CN105368838A - Microlunatus phosphovorus engineering strain capable of efficiently accumulating polyphosphate and application thereof - Google Patents
Microlunatus phosphovorus engineering strain capable of efficiently accumulating polyphosphate and application thereof Download PDFInfo
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Abstract
The invention discloses a microlunatus phosphovorus engineering strain capable of efficiently accumulating polyphosphate. The microlunatus phosphovorus engineering strain is microlunatus phosphovorus JN459-M571 and is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 11770 on December 1, 2015. A continuous error-prone PCR method is adopted to mutate a promoter of a ppk2 gene in vitro, JN459 is used as a starting strain and a strain library containing the mutant of the promoter of the ppk2 gene is established through a gene recombination technology, and the JN459-M571 strain, of which, the capacity of decomposing polyphosphate under anaerobic conditions is reduced and the capacity of synthesizing polyphosphate under aerobic conditions has no significant difference, is obtained through screening. The Poly-P (polyphosphate) content of the strain disclosed by the invention is 2.4 times higher than that of an original strain JN459 in an anaerobic operation process and the strain disclosed by the invention can be applied to an EBPR (Enhanced biological phosphorus removal) process in a sewage treatment plant.
Description
Technical field
The invention belongs to Microbial Breeding and biological technical field, be specifically related to long-pending phosphorus solar month of 30 days bacterium engineering bacteria and the application thereof of a strain efficient accumulation polyphosphoric acid salt.
Background technology
Current body eutrophication problem is quite serious, and research confirms that phosphorus is one of key factor causing body eutrophication, and therefore the phosphorus element content reduced in sewage disposal plant effluent is the important measures controlling body eutrophication.Current sewage work generally adopts enhancement type Biological Phosphorus Removal System (Enhancebiologicalphosphorusremoval, EBPR) as first-selected biological phosphate-eliminating technology.The realization of EBPR biological phosphorus removal functional depends on the poly-phosphorus effect of poly-phosphorus microorganism in active sludge, poly-phosphorus microorganism is the microorganism general name that a class can synthesize polyphosphoric acid salt, has titanium pigment in excessive consumption water body and is stored in the feature of thalline with polyphosphoric acid salt form.
Amass the representative that phosphorus solar month of 30 days bacterium (Microlunatusphosphovorus) is poly-phosphorus microorganism, the polyphosphoric acid salt (Poly-P) of its accumulation can reach more than 10% of dry cell weight, in EBPR system, have good phosphor-removing effect.But it is the same with other poly-phosphorus microorganisms, long-pending phosphorus solar month of 30 days there is the circulation of a synthesis Sum decomposition polyphosphoric acid salt in bacterium, namely in aerobic, synthesize polyphosphoric acid salt, decompose polyphosphoric acid salt when anaerobism to obtain energy, maintain thalline to the demand of GTP and ATP.Long-pending phosphorus solar month of 30 days bacterium its effect in EBPR of this properties influence, particularly when the emission standard for water outlet total phosphorous being caused can not to reach legal during hypoxgia appears in EBPR system.Therefore, develop a kind of long-pending phosphorus solar month of 30 days bacterium of under anaerobic decomposing that polyphosphoric acid salt ability significantly reduces and there is very important actual application value.
Polyphosphate kinase PPK2 is the key enzyme that reversible reaction between phosphorus solar month of 30 days bacterium Poly-P and ATP/GTP is amassed in catalysis.Under aerobic condition, cellular energy is sufficient, and titanium pigment is catalyzed and synthesized Poly-P by PPK2 under the driving of ATP/GTP, and under anaerobic condition, PPK2 catalysis Poly-P is decomposed to form ATP/GTP, maintains the Nucleotide storehouse balance of cell, meets the normal growth demand of cell.The special role of PPK2 determines and the mode of inactivation ppk2 gene can not be adopted to transform long-pending phosphorus solar month of 30 days bacterium, also not yet has at present and obtains about by transformation ppk2 gene promoter the report that high-performance amasss phosphorus solar month of 30 days bacterium.
Summary of the invention
For above-mentioned prior art, the object of this invention is to provide long-pending phosphorus solar month of 30 days bacterium engineering bacteria and the application thereof of a strain efficient accumulation polyphosphoric acid salt.By the promotor of transformation ppk2 gene, weaken the activation of hypoxia condition to ppk2 gene, reduce the expression level of ppk2, reduce PPK2 to the decomposition of Poly-P, the final performance improving long-pending phosphorus solar month of 30 days bacterium accumulation Poly-P.
For achieving the above object, the present invention adopts following technical scheme:
One aspect of the present invention relates to a kind of ppk2 gene promoter through directional transformation, and it has the nucleotide sequence as shown in SEQIDNO.1, or the nucleotide sequence with same function that this sequence is formed through replacing, lacking or add one or several base.
Another aspect of the present invention relates to this promotor and reduces application in ppk2 expression level under low oxygen conditions.
The one that also relates in one aspect to of the present invention amasss phosphorus solar month of 30 days bacterium, and it contains the above-mentioned ppk2 gene promoter through directional transformation.
Another aspect of the invention relates to the long-pending phosphorus solar month of 30 days bacterium engineering bacteria of a strain efficient accumulation polyphosphoric acid salt, and this bacterial strain is specially long-pending phosphorus solar month of 30 days bacterium (Microlunatusphosphovorus) JN459-M571.Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 1st, 2015 and (be called for short CGMCC, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number is CGMCCNO.11770.
Compared with long-pending phosphorus solar month of 30 days bacterium (Microlunatusphosphovorus) JN459-M571 of the present invention amasss phosphorus solar month of 30 days bacterium JN459 with wild-type, ppk2 gene promoter has the change of 3 bases, is that-46 ,-76 and-79 bit bases all become A by G respectively.
Another object of the present invention is to provide a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is long-pending phosphorus solar month of 30 days bacterium (Microlunatusphosphovorus) JN459-M571.
Described long-pending phosphorus solar month of 30 days bacterium (Microlunatusphosphovorus) JN459-M571 and/or described microbial inoculum also belong to protection scope of the present invention preparing the application in biological phosphate-eliminating agent.
A present invention also object is to provide a pair primer for the fallibility PCR of directional transformation (i.e. external sudden change) ppk2 gene promoter, and its primer sequence is respectively as shown in SEQ ID NO.2 and SEQIDNO.3.
Another object of the present invention is to provide the preparation method of this long-pending phosphorus solar month of 30 days bacterium (Microlunatusphosphovorus) JN459-M571, the external promotor to ppk2 gene of continuous error-prone PCR method is adopted to suddenly change, with existing long-pending phosphorus solar month of 30 days bacterium JN459 for the bacterium that sets out, the strain storehouse containing ppk2 transgenation promotor is set up by homologous recombination technique, measure change that is aerobic and Poly-P content under anaerobism operational conditions, screening obtains high-performance and amasss phosphorus solar month of 30 days bacterium JN459-M571.
Upstream primer ppk2ProForward sequence: GTCGAAATGTCGGAAGCCGTGC (SEQIDNO.2);
Downstream primer ppk2ProReverse sequence: TAACCCATTGTTCGCCCGGCAG (SEQIDNO.3).
The concrete grammar of described continuous error-prone PCR method external sudden change ppk2 gene promoter is:
(1) PCR reaction system: template DNA 2ul, PCR damping fluid 5ul, Taq DNA polymerase 1ul, dATP1 ~ 3ul, dTTP1 ~ 3ul, dGTP1 ~ 3ul, dCTP1 ~ 3ul, upstream primer 2ul, downstream primer 2ul, 25mMMgCl
21 ~ 8ul, 5mMMnCl
20 ~ 1ul, adds ultrapure water to 50ul.
(2) PCR condition: denaturation 95 DEG C 5 minutes, sex change 95 DEG C 30 seconds, anneal 52 ~ 60 DEG C 30 seconds, extend 72 DEG C 2 minutes, circulate 30 times, extend 8 minutes eventually.
The template DNA of the 1st PCR derives from long-pending phosphorus solar month of 30 days bacterium JN459, and the template DNA of the 2nd PCR and follow-up PCR adopts PCR primer last time.
The construction process of the described strain storehouse containing ppk2 transgenation promotor, concrete steps are as follows:
(1) the shuttle vectors pMV306K carrying ppk2 gene upstream and downstream homology arm is first built, then PCR primer is reclaimed by after continuous error-prone PCR product Agar Gel sugar electrophoretic separation, under GibsonCloningMasterMix effect, vitro recombination is carried out with above-mentioned shuttle vectors pMV306K, transformation of E. coli DH5a, picking transformant extracts plasmid and carries out double digestion qualification;
(2) adopt electroporated technology that restructuring pMV306K plasmid is proceeded to JN459 bacterial strain, under homologous recombination effect, obtain a series of transformant, build the strain storehouse containing ppk2 gene promoter mutant.
Beneficial effect of the present invention:
(1) the present invention adopts the mode of orthomutation to screen the mutant of Polyphosphate kinase ppk2 gene promoter first, amass phosphorus solar month of 30 days bacterium JN459-M571 by detection is aerobic with Poly-P content acquisition high-performance under anaerobism operational conditions simultaneously, after its anaerobic condition runs 24 hours, Poly-P content is than wild-type bacteria plant height 2.4 times, can be applicable to the EBPR Biological Phosphorus Removal Processes of sewage work, there is important economic worth and social value.
(2) the present invention adopts the promoter sequence of continuous error-prone PCR method external sudden change Polyphosphate kinase ppk2, reduce the activation of hypoxia condition to ppk2 gene, reduce the expression level of ppk2, decrease the decomposition of PPK2 to Poly-P, ultimately improve the performance of long-pending phosphorus solar month of 30 days bacterium accumulation Poly-P.
(3) long-pending phosphorus solar month of 30 days bacterium JN459-M571 of the present invention under anaerobic Poly-P decomposition rate significantly reduce, and synthesize Poly-P performance under aerobic condition without significant difference.
(4) preparation method of long-pending phosphorus solar month of 30 days bacterium engineering bacteria of the present invention, the efficiency of sudden change work is high, and specific aim is very strong, reduces randomness and the blindness of selection by mutation, meets the breeding needs of microbial strains.
Accompanying drawing explanation
Fig. 1: the removal effect to titanium pigment in sewage under aerobic and anaerobism operational conditions;
Fig. 2: Poly-P content under aerobic and anaerobism operational conditions.
Embodiment
The present invention is further illustrated in conjunction with the embodiments, should be noted that following explanation is only to explain the present invention, not limiting its content.
Embodiment 1: continuous error-prone PCR obtains the ppk2 promotor containing mutating alkali yl
Continuous error-prone PCR is adopted to suddenly change to the promotor of ppk2 gene in vitro.Be specially:
1. the primer of fallibility PCR is:
Upstream primer ppk2ProForward sequence: GTCGAAATGTCGGAAGCCGTGC (SEQIDNO.2);
Downstream primer ppk2ProReverse sequence: TAACCCATTGTTCGCCCGGCAG (SEQIDNO.3).
(1) PCR reaction system: template DNA 2ul, PCR damping fluid 5ul, Taq DNA polymerase 1ul, dATP1 ~ 3ul, dTTP1 ~ 3ul, dGTP1 ~ 3ul, dCTP1 ~ 3ul, upstream primer 2ul, downstream primer 2ul, 25mMMgCl
21 ~ 8ul, 5mMMnCl
20 ~ 1ul, adds ultrapure water to 50ul.
(2) PCR condition: denaturation 95 DEG C 5 minutes, sex change 95 DEG C 30 seconds, anneal 52 ~ 60 DEG C 30 seconds, extend 72 DEG C 2 minutes, circulate 30 times, extend 8 minutes eventually.
The template DNA of the 1st PCR derives from long-pending phosphorus solar month of 30 days bacterium JN459, and the template DNA of the 2nd PCR and follow-up PCR adopts PCR primer last time.
PCR primer directly send order-checking, and result shows: as dATP3ul, dTTP3ul, dGTP1ul, dCTP1ul, upstream primer 2ul, downstream primer 2ul, 25mMMgCl in PCR system
23ul, 5mMMnCl
20.5ul, PCR annealing temperature is 56 DEG C, and during 3 consecutive PCRs, base mismatch number is 3 ~ 6, and mutation frequency is relatively applicable to carrying out directional transformation.
Embodiment 2: the mutant library of long-pending phosphorus solar month of 30 days bacterium ppk2 gene promoter builds
Build the shuttle vectors pMV306K carrying ppk2 gene upstream and downstream homology arm, PCR primer is reclaimed after being separated by continuous error-prone PCR product agarose gel electrophoresis in embodiment 1, under GibsonCloningMasterMix effect, vitro recombination is carried out with above-mentioned shuttle vectors pMV306K, transformation of E. coli DH5a, picking transformant extracts plasmid and carries out double digestion qualification (pMV306K is conventional plasmid of the prior art).
By following formulated liquid nutrient medium: glucose 0.5g, peptone 0.5g, yeast powder 0.5g, Sodium Glutamate 0.5g, ammonium sulfate 0.1g, dipotassium hydrogen phosphate 0.44g, magnesium sulfate 0.1g, purified water 1L, pH7.0,0.15MPa sterilizing 30min.The bottled 50ml liquid nutrient medium of every 250ml triangle.
Phosphorus solar month of 30 days bacterium JN459 inoculation will be amassed to aforesaid liquid substratum, 25 DEG C, 200rpm shaking table is cultured to logarithmic phase, collected by centrifugation thalline, and wash 3 times with 4 DEG C of 10% glycerine, be finally diluted to 10 with 4 DEG C of 10% glycerine
8individual/ml, as electroporated competent cell.
Electroporated technology is adopted restructuring pMV306K plasmid to be proceeded to long-pending phosphorus solar month of 30 days bacterium JN459 bacterial strain, a series of transformant is obtained under homologous recombination effect, build the strain storehouse containing ppk2 gene promoter mutant, altogether acquisition 1035 recombinant bacterial strains, numbering JN459-M1 to JN459-M1035.
The gene order of above-mentioned ppk2 gene and upstream and downstream thereof and gene location are well known in the prior art, be documented in KawakoshiA., NakazawaH., FukadaJ., SasagawaM., KatanoY., NakamuraS., HosoyamaA., SasakiH., IchikawaN., HanadaS., KamagataY., NakamuraK., YamazakiS., FujitaN.DecipheringthegenomeofpolyphosphateaccumulatingA ctinobacteriumMicrolunatusphosphovorus.DNAResearch.2012,19:383-394.The method that the structure of recombinant shuttle vector pMV306K adopts is general genetic engineering technique (as reference " Principles of Gene Engineering and technology ", Zou Keqin, press of Zhejiang University), is the routine techniques means of prior art.
Long-pending phosphorus solar month of 30 days bacterium JN459 bacterial strain is documented in document " separation of polyP bacteria JN459 and poly-phosphorus characteristic research. ", Zhong Chuanqing, Jiang Tianyi, Wang Jing, Zhang Chunming. Journal of Shandong Jianzhu University .201530 (1): in 25-28.
Embodiment 3: the poly-phosphorus screening active ingredients of mutant strain
Recombinant bacterial strain embodiment 2 built above-mentioned liquid nutrient medium (embodiment 2) is cultured to logarithmic phase, collected by centrifugation thalline, and washs with sterilized water, be diluted to 10
8individual/ml, is inoculated into new aforesaid liquid substratum by 5% inoculum size, and each recombinant bacterial strain inoculates a 50ml/250ml shaking flask.25 DEG C, 200rpm shaking table cultivates 48 hours, draws 10ml bacterium liquid, the centrifugal 10min of 3000rpm collects thalline, measures Poly-P content.Remaining nutrient solution air in sterile nitrogen displacement shaking flask, then seals bottleneck, 25 DEG C, 200rpm shaking table continues cultivation 24 hours, and draw 10ml bacterium liquid, the centrifugal 10min of 3000rpm collects thalline, measures Poly-P content.The results are shown in Table 1.
Result show 17 plant mutant strains anaerobism after 24 hours Poly-P content be significantly higher than starting strain JN459, wherein the Poly-P content of JN459-M571 mutant bacteria is the highest, reach 8.62%, illustrate these mutant strains under anaerobic decomposed P oly-P ability decline.
When table 1 is aerobic and anaerobism is run, Poly-P accounts for dry cell weight (%)
Embodiment 4: the genetic stability of long-pending phosphorus solar month of 30 days bacterium JN459-M571 bacterial strain is investigated
By following formulated solid medium: glucose 0.5g, peptone 0.5g, yeast powder 0.5g, Sodium Glutamate 0.5g, ammonium sulfate 0.1g, dipotassium hydrogen phosphate 0.44g, magnesium sulfate 0.1g, agar powder 20g, purified water 1L, pH7.0,0.15MPa sterilizing 30min.
By JN459-M571 strain suspensions dilution spread on solid medium 25 DEG C cultivate 7 days, then be separated single bacterium colony of JN459-M571 and carry out enlarged culturing, mode continuous passage is cultivated and is separated monospore six times according to this, and gather phosphorus activity according to the method validation of embodiment 3, the results are shown in Table 2, result shows that JN459-M571 bacterial strain has good genetic stability, meets the demand of EBPR Biological Phosphorus Removal System.
The poly-phosphorus expression activitiy of the different passage number of table 2
The determined dna sequence of embodiment 5:ppk2 gene promoter mutant
Extract the genomic dna of engineering bacteria JN459-M571 as template, with ppk2ProForward and ppk2ProReverse for primer carries out high-fidelity PCR amplification, PCR primer is separated through agarose gel electrophoresis and reclaims, be connected transformation of E. coli DH5a with pCR-Blunt carrier, the several positive colony of picking carries out DNA sequencing.In JN459-M571, the DNA sequence dna of ppk2 promotor is as shown in SEQ ID NO.1, and in JN459, the DNA sequence dna of ppk2 promotor is as shown in SEQ ID NO.4.Result shows, and in JN459-M571, ppk2 gene promoter has the change of 3 bases, is that-46 ,-76 and-79 bit bases become A by G respectively.
Bacterial strain JN459-M571 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 1st, 2015 and (is called for short CGMCC, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number is CGMCCNO.11770.
Embodiment 6:JN459-M571 verifies the removal effect of titanium pigment in sewage
By following ingredients preparation synthetic sewage: glucose 0.3g, peptone 0.1g, yeast powder 0.01g, sodium-acetate 0.15g, ammonium chloride 0.2g, sodium-chlor 0.05g, dipotassium hydrogen phosphate 0.1g, magnesium sulfate 0.12g, purified water 1L.Actual measurement NH
4 +-N is 65.1mg/L, PO
4 3--P is 14.2mg/L.
JN459-M571 strain liquid is cultured to logarithmic phase, collected by centrifugation thalline, and with sterilized water washing, is then inoculated in 10LSBR reactor.Adopt aerobic and Anoxic technique, in each working cycle, aerobic time is 12h, dissolved oxygen concentration is about 4mg/L, hypoxic exposure is 12h, with sterile nitrogen displacement system hollow gas, maintain dissolved oxygen concentration lower than 0.2mg/L, whole technique maintains about 3d, sampling detects titanium pigment content in aqueous phase, and result as shown in Figure 1; Run Poly-P content in late detection thalline, with JN459 bacterial strain for contrast, result as shown in Figure 2.Operation result show, JN459-M571 bacterial strain when anaerobism is run titanium pigment ascensional range between 7.7%-10.1%, significantly lower than JN459 bacterial strain (between 23.9%-31.3%).Simultaneously JN459-M571 bacterial strain reaches 79.8% at the titanium pigment clearance of 72 hours, is significantly higher than 59.8% clearance of JN459 bacterial strain; In cell, Poly-P content reaches 8.6% of dry cell weight, also higher than 4.3% of N459 bacterial strain.
Claims (10)
1. through a ppk2 gene promoter for directional transformation, it is characterized in that, it has the nucleotide sequence as shown in SEQIDNO.1, or the nucleotide sequence with same function that this sequence is formed through replacing, lacking or add one or several base.
2. promotor according to claim 1 reduces the application of ppk2 expression level under low oxygen conditions.
3. long-pending phosphorus solar month of 30 days a bacterium, it contains the ppk2 gene promoter through directional transformation according to claim 1.
4. the long-pending phosphorus solar month of 30 days bacterium engineering bacteria of a strain efficient accumulation polyphosphoric acid salt, this bacterial strain is long-pending phosphorus solar month of 30 days bacterium (Microlunatusphosphovorus) JN459-M571, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 1st, 2015, deposit number is CGMCCNO.11770.
5. a microbial inoculum, is characterized in that, the activeconstituents of this microbial inoculum is long-pending phosphorus solar month of 30 days bacterium JN459-M571 according to claim 4.
6. amass microbial inoculum described in phosphorus solar month of 30 days bacterium JN459-M571 and/or claim 5 described in claim 4 and prepare the application in biological phosphate-eliminating agent.
7. for the primer of the fallibility PCR of ppk2 gene promoter described in directional transformation claim 1, it is characterized in that, its primer sequence is respectively as shown in SEQIDNO.2 and SEQIDNO.3.
8. the preparation method of long-pending phosphorus solar month of 30 days bacterium JN459-M571 according to claim 4, it is characterized in that, the external promotor to ppk2 gene of continuous error-prone PCR method is adopted to suddenly change, with existing long-pending phosphorus solar month of 30 days bacterium JN459 for the bacterium that sets out, the strain storehouse containing ppk2 transgenation promotor is set up by homologous recombination technique, measure change that is aerobic and Poly-P phosphate content under anaerobism operational conditions, screening obtains high-performance and amasss phosphorus solar month of 30 days bacterium JN459-M571.
9. preparation method as claimed in claim 8, is characterized in that, the method for described continuous error-prone PCR method external sudden change ppk2 gene promoter is:
(1) PCR reaction system: template DNA 2ul, PCR damping fluid 5ul, Taq DNA polymerase 1ul, dATP1 ~ 3ul, dTTP1 ~ 3ul, dGTP1 ~ 3ul, dCTP1 ~ 3ul, upstream primer 2ul, downstream primer 2ul, 25mMMgCl
21 ~ 8ul, 5mMMnCl
20 ~ 1ul, adds ultrapure water to 50ul;
(2) PCR condition: denaturation 95 DEG C 5 minutes, sex change 95 DEG C 30 seconds, anneal 52 ~ 60 DEG C 30 seconds, extend 72 DEG C 2 minutes, circulate 30 times, extend 8 minutes eventually;
The template DNA of the 1st PCR derives from long-pending phosphorus solar month of 30 days bacterium JN459, and the template DNA of the 2nd PCR and follow-up PCR adopts PCR primer last time;
Described upstream primer and downstream primer are the primer of fallibility PCR according to claim 7.
10. preparation method as claimed in claim 8, is characterized in that, the construction process of the described strain storehouse containing ppk2 transgenation promotor is:
(1) the shuttle vectors pMV306K carrying ppk2 gene upstream and downstream homology arm is built, PCR primer is reclaimed after being separated by continuous error-prone PCR product agarose gel electrophoresis, under GibsonCloningMasterMix effect, vitro recombination is carried out with the shuttle vectors pMV306K carrying ppk2 gene upstream and downstream homology arm, transformation of E. coli DH5a, picking transformant extracts plasmid and carries out double digestion qualification;
(2) restructuring pMV306K plasmid is proceeded to JN459 bacterial strain, under homologous recombination effect, obtain a series of transformant, namely build the strain storehouse obtained containing ppk2 gene promoter mutant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510969862.XA CN105368838B (en) | 2015-12-21 | 2015-12-21 | The product phosphorus solar month of 30 days bacterium engineering bacteria of one plant of efficient accumulation polyphosphate and its application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510969862.XA CN105368838B (en) | 2015-12-21 | 2015-12-21 | The product phosphorus solar month of 30 days bacterium engineering bacteria of one plant of efficient accumulation polyphosphate and its application |
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| Publication Number | Publication Date |
|---|---|
| CN105368838A true CN105368838A (en) | 2016-03-02 |
| CN105368838B CN105368838B (en) | 2018-04-13 |
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Cited By (7)
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| CN106479928A (en) * | 2016-11-02 | 2017-03-08 | 中国科学院微生物研究所 | The resistance to high COD salt solution meningitidis strains of one plant of resistance to high salt and the indigenous plasmid of the bacterial strain of originating |
| CN107162188A (en) * | 2017-05-18 | 2017-09-15 | 北京工业大学 | The apparatus and method of the synchronous enhanced biological phosphorus removal of integrated autotrophic denitrification |
| CN110904069A (en) * | 2019-12-04 | 2020-03-24 | 天津市职业大学 | PPK2 protein and application thereof in polyacrylamide gel electrophoresis 35kd standard substance |
| CN111170471A (en) * | 2020-01-16 | 2020-05-19 | 浙江永续环境工程有限公司 | Composite phosphorus-accumulating bacteria flowing biological bed membrane method |
| CN112322664A (en) * | 2020-11-02 | 2021-02-05 | 江苏南创化学与生命健康研究院有限公司 | Method for producing polyphosphate with high polymerization degree |
| CN112779021A (en) * | 2021-02-03 | 2021-05-11 | 中南大学 | Phosphorus-containing heavy metal contaminated soil remediation material and preparation method and application thereof |
| CN114561560A (en) * | 2022-03-02 | 2022-05-31 | 广西惟邦环境科技有限公司 | Dephosphorization treatment method for extracting high-grade and low-grade manganese ores |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479928A (en) * | 2016-11-02 | 2017-03-08 | 中国科学院微生物研究所 | The resistance to high COD salt solution meningitidis strains of one plant of resistance to high salt and the indigenous plasmid of the bacterial strain of originating |
| CN106479928B (en) * | 2016-11-02 | 2019-10-11 | 中国科学院微生物研究所 | The indigenous plasmid of one plant of resistance to resistance to high COD salt water meningitidis strains and the source bacterial strain with high salt |
| CN107162188A (en) * | 2017-05-18 | 2017-09-15 | 北京工业大学 | The apparatus and method of the synchronous enhanced biological phosphorus removal of integrated autotrophic denitrification |
| CN107162188B (en) * | 2017-05-18 | 2020-07-03 | 北京工业大学 | Device and method for integrated autotrophic nitrogen removal and synchronous reinforcement of biological phosphorus removal |
| CN110904069A (en) * | 2019-12-04 | 2020-03-24 | 天津市职业大学 | PPK2 protein and application thereof in polyacrylamide gel electrophoresis 35kd standard substance |
| CN111170471A (en) * | 2020-01-16 | 2020-05-19 | 浙江永续环境工程有限公司 | Composite phosphorus-accumulating bacteria flowing biological bed membrane method |
| CN112322664A (en) * | 2020-11-02 | 2021-02-05 | 江苏南创化学与生命健康研究院有限公司 | Method for producing polyphosphate with high polymerization degree |
| CN112322664B (en) * | 2020-11-02 | 2021-07-27 | 江苏南创化学与生命健康研究院有限公司 | Method for producing polyphosphate with high polymerization degree |
| CN112779021A (en) * | 2021-02-03 | 2021-05-11 | 中南大学 | Phosphorus-containing heavy metal contaminated soil remediation material and preparation method and application thereof |
| CN112779021B (en) * | 2021-02-03 | 2021-10-15 | 中南大学 | Phosphorus-containing heavy metal contaminated soil remediation material and preparation method and application thereof |
| CN114561560A (en) * | 2022-03-02 | 2022-05-31 | 广西惟邦环境科技有限公司 | Dephosphorization treatment method for extracting high-grade and low-grade manganese ores |
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