CN106542643A - The method that the oily microbial inoculum of efficient piezophilic drop with Woelm Alumina as carrier processes deep-sea oil spilling - Google Patents
The method that the oily microbial inoculum of efficient piezophilic drop with Woelm Alumina as carrier processes deep-sea oil spilling Download PDFInfo
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Abstract
The invention provides a kind of efficiently drop the method that oily microbial inoculum processes deep-sea oil spilling by carrier apposition growth of Woelm Alumina, porous alumina carrier is prepared using gel injection-moulding method, it is characterised in that：(1) cylindric porous alumina carrier, a diameter of 1.60-1.70cm, a height of 1.30-1.40cm, porosity are 52-59%, and bulk density is 2.57-2.67g/cm3, pore-size distribution is micron order, and the load bacterium amount of carrier is up to 1.1 × 108-1.3×108Cfu/；(3) good pressure-resistant performance, can bear 0.4MPa pressure below.The described oily microbial inoculum of efficient piezophilic drop is the efficient drop oil microbial inoculum for being resistant to 0.3-0.4MPa pressure.After described porous alumina carrier apposition growth microbial inoculum, it is about 1cm using 1350-1400 aperture, the length of side is about the square net about 9-10 Woelm Alumina of loading of 20cm and is placed in the large-scale trawlnet with buoy and is bound to its bottom, Jing ships are transported and are thrown in the deep-sea oil spill marine site basic sediment within 30-40 rice depth, can effectively realize the recycling of degraded and Woelm Alumina of the microbial inoculum to deposit petrochina hydro carbons.
Description
Technical field
The invention belongs to deep-sea oil spilling technical field of biological remediation, and in particular to one kind is with Woelm Alumina as carrier
The method that the oily microbial inoculum of the efficient piezophilic drop of apposition growth processes deep-sea oil spilling.
Background technology
As the world is to oil and its growing, exploitation at sea, transport, handling and the stone of product demand
Oil spill accident during oily use is also increasing, causes serious environmental pollution.Crude Oil at Sea leakage etc.
Accident is caused to China's immediate offshore area ecological environment and is had a strong impact on, and oil spill accident process in take add
The measures such as detergent are that the oil slick of ocean surface is transferred in marine sediment, and this method thoroughly can not be solved
The harm that certainly marine oil spill is caused to marine ecosystems.
Microbial degradation be remove environment petrochina pollutant main path, relative to either physically or chemically it
The characteristics of with low cost, small investment, efficiency high.Microbe carrys out catalytic degradation petroleum hydrocarbon, reduce or
It is final to eliminate oil pollution just by the most attention of countries in the world.Many microorganisms can be with hydro carbons as sole carbon source
Growth, the microorganism of decomposing petroleum hydrocarbon are present in a large number by petroleum hydrocarbon contaminated water body and basic sediment.For
The petroleum hydrocarbon in marine bottom deposit is eliminated, and generally oil degradation bacteria is thrown in using to oil spilling marine site at present
Agent is come the bioremediation technology of oil spilling of degrading, and its key problem in technology is the fixation rate for improving seabed microorganism.Due to
Biological restoration microbial inoculum density itself is low and relatively decentralized, and the microbial inoculum of input is often floated across the sea, it is difficult to seabed
Contaminated deposit plays Degradation.And adopt immobilized microorganism technology, will microorganism be fixed on
In effect carrier, keep the activity of microorganism and the cell density of unit volume microorganism can be improved, effectively can solve
The biological restoration problem of environment is polluted certainly.Immobilized microorganism energy stay active for long periods, immobilized microorganism are carried
The microenvironment of body also helps the indigenous bacterium of shielding, phage and toxicant harmful competition, phagocytosis to microorganism
And murder by poisoning, mitigate infringement of the shearing force of wave to microorganism.Carrier is immobilized key, domestic outside water
The kind of carrier adopted in process is a lot, mainly includes machine material support and Inorganic material carrier.As peat,
Filler, activated carbon, coke fines, zeolite, fine quartz sand, Vermiculitum etc., but these carrier application Yu Haiyangs are overflow
The biological restoration of oil all has certain defect.Such as peat, filler, activated carbon, coke fines, zeolites
These carriers are difficult to sink to seabed with contaminated deposition as density is little, comprcssive strength is little, mechanical strength is low
Thing is contacted, it is more difficult to bear subsea pressure and water flow impact pressure.And fine quartz sand carrier granular little Yi is rushed by wave
Dissipate, Vermiculitum class carrier mainly uses its cation exchange property, as remove harmful substance adsorbent and from
Sub- commutativity inorganic agent, for wastewater treatment；The interlamellar spacing of Vermiculitum class carrier is little for nanoscale, is unfavorable for micro- life
The load growth of thing, diminishes and is difficult to reach although high-temperature heating can load microbial inoculum density after volumetric expansion
Load bacterium to deep-sea crude Treatment requires.Even if expending a large amount of financial resource and material resource, affected by the special environment in seabed,
Utilization these the carrier loaded high efficient petroleum degrading bacteria agent thrown in oil spilling marine site are easily broken up and are hardly entered
And anchor in deposit, cause the content of microorganisms in deposit less, microorganism is weakened in deposit
The degradation capability of petroleum hydrocarbon.Therefore, research density is big, compressive resistance is high and is capable of attached microbial growth
The preparation of porous inorganic material carrier seems very necessary.Such as list of references [1] Zhu Zhu, Li Heping, Gu Zheng Zegen.
Surely the carrier material in cell technology and its application [J] in environmental improvement are changed. Chongqing Univ. of Architecture's journal,
2000,22(5):99-100；[2] Li Hui, Wang Ping, Xiao Ming. kieselguhr and Pulvis Talci are used as pseudomonas fluorescens P13
The study on the carrier [J] of microbial inoculum. Chinese biological is prevented and treated, and 2009,25 (3):239-244；[3] Xu Jinlan, Huang Tinglin, Tang Zhi
It is new etc. the screening of high efficient petroleum degrading bacteria and the research [J] of oil-polluted soils biological restoration characteristic. environment section
Journal, 2007,27 (4):622-625；[5] Liu Yun, Wu Pingxiao, Dang Zhi. a huge sum of money in pillared vermiculite Adsorption waste water
The experimentation [J] of category ion. Minerals And Rocks, 2006,26 (4):8-13.
Porous ceramicss are that a kind of Jing high temperature burns till, a large amount of phases each other are formed in shaping with material body in sintering process
New ceramic material that is logical or closing pore.Porous ceramicss have porosity big compared with high, specific surface area, mechanical strong
The features such as higher, chemical stability of degree is good allows porous material as the good load of deep-sea oil spilling remediation microbial inoculum
Body.Wherein, porous zirconia and porous oxidation aluminum are respectively provided with excellent process based prediction model, are porous
The emphasis and focus of ceramic research.Compared with porous oxidation zirconia material, porous oxidation aluminum is possessing higher hole
While the features such as gap rate, high density, high pressure resistant, anti-shearing and stable chemical nature, with more cost
Cheap advantage, it is adaptable to the widespread adoption of actual deep-sea oil spilling degraded.Such as list of references [4] Wang Lili, king
Elegant peak, Jiang Hongtao etc. preparation and the performance [J] of hydroxyapatite coating layer material are coated on porous zirconia matrix.
Mechanical engineering material, 2008,32 (12):5-7；[5] Yang Fei, Yang Rentang, Li Jun etc. inorganic Zeolite support antibacterial
Preparation and its anti-microbial property [J]. paper and papermaking, 2009,28 (3):28-31；[6] Tang Lin. macropore alumina supporter
The research [J] of preparation condition. Chemical Engineer, 2014,01:56-57；[7] Xu Huanhuan. multiaperture pellumina
Prepare and its applied research [D]. Nanjing, Nanjing Normal University .2013.
Based on the above-mentioned advantage of porous oxidation aluminum, the present invention is selected as the load of the oily microbial inoculum of efficient piezophilic drop
Body simultaneously is prepared for meeting the Woelm Alumina of microbial inoculum carrier requirement.The present invention is using porous oxidation aluminum as carrier
Deep-sea oil spilling marine site of the 30-40 rice with is thrown to after the oily microbial inoculum of the efficient piezophilic drop of apposition growth, bacterium is realized
Agent processes deposition to effective degraded of marine sediments petrochina and targetedly, centrality, non-secondary pollution
Petroleum hydrocarbon in thing, and the mesh that the immediate offshore area ecological environment harm that marine oil spill accident is caused is minimized
Mark.
The content of the invention
In order to overcome the deficiencies in the prior art, the invention provides one kind is with Woelm Alumina as carrier apposition growth
The method that efficiently the oily microbial inoculum of piezophilic drop processes deep-sea oil spilling.
The technical solution adopted in the present invention is：High according to oil spilling marine site subsea pressure, ocean current impulsive force is larger
Special environment, is prepared for using gel injection-moulding method that porosity is higher, bulk density is larger and compressive resistance is higher
Woelm Alumina, the Woelm Alumina of preparation meets the requirement of microbial inoculum carrier and cost is cheap.
Adopt Woelm Alumina prepared by gel injection-moulding method for cylindric, a diameter of 1.60-1.70cm is a height of
1.30-1.40cm, porosity are 52-59%, and bulk density is 2.57-2.67g/cm3, pore-size distribution is micron order,
The load bacterium amount of carrier is up to 1.1 × 108-1.3×108Cfu/.
Compared with prior art, the invention has the beneficial effects as follows can be by the porous alumina carrier of preparation come attached
The oily microbial inoculum of the efficient piezophilic drop of growth and support density is larger, the depth of below sea level 30-40 rice can be rendered to
Extra large oil spill marine site bottom, improves degradation efficiency of the microbial inoculum to marine sediments petrochina hydro carbons.Its characteristic
Can be to bear oil spilling marine site bottom not rupture less than the pressure of 0.4MPa, be difficult to be impacted and occurred by ocean current
Migrate, appointed place can be sunk to without the need for external force, it is achieved thereby that efficiently the oily microbial inoculum of piezophilic drop pollutes heavy to seabed
Effective, the targetedly processing intent of product thing petrochina hydro carbons.
Description of the drawings
Fig. 1 dehydrogenase activity standard curves
The size of the oily microbial inoculum dehydrogenase activity of efficient piezophilic drop is represented with TTC values, absorbance represents the density of bacterium solution,
Determine under different absorbances the size of TTC values and map, the dependency of matched curve is fine.
Fig. 2 dehydrogenase activities-bacterium solution volume relation curve
Fig. 3 porous alumina carriers
Take the logarithm trophophase bacterium solution it is each 1,2,3,4,5,6ml, according to measuring desaturase liveness method, respectively
Its dehydrogenase activity is surveyed, dehydrogenase activity-bacterium solution volume curve is drawn, as shown in Figure 2.Relative index
R2=0.9971, illustrate that dehydrogenase activity is fine with the dependency of bacterium solution volume.
Specific embodiment
1. the preparation of Woelm Alumina and sign
Porous oxidation aluminum is prepared using gel injection molding and forming technology, its thinking is first to prepare low viscosity, height
The concentrate suspension of solid volume fraction；Then, addition initiator and catalyst in suspension；To finally prepare
The slurry cast for going out in model, in the presence of initiator and catalyst, send out by the organic monomer in concentrate suspension
Raw cross-linking polymerization, forms tridimensional network；In suitable temperature, humidity and atmosphere protection low suspension body
Solid particle coagulation forming in the original location, and then obtain even structure, highdensity base substrate；In appropriate sintering
Further sintered under system, finally given with the stable porous of certain porosity, high intensity, character
Alumina support.Key step is as follows：
1) the alumina powder jointed of Nano Particle is scattered in deionized water, is gradually added a certain proportion of list
Body acrylamide and cross-linking agent methylene-bisacrylamide, the premixed liquid Jing 260r/min ball milling 4-6 for obtaining are little
When, obtain suspension；
2) a certain amount of initiator and catalyst are added in suspension, carries out appropriate mechanical agitation and obtain slurry；
3) slurry is injected into columned mould, is placed on after spontaneously drying 2-3 days under room temperature, the demoulding is obtained
To even structure, highdensity base substrate.The basal diameter of cylindrical mold is 1.7cm, high 1.4cm；
4) base substrate is dried 2 hours at a temperature of 60 DEG C, is dried 2 hours at 100 DEG C；
5) base substrate after drying is sintered, specific sintering schedule such as following table, finally gives and have necessarily
The stable porous alumina carrier of porosity, high intensity, high density and character.
1 blank sintering system table of table
Sample after burning till makes standard specimen, Archimedes method is respectively adopted and determines its bulk density, mercury injection apparatus
Method determines its pore-size distribution, SEM methods and surveys its pore size, and determines porous oxygen using Related Computational Methods
Change high aluminum, basal diameter and comprcssive strength.A diameter of 1.60-1.70cm of Woelm Alumina is calculated, it is high
For 1.30-1.40cm, porosity is 52-59%, and bulk density is 2.57-2.67g/cm3, pore-size distribution is micro-
Meter level.Porous alumina carrier cost and low raw-material cost that firing is obtained, can carry out batch production preparation.
2. Woelm Alumina carries bacterium step and carries the calculating of bacterium amount
The dehydrogenase activity of microorganism reflects the ability of its degradation of organic substances, contained bacterium in certain fluid medium
The dehydrogenase activity value of agent is suitable with the dehydrogenase activity value of carrier loaded microbial inoculum, can substantially estimate carrier
Load bacterium amount.
1) Woelm Alumina carries bacterium step and the calculation procedure of load bacterium amount is as follows：
1ml bacterium solutions are taken in superclean bench adds what is sterilized to fill 100ml beef extract-peptone liquid cultures
Base (Carnis Bovis seu Bubali cream 3g/L, peptone 10g/L, Sodium Chloride 30g/L, pH 7.0-7.2,121 DEG C of sterilizing 20min)
In 250ml conical flasks, while the Woelm Alumina of sterilizing is put into wherein, shake up after sealing and be placed in 25 DEG C -28 DEG C
Pressure for 0.4MPa pressure vessel in cultivate；In 23h-25h, (exponential phase of bacterium solution) takes
Go out aluminium oxide to be placed in the centrifuge tube of 50ml sterilizings.
2) the dehydrogenase activity concrete operations for determining load bacterium Woelm Alumina are as follows：
(1) measure (TTC methods) of Woelm Alumina dehydrogenase activity
1. successively in the centrifuge tube for be loaded with Woelm Alumina add 4mLTris-HCl buffer, Na2SO3
Solution 1mL, TTC solution 1mL so as to do not have the upper surface of peroxidating aluminum.
2. it is immediately placed in after shaking up in 37 DEG C of thermostat water baths, and is shaken gently for, writes down the response time
(10min-60min, depending on colour developing situation).
Centrifuge tube is taken out from water-bath, in pipe adds one to drip concentrated sulphuric acid terminating reaction immediately.
3. 5mL acetone is added in each centrifuge tube, fully mixed, extract TF.
4. each centrifuge tube is centrifuged 2min at 3,000 rpm.
5. draw out supernatant, (absorbance of measure should be to determine absorbance under 486nm wavelength
Less than 0.8, colorimetric again must be diluted during colourity overrich).
6. dehydrogenase activity is represented with TF values, is calculated by following equation：
TF μ g/ (mLh)=A × B × C
A- standard curve respective value, μ g/mL
B- incubation times calibration=60min/ incubation time min, h-1
Extension rate during C- colorimetrics
By being calculated the TF values (dehydrogenase activity) for carrying bacterium Woelm Alumina up to about
74-81μg/(mL·h)。
(2) Woelm Alumina carries the calculating of bacterium amount
Porous can be obtained according to Fig. 2 curves and with reference to the TF peaks (dehydrogenase activity) for carrying bacterium Woelm Alumina
The load thalline product of aluminium oxide is about 2.0-2.1ml.According to colony counting method obtain bacterium solution density be 5.5 ×
107cfu/ml-6.0×107Cfu/ml, thus obtain prepare Woelm Alumina load bacterium amount be 1.1 ×
108cfu-1.3×108Cfu/.
3. the deep-sea put-on method of bacterium Woelm Alumina is carried
Step one, the making for loading load bacterium Woelm Alumina mesh bag
1) several apertures are prepared and is about 1cm, the length of side is about the square net of 20cm, and the aperture of net is less than
The basal diameter of aluminium oxide is missed with preventing the Woelm Alumina for loading.About 9-10 can be loaded using the net
Woelm Alumina and in net can sprawl and come, can avoid overlapping so as to effective utilization space, raising oxygen
Change the contact area of aluminum and deposit.
2) large-scale trawlnet is prepared, the quality of each aluminium oxide is about 7-9g, is calculated according to the useful load of 100Kg
Understand about to load 12000-14000 aluminium oxide, sprawl floor space and be about 2.7-3.2m2, the floor space of trawlnet
Should be greater than 3.2m2.Therefore the trawlnet aperture for preparing is about 5cm, and spreading area is about 4-5m2。
Step 2, the stowage for carrying bacterium Woelm Alumina mesh bag
The Woelm Alumina of about 9-10 load bacterium is placed in net, then the corner of net is aggregating and is used cord
Sealing, so makes several mesh bags for being mounted with Woelm Alumina.Each little mesh bag is attached in sweep net,
With an end closure of rope, there is buoy in other end system, is easy to the position for indicating the Woelm Alumina thrown in while can
To realize that the repetition of aluminium oxide is recycled.
Step 3, the deep-sea put-on method for carrying bacterium Woelm Alumina
Bacterium Woelm Alumina will be carried it will be loaded into 1350-1400 aperture and be about 1cm, the length of side about 20cm will be just
It is placed in inside square mesh bag in large-scale trawlnet, little mesh bag is tied up in large-scale trawlnet bottom with cord, make bottom oxide
Alumina supporter is farthest sprawled, and is placed on ship, and ship is docked in the marine site by oil spill then,
Trawlnet is thrown to the deep-sea oil spill marine site within 30-40 rice depth, and trawlnet can sink down into automatically and spread over sea
On bottom sediment, buoy is fastened on rope with mark position.Can be the porous oxidation for carrying bacterium using the method
Aluminum be thrown to specify by oil spill marine site basic sediment in, realize oil degradation microbial inoculum and bottom sediment
Mixing, be degradation bacterial agent degraded deposit in petroleum hydrocarbon create favorable conditions, while porous oxygen can be realized
Change the recycling of alumina supporter.
Claims (6)
1. the method that the oily microbial inoculum of efficient piezophilic drop with Woelm Alumina as carrier processes deep-sea oil spilling, is characterized in that：Institute
It is cylindric to state carrier, the carrier of oily microbial inoculum apposition growth can be dropped as efficient piezophilic, aid in which to process deep-sea
Oil spilling.
2. the oily microbial inoculum of efficient piezophilic drop as described in claim 1, is characterized in that：It is resistant to 0.3-0.4MPa pressure.
3. the carrier as described in claim 1, is characterized in that：A diameter of 1.60-1.70cm, a height of 1.30-1.40cm,
Porosity is 52-59%, and bulk density is 2.57-2.67g/cm3, pore-size distribution is micron order.
4. the carrier as described in claim 3, is characterized in that：Micron-sized pore-size distribution is drop with higher porosity
The apposition growth of oily microbial inoculum creates condition；Bulk density overcomes other inorganic carriers more than the density of sea water
The little shortcoming for being difficult to, being easily unable to reach by sea water water impact appointed place of density, realizes microbial inoculum to dirt
Effective degraded of dye deposit petrochina；It is high with compressive resistance, the characteristics of anti-shear ability is strong, can adapt to
The special environment of seabed high pressure, overcomes that general inorganic carrier density is little, material is loose is difficult to sink to seabed and holds
By the shortcoming of high pressure；Cost is cheap, nontoxic stable chemical nature, repeats and utilizes.
5. the carrier as described in claims 4, is characterized in that：The load bacterium amount of porous alumina carrier up to 1.1 ×
108-1.3×108Cfu/.
6. the carrier as described in claims 3,4, is characterized in that：Porous alumina carrier apposition growth barophilic bacteria
After agent, it is about 1cm using 1350-1400 aperture, the length of side is about the square net of 20cm and loads about 9-10
Individual Woelm Alumina is placed in the large-scale trawlnet with buoy and is bound to its bottom, and Jing ships are transported and are thrown to
In deep-sea oil spill marine site basic sediment within 30-40 rice depth, can effectively realize microbial inoculum to deposit
The degraded of petrochina hydro carbons and the recycling of Woelm Alumina.
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