As can be seen from Table 2, material add-on porous medium in 100~1000g/L has more than 75% adsorption to somatic cells.More than adding material 500g/L, thalline adsorption rate just can reach more than 80%.
Embodiment 3, acinetobacter calcoaceticus ATCC19194 solidify the preparation method of absorption microbial inoculum
1, the screening of degraded bacterial classification
To pollute region, collect crude oil (the present embodiment be take extra large two station commingled crudes temporarily as example, hereinafter to be referred as crude oil), the mass volume ratio with 2% joins minimal medium (KH 2pO 43.48 g/L, Na 2hPO 41.5g/L, MgSO 40.70g/L, (NH 4) 2sO 44g/L, NaCl20g/L, yeast powder 0.05g/L, pH7.0 ~ 7.2) in, screening the bacterial classification good to oil degradation performance, culture condition is 25 ℃, 150r/min, inoculum size 2%, incubation time is 7d.
The present embodiment is selected acinetobacter calcoaceticus (Acinetobacter haemolyticus ATCC 19194 from 30 kinds of bacterial strains to be selected, buy in U.S.'s typical case's culture center, its article No. is ATCC19194, hereinafter to be referred as ATCC 19194), be seeded to the crude oil minimal medium of bacterium, measured the oil degradation rate of bacterial classification in degradation process, wherein: the rear crude content of crude content-degraded before oil degradation rate %=(degraded) former oil concentration * 100% before/degraded, crude content is measured and measured with reference to GB GB17378.4-2007 method.The results are shown in Figure shown in 1.As can be seen from the figure, ATCC19194 is 63% to the degradation rate of crude oil.
2, the enlarged culturing of bacterial classification
Second step with embodiment 1.
3, solidify the absorption of absorption microbial inoculum
Following public be bacterial classification ATCC19194 and solid material with the preparation process of several different ratioss, do not limit the present invention.
ATCC19194 seed liquor and zeolite (or haydite) are carried out to adsorption experiment with the listed ratio of table 3, after reinforcing body adsorption medium, stir 2min static 1.5h again, system after absorption is got supernatant liquor 100 μ l dilutions and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in initial seed liquid dense (cfu2), calculate biomass (cfu3) and the adsorption rate (%) of zeolite adsorption, wherein the method for calculation of adsorption rate are as follows: cfu3=cfu2-cfu1, adsorption rate %=cfu3/cfu2 * 100%.The microbial inoculum that the 25th group of experiment of take forms is example No. 25, and adsorption rate reaches 88.5%.
The adsorption of table 3 porous medium to ATCC19194 somatic cells
Group Bacterium number Bacterium liquid ml Zeolite g Haydite g Potassium aluminium sulfate g Adsorption rate % Bacterium material ratio
21 ATCC19194 25 2.5 / / 63.2 10∶1
22 ATCC19194 25 7.5 / / 74.8 10∶3
23 ATCC19194 25 12.5 / / 79.8 2∶1
24 ATCC19194 25 15 / / 86.7 5∶3
25 ATCC19194 25 17.5 / / 88.5 10∶7
26 ATCC19194 25 20 / / 89.7 5∶4
27 ATCC19194 25 25 / / 91.2 1∶1
28 ATCC19194 25 / 15 / 88.2 5∶3
29 ATCC19194 25 / 20 / 89.5 5∶4
As can be seen from Table 3, material add-on is in 100 ~ 1000g/L, and porous medium has more than 60% adsorption to somatic cells.More than adding material 500g/L, thalline adsorption rate just can reach more than 80%.
The preparation method that embodiment 4, aeruginosa atcc 15692 solidify absorption microbial inoculum
1, the screening of degraded bacterial classification
To pollute region, collect crude oil (the present embodiment be take extra large two station commingled crudes temporarily as example, hereinafter to be referred as crude oil), the mass volume ratio with 2% joins minimal medium (KH 2pO 43.48 g/L, Na 2hPO 41.5 g/L, MgSO 40.70 g/L, (NH 4) 2sO 44 g/L, NaCl 20g/L, yeast powder 0.05 g/L, pH 7.0 ~ 7.2) in, screening the bacterial classification good to oil degradation performance, culture condition is 25 ℃, 150r/min, inoculum size 2%, incubation time is 7d.
The present embodiment is selected pseudomonas (Pseudomonas aeruginosa PA01 from 30 kinds of bacterial strains to be selected, buy in U.S.'s typical case's culture center, its article No. is ATCC15692, hereinafter to be referred as ATCC 15692), be seeded to the crude oil minimal medium of bacterium, measured the oil degradation rate of bacterial classification in degradation process, wherein: the rear crude content of crude content-degraded before oil degradation rate %=(degraded) former oil concentration * 100% before/degraded, crude content is measured and measured with reference to GB GB17378.4-2007 method.The results are shown in Figure shown in 1.As can be seen from the figure, ATCC15692 is 56% to the degradation rate of crude oil.
2, the enlarged culturing of bacterial classification
Second step with embodiment 1.
3, solidify the absorption of absorption microbial inoculum
Following public be bacterial classification ATCC15692 and solid material with the preparation process of several different ratioss, do not limit the present invention.
ATCC15692 seed liquor and zeolite (or haydite) are carried out to adsorption experiment with the listed ratio of table 4, after reinforcing body adsorption medium, stir 2min static 1.5h again, system after absorption is got supernatant liquor 100 μ l dilutions and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in initial seed liquid dense (cfu2), calculate biomass (cfu3) and the adsorption rate (%) of zeolite adsorption, wherein the method for calculation of adsorption rate are as follows: cfu3=cfu2-cfu1, adsorption rate %=cfu3/cfu2 * 100%.The microbial inoculum that the 35th group of experiment of take forms is example No. 35, and adsorption rate reaches 82.5%.
The adsorption of table 4 porous medium to ATCC15692 somatic cells
Group Bacterium number Bacterium liquid ml Zeolite g Haydite g Potassium aluminium sulfate g Adsorption rate % Bacterium material ratio
31 ATCC15692 25 2.5 / / 65.2 10∶1
32 ATCC15692 25 7.5 / / 68.8 10∶3
33 ATCC15692 25 12.5 / / 76.5 2∶1
34 ATCC15692 25 15 / / 76.7 5∶3
35 ATCC15692 25 17.5 / / 82.5 10∶7
36 ATCC15692 25 20 / / 85.7 5∶4
37 ATCC15692 25 25 / / 89.2 1∶1
38 ATCC15692 25 / 15 / 87.2 5∶3
39 ATCC15692 25 / 20 / 88.5 5∶4
As can be seen from Table 4, material add-on is in 100 ~ 1000g/L, and porous medium has more than 65% adsorption to somatic cells.More than adding material 500g/L, thalline adsorption rate just can reach more than 75%.
The preparation method that embodiment 5, mixed bacterium are solidified absorption microbial inoculum
1, the screening of degraded bacterial classification
To pollute region, collect crude oil (the present embodiment be take extra large two station commingled crudes temporarily as example, hereinafter to be referred as crude oil), the mass volume ratio with 2% joins minimal medium (KH 2pO 43.48g/L, Na 2hPO 41.5g/L, MgSO 40.70g/L, (NH 4) 2sO 44g/L, NaCl20g/L, yeast powder 0.05g/L, pH7.0 ~ 7.2) in, screening the bacterial classification good to oil degradation performance, culture condition is 25 ℃, 150r/min, inoculum size 2%, incubation time is 7d.
The present embodiment is selected pseudomonas (Pseudomonas aeruginosa PA01 from 30 kinds of bacterial strains to be selected, buy in U.S.'s typical case's culture center, its article No. is ATCC15692, hereinafter to be referred as ATCC15692), acinetobacter calcoaceticus (Acinetobacter haemolyticusATCC19194, buy in U.S.'s typical case's culture center, its article No. is ATCC19194, hereinafter to be referred as ATCC19194), burkholderia (Burkholderia mallei ATCC23344, buy in U.S.'s typical case's culture center, its article No. is ATCC23344, hereinafter to be referred as ATCC23344), and rhodococcus (Rhodococcus erythoropolisT7-3, be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number is CGMCCNo.6104, hereinafter to be referred as T7-3) equal-volume fermenting mixture (hereinafter to be referred as mixed bacterium), be seeded to the crude oil minimal medium of bacterium, measured the oil degradation rate of bacterial classification in degradation process, wherein: the rear crude content of crude content-degraded before oil degradation rate %=(degraded) former oil concentration * 100% before/degraded, crude content is measured and is measured with reference to GB GB17378.4-2007 method.The results are shown in Figure shown in 1.As can be seen from the figure, mixed bacterium is 85.6% to the degradation rate of crude oil.
2, solidify the absorption of absorption microbial inoculum
Following public be mixed bacterium and solid material with the preparation process of several different ratioss, do not limit the present invention.
Mixed fermentation liquid and zeolite (or haydite) are carried out to adsorption experiment with the listed ratio of table 5, after 40-48 group reinforcing body adsorption medium, stir 2min static 1.5h again, after 49-50 group reinforcing body adsorption medium, add again 1 ~ 2g/l potassium aluminium sulfate, shake rear static 1.5h, system after absorption is got supernatant liquor 100 μ l dilutions and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in initial seed liquid dense (cfu2), calculate biomass (cfu3) and the adsorption rate (%) of zeolite adsorption, wherein the method for calculation of adsorption rate are as follows: cfu3=cfu2-cfu1, adsorption rate %=cfu3/cfu2 * 100%.The microbial inoculum that the 42nd group of experiment of take forms is example No. 42, and adsorption rate reaches 82.5%.The microbial inoculum that the 49th group of experiment of take forms is example No. 49, and adsorption rate reaches 98.5%.
The adsorption of table 5 porous medium to mixed bacterium somatic cells
Group Bacterium number Bacterium liquid ml Zeolite g Haydite g Potassium aluminium sulfate g Adsorption rate % Bacterium material ratio
40 Mixed bacterium 25 2.5 / / 76.8 10∶1
41 Mixed bacterium 25 7.5 / / 78.4 10∶3
42 Mixed bacterium 25 12.5 / / 81.1 2∶1
43 Mixed bacterium 25 15 / / 84.6 5∶3
44 Mixed bacterium 25 17.5 / / 85.5 10∶7
45 Mixed bacterium 25 20 / / 88.7 5∶4
46 Mixed bacterium 25 25 / / 91.2 1∶1
47 Mixed bacterium 25 / 15 / 92.2 5∶3
48 Mixed bacterium 25 / 20 / 85.5 5∶4
49 Mixed bacterium 25 12.5 ? 0.2 98.5 2∶1
50 Mixed bacterium 25 12.5 ? 0.4 98.6 2∶1
As can be seen from Table 5, as long as more than the volume ratio of mixed bacterium bacterium liquid and material reaches 2:1, thalline adsorption rate just can reach more than 80%, adds flocculation agent potassium aluminium sulfate and can improve the effect that solid material adsorbs thalline, thalline adsorption rate can reach 98%.
Data in consolidated statement 1 ~ 5, obtain the best preparation method of curing absorption microbial inoculum provided by the invention: every 1 part of zymocyte liquid adds respectively 100 ~ 1000g/L porous medium (zeolite or haydite), 0.15% Secondary ammonium phosphate and 0.4% SODIUMNITRATE, fully be uniformly mixed, standing 1.5h, can make the curing absorption microbial inoculum of thalline adsorption rate >=75%.If add the potassium aluminium sulfate of 1 ~ 2g/L after stirring, mix rear standing 1.5h, can make the curing absorption microbial inoculum of thalline adsorption rate >=98%.
Embodiment 6, the effect of curing absorption microbial inoculum 7 provided by the invention in lab simulation marine bottom sediment is repaired
What the present embodiment was narrated is the 7th group of simulation repairing effect that solidifies absorption microbial inoculum in table 1, does not limit the present invention.
In 3 stainless cylinder of steels of 10L (d=40cm at the bottom of tank, the high 80cm of tank), add respectively 3L artificial seawater (NaCl24.53g, KCl0.695g, CaCl 21.16g, MgCl 25.2g, Na 2sO 44.09g, NaHCO 30.201g, KBr0.101g, H 3bO 30.027g, SrCl 20.025g, NaF0.003g, pH7.8, prepares with distilled water), 121 ℃ of sterilizing 20min, add respectively 3kg to be mixed with the marine bottom sediment of 3g sea two station commingled crudes, after standing over night, add respectively 1kg/m 2, 1.5kg/m 2, 2kg/m 2the solid absorption microbial inoculum of repaired area, then add respectively and be equivalent to 25% microbial inoculum amount nutritive salt fertilizer (ammonium sulfate: Sodium phosphate dibasic=1:1), cultivate 15d for 20-25 ℃, every day, each stirred once with stainless steel bar sooner or later.Every day is with 5 point sampling methods, with sterile sampler, get each 5g left and right of settling, deposit in sterile chamber, again 5 samples are fully mixed, get mixed soil sample 2.5g, add the sterile distilled water 22.5mL dilution of filtering with 0.2 μ m filter, fully the equal liquid of sample of 1:10 is made in vibration, detects total plate count (SY/T0532-93), petroleum hydrocarbon degradation bacterium number (MPN method) and oils fingerprinting (GB/T21247-2007) in settling.
Wherein the measuring method of petroleum hydrocarbon degradation bacterium number is as follows: testing sample is done to a series of 10 times of dilutions, and each extent of dilution is got 3-5 repeated inoculation in suitable liquid nutrient medium (whiteruss 2g/L, MgSO 40.2g/L, CaCl 20.02g/L, KH 2pO 41.0g/L, (NH 4) 2hPO 41.0g/L, KNO 31.0g/L, FeCl 30.05g/L, pH7.2) in, at 25 ℃, cultivate 7-10 days, the pipe number that occurs bacterial growth in having last 3 extent of dilution (being critical progression) of bacterium liquid growth is as quantitative index (during counting, observation index is substratum turbidity and hydrocarbons outward appearance emulsion dispersion degree), by finding approximation on maximum most probable number (MPN) table, be multiplied by again the extension rate of quantitative index the first figure place, be in original bacteria liquid containing bacterium number.
The measurement result of total plate count and alkane degradation bacterium quantity as shown in Figure 5 and Figure 6, can find out that the bacterium number of the petroleum hydrocarbon degradation bacterium in experiment is occupied an leading position all the time, illustrates that the bacterium of alkane degradation bacterium is dense higher, is more conducive to improve petroleum degradation rate.Solidify absorption microbial inoculum at 25 ℃, degradation efficiency temporal evolution enlarges markedly, and especially, since the 10th day, degradation efficiency has had large increase, at the 13d of experimental phase, reached 50% left and right (Fig. 7), the petroleum hydrocarbon that is conducive to degrade in settling that adds that solidifies absorption microbial inoculum has been described.
Embodiment 7, the effect of curing absorption microbial inoculum 42 provided by the invention in lab simulation marine bottom sediment is repaired
What the present embodiment was narrated is the 42nd group of simulation repairing effect that solidifies absorption microbial inoculum in table 5, does not limit the present invention.
In 3 stainless cylinder of steels of 10L (d=40cm at the bottom of tank, the high 80cm of tank), add respectively 3L artificial seawater (NaCl24.53g, KCl0.695g, CaCl 21.16g, MgCl 25.2g, Na 2sO 44.09g, NaHCO 30.201g, KBr0.101g, H 3bO 30.027g, SrCl 20.025g, NaF0.003g, pH7.8, prepares with distilled water), 121 ℃ of sterilizing 20min, add respectively 3kg to be mixed with the marine bottom sediment of 3g sea two station commingled crudes, after standing over night, add respectively 1kg/m 2, 1.5kg/m 2, 2kg/m 2the solid absorption microbial inoculum of repaired area, then add respectively and be equivalent to 25% microbial inoculum amount nutritive salt fertilizer (ammonium sulfate: Sodium phosphate dibasic=1:1), cultivate 15d for 20-25 ℃, every day, each stirred once with stainless steel bar sooner or later.Every day is with 5 point sampling methods, with sterile sampler, get each 5g left and right of settling, deposit in sterile chamber, again 5 samples are fully mixed, get mixed soil sample 2.5g, add the sterile distilled water 22.5mL dilution of filtering with 0.2 μ m filter, fully the equal liquid of sample of 1:10 is made in vibration, detects total plate count (SY/T0532-93), petroleum hydrocarbon degradation bacterium number (MPN method) and oils fingerprinting (GB/T21247-2007) in settling.
Wherein the measuring method of petroleum hydrocarbon degradation bacterium number is as follows: testing sample is done to a series of 10 times of dilutions, and each extent of dilution is got 3-5 repeated inoculation in suitable liquid nutrient medium (whiteruss 2g/L, MgSO 40.2g/L, CaCl 20.02g/L, KH 2pO 41.0g/L, (NH 4) 2hPO 41.0g/L, KNO 31.0g/L, FeCl 30.05g/L, pH7.2) in, at 25 ℃, cultivate 7-10 days, the pipe number that occurs bacterial growth in having last 3 extent of dilution (being critical progression) of bacterium liquid growth is as quantitative index (during counting, observation index is substratum turbidity and hydrocarbons outward appearance emulsion dispersion degree), by finding approximation on maximum most probable number (MPN) table, be multiplied by again the extension rate of quantitative index the first figure place, be in original bacteria liquid containing bacterium number.
The bacterium number of the petroleum hydrocarbon degradation bacterium in experiment is occupied an leading position all the time, and the bacterium of alkane degradation bacterium is dense higher, is more conducive to improve petroleum degradation rate.Solidify absorption microbial inoculum at 25 ℃, degradation efficiency temporal evolution enlarges markedly, and especially, since the 10th day, degradation efficiency has had large increase, within the 13rd day in the experimental phase, reached 60% left and right, the petroleum hydrocarbon that is conducive to degrade in settling that adds that solidifies absorption microbial inoculum has been described.
Embodiment 8, the effect of curing absorption microbial inoculum 42 provided by the invention in beach is repaired
What the present embodiment was narrated is the 42nd group of curing absorption microbial inoculum and the effect of corresponding fermented liquid mixture in beach is repaired thereof in table 5, does not limit the present invention.
In Bohai Sea Gulf, selected petroleum pollution shoal area delimited the experiment block of 3 4m * 4m, and wherein 2 blocks are made as test site, and wherein 1 block (T1) is pressed 3L/m 2beach area adds mixed bacteria liquid, by 0.6kg/m 2beach area adds nutrition agent (sulfate of ammoniac, Sodium phosphate dibasic); Another 1 block (T2) is pressed 1.5kg/m 2beach area adds curing absorption microbial inoculum (add 500 ~ 1000g/L zeolite in bacterium liquid, add the auxiliary absorption of flocculation agent of 1 ~ 2g/L), by 0.6kg/m 2beach area adds nutritive salt fertilizer (sulfate of ammoniac, Sodium phosphate dibasic), and after above two block microbial inoculum addition manners are and mix, ploughing and weeding formula is broadcasted sowing; All the other 1 blocks (T3) are as blank assay.According to the method for embodiment 6, experiment block deposition medium is carried out to tracking monitor, monitoring project comprises total petroleum hydrocarbon (GB/T16488-1996), total plate count (SY/T0532-93), petroleum hydrocarbon degradation bacterium quantity (MPN method, with embodiment 4) and oils fingerprinting (GB/T21247-2007), using site operation same day as 0d, at 3d, 9d, 18d, 30d, 60d, carry out tracking monitor respectively afterwards.
The results are shown in Figure shown in 8-10, test the initial stage as seen, oil degradation speed is slower, and the effect that adds bacterium liquid (T1) is better than and adds the effect of solidifying absorption microbial inoculum (T2), this with add one group, zeolite and make the quantity of bacterium lose relevant when the zeolite adsorption bacterium liquid; In the experiment later stage, the effect that adds curing absorption microbial inoculum (T2) is better than the effect of spraying bacterium liquid (T1).Therefore, solidify absorption microbial inoculum and be conducive to hold for a long time bacterium, can be used for the reparation of the on-the-spot petroleum pollution of beach.
Embodiment 9, the effect of curing absorption microbial inoculum 7 provided by the invention and 14 in beach is repaired
The present embodiment narration be in table 1 the 7th group solidify in absorption microbial inoculum and table 2 the 14th group and solidify the effect of absorption microbial inoculum in beach is repaired, do not limit the present invention.
In Bohai Sea Gulf, selected petroleum pollution shoal area delimited the experiment block of 3 4m * 4m, and wherein 2 blocks are made as test site, are respectively T4: by 1.5kg/m 2beach area adds the 7th group to solidify absorption microbial inoculum, by 0.6kg/m 2beach area adds nutrition agent (sulfate of ammoniac, Sodium phosphate dibasic); T5 presses 1.5kg/m 2beach area adds the 14th group to solidify absorption microbial inoculum, by 0.6kg/m 2beach area adds nutritive salt fertilizer (sulfate of ammoniac, Sodium phosphate dibasic), and after above two block microbial inoculum addition manners are and mix, ploughing and weeding formula is broadcasted sowing; All the other 1 block T6 are as blank assay.According to the method for embodiment 6, within 60 days, testing afterwards the variation of block deposition medium monitoring total petroleum hydrocarbon (GB/T16488-1996), take T6 as contrast is to calculate petroleum hydrocarbon degradation rate.
Experimental result as shown in figure 11.The beach petroleum hydrocarbon degradation rate that adds No. 7 remediation microbial inoculums after 60 days is 56.2%, and the beach petroleum hydrocarbon degradation rate that adds No. 7 remediation microbial inoculums is 47.8%.In addition this curing absorption microbial inoculum is conducive to hold for a long time bacterium, can be used for the reparation of the on-the-spot petroleum pollution of beach.
The ecological security evaluation of embodiment 10, curing remediation microbial inoculum provided by the invention
What use is 42 groups of ecological security evaluations of solidifying absorption microbial inoculum in his-and-hers watches 5, does not limit the present invention.
According to < < offshore oil exploration and exploitation pollutent bio-toxicity > > (GB/T18420.2-2001), utilize halogen worm as biological subject, carry out preliminary indoor toxicity tests, try to achieve the optimum concn of microorganism renovation agent field usage, for field experiment lays the first stone.
Get appropriate halogen worm (Artemiidae) worm's ovum, add 1L seawater to cultivate 24h under 26 ℃ of room temperatures, illumination condition.According to adding than requiring, get successively tested mixed bacterium and solidify absorption microbial inoculum, according to the concentration (volume ratio 1.5 ‰, 3 ‰, 6 ‰, 12 ‰, 24 ‰) of setting, the microbial inoculum of getting respective amount adds seawater, is mixed with the microbial inoculum of the requirement of experiment concentration of respective concentration.In each quantitative ware, select 10 healthy halogen worms and put into, then add appropriate each concentration sample, under 26 ℃ of room temperatures, illumination condition, cultivate.And each concentration establish one in contrast parallel.Every 24h observes, and records the dead number of halogen worm.
The results are shown in Table in 6,72h incubation time, the halogen worm survival rate of each volumetric concentration is all more than 56%.In 96h incubation time, in the microbial inoculum that volumetric concentration is 6 ‰, halogen worm survival number is maximum, and survival rate reaches more than 90%.Show to solidify absorption microbial inoculum and can not affect the marine eco-environment.
Table 6 solidifies absorption microbial inoculum ecological safety assessment halogen worm survival result
Concentration The 24h number of surviving The 48h number of surviving The 72h number of surviving The 96h number of surviving
Contrast 10 9 9 8
1.5‰ 9 8 7 6
3‰ 8 7 6 6
6‰ 9 9 8 7
12‰ 7 6 6 5
24‰ 6 5 5 3

Claims (4)

1. the microorganism that is applicable to Marine shoal and the petroleum pollution of thalassogenic sedimentation substance environment is repaired a preparation method of solidifying absorption microbial inoculum, it is characterized in that the preparation process of described curing absorption microbial inoculum is:
1st, by preserving number, be CGMCC No.6104 rhodococcus erythropolis ( rhodococcuserythropolis) T7-3; Preserving number be ATCC 23344 burkholderia ( burkholderia mallei); Preserving number be ATCC 19194 acinetobacter calcoaceticus ( acinetobacter haemolyticus); And the preserving number pseudomonas that is ATCC15692 ( pseudomonas aeruginosa) PA01 is respectively through slant culture, shake-flask seed fermentation and fermentor tank cascade amplification, finally all obtaining concentration is 10 8~ 10 10the fermented liquid of individual/mL;
2nd, each bacterium liquid the 1st step being obtained by volume equal proportion mixes;
3rd,, to the zeolite or the haydite that add 100 ~ 1000g/L in the mixed bacteria liquid of the 2nd step, fully mix;
4th, in the mixture of the 3rd step, add flocculation agent 1 ~ 2g/L, described flocculation agent is potassium aluminium sulfate, fully mixes, and mixes rear standing 0.5 ~ 24h, makes the curing absorption microbial inoculum of thalline adsorption rate >=90%.
2. the microorganism that is applicable to Marine shoal and the petroleum pollution of thalassogenic sedimentation substance environment is repaired a curing absorption microbial inoculum, it is characterized in that described curing absorption microbial inoculum is made by method claimed in claim 1.
3. the application of curing absorption microbial inoculum claimed in claim 2 in the biological restoration of Marine shoal or the petroleum pollution of thalassogenic sedimentation substance environment.
4. application according to claim 3, is characterized in that by 1 ~ 2kg/m 2repaired area adds described curing absorption microbial inoculum, by 0.2 ~ 1kg/m 2repaired area adds ammonium sulfate or Sodium phosphate dibasic.
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