CN103013866A - Nitrate-resistant sulfate reducing bacteria - Google Patents
Nitrate-resistant sulfate reducing bacteria Download PDFInfo
- Publication number
- CN103013866A CN103013866A CN2012105122167A CN201210512216A CN103013866A CN 103013866 A CN103013866 A CN 103013866A CN 2012105122167 A CN2012105122167 A CN 2012105122167A CN 201210512216 A CN201210512216 A CN 201210512216A CN 103013866 A CN103013866 A CN 103013866A
- Authority
- CN
- China
- Prior art keywords
- clostridium
- nitrate
- reducing bacteria
- taihun5
- sulfate reducing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 53
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 title claims abstract description 42
- 229910002651 NO3 Inorganic materials 0.000 title claims abstract description 29
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 241000193403 Clostridium Species 0.000 claims abstract description 55
- 230000001580 bacterial effect Effects 0.000 claims abstract description 26
- 239000002351 wastewater Substances 0.000 claims abstract description 17
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 13
- 244000005700 microbiome Species 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000004065 wastewater treatment Methods 0.000 claims abstract description 4
- 241000193464 Clostridium sp. Species 0.000 claims abstract description 3
- 229910021653 sulphate ion Inorganic materials 0.000 claims description 25
- 238000000034 method Methods 0.000 abstract description 14
- 230000008569 process Effects 0.000 abstract description 10
- 230000004060 metabolic process Effects 0.000 abstract description 7
- 230000009467 reduction Effects 0.000 abstract description 5
- 238000012545 processing Methods 0.000 abstract description 4
- 239000002131 composite material Substances 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 15
- 108020004465 16S ribosomal RNA Proteins 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 238000000926 separation method Methods 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 3
- 108010027912 Sulfite Oxidase Proteins 0.000 description 3
- 102000043440 Sulfite oxidase Human genes 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000010949 copper Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000013081 phylogenetic analysis Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000426018 Clostridium sp. taihuN5 Species 0.000 description 2
- 241000605739 Desulfovibrio desulfuricans Species 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- 241000605909 Fusobacterium Species 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 108010028074 Hydrogensulfite Reductase Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 101150095420 dsr gene Proteins 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 2
- 239000000976 ink Substances 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 238000010079 rubber tapping Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- HNBDQABBWNOTRU-UHFFFAOYSA-N thalline Chemical compound C1=CC=[Tl]C=C1 HNBDQABBWNOTRU-UHFFFAOYSA-N 0.000 description 2
- 108010032655 Adenylyl-sulfate reductase Proteins 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000605716 Desulfovibrio Species 0.000 description 1
- 241000605747 Desulfovibrio africanus Species 0.000 description 1
- 241000605762 Desulfovibrio vulgaris Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000003723 Smelting Methods 0.000 description 1
- 102000004523 Sulfate Adenylyltransferase Human genes 0.000 description 1
- 108010022348 Sulfate adenylyltransferase Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 229910052770 Uranium Inorganic materials 0.000 description 1
- 241001147804 [Clostridium] celerecrescens Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229910001448 ferrous ion Inorganic materials 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003295 industrial effluent Substances 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- GNBVPFITFYNRCN-UHFFFAOYSA-M sodium thioglycolate Chemical compound [Na+].[O-]C(=O)CS GNBVPFITFYNRCN-UHFFFAOYSA-M 0.000 description 1
- NGSFWBMYFKHRBD-UHFFFAOYSA-N sodium;2-hydroxypropanoic acid Chemical compound [Na+].CC(O)C(O)=O NGSFWBMYFKHRBD-UHFFFAOYSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- JFALSRSLKYAFGM-UHFFFAOYSA-N uranium(0) Chemical compound [U] JFALSRSLKYAFGM-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses nitrate-resistant sulfate reducing bacteria. The nitrate-resistant sulfate reducing bacteria are named as clostridium (Clostridium sp.) taihuN5 in classification, wherein the bacterial strain is preserved in General Microbiology Centre of China Committee for Culture Collection of Microorganisms; the preservation number is 23 Nov 2012; and the preservation number is CGMCC No.6887. Application of the nitrate-resistant sulfate reducing bacteria in heavy metal wastewater treatment is also disclosed by the invention. The nitrate-resistant sulfate reducing bacteria provided by the invention can keep normal growth and metabolism under the condition of high-concentration nitrate, carry out sulfte reduction, and are good bacterial sources of the process for processing heavy metal wastewater by composite sulfate reducing bacteria.
Description
Technical field
The present invention relates to a kind of sulphate reducing bacteria, belong to the environmental microorganism field.
Background technology
Sulphate reducing bacteria (sulfate reducing bacteria, SRB) is take organic compound or mineral compound as electron donor, and the class prokaryotic micro-organisms that sulphate reducing produces sulfide has various phylogeny branch and physiology characteristic.Sulphate reducing bacteria under anaerobic produces sulfide by the alienation sulfate reduction, and this process can consume the sulfate radical in the solution, can be for the treatment of sulfate wastewater; Born of the same parents' foreign object of the electronegativity of sulphate reducing bacteria cell surface and secretion has stronger electrostatic adhesion and biological flocculation to heavy metal ion; Can consume hydrogen ion in the time of its metabolism sulfate ion, reduce solution acidity, can make metal ion form precipitation of hydroxide, metabolism produces a large amount of sulfide and can be combined with heavy metal cation, form insoluble metallic sulfide precipitation, thereby reduce the free metal concentration in the water body.Sulphate reducing bacteria processing waste water suitability is stronger, cheap, treatment effect better, non-secondary pollution, be widely used in sulfate wastewater all the time, the improvement of heavy metal industrial effluent and acidic mine waste water is also developed preferably, process bacterial classification and develop into the complex vitriolate reducing bacteria from original single sulphate reducing bacteria, technique develops into anaerobic mud bed, fluidized-bed process and immobilization technology etc. by batch precipitation.
A large amount of nitric acid commonly used is washed copper etc. such as nitre leaching-ore and nitric acid in the exploitation of metalliferous mineral and the smelting process, so that heavy metal polluted waste water often contains high concentration nitrate simultaneously, adopts such as in-situ leaching that nitrate concentration reaches 700mg/L in the uranium ore waste water.The existence meeting of nitrate produces strong restraining effect to sulphate reducing bacteria itself, and then affects the removal effect of heavy metal, is containing the NaNO of 70mM/L such as the sulphate reducing bacteria (Desulfovibrio vulgaris) of a strain Desulfovibrio
3Substratum in cultivate, the highest bacterial concentration of its growth has descended 50%, the restraining effect of nitrate is obvious.Therefore sulphate reducing bacteria that can enduring high-concentration nitrate provides good bacterial classification source for the microbial augmentation waste water treatment process.
Summary of the invention
Technical problem to be solved by this invention provides the sulphate reducing bacteria of a strain nitrate resisting, for sulphate reducing bacteria treatment of nitric acid salt waster water process provides the bacterial classification source.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The sulphate reducing bacteria of one strain nitrate resisting, its Classification And Nomenclature is clostridium (Clostridium sp.) taihuN5, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC), preservation address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101, preservation date is on November 23rd, 2012, and deposit number is CGMCC No.6887.This bacterial strain is that the contriver separates from the lake bed sediment of Zhu Shan gulf, Taihu Lake in July, 2011 and obtains.
The sulphate reducing bacteria of above-mentioned nitrate resisting is gram-positive microorganism, and it is shaft-like that thalline is, and long is 2.5-4.3 μ m, and wide is 0.8-1.2 μ m, the hydrogen sulfide that growth metabolism produces and the Fe on the solid medium
2+Make bacterium colony be black, circle, neat in edge, diameter 1 ~ 3mm.
The sulphate reducing bacteria of above-mentioned nitrate resisting, useful length is about the nucleotide sequence of 1500kb (the sequence accession number among the GenBank is: JX847660) on its 16SrRNA gene, with this sequence input GenBank, compare with Blast software and database sequence, the result shows with the 16S rDNA sequence similarity of Clostridium celerecrescens strain DSM 5628 higher, is 99%.Based on the Phylogenetic Analysis result of 16S rDNA gene, be accredited as the new bacterial strain of a strain of fusobacterium (Clostridium).The 16S rDNA sequence of bacterial strain Clostridium sp.taihuN5 of the present invention is shown in SEQ ID No.1.。
The application of the sulphate reducing bacteria of above-mentioned nitrate resisting in wastewater treatment.
Wherein, described waste water is the waste water that contains nitrate and vitriol.
Wherein, described heavy metal is Cu
2+, Zn
2+, Ni
2+, Pb
2+And Cd
2+In any one or a few.
Bacterial strain of the present invention can be in high concentration nitrate solution normal growth, metabolism vitriol makes it to be reduced into H
2S, H
2S easily and the Cu in the waste water
2+, Zn
2+, Ni
2+, Pb
2+, Cd
2+In conjunction with, CuS, the ZnS, NiS, PbS, the CdS that generate indissoluble precipitate, and heavy metal ion is separated from waste water.
Beneficial effect: sulfate reduction bacterial strain provided by the invention (Clostridium sp.taihuN5) can keep normal growth metabolism under the high concentration nitrate condition, carry out sulfate reduction, process the bacterial classification source of heavy metal wastewater thereby technique for good complex vitriolate reducing bacteria.
Description of drawings
Fig. 1 is the colonial morphology figure of clostridium (Clostridium) taihuN5 on solid medium.
Fig. 2 is clostridium (Clostridium) taihuN5 electron micrograph behind gramstaining.
Fig. 3 is that the 16S rDNA sequence of clostridium (Clostridium) taihuN5 and close bacterial strain is carried out the phylogenetic evolution tree that sequence analysis makes up.
Fig. 4 is sulfate concentration in clostridium (Clostridium) taihuN5 growth curve and the culture system.
Fig. 5 is that clostridium (Clostridium) taihuN5 is at 1g/LNaNO
3Sulfate concentration in growth curve and the culture system under the condition.
Fig. 6 is the growth curve of clostridium (Clostridium) taihuN5 under different concns nitrate condition.
Fig. 7 is clostridium (Clostridium) taihuN5 sulfite reductase (DSR) gene PCR amplified production electrophorogram.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described content of embodiment only is used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
Separation and the evaluation of embodiment 1 bacterial strain of the present invention.
(1) separation and the purifying of clostridium (Clostridium) taihuN5.
As indicator, the meta-bolites hydrogen sulfide of sulphate reducing bacteria can form the prepared Chinese ink look with ferrous ion to the employing liquid nutrient medium, obtains the sign of sulphate reducing bacteria flora as enrichment with ferrous sulfate.Employing has the small-caliber vessel (500ml serum bottle) of spiral mouth stopper, autoclaving behind the enrichment medium of packing certain volume., cover tightly to be placed in 30 ℃ of thermostat containers and cultivate to full state by 20% inoculum size inoculation bottom mud in Taihu Lake, lucifuge leaves standstill an about week, and the color for the treatment of pregnant solution becomes prepared Chinese ink look bottle mouth position and gives out H
2The rotten-egg odour of S namely represents the enrichment success.
The bacterium liquid that enrichment is good adopts respectively aseptic technique to make different dilution bacteria suspensions.Culture dish is aseptic one deck substratum of pouring into after sterilization, after solidifying, takes respectively 10
-5～10
-9Dilution bacteria suspension 0.1ml is coated with at each agar plate.After being coated with, allow bacterium liquid permeate five minutes, open culture dish and on one side solid medium is poured into once again.Under 30 ℃ condition, cultivated 5-6 days, the spherical small colonies of many black that grows in the flat board is required bacterium colony (Fig. 1), selects suitable dilution training ware, and each bacterium colony separates to such an extent that open, throw off the upper strata substratum, in the new liquid nutrient medium of toothpick picking list bacterium colony access.
Repeat dilution spread, sandwich culfure, select etc. twice, can reach the separation and purification to sulphate reducing bacteria, the pure bacterial strain that acquisition can be identified, be preserved, transfers.
TaihuN5 carries out film-making, gramstaining to separation of bacterial (Clostridium).Microscopy is gram-positive microorganism, and it is shaft-like that thalline is, and long is 2.5-4.3 μ m, and wide is 0.8-1.2 μ m(Fig. 2)
(2) enrichment and isolation medium.
KH
2PO
40.5g, NH
4Cl 1.0g, CaCl
22H
2O 0.1g, Na
2SO
41.0g, MgSO
47H
2O 2.0g, Sodium.alpha.-hydroxypropionate (80%) 3.5ml, yeast extract 1.0g, FeSO
47H
2O 0.5g, Vc 0.1g, Thioglycolic acid sodium salt 0.1g, distilled water 1000ml transfers about pH to 7.5.
Above-mentioned substratum then adds 1.6% agar as making solid medium.
(3) pcr amplification of 16S rDNA and order-checking.
Take total DNA of the extraction bacterium of extracting as template, use 16S rDNA universal primer (27F:5 '-AGAGTTTGATCCTGGCTCAG-3 '; 1492R:5 '-GGTTACCTTGTTACGACTT-3 ') amplification, PCR reaction system: 10 μ l systems, 10 * Buffer, 1 μ l, dNTP 0.5 μ l, Mg
2+0.75 μ l, each 0.25 μ l of primer, template dna 0.25 μ l, rTaq 0.0625 μ l, ddH2O 6.9375 μ l.Pcr amplification condition: 94 ℃ of 4min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, 30 circulations; 72 ℃ of 10min.With 1% sepharose the PCR product of 16S rDNA is done electrophoresis detection, find the target stripe of 1500bp, the recovery of tapping rubber to being the TA clone with the PMD19-T carrier behind the PCR product purification, is entrusted the large gene sequencing of China after the sub-empirical tests of picking positive colony.
The 16S rDNA length that obtains clostridium (Clostridium) taihuN5 after the order-checking is the sequence of 1414bp, be submitted to GenBank and other existing bacterial strain sequences are compared, and the 16SrRNA sequence of bacterial strain is classified and the Phylogenetic Analysis (see figure 3) by the NJ method with MEGA software, the 16S rDNA sequence similarity of order-checking institute's calling sequence and Clostridium celerecrescensstrain DSM 5628 is higher, is 99%.Based on the Phylogenetic Analysis result of 16S rDNA gene, be accredited as the new bacterial strain of a strain of fusobacterium (Clostridium).
Embodiment 2: the growth characteristics of bacterial strain of the present invention.
In the anaerobism pipe, add the 9.8ml substratum, clostridium (Clostridium) the taihuN5 bacterium liquid of inoculation 0.2ml logarithmic phase, 30 ℃ of lucifuges are cultivated in anaerobic box.(culture medium prescription is with above-mentioned concentration and separation substratum) respectively at postvaccinal 0,10,15,20,25,30,35,40,45,50,60,70h, 80h, 90h, 100h sampling, measures bacterium liquid in the absorbancy at 600nm place.With 0.22 μ m membrane filtration sample, with SO in the ion chromatography sample
4 2-Concentration.
SO in clostridium (Clostridium) taihuN5 growth curve and the substratum
4 2-Concentration curve as shown in Figure 4, postvaccinal 0-30 hour is lag period, 30-60 hour is logarithmic phase, sulfate concentration reduces, the sulfate concentration after the logarithmic phase in the substratum does not almost change.Bacterium liquid absorbancy descends after cultivating 100h, SO when finishing to cultivation
4 2-The concentration minimizing is about 10%.
Embodiment 3: bacterial strain of the present invention is to the nitrate tolerance.
(1) clostridium (Clostridium) taihuN5 is containing 1g/LNaNO
3Growth in the substratum.
In the anaerobism pipe, add the 9.8ml substratum, clostridium (Clostridium) the taihuN5 bacterium liquid of inoculation 0.2ml logarithmic phase, 30 ℃ of lucifuges are cultivated in anaerobic box.(culture medium prescription is for adding 1g/LNaNO
3The concentration and separation substratum), respectively at postvaccinal 0,10,15,20,25,30,35,40,45,50,60,70h, 80h, 90h, 100h sampling, measure bacterium liquid in the absorbancy at 600nm place.With 0.22 μ m membrane filtration sample, with SO in the ion chromatography sample
4 2-Concentration.
Clostridium (Clostridium) taihuN5 is at 1g/LNaNO
3SO in growth curve under the condition and the substratum
4 2-Concentration curve as shown in Figure 5, at the front 80h that cultivates, growing state basic with do not add NaNO
3Growth curve is consistent, but obviously do not descend in late stage of culture bacterium liquid absorbancy, but still remain on maximum absorbance, sulfate concentration is in whole incubation period continuous decrease, illustrate that the nitrate that adds in the substratum does not suppress the growth of taihuN5, has promoted SO on the contrary
4 2-Reduction.
(2) clostridium (Clostridium) taihuN5 is at different concns NO
3 -Under tolerance.
Four treatment group are set in the concentration and separation substratum, add NaNO
3, make NaNO in the substratum
3Final concentration be 0,30,70,100mM/L, in the anaerobism pipe, add the 9.8ml substratum, clostridium (Clostridium) the taihuN5 bacterium liquid of inoculation 0.2ml logarithmic phase, 30 ℃ of lucifuges are cultivated in anaerobic box.Every the 24h sampling, measure the absorbancy of bacterium liquid under wavelength 600nm in the anaerobism pipe.
Clostridium (Clostridium) taihuN5 0,30,70,100mM/LNaNO
3Under upgrowth situation as shown in Figure 6, add 30mM/L NaNO
3The treatment group upgrowth situation basic be consistent and 70 and 100mM/LNaNO without the interpolation group
3Prolong the lag period for the treatment of group, but maximum absorbance value and other two groups are without significant difference, illustrate that clostridium (Clostridium) taihuN5 has been subject to the impact of high concentration nitrate at early growth period, but the metabolism adjusting by self can be eliminated nitrate fully on its impact, therefore think that clostridium (Clostridium) taihuN5 is the bacterial strain that a strain can tolerate nitrate, processing in the waster water process for sulphate reducing bacteria has preferably application prospect.
Embodiment 4: amplification and the order-checking of clostridium (Clostridium) taihuN5 sulfite reductase (DSR) gene.
Take total DNA of the extraction bacterium of extracting as template, use the DSR primer (DSR1F:5 '-ACSCACTGGAAGCACG-3 '; DRS4R:5 '-GTGTAGCAGTTACCGCA-3 ') amplification.PCR system: 10 μ l systems, 10 * Buffer, 1 μ l, dNTP 0.5 μ l, Mg2+0.75 μ l, each 0.25 μ l of primer, template dna 0.25 μ l, rTaq 0.0625 μ l, ddH2O 6.9375 μ l.PCR condition: 94 ℃ of 4min; 94 ℃ of 30s, 53 ℃ of 50s, 72 ℃ of 1.5min, 30 circulations; 72 ℃ of 10min.With 1% sepharose the PCR product of 16S rDNA is done electrophoresis detection, find length 1900bp fragment (Fig. 7) recovery of tapping rubber, to being the TA clone with the PMD19-T carrier behind the PCR product purification, entrust the large gene sequencing of China after the sub-empirical tests of picking positive colony.The DSR length that obtains clostridium (Clostridium) taihuN5 after the order-checking is the sequence of 1992bp (shown in SEQ ID No.2), use the Blast retrieval analysis to confirm that this sequence is a part of sequence of sulfite reductase gene cluster (dsr), and higher with the homology of the DSR gene regions of following bacteriums: Desulfovibrio desulfuricans isolate SRDQC(97%), Desulfovibriodesulfuricans strain F28-1(93%) and Desulfovibrio africanus(91%).
Dissimilatory sulfite reductase (dissimilatory sulfite reductase, DSR) is sulphate reducing bacteria reduction SO
4 2-Produce H
2One of key enzyme of S, SO
4 2-At first under the effect of ATP sulfurylase and APS reductase, obtain 2 electronics, become SO
3 2-, SO
3 2-Under the catalysis of DSR, finally reduce by a series of electronic transfer process and to form S
2-.DSR extensively is present in the various sulphate reducing bacterias, and the DSR gene is quite conservative in the SRB of same monoid, but has than big difference in Different groups SRB, can be used as the index of classification and the evolution of SRB.
Embodiment 5: the application of clostridium (Clostridium) taihuN5 in processing metallic wastewater.
Clostridium (Clostridium) the taihuN5 bacterial classification inoculation of cryopreservation is activated in the anaerobism pipe that the concentration and separation liquid nutrient medium is housed, with the enlarged culturing in the 500ml concentration and separation liquid nutrient medium of the bacterial classification inoculation after the activation, logarithmic phase to be grown to is inoculated in the bacterium liquid of enlarged culturing the Zn that contains of preparation
2+Among the artificial wastewater, add simultaneously isopyknic concentration and separation substratum, 30 ℃ of sealings are cultivated and are processed metallic wastewater (every 3 days supplemented mediums).Zn in this water
2+Concentration 50mg/L, SO
4 2-Concentration 800mg/L, the H of after treatment clostridium (Clostridium) taihuN5 generation
2S makes the Zn in the solution
2+Form the ZnS precipitation, water outlet Zn
2+Density loss is to 1.2mg/L, SO
4 2-Concentration 150mg/L reaches national industrial wastewater discharge standard, Zn
2+Clearance is 97.6%, SO
4 2-Clearance is 81.5%.
Claims (4)
1. the sulphate reducing bacteria of a strain nitrate resisting, its Classification And Nomenclature is clostridium (Clostridium sp.) taihuN5, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preservation date is on November 23rd, 2012, and deposit number is CGMCC No.6887.
2. the application of the sulphate reducing bacteria of nitrate resisting claimed in claim 1 in heavy metal containing wastewater treatment.
3. application according to claim 2 is characterized in that, described waste water is the heavy metal wastewater thereby that contains nitrate and vitriol.
4. application according to claim 2 is characterized in that, described heavy metal is Cu
2+, Zn
2+, Ni
2+, Pb
2+And Cd
2+In any one or a few.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210512216.7A CN103013866B (en) | 2012-12-04 | 2012-12-04 | Nitrate-resistant sulfate reducing bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210512216.7A CN103013866B (en) | 2012-12-04 | 2012-12-04 | Nitrate-resistant sulfate reducing bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103013866A true CN103013866A (en) | 2013-04-03 |
| CN103013866B CN103013866B (en) | 2014-12-31 |
Family
ID=47962984
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210512216.7A Expired - Fee Related CN103013866B (en) | 2012-12-04 | 2012-12-04 | Nitrate-resistant sulfate reducing bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103013866B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112543A (en) * | 2015-09-22 | 2015-12-02 | 北京世纪金道石油技术开发有限公司 | Molecular detection method for desulfovibro |
| CN107287129A (en) * | 2016-04-01 | 2017-10-24 | 兰州大学 | One plant can make heavy metal settle sulfate reducing bacteria and its application |
| CN107699519A (en) * | 2017-10-18 | 2018-02-16 | 三峡大学 | One strain of sulfate reduction bacteria, isolation and identification method and its application |
| CN110494552A (en) * | 2018-08-09 | 2019-11-22 | 中国石油大学(北京) | One plant of bacterial strain and its application with extremely strong sulfate reduction ability |
| CN114437987A (en) * | 2022-02-28 | 2022-05-06 | 中国科学院地理科学与资源研究所 | Sulfate reducing bacteria and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102534210A (en) * | 2012-01-17 | 2012-07-04 | 江西理工大学 | Metal ore heap leaching, anaerobic enrichment transformation and biological leaching extraction process |
-
2012
- 2012-12-04 CN CN201210512216.7A patent/CN103013866B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102534210A (en) * | 2012-01-17 | 2012-07-04 | 江西理工大学 | Metal ore heap leaching, anaerobic enrichment transformation and biological leaching extraction process |
Non-Patent Citations (3)
| Title |
|---|
| LIU Y: "Genbank:JX847660", 《GENBANK》 * |
| 任军俊等: "硫酸盐还原菌处理废水的研究进展与展望", 《水资源与水工程学报》 * |
| 胡凯光等: "硫酸根和硝酸根对ZVI-SRB处理铀废水的影响研究", 《矿业工程研究》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112543A (en) * | 2015-09-22 | 2015-12-02 | 北京世纪金道石油技术开发有限公司 | Molecular detection method for desulfovibro |
| CN107287129A (en) * | 2016-04-01 | 2017-10-24 | 兰州大学 | One plant can make heavy metal settle sulfate reducing bacteria and its application |
| CN107287129B (en) * | 2016-04-01 | 2021-05-28 | 兰州大学 | Sulfate reducing bacteria capable of settling heavy metals and application thereof |
| CN107699519A (en) * | 2017-10-18 | 2018-02-16 | 三峡大学 | One strain of sulfate reduction bacteria, isolation and identification method and its application |
| CN107699519B (en) * | 2017-10-18 | 2020-10-02 | 三峡大学 | Sulfate reducing bacteria, separation and identification method and application thereof |
| CN110494552A (en) * | 2018-08-09 | 2019-11-22 | 中国石油大学(北京) | One plant of bacterial strain and its application with extremely strong sulfate reduction ability |
| CN110494552B (en) * | 2018-08-09 | 2021-11-26 | 中国石油大学(北京) | Bacterial strain with extremely strong sulfate reducing capability and application thereof |
| CN114437987A (en) * | 2022-02-28 | 2022-05-06 | 中国科学院地理科学与资源研究所 | Sulfate reducing bacteria and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103013866B (en) | 2014-12-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103013868B (en) | Sulfate reducing bacteria with tolerance on arsenic | |
| Bai et al. | Bioremediation of cadmium by growing Rhodobacter sphaeroides: kinetic characteristic and mechanism studies | |
| CN105255782B (en) | There is fiber bacterium and the purposes of reducing power to Cr VI | |
| CN103013866B (en) | Nitrate-resistant sulfate reducing bacteria | |
| CN107287129B (en) | Sulfate reducing bacteria capable of settling heavy metals and application thereof | |
| CN112592855B (en) | Bacillus subtilis and method for treating uranium and cadmium polluted water body by using same | |
| CN112322550B (en) | Stenotrophomonas maltophilia and application thereof in heavy metal treatment | |
| CN102337229A (en) | Separation of heavy metal tolerant bacteria and application thereof in reclamation of gangue | |
| CN106867921A (en) | The method that thiobacillus ferrooxidans and bacterium treatment acid wastewater in mine reclaim iron resource | |
| CN102533616A (en) | Ralstonia pickettii highly resistant to cadmium and extraction method and application thereof | |
| CN103833144B (en) | A kind of method utilizing flcos producing bacteria fermented liquid to remove heavy metal ion in water | |
| CN113980830B (en) | Pseudomonas stutzeri, culture thereof and application thereof | |
| CN105400718B (en) | One plant of nitrate relies on ferrous oxidation bacterial strain and its application | |
| Karnachuk et al. | Bacteria of the sulfur cycle in the sediments of gold mine tailings, Kuznetsk Basin, Russia | |
| CN107699519B (en) | Sulfate reducing bacteria, separation and identification method and application thereof | |
| CN107058189B (en) | Bacillus megaterium and its processing heavy metal-glyphosate combined pollution in apply | |
| CN102162029B (en) | Microbiological oxidation and reduction coupling leaching method for valuable metal in manganese oxide ore | |
| WO2017035856A1 (en) | Selenophilic microbe wautersiella enshiensis ylx-1 and application thereof | |
| CN111378592B (en) | Bacillus licheniformis and method for treating malodorous organic wastewater by using same to purify water | |
| CN101434919B (en) | A strain of sulfate reduction bacteria and process for using the same for treating acidic mine wastewater with straw as carbon nitrogen source | |
| CN113249276B (en) | Bacillus cereus and application thereof | |
| CN105779327B (en) | One bacillus and culture medium and in alkaline ore dressing wastewater treatment reduce pH value method | |
| RU2355756C1 (en) | Desulfovibrio oxamicus BACTERIA STRAIN, USED FOR PURIFYING SEWAGE WATER FROM SULPHATES AND IONS OF HEAVY METALS | |
| CN108265013B (en) | Sulfate reducing bacteria and process for recovering valuable metals in zinc smelting wastewater by using same | |
| CN108102988B (en) | Heavy metal resistant bacillus, microbial preparation and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141231 Termination date: 20161204 |