CN103013866B - Nitrate-resistant sulfate reducing bacteria - Google Patents
Nitrate-resistant sulfate reducing bacteria Download PDFInfo
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- CN103013866B CN103013866B CN201210512216.7A CN201210512216A CN103013866B CN 103013866 B CN103013866 B CN 103013866B CN 201210512216 A CN201210512216 A CN 201210512216A CN 103013866 B CN103013866 B CN 103013866B
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- clostridium
- nitrate
- reducing bacteria
- taihun5
- sulfate reducing
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- 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 30
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 title claims abstract description 30
- 241000193403 Clostridium Species 0.000 claims abstract description 55
- 230000001580 bacterial effect Effects 0.000 claims abstract description 21
- 239000002351 wastewater Substances 0.000 claims abstract description 18
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- 244000005700 microbiome Species 0.000 claims abstract description 8
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- 238000000034 method Methods 0.000 abstract description 18
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- 238000001556 precipitation Methods 0.000 description 5
- 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
- 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
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- 241000426018 Clostridium sp. taihuN5 Species 0.000 description 2
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- 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
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- 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
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- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 241000209094 Oryza Species 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
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- 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
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
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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 field of environment microorganism.
Background technology
Sulphate reducing bacteria (sulfate reducing bacteria, SRB) is with organic compound or mineral compound for 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 alienation sulfate reduction, and this process can consume the sulfate radical in solution, may be used for processing sulfate wastewater; The electronegativity of sulphate reducing bacteria cell surface and born of the same parents' foreign object heavy metal ion of secretion have stronger electrostatic adhesion and biological flocculation; Hydrogen ion can be consumed while its metabolism sulfate ion, reduce solution acidity, metal ion can be made to form precipitation of hydroxide, and metabolism produces a large amount of sulfide and can be combined with heavy metal cation, form insoluble metallic sulfide precipitation, thus reduce the free metal concentration in water body.Sulphate reducing bacteria process 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 develops into complex vitriolate reducing bacteria from original single sulphate reducing bacteria, and technique develops into anaerobic mud bed, fluidized-bed process and immobilization technology etc. by batch precipitation.
Commonly use a large amount of nitric acid in the exploitation of metalliferous mineral and smelting process, as nitre leaching-ore and nitric acid wash copper etc., make heavy metal polluted waste water often simultaneously containing high concentration nitrate, adopt nitrate concentration in uranium ore waste water as in-situ leaching and reach 700mg/L.The existence of nitrate can produce strong restraining effect to sulphate reducing bacteria itself, and then affects the removal effect of heavy metal, if the sulphate reducing bacteria (Desulfovibrio vulgaris) of a strain Desulfovibrio is at the NaNO containing 70mM/L
3substratum in cultivate, its growth the highest bacterial concentration have dropped 50%, the restraining effect of nitrate is obvious.Good bacterial classification source is provided therefore, it is possible to the sulphate reducing bacteria of enduring high-concentration nitrate is microbial augmentation waste water treatment process.
Summary of the invention
Technical problem to be solved by this invention is to provide the sulphate reducing bacteria of a strain nitrate resisting, for sulphate reducing bacteria treatment of nitric acid salt waster water process provides bacterial classification to originate.
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's 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 contriver is separated in July, 2011 and obtains from the lake bed sediment of Zhu Shan gulf, Taihu Lake.
The sulphate reducing bacteria of above-mentioned nitrate resisting is gram-positive microorganism, and thalline is shaft-like, and long is 2.5-4.3 μm, and wide be 0.8-1.2 μm, the Fe on the hydrogen sulfide of growth metabolism generation and solid medium
2+bacterium colony is made to be black, circular, neat in edge, diameter 1 ~ 3mm.
The sulphate reducing bacteria of above-mentioned nitrate resisting, on its 16SrRNA gene, useful length is about the nucleotide sequence (sequence accession number in GenBank is: JX847660) of 1500kb, by this sequence inputting GenBank, compare with Blast software and database sequence, 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 a strain new strains of fusobacterium (Clostridium).The 16S rDNA sequence of bacterial strain Clostridium sp.taihuN5 of the present invention is as shown in SEQ ID No.1.。
The sulphate reducing bacteria application in the treatment of waste water of above-mentioned nitrate resisting.
Wherein, described waste water is the waste water containing 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 in high concentration nitrate solution normal growth, metabolism vitriol makes it to be reduced into H
2s, H
2the Cu of S easily and in waste water
2+, Zn
2+, Ni
2+, Pb
2+, Cd
2+in conjunction with, generate CuS, ZnS, NiS, PbS, CdS precipitation of indissoluble, 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 high concentration nitrate condition, carry out sulfate reduction, for the bacterial classification of good complex vitriolate reducing bacteria process heavy metal wastewater thereby technique is originated.
Accompanying drawing explanation
Fig. 1 is the colonial morphology figure of clostridium (Clostridium) taihuN5 on solid medium.
Fig. 2 is clostridium (Clostridium) taihuN5 electron micrograph after gramstaining.
Fig. 3 is the phylogenetic evolution tree that the 16S rDNA sequence of clostridium (Clostridium) taihuN5 and close bacterial strain carries out sequence analysis structure.
Fig. 4 is sulfate concentration in clostridium (Clostridium) taihuN5 growth curve and culture system.
Fig. 5 is that clostridium (Clostridium) taihuN5 is at 1g/LNaNO
3sulfate concentration in growth curve and culture system under 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.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
The Isolation and ldentification of embodiment 1 bacterial strain of the present invention.
(1) abstraction and purification of clostridium (Clostridium) taihuN5.
Adopt liquid nutrient medium using ferrous sulfate as indicator, the meta-bolites hydrogen sulfide of sulphate reducing bacteria can form prepared Chinese ink look with ferrous ion, obtains the mark of sulphate reducing bacteria flora as enrichment.Adopt the small-caliber vessel (500ml serum bottle) having spiral mouth stopper, autoclaving after the enrichment medium of packing certain volume.Inoculum size inoculation bottom mud in Taihu Lake by 20% is to full state, and cover tightly to be placed in 30 DEG C of thermostat containers and cultivate, lucifuge leaves standstill about one week, treats that the color of pregnant solution becomes prepared Chinese ink look bottle mouth position and gives out H
2the rotten-egg odour of S, namely represents enrichment success.
Aseptic technique is adopted to make different dilution bacteria suspension respectively bacterium liquid good for enrichment.Culture dish is aseptic after sterilizing pours one deck substratum into, after to be solidified, takes 10 respectively
-5~ 10
-9dilution bacteria suspension 0.1ml is coated with on each agar plate.After being coated with, allowing bacterium liquid permeate five minutes, open culture dish and solid medium is poured into once again.5-6 days is cultivated under the condition of 30 DEG C, the spherical small colonies of many black grown in flat board is required bacterium colony (Fig. 1), and select the training ware of appropriate dilution, each bacterium colony is separated comparatively open, throw off upper strata substratum, access in new liquid nutrient medium with toothpick picking list bacterium colony.
Repeat dilution spread, sandwich culfure, to select etc. twice, the separation and purification to sulphate reducing bacteria can be reached, obtain the pure bacterial strain can identified, preserve, transfer.
Film-making is carried out, gramstaining to separation of bacterial (Clostridium) taihuN5.Microscopy is gram-positive microorganism, and thalline is shaft-like, and long is 2.5-4.3 μm, and wide is 0.8-1.2 μm (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, adjust pH to about 7.5.
Above-mentioned substratum as made solid medium, then adds 1.6% agar.
(3) pcr amplification of 16S rDNA and order-checking.
With the STb gene of extraction bacterium extracted for 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, primer each 0.25 μ l, template dna 0.25 μ l, rTaq 0.0625 μ l, ddH2O 6.9375 μ l.Pcr amplification condition: 94 DEG C of 4min; 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1.5min, 30 circulations; 72 DEG C of 10min.Do electrophoresis detection by the PCR primer of 1% sepharose to 16S rDNA, find the target stripe of 1500bp, carry out rubber tapping and reclaim, be TA clone to after PCR primer purifying with PMD19-T carrier, after the sub-empirical tests of picking positive colony, entrust Hua Da gene sequencing.
The 16S rDNA length obtaining clostridium (Clostridium) taihuN5 after order-checking is the sequence of 1414bp, be submitted to GenBank and other existing strain sequence are compared, and classified and Phylogenetic Analysis (see figure 3) by the 16SrRNA sequence of NJ method to bacterial strain with MEGA software, the 16S rDNA sequence similarity of order-checking gained sequence and Clostridium celerecrescensstrain DSM 5628 is higher, is 99%.Based on the Phylogenetic Analysis result of 16S rDNA gene, be accredited as a strain new strains of fusobacterium (Clostridium).
Embodiment 2: the growth characteristics of bacterial strain of the present invention.
In anaerobism pipe, add 9.8ml substratum, clostridium (Clostridium) the taihuN5 bacterium liquid of inoculation 0.2ml logarithmic phase, in anaerobic box, 30 DEG C of lucifuges are cultivated.(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, measure the absorbancy of bacterium liquid at 600nm place.With 0.22 μm of membrane filtration sample, with SO in ion chromatography sample
4 2-concentration.
SO in clostridium (Clostridium) taihuN5 growth curve and substratum
4 2-as shown in Figure 4, postvaccinal 0-30 hour is lag period to concentration curve, within 30-60 hour, is logarithmic phase, and sulfate concentration reduces, and the sulfate concentration in the wild Oryza species of logarithmic phase has almost no change.After cultivating 100h, bacterium liquid absorbancy declines, and arrives SO at the end of cultivating
4 2-concentration minimizing is about 10%.
Embodiment 3: bacterial strain of the present invention is to nitrate tolerance.
(1) clostridium (Clostridium) taihuN5 is containing 1g/LNaNO
3growth in substratum.
In anaerobism pipe, add 9.8ml substratum, clostridium (Clostridium) the taihuN5 bacterium liquid of inoculation 0.2ml logarithmic phase, in anaerobic box, 30 DEG C of lucifuges are cultivated.(culture medium prescription is for adding 1g/LNaNO
3concentration and separation substratum), respectively at postvaccinal 0,10,15,20,25,30,35,40,45,50,60,70h, 80h, 90h, 100h sampling, measure the absorbancy of bacterium liquid at 600nm place.With 0.22 μm of membrane filtration sample, with SO in ion chromatography sample
4 2-concentration.
Clostridium (Clostridium) taihuN5 is at 1g/LNaNO
3sO in growth curve under condition and substratum
4 2-as shown in Figure 5,80h before cultivation, growing state is basic and do not add NaNO for concentration curve
3growth curve is consistent, but phase bacterium liquid absorbancy does not obviously decline after incubation, but still remains on maximum absorbance, sulfate concentration continues to reduce at whole incubation period, illustrate that the nitrate added in substratum does not suppress the growth of taihuN5, facilitate 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 concentration and separation substratum, add NaNO
3, make NaNO in substratum
3final concentration be 0,30,70,100mM/L, in anaerobism pipe, add 9.8ml substratum, clostridium (Clostridium) the taihuN5 bacterium liquid of inoculation 0.2ml logarithmic phase, in anaerobic box, 30 DEG C of lucifuges are cultivated.Every 24h sampling, measure the absorbancy of bacterium liquid under wavelength 600nm in anaerobism pipe.
Clostridium (Clostridium) taihuN5 0,30,70,100mM/LNaNO
3under upgrowth situation as shown in Figure 6, add 30mM/L NaNO
3treatment group upgrowth situation basic be consistent without interpolation group, and 70 and 100mM/LNaNO
3the lag period for the treatment of group extends, but maximum absorbance value and other two groups are without significant difference, illustrate that clostridium (Clostridium) taihuN5 receives the impact of high concentration nitrate at early growth period, but the impact of nitrate on it can be eliminated completely by the Metabolism regulation of self, therefore think that clostridium (Clostridium) taihuN5 is the bacterial strain that a strain can tolerate nitrate, for having good application prospect in sulphate reducing bacteria process waster water process.
Embodiment 4: the amplification of clostridium (Clostridium) taihuN5 sulfite reductase (DSR) gene and order-checking.
With the STb gene of the extraction bacterium of extracting for template, and use 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, primer each 0.25 μ l, template dna 0.25 μ l, rTaq 0.0625 μ l, ddH2O 6.9375 μ l.PCR condition: 94 DEG C of 4min; 94 DEG C of 30s, 53 DEG C of 50s, 72 DEG C of 1.5min, 30 circulations; 72 DEG C of 10min.Do electrophoresis detection by the PCR primer of 1% sepharose to 16S rDNA, find length 1900bp fragment (Fig. 7) to carry out rubber tapping and reclaim, be TA clone to after PCR primer purifying with PMD19-T carrier, after the sub-empirical tests of picking positive colony, entrust Hua Da gene sequencing.The sequence (as shown in SEQ ID No.2) that the DSR length of clostridium (Clostridium) taihuN5 is 1992bp is obtained after order-checking, Blast retrieval analysis is used 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 region 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-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 reduced by a series of electronic transfer process and form S
2-.DSR is extensively present in various sulphate reducing bacteria, and DSR gene is quite conservative in the SRB of same monoid, in Different groups SRB, but have bigger difference, can as the index of the classification of SRB and evolution.
Embodiment 5: the application of clostridium (Clostridium) taihuN5 in process metallic wastewater.
Clostridium (Clostridium) the taihuN5 strain inoculation of cryopreservation is activated in the anaerobism pipe that concentration and separation liquid nutrient medium is housed, by the strain inoculation after activation to enlarged culturing in 500ml concentration and separation liquid nutrient medium, logarithmic phase to be grown to, what the bacterium liquid of enlarged culturing is inoculated in preparation contains Zn
2+in artificial wastewater, add isopyknic concentration and separation substratum, process metallic wastewater (every 3 days supplemented mediums) is cultivated in 30 DEG C of sealings simultaneously.Zn in this water
2+concentration 50mg/L, SO
4 2-concentration 800mg/L, the H of clostridium (Clostridium) taihuN5 generation after treatment
2s makes the Zn in solution
2+form 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 (1)
1. the application of sulphate reducing bacteria in heavy metal containing wastewater treatment of a strain nitrate resisting;
Described waste water is the heavy metal wastewater thereby containing nitrate and vitriol;
Described heavy metal is Cu
2+, Zn
2+, Ni
2+, Pb
2+and Cd
2+in any one or a few;
The sulphate reducing bacteria of described nitrate resisting, its Classification And Nomenclature is clostridium (Clostridium sp.) taihuN5, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on November 23rd, 2012, and deposit number is CGMCC No.6887;
Wherein, described nitrate concentration is 100mmol/L.
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| CN105112543B (en) * | 2015-09-22 | 2017-07-07 | 北京世纪金道石油技术开发有限公司 | A kind of molecular detecting method of sulfate reducing bacteria |
| CN107287129B (en) * | 2016-04-01 | 2021-05-28 | 兰州大学 | Sulfate reducing bacteria capable of settling heavy metals and application thereof |
| CN107699519B (en) * | 2017-10-18 | 2020-10-02 | 三峡大学 | Sulfate reducing bacteria, separation and identification method and application thereof |
| WO2020029149A1 (en) * | 2018-08-09 | 2020-02-13 | 中国石油大学(北京) | Bacterial strain having very strong sulfate reduction ability and use thereof |
| CN114437987B (en) * | 2022-02-28 | 2023-07-21 | 中国科学院地理科学与资源研究所 | Sulfate reducing bacteria and application thereof |
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| 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 |
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| Genbank:JX847660;Liu Y;《Genbank》;20120930;全文 * |
| 硫酸根和硝酸根对ZVI-SRB处理铀废水的影响研究;胡凯光等;《矿业工程研究》;20100930;第25卷(第3期);全文 * |
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