CN112522139B

CN112522139B - Achromobacter and application thereof in degradation of p-toluenesulfonic acid and quizalofop-p-ethyl

Info

Publication number: CN112522139B
Application number: CN202011347094.1A
Authority: CN
Inventors: 蔡天明; 蔡舒; 陈立伟
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2022-07-15
Anticipated expiration: 2040-11-26
Also published as: CN112522139A

Abstract

The invention discloses an Achromobacter strain, which is classified and named as Achromobacter sp, has the strain name of MBZ-2, is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, has the preservation date of 2020, 8 and 13 days, and has the preservation number of CGMCC No. 20529. The achromobacter provided by the invention can effectively degrade p-toluenesulfonic acid and a downstream product thereof, namely the herbicide quizalofop-p-ethyl.

Description

Achromobacter and application thereof in degradation of p-toluenesulfonic acid and quizalofop-p-ethyl

Technical Field

The invention relates to the field of biology, and in particular relates to achromobacter and application thereof in degradation of p-toluenesulfonic acid and quizalofop-p-ethyl.

Background

P-toluenesulfonic acid (PTS) has a molecular formula: c₇H₈O₃S, the structural formula is shown as formula I. The p-toluenesulfonic acid is white needle-shaped or powdery crystal, has a melting point of 106 ℃ and a boiling point of 140 ℃/2.67kPa, is easily soluble in polar solvents such as water, alcohol and ether, is difficultly soluble in non-polar solvents such as benzene and toluene, is strong organic acid without oxidability, and has the acidity 100 ten thousand times that of benzoic acid. It is an important chemical synthesis raw material, and is widely used as an intermediate for synthesizing medicines (such as naproxen, doxycycline and the like), pesticides (such as quizalofop-p-ethyl, dicofol and the like) and dyes (such as acid orange 7 and the like); also can be used as a catalyst for synthesizing esters (such as benzyl propionate, benzyl butyrate and the like), a stabilizer for polymerization reaction and a curing agent for industries of resin, paint, coating, casting, artificial board and the like. The p-toluenesulfonic acid has strong irritation on eyes, skin, mucosa and the like, is harmful to human bodies after being inhaled, ingested or absorbed through skin, and can cause spasm and edema of throats and bronchi, chemical pneumonia or pulmonary edema after being inhaled. Meanwhile, the substance has stronger biotoxicity. The half lethal concentration of the oral cavity of a rat is 2500mg/kg, and the half lethal concentration of the oral cavity of a mouse is 400 mg/kg.

Quizalofop-p-ethyl (Quizalofop-p-ethyl), chemical name ((R) -2- [4- (6-chloroquinoxaline-2-yloxy) ] ethyl propionate), is an aryloxy phenoxy propionate (AOPP) herbicide widely applied to control of annual or perennial weeds in broadleaf crop fields such as soybean, cotton, rape and the like after emergence of seedlings. After the quizalofop-p-ethyl is absorbed by weeds, the growth of meristematic tissues is damaged by inhibiting acetyl CoA carboxylase, and the weeds are necrotized. Although quizalofop-p-ethyl is considered as a low-toxicity pesticide, the widespread use and the large amount of residue and accumulation in the environment cause global environmental pollution and ecological destruction, and besides directly causing pollution to soil environment, water environment and atmospheric environment, the quizalofop-p-ethyl is also enriched in organisms through a food chain, and has serious influence on the safety of people and livestock. Quizalofop-p-ethyl with concentration more than 0.05mmol/L seriously damages the cell wall of diatom cell; the chromosomal aberration rate of the onion root meristem cells is increased along with the increase of the concentration of quizalofop-p-ethyl. Quizalofop-p-ethyl causes cholestasis and liver damage to farmers exposed to quizalofop-p-ethyl. Therefore, the maximum residue of quizalofop-p-ethyl is limited in many countries, but quizalofop-p-ethyl is still widely produced and used in China.

P-toluenesulfonic acid is one of important intermediates in the quizalofop-p-ethyl synthesis process, and due to high water solubility and difficulty in recycling from wastewater, wastewater containing high-concentration p-toluenesulfonic acid is generated in the quizalofop-p-ethyl synthesis process; meanwhile, quizalofop-p-ethyl as a synthetic product also has a small amount of residues in production wastewater. The microbial degradation has the advantages of low cost, high efficiency, no secondary pollution, good ecological restoration performance and the like, and is applied to many aspects, so that the screening of the high-efficiency degrading strain capable of efficiently degrading the p-toluenesulfonic acid and the quizalofop-p-ethyl at the same time has important environmental significance for treating the production wastewater of the quizalofop-p-ethyl.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing the achromobacter aiming at the defects of the prior art.

The invention also aims to solve the technical problem of providing the application of the achromobacter in degrading p-toluenesulfonic acid or quizalofop-p-ethyl.

The present invention also provides a microbial agent containing the above Achromobacter.

The invention also aims to solve the technical problem of providing the application of the microbial agent in degrading p-toluenesulfonic acid and quizalofop-p-ethyl.

In order to solve the first technical problem, the invention discloses an Achromobacter strain, which is classified and named as Achromobacter sp (Achromobacter sp), has the strain name of MBZ-2, is deposited in the China general microbiological culture Collection center, and has the address: no. 3 of Xilu No.1 of Beijing, Chaoyang, Chao province, the institute of microbiology of Chinese academy of sciences, the postal code 100101, the preservation date of 2020, 8 months and 13 days, and the preservation number of CGMCC No. 20529.

Wherein the Achromobacter sp MBZ-2 has the following morphological characteristics: gram-negative, rod-shaped, spore-free, liquefied gelatin, no reduction of nitrate and no production of acid from glucose; as shown in figure 1, after the bacterial strain is cultured in an LB solid culture medium for 2d, the bacterial colony is dry and round, slightly raised, light yellow, moist, semitransparent, neat and smooth in edge; the 16S rDNA sequence is shown in SEQ ID No. 1.

In order to solve the second technical problem, the invention discloses application of the leucobacter in degrading p-toluenesulfonic acid or quizalofop-p-ethyl.

In order to solve the third technical problem, the invention discloses a microbial agent of a microorganism prepared by the achromobacter.

Wherein, the achromobacter is inoculated in an LB liquid culture medium, cultured and centrifuged, and the precipitate is washed and then is resuspended in water, thus obtaining the achromobacter.

Wherein the culture is carried out at 37 ℃ and shaking at 180rpm until the OD600 is 1.0.

Wherein, the resuspension is to resuspend the washed precipitate in water in the same volume.

In order to solve the fourth technical problem, the invention discloses application of the microbial agent in degradation of p-toluenesulfonic acid.

The invention further discloses an application of the microbial agent in degrading quizalofop-p-ethyl.

Wherein the microbial agent is inoculated in a quizalofop-p-ethyl-containing material to degrade the quizalofop-p-ethyl.

Wherein the dosage ratio of the microbial agent to the quizalofop-p-ethyl is 0.2 mL/mg.

Wherein the degradation is the degradation by culturing in a shaker at 37 ℃ and 180 rpm.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the achromobacter provided by the invention can effectively degrade p-toluenesulfonic acid and a downstream product thereof, namely the herbicide quizalofop-p-ethyl.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a photograph of the strain morphology of the strain MBZ-2, left: photograph of LB solid plate coating; and (3) right: photograph of crystal violet staining.

FIG. 2 is a graph comparing the effect of 0h and 36h on the MSM medium of toluene sulfonic acid when MBZ-2 is inoculated.

FIG. 3 is a graph comparing the effect of quizalofop-p-ethyl MSM medium inoculated with MBZ-2 at 0h and 36 h.

FIG. 4 shows the effect of MBZ-2 on the degradation of 50mg/L quizalofop-p-ethyl.

FIG. 5 shows the effect of MBZ-2 on the degradation of 500mg/L p-toluenesulfonic acid.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1: isolation of Achromobacter (Achromobacter sp) MBZ-2

Collecting sludge sample from activated sludge tank of wastewater treatment plant of certain pesticide production enterprise in Jiangsu, and adopting connectionPerforming enrichment culture by continuous enrichment culture method, testing degradation effect by high performance liquid chromatography, diluting and coating effective enrichment solution on solid inorganic salt (culture medium formula: p-toluenesulfonic acid 0.5g/L, NH) containing 0.5g/L p-toluenesulfonic acid₄Cl 1.0g/L，KH₂PO₄ 0.6g/L，K₂HPO₄ 1.3g/L，NaCl 0.5g/L，MgSO₄·7H₂O0.2 g/L, pH7.0. ) Culturing on the culture medium in a constant temperature incubator at 30 ℃ until bacterial colonies grow out. According to the difference of the size, color and shape of the colony, different single colonies are picked and streaked for purification. The strain with the degradation efficiency of 98.4% after 72h was selected and named as MBZ-2.

Example 2: determination of degradation effect of Achromobacter sp (Achromobacter sp) MBZ-2 on quizalofop-p-ethyl

Achromobacter (Achromobacter sp) MBZ-2 is streaked on an LB solid plate, then a single colony is picked from the LB solid plate, inoculated in an LB liquid culture medium, cultured in a shaker at 37 ℃ and 180rpm until OD600 is 1.0, centrifuged at 10000rpm, and then a supernatant is discarded, washed three times with sterile water and resuspended in sterile water with the same volume to be used as a seed solution. The MBZ-2 seed liquid is inoculated in an inorganic salt culture medium with the quizalofop-p-ethyl concentration of 50mg/L in an inoculation amount of 1 vt%, the culture is carried out in a shaking table at 37 ℃ and 180rpm, the concentration of the quizalofop-p-ethyl in the solution is measured after sampling and filtering every 4 hours, the effect is shown in figure 3, and the degradation curve is shown in figure 4.

Example 3: determination of Effect of Achromobacter (Achromobacter sp) MBZ-2 on degradation of p-toluenesulfonate

Achromobacter (Achromobacter sp) MBZ-2 is streaked on an LB solid plate, then a single colony is picked from the LB solid plate, inoculated in an LB liquid culture medium, cultured in a shaker at 37 ℃ and 180rpm until OD600 is 1.0, centrifuged at 10000rpm, and then a supernatant is discarded, washed three times with sterile water and resuspended in sterile water with the same volume to be used as a seed solution. The MBZ-2 seed solution is inoculated in an inorganic salt culture medium with the concentration of the p-toluenesulfonic acid of 500mg/L in an inoculation amount of 1 vt%, the culture is carried out in a shaker at 37 ℃ and 180rpm, the p-toluenesulfonic acid concentration in the solution is measured after sampling and filtering every 4h, the effect is shown in figure 2, and the degradation curve is shown in figure 5.

Example 4: herbicide pollutant degradation substrate spectrum of Achromobacter sp MBZ-2

Respectively adding 50mg/L alachlor, acetochlor, pretilachlor, 2, 4-dichlorophenoxyacetic acid and fluazifop-p-butyl into inorganic salt culture media, inoculating MBZ-2 seed liquid into each culture medium by the inoculation amount of 1 vt%, performing shake culture at 37 ℃ and 180rpm for 36h, measuring the residual concentration of pollutants by utilizing high performance liquid chromatography, and calculating the degradation rate. The results are given in Table 1 below.

TABLE 1

Contamination of the body	Degradation rate
		Alachlor	14.5％
Acetochlor	11.3％
		Pretilachlor	5.5％
2, 4-Dichlorophenoxyacetic acid	77.2％
		Fluazifop-p-butyl	92.3％

The invention provides a thought and a method for achromobacter and application thereof in degrading p-toluenesulfonic acid and quizalofop-p-ethyl, and a plurality of methods and ways for realizing the technical scheme are provided. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Nanjing university of agriculture

<120> achromobacter and application thereof in degradation of p-toluenesulfonic acid and quizalofop-p-ethyl

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1436

<212> DNA

<213> Achromobacter (Achromobacter sp)

<400> 1

gggaaagcgg gagcttaaca tgcaagtcga acggcagcac ggacttcggt ctggtggcga 60

gtggcgaacg ggtgagtaat gtatcggaac gtgcccagta gcgggggata actacgcgaa 120

agcgtagcta ataccgcata cgccctacgg gggaaagcag gggatcgcaa gaccttgcac 180

tattggagcg gccgatatcg gattagctag ttggtggggt aacggctcac caaggcgacg 240

atccgtagct ggtttgagag gacgaccagc cacactggga ctgagacacg gcccagactc 300

ctacgggagg cagcagtggg gaattttgga caatggggga aaccctgatc cagccatccc 360

gcgtgtgcga tgaaggcctt cgggttgtaa agcacttttg gcaggaaaga aacgtcgcgg 420

gttaatacct cgcgaaactg acggtacctg cagaataagc accggctaac tacgtgccag 480

cagccgcggt aatacgtagg gtgcaagcgt taatcggaat tactgggcgt aaagcgtgcg 540

caggcggttc ggaaagaaag atgtgaaatc ccagagctta actttggaac tgcattttta 600

actaccgggc tagagtgtgt cagagggagg tggaattccg cgtgtagcag tgaaatgcgt 660

agatatgcgg aggaacaccg atggcgaagg cagcctcctg ggataacact gacgctcatg 720

cacgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc ctaaacgatg 780

tcaactagct gttggggcct tcgggccttg gtagcgcagc taacgcgtga agttgaccgc 840

ctggggagta cggtcgcaag attaaaactc aaaggaattg acggggaccc gcacaagcgg 900

tggatgatgt ggattaattc gatgcaacgc gaaaaacctt acctaccctt gacatgtctg 960

gaatgccgaa gagatttggc agtgctcgca agagaaccgg aacacaggtg ctgcatggct 1020

gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtca 1080

ttagttgcta cgaaagggca ctctaatgag actgccggtg acaaaccgga ggaaggtggg 1140

gatgacgtca agtcctcatg gcccttatgg gtagggcttc acacgtcata caatggtcgg 1200

gacagagggt cgccaacccg cgagggggag ccaatcccag aaacccgatc gtagtccgga 1260

tcgcagtctg caactcgact gcgtgaagtc ggaatcgcta gtaatcgcgg atcagcatgt 1320

cgcggtgaat acgttcccgg gtcttgtaca caccgcccgt cacaccatgg gagtgggttt 1380

taccagaagt agttagccta accgcaaggg gggcgatacc acggtagatc gttgcg 1436

Claims

1. Achromobacter strain classified and named Achromobacter (A)Achromobacter sp) The strain is named as MBZ-2, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation date of 2020, 8 months and 13 days, and has the preservation number of CGMCC number 20529.

2. The use of the Achromobacter bacterium of claim 1 for degrading p-toluenesulfonic acid or quizalofop-p-ethyl in vitro.

3. A microbial preparation comprising the Achromobacter bacterium according to claim 1.

4. The method for preparing the microbial agent according to claim 3, wherein the Achromobacter strain according to claim 1 is inoculated into an LB liquid medium, cultured, centrifuged, and the precipitate is washed and then resuspended in water.

5. The method of claim 4, wherein the resuspension is performed by resuspending the washed pellet in water in equal volume.

6. Use of the microbial inoculant of claim 3 for in vitro degradation of p-toluenesulfonic acid.

7. The use of the microbial inoculant of claim 3 for the in vitro degradation of quizalofop-p-ethyl.

8. The use as claimed in claim 7, wherein the microbial inoculum is inoculated into quizalofop-p-ethyl-containing material to degrade quizalofop-p-ethyl.

9. The use of claim 8, wherein the ratio of the amount of the microbial agent to the amount of quizalofop-p-ethyl is 0.2 mL/mg.

10. The use according to claim 8, wherein the degradation is a degradation by incubation in a shaker at 37 ℃ and 180 rpm.