CN109913387A - Degrade N-Methyl pyrrolidone enterobacteria and application in the treatment of waste water - Google Patents
Degrade N-Methyl pyrrolidone enterobacteria and application in the treatment of waste water Download PDFInfo
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- CN109913387A CN109913387A CN201910209582.7A CN201910209582A CN109913387A CN 109913387 A CN109913387 A CN 109913387A CN 201910209582 A CN201910209582 A CN 201910209582A CN 109913387 A CN109913387 A CN 109913387A
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Abstract
The invention discloses the enterobacteria of one plant of N-Methyl pyrrolidone of degrading and applications in the treatment of waste water.The present invention is using activated sludge as bacterium source, it is the minimal medium of sole carbon source as screening and culturing medium using N-Methyl pyrrolidone, isolate and purify to obtain one plant using N-Methyl pyrrolidone as the denitrifying enterobacteria of electron donor progress, deposit number is CCTCC NO:M2019128.Enterobacteria of the invention can carry out anoxic denitrification reaction by sole electron donor of N-Methyl pyrrolidone, the synchronous mineralizing and degrading for realizing N-Methyl pyrrolidone, nitrate nitrogen and N-Methyl pyrrolidone are realized in 15 hours and 18 hours respectively to be completely removed, with efficient organic matter degradation ability and denitrifying capacity, the removal suitable for Coal Gas Washing Cycling Water nitrate nitrogen and persistent organic pollutants is handled.
Description
Technical field
The invention belongs to environmental organic pollutant biologic treating technique fields, are related to one plant of degradation N-Methyl pyrrolidone
Enterobacteria, and in particular to one plant carries out denitrifying intestines bar by electron donor of persistent organic pollutants N-Methyl pyrrolidone
Bacterium and its application in the wastewater treatment containing N-Methyl pyrrolidone.
Background technique
N-Methyl pyrrolidone (N-methylpyrrolidone, NMP), as a kind of typical organic polar solvent, by
In its good water miscibility, low volatility, it is highly polar and non-corrosive the features such as, be widely used in coating, adhesive, combustion
In the industries such as material and drug manufacture.N-Methyl pyrrolidone stable structure, facile hydrolysis, does not turn in soil and between underground water easily
It moves, there is bio-toxicity and teratogenesis characteristic.It is reported that having more than 2400 tons of N-Methyl pyrrolidone every year passes through industrial wastewater
It is discharged into environment.Therefore, the water area ecological environment polluted by N-Methyl pyrrolidone is repaired, it has also become environmental improvement at present
One of the important topic in field.
Currently, the processing method of the waste water containing N-Methyl pyrrolidone include the advanced oxidation processes such as photocatalytic method, ozone oxidation,
Membrane separation process, biological treatment etc..Wherein, the physico-chemical process such as advanced oxidation and UF membrane processing contains N-Methyl pyrrolidone
Waste water higher cost, secondary pollution are serious.Biologic treating technique has many advantages, such as economic, efficient, secondary pollution is small, can be real
Existing innoxious governance, is most widely used wastewater processing technology.But due to the toxicity of N-Methyl pyrrolidone and spy difficult to degrade
Property, need searching to be resistant to N-Methyl pyrrolidone bio-toxicity, the bacterial strain with degradation function.
Due to the toxicity and characteristic difficult to degrade of N-Methyl pyrrolidone, N-Methyl pyrrolidone pollutes the premise of biological treatment
It is to obtain the special bacterial strain for being resistant to N-Methyl pyrrolidone bio-toxicity and being able to achieve N-Methyl pyrrolidone mineralizing and degrading.
Kr í zek et al. reports pseudomonad, secondary coccus, acinetobacter calcoaceticus and the degradable N-Methyl pyrrolidone of Rhodococcus sp synergistic effect,
Degradation rate is 4.17mg L-1h-1, degradation cycle is 72 hours (Kr í zek, K., et al.N-methyl-2-
pyrrolidone-degrading bacteria from activated sludge.Water Sci.Technol.2015,
71,776-782.).Cai Shu et al. reports the degradable N-Methyl pyrrolidone of secondary coccus, and degradation rate is 17.86mg L-1h-1, degradation cycle is 28 hours (Cai, S., et al.Biodegradation of N-methylpyrrolidone by
Paracoccus sp.NMD-4 and its degradation pathway.Int.Biodeter.Biodegr.2014,93,
70-77.).The bacterial strain of existing degradable N-Methyl pyrrolidone, not only degradation cycle is longer, degradation efficiency is not high, need to also be
It is cultivated under the conditions of oxygen consumption, considerably increases the process operations costs such as power consumption aeration in practical applications.In view of the pyrrole of methyl containing N-
The nitrate of higher concentration is usually contained in the industrial wastewater of pyrrolidone, as can obtaining can supply by electronics of N-Methyl pyrrolidone
Body, the high efficient strain that nitrate nitrogen is electron acceptor, realize the degradation of N-Methyl pyrrolidone while anoxic denitrification denitrogenation,
It will be to low cost and harmless treatment generation significance containing N-Methyl pyrrolidone and the industrial wastewater of nitrate nitrogen.
Summary of the invention
The purpose of the present invention is to provide one plant degrade N-Methyl pyrrolidone enterobacteria (Enterobacte) bacterial strain,
Headed by the bacterial strain strain using N-Methyl pyrrolidone as electron donor, nitrate nitrogen be electron acceptor carry out denitrification denitrogenation Black Liquor with Efficient Bacteria
The degradation of N-Methyl pyrrolidone is realized in strain while anoxic denitrification denitrogenation.
Inventor utilizes using the activated sludge for denitrification denitrogenation as bacterium source using N-Methyl pyrrolidone as sole carbon source
Screening and culturing medium, carry out the purifies and separates of bacterial strain, obtained one plant can use N-Methyl pyrrolidone be electron donor into
The denitrifying enterobacteria of row, is Enterobacter through molecular biology identification, is named as Enterobacter
sp.NJUST50.In on 03 06th, 2019 in Chinese Typical Representative object collection (CCTCC) preservation, preservation address is the bacterial strain
Wuhan University, Wuhan, China city, deposit number are CCTCC NO:M2019128.
The present invention also provides the cultural method of above-mentioned enterobacteria Enterobacter sp.NJUST50, specific steps are as follows: will
Enterobacter sp.NJUST50 is inoculated in the medium, and medium pH is 6~8, and cultivation temperature is 20 DEG C~35 DEG C.
Preferably, the culture medium also contains N-Methyl pyrrolidone and sodium nitrate, sodium nitrate and N- crassitude
The molar ratio of ketone is 1.0~2.0:1.0, and N-Methyl pyrrolidone concentration is 2800~3100mg L-1, sodium nitrate concentration 4800
~5300mg L-1。
The present invention also provides above-mentioned enterobacteria Enterobacter sp.NJUST50 useless containing N-Methyl pyrrolidone
Application in water process.
Further, the present invention also provides above-mentioned enterobacteria Enterobacter sp.NJUST50 to contain N- methyl pyrrole
Application in the lithium battery production wastewater treatment of pyrrolidone.
Specifically, above-mentioned enterobacteria Enterobacter sp.NJUST50 is containing N-Methyl pyrrolidone wastewater treatment
In application, method particularly includes: enterobacteria Enterobacter sp.NJUST50 seed liquor is inoculated into containing N- methylpyrrole
In the waste water of alkanone, anoxic culture, cultivation temperature is 20 DEG C~35 DEG C, and culture pH is 6~8.
Preferably, the inoculum concentration of the enterobacteria Enterobacter sp.NJUST50 seed liquor is 3%~10%.
Enterobacteria Enterobacter sp.NJUST50 provided by the invention can use N- methyl under anoxic conditions
Pyrrolidones is sole electron donor, and nitrate nitrogen is that sole electron acceptor is metabolized and is grown, while having efficient N- first
Base pyrrolidones degradation capability and denitrification denitrogenation ability.Compared to oxygen consumption condition, Enterobacter sp.NJUST50 is to life
The adaptability and tolerance of dis environment are stronger, are aerated workshop section applied to that can reduce oxygen consumption in the process of industrial wastewater,
Save economic cost.It is added in the practical lithium battery production waste water containing N-Methyl pyrrolidone after pretreatment
Enterobacter sp.NJUST50 is handled, and nitrate nitrogen and N-Methyl pyrrolidone are respectively in 15 hours and 18 hours
Realization completely removes.
Detailed description of the invention
Fig. 1 is the scanning electron microscope (SEM) photograph of enterobacteria Enterobacter sp.NJUST50.
It is 2800- that Fig. 2, which is enterobacteria Enterobacter sp.NJUST50 in N-Methyl pyrrolidone initial concentration,
3100mg L-1, nitrate nitrogen initial concentration be 790-870mg L-1Fluid nutrient medium in the degradation of N-Methyl pyrrolidone is imitated
Fruit and denitrification effect figure.
Fig. 3 is enterobacteria Enterobacter sp.NJUST50 after pretreatment while containing nitrate nitrogen and N- methyl pyrrole
Denitrification effect in the lithium battery production waste water of pyrrolidone and the degradation effect figure to N-Methyl pyrrolidone.
Specific embodiment
Below by specific embodiments and the drawings, the invention will be further described, makes those skilled in the art more fully
Understand the present invention, but do not limit the invention in any way.
Embodiment 1
The screening of Enterobacter sp.NJUST50 separates and identification.
(1) screening and separation of bacterial strain
5g is sampled from the existing activated sludge for denitrification denitrogenation, is added in 100mL physiological saline and stirs
Two hours are stood after even.In minimal medium after taking 1mL supernatant to be added to 121 DEG C of high-temperature sterilizations, 180 revs/min
Shaking table enrichment culture three days, after being continuously enriched with three times, take culture solution sterile water gradient dilution to 10-4-10-10Times.It prepares inorganic
20 μ L of culture solution after dilution is respectively coated on inorganic salts Solid agar culture, is placed in life by salt Solid agar culture
Change in incubator and cultivates three days for 30 DEG C.Select the single colonie on culture dish with notable difference, the side separated using plate streaking
Method carries out purifying culture, after continuous purification five times, obtains single bacterial strain, carries out inclined-plane preservation.It prepares and contains nitrate nitrogen and N- first
The inorganic salt liquid culture medium of base pyrrolidones is fitted into serum bottle, is aerated with pure helium to remove dissolved oxygen, inoculation point
From the bacterial strain that purifying obtains, 180 revs/min and 30 DEG C of the condition anoxic culture in isothermal vibration incubator monitors nitrate nitrogen
And the concentration variation of N-Methyl pyrrolidone.Choose the bacterium that can effectively remove nitrate nitrogen and N-Methyl pyrrolidone in culture medium
Strain, is named as NJUST50, is carried out inclined-plane and saved and -80 DEG C of cryo-conservations.
The composition of LB culture medium is as follows: tryptone (10g L-1), yeast extract (5g L-1), sodium chloride (10g L-1)。
The composition of minimal medium is as follows: NaHPO4·12H2O(1.53g L-1), KH2PO4(0.38g L-1), MgSO4
(0.1g L-1), CaCl2(0.05g L-1), trace element solution SL-4 (10mL).Microelement SL-4 composition: EDTA (0.5gL-1), FeSO4·7H2O(0.2g L-1), microelement SL-6 (100mL L-1).Microelement SL-6 composition: ZnSO4·7H2O
(0.01g L-1), MnCl2·4H2O(0.03g L-1), H3BO4(0.3g L-1), CoCl2·6H2O(0.2g L-1), CuCl2·
2H2O(0.01g L-1), NiCl2·6H2O(0.02g L-1), Na2MoO4·2H2O(0.03g L-1), N-Methyl pyrrolidone and
The amount of sodium nitrate needs to add according to experiment.
On the basis of liquid medium within be added 20g/L agar, in autoclave 121 DEG C of high pressure sterilizations after twenty minutes,
Acquisition inorganic salts Solid agar culture after being cooled to room temperature is poured into sterile petri dish.
(2) identification of bacterial strain
Morphology, Physiology and biochemistry test are carried out to bacterial strain.The 16S rRNA gene order for measuring bacterial strain, by the 16S of bacterial strain
Gene order in rRNA gene order and GenBank database carries out tetraploid rice and analyzes as a result, from molecular biology
The kind of the bacterium is determined in level.
(2.1) morphological feature: NJUST50 bacterium colony is at milky, and surface glossy clear, neat in edge is glossy, in liquid
Body culture medium intermediate range diffusivity is muddy.The strain cell is in the shape of a rod, having a size of 1.1-1.5 μm of 0.2-0.4 μ m.Fig. 1 is bacterium
The stereoscan photograph of NJUST50.
(2.2) physiological and biochemical property: Gram-negative, non-fermented type bacterium.
(2.3) molecular biology identification: using the core DNA of NJUST50 bacterial strain as template, with bacterium amplification universal primer into
Row PCR amplification measures the gene order of bacterial strain NJUST50.By the 16S rRNA gene order of bacterial strain and GenBank database into
Row tetraploid rice, the results showed that, the sequence similarity of NJUST50 and Enterobactersp.CZBSA2 is up to 96% or more.
It is accredited as according to the morphology of NJUST50, Physiology and biochemistry test and molecular biological analysis, NJUST50
Enterobacter is named as Enterobacter sp.NJUST50.
Embodiment 2
Bacterial strain Enterobacter sp.NJUST50 denitrification denitrogenation and the degradation property to N-Methyl pyrrolidone.
Enterobacter sp.NJUST50 is seeded to containing 500mg L-1The LB culture medium of N-Methyl pyrrolidone
In, 180 revs/min of shaking table cultures under the conditions of 30 DEG C carry out the enrichment of NJUST50 bacterial strain, after bacterial strain enters logarithmic growth phase (about
48 hours), gained bacterium solution centrifuge is centrifuged 10 minutes (6000 revs/min), deposition thallus is obtained, with inorganic after sterilizing
Salt fluid nutrient medium is resuspended, and centrifugation, repeated washing three times, thallus is resuspended in sterile liquid minimal medium, is planted
Sub- liquid (control OD600About 1.5-2.0).
Preparation initial concentration is 790-870mg L-1Nitrate nitrogen and 2800-3100mg L-1N-Methyl pyrrolidone it is inorganic
Above-mentioned seed liquor is added to simulation nitrate nitrogen and N- methyl through overexposure helium deoxygenation as simulated wastewater by salt fluid nutrient medium
In the waste water of pyrrolidones, inoculum concentration 5%, anoxic culture under conditions of 30 DEG C, 180 revs/min monitors nitre state in waste water
The variation of the concentration of nitrogen and N-Methyl pyrrolidone.Set up the blank control for not being inoculated with NJUST50.Experimental result is as shown in Figure 2.Knot
Fruit shows, 790-870mg L-1Nitrate nitrogen realizes complete denitrogenation, 2800-3100mg L in 50 hours-1N-Methyl pyrrolidone
It is completely degraded in 55 hours.And in the blank control for not being inoculated with NJUST50, nitrate nitrogen and N-Methyl pyrrolidone do not have
Apparent variation.
Embodiment 3
Enterobacter sp.NJUST50 produces waste water containing the lithium battery of nitrate nitrogen and N-Methyl pyrrolidone in reality
In to the degradation effect of N-Methyl pyrrolidone.
Enterobacter sp.NJUST50 seed liquor is accessed with 3%-10% inoculum concentration and contains nitre simultaneously after pretreatment
The practical lithium battery of state nitrogen and N-Methyl pyrrolidone produces the waste water (L of 260-350mg containing nitrate nitrogen-1, N-Methyl pyrrolidone
900-1100mg L-1) in, anoxic culture under conditions of 30 DEG C, 180 revs/min.Monitor wastewater treatment front and back nitrate nitrogen and N-
The concentration of methyl pyrrolidone changes.
As shown in figure 3, practical after pretreatment produce waste water containing the lithium battery of nitrate nitrogen and N-Methyl pyrrolidone simultaneously
Middle inoculation Enterobacter sp.NJUST50 bacterial strain, after processing in 15 hours or so, nitrate nitrogen removal rate is 100%, warp
N-Methyl pyrrolidone is completely removed after processing in 18 hours or so.
This example demonstrates that isolated Enterobacter sp.NJUST50 can be successfully applied to contain nitre state simultaneously
The lithium battery of nitrogen and N-Methyl pyrrolidone produces the biochemical treatment of waste water, realizes nitrate nitrogen and N-Methyl pyrrolidone in waste water
Efficient removal.
Sequence table
<110>Institutes Of Technology Of Nanjing
<120>degrade N-Methyl pyrrolidone enterobacteria and application in the treatment of waste water
<141> 2019-03-19
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1306
<212> DNA
<213> Enterobacter
<400> 1
gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gtgacattct gattcacgat 60
tactagcgat tccgacttca tggagtcgag ttgcagactc caatccggac tacgacgcac 120
tttatgaggt ccgctagctc tcgcgagatt gcttctcttt gtatgcgcca ttgtagcacg 180
tgtgtagccc tggtcgtaag ggccatgatg acttgacgtc atccccacct tcctccagtt 240
tatcactggc agtctccttt gagttcccgg cctaaccgct ggcaacaaag gataagggtt 300
gcgctcgttg cgggacttaa cccaacattt cacaacacga gctgacgaca gccatgcagc 360
acctgtctca cagttcccga aggcaccaat ccatctctgg aaagttctgt ggatgtcaag 420
accaggtaag gttcttcgcg ttgcatcgaa ttaaaccaca tgctccaccg cttgtgcggg 480
cccccgtcaa ttcatttgag ttttaacctt gcggccgtac tccccaggcg gtcgacttaa 540
cgcgttagct ccggaagcca cgcctcaagg gcacaacctc caagtcgaca tcgtttacgg 600
cgtggactac cagggtatct aatcctgttt gctccccacg ctttcgcacc tgagcgtcag 660
tcttcgtcca gggggccgcc ttcgccaccg gtattcctcc agatctctac gcatttcacc 720
gctacacctg gaattctacc cccctctacg agactcaagc ctgccagttt cggatgcagt 780
tcccaggttg agcccgggga tttcacatcc gacttgacag accgcctgcg tgcgctttac 840
gcccagtaat tccgattaac gcttgcaccc tccgtattac cgcggctgct ggcacggagt 900
tagccggtgc ttcttctgcg ggtaacgtca atcgacgcgg ttattaaccg catcgccttc 960
ctccccgctg aaagtacttt acaacccgaa ggccttcttc atacacgcgg catggctgca 1020
tcaggcttgc gcccattgtg caatattccc cactgctgcc tcccgtagga gtctggaccg 1080
tgtctcagtt ccagtgtggc tggtcatcct ctcagaccag ctagggatcg tcgcctaggt 1140
gagccgttac cccacctact agctaatccc atctgggcac atctgatggc aagaggcccg 1200
aaggtccccc tctttggtct tgcgacgtta tgcggtatta gctaccgttt ccagtagtta 1260
tccccctcca tcaggcagtt tcccagacat tactcacccg tccgcc 1306
Claims (10)
1. the enterobacteria for N-Methyl pyrrolidone of degrading is Enterobacter sp.NJUST50, deposit number CCTCC
NO:M2019128.
2. the cultural method of enterobacteria according to claim 1, which is characterized in that specific steps are as follows: will
Enterobacter sp.NJUST50 is inoculated in the medium, and medium pH is 6~8, and cultivation temperature is 20 DEG C~35 DEG C.
3. cultural method according to claim 2, which is characterized in that the culture medium also contains N-Methyl pyrrolidone
And sodium nitrate.
4. cultural method according to claim 3, which is characterized in that in the culture medium, sodium nitrate and N- methyl pyrrole
The molar ratio of pyrrolidone is 1.0~2.0:1.0.
5. cultural method according to claim 3 or 4, which is characterized in that the N-Methyl pyrrolidone concentration is
2800~3100mg L-1。
6. cultural method according to claim 3 or 4, which is characterized in that the sodium nitrate concentration be 4800~
5300mg L-1。
7. application of the enterobacteria according to claim 1 in the wastewater treatment containing N-Methyl pyrrolidone.
8. enterobacteria according to claim 1 answering in the lithium battery production wastewater treatment containing N-Methyl pyrrolidone
With.
9. application according to claim 7 or 8, which is characterized in that method particularly includes: by enterobacteria Enterobacter
Sp.NJUST50 seed liquor is inoculated into the waste water containing N-Methyl pyrrolidone, anoxic culture, and cultivation temperature is 20 DEG C~35
DEG C, culture pH is 6~8.
10. application according to claim 9, which is characterized in that the enterobacteria Enterobacter sp.NJUST50
The inoculum concentration of seed liquor is 3%~10%.
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Cited By (6)
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| CN111762889A (en) * | 2020-07-23 | 2020-10-13 | 南京理工大学 | Biological enhanced treatment process for lithium battery production wastewater |
| CN113293100A (en) * | 2021-04-23 | 2021-08-24 | 东莞市科绿智能环保科技有限公司 | Method for culturing special microorganisms for lithium battery wastewater treatment |
| CN114806921A (en) * | 2022-03-02 | 2022-07-29 | 南京理工大学 | Denitrifying bacterium taking N-methyl pyrrolidone as electron donor and application thereof |
| CN114933990A (en) * | 2022-05-31 | 2022-08-23 | 南京理工大学 | N-methylpyrrolidone degradation synchronous denitrifying bacteria and application thereof |
| CN117185479A (en) * | 2023-10-09 | 2023-12-08 | 深圳市睿维盛环保科技有限公司 | Bacteria-carrying slow-release carbon source and preparation method and application thereof |
| CN117887640A (en) * | 2024-03-13 | 2024-04-16 | 南京农业大学 | N-methyl pyrrolidone degradation strain, microbial inoculum and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111762889A (en) * | 2020-07-23 | 2020-10-13 | 南京理工大学 | Biological enhanced treatment process for lithium battery production wastewater |
| CN111762889B (en) * | 2020-07-23 | 2021-03-23 | 南京理工大学 | Biological enhanced treatment process for lithium battery production wastewater |
| WO2022016769A1 (en) * | 2020-07-23 | 2022-01-27 | 南京理工大学 | Biological enhancement processing process for lithium battery production wastewater |
| CN113293100A (en) * | 2021-04-23 | 2021-08-24 | 东莞市科绿智能环保科技有限公司 | Method for culturing special microorganisms for lithium battery wastewater treatment |
| CN114806921A (en) * | 2022-03-02 | 2022-07-29 | 南京理工大学 | Denitrifying bacterium taking N-methyl pyrrolidone as electron donor and application thereof |
| CN114806921B (en) * | 2022-03-02 | 2023-09-01 | 南京理工大学 | Denitrifying bacteria with N-methylpyrrolidone as electron donor and application thereof |
| CN114933990A (en) * | 2022-05-31 | 2022-08-23 | 南京理工大学 | N-methylpyrrolidone degradation synchronous denitrifying bacteria and application thereof |
| CN114933990B (en) * | 2022-05-31 | 2023-09-05 | 南京理工大学 | N-methylpyrrolidone degradation synchronous denitrifying bacterium and application thereof |
| CN117185479A (en) * | 2023-10-09 | 2023-12-08 | 深圳市睿维盛环保科技有限公司 | Bacteria-carrying slow-release carbon source and preparation method and application thereof |
| CN117185479B (en) * | 2023-10-09 | 2024-07-12 | 深圳市睿维盛环保科技有限公司 | Bacteria-carrying slow-release carbon source and preparation method and application thereof |
| CN117887640A (en) * | 2024-03-13 | 2024-04-16 | 南京农业大学 | N-methyl pyrrolidone degradation strain, microbial inoculum and application thereof |
| CN117887640B (en) * | 2024-03-13 | 2024-05-14 | 南京农业大学 | N-methyl pyrrolidone degradation strain, microbial inoculum and application thereof |
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