CN109913387B - Enterobacter for degrading N-methylpyrrolidone and application of enterobacter in wastewater treatment - Google Patents
Enterobacter for degrading N-methylpyrrolidone and application of enterobacter in wastewater treatment Download PDFInfo
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Abstract
The invention discloses an enterobacter for degrading N-methylpyrrolidone and application thereof in wastewater treatment. The invention uses activated sludge as a bacteria source, uses an inorganic salt culture medium with N-methyl pyrrolidone as a unique carbon source as a screening culture medium, separates and purifies to obtain a strain of enterobacter for denitrification by using N-methyl pyrrolidone as an electron donor, and has a preservation number of CCTCC NO: m2019128. The enterobacter can carry out anoxic denitrification reaction by taking N-methylpyrrolidone as the only electron donor, synchronously realize mineralization and degradation of the N-methylpyrrolidone, and completely remove nitrate nitrogen and the N-methylpyrrolidone within 15 hours and 18 hours respectively, has high-efficiency organic matter degradation capability and denitrification capability, and is suitable for removal treatment of high-concentration nitrate nitrogen and refractory organic pollutants in wastewater.
Description
Technical Field
The invention belongs to the technical field of biological treatment of environmental organic pollutants, relates to an enterobacter strain for degrading N-methylpyrrolidone, and particularly relates to an enterobacter strain for carrying out denitrification by taking the nondegradable organic pollutant N-methylpyrrolidone as an electron donor and application thereof in treatment of wastewater containing the N-methylpyrrolidone.
Background
N-methylpyrrolidone (NMP), a typical organic polar solvent, is widely used in the industries of coatings, adhesives, fuels, pharmaceutical manufacturing, and the like due to its good water miscibility, low volatility, high polarity, non-corrosiveness, and the like. The N-methyl pyrrolidone has stable structure, is not easy to hydrolyze, is easy to transfer between soil and underground water, and has biotoxicity and teratogenicity. Over 2400 tons of N-methylpyrrolidone are reported to be discharged into the environment by industrial waste water every year. Therefore, restoring the ecological environment of water areas polluted by N-methyl pyrrolidone has become one of the important issues in the field of environmental management at present.
At present, the treatment method of the wastewater containing N-methyl pyrrolidone comprises a photocatalysis method, an advanced oxidation method such as ozone oxidation, a membrane separation method, a biological treatment method and the like. Wherein, the cost for treating the waste water containing the N-methyl pyrrolidone by the physical and chemical methods such as advanced oxidation, membrane separation and the like is higher, and the secondary pollution is serious. The biological treatment technology has the advantages of economy, high efficiency, small secondary pollution and the like, can realize harmless treatment, and is the most widely applied wastewater treatment technology. However, due to the toxic and difficult-to-degrade properties of N-methylpyrrolidone, it is necessary to find strains with degradation function that are able to withstand the biotoxicity of N-methylpyrrolidone.
Due to the toxicity and the difficult degradation characteristic of the N-methyl pyrrolidone, the precondition of biological treatment of N-methyl pyrrolidone pollution is to obtain a special strain which can resist the biotoxicity of the N-methyl pyrrolidone and can realize the mineralization degradation of the N-methyl pyrrolidone. Kri i zek et al reported that Pseudomonas, Paracoccus, Acinetobacter and Rhodococcus act synergistically to degrade N-methylpyrrolidone at a rate of 4.17 mg.L-1·h-1The degradation period was 72 hours (Kr i zek, K., et al, N-methyl-2-pyrrolidone-degrading bacteria from activated slurry. Water Sci. technol.2015,71, 776-782.). Chuasiu et al reported that Paracoccus could degrade N-methylpyrrolidone at a rate of 17.86 mg.L-1·h-1Degradation ofThe period is 28 hours (Cai, s., et al. biodegradation of N-methylpyrrolidone by Paracoccus sp.nmd-4 and its degradation path. int.biodeter.biodegr.2014,93, 70-77.). The existing bacterial strain capable of degrading the N-methylpyrrolidone has the advantages of longer degradation period and low degradation efficiency, and needs to be cultured under the condition of oxygen consumption, so that the operation cost of power consumption aeration and other processes is greatly increased in practical application. Considering that the industrial wastewater containing the N-methyl pyrrolidone generally contains nitrate with higher concentration, if high-efficiency strains which can use the N-methyl pyrrolidone as an electron donor and nitrate nitrogen as an electron acceptor can be obtained, the degradation of the N-methyl pyrrolidone is realized while the nitrogen is denitrified by anoxic denitrification, and the method has important significance for the low-cost and harmless treatment of the industrial wastewater containing the N-methyl pyrrolidone and the nitrate nitrogen.
Disclosure of Invention
The invention aims to provide an enterobacter (Enterobacter) strain for degrading N-methylpyrrolidone, which is a high-efficiency strain for carrying out denitrification by taking the N-methylpyrrolidone as an electron donor and nitrate nitrogen as an electron acceptor and realizes the degradation of the N-methylpyrrolidone while carrying out anoxic denitrification.
The inventor uses activated sludge for denitrification as a bacterial source and utilizes a screening culture medium which uses N-methylpyrrolidone as a unique carbon source to purify and separate bacterial strains to obtain a strain of Enterobacter which can use N-methylpyrrolidone as an electron donor for denitrification, and the Enterobacter is identified as Enterobacter sp. The strain is preserved in the China typical collection center (CCTCC) at 03 and 06 months in 2019, the preservation address is Wuhan university in Wuhan City, China, and the preservation number is CCTCC NO: m2019128.
The invention also provides a culture method of the Enterobacter sp.NJUST50, which comprises the following specific steps: inoculating Enterobacter sp.NJUST50 into a culture medium, wherein the pH value of the culture medium is 6-8, and the culture temperature is 20-35 ℃.
Preferably, the culture medium further contains N-methylpyrrolidone and sodium nitrate, and the molar ratio of the sodium nitrate to the N-methylpyrrolidone is 1.0-2.0: 1.0, the concentration of N-methyl pyrrolidone is 2800-3100mg/L, and the concentration of sodium nitrate is 4800-5300 mg/L.
The invention also provides application of the Enterobacter sp.
Further, the invention also provides application of the Enterobacter sp.NJUST50 in treatment of wastewater generated in production of lithium batteries containing N-methylpyrrolidone.
Specifically, the application of the Enterobacter sp.njustt 50 in the treatment of wastewater containing N-methylpyrrolidone comprises the following specific steps: inoculating Enterobacter sp.NJUST50 seed liquid into wastewater containing N-methylpyrrolidone, and carrying out anoxic culture at the culture temperature of 20-35 ℃ and the culture pH of 6-8.
Preferably, the inoculation amount of the Enterobacter sp.njustt 50 seed liquid is 3% -10%.
The Enterobacter sp.NJUST50 provided by the invention can utilize N-methylpyrrolidone as a unique electron donor and nitrate nitrogen as a unique electron acceptor to carry out metabolism and growth under the anoxic condition, and has efficient N-methylpyrrolidone degradation capability and denitrification capability. Compared with the oxygen consumption condition, the Enterobacter sp.NJUST50 has stronger adaptability and tolerance to the living environment, can reduce the oxygen consumption aeration section when being applied to the process treatment of industrial wastewater, and saves the economic cost. Adding Enterobacter sp.NJUST50 into the pretreated lithium battery production wastewater actually containing the N-methylpyrrolidone for treatment, and completely removing nitrate nitrogen and the N-methylpyrrolidone within 15 hours and 18 hours respectively.
Drawings
Fig. 1 is a scanning electron micrograph of Enterobacter sp.
FIG. 2 is a graph showing the degradation effect and denitrification effect of Enterobacter sp.NJUST50 on N-methylpyrrolidone in a liquid medium having an initial concentration of 2800-3100mg/L and an initial concentration of nitrate nitrogen of 790-870 mg/L.
Fig. 3 is a graph showing the denitrification effect of Enterobacter sp.njustt 50 in pretreated lithium battery production wastewater containing both nitrate nitrogen and N-methylpyrrolidone, and the degradation effect of N-methylpyrrolidone.
Detailed Description
The present invention is further described below by way of specific examples and figures to provide a more complete understanding of the invention to those skilled in the art, but not to limit the invention in any way.
Example 1
Screening, isolation and identification of Enterobacter sp.
(1) Screening and isolation of strains
5g of the existing activated sludge for denitrification was sampled and added to 100mL of physiological saline, and the mixture was stirred uniformly and then allowed to stand for two hours. Adding 1mL of supernatant into the inorganic salt culture medium sterilized at 121 deg.C, performing shake culture at 180 rpm for three days, performing three times of enrichment, and diluting the culture solution with sterile water to 10-4-10-10And (4) doubling. Preparing an inorganic salt agar solid culture medium, respectively coating 20 mu L of the diluted culture solution on the inorganic salt agar solid culture medium, and placing the culture medium in a biochemical incubator for three days at 30 ℃. And selecting single colonies with obvious differences on a culture dish, performing purification culture by adopting a plate streaking separation method, continuously purifying for five times to obtain a single strain, and performing slant storage. Preparing an inorganic salt liquid culture medium containing nitrate nitrogen and N-methylpyrrolidone, filling the inorganic salt liquid culture medium into a serum bottle, aerating with pure helium to remove dissolved oxygen, inoculating, separating and purifying the obtained strain, culturing the strain in a constant-temperature shaking incubator under the anoxic condition of 180 revolutions per minute and 30 ℃, and monitoring the concentration change of the nitrate nitrogen and the N-methylpyrrolidone. Selecting a strain capable of effectively removing nitrate nitrogen and N-methylpyrrolidone in a culture medium, and carrying out slant storage and low-temperature storage at minus 80 ℃, wherein the strain is named as NJUST 50.
The composition of the LB medium was as follows: tryptone (10g/L), yeast extract (5g/L), sodium chloride (10 g/L).
The composition of the inorganic salt culture medium is as follows: NaHPO4·12H2O(1.53g/L)，KH2PO4(0.38g/L)，MgSO4(0.1g/L)，CaCl2(0.05g/L), microelement solution SL-4(10 mL). Microelement SL-4: EDTA (0.5g/L), FeSO4·7H2O (0.2g/L) and SL-6(100 mL/L). Microelement SL-6: ZnSO4·7H2O(0.01g/L)，MnCl2·4H2O(0.03g/L)，H3BO4(0.3g/L)，CoCl2·6H2O(0.2g/L)，CuCl2·2H2O(0.01g/L)，NiCl2·6H2O(0.02g/L)，Na2MoO4·2H2The amounts of O (0.03g/L), N-methylpyrrolidone and sodium nitrate are added according to the experimental requirements.
Adding 20g/L agar into liquid culture medium, autoclaving at 121 deg.C for 20 min in a sterilizer, and cooling to room temperature in a sterile culture dish to obtain inorganic salt agar solid culture medium.
(2) Identification of strains
The strain is subjected to morphological, physiological and biochemical tests. Determining the 16S rRNA gene sequence of the strain, comparing the 16S rRNA gene sequence of the strain with the gene sequences in a GenBank database, analyzing the results, and determining the species of the strain on the molecular biology level.
(2.1) morphological characteristics: NJUST50 colonies are milky white, smooth and transparent in surface, neat in edge, glossy, and diffusively turbid in liquid culture medium. The strain cell is rod-shaped, and the size is 0.2-0.4 μm × 1.1-1.5 μm. FIG. 1 is a scanning electron micrograph of bacterium NJUST 50.
(2.2) physiological and biochemical characteristics: gram-negative, non-fermentative bacteria.
(2.3) molecular biology identification: taking nuclear DNA of the NJUST50 strain as a template, carrying out PCR amplification by using a universal primer for bacterial amplification, and determining the gene sequence of the NJUST50 strain. Homology comparison of the 16S rRNA gene sequence of the strain and a GenBank database shows that the sequence similarity of NJUST50 and Enterobactersp.CZBSa2 reaches over 96 percent.
Based on morphological, physiological and biochemical tests and molecular biological analysis of NJUST50, NJUST50 was identified as Enterobacter, named Enterobacter sp.
Example 2
Bacterial strain Enterobacter sp. NJUST50 denitrification and degradation performance to N-methyl pyrrolidone.
Inoculating Enterobacter sp.NJUST50 into LB culture medium containing 500 mg/LN-methyl pyrrolidone, shaking-culturing at 30 deg.C for 180 r/min, enriching NJUST50 strain, after the strain has entered logarithmic growth phase (about 48 hr), centrifuging the obtained bacterial liquid with centrifuge for 10 min (6000 r/min) to obtain deposited thallus, re-suspending with sterilized inorganic salt liquid culture medium, centrifuging, washing repeatedly for three times, re-suspending the thallus in sterile liquid inorganic salt culture medium to obtain seed liquid (controlling OD)600About 1.5-2.0).
Preparing an inorganic salt liquid culture medium with initial concentration of 790-870mg/L of nitrate nitrogen and 2800-3100 mg/LN-methyl pyrrolidone as simulated wastewater, adding the seed liquid into wastewater of simulated nitrate nitrogen and N-methyl pyrrolidone which are exposed with helium and deoxidized, wherein the inoculation amount is 5%, carrying out anoxic culture at 30 ℃ and 180 r/min, and monitoring the concentration change of the nitrate nitrogen and the N-methyl pyrrolidone in the wastewater. A blank was set up which was not inoculated with NJUST 50. The results of the experiment are shown in FIG. 2. The results show that 790-870mg/L nitrate nitrogen achieves complete denitrification within 50 hours, and 2800-3100 mg/LN-methylpyrrolidone is completely degraded within 55 hours. In contrast, nitrate nitrogen and N-methylpyrrolidone did not change significantly in the blank not inoculated with NJUST 50.
Example 3
NJUST50 has the degradation effect on N-methylpyrrolidone in the actual wastewater containing nitrate nitrogen and N-methylpyrrolidone in the lithium battery production.
Inoculating the Enterobacter sp.NJUST50 seed solution into the pretreated actual lithium battery production wastewater (containing 350mg/L of nitrate nitrogen 260-containing and 1100mg/L of N-methylpyrrolidone 900-containing) containing both nitrate nitrogen and N-methylpyrrolidone at the inoculation amount of 3-10%, and carrying out anoxic culture at the temperature of 30 ℃ and under the condition of 180 r/min. The concentration changes of nitrate nitrogen and N-methyl pyrrolidone before and after wastewater treatment are monitored.
As shown in fig. 3, after pretreatment, the ammonium nitrate sp.njustt 50 strain was inoculated into lithium battery production wastewater containing nitrate nitrogen and N-methylpyrrolidone, and after about 15 hours of treatment, the removal rate of nitrate nitrogen was 100%, and after about 18 hours of treatment, N-methylpyrrolidone was completely removed.
The embodiment shows that the separated Enterobacter sp.NJUST50 can be successfully applied to the biochemical treatment of the lithium battery production wastewater containing nitrate nitrogen and N-methylpyrrolidone at the same time, and the high-efficiency removal of the nitrate nitrogen and the N-methylpyrrolidone in the wastewater is realized.
Sequence listing
<110> Nanjing university of science and technology
<120> Enterobacter capable of degrading N-methylpyrrolidone and application thereof in wastewater treatment
<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 Enterobacter for degrading the N-methylpyrrolidone is Enterobacter sp.NJUST50, and the preservation number is CCTCC NO: m2019128.
2. The method for culturing Enterobacter according to claim 1, comprising the steps of: inoculating Enterobacter sp.NJUST50 into a culture medium, wherein the pH value of the culture medium is 6-8, and the culture temperature is 20-35 ℃.
3. The method according to claim 2, wherein the culture medium contains N-methylpyrrolidone and sodium nitrate.
4. The culture method according to claim 3, wherein the molar ratio of sodium nitrate to N-methylpyrrolidone in the culture medium is 1.0-2.0: 1.0.
5. the culture method according to claim 3 or 4, wherein the concentration of N-methylpyrrolidone is 2800 to 3100 mg/L.
6. The culture method according to claim 3 or 4, wherein the concentration of sodium nitrate is 4800 to 5300 mg/L.
7. Use of enterobacteria according to claim 1 for the treatment of waste water containing N-methylpyrrolidone.
8. The use of enterobacter according to claim 1 in the treatment of lithium battery production wastewater containing N-methylpyrrolidone.
9. The use according to claim 7 or 8, characterized in that the specific method is: inoculating Enterobacter sp.NJUST50 seed liquid into wastewater containing nitrate nitrogen and N-methylpyrrolidone, and carrying out anoxic culture at the culture temperature of 20-35 ℃ and the culture pH of 6-8.
10. The use according to claim 9, wherein the inoculum size of the Enterobacter sp.
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| CN111762889B (en) * | 2020-07-23 | 2021-03-23 | 南京理工大学 | Biological enhanced treatment process for lithium battery production wastewater |
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| 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 |
| CN114933990B (en) * | 2022-05-31 | 2023-09-05 | 南京理工大学 | N-methylpyrrolidone degradation synchronous denitrifying bacterium 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 |
| 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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| CN106591169A (en) * | 2016-02-02 | 2017-04-26 | 江苏南资环保股份有限公司 | NMP (N-Methyl Pyrrolidone) degradation bacillus NMP-2 and application thereof |
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| N-甲基吡咯烷酮在制革二级出水中的生物降解性研究;解书冬等;《皮革科学与工程》;20151231;第25卷(第1期);第54-59页 * |
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