CN104357435A - Immobilized bacteria agent for biologically repairing spilled oil-polluted shore - Google Patents
Immobilized bacteria agent for biologically repairing spilled oil-polluted shore Download PDFInfo
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- CN104357435A CN104357435A CN201410606305.7A CN201410606305A CN104357435A CN 104357435 A CN104357435 A CN 104357435A CN 201410606305 A CN201410606305 A CN 201410606305A CN 104357435 A CN104357435 A CN 104357435A
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Abstract
The invention aims at providing an immobilized bacteria agent for biologically repairing a spilled oil-polluted shore and a preparation technology of the immobilized bacteria agent, and effectively solves the problem that thalli applied in a shore oil-spilling biological repairing process are easily affected by the environmental conditions such as sea waves, ultraviolet and temperature. An immobilizing material for preparing the immobilized bacteria agent comprises sodium alginate, polyvinyl alcohol and active carbon according to the mass ratio of (4-6):(8-12):(1-2). The immobilized bacteria agent is prepared by an embedding method; compared with free thalli, the immobilized bacteria agent has higher petroleum degradation capacity, washing resistance and reusability. The application practice in repairing of a spilled oil-polluted shore '11.22' of Huangdao in 2013 shows that the immobilized bacteria agent keeps high activity in an actual shore environment, and the shore oil pollution repairing effect is remarkable; no identical oil-spilling biological repairing technology is reported in China; the immobilized bacteria agent has an extremely good development and application prospect.
Description
Technical field
The invention belongs to the technical field of biological remediation that oil and petroleum products pollute shoreline, be specifically related to a kind of immobilized microbial inoculum for oil spill seashore biological restoration and technology of preparing thereof and application.
Background technology
In recent years, day by day serious marine oil spill pollutes the concern causing countries in the world ocean management department and ecologist.Bioremediation technology have safe and efficient, repair thoroughly, the advantage such as non-secondary pollution, environmental benefit are good, be acknowledged as and process marine oil spill and particularly clear up one of most effective means of seashore greasy dirt.But biological restoration also exists some problems in actual applications at present, particularly oil spill seashore complex environment condition has had a strong impact on the survival rate and the degrading activity that apply oil degradation bacteria.From late 1980s, begin one's study by the combination of physics, chemistry and biological method both at home and abroad, strengthen biological restoration effect.Therefore, seek new technology further and become the key of dealing with problems to reduce the disadvantageous effect of ocean environment to biological restoration process.
Immobilized microorganism technology is the technology developed rapidly from the sixties in 20th century, this technology free microorganism cell can be fixed on restriction space, avoid thalline to run off, improve thalline utilization ratio and provide applicable microenvironment to keep microbial activity for a long time.From 20 century 70s, this technology just starts to be applied to water treatment field, have microbe density high, run off less, the advantage such as efficiency is high, stability is strong, resistance to murder by poisoning ability is strong, significantly can strengthen the effect of biological restoration.In ocean environment biological restoration process, need the impact reducing the adverse environment condition such as alluvion, seawater dilution, uviolizing and tideland thermohaline change, and microorganism embedded immobilization technology can be used as a kind of Main Means and solves the problem.At present, there is not been reported for the domestic research and development for marine oil surfactant hydrocarbon degradation bacteria immobilized microbial inoculum and relevant technology of preparing.This patent is based on ocean environment original inhabitants petroleum hydrocarbon degradation bacterium, and establish immobilized microbial inoculum technology of preparing, the biological restoration polluting seashore for marine oil spill provides tachnical storage and theoretical direction.
Summary of the invention
The object of this invention is to provide a kind of immobilized microbial inoculum for oil spill seashore biological restoration, apply the problem that microbial inoculum is subject to the environmental influences such as wave, ultraviolet, temperature in effective solution oil spilling seashore biological restoration process, make up the deficiencies in the prior art.
First the present invention provides a kind of immobilization material for the preparation of immobilized microbial inoculum, and described immobilization material comprises sodium alginate, polyvinyl alcohol and gac, and its mass ratio is 4 ~ 6:8 ~ 12:1 ~ 2;
Above-mentioned immobilization material pollutes the immobilized microbial inoculum of the biological restoration of seashore for the preparation of being applicable to marine oil spill;
The present invention also provides a kind of technology of preparing of immobilized microbial inoculum, comprises and uses above-mentioned immobilization material and petroleum hydrocarbon degradation bacterium, adopts embedding method to be prepared;
Petroleum hydrocarbon degradation bacterium of the present invention mainly from the native bacterium of ocean environment screening, comprises alkane eating bacteria (Alcanivorax sp.), extra large bacillus (Marinobacter sp.), bacterium of unlinking (Cycloclasticus sp.) etc.
The cell concn of immobilized microbial inoculum Petroleum Hydrocarbon degradation bacteria is preferably 10
9cFU/g ~ 10
11cFU/g.
The preparation method of immobilized microbial inoculum of the present invention is as follows: after polyvinyl alcohol, sodium alginate and gac sterilising treatment; Adopt 80 DEG C of waters bath with thermostatic control to be dissolved in antiseptic sea water, be cooled to 40 DEG C, then with the bacteria suspension Homogeneous phase mixing of petroleum hydrocarbon degradation bacterium; By peristaltic pump, above-mentioned liquid formulation is instilled saturated boric acid and 3% ~ 8%CaCl
2mixing solutions in and stir, be cross-linked to preparation and solidify; Be separated immobilized microbial inoculum particle, and carry out vacuum lyophilization.
Immobilized microbial inoculum of the present invention can be used for the biological restoration that marine oil spill pollutes seashore, and its application method is as follows: be loaded into by immobilized microbial inoculum in mesh bag, and be then laid in oil spill seashore surface, its usage quantity is 1.0kg/m
2~ 1.5kg/m
2.
Immobilized microbial inoculum involved in the present invention, for oil spilling shoal environment feature, takes entrapping method to prepare immobilized microbial inoculum, has better oil degradation ability, flushing resistance and repeat performance compared with free thalline.In the application practice of Huang Island " 11.22 " oil spilling seashore in 2013 reparation, also demonstrate this immobilized microbial inoculum can keep good activity in actual shoal environment, its oil spill seashore repairing effect is remarkable, has good development prospect.
Accompanying drawing explanation
Fig. 1 is immobilized microbial inoculum scanning electron microscope (SEM) photograph;
Fig. 2 is that immobilized microbial inoculum mechanical property compares schematic diagram;
Fig. 3 is that immobilized microbial inoculum petroleum degradation rate compares schematic diagram;
Fig. 4 is that the secondary petroleum degradation rate reclaiming immobilized microbial inoculum compares schematic diagram;
Fig. 5 is that the Determination of Alkane Content after immobilized microbial inoculum biological degradation in unrecovered oil compares schematic diagram;
Fig. 6 is that the aromaticity content after immobilized microbial inoculum biological degradation in unrecovered oil compares schematic diagram;
Fig. 7 is Huang Island " 11.22 " oil spill seashore live biometric reparation figure;
Fig. 8 is that Huang Island " 11.22 " oil spill seashore biological restoration district compares schematic diagram with the oil content in weathering district settling.
Wherein:
Sn in Fig. 2, Fig. 3, Fig. 4: the immobilized microbial inoculum representing different group; FB: represent free thalline process; NC: represent negative control process; Different letter representation significant differences above post, p < 0.05.
N-Cm in Fig. 5: represent m carbon normal paraffin, if n-C16 is 16 carbon normal paraffins.
C0-N, C0-F, C0-D, C0-P, C0-C in Fig. 6 are respectively naphthalene, fluorenes, dibenzothiophene, phenanthrene, bend; C1 (C2, C3, C4)-naphthalene represents be modified with 1 (2,3,4) individual alkyl on carbon atom; C1 (C2, C3)-fluorenes represents be modified with 1 (2,3) individual alkyl on carbon atom; C1 (C2, C3)-dibenzothiophene represents be modified with 1 (2,3) alkyl on carbon atom; C1 (C2, C3, C4)-Fei represents be modified with 1 (2,3,4) individual alkyl on carbon atom; C1 (C2)-bend expression to be modified with 1 (2) individual alkyl on carbon atom.Sn: the immobilized microbial inoculum representing different group; FB: represent free thalline process; NC: represent negative control process.
Embodiment
Substratum used by the present invention is respectively ONR7a substratum and M8 substratum, and wherein ONR7a substratum often rises containing 22.79g NaCl, 11.18g MgCl
26H
2o, 3.98g Na
2sO
4, 1.46gCaCl
22H
2o, 1.30g TAPSO, 0.72g KCl, 0.27g NH
4cl, 89.00mg Na
2hPO
47H
2o, 83.00mg NaBr, 31.00mg NaHCO
3, 27.00mg H
3bO
3, 24.00mg SrCl
26H
2o, 2.60mgNaF, 2.00mg FeCl
24H
2o, regulates pH to 7.6.
M8 substratum is often liter of interpolation 2g sodium acetate on the basis of ONR7a substratum, 0.5g peptone, 0.5g yeast extract, and 0.5g potato leaches powder, 0.2g glucose, 0.2g sucrose, 0.05g sodium malate, 0.05g trisodium citrate, 0.05g Seignette salt, regulates pH to 7.6.
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1 immobilized microbial inoculum prepares determination and the technology of preparing thereof of material composition
The marine microorganism used comprises alkane eating bacteria Alcanivorax sp.97CO-5 (CGMCC No.3735), Alcanivorax sp.97CO-6 (CGMCC No.3736) and extra large bacillus Marinobacter sp.PY97S (CGMCC No.3244).
Get bacterial strain that-80 DEG C of glycerine pipes hide activate in M8 substratum also enlarged culturing to OD
630=1.2; Under 7,500rpm condition, centrifugal 8min obtains thalline, and antiseptic sea water washes twice, and adopts antiseptic sea water resuspended in 1:10 (w/v) ratio; Three kinds of bacteria suspensions are built the bacteria suspension of oil degradation flora DC10 by 1 ~ 3:1 ~ 2:2 ~ 4 ratio uniform mixing, 4 DEG C of preservations are stand-by.Immobilized microbial inoculum is prepared with peristaltic pump drop method.The proportion of composing of solidifying agent is determined according to table 1.Polyvinyl alcohol and sodium alginate and gac are placed in test tube, and sterilizing (121 DEG C, 15min) processes; Be dissolved in antiseptic sea water in 80 DEG C of waters bath with thermostatic control, then be cooled to 40 DEG C, mix according to the ratio uniform of bacteria suspension with immobilized reagent (v/v) 1: 10.Peristaltic pump instills saturated boric acid and 3% ~ 8%CaCl
2mixing solutions (linking agent) in, overhead stirrer (rotating speed 100rpm) stirs, crosslinked 0.5h ~ 4h, is separated immobilization particle.After the freezing 12h of immobilization particle-20 DEG C of separated and collected, carry out vacuum lyophilization, obtained immobilized microbial inoculum.
Table 1: the orthogonal experimental design preparing the curing agent component that immobilized microbial inoculum adopts
Although find in experiment that sodium alginate simple and efficiently can prepare immobilized microbial inoculum, prepared immobilized microbial inoculum is easily broken, is unfavorable for that marine oil spill is repaired on the spot, and transports and preserve difficulty for a long time; And polyvinyl alcohol easy adhesion in immobilization process is agglomerating, balling-up is poor, but immobilization particle (agglomerate) good stability, physical strength are high.So, experimental selection sodium alginate and polyvinyl alcohol composite material.In addition, add proper amount of active carbon can reduce the hardness of immobilized microbial inoculum, improve the spacial framework of granule interior; Utilize the adsorptive power of gac, the petroleum hydrocarbon composition in adsorbable contaminate environment simultaneously, promote that microorganism is to the degraded of petroleum hydrocarbon.By the proportioning between said components, both ensured the stability of particle, the particular surroundings requirement of oil spill seashore can have been adapted to again.
According to 9 groups of different combinations of materials, prepare each group of immobilized microbial inoculum.By the optimum proportioning in orthogonal test determination immobilized microbial inoculum between each composition.Result shows, group S1, S4 cannot balling-up, only forms floating membranoid substance on linking agent surface; Although group S7 has part to sink, major part still floats on linking agent surface.Except above-mentioned 3 groups, other each group all can balling-up.To sum up, when adopting matrix material to prepare immobilized microbial inoculum, the concentration of sodium alginate is 20gL
-1~ 30gL
-1; Polyvinyl alcohol consumption is 40gL
-1~ 60gL
-1; Activated carbon dosage is 5gL
-1~ 10gL
-1.
Specific as follows to the evaluation method of the immobilized microbial inoculum of preparation:
The microscopic morphology of immobilized microbial inoculum is observed: random picking 2 ~ 3 immobilization particles, fixes 1h ~ 2h respectively, PBS buffer solution for cleaning 3 times, each 15min ~ 20min with 2.5% glutaraldehyde-2% osmic acid; Each once with 30%, 50%, 70%, 80%, 90% ethanol dehydration, each 15min ~ 20min, then use 100% ethanol dehydration 3 times; Wash 1 time, 15min ~ 20min with the trimethyl carbinol and ethanol (1:1), the trimethyl carbinol washs 2 times, each 15min ~ 20min; Carbon dioxide critical point is dry, ion beam sputtering deposition gold, and Hitachi S-4800 field emission microscopy observation, takes a picture.
Physical strength: get 2g immobilized microbial inoculum particle and put into 50mL antiseptic sea water containing equivalent sandstone (diameter 3mm ~ 5mm), 28 DEG C, 125rpm shaking table vibration 15d, record grain breakage quantity also calculates percentage of damage.And gravimetry change, check abrasion of particles situation.
Mass-transfer performance: the immobilized microbial inoculum particle selecting the equal size of 2g puts into the blue-black ink (2) that 30mL dilutes on an equal basis, 28 DEG C, the vibration of 125rpm shaking table, take antiseptic sea water as reference, measure solution A 406 (initial A406 ≈ 0.25), until absorbancy is constant, judged the difference of mass transfer performances by the size of light absorption value.
Particle diameter: the immobilized microbial inoculum (n>20) that random selecting prepares closely is arranged in a row, adopt vernier caliper measurement particle diameter, repeat 3 times, ask its mean value to obtain the particle diameter of particle, observe simultaneously and record outward appearance and the roundness of immobilized microbial inoculum.
Adopt sodium alginate, polyvinyl alcohol and gac, its mass ratio be immobilization particle prepared by the condition of 4 ~ 6:8 ~ 12:1 ~ 2 microscopic morphology observe and physical index as follows:
(1) choose S8 group immobilized microbial inoculum to observe under scanning electron microscope through respective handling, immobilized microbial inoculum bead inner void is intensive, is interconnected in cellular, can be thalline and provides enough living spaces.PVA constitutes main force's skeleton of bead, and gac and sodium alginate then play the effect of loose internal network, and thalline is adsorbed in network skeleton, and form is intact, and be evenly distributed (Fig. 1).
(2) immobilized microbial inoculum mechanical property as shown in Figure 2, and group S2, S3 immobilized microbial inoculum wear rate and percentage of damage are all up to more than 50%, and mechanical property is poor, can not reach application request.The wear rate of group S5, S6, S8, S9 immobilized microbial inoculum only about 10%, has good mechanical property.In conjunction with cost factor, select 40gL
-1~ 60gL
-1polyvinyl alcohol as the optimum concn of preparation immobilization preparation.
(3) particle diameter of immobilized microbial inoculum, mass-transfer performance and thalline content are as shown in table 2.Research finds that the particle diameter of each immobilized microbial inoculum is all at about 5mm, and increases with polyvinyl alcohol concentration, and particle diameter also increases.Except group S2, all the other particle diameters respectively organized are close, are 5.5mm ~ 6mm.Except S2, S3, other each group all has good mass-transfer performance, and under 406nm, its absorbance can be down to 0.15 ~ 0.2.The thalline content of S5 and S8 is the highest, and the thalline content of its correspondence is all 1 × 10
9cFUg
-1above.Consider above index, the optimum formula of solidifying agent is: the concentration 20gL of sodium alginate
-1~ 30gL
-1; Polyvinyl alcohol consumption is 40gL
-1~ 60gL
-1; Activated carbon dosage is 5gL
-1~ 10gL
-1.
The leading indicator of table 2 immobilized microbial inoculum particle
Sn: the immobilized microbial inoculum representing different group.
Embodiment 2: the preparation of immobilized microbial inoculum
The marine microorganism used comprises alkane eating bacteria Alcanivorax sp.97CO-5 (CGMCC No.3735), Alcanivorax sp.97CO-6 (CGMCC No.3736) and extra large bacillus Marinobacter sp.PY97S (CGMCC No.3244).
The preparation process of immobilized microbial inoculum is as follows:
The above-mentioned bacterial strains getting-80 DEG C of glycerol stocks activate in M8 substratum and enlarged culturing to logarithmic growth later stage (OD
630=1.0 ~ 1.5), use 500mL Erlenmeyer flask to carry out the cultivation of seed liquor, 30L fermentor tank is for obtaining bacterium liquid, and culture condition is: mixing speed 90rpm, temperature 25 DEG C, pH 7.0, and air flow is 0.25m
3/ h, body of ventilating be dry air.Under the condition of 7,500rpm, centrifugal 8min obtains thalline, washes twice, by the resuspended thalline of 1:10 (thalline: seawater, w/v) ratio with antiseptic sea water; Then, 3 kinds of bacteria suspensions are obtained the bacteria suspension of oil degradation flora by 1 ~ 3:1 ~ 2:2 ~ 4 ratio uniform mixing, 4 DEG C of preservations are stand-by.
Polyvinyl alcohol, sodium alginate and gac are placed in test tube, and sterilizing (121 DEG C, 15min) processes; In 80 DEG C of waters bath with thermostatic control, antiseptic sea water is cooled to 40 DEG C after dissolving, and mixes according to the ratio uniform of bacteria suspension with immobilized reagent 1: 10 (v/v).Linking agent (saturated boric acid and 3% ~ 8%CaCl is instilled by peristaltic pump
2mixing solutions) in, overhead stirrer (rotating speed 100rpm) stirs, crosslinked 2h, is separated immobilized microbial inoculum particle.After freezing for particle-20 DEG C 12h, carry out vacuum lyophilization, the immobilized microbial inoculum of obtained degradation flora.
The performance index of the immobilized microbial inoculum obtained by aforesaid method are as follows: wear rate and breakage rate are all lower than 30%; Mass-transfer performance good (absorbance after absorption under 406nm can be down to 0.15 ~ 0.2); Particle diameter is 5.5mm ~ 6.0mm; Thalline content >1 × 10
9cFU/g.
Embodiment 3: immobilized microbial inoculum oil degradation performance measurement
ONR7a substratum, for preparation and the experiment of immobilized microbial inoculum measuring mechanical property of oil degradation assay medium;
Oil substratum: add 1g crude oil in 100mL ONR7a substratum.This experiment adopt oil sample to be originate from the light crude at the South Sea (at 25 DEG C, oil sample density is 826.7kgm
-3, kinematic viscosity is 9.36mm
2s
-1).
By Integrated comparative immobilized microbial inoculum physical properties parameter, choose combination of materials S5, S6 and S8 described in embodiment 1 and prepare immobilized microbial inoculum and carry out oil degradation performance test.With the degradation flora (free thalline process) described in embodiment 2 for positive control, with the oil substratum do not inoculated for negative control, get 10g immobilized microbial inoculum and carry out the biological degradation experiment of oil, culture condition is 20 DEG C, 125rpm, 12d.Above-mentioned often kind of process establish 3 parallel.The methods such as sample pre-treatments, petroleum degradation rate mensuration, the analysis of petroleum hydrocarbon change of component are as follows:
After degraded 12d, filtration, centrifugal (10,000rpm, 12min) remove immobilized microbial inoculum and free thalline, 50mL CH
2cl
2divide three washing triangular flasks, after merging, pour extraction oil residues wherein in separating funnel into.Get 20mL organic extractant phase liquid to transfer in point end flask, 40 DEG C of concentrating under reduced pressure, according to following formulae discovery degradation rate.
Petroleum degradation rate=[(m
0-2.5m)/m
0] × 100%
In formula, m
0for adding the oil weight in substratum at first; M is greasy dirt weight residual after extraction.Another from CH
2cl
2accurately measure 2mL solution mutually, use anhydrous Na
2sO
4dehydration, crosses 0.22 μm of organic solvent-resistant filter membrane.Nitrogen dries up, and again dissolves with 1mL normal hexane, moves in GC sample bottle, with GC-FID, GC-MS, alkane residual after oil degradation, aromatic component is carried out to analysis and measures.GC-FID GC conditions is: chromatographic column HP-5MS (30m × 0.25mm × 0.25m μm); Injector temperature 260 DEG C, carrier gas is high-purity He, flow velocity 1.0mLmin
-1, constant current mode, Splitless injecting samples 1 μ L.Column oven adopts heating schedule: initial 50 DEG C, keeps 2min, with 6.0 DEG C of min
-1rise to 300 DEG C and keep 16min, adopt Selective ion mode scan pattern (SIM).The analysis condition of GC-MS is: chromatographic column HP-5MS (30m × 0.25mm × 0.25m μm); Injector temperature 280 DEG C; Ion source temperature 250 DEG C.Carrier gas is He (99.999%), flow velocity 1mLmin
-1.Temperature programming: 50 DEG C keep 2min, with 6 DEG C of min
-1rise to 300 DEG C, keep 10min.Scan pattern: SCAN, SIM pattern; Quality of scanning scope: 50 ~ 500.Sample introduction: HP7683 automatic sampler, Splitless injecting samples 1 μ L.Data acquisition and processing (DAP): HP3365 chem workstation.
Gravimetric determination degradation rate experimental result shows, immobilized microbial inoculum S8 group can reach 40% at the petroleum degradation rate of 12d, phase specific ionization thalline (33%) has obvious raising, compares negative control process (its natural loss rate is 7.9%) and then improves 32.1%.In addition, immobilized microbial inoculum group S5 and S6 is respectively 37% and 36% at the petroleum degradation rate of 12d, a little more than free thalline (Fig. 3).
By reclaiming immobilized microbial inoculum particle and the experiment of secondary oil degradation, that has investigated this immobilized microbial inoculum reuses effect.Result shows, in the experiment of secondary oil degradation, immobilized microbial inoculum still has certain degradation effect, and the 12d degradation rate wherein organizing S8 is 21%, compares negative control process and improves nearly 15% (Fig. 4).
Cultivate through 12d, from alkane component change, free thalline and immobilized microbial inoculum process all have good degradation effect to alkane (C16 ~ C32), but the degradation effect of immobilized microbial inoculum is better.Immobilized microbial inoculum is 45% to the degradation rate of C16 ~ C32 alkane, higher than free thalline process (35%).Wherein, the degradation rate of immobilized microbial inoculum to long chain alkane (> C30) reaches 50%, apparently higher than free thalline process (Fig. 5).
The degradation effect (40% ~ 50%) of immobilized microbial inoculum to aromatic hydrocarbons is also obviously better than free thalline (35%).Wherein, group S8 immobilized microbial inoculum all has good degradation effect to naphthalene (N0-N4), luxuriant and rich with fragrance (P0-P3) and (C0-C2) series matter in the wrong, is respectively 48%, 50% and 53%; The degradation effect of dibenzothiophene (D0-D3) and fluorenes (C0-C2) series matter is taken second place, is respectively 41% and 38%.This illustrates that bacterium is obtained for raising to the avidity of long chain alkane and arene material and mass-transfer efficiency after immobilization process, and its degradation property have also been obtained raising (Fig. 6).
Consider the factors such as mechanical property, particle diameter, mass-transfer performance, bacterial content and oil degradation effect, the immobilization effect of group S5, S6 and S8 is best, and determining the optimum formula preparing immobilized microbial inoculum is: sodium alginate 20 ~ 30gL
-1; Polyvinyl alcohol 40 ~ 60gL
-1; Gac 5 ~ 10gL
-1.In addition, under overhead stirrer (100rpm) stirs, peristaltic pump (100rpm) a large amount of immobilized microbial inoculum particle of preparation fast can be utilized.
Embodiment 4: the biological restoration application of oil spilling seashore
On November 22nd, 2013, Huang Island pipe laying is blasted, and part Jiaozhou Bay seashore is subject to serious oil spill.Oil spilling seashore is located in remote, the more difficult cleaning of engineering truck, thus uses immobilized microbial inoculum described in this patent to carry out live biometric reparation to oil spilling seashore.The experiment of this place amounts to zymocyte liquid 200L, prepares immobilized microbial inoculum according to step described in embodiment 2, for site remediation experiment (Fig. 7).
First by immobilized microbial inoculum particle packing in mesh bag (this mesh bag has wear resisting property and can isolate ultraviolet effect to a certain extent), then use rope, weight fixed.In oil spill seashore region, selected 2 experiments block (1m × 1m) are as biological restoration treatment zone, the mesh bag being filled with immobilized microbial inoculum are positioned over oil spill seashore surface, neighbouring untreated areas treatment zone in contrast.The sediment sample (3 parallel sample, independent masking foil parcel) repairing district and check plot is gathered respectively at 7d, 14d, 30d; Then oil content is measured according to method described in embodiment 3.
Calculate through weighting method, after 14d, the degradation rate in control treatment district is 13.5%, and the petroleum degradation rate in repair process district is 30.7%.After 30d, crude content in the every 100g in control treatment district settling is 1.03g (its corresponding degradation rate is 2.3%), the every 100g in repair process district settling Crude Oil content is 0.55g (its corresponding degradation rate is 48%), and repair process district crude content is only 53.4% (Fig. 8) of control treatment district crude content.Repair process district crude content, significantly lower than control treatment district, shows that immobilized microbial inoculum maintains good degrading activity in oil spill shoal environment, effectively can accelerate the recovering process of oil spill shoal environment.
Claims (9)
1. an immobilization material, is characterized in that, described solidify material includes sodium alginate, polyvinyl alcohol and gac, and its mass ratio is 4 ~ 6:8 ~ 12:1 ~ 2.
2. immobilization material according to claim 1 is preparing the application in immobilized microbial inoculum.
3. apply as claimed in claim 2, it is characterized in that, described immobilized microbial inoculum is be applicable to the immobilized microbial inoculum that marine oil spill pollutes the biological restoration of seashore.
4. an immobilized microbial inoculum, is characterized in that, described immobilized microbial inoculum includes immobilization material according to claim 1, and marine oil surfactant hydrocarbon degradation bacteria.
5. immobilized microbial inoculum as claimed in claim 4, it is characterized in that, the cell concn of described immobilized microbial inoculum Petroleum Hydrocarbon degradation bacteria is 10
9~ 10
11cFU/g.
6. immobilized microbial inoculum as claimed in claim 4, is characterized in that, described thalline comprise alkane eating bacteria, extra large bacillus, unlink in bacterium one or more.
7. the preparation method of immobilized microbial inoculum according to claim 4, is characterized in that, described method is by after polyvinyl alcohol, sodium alginate and gac sterilising treatment; Antiseptic sea water is adopted to dissolve in 80 DEG C of waters bath with thermostatic control and be cooled to 40 DEG C, then with the bacteria suspension Homogeneous phase mixing of petroleum hydrocarbon degradation bacterium; By peristaltic pump, liquid formulation is instilled saturated boric acid and 4%CaCl
2solution in stir, be cross-linked to preparation and solidify, be separated immobilization particle, then carry out vacuum lyophilization.
8. immobilized microbial inoculum described in claim 4 pollutes the application in the biological restoration of seashore at marine oil spill.
9. applying as claimed in claim 4, it is characterized in that, is be loaded in mesh bag by immobilized microbial inoculum according to claim 4, and be positioned over oil spill seashore surface, its usage quantity is 1.0 ~ 1.5kg/m
2.
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| CN105087538B (en) * | 2015-07-29 | 2018-05-29 | 浙江大学 | A kind of oil degradation microbial inoculum and its preparation method and application |
| CN105018458A (en) * | 2015-07-31 | 2015-11-04 | 湖南大学 | Compound microbial agent as well as preparation method and application thereof |
| CN107151663A (en) * | 2017-06-30 | 2017-09-12 | 国家海洋局第海洋研究所 | It is a kind of to utilize the immobilized microbial inoculum repaired for oil pollution prepared by kelp residue |
| CN109402105A (en) * | 2018-10-15 | 2019-03-01 | 山东交通学院 | A kind of microbe carrier that floatability is degradable and preparation method and application |
| CN109701200A (en) * | 2018-12-25 | 2019-05-03 | 广东省石油与精细化工研究院 | One kind is for the remaining composition of Pesticide Residue in Soil of degrading |
| CN112251368A (en) * | 2020-04-03 | 2021-01-22 | 太原理工大学 | Immobilized microbial cell for degrading grease in high-concentration kitchen wastewater and preparation method and application thereof |
| CN111534549A (en) * | 2020-04-15 | 2020-08-14 | 浙江省海洋水产养殖研究所 | Immobilized microbial inoculum for repairing ocean oil spill pollution |
| CN111534549B (en) * | 2020-04-15 | 2021-09-21 | 浙江省海洋水产养殖研究所 | Immobilized microbial inoculum for repairing ocean oil spill pollution |
| CN114181928A (en) * | 2021-12-14 | 2022-03-15 | 浙江省海洋水产研究所 | Immobilized microbial agent for offshore oil pollution remediation |
| CN114181928B (en) * | 2021-12-14 | 2023-10-31 | 浙江省海洋水产研究所 | Immobilized microbial agent for repairing marine petroleum pollution |
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