CN106542643B - Method for treating deep sea spilled oil by using high-efficiency pressure-philic oil-reducing microbial inoculum with porous alumina as carrier - Google Patents
Method for treating deep sea spilled oil by using high-efficiency pressure-philic oil-reducing microbial inoculum with porous alumina as carrier Download PDFInfo
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Abstract
The invention provides a method for treating deep sea spilled oil by using a high-efficiency oil-reducing microbial inoculum attached to and grown by using porous alumina as a carrier, which adopts a gel injection molding method to prepare the porous alumina carrier and is characterized in that: (1) a cylindrical porous alumina carrier with the diameter of 1.60-1.70cm and the height of 1.30-1.40cm, porosity of 52-59%, and bulk density of 2.57-2.67g/cm3The pore size distribution is micron grade, the bacteria-carrying capacity of the carrier can reach 1.1 × 108‑1.3×108cfu/piece; (3) the pressure resistance is good, and the pressure can be born below 0.4 MPa. The high-efficiency oil-lowering agent capable of enduring pressure of 0.3-0.4MPa is used. After the porous alumina carrier is attached with the growth microbial inoculum, 1350-1400 square nets with the pore diameter of about 1cm and the side length of about 20cm are used for loading about 9-10 porous aluminas, the porous aluminas are placed in a large towing net with a buoy and bound at the bottom of the large towing net, the large towing net is transported by a ship and thrown into bottom sediments at the bottom of a deep sea area polluted by deep sea oil spill within the depth of 30-40 meters, and the degradation of petroleum hydrocarbons in the sediments and the reutilization of the porous aluminas by the microbial inoculum can be effectively realized.
Description
Technical Field
The invention belongs to the technical field of deep sea oil spill bioremediation, and particularly relates to a method for treating deep sea oil spill by using a porous alumina as a carrier to adhere and grow a high-efficiency pressure-drop oil-philic microbial inoculum.
Background
With the increasing demand of the world for oil and products thereof, oil spill accidents in the process of offshore mining, transportation, loading and unloading and oil use are increasing, and serious environmental pollution is caused. Accidents such as offshore crude oil leakage and the like cause serious influence on the ecological environment of offshore areas in China, and measures such as adding oil dispersing agents and the like adopted in the oil spill accident treatment are to transfer floating oil on the surface of ocean into ocean sediments, so that the method cannot thoroughly solve the problem of harm of ocean oil spill on ocean ecological systems.
The microbial degradation is a main way for removing petroleum pollutants in the environment, and has the characteristics of low cost, less investment and high efficiency compared with a physical or chemical method. The use of microorganisms to catalytically degrade petroleum hydrocarbons to reduce or ultimately eliminate petroleum pollution is receiving widespread attention from countries throughout the world. Many microorganisms can grow on hydrocarbons as the sole carbon source, and the microorganisms that degrade petroleum hydrocarbons are present in large quantities in the water bodies and bottom sediments contaminated with petroleum hydrocarbons. In order to eliminate petroleum hydrocarbons in bottom sediments of oceans, a bioremediation technology for degrading spilled oil by putting a petroleum degrading microbial inoculum into an oil spilled sea area is generally adopted at present, and the technical key is to improve the fixation rate of submarine microorganisms. Because the bioremediation microbial inoculum is low in density and relatively dispersed, the thrown microbial inoculum always floats on the sea surface, and the degradation effect on polluted sediments on the seabed is difficult to perform. And the microorganism immobilization technology is adopted, namely, the microorganism is immobilized in an effective carrier, the activity of the microorganism is maintained, the cell density of the microorganism in unit volume can be improved, and the bioremediation problem of the polluted environment can be effectively solved. The immobilized microorganism can keep activity for a long time, the microenvironment of the immobilized microorganism carrier is also beneficial to shielding malignant competition, phagocytosis and poison of indigenous bacteria, bacteriophage and toxic substances to the microorganism, and the damage of the shearing force of sea waves to the microorganism is reduced. The carrier is the key of immobilization, and the carriers adopted in water treatment at home and abroad are various, and mainly comprise organic material carriers and inorganic material carriers. Such as peat, filler, activated carbon, coke powder, zeolite, fine quartz sand, vermiculite and the like, but the application of the carriers to the bioremediation of ocean oil spill has certain defects. Such as peat, fillers, activated carbon, coke breeze, zeolites, are difficult to sink to the sea floor and contact with contaminated sediments due to their low density, compressive strength and mechanical strength, and are more difficult to withstand the pressure of the sea floor and the impact of water currents. The particle of the fine quartz sand carrier is small and easy to be dispersed by sea waves, and the vermiculite carrier is mainly used as an adsorbent for removing harmful substances and an ion exchange treating agent for treating wastewater by utilizing the cation exchange performance of the vermiculite carrier; the vermiculite carrier has small interlayer spacing of nanometer level, is not beneficial to the load growth of microorganisms, can load microbial inoculum after volume expansion by high-temperature heating, but has small density and is difficult to meet the requirement of carrying bacteria for deep sea crude oil treatment. Even if a large amount of financial resources and material resources are consumed and the influence of special environment of the seabed is caused, the high-efficiency petroleum degradation microbial inoculum loaded by the carriers and thrown into the oil spill sea area is easy to disperse and difficult to enter and fix in the sediments, so that the content of microorganisms in the sediments is low, and the degradation capability of the microorganisms on petroleum hydrocarbons in the sediments is weakened. Therefore, it is necessary to research the preparation of a porous inorganic material carrier which has high density, high compressive strength and can adhere microorganisms to grow. Such as reference [1] cinnabar, plum and peace, zheng zhenzhi, carrier materials in the immobilized cell technology and their use in environmental management [ J ]. proceedings of Chongqing university of construction, 2000,22(5): 99-100; [2] researches on the carrier of Pseudomonas fluorescens P13 microbial inoculum by using Li Hui, Wang Ping, Xiaoming, diatomite and talcum powder [ J ]. Chinese biological control, 2009,25(3): 239-244; [3] xujinlan, Huangting Lin, Tangzhixin, etc. screening of high-efficiency petroleum degrading bacteria and research on bioremediation characteristics of petroleum-polluted soil [ J ]. environmental science bulletin, 2007,27(4): 622-; [5] experimental study on adsorption removal of heavy metal ions in wastewater by using Liuyun, Wu Pingxiao and Dang Zhi pillared vermiculite [ J ]. mineral rock, 2006,26(4): 8-13.
The porous ceramic is a novel ceramic material which is formed into a large number of communicated or closed pores in a material body in the forming and sintering processes after being sintered at high temperature. The porous ceramic has the characteristics of high porosity, large specific surface area, high mechanical strength, good chemical stability and the like, so that the porous material can be used as a good carrier of a deep sea oil spill repairing microbial inoculum. Among them, both porous zirconia and porous alumina materials have excellent physical and chemical properties, and are the focus and hot spot of porous ceramic research. Compared with the porous zirconia material, the porous alumina material has the characteristics of higher porosity, high density, high pressure resistance, shear resistance, stable chemical property and the like, has the advantage of low manufacturing cost, and is suitable for large-scale application of actual deep-sea oil spill degradation. For example, reference [4] Wanglili, Wangxufeng, Jianghuotao, etc. preparation and performance of hydroxyapatite coating material coated on porous zirconia base [ J ] mechanical engineering material 2008,32(12): 5-7; [5] preparation of inorganic zeolite carrier antibacterial agent and its antibacterial property [ J ] paper and paper making, 2009,28(3): 28-31; [6] study of preparation conditions of Tanglin, macroporous alumina support [ J ] chemical Engineer, 2014,01: 56-57; [7] xuhuan, preparation of porous alumina film and application research [ D ]. Nanjing, university of Nanjing teachers.2013.
Based on the advantages of the porous alumina material, the porous alumina material is selected as a carrier of the efficient oil-philic degradation microbial inoculum and the porous alumina meeting the requirements of the microbial inoculum carrier is prepared. The invention takes the porous alumina material as a carrier to attach and grow the high-efficiency pressure-drop oleophilic microbial inoculum, and then puts the oleophilic microbial inoculum into a deep sea oil spill sea area within 30-40 meters, thereby realizing the purposes of effectively degrading petroleum in polluted sediments by the microbial inoculum, treating petroleum hydrocarbons in the sediments with pertinence, centralization and no secondary pollution, and reducing the ecological environment hazard of the offshore area caused by ocean oil spill accidents to the minimum.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for treating deep sea spilled oil by using a high-efficiency pressure-lowering oleophilic microbial inoculum attached and grown by using porous alumina as a carrier.
The technical scheme adopted by the invention is as follows: according to the special environment that the sea bottom pressure of the oil spilling sea area is high and the ocean current impact force is large, the porous alumina with high porosity, high volume density and high compressive strength is prepared by a gel injection molding method, and the prepared porous alumina meets the requirements of a microbial inoculum carrier and has low manufacturing cost.
The porous alumina prepared by the gel injection molding method is cylindrical, the diameter is 1.60-1.70cm, the height is 1.30-1.40cm, the porosity is 52-59%, and the volume density is 2.57-2.67g/cm3The pore size distribution is micron grade, the bacteria-carrying capacity of the carrier can reach 1.1 × 108-1.3×108cfu/piece.
Compared with the prior art, the invention has the beneficial effects that the high-efficiency pressure-drop-philic oil microbial inoculum can be attached and grown through the prepared porous alumina carrier, the density of the carrier is higher, the carrier can be thrown to the bottom of a deep sea area polluted by deep sea oil spill below 30-40 meters of sea level, and the degradation efficiency of the microbial inoculum on petroleum hydrocarbons in polluted sediments is improved. The method has the characteristics that the pressure of the bottom of the oil spilling sea area is less than 0.4MPa, the pressure is not broken, the oil spilling sea area is not easy to be impacted by ocean currents to migrate, and the oil spilling sea area can sink to a specified place without external force, so that the purpose of effectively and specifically treating petroleum hydrocarbons in the seabed polluted sediments by the efficient oil philic-pressure reducing microbial inoculum is achieved.
Drawings
FIG. 1 Standard Curve of dehydrogenase Activity
And the TTC value represents the activity of the dehydrogenase of the high-efficiency pressure-drop oleophilic bacterium agent, the absorbance value represents the density of the bacterium liquid, the TTC values under different absorbances are measured and plotted, and the correlation of a fitting curve is good.
FIG. 2 is a graph showing the relationship between dehydrogenase activity and bacterial liquid volume
FIG. 3 porous alumina support
The dehydrogenase activity was measured for each of 1, 2, 3, 4, 5 and 6ml of the bacterial suspension in the logarithmic growth phase according to the dehydrogenase activity measuring method, and a dehydrogenase activity-bacterial suspension volume curve was plotted as shown in FIG. 2. Correlation index R20.9971 shows that the dehydrogenase activity correlates well with the volume of the bacterial suspension.
Detailed Description
1. Preparation and characterization of porous alumina
Preparing the porous alumina material by adopting a gel casting process, wherein the idea is to prepare a concentrated suspension with low viscosity and high solid phase volume fraction; then, adding an initiator and a catalyst into the suspension; finally, pouring the prepared slurry into a model, and carrying out cross-linking polymerization reaction on the organic monomers in the thick suspension under the action of an initiator and a catalyst to form a three-dimensional network structure; under the protection of proper temperature, humidity and atmosphere, the solid particles of the suspension are solidified and formed in situ, and then a blank body with uniform structure and high density is obtained; further sintering is carried out under a proper sintering system, and finally the porous alumina carrier with certain porosity, high strength and stable properties is obtained. The method mainly comprises the following steps:
1) dispersing alumina powder with a nano-scale particle size in deionized water, gradually adding monomer acrylamide and a cross-linking agent methylene bisacrylamide in a certain proportion, and performing ball milling on the obtained premixed liquid at 260r/min for 4-6 hours to obtain a suspension;
2) adding a certain amount of initiator and catalyst into the suspension, and carrying out proper mechanical stirring to obtain slurry;
3) and injecting the slurry into a cylindrical mold, naturally drying the cylindrical mold at room temperature for 2-3 days, and demolding to obtain a blank with a uniform structure and high density. The diameter of the bottom surface of the cylindrical mold is 1.7cm, and the height of the bottom surface of the cylindrical mold is 1.4 cm;
4) drying the blank at 60 ℃ for 2 hours, and drying at 100 ℃ for 2 hours;
5) and sintering the dried green body, wherein the specific sintering system is as shown in the following table, and finally obtaining the porous alumina carrier with certain porosity, high strength, high density and stable properties.
TABLE 1 Green body sintering preparation table
Preparing the sintered sample into a standard sample, respectively measuring the volume density of the sample by adopting an Archimedes method, measuring the pore size distribution of the sample by adopting a mercury porosimeter method, measuring the pore size of the sample by adopting an SEM method, and measuring the height, the bottom surface diameter and the compressive strength of the porous alumina by utilizing a related calculation method. The porous alumina with the diameter of 1.60-1.70cm, the height of 1.30-1.40cm, the porosity of 52-59 percent and the volume density of 2.57-2.67g/cm is obtained by calculation3The pore size distribution is in the order of microns. The porous alumina carrier obtained by firing has low manufacturing cost and low raw material cost, and can be prepared by batch production. 2. Step of loading porous alumina with bacteria and calculation of bacteria loading amount
The dehydrogenase activity of the microorganism reflects the ability of the microorganism to degrade organic substances, and the amount of the microorganism carried on the carrier can be estimated approximately when the value of the dehydrogenase activity of the microorganism contained in a certain liquid medium is equivalent to the value of the dehydrogenase activity of the microorganism carried on the carrier.
1) The steps of loading the porous alumina with bacteria and calculating the bacteria loading amount are as follows:
taking 1ml of bacterial liquid from a clean bench, adding the bacterial liquid into a sterilized 250ml conical flask containing 100ml of beef extract peptone liquid culture medium (beef extract 3g/L, peptone 10g/L, sodium chloride 30g/L, pH 7.0-7.2,121 ℃ and sterilizing for 20min), putting the sterilized porous alumina into the conical flask, sealing, shaking uniformly, and culturing in a pressure container with the pressure of 0.4MPa at the temperature of 25-28 ℃; alumina was taken out at 23h-25h (logarithmic growth phase of the inoculum) and placed in 50ml sterilized centrifuge tubes.
2) The specific operation for measuring the dehydrogenase activity of the bacteria-carrying porous alumina is as follows:
(1) determination of the Activity of porous alumina dehydrogenase (TTC method)
① adding 4ml Tris-HCl buffer solution and Na sequentially into a centrifuge tube loaded with porous alumina2SO31mL of solution and 1mL of TTC solution were allowed to stand clear of the upper surface of the aluminum oxide.
② was shaken well and immediately placed in a 37 ℃ constant temperature water bath, and gently shaken to record the reaction time (10min-60min, depending on the color development).
The tube was removed from the water bath and a drop of concentrated sulfuric acid was immediately added to the tube to terminate the reaction.
③ Add 5mL of acetone to each tube, mix well, and extract TF.
④ the tubes were centrifuged at 3000rpm for 2 min.
⑤ and sucking out the supernatant, and measuring the absorbance value at 486nm wavelength (the measured absorbance value should be below 0.8, and the color should be diluted and re-compared when the color is too concentrated).
⑥ dehydrogenase activity is expressed as TF values and calculated by the following formula:
TFμg/(mL·h)＝A×B×C
a-value corresponding to standard curve,. mu.g/mL
B-culture time is adjusted to 60 min/culture time min, h-1
Dilution factor in C-color comparison
The TF value (dehydrogenase activity) of the bacteria-carrying porous alumina is calculated to be about 74-81 mu g/(mL & h) at most.
(2) Calculation of the bacterial load of the porous alumina
According to the curve of figure 2 and the combination of the TF maximum value (dehydrogenase activity) of the porous alumina, the bacteria-carrying volume of the porous alumina is about 2.0-2.1ml, and the density of the bacteria liquid obtained by the plate counting method is 5.5 × 107cfu/ml-6.0×107cfu/ml, the bacteria carrying capacity of the prepared porous alumina is 1.1 × 108cfu-1.3×108cfu/piece.
3. Deep sea putting method of bacteria-carrying porous alumina
Step one, manufacturing of porous alumina mesh bag loaded with bacteria
1) Several square nets with a pore size of about 1cm and a side length of about 20cm were prepared, the pore size of the net being smaller than the diameter of the bottom surface of the alumina to prevent the loaded porous alumina from leaking out. The net can be used for loading about 9-10 porous aluminas and spreading in the net, so that the overlapping can be avoided, the space can be effectively utilized, and the contact area of the alumina and the sediment can be increased.
2) Preparing a large trawl net, wherein the mass of each alumina is about 7-9g, and 12000-14000 aluminas are loaded according to the calculation of 100Kg of loading amount, and the spreading base area is about 2.7-3.2m2The bottom area of the trawl net should be more than 3.2m2. The trawl thus prepared has a pore size of about 5cm and a spreading area of about 4-5m2。
Step two, loading method of bacterium-carrying porous alumina mesh bag
About 9-10 porous alumina carrying bacteria are placed in the net, then the four corners of the net are polymerized and sealed by strings, and thus a plurality of net bags carrying the porous alumina are manufactured. Each small net bag is packed in the large trawl net, one end of the rope is used for sealing, and the other end of the rope is tied with a buoy, so that the position of the put porous alumina can be conveniently indicated, and the alumina can be repeatedly recycled.
Step three, deep sea putting method of bacteria-carrying porous alumina
The bacterial-carrying porous alumina is loaded into 1350-1400 square mesh bags with the aperture of about 1cm and the side length of about 20cm and placed in a large trawl, a small mesh bag is tied at the bottom of the large trawl by a thin rope, so that an alumina carrier at the bottom is spread to the maximum extent and placed on a ship, then the ship is moored in a sea area polluted by oil spill, the trawl is thrown into a deep sea area polluted by the oil spill within the depth of 30-40 meters, the trawl can automatically sink to spread on seabed sediments, and a buoy is tied on the rope to mark the position. By using the method, the porous alumina carrying bacteria can be put into the bottom sediment of the designated sea area polluted by the spilled oil, so that the mixing of the petroleum degrading bacteria and the seabed sediment is realized, favorable conditions are created for degrading petroleum hydrocarbon in the sediment by the degrading bacteria, and the reutilization of the porous alumina carrier can be realized.
Claims (5)
1.The method for treating deep sea spilled oil by using the high-efficiency pressure-philic oil-reducing microbial inoculum with porous alumina as a carrier is characterized by comprising the following steps of: the carrier is cylindrical, the diameter is 1.60-1.70cm, the height is 1.30-1.40cm, the porosity is 52-59%, and the volume density is 2.57-2.67g/cm3The pore size distribution is micron-sized, the porous alumina carrier can be used as a carrier for the attachment growth of a high-efficiency extreme pressure oil-lowering microbial inoculum and is used for assisting the carrier in treating deep sea spilled oil, and the specific preparation method of the porous alumina carrier comprises the following steps:
1) dispersing alumina powder with a nano-scale particle size in deionized water, gradually adding monomer acrylamide and cross-linking agent methylene bisacrylamide, and ball-milling the obtained premixed solution for 4-6 hours at 200r/min to obtain a suspension;
2) adding an initiator and a catalyst into the suspension, and mechanically stirring to obtain slurry;
3) injecting the slurry into a cylindrical mold, naturally drying the cylindrical mold at room temperature for 2-3 days, and demolding to obtain a blank, wherein the ground diameter of the cylindrical mold is 1.7cm, and the height of the cylindrical mold is 1.4 cm;
4) drying the blank at 60 ℃ for 2 hours, and drying at 100 ℃ for 2 hours;
5) sintering the dried blank body, and obtaining the porous alumina carrier after sintering, wherein the sintering is as follows: setting the sintering initial temperature at 25 ℃, heating to 60 min-220 ℃, heating to 460 ℃ again, keeping the temperature at 460 ℃ for 120min, then continuing heating to 580 ℃ for 60min, heating to 1000 ℃ again, and keeping the temperature at 1000 ℃ for 480 min.
2. The method for treating deep sea spilled oil by using the high-efficiency pressure-philic oil microbial inoculum with the porous alumina as the carrier according to claim 1, which is characterized in that: the high-efficiency oil-philic pressure-reducing microbial inoculum can resist the pressure of 0.3-0.4 MPa.
3. The method for treating deep sea spilled oil by using the high-efficiency pressure-philic oil microbial inoculum with the porous alumina as the carrier according to claim 1, which is characterized in that: the micron-sized pore size distribution and the higher porosity in the carrier create conditions for the attachment growth of the oil reducing microbial inoculum; the volume density is greater than that of seawater, so that the defects that other inorganic carriers are low in density, difficult to sink and easy to impact by seawater flow and cannot reach a specified place are overcome, and the effective degradation of petroleum in polluted sediments by the microbial inoculum is realized; the high-pressure-resistant high-shear-resistant high-pressure-; low cost, no toxicity, no harm, stable chemical property and reusability.
4. The method for treating deep sea oil spill by using the high-efficiency pressure-drop oil microbial inoculum with the porous alumina as the carrier according to claim 3, wherein the bacterial loading capacity of the porous alumina carrier can reach 1.1 × 108-1.3×108cfu/piece.
5. The method for treating deep sea spilled oil by using the high-efficiency pressure-philic oil microbial inoculum with the porous alumina as the carrier according to claim 4, which is characterized in that: after the porous alumina carrier is attached with the growth barotropic bacteria agent, 1350-1400 square nets with the aperture of 1cm and the side length of 20cm are used for loading 9-10 porous aluminas, the porous aluminas are placed in a large trawl with a buoy and bound at the bottom of the large trawl, the large trawl is transported by a ship and thrown into bottom sediments at the bottom of a deep sea area polluted by deep sea oil spill within the depth of 30-40 meters, and the degradation of petroleum hydrocarbons in the sediments and the reutilization of the porous aluminas by the bacteria agent can be effectively realized.
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