CN1132933C - Brachybacillus strain and its application in removing sulfur from sulfur organic compound - Google Patents
Brachybacillus strain and its application in removing sulfur from sulfur organic compound Download PDFInfo
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Abstract
The present invention relates to a bacillus brevis R-6 strain. The strain is preserved in the China General Microbiological Culture Collection Center on Apr. 29th, 2001, and the preservation number is CGMCC NO. 0571. The strain is obtained from oxygen consumption activated sludge in a treatment pool for waste water containing oil, soil polluted by oil around an oil well and a soil sample with the high sulfur content around a coal mine pit via separation. The bacillus brevis R-6 strain and the static cell sap in a non-growing period, immobilized cells, cell sap cultures or a coarse enzyme extract of the strain can eliminate sulfur in sulfur-containing organic compounds by an approach of fracturing C-S bonds via oxidization.
Description
The present invention relates to the bacillus brevis bacterial strain and the application of sulphur in removing organic compounds containing sulfur.
Oil and coal are the most important in the world energy, contain sulfide in these mineral fuel, can produce a large amount of sulfur oxides emissions during burning in atmospheric environment, form acid rain, serious environment pollution destroys the eubiosis, is directly threatening earth human existence space.According to relevant, the whole world enters the SO in the atmosphere every year
2Nearly 200,000,000 tons, at Chinese SO
2Quantity discharged reached 1,795 ten thousand tons.The existence of sulfide simultaneously also can influence outward appearance, erosive burning and the transportation equipment of fuel and products thereof.Have the harm that causes for fear of poisonous sulfide, before fuel combustion, must the sulphur in the fuel be removed in the combustion processes or after the burning, this has become whole world key subjects extremely to be solved.In the face of increasingly serious environmental protection situation, people's environmental consciousness constantly strengthens, and becomes more and more stricter for environmental protection requirement.Therefore, developed country more and more payes attention to the raising of fuel oil quality, and the U.S. has just passed through Clean Air Act Amendment nineteen ninety, and Environmental Protection Agency has proposed to use the requirement of reformulated gasoline.From 1998, EPA adopted complex model, further reduces vehicle emission pollution, and European Parliament's legislation once in 1998 requires enforcement clean gasoline prescription in 2000.In a word, in 10 years from now on, the composition and the quality index of transport fuel (particularly gasoline and diesel oil) will have bigger change, and the H/C that promptly requires fuel is than rising to some extent, and sulphur and aromaticity content will reduce greatly.North America and European countries require that sulphur content will be lower than 50ppm in gasoline in 2005, the diesel oil, and sulphur content will be lower than 30ppm in gasoline in 2010, the diesel oil, even 10ppm.China is a developing country, and oil production and fuel consumption have become one of great powers in the world, SO
2Quantity discharged considerably beyond world average level.In the face of increasingly serious environmental protection situation, China has implemented the strategy of sustainable development, carries out cleaner production energetically, and has issued a series of environmental regulations.The preliminary proposition is reduced to present diesel oil sulphur content below the 2000ppm from 5000ppm; sulphur content is lower than the requirement of 500ppm in the derv fuel oil of part big city; therefore; exploitation and strengthen efficiently, fuel desulfurization before combustion technology cheaply, will produce positive and far-reaching influence to the environment protection and the strategy of sustainable development that China implements.
The method that reduces sulphur content in oil, petroleum products and the coal has physics, chemistry and biological process.The desulfurization before combustion technology of physics and chemical method can remove the inorganic sulfur in the fuel effectively, but the poor effect of organic sulfide removal, because the organic sulfide complicated component that contains in the mineral fuel, major part is a heterogeneous ring compound, C-S chemistry covalent linkage wherein is very firm, can't effectively remove with conventional physics and chemical process.
Hydrogenation catalyst commonly used at present removes organosulfur in the fuel (HDB) technology must be at high temperature (〉=300 ℃), high pressure (〉=100atm), desulfurization under the severe condition that hydrogenation and metal catalyst exist, process cost is higher, and metal catalyst is subject to the sterically hindered influence of substituting group around the substrate sulphur atom, so lower for the decreasing ratio of baroque heterocycle sulfide such as a large amount of methyl substituted thionaphthenes that exist, dibenzothiophene in the fuel.(e.g.Houalla, M., Broderick, D.H., Sapre, A.V., Nag, N.K., de Beer, V.H.J., Gates, B.C., Kwart, H.J.Catalt., 61,523-527 (1980)). in fact, in petroleum naphtha and diesel oil, still exist alkyl substituent (e.g.Kabe, T., the Ishihara of multiple dibenzothiophene, A.and Tajima, H.Ind.Eng.Chem.Res., 31,1577-1580 (1992)).The requirement of low sulfur content makes HDS process reaction condition more and more harsher, and equipment and process cost increase, and the simultaneous olefin saturation causes the loss of fuel combustion value.Therefore, pressing for a kind of sulphur that can remove of searching from oil or petroleum products does not but cause the oil combustion value to reduce economically viable novel process.
Biocatalytic desulfurization (BDS) method, with its selectivity height that has, side reaction is few, the reaction conditions gentleness, equipment is simple, and working cost is low, less investment, little to the fuel value influence, do not cause advantages such as secondary pollution, become the cleaning oil fuel production technology that attracts people's attention gradually, the auxiliary approach that promises to be traditional certain embodiments carries out the precision work of sulfurous fuels, make them reach the requirement level, so caused numerous scientists and engineers' interest.
At present, the microorganism that has separated multiple energy removal of organic sulfur from.Aerobic bacteria or the anerobe main difficult Compound D BT in can both metabolism diesel oil wherein, as sulfate-reducing bacteria, Desulfovibrio (Desulfovibrio) desulfuricans M6, anaerobism ground degraded DBT, main product is a biphenyl, degradation rate is 42%.It is reported that other sulfate-reducing bacteria also can be converted into biphenyl with DBT to anaerobism, but transformation efficiency is extremely low.Anaerobic process are because there is the adding that does not need oxygen, and the advantage that can be applied directly in the oil well has bigger magnetism, but also do not find to be used for the efficient anaerobic microorganism of actual petroleum sweetening system.
Microorganism by aerobic approach metabolism DBT has pseudomonas (pseudomonas sp.), rhodococcus (Rhodococcus sp.), excellent bacillus (Corynebacterium sp.) and tyrothricin (Brevibacterium sp.) etc.By to various microbial metabolism Study on Mechanism; the desulfurization approach of learning microorganism mainly contains three kinds: a kind of is the carbon skeleton (C-C key) of single-minded oxidation DBT; and the C-S key still keeps; this approach is isolated pseudomonas (Pseudomonas) from soil; find in the mixed culture of Bai Yelinke Salmonella (Beijerinckia) and acinetobacter calcoaceticus (Acinetobacter) and root nodule bacterium (Rhizobium); DBT phenyl ring becomes earlier before fracture and is oxy-compound in the metabolic process; sulphur atom is not released; cis 4-[2-(3-hydroxyl)-thienyl]-2-oxygen-3-butenoic acid (tran-HTOB) and 3-hydroxyl-2-formyl radical benzene thiophene (HFBT) accumulate in substratum; other bacterium such as P.eruginosa ERC-8; Beijerinckia sp., Pseudomonas; Rhizobium melioti and Pseudomonas sp.C18 etc. also have such pathways metabolism.The Zhong Huifang research group of Institute of Microorganism, Academia Sinica also once was separated to the bacterial strain that DBT is decomposed into water-soluble organic sulfide, can remove organosulfur 22.2%～32.0% in the coal, (microorganism journal 35 (2): 130-135 such as Zhong Huifang, 1995).Such metabolism can be removed owing to containing carbon structure, and causes the loss of fuel energy.
Another kind of approach is sole carbon source, sulphur source, the energy with DBT, the C-S key of direct oxidation DBT generates benzoate, the bacterial classification of having reported has tyrothricin (Brevibacterium) and Arthrobacter (Arthobacter), they are with the DBT-sulfoxide, the desulfurization of DBT-sulfone finally generates benzoate and vitriol, and tyrothricin (Brevibacterium) can slough the sulphur of DBT in the thick oil and not attack non-sulphur hydrocarbon polymer.In this pathways metabolism, not only remove the sulphur in the heterogeneous ring compound, also destroyed the carbon skeleton of hydrocarbon, caused the loss of fuel value equally.
Article three, in the approach, the C-S key of the single-minded oxidation scission DBT of microorganism, and do not open the C-C key, and with distinctive enzyme system sulphur is taken off to specificity from heterocycle, do not cause the loss of fuel value.Atlantic ResearchCorporation reports that the earliest microorganism can slough sulphur and do not change the structure of hydrocarbon from DBT.Kilbane in 1989 etc. have isolated red rhodococcus (Rhodococcus erythropolis) IGTS8 (being called R.rhodochrous in the past), this bacterium has obtained extensive studies, particularly in recent years, by gene engineering method the desulfurization gene (dsz) of this quasi-microorganism is cloned, express, order-checking, and successfully transform and made up the reorganization bacterium, improved desulfuration efficiency, make it have wide prospect in industrial application (Denome S.A.et al.J Bacteriol.176 (21): 6707-6716 more, 1994), and proposed the desulfurization approach of DBT: this approach is through DBT5-sulfoxide (DBTO), DBT5-sulfone (DBTO
2) and 2-Hydroxybiphenyl-2--sulfinate (HBPS), the DBT metabolism is 2-xenol (2-HBP) the most at last, so this approach also claims " 4S " approach." 4S " approach of single-minded desulfurization is more satisfactory at present, causes the research field of everybody extensive concern.After R.erythropolis IGTS8, reported that again other several bacteriums have similar pathways metabolism, as corynebacterium genus bacteria (Corynebacterium) SY1, R.erythropolis D-1, nocardia globerula (Nocardia globelula), Agrobacterium (Agrobacter) MC501, Mycobacterium (Mycobaterium) G3, Xanthomonas (Xanthomonas), all these bacteriums all are gram-positive microorganisms.
Pseudomonas (Pseudomonas) CB1 is that people such as Isbister separate from soil and colliery sample, can be to grow in the substratum that exists of the sole carbon source and the energy and high DBT with the benzoate.With
35S-DBT and
14C-DBT specializes in DBT heterocycle sulphur tracer experiment as CB1, measures generation
35S vitriol, 2-2 '-dihydroxybiphenyl reach
14CO
2But what it's rather a pity is that they claimed afterwards that the CB1 bacterial strain was unstable and lost (e.g.Isbister J.D1993), so can not further confirm the pathways metabolism that this pseudomonas is definite.
The objective of the invention is to overcome and above-mentionedly remove the defective that the organosulfur technology exists in the fossil oil, have the bacillus brevis bacterial strain of C-S bond energy power the specificity catalysis fracture organic compound and the application of sulphur in removing organic compounds containing sulfur and provide from occurring in nature is isolated.
Technical scheme of the present invention is as follows:
Bacillus brevis bacterial strain provided by the invention, it is characterized in that: this bacterial strain is Bacillus brevis R-6, in April 29 calendar year 2001 be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC NO.0571;
This Bacillus brevis R-6 bacterial strain is separated in the soil sample around the higher pit, colliery of the soil of oil pollution and sulphur content around the oxygen consumption active sludge, oil well from the oily water treatment pond to obtain;
Its concrete screening step is as follows:
Gather the soil sample sample around from pit, colliery, Mentougou, BeiJing, China, sample thief 5 grams are suspended in no sulfenyl basal culture medium, and (substratum is formed: 1000ml water, KH
2PO
4, 2.44g; Na
2HPO
412H
2O 14.03g; NH
4Cl, 2.00g; MgCl
26H
2O, 0.36g; CaCl
22H
2O, 1; FeCl
31mg; MnCl4H
2O, 4mg; CuCl
2, 0.755mg; Glycerol 0.8ml or glucose 1-3g, 20 minutes 121 ℃ of autoclavings) in, put in the shaking table and mix after 30 minutes, leave standstill, draw upper strata suspension and be transferred to nutritional medium (composition g/g:NaCl 1.0%; Yeast extract powder 0.5%, 1%, 20 minute autoclaving of Tryptones) in, put in the shaking table and cultivated 24 hours with 150 rev/mins.Draw 5ml bacterium liquid and add in the 100ml sterilization basic medium, add dibenzothiophene DBT-ethanolic soln more therein and make that DBT concentration is 0.1mM in the nutrient solution.In shaking table, be sprayed with line separation on the nutrient agar of DBT after 2-3 days with 150 rev/mins of cultivations.Single bacterium colony that separation obtains is inoculated in respectively and contains in the basic medium that DBT concentration is 0.2mM, acts on 2-3 days with 150 rev/mins 30 ℃ in shaking table.It is 1.0 that sample after biological action is regulated pH with 1N hydrochloric acid, 5000 rev/mins centrifugal 20 minutes down, supernatant liquor is with isopyknic ethyl acetate oscillation extraction.Extracted organic phase refluxes and is concentrated into 1ml, carries out GC-MS and analyzes.Select dibenzothiophene sulfone DBTO is arranged in the product
2And the bacterial strain of xenol HBP carries out the progressively domestication of strain excellent.
This Bacillus brevis R-6 bacterial strain normal temperature that suits in neutrality and utmost point slightly acidic developing medium is cultivated, and its bacteriology morphological specificity: R-6 is a Gram-positive bacillus, and obligate is aerobic, the acid-fast stain feminine gender.Endogenous spore is arranged, give birth in the gemma.The sporocyst cell is little to be had and expands but not obvious; In a cell, has only a statospore; The cell walls chemical composition belongs to the II type; The sporocyst cell of bacterial strain R-6 is a rod-short, and its length is 0.5-0.7 * 1.2-1.4 μ m, does not form spore chain, no spirobacteria filament; The statospore of rod-short makes the sporocyst cell show slightly expansion; Give birth in the statospore, can move, the flagellum of growing thickly is arranged.
This Bacillus brevis R-6 strain culturing feature:
This Bacillus brevis R-6 bacterial strain can be cultivated 2-5 days for 30 ℃ on nutrient agar medium, Sang Tasi agar, the female cream agar of malt extract alcohol, ox meat extract agar and five kinds of substratum of inorganic blue starch agar.Cultural characteristic sees Table 1:
Table 1
| Substratum | The bacterium colony colour generation | The colony edge shape | The bacterium colony surface shape |
| Nutrient agar medium | The straw yellow | The edge is smooth | Smooth |
| Ox meat extract agar | Creamy | The edge is smooth | Projection |
| The female cream agar of malt extract alcohol | The almond yellow | The edge splintery | The mucus shape |
| The inorganic salt Starch Agar | Clear, colorless | The edge is smooth | Slightly coarse |
| Sang Tasi agar | The ivory yellow | There is projection at the edge | Smooth |
The result who carries out the analysis of full cell hydrolyzed solution according to instant thin-layer chromatography methods such as Hasegawa T. shows: this Bacillus brevis R-6 strain cell wall chemical composition is: bacterial strain Bacillus brevis R-6 contains meso-DAP (diaminopimelic acid Diaminopimelicacicl acid), glycine, semi-lactosi, glucose, seminose, and the cell walls chemical composition belongs to the II type.
This Bacillus brevis R-6 bacterial strain physiological and biochemical property sees Table 2:
Table 2
| The experiment title | The result | Utilize sugar to produce acid | The result |
| Gelatine liquefication | + | Melibiose | - |
| The starch hydrolysis | - | Semi-lactosi | + |
| Indole reaction | - | Ribose | - |
| Nitrate reduction | - | Maltose | - |
| The catalase reaction | + | N.F,USP MANNITOL | + |
| Grow on the Mierocrystalline cellulose | - | Inositol | - |
| The VP test | + | Sucrose | - |
| The casein hydrolysis | + | Wood sugar | - |
| Tyrosine | - | Pectinose | + |
| The yolk growth | - | Glucose | + |
| Anaerobic growth | - | ||
| 5％Nacl | + | ||
| The growth of 0.001% N,O-Diacetylmuramidase | - |
Content with reference to " Bergy ' s Mannual of Systematic Bacteriology " Vol.VIII, surely the principle that belongs to according to morphological specificity and cell walls chemical composition, Bacillus brevis bacterial strain R-6 is a Gram-positive bacillus, and obligate is aerobic, the acid-fast stain feminine gender; Endogenous spore is arranged, give birth in the gemma; The sporocyst cell is little to be had and expands but not obvious; In a cell, has only a statospore; The cell walls chemical composition belongs to the II type; The sporocyst cell of Bacillus brevis R-6 bacterial strain is a rod-short, and its length is 0.5-0.7 * 1.2-1.4 μ m, does not form spore chain, no spirobacteria filament; The statospore of rod-short makes the sporocyst cell show slightly expansion; Give birth in the statospore, can move, the flagellum of growing thickly is arranged.
Surely plant principle according to cultural characteristic and physiological and biochemical property, prove that this bacterial classification and bacillus brevis are quite similar, be bacillus brevis (Bacillus brevis) so name.(Bacillus sphaericus ATCCNo.53969) is different with Bacillus sphaericus.Bacillus brevis R6 bacterial strain of the present invention both can be cultivated in nutritional medium, also can be at sulfur-bearing source (MgSO
4, dimethyl sulfoxide (DMSO), DBT or other organosulfur compounds) basic medium in cultivate.And the former can only grow in the nutrition substratum, can not grow in the basic medium that is added with organic carbon source and sulphur source.
Before Bacillus brevis R-6 bacterial strain of the present invention is used, tame under the condition of organosulfur source existence, bacterial strain carries out neutral aerobic cultivation under the proper temperature between 25-35 ℃.
This Bacillus brevis R-6 bacterial strain sweetening power is strong, and desulfurization substrate scope is wide.
Bacillus brevis R-6 bacterial strain of the present invention, can be with the bacterial classification or have desulphurizing activated freezing non-growing period resting cell and dissociant thereof to make desulfurization biocatalyst in vegetative period of fresh culture, one or several the desulphurizing activated enzyme that has that obtains in the cell of also available this bacterial strain or its dissociant is made desulfurization biocatalyst, and also available absorption or the immobilized cell that is embedded on the carrier are made desulfurization biocatalyst.
Bacillus brevis R-6 bacterial strain provided by the invention can be used as C-S key in the catalyst selectivity fracture organic compounds containing sulfur, removes sulphur atom, is particularly useful for removing of the middle organic heterocyclic sulphur of fossil oil (as coal, oil and products thereof); Solve existing oil hydrogenation technique processing cost height, be not easy to remove shortcoming such as sulphur in the heterogeneous ring compound; This bacterial strain removes the heterocycle sulphur in the organic compound in the fuel by specificity ' 4S ' approach, and do not destroy the C-C skeleton of organic compound, do not reduce the combustion heat value of fuel, this bacterium extensively is present in the crude oil, have many good characteristics as the industrial application bacterial strain, the ability of organic solvent-resistant is strong, the accumulation of no intermediate product in the grown cell sweetening process, metabolism generates final product HBP soon, and do not detect the existence of inorganic sulfur, the sulphur that produces is considered to combination or is blended in the cell of grown cell, therefore can not take place because of the sulfate ion accumulation suppresses the desulphurizing activated phenomenon of bacterial classification, and bacterial strain has wide industrial application potentiality.
Further describe the present invention below in conjunction with drawings and the specific embodiments:
Accompanying drawing 1 for R-6 of the present invention remove the result of sulphur among the DBT-●-dispersion agent be dimethylformamide-■-dispersion agent be n-Hexadecane-▲-dispersion agent is an ethanol
Embodiment 1: the screening step of Bacillus brevis R-6 bacterial strain provided by the invention is as follows:
Get the aerobic activated sludge sample from Chinese Yanshan Petrochemical Co. sewage from oil refinery treating pond, sample thief 5 grams are suspended in no sulfenyl basal culture medium and (form: 1000ml water, K
2HPO
43H
2O, 4g; NaH
2PO
42H
2O, 4.52g; NH
4Cl, 2.00g; MgCl
26H
2O, 0.2g; CaCl
22H
2O, 1mg; FeCl
36H
2O, 1mg; MnCl4H
2O, 4mg; Glycerol 0.2% (v/v) or glucose 1-3g, 20 minutes 121 ℃ of autoclavings) in, put in the shaking table and mix after 30 minutes, leave standstill, draw upper strata suspension and be transferred to nutritional medium (composition g/g:NaCl 1.0%; Yeast extract powder 0.5%, 1%, 20 minute autoclaving of Tryptones) in, puts in the shaking table, cultivated 24 hours for 30 ℃ with 150 rev/mins.Draw 5ml bacterium liquid and add in the 100ml sterilization basic medium, add dibenzothiophene DBT-ethanolic soln more therein and make that DBT concentration is 0.1mM in the nutrient solution.In shaking table,, cultivate after 2-3 days for 30 ℃ and be sprayed with line separation on the nutrient agar of DBT with 150 rev/mins.Single bacterium colony that separation obtains is inoculated in respectively and contains in the basic medium that DBT concentration is 0.2mM, acts on 2-3 days with 150 rev/mins 30 ℃ in shaking table.It is 1.0 that sample after biological action is regulated pH with 1N hydrochloric acid, 5000 rev/mins centrifugal 20 minutes down, supernatant liquor is with isopyknic ethyl acetate oscillation extraction.Extracted organic phase refluxes and is concentrated into 1ml, carries out GC-MS and analyzes.Select dibenzothiophene sulfone DBTO is arranged in the product
2And the bacterial strain of xenol carries out the progressively domestication of strain excellent.
The result, chosen the bacillus brevis bacterial strain R-6 bacterial strain that dibenzothiophene is had the specificity sweetening power, and in be preserved in April 29 calendar year 2001 " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC NO.0571;
This Bacillus brevis R-6 bacterial strain normal temperature that suits in neutrality and utmost point slightly acidic developing medium is cultivated, and its bacteriology morphological specificity: R-6 is a Gram-positive bacillus, and obligate is aerobic, the acid-fast stain feminine gender.Endogenous spore is arranged, give birth in the gemma.The sporocyst cell is little to be had and expands but not obvious; In a cell, has only a statospore; The cell walls chemical composition belongs to the II type; The sporocyst cell of bacterial strain R-6 is a rod-short, and its length is 0.5-0.7 * 1.2-1.4 μ m, does not form spore chain, no spirobacteria filament; The statospore of rod-short makes the sporocyst cell show slightly expansion; Give birth in the statospore, can move, the flagellum of growing thickly is arranged.
Embodiment 2: the cell liquid culture of preparation Bacillus brevis R-6 bacterial strain:
Picking nutrient agar medium slant culture bacillus brevis Bacillus brevis R-6 bacterial strain one platinum loop is seeded in the test tube that sterilization is housed, add sterilized DBT-ethanolic soln, making DBT concentration is 0.1mmol, in 30 ℃, cultivates 16-24 hour in bio-incubator under 150 rev/mins.Draw the bacterium liquid 1ml that cultivates acquisition and be seeded in the 300ml Erlenmeyer flask that 100ml basis minimal medium (pH=7.0) is housed, add sterilized DBT-ethanolic soln again, making DBT concentration is 0.1-0.5mmol.This Erlenmeyer flask is placed in the bio-incubator in 30 ℃, cultivated 48 hours for 150 rev/mins, make the liquid medium of Bacillus brevis bacterial strain R-6 cell.
Embodiment 3: the non-growing period resting cell liquid of preparation Bacillus brevis R-6 bacterial strain:
The liquid medium centrifugal (8000 rev/mins) of the bacterial strain R-6 cell that method obtained that will be by embodiment 2 10 minutes removes upper strata suspension.With an amount of physiological saline washing centrifugation 3 times, the gained centrifugal sediment is suspended in the physiological saline, freezing promptly makes the non-growing period resting cell liquid of Bacillus brevis bacterial strain R-6 in refrigerator then.
Embodiment 4: the thick enzyme extraction liquid of preparation Bacillus brevis R-6 bacterial strain:
The eccentric cell lyophilize of the bacterial strain R-6 that method obtained that will be by embodiment 3, cell after the lyophilize grinds in homogenizer earlier, ultrasonication under 20Hz then, add behind an amount of physiological saline centrifugal 30 minutes with 8000 rev/mins, discard the solid residue of centrifugation, its upper strata thick enzyme Centrifugical extraction liquid is the thick enzyme Centrifugical extraction liquid of the Bacillus brevis bacterial strain R-6 that makes.
Embodiment 5: the immobilized cell of preparation Bacillus brevis R-6 bacterial strain:
Physical adsorption: will clean, solid carriers such as the diatomite after dry, the sterilization, perforated brick, polyvinyl chloride thin slice add the sterilization developing medium, the Bacillus brevis bacterial strain R-6 of inoculation fresh culture, normal temperature is cultivated, and attached on the carrier, forms immobilized cell in the growth process
Gel embedding: (1) polyacrylamide gel embedding: the Bacillus brevis bacterial strain R-6 cell suspension of 3ml fresh culture, add 12ml physiological saline, add acrylamide 2.25g again, N, N '-methylene diacrylamide 0.12g, 5% dimethylamino, third fine 1.5ml and 2.5% persulfuric acid 1.5ml, finish polymerization after, obtain 15% polyacrylamide gel, be cut into small pieces, with distilled water and each washed twice of physiological saline, drain, promptly make immobilized cell.
(2) Lalgine embedding: the Bacillus brevis bacterial strain R-6 cell suspension of 3ml fresh culture, 2% the sodium alginate soln that adds that 20ml prepares stirs evenly mixture.Be squeezed into thick needle in 2% the gegenion solution (calcium salt or aluminium salt), placed refrigerator 10 hours, with distilled water and each washed twice of physiological saline, form be surrounded by Bacillus brevis bacterial strain R-6 immobilized cell evenly, spherical, gel that the height microvoid structure is arranged.
Embodiment 6: the application of the present invention's organosulfur in removing dibenzothiophene (DBT):
(1) sterilized DBT is made into DBT-dimethylformamide (also can the be ethanol) liquid storage of 100mmol/L;
(2) add sterilising medium 30ml, DBT-dimethylformamide liquid storage 0.06ml successively in the 100ml Erlenmeyer flask, the concentration that makes DBT in the Erlenmeyer flask is 0.2mmol/L;
The cell culture fluid of the Bacillus brevis R-6 bacterial strain that (3) embodiment 2 is made, or the non-growing period resting cell liquid of the Bacillus brevis R-6 bacterial strain that makes of embodiment 3, or thick enzyme extraction liquid (containing NADH in the thick enzyme extraction liquid) 3ml of the Bacillus brevis R-6 bacterial strain that makes of embodiment 4 adds above-mentioned steps
(4) in the Erlenmeyer flask, Erlenmeyer flask is placed in the bio-incubator, in 30 ℃, 150 rev/mins of cultivations were every 12 hours sampling and measuring.The measurement result of its DBT degraded is seen Fig. 1, its result shows, under the situation that different DBT dispersion agents exists, bacterial strain R-8 can remove the sulphur among the DBT preferably, n-Hexadecane is during as dispersion agent, and desulfurization rate is the fastest, is the 0.0037mM/hg dry cell weight, dimethylformamide and ethanol are during as dispersion agent, and sweetening effectiveness is close.This is because the existence meeting of n-Hexadecane forms a skim on the DBT surface, helps and microbial process.
Embodiment 7: the application of the sulphur of the present invention in removing dibenzothiophene sulfone (DBTO2):
Sterilized DBTO
2Be made into DBT-dimethylformamide (also can the be ethanol) liquid storage of 100mmol/L; Sulphur source DBT among the embodiment 6 is changed to DBTO
2, other operation stepss and method be with embodiment 6, its DBTO
2That degrades the results are shown in Table 3:
Table 3
| Time (hour) | 0 | 12 | 24 | 36 | 48 |
| DBTO 2Concentration mM | 0.2 | 0.101 | 0.062 | 0.036 | 0.018 |
| DBTO 2Degradation rate % | 49.5 | 69 | 82 | 91 |
Embodiment 8: the present invention is removing 4, the application of the sulphur in the 6-dimethyl Dibenzothiophene sulfone (DMDBT):
Press the method for embodiment 6, the sulphur source is changed to DMDBT-dimethylformamide (also can be ethanol or n-Hexadecane) solution, after the condition of pressing embodiment 6 was cultivated, the method for press embodiment 1 again was every 12 hours sampling and measuring, and the measurement result that DMDBT degrades sees Table 4:
Table 4
| Time (hour) | 0 | 12 | 24 | 36 | 48 |
| DMDBT concentration (mM) | 0.20 | 0.118 | 0.057 | 0.021 | 0 |
| DMDBT degradation rate % | 41 | 71.4 | 89.5 | 100 |
Embodiment 9: the present invention in removing thionaphthene (TPT) sulphur application:
Press the method for embodiment 6, catalytic substrate is changed to thionaphthene-dimethylformamide (also can be ethanol or n-Hexadecane) solution, after the condition of pressing embodiment 6 was cultivated, the method for pressing embodiment 1 again was every 12 hours sampling and measuring.The measurement result of thionaphthene degraded sees Table 5:
Table 5
| Time (hour) | 0 | 12 | 24 | 36 | 48 |
| TPT concentration mM | 0.2 | 0.207 | 0.176 | 0.122 | 0.082 |
| TPT degradation rate % | 0 | 12 | 39 | 59 |
Embodiment 10: the application of the present invention's sulphur in removing hexichol sulfide:
Press the method for embodiment 6, the sulphur source is changed to hexichol sulfide-dimethylformamide (also can be ethanol or n-Hexadecane) solution, after the condition of pressing embodiment 6 was cultivated, the method for pressing embodiment 1 again was every 12 hours sampling and measuring.The measurement result of hexichol sulfide degraded sees Table 6:
Table 6
| Time (hour) | 0 | 12 | 24 | 36 | 48 |
| DPS concentration (mM) | 0.2 | 0.124 | 0.122 | 0.066 | 0.021 |
| DPS degradation rate (%) | 38 | 39 | 67 | 90 |
Claims (7)
1. a bacillus brevis strain, it is characterized in that: this bacterial strain is Bacillus brevis R-6, is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC NO.0571 April 29 calendar year 2001.
2. by the described bacillus brevis bacterial strain of claim 1, it is characterized in that: separated obtaining around the oxygen consumption active sludge of this bacterial strain from the oily water treatment pond, the oil well in the soil sample around the higher pit, colliery of the soil of oil pollution and sulphur content; This bacterial strain normal temperature that suits in neutrality and utmost point slightly acidic developing medium is cultivated; Its bacteriology morphological specificity: belong to Gram-positive bacillus, obligate is aerobic, and the acid-fast stain feminine gender has endogenous spore, gives birth in the gemma, and the sporocyst cell is little to be had and expand but not obvious, in a cell, has only a statospore, and the cell walls chemical composition belongs to the II type;
The sporocyst cell of this Bacillus brevisR-6 bacterial strain is a rod-short, and its length is 0.5-0.7 * 1.2-1.4 μ m, does not form spore chain, no spirobacteria filament; The statospore of rod-short makes the sporocyst cell show slightly expansion; Give birth in the statospore, can move, the flagellum of growing thickly is arranged.
The application of the described bacillus brevis bacterial strain of 3 claims 1 sulphur in removing organic compounds containing sulfur is characterized in that: this Bacillus brevis R-6 bacterial strain removes sulphur in the organic compounds containing sulfur by oxidation scission C-S key approach.
4. remove the application of sulphur in the organic compounds containing sulfur by the described bacillus brevis bacterial strain of claim 3, it is characterized in that: the non-growing period resting cell liquid of this Bacillus brevis R-6 bacterial strain removes sulphur in the organic compounds containing sulfur by oxidation scission C-S key approach.
5. remove the application of sulphur in the organic compounds containing sulfur by the described bacillus brevis bacterial strain of claim 3, it is characterized in that: the immobilized cell of this Bacillus brevis R-6 bacterial strain removes sulphur in the organic compounds containing sulfur by oxidation scission C-S key approach.
6. remove the application of sulphur in the organic compounds containing sulfur by the described bacillus brevis bacterial strain of claim 3, it is characterized in that: the cell liquid culture of this Bacillus brevis R-6 bacterial strain removes sulphur in the organic compounds containing sulfur by oxidation scission C-S key approach.
7. remove the application of sulphur in the organic compounds containing sulfur by the described bacillus brevis bacterial strain of claim 3, it is characterized in that: the thick enzyme extraction liquid that contains NADH of this Bacillus brevis R-6 bacterial strain removes sulphur in the organic compounds containing sulfur by oxidation scission C-S key approach, contains NADH in the thick enzyme extraction liquid of Bacillus brevis R-6 bacterial strain.
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| CN 01115920 CN1132933C (en) | 2001-05-22 | 2001-05-22 | Brachybacillus strain and its application in removing sulfur from sulfur organic compound |
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| CN 01115920 CN1132933C (en) | 2001-05-22 | 2001-05-22 | Brachybacillus strain and its application in removing sulfur from sulfur organic compound |
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| US11879101B2 (en) | 2020-08-13 | 2024-01-23 | Yangtze University | Brevibacillus agri, preparation thereof, method for preparing surfactant and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100345628C (en) * | 2004-11-17 | 2007-10-31 | 中国科学院过程工程研究所 | Regeneration of desulfurizing adsorbent |
| CN100523138C (en) * | 2005-11-11 | 2009-08-05 | 中国科学院过程工程研究所 | Desulfurization regeneration method for sulfur-adsorbent ion liquid |
| CN101560483B (en) * | 2009-05-31 | 2010-11-17 | 南京农业大学 | Lipopeptide-producing bacillus pumilus and application thereof |
| CN102260568A (en) * | 2010-05-25 | 2011-11-30 | 上海彤微环保科技有限公司 | Combined microbial desulfurization method and process of coal |
| CN101993177B (en) * | 2010-10-13 | 2013-05-01 | 江苏博大环保股份有限公司 | Method for treating polymer-containing sewage of oil field |
| CN106433855B (en) * | 2016-10-28 | 2019-02-22 | 中南大学 | A kind of efficient flotation separation of coal-biological coupling sulfur method |
| CN108660095B (en) * | 2018-05-18 | 2021-07-13 | 中国石油化工股份有限公司 | Bacillus PY-3, microbial inoculum and application thereof |
| CN115074272B (en) * | 2022-06-10 | 2023-03-24 | 广西科学院 | Biological desulfurization bacillus aryabhattai and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US11879101B2 (en) | 2020-08-13 | 2024-01-23 | Yangtze University | Brevibacillus agri, preparation thereof, method for preparing surfactant and use thereof |
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