CN112522131B - Rhizobium and application thereof in degradation of nonyl phenol in water environment - Google Patents
Rhizobium and application thereof in degradation of nonyl phenol in water environment Download PDFInfo
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- CN112522131B CN112522131B CN202011161526.XA CN202011161526A CN112522131B CN 112522131 B CN112522131 B CN 112522131B CN 202011161526 A CN202011161526 A CN 202011161526A CN 112522131 B CN112522131 B CN 112522131B
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
- C02F2101/345—Phenols
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Abstract
The invention discloses a Rhizobium, named as Rhizobium sp (NPLJ-2), which is preserved in China center for type culture Collection (CCTCC M2020603) in 10-19 months of 2020. The invention also discloses application of the rhizobium in degrading nonyl phenol in a water environment. The rhizobium of the invention can reach more than 80 percent of biodegradation rate to nonyl phenol with initial concentration of 100mg/L in water under aerobic condition, effectively shortens biodegradation time, and effectively reduces COD contributed by nonyl phenol in sewage; the method quickly and efficiently degrades nonyl phenol in the sewage by using a biological method, and has important significance for treating high-concentration environmental hormone-nonyl phenol in the sewage.
Description
Technical Field
The invention relates to rhizobia and application of the rhizobia in degrading nonylphenol in water.
Background
The printing and dyeing industry is a large household for industrial water and wastewater discharge in China, and according to incomplete statistics, the discharge amount of printing and dyeing wastewater in China is about 300-400 ten thousand meters3And/d, which accounts for about 35% of the total industrial wastewater discharge. For a long time, the printing and dyeing wastewater becomes a difficult point in the field of industrial wastewater treatment due to high concentration and high salt content of organic matters and difficult biochemical degradation of the organic matters.
Nonylphenol ethoxylates (NPEOs) are widely used in textile processing aids as important industrial nonionic surfactants. The concentration range of NPEO in the textile wastewater is as high as 450-5680 mug/L. NPEO is generated by polymerizing NP and ethylene oxide, the instability of NPEOs is related to a polyethoxylate chain consisting of 1-20 ethoxy units, the NPEOs can be converted into NP, NP (1-3) EO and NP (1-3) EC through various ways after entering the environment, and the substances have a certain estrogen effect and can generate toxicity to organisms in a water body, so the NPEO is widely concerned.
Nonylphenol (NP) is one of the major products of NPEOs, which has estrogenic, neurotoxic, immunotoxic and reproductive toxicities, etc., with half-lives in the environment varying from a few days to a hundred days. The NP in the environment has wide distribution range, biological enrichment property and higher cost when being treated by physical adsorption and chemical photolysis, so the NP is not suitable for large-scale application. The currently reported microorganisms (Pseudomonas stutzeri, Leclercia adecarboxylata and the like) mostly have a certain removal effect on low-concentration NP, when the concentration of the NP is less than 10mg/L, the degradation rate is 60-75%, when the concentration of the NP is 50mg/L, the degradation rate is 20-40%, when the concentration of the NP is 100mg/L, the degradation rate is only 10-20%, and residual NP can be continuously accumulated in the environment and enter organisms.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides rhizobium which can rapidly and efficiently degrade high-concentration nonyl phenol in sewage, aiming at the problems of low treatment concentration, low removal rate, low degradation rate and the like of nonyl phenol in a microorganism treatment water body in the prior art.
The technical scheme is as follows: the rhizobia is named as Rhizobium (Rhizobium sp.) NPLJ-2, is classified and named as Rhizobium (Rhizobium sp.), has the strain number of NPLJ-2, is preserved in China center for type culture Collection in Wuhan, China, has the preservation number of CCTCC M2020603, and has the preservation date of 2020, 10 months and 19 days.
The Rhizobium (Rhizobium sp.) NPLJ-2 strain grows well in an LB culture medium under the aerobic condition of 30 ℃, the colony is circular, the diameter is 0.2-1 mm, the color of the colony is white, the colony is opaque, the surface is dry, and gram staining is negative.
The invention also aims to solve the technical problem of providing the application of the rhizobium in degrading nonyl phenol in water environment.
The application is as follows: inoculating rhizobium into an expanded culture medium, carrying out aerobic culture at 28-30 ℃ for 24-48 h to obtain an NPLJ-2 expanded culture product, wherein the inoculation amount of the rhizobium is 1% -3% according to volume fraction; inoculating the culture-expanding product into sewage containing nonyl phenol for treatment.
Wherein, the inoculation amount of Rhizobium (Rhizobium sp) NPLJ-2 in the sewage is 3-5 per mill (mass fraction), and the mass concentration of nonyl phenol in the sewage is not lower than 100 mg/L.
When the bacteria are applied, the temperature of the nonylphenol-containing sewage is 25-30 ℃, the pH value is 7.0-7.5, and the degradation rate of Rhizobium (Rhizobium sp) NPLJ-2 paranonylphenol is accelerated under the condition.
Has the advantages that: the rhizobium of the invention can achieve a biodegradation rate of more than 80% to nonyl phenol with an initial concentration of 100mg/L in a water body under an aerobic condition, effectively shortens biodegradation time, and simultaneously effectively reduces COD concentration in water by directly converting nonyl phenol in the water body and removing nonyl phenol from the water body; the invention can rapidly and efficiently degrade high-concentration nonyl phenol in sewage by using a biological method, thereby having important significance for treating the high-concentration environmental hormone-nonyl phenol in sewage.
Drawings
FIG. 1 is a phylogenetic tree of Rhizobium sp (NPLJ-2) of the present invention;
FIG. 2 is a graph showing the degradation rate of Rhizobium sp NPLJ-2 to nonylphenol and COD in wastewater according to the present invention.
Detailed Description
The technical solution of the present invention is further explained with reference to the accompanying drawings and specific embodiments.
In the invention, Rhizobium (Rhizobium sp) NPLJ-2 is obtained by screening from activated sludge of certain landfill leachate biological treatment aerobic section in Nanjing.
The specific process is as follows:
(1) collecting active sludge of an aerobic section from a percolate biological treatment section of a certain refuse incineration plant of Nanjing;
(2) 250mL of the obtained mud-water mixture is contained in a conical flask, the mixture is stirred on a magnetic stirrer at normal temperature, and 100mg of nonyl phenol is added into the mixture every day; pouring out the supernatant (detecting whether the supernatant contains the target pollutant, namely nonyl phenol) before adding the medicine each time, then supplementing the supernatant with tap water, and performing acclimatization until the target pollutant cannot be detected in the solution, thus completing the acclimatization;
(3) taking out the sludge obtained in the step (2), respectively coating the sludge after gradient dilution in an inorganic salt culture medium containing nonyl phenol, and statically culturing for 3-5 days at the temperature of 30 ℃ to obtain single colonies; the mass concentration of the nonyl phenol in the inorganic salt culture medium is not less than 100 mg/L;
(4) and (4) selecting the single colony in the step (3), streaking and purifying, and streaking and storing on a slope to obtain a pure strain.
The culture medium adopted by the amplification culture of the pure strain is one of LB culture medium, inorganic salt culture medium or beef extract peptone culture medium.
The LB culture medium comprises the following components by mass: each liter of water contains 5.0g of yeast powder, 10g of sodium chloride and 10g of peptone, and the pH value of the culture medium is 7.0-7.5.
The inorganic salt culture medium comprises the following components in parts by mass: the water per liter contains 100mg of nonyl phenol, 0.262g of ammonium sulfate and 0.025g of disodium hydrogen phosphate, and the pH value of the culture medium is 7.0-7.5.
The beef extract peptone medium comprises the following components in parts by mass: 3g of beef, 10g of peptone and 5g of sodium chloride are contained in each liter of water, and the pH value of the culture medium is 7.2-7.6.
Inoculating the rhizobia (Rhizobium sp.) NPLJ-2 obtained in the step (4) (the inoculation amount is 1-3% (volume fraction)) into an expanding culture medium for expanding culture, carrying out aerobic culture at 28-30 ℃ for 24-48 h, and obtaining an expanding culture product of the NPLJ-2 after culture.
And (4) identifying the screened strains (the pure strains obtained in the step (4)).
The NPLJ-2 with the best degradation effect and the fastest growth speed is obtained by screening. Wherein, the NPLJ-2 is identified as Rhizobium sp.
Rhizobium sp NPLJ-2 grows well in LB culture medium under aerobic condition of 30 ℃, the colony is circular, the diameter is 0.2-1 mm, the color of the colony is white, the colony is opaque, the surface is dry, and gram stain is negative.
The NPLJ-2 strain 16S rRNA complete sequence obtained by PCR amplification and Sanger sequencing is as follows (SEQ ID NO. 1):
NPLJ-2 sequencing results are submitted to GenBank for BLAST comparison, strain sequences with the similarity of more than 99% to NPLJ-2 base sequences are selected from the BLAST results, multi-series comparison is carried out through Clustal W, and a phylogenetic tree is constructed by using MAGE to obtain the NPLJ-2 and Rhizobium sp.strainank13 (the accession number is MN662624.1), the genetic relationship is nearest, so that the strain NPLJ-2 is identified as Rhizobium.
The Rhizobium sp (Rhizobium sp.) NPLJ-2 provided by the invention is used for measuring the degradation rate of nonyl phenol pollutant, and the specific steps are as follows:
(1) centrifuging the bacterial liquid obtained after the NPLJ-2 is subjected to amplification culture at 6000rpm for 10min, and pouring out the supernatant to obtain thalli;
(2) taking the thalli obtained in the step (1) according to the inoculation amount of 3 per mill (mass fraction), scattering, and adding the thalli into simulated nonylphenol wastewater, wherein the mass concentration of nonylphenol in water is 100 mg/L; and the treatment group without NPLJ-2 is used as a reference;
(3) putting the control group and the treatment group in the step (2) into a shaking table, and carrying out oscillation reaction at 180rpm and 28 ℃ under aerobic conditions;
(4) and (3) sampling at regular time to detect the removal rate of the nonyl phenol and the COD concentration in the water body in the treatment group and the control group in the step (3), and the result is shown in figure 2.
As shown in figure 2, NPLJ-2 can efficiently degrade nonyl phenol in water, 100mg/L of nonyl phenol is rapidly degraded within 24h, the degradation rate is reduced along with the reduction of the concentration of nonyl phenol, and the removal rate of nonyl phenol reaches 80% after 48 h. The COD concentration in the sewage is obviously reduced while the concentration of the nonyl phenol in the water is reduced, which shows that the nodulation bacterium (Rhizobium sp.) NPLJ-2 can directly discharge the nonyl phenol from the water, thereby completely removing the nonyl phenol from the water.
According to the invention, Rhizobium (Rhizobium sp) NPLJ-2 with high-efficiency removal efficiency on nonyl phenol is obtained after activated sludge in the aerobic section of the landfill leachate is acclimated step by step. The rhizobium of the invention can achieve a biodegradation rate of more than 80% to 100mg/L nonyl phenol under aerobic conditions, and the biodegradation time is obviously shortened; meanwhile, the method can also remove the nonyl phenol directly from the water body, thereby effectively reducing the concentration of COD in the water body.
Sequence listing
<110> Nanjing Jiang island environmental science and technology research institute Co., Ltd
<120> rhizobium and application thereof in degradation of nonyl phenol in water environment
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1312
<212> DNA
<213> Rhizobium (2 Ambystrhizobium)
<400> 1
gggagtggca gacgggtgag taacgcgtgg gaacataccc tttcctgcgg aatagctccg 60
ggaaactgga attaataccg catacgccct acgggggaaa gatttatcgg ggaaggattg 120
gcccgcgttg gattagctag ttggtggggt aaaggcctac caaggcgacg atccatagct 180
ggtctgagag gatgatcagc cacattggga ctgagacacg gcccaaactc ctacgggagg 240
cagcagtggg gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgagtga 300
tgaaggcctt agggttgtaa agctctttca ccggagaaga taatgacggt atccggagaa 360
gaagccccgg ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc 420
ggaattactg ggcgtaaagc gcacgtaggc ggatatttaa gtcaggggtg aaatcccaga 480
gctcaactct ggaactgcct ttgatactgg gtatcttgag tatggaagag gtaagtggaa 540
ttccgagtgt agaggtgaaa ttcgtagata ttcggaggaa caccagtggc gaaggcggct 600
tactggtcca ttactgacgc tgaggtgcga aagcgtgggg agcaaacagg attagatacc 660
ctggtagtcc acgccgtaaa cgatgaatgt tagccgtcgg gcagtatact gttcggtggc 720
gcagctaacg cattaaacat tccgcctggg gagtacggtc gcaagattaa aactcaaagg 780
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgcaga 840
accttaccag ctcttgacat tcggggtatg ggcattggag acgatgtcct tcagttaggc 900
tggccccaga acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 960
gtcccgcaac gagcgcaacc ctcgccctta gttgccagca tttagttggg cactctaagg 1020
ggactgccgg tgataagccg agaggaaggt ggggatgacg tcaagtcctc atggccctta 1080
cgggctgggc tacacacgtg ctacaatggt ggtgacagtg ggcagcgaga cagcgatgtc 1140
gagctaatct ccaaaagcca tctcagttcg gattgcactc tgcaactcga gtgcatgaag 1200
ttggaatcgc tagtaatcgc agatcagcat gctgcggtga atacgttccc gggccttgta 1260
cacaccgccc gtcacaccat gggagttggt tttacccgaa ggtagtgcgc ta 1312
Claims (8)
1. A rhizobium, which is characterized in that: named as Rhizobium sp (Rhizobium sp.) NPLJ-2, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2020603 in 10 months and 19 days of 2020.
2. Use of the rhizobia of claim 1 to degrade nonylphenol in an aqueous environment.
3. Use of rhizobia according to claim 2 to degrade nonylphenol in an aqueous environment, wherein: the degradation of nonyl phenol in water by rhizobia is carried out under aerobic condition.
4. Use of rhizobia according to claim 2 to degrade nonylphenol in an aqueous environment, wherein: inoculating rhizobium into an expanded culture medium, carrying out aerobic culture at 28-30 ℃ for 24-48 h to obtain an NPLJ-2 expanded culture product, wherein the inoculation amount of the rhizobium is 1% -3% according to volume fraction; inoculating the culture-expanding product into sewage containing nonyl phenol for treatment.
5. Use of rhizobia according to claim 4 to degrade nonylphenol in an aqueous environment, wherein: the inoculation amount of the rhizobia in the sewage is 3-5 per mill calculated by mass fraction.
6. Use of rhizobia according to claim 4 to degrade nonylphenol in an aqueous environment, wherein: the mass concentration of nonyl phenol in the sewage is not lower than 100 mg/L.
7. Use of rhizobia according to claim 4 to degrade nonylphenol in an aqueous environment, wherein: when the thalli is applied, the temperature of sewage is 25-30 ℃, and the pH value is 7.0-7.5.
8. Use of rhizobia according to claim 4 to degrade nonylphenol in an aqueous environment, wherein: the expanding culture medium is one of LB culture medium, inorganic salt culture medium or beef extract peptone culture medium.
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| CN202011161526.XA CN112522131B (en) | 2020-10-26 | 2020-10-26 | Rhizobium and application thereof in degradation of nonyl phenol in water environment |
| PCT/CN2020/124251 WO2022087870A1 (en) | 2020-10-26 | 2020-10-28 | Rhizobium and use thereof in degradation of nonylphenol in water environment |
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| CN202011161526.XA CN112522131B (en) | 2020-10-26 | 2020-10-26 | Rhizobium and application thereof in degradation of nonyl phenol in water environment |
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| CN103382446B (en) * | 2013-05-31 | 2015-02-25 | 南京农业大学 | Rhizobium capable of degrading polychlorinated biphenyl and application thereof |
| CN103343100B (en) * | 2013-07-01 | 2014-08-13 | 中国农业科学院农业资源与农业区划研究所 | Bacterium capable of degrading pesticides chlorimuron-ethyl and carbendazim and application thereof |
| CN103540544B (en) * | 2013-08-23 | 2015-04-29 | 南京理工大学 | Bacillus radicicola capable of degrading pyridine as well as breeding method and application thereof |
| CN104724896A (en) * | 2015-03-04 | 2015-06-24 | 同济大学 | Method for promoting anaerobic degradation of estrogen nonylphenol in sludge through alkali treatment |
| CN107937310B (en) * | 2017-12-19 | 2021-04-27 | 中国科学院烟台海岸带研究所 | Growth-promoting rhizobacteria and application thereof |
| CN109234353B (en) * | 2018-09-14 | 2021-10-29 | 暨南大学 | Experimental method for effectively degrading nonyl phenol by microalgae |
| CN111087149A (en) * | 2019-12-25 | 2020-05-01 | 盐城师范学院 | Method for realizing efficient anaerobic degradation of nonyl phenol in sludge under fermentation acid production condition |
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