CN103045579A - 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 PDFInfo
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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
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 the 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 fully, some irreducible oils are difficult to degraded under field conditions (factors), and poisonous, objectionable constituent have accumulation, thereby marine ecology is caused long-term infringement.National monitoring result showed in 2005, 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, the 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 the petroleum pollution event is very urgent by the technical study of petroleum pollution.Especially the oil spill accident that occured in Dalian on July 16th, 2010, and afterwards behind the oil spill accident that Penglai, shandong Province occurs, the fact that ocean year increases is carried 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, can remove the part surface oil spilling through physical method (such as the machinery recovery etc.), remaining oil spilling can drift to beach under the effect of wind, wave, stream, and the seabed is sunk in the meeting that density is larger.The aromatic component of difficult degradation (carcinogenic substance) prolonged stay causes huge persistence infringement to Marine ecosystems and HUMAN HEALTH in the oil in beach soil and thalassogenic sedimentation substance environment.
The microbial treatment technology is the efficient remedying oil-polluted new technology of generally acknowledging in the world, refers to utilize microorganism to come the 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 the 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 fit to the national conditions of China, and to protection China marine eco-environment, the sustainable development of keeping oceanic resources has great scientific meaning and using value.
In the process that the petroleum pollution in ocean biological restoration is processed, 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 the bio-augmentation method has two development trends: the one, and for the ecosystem of microbiological deterioration scarce capacity, add inoculating microbe and do additional; The 2nd, the degrading microorganism of premium properties is selected or transformed to the crude oil type that can not degrade for indigenous microorganism or important oil composition." nature and acceleration biological treating research project " that United States Government subsidizes tries hard to by the modern molecular biology means, illustrating the suitable molecular regulation means of basis development of pollutent metabolic mechanism, to reduce nutrition addition and the biological accumulation amount of degradation bacteria in the biological restoration process; Also have the scholar with Pseudomonas aeruginosa as acceptor, the various plasmids that pseudomonas putida etc. is carried change over to wherein, consisted of " super have a liking for oily engineering bacteria " with multiple plasmid, be used for marine oil overflow and process, its high tens times of energy force rate wild mushroom removing greasy dirt arrives hundred times.Domestic also have the scholar to utilize the mixed bacterium synergy to strengthen the degradation effect of petroleum hydrocarbon, the 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: CN 101050435A), 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: CN 101717725A), utilize the microbiobacterial agents such as the logical bacterium at least a fixation support embedding such as diatomite, kaolin, the peat composed of rotten mosses and chitin bacillus pumilus, Venice acinetobacter calcoaceticus, pseudomonas mendocina and Pi Shi Rolls, to improve oil spill shoreline biological degradation effect.These inventions can improve the biological restoration efficient 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 that uses 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 the 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 the absorption microbial inoculum, be dense by bacterium be 10
8~ 10
10Add 100 ~ 1000g/L porous medium and 1 ~ 2g/L flocculation agent in the microbial fermentation bacterium liquid of the suitable degraded target oil product of individual/mL, fully be 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 preserving number is CGMCC No.6104; hereinafter to be referred as T7-3); or burkholderia (Burkholderia mallei ATCC 23344; buy in U.S. typical case's culture center; its article No. is ATCC23344, hereinafter to be referred as ATCC23344), or acinetobacter calcoaceticus (Acinetobacter haemolyticus ATCC 19194, buy in U.S. typical case's culture center, its article No. is ATCC19194, hereinafter to be referred as ATCC 19194), or genus bacillus (Bacillus licheniformis ATCC 14580, buy in U.S. typical case's culture center, its article No. is ATCC14580, hereinafter to be referred as ATCC 14580), or pseudomonas (Pseudomonas aeruginosa PA01, buy in U.S. typical case's culture center, its article No. is ATCC15692, hereinafter to be referred as ATCC 15692), 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 fit to pollution degradation zone crude oil;
2nd, obtaining bacterium dense through slant culture, shake-flask seed fermentation and fermentor tank cascade amplification is 10
8~ 10
10The zymocyte liquid of individual/mL;
3rd, in the zymocyte liquid in the 2nd step, add 100 ~ 1000g/L porous medium, fully mix; Described porous medium is zeolite or haydite;
4th, in the mixture in the 3rd step, add flocculation agent 1 ~ 2g/L, fully mix, leave standstill 0.5 ~ 24h after the mixing, 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 the absorption microbial inoculum and all is 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 the marine bottom sediment, and can prolong 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, enlarge the range of application of remediation microbial inoculum, therefore had Marine shoal and marine bottom sediment petroleum pollution biological restoration ability.
Description of drawings
Fig. 1 is that the alkane degradation bacterium is to the degradation rate temporal evolution graphic representation of crude oil.
Fig. 2 is the residual microorganism effect comparison diagram of supernatant liquor before and after the solid material absorption (A is coated with dull and stereotyped effect for the fermented liquid supernatant 100 μ l of not absorption, and B is that the fermented liquid supernatant 100 μ l after the absorption are coated with dull and stereotyped effect).
Fig. 3 is fermented liquid outside drawing before and after the solid material absorption (A is the fermented liquid that does not adsorb for the fermented liquid after adsorbing, B).
Fig. 4 is scanning electron microscope (SEM) figure before and after the solid material absorption (A is that the material porous medium before the absorption is inner, and B is inner for the material porous medium after adsorbing).
Fig. 5 is total plate count change curve in the simulating lab test settling.
Fig. 6 is alkane degradation bacterium sum change curve in the 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 the beach field experiment settling.
Fig. 9 is total count change curve in the beach field experiment settling.
Figure 10 is the change curve of alkane degradation bacterium in the beach field experiment settling.
Figure 11 is 60 days degradation rate histograms of total petroleum hydrocarbon in the beach test in place settling.
Embodiment
1, the screening of degraded bacterial classification
Collect crude oil (the present embodiment temporarily take extra large two station commingled crudes as example, hereinafter to be referred as crude oil) to pollute the region, 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, NaCl 20g/L, yeast powder 0.05g/L, pH 7.0 ~ 7.2) in, forming the crude oil minimal medium, 121 ℃ of sterilization 20min screen the bacterial classification good to the 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 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; its preserving number is CGMCC No.6104, suggestion Classification And Nomenclature are that rhodococcus erythropolis (Rhodococcus erythoropolis) is hereinafter to be referred as T7-3).The crude oil minimal medium that is seeded to sterilize, measured the oil degradation rate of bacterial classification in degradation process, wherein: crude content after oil degradation rate %=(degraded front crude content-degraded)/the front former oil concentration of degraded * 100%, crude content is measured and is measured with reference to GB GB17378.4-2007 method.The results are shown in shown in Figure 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 the mL.The below selects is a kind of in the microorganism described in the summary of the invention, does not limit the present invention.
1), flat board divides pure
T bacteria 7-3 with on disinfection inoculation ring picking list colony inoculation to the 1 solid medium flat board, is rule repeatedly, and behind 25 ℃ of lower cultivation 72h, observation has or not miscellaneous bacteria, with the purity of checking object bacteria.
Wherein, No. 1 the dull and stereotyped preparation method of solid medium is as follows: take by weighing respectively the 5.0g peptone, 1.0g yeast powder, then stirring and dissolving takes by weighing the 18g agar powder in 1000mL ageing seawater successively, boil dissolving after, transfer pH7.6 ~ 7.8, behind 121 ℃ of lower sterilization 20min, be down to 50 ℃, pour and be tiled in culture dish (d=10cm) bottom into every 20mL, obtain the solid medium flat board after the cooling.
2), slant culture
Will be on the above-mentioned flat board the correct single bacterium colony of checking with disinfection inoculation ring picking one ring, be inoculated on No. 1 culture medium slant, repeatedly line, 25 ℃ lower cultivate 72h after, for subsequent use.
Wherein, No. 1 the culture medium slant preparation method is as follows: take by weighing respectively the 5.0g peptone, 1.0g yeast powder, then stirring and dissolving takes by weighing the 18g agar powder in 1000mL ageing seawater successively, after boiling dissolving, transfer pH7.6 ~ 7.8, behind 121 ℃ of lower sterilization 20min, be down to 50 ℃, be sub-packed in 180mm * 18mm(length * diameter with every 5mL) in the test tube, 10 ° of-20 ° of inclination room temperatures are placed, and obtain culture medium slant after the cooling.
3), shake-flask seed is cultivated
With the bacterial classification on the above-mentioned inclined-plane, aseptic technique is inoculated in the 500mL Erlenmeyer flask of No. 1 liquid nutrient medium that 200mL is housed on Bechtop, and 25 ℃, 200r/min cultivates cell density and just reached 5 * 10
8Individual/mL namely stops to cultivate, and is for subsequent use.
Wherein No. 1 liquid nutrient medium preparation method is as follows: take by weighing respectively the 5.0g peptone, and the 1.0g yeast powder, stirring and dissolving is in 1000mL ageing seawater successively, and every 200mL divides and installs in the 500mL Erlenmeyer flask, in 121 ℃ of lower sterilization 20min.
4), fermentor cultivation
Above-mentioned seed liquor nutrient solution being housed, 40 ~ 60% liquid measures is amplified fermentation culture in the fermentor tank of sterilization fermentation substratum respectively step by step with the inoculum size of 3-5%.In the fermenting process, control 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 the mL.
The preparation method of above-mentioned fermention medium is as follows: take by weighing 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 sal epsom, 0.01g calcium chloride, 0.005g vitamin H and add to successively in the distilled water of 1000mL, transfer pH to 7.2 after the stirring and dissolving, in 115 ℃ of sterilization 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 adsorption experiment with the listed ratio of table 1, wherein stir again static 1.5h of 2min behind the 1-9 group reinforcing body adsorption medium, system after the absorption is got supernatant liquor 100 μ l dilution and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in the 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%.No. 3, the microbial inoculum that forms take the 3rd group of experiment is as example, and the T7-3 cell concentration is 9 * 10 before the zeolite adsorption
8CFU/mL, cell concentration is 1.9 * 10 after the absorption
8CFU/mL, adsorption rate reaches 78.9%, average every g zeolite adsorption 1.8 * 10
9The CFU thalline.
Table 1 porous medium is to the adsorption of T7-3 somatic cells
| Group | Bacterium number | Bacterium liquid ml | Zeolite g | Haydite g | Potassium aluminium sulfate g | Adsorption rate % | |
| 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, take 25ml bacterium liquid as example, material add-on 2.5g, namely during mass volume ratio 100g/L, corresponding bacterium material is than being 10:1; Material add-on 12.5g, namely 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.Add more than the material 500g/L, the thalline adsorption rate just can reach more than 75%, and the fermented liquid outside drawing illustrates that the solid materials such as zeolite or haydite are higher to the thalline adsorption efficiency as shown in Figure 3 before and after the solid material absorption, can be applied to the preparation of solid fungicide.Solid microbial sample after blank zeolite and the absorption is carried out scanning electron microscope (SEM) to be detected, as shown in Figure 4, can obviously see the thalline that is adsorbed on the zeolite, and cell density is larger, illustrates that it is feasible using zeolite to carry out thalline absorption preparation solid microbial as carrier.
1, the screening of degraded bacterial classification
Collect crude oil (the present embodiment temporarily take extra large two station commingled crudes as example, hereinafter to be referred as crude oil) to pollute the region, 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, NaCl 20g/L, yeast powder 0.05g/L, pH 7.0 ~ 7.2) in, screening the bacterial classification good to the oil degradation performance, culture condition is 25 ℃, 150r/min, inoculum size 2%, incubation time is 7d.
The present embodiment is selected burkholderia (Burkholderia mallei ATCC 23344 from 30 kinds of bacterial strains to be selected, buy in U.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: crude content after oil degradation rate %=(degraded front crude content-degraded)/the front former oil concentration of degraded * 100%, crude content is measured and is measured with reference to GB GB17378.4-2007 method.The results are shown in shown in Figure 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 adsorption experiment with the listed ratio of table 2, stir again static 1.5h of 2min behind the reinforcing body adsorption medium, system after the absorption is got supernatant liquor 100 μ l dilution and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in the 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%.No. 14, the microbial inoculum that forms take the 14th group of experiment is as example, and adsorption rate reaches 86.7%.
Table 2 porous medium is to the adsorption of ATCC23344 somatic cells
| Group | Bacterium number | Bacterium liquid ml | Zeolite g | Haydite g | Potassium aluminium sulfate g | Adsorption rate % | |
| 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 adsorption more than 75% to somatic cells.Add more than the material 500g/L, the thalline adsorption rate just can reach more than 80%.
1, the screening of degraded bacterial classification
Collect crude oil (the present embodiment temporarily take extra large two station commingled crudes as example, hereinafter to be referred as crude oil) to pollute the region, 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, NaCl 20g/L, yeast powder 0.05g/L, pH 7.0 ~ 7.2) in, screening the bacterial classification good to the 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. 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: crude content after oil degradation rate %=(degraded front crude content-degraded)/the front former oil concentration of degraded * 100%, crude content is measured and is measured with reference to GB GB17378.4-2007 method.The results are shown in shown in Figure 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 adsorption experiment with the listed ratio of table 3, stir again static 1.5h of 2min behind the reinforcing body adsorption medium, system after the absorption is got supernatant liquor 100 μ l dilution and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in the 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%.No. 25, the microbial inoculum that forms take the 25th group of experiment is as example, and adsorption rate reaches 88.5%.
Table 3 porous medium is to the adsorption of 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, the material add-on is in 100 ~ 1000g/L, and porous medium has adsorption more than 60% to somatic cells.Add more than the material 500g/L, the thalline adsorption rate just can reach more than 80%.
The preparation method that embodiment 4, aeruginosa atcc 15692 solidify the absorption microbial inoculum
1, the screening of degraded bacterial classification
Collect crude oil (the present embodiment temporarily take extra large two station commingled crudes as example, hereinafter to be referred as crude oil) to pollute the region, 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, NaCl 20g/L, yeast powder 0.05g/L, pH 7.0 ~ 7.2) in, screening the bacterial classification good to the 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. 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: crude content after oil degradation rate %=(degraded front crude content-degraded)/the front former oil concentration of degraded * 100%, crude content is measured and is measured with reference to GB GB17378.4-2007 method.The results are shown in shown in Figure 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 adsorption experiment with the listed ratio of table 4, stir again static 1.5h of 2min behind the reinforcing body adsorption medium, system after the absorption is got supernatant liquor 100 μ l dilution and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in the 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%.No. 35, the microbial inoculum that forms take the 35th group of experiment is as example, and adsorption rate reaches 82.5%.
Table 4 porous medium is to the adsorption of 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, the material add-on is in 100 ~ 1000g/L, and porous medium has adsorption more than 65% to somatic cells.Add more than the material 500g/L, the thalline adsorption rate just can reach more than 75%.
The preparation method that embodiment 5, mixed bacterium are solidified the absorption microbial inoculum
1, the screening of degraded bacterial classification
Collect crude oil (the present embodiment temporarily take extra large two station commingled crudes as example, hereinafter to be referred as crude oil) to pollute the region, 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, NaCl 20g/L, yeast powder 0.05g/L, pH 7.0 ~ 7.2) in, screening the bacterial classification good to the 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. typical case's culture center, its article No. is ATCC15692, hereinafter to be referred as ATCC 15692), acinetobacter calcoaceticus (Acinetobacterhaemolyticus ATCC 19194, buy in U.S. typical case's culture center, its article No. is ATCC19194, hereinafter to be referred as ATCC 19194), burkholderia (Burkholderia mallei ATCC 23344, buy in U.S. typical case's culture center, its article No. is ATCC23344, hereinafter to be referred as ATCC23344), and rhodococcus (Rhodococcus erythoropolis T7-3, 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); crude content after oil degradation rate %=(degraded front crude content-degraded)/the front former oil concentration of degraded * 100% be seeded to the crude oil minimal medium of bacterium; measured the oil degradation rate of bacterial classification in degradation process; wherein:, crude content mensuration is measured with reference to GB GB17378.4-2007 method.The results are shown in shown in Figure 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 adsorption experiment with the listed ratio of table 5, stir again static 1.5h of 2min behind the 40-48 group reinforcing body adsorption medium, add again 1 ~ 2g/l potassium aluminium sulfate behind the 49-50 group reinforcing body adsorption medium, shake rear static 1.5h, system after the absorption is got supernatant liquor 100 μ l dilution and is coated with nutrient agar plate calculating residue bacterium dense (cfu1), and compare with the bacterium in the 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%.No. 42, the microbial inoculum that forms take the 42nd group of experiment is as example, and adsorption rate reaches 82.5%.No. 49, the microbial inoculum that forms take the 49th group of experiment is as example, and adsorption rate reaches 98.5%.
Table 5 porous medium is to the adsorption of mixed bacterium somatic cells
| Group | Bacterium number | Bacterium liquid ml | Zeolite g | Haydite g | Potassium aluminium sulfate g | Adsorption rate % | |
| 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 the volume ratio of mixed bacterium bacterium liquid and material reaches more than the 2:1, the thalline adsorption rate just can reach more than 80%, adds the effect that the flocculation agent potassium aluminium sulfate can improve solid material absorption thalline, and the thalline adsorption rate can reach 98%.
Data in the consolidated statement 1 ~ 5, obtain the best preparation method of curing absorption microbial inoculum provided by the invention: namely per 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 mix, leave standstill 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, leave standstill 1.5h behind the mixing, can make the curing absorption microbial inoculum of thalline adsorption rate 〉=98%.
What the present embodiment was narrated is the 7th group of simulation repairing effect that solidifies the absorption microbial inoculum in the table 1, does not limit the present invention.
In 3 stainless cylinder of steels of 10L (d=40cm at the bottom of the tank, the high 80cm of tank), add respectively 3L artificial seawater (NaCl 24.53g, KCl 0.695g, CaCl
21.16g, MgCl
25.2g, Na
2SO
44.09g, NaHCO
30.201g, KBr 0.101g, H
3BO
30.027g, SrCl
20.025g, NaF 0.003g, pH 7.8, prepare with distilled water), 121 ℃ of sterilization 20min add respectively the marine bottom sediment that 3kg is mixed with 3g sea two station commingled crudes, after the standing over night, add respectively 1kg/m
2, 1.5kg/m
2, 2kg/m
2The solid absorption microbial inoculum of repaired area adds respectively and is 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, get about each 5g of settling with sterile sampler, deposit in the 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/T 21247-2007) in the settling.
Wherein the measuring method of petroleum hydrocarbon degradation bacterium number is as follows: testing sample is done 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, pH 7.2) in 25 ℃ of lower cultivations 7-10 days, in last 3 extent of dilution (being critical progression) that will have bacterium liquid to grow the pipe number of bacterial growth appears as quantitative index (observation index is substratum turbidity and hydrocarbons outward appearance emulsion dispersion degree during counting), by finding approximation on the maximum most probable number (MPN) table, multiply by again the extension rate of quantitative index the first figure place, be and contain the bacterium number in the original bacteria liquid.
The measurement result of total plate count and alkane degradation bacterium quantity can find out that the bacterium number of the petroleum hydrocarbon degradation bacterium in the experiment is occupied an leading position all the time as shown in Figure 5 and Figure 6, illustrates that the bacterium of alkane degradation bacterium is dense higher, more is conducive to improve petroleum degradation rate.Solidify the absorption microbial inoculum at 25 ℃, the degradation efficiency temporal evolution enlarges markedly, and especially since the 10th day, degradation efficiency had large increase, about the 13d of experimental phase has reached 50% (Fig. 7), the adding of solidifying the absorption microbial inoculum be conducive to degrade petroleum hydrocarbon in the settling is described.
What the present embodiment was narrated is the 42nd group of simulation repairing effect that solidifies the absorption microbial inoculum in the table 5, does not limit the present invention.
In 3 stainless cylinder of steels of 10L (d=40cm at the bottom of the tank, the high 80cm of tank), add respectively 3L artificial seawater (NaCl 24.53g, KCl 0.695g, CaCl
21.16g, MgCl
25.2g, Na
2SO
44.09g, NaHCO
30.201g, KBr 0.101g, H
3BO
30.027g, SrCl
20.025g, NaF 0.003g, pH 7.8, prepare with distilled water), 121 ℃ of sterilization 20min add respectively the marine bottom sediment that 3kg is mixed with 3g sea two station commingled crudes, after the standing over night, add respectively 1kg/m
2, 1.5kg/m
2, 2kg/m
2The solid absorption microbial inoculum of repaired area adds respectively and is 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, get about each 5g of settling with sterile sampler, deposit in the 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/T 21247-2007) in the settling.
Wherein the measuring method of petroleum hydrocarbon degradation bacterium number is as follows: testing sample is done 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, pH 7.2) in 25 ℃ of lower cultivations 7-10 days, in last 3 extent of dilution (being critical progression) that will have bacterium liquid to grow the pipe number of bacterial growth appears as quantitative index (observation index is substratum turbidity and hydrocarbons outward appearance emulsion dispersion degree during counting), by finding approximation on the maximum most probable number (MPN) table, multiply by again the extension rate of quantitative index the first figure place, be and contain the bacterium number in the original bacteria liquid.
The bacterium number of the petroleum hydrocarbon degradation bacterium in the experiment is occupied an leading position all the time, and the bacterium of alkane degradation bacterium is dense higher, more is conducive to improve petroleum degradation rate.Solidify the absorption microbial inoculum at 25 ℃, the degradation efficiency temporal evolution enlarges markedly, and especially since the 10th day, degradation efficiency had large increase, reached about 60% in the 13rd day in the experimental phase, the adding of solidifying the absorption microbial inoculum be conducive to degrade petroleum hydrocarbon in the settling has been described.
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 the table 5, does not limit the present invention.
Selected petroleum pollution shoal area delimited the experiment block of 3 4m * 4m in Bohai Sea Gulf, and wherein 2 blocks are made as the test site, and wherein 1 block (T1) is pressed 3L/m
2The beach area adds mixed bacteria liquid, presses 0.6kg/m
2The beach area adds nutrition agent (sulfate of ammoniac, Sodium phosphate dibasic); 1 block (T2) is pressed 1.5kg/m in addition
2The beach area adds curing absorption microbial inoculum (add 500 ~ 1000g/L zeolite in the bacterium liquid, add the auxiliary absorption of flocculation agent of 1 ~ 2g/L), presses 0.6kg/m
2The beach area adds nutritive salt fertilizer (sulfate of ammoniac, Sodium phosphate dibasic), and the ploughing and weeding formula was broadcasted sowing after above two block microbial inoculum addition manners were and mix; All the other 1 blocks (T3) are as blank assay.Method according to embodiment 6 is carried out tracking monitor to experiment block deposition medium, 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/T 21247-2007), with site operation same day as 0d, carry out tracking monitor at 3d, 9d, 18d, 30d, 60d respectively afterwards.
The results are shown in Figure shown in the 8-10, as seen test the initial stage, 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 in zeolite and make the quantity of bacterium lose relevant when the zeolite adsorption bacterium liquid; In the experiment later stage, add the effect of solidifying absorption microbial inoculum (T2) and be better than the effect of spraying bacterium liquid (T1).Therefore, solidify the 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.
What the present embodiment was narrated is that the effect of the 14th group of curing absorption microbial inoculum in the beach reparation in microbial inoculum and the table 2 adsorbed in the 7th group of curing in the table 1, does not limit the present invention.
Selected petroleum pollution shoal area delimited the experiment block of 3 4m * 4m in Bohai Sea Gulf, and wherein 2 blocks are made as the test site, are respectively T4: press 1.5kg/m
2The beach area adds the 7th group and solidifies the absorption microbial inoculum, presses 0.6kg/m
2The beach area adds nutrition agent (sulfate of ammoniac, Sodium phosphate dibasic); T5 presses 1.5kg/m
2The beach area adds the 14th group and solidifies the absorption microbial inoculum, presses 0.6kg/m
2The beach area adds nutritive salt fertilizer (sulfate of ammoniac, Sodium phosphate dibasic), and the ploughing and weeding formula was broadcasted sowing after above two block microbial inoculum addition manners were and mix; All the other 1 block T6 are as blank assay.Method according to embodiment 6 was tested the variation that the block deposition medium is monitored total petroleum hydrocarbon (GB/T16488-1996) afterwards to 60 days, take T6 as contrasting with calculation of oil alkane 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%.This curing absorption microbial inoculum is conducive to hold for a long time bacterium in addition, 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 the absorption microbial inoculum in the his-and-hers watches 5, does not limit the present invention.
According to " offshore oil exploration and exploitation pollutent bio-toxicity " (GB/T 18420.2-2001), utilize the 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 an amount of halogen worm (Artemiidae) worm's ovum, add the 1L seawater and under 26 ℃ of room temperatures, illumination condition, cultivate 24h.According to adding than requiring, get successively tested mixed bacterium and solidify the 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 an amount of each concentration sample, under 26 ℃ of room temperatures, illumination condition, cultivate.And each concentration establish one in contrast parallel.Every 24h observes, the dead number of record halogen worm.
The results are shown in Table in 6, the 72h incubation time, the halogen worm survival rate of each volumetric concentration is all more than 56%.In the 96h incubation time, volumetric concentration is that halogen worm survival number is maximum in 6 ‰ the microbial inoculum, and survival rate reaches more than 90%.Show that solidifying the absorption microbial inoculum 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 |
| |
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 (8)
1. a microorganism that is applicable to Marine shoal and the petroleum pollution of thalassogenic sedimentation substance environment is repaired the preparation method of solidifying the absorption microbial inoculum, it is characterized in that the preparation process of described curing absorption microbial inoculum is:
1st, choose the microbial strains that is fit to pollution degradation zone crude oil;
2nd, obtaining bacterium dense through slant culture, shake-flask seed fermentation and fermentor tank cascade amplification is 10
8~ 10
10The zymocyte liquid of individual/mL;
3rd, in the zymocyte liquid in the 2nd step, add 100 ~ 1000g/L porous medium, fully mix;
4th, in the mixture in the 3rd step, add flocculation agent 1 ~ 2g/L, fully mix, leave standstill 0.5 ~ 24h after the mixing, make the curing absorption microbial inoculum of thalline adsorption rate 〉=90%.
2. method according to claim 1 is characterized in that described porous medium is zeolite or haydite.
3. a microorganism that is applicable to Marine shoal and the petroleum pollution of thalassogenic sedimentation substance environment is repaired and solidifies the absorption microbial inoculum, it is characterized in that described curing absorption microbial inoculum be dense by bacterium be 10
8~ 10
10Add 100 ~ 1000g/L porous medium and 1 ~ 2g/L flocculation agent in the microbial fermentation bacterium liquid of the suitable degraded target oil product of individual/mL, fully be mixed.
4. microbial inoculum is adsorbed in curing according to claim 3, it is characterized in that described microorganism comprises:
Rhodococcus (Rhodococcus erythoropolis T7-3), preserving number is CGMCC No.6104; Or burkholderia (Burkholderia mallei ATCC23344); Or acinetobacter calcoaceticus (Acinetobacter haemolyticus ATCC19194); Or genus bacillus (Bacillus licheniformis ATCC14580); Or pseudomonas (Pseudomonas aeruginosa PA01); Or the equal proportion mixture of these bacterium.
5. according to claim 3 or 4 described curing absorption microbial inoculums, it is characterized in that described porous medium is zeolite or haydite.
The preparation of the described method of claim 1 or the application of curing absorption microbial inoculum claimed in claim 3 in the biological restoration of Marine shoal or the petroleum pollution of thalassogenic sedimentation substance environment.
7. application according to claim 6 is characterized in that by 1 ~ 2kg/m
2Repaired area adds described curing absorption microbial inoculum, by 0.2 ~ 1kg/m
2Repaired area adds nutritive salt fertilizer.
8. application according to claim 7 is characterized in that described nutritive salt fertilizer is sulfate of ammoniac or Sodium phosphate dibasic.
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