CN110591981A

CN110591981A - Paracoccus P-2 for degrading deltamethrin and application thereof

Info

Publication number: CN110591981A
Application number: CN201911016671.6A
Authority: CN
Inventors: 孟庆国; 顾伟; 王文; 唐梦月; 郝文静
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2019-10-24
Filing date: 2019-10-24
Publication date: 2019-12-20
Anticipated expiration: 2039-10-24
Also published as: CN110591981B

Abstract

The invention provides a paracoccus P-2 for degrading deltamethrin and application thereof, relating to the technical field of biological engineering. The preservation number of the paracoccus P-2 is CCTCC M2019721. The paracoccus P-2 has a good degradation effect on deltamethrin, the residual quantity of the deltamethrin in the hepatopancreas and muscle tissues of the eriocheir sinensis can be remarkably reduced, the degradation rate of the deltamethrin in the hepatopancreas by the paracoccus P-2 is 60-80%, the degradation efficiency in the muscle is more than 60%, and the paracoccus P-2 can be prepared into a deltamethrin degradation agent and applied to reducing the content of the deltamethrin in the eriocheir sinensis.

Description

Paracoccus P-2 for degrading deltamethrin and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a paracoccus P-2 for degrading deltamethrin and application thereof.

Background

Chinese mitten crabs (Eriocheir sinensis) also called river crabs are popular among people because of delicious taste, rich nutritional value, simple cooking and the like. At present, the breeding quantity and the market demand of river crabs in China are increased at a high speed, and the problems of quality safety and the like are also concerned.

For a long time, the pyrethroid is always considered as a bionic pesticide with high insecticidal efficiency and low toxicity, and becomes organic phosphorus and chlorine after being put into the market; the pesticide-like is effectively replaced. Especially, in the process of culturing economic aquatic animals, the pyrethroid pesticide is introduced to treat fish parasitic diseases, and the effect is very obvious. Therefore, with the continuous expansion of the application range of the pesticide, the disadvantages of the pesticide are gradually revealed, for example, the pyrethroid pesticide is fat-soluble, can invade into aquatic animals such as gills and muscles, and influences the metabolic activity of the aquatic animals through the gills and the like, so that the pyrethroid pesticide residue seriously exceeds the standard, a large amount of death of the aquatic animals is caused, and the pesticide also causes great troubles to the life of human beings and the bearing capacity of the environment.

Disclosure of Invention

In view of the above, the invention aims to provide a paracoccus P-2 for degrading deltamethrin and an application thereof, which can reduce or eliminate the problem of high residual deltamethrin in aquatic economic animals.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides Paracoccus sp-2 for degrading deltamethrin, wherein the preservation number of the Paracoccus P-2 is CCTCC M2019721.

Preferably, the 16S rDNA sequence of paracoccus P-2 is as shown in SEQ ID NO: 1 is shown.

The invention also provides a deltamethrin degrading agent which comprises the paracoccus P-2.

The invention also provides application of the paracoccus P-2 or the deltamethrin degrading agent in reducing the content of deltamethrin in the eriocheir sinensis.

Preferably, in the application, the Paracoccus P-2 is added into the culture pond of the Eriocheir sinensis, and the addition amount of the Paracoccus P-2 is10⁸CFU/L。

Preferably, the pH value of the water in the culture pond is 7-8.

Preferably, the concentration of deltamethrin in the culture pond is less than 150 mg/L.

The invention provides Paracoccus sp-2 for degrading deltamethrin, wherein the preservation number of the Paracoccus P-2 is CCTCC M2019721. The degradation characteristics of the paracoccus P-2 are analyzed, the degradation characteristics of the strain are researched by a method for controlling a single-factor variable, and the influence of different inoculation amounts, pH values and initial pesticide concentrations on the degradation capability of the strain is determined. The results show that the bacterial strain has good degradation effect when the pH is 7, the substrate concentration is low and the inoculation amount is large.

Meanwhile, the degradation effect of the strain P-2 on the deltamethrin is verified under the laboratory culture condition. Adding deltamethrin pesticide into the culture water body of the eriocheir sinensis to ensure that the initial concentration reaches 3.0 mu g/L, and then adding separated paracoccus P-2 to ensure that the concentration of the bacterial liquid in the water body is 6.0 multiplied by 10⁸CFU/L. The result shows that in the experimental group added with the degrading strain, the residual quantity of deltamethrin in the hepatopancreas and muscle tissues of the eriocheir sinensis is obviously reduced, the degrading rate of the paracoccus P-2 to the deltamethrin in the hepatopancreas is between 60 and 80 percent, and the degrading efficiency in the muscle is over 60 percent. The bacterial strain is proved to be capable of effectively reducing the residue of deltamethrin in the eriocheir sinensis body, can be used for preparing deltamethrin degradation agent and is applied to reducing the content of deltamethrin in the eriocheir sinensis body.

Drawings

FIG. 1 shows the morphology of strain P-2 on solid MCM medium;

FIG. 2 is a gram stain of strain P-2;

FIG. 3 is a phylogenetic tree of strain P-2;

FIG. 4 is a linear equation of a standard curve of deltamethrin concentration and response value;

FIG. 5 is a graph of the effect of initial concentrations of deltamethrin on degradation;

FIG. 6 is the effect of pH on degradation;

FIG. 7 is a graph of the effect of inoculum size on degradation;

FIG. 8 shows the residual amount of deltamethrin in the hepatopancreas of Eriocheir sinensis;

FIG. 9 shows the residual amount of deltamethrin in the muscle of Eriocheir sinensis.

Biological preservation information

Paracoccus (Paracoccus sp.) for degrading deltamethrin, the serial number of the strain is P-2, the preservation time is 2019.9.12, the preservation place is China Center for Type Culture Collection (CCTCC), the specific address is Bayioney Lojia mountain in Wuchang district, Wuhan City, Hubei province, and the preservation number is CCTCC M2019721.

Detailed Description

The paracoccus P-2 is separated from bottom mud soil of a eriocheir sinensis culture pond polluted by pesticide, and the paracoccus P-2 can grow by taking deltamethrin as a unique carbon source. The bacterial colony of the paracoccus P-2 is round and convex, milky, glossy and viscous, and the paracoccus P-2 is gram-negative bacteria and spherical; the paracoccus P-2 is unable to hydrolyze starch and not liquefy gelatin. The 16S rDNA sequence of paracoccus P-2 is shown as SEQ ID NO: 1, and the following components: AACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCCTTCGGGGTGAGCGGCGGACGGGTGAGTAACGCGTGGGAATATGCCCTTTGGTACGGAATAGTCCTGGGAAACTGGGGGTAATACCGTATGCGCCCTTCGGGGGAAAGATTTATCGCCAAAGGATTAGCCCGCGTTGGATTAGGTAGTTGGTGGGGTAATGGCCTACCAAGCCGACGATCCATAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAATGGGGGCAACCCTGATCTAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCAGCTGGGAAGATAATGACGGTACCAGCAGAAGAAGCCCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGACCGGAAAGTTGGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTCAAAACTATCGGTCTGGAGTTCGAGAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACAGGCTCGATACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCCAGTCGTCGGGCAGCATGCTGTTCGGTGACACACCTAACGGATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAACCCTTGACATCCCAGGACCGGCCCGGAGACGGGTCTTTCACTTCGGTGACCTGGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTCGGTTAAGTCCGGCAACGAGCGCAACCCACACTCTTAGTTGCCAGCATTTGGTTGGGCACTCTAAGAGAACTGCCGATGATAAGTCGGAGGAAGGTGTGGATGACGTCAAGTCCTCATGGCCCTTACGGGTTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGTTAATCCCCAAAAGCCATCTCAGTTCGGATTGGGGTCCGCAACTCGACCCCATGAAGTTGGAATCGCTAGTAATCGCGGAACAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGGTCTACCCGACGGCCGTGCGCTAACCAGCAATGGGGGCAGCGGACCACGGTAGGCTCAGCGACTGGGGTG are provided.

The invention also provides a deltamethrin degrading agent which comprises the paracoccus P-2. The concentration of the paracoccus P-2 in the deltamethrin degradation agent is not particularly limited, and the preferable working concentration reaches 6 x 10⁸And (5) CFU/L. The source and the type of the auxiliary materials contained in the deltamethrin degradation agent are not specially limited.

In the application, the Paracoccus P-2 is preferably added into the culture pond of the Eriocheir sinensis, and the addition amount of the Paracoccus P-2 is 10⁸CFU/L. When the deltamethrin is degraded, the pH value of water in the culture pond is preferably 7-8, and more preferably 7. In the invention, when the concentration of deltamethrin in the culture pond is less than 150mg/L, the degradation effect is higher.

The invention provides a method for degrading deltamethrin by using the paracoccus P-2 and the application thereof, which are described in detail in the following examples, but the invention is not limited to the scope of the invention.

Example 1

1. Sample collection

Collecting a bottom mud sample from bottom mud of a eriocheir sinensis culture pond polluted by pesticide for a long time. Removing non-soil substances such as gravel, algae and the like after obtaining the substrate sludge sample, filling the substrate sludge sample into a sample bottle, labeling and marking the collection time, and storing the sample in a refrigerator at 4 ℃ for strain screening.

2. Drugs and reagents

The drugs and reagents used in this example are shown in table 1:

TABLE 1 reagent name and Source

Name of reagent	Purity of	Manufacturer of the product
			NH₄Cl	A·R	Xilong chemical corporation
KH₂PO₄	A·R	Xilong chemical corporation
			K₂HPO₄	A·R	Xilong chemical corporation
(NH₄)₂SO₄	A·R	Xilong chemical corporation
			NH₄NO₃	A·R	Xilong chemical corporation
NaNO₃	A·R	Xilong chemical corporation
			MgSO₄·7H₂O	A·R	Chemical reagents of national drug group Co Ltd
Yeast cream	A·R	Chemical reagents of national drug group Co Ltd
			Peptone	A·R	Beijing Oobo Star Biotechnology Limited liability company
Beef extract	A·R	Shanghai Bo microbial science and technology Co., Ltd
			Agar powder	A·R	Beijing Solaibao Tech & ltTech & gt Ltd
Deltamethrin standard substance	99.5％	National pesticide quality supervision and inspection center

3. Apparatus and device

The instruments and equipment used in this example are shown in table 2:

TABLE 2 names and sources of instruments and devices

4. Preparation of culture medium

Enrichment culture medium: peptone 10.0g, NaCl 1.0g, KH₂PO41.0 g and 1000mL of water, and adjusting the pH value to 7.0-8.0.

Basal salt medium (MCM): NH (NH)₄NO₃ 1.0g，MgSO₄·7H₂O 0.5g，(NH₄)₂SO₄ 0.5g，KH₂PO₄0.5g，NaCl 0.5g，K₂HPO₄1.5g, 0.05g of yeast extract and 1000mL of water, and adjusting the pH value to 7.0-8.0.

MCM solid medium: adding agar about 2% to the culture medium to obtain corresponding solid culture medium.

Starch hydrolysis experiment culture medium: 0.5g of beef extract, 0.5g of NaCl, 1.0g of peptone, 0.2g of starch, 2.0g of agar powder and 100mL of distilled water. The tubes were separated in 5mL tubes to a height of about 1/4. Iodine solution is used as indicator.

Gelatin culture medium: 1.0g of peptone, 12.0g of gelatin, 0.5g of beef extract, 0.5g of NaCl, 7.0-7.6 of pH value, 100mL of distilled water and 2.0g of agar powder. The tubes were separated in 5mL tubes to a height of about 1/4.

Sugar fermentation medium: peptone 1.0g, NaCl 0.5g, 1.6% bromocresol purple ethanol solution 0.2mL, pH 7.0, distilled water 100 mL. 1mL of each 20% sugar solution of different types was prepared and aliquoted in 5mL portions to a height of about 1/4 in a test tube.

Indole culture medium: peptone 0.5g, glucose 0.5g, K₂HPO₄0.2g and 100mL of distilled water, and the pH was adjusted to 7.0. The tubes were separated in 5mL tubes to a height of about 1/4. Indole reagent: 1.0g of p-dimethylaminobenzaldehyde; 95mL of ethanol (95%); concentrated hydrochloric acid 20 mL.

M.R culture medium: 0.5g of glucose, 0.5g of peptone and 0.5g of NaCl, adjusting the pH value to 7.0-7.2, and 100mL of distilled water. The tubes were separated in 5mL tubes to a height of about 1/4. Indicator (b): methyl red 0.10g, 95% ethanol 300mL, distilled water 200 mL.

V.P experiments identify the culture media: peptone 0.5g, glucose 0.5g, K₂HPO₄0.2g, 100mL of distilled water, and pH 7.0-7.2. The tubes were separated in 5mL tubes to a height of about 1/4.

The above culture media were sterilized at 121 deg.C for 30min before use.

5. Experimental methods

5.1 isolation and screening of strains

(1) Weighing 1.0g of bottom mud of the eriocheir sinensis pond, adding the bottom mud into 100mL of enrichment medium with 100mg/L of deltamethrin, and uniformly mixing the bottom mud and the enrichment medium by oscillation;

(2) culturing at 30 ℃ for 7d under the culture condition of 180 r/min;

(3) taking out the culture solution, inoculating the culture solution into a fresh enrichment culture medium by 10 percent of inoculation amount, and increasing the concentration of deltamethrin to 200 mg/L;

(4) repeating the step (3), and increasing the concentration of deltamethrin until the final concentration is 500mg/L to obtain an enrichment culture solution;

(5) taking out the enrichment culture solution, inoculating the enrichment culture solution into a basic salt (MCM) culture medium by 10 percent of inoculation amount, adding deltamethrin to ensure that the final concentration is 50mg/L, and culturing for 7d under the constant temperature culture condition of 180r/min at 30 ℃;

(6) repeating the step (5) to obtain a culture solution after domestication;

(7) 1mL of domesticated culture solution is taken for gradient dilution and diluted into 5 concentrations: 10^-4～10^-8；

(8) Preparing an MCM solid culture medium (deltamethrin is a unique carbon source) with the deltamethrin concentration of 500mg/L, coating the diluent on the solid culture medium, carrying out constant-temperature culture in an oven at 30 ℃ for 3d, and observing the growth condition of colonies;

(9) selecting a flat plate with proper growing density of bacterial colonies for observation, and selecting bacterial colonies with different colors, different smoothness degrees and good growing conditions for purification;

(10) dipping a small amount of strains by using an inoculating loop, repeatedly streaking and purifying on an MCM solid culture medium by adopting a Z-shaped streaking method until a pure single colony is obtained;

(11) and selecting a single colony, adding the single colony into an LB culture medium for amplification culture, and obtaining pure bacterial liquid for storage.

Short-term preservation: the strain is streaked and cultured on MCM solid nutrient medium with the concentration of the deltamethrin being 500mg/L, sealed by a sealing film, stored in a refrigerator at 4 ℃, and the storage time is recorded.

And (3) long-term storage: the bacterial liquid was mixed with equal amount of 40% (W/V) sterilized glycerol, stored in an ultra-low temperature refrigerator at-80 ℃ and the storage time was recorded.

The shape of Paracoccus P-2 on MCM solid medium is shown in figure 1, and the Paracoccus P-2 is round and convex, milky white, glossy and sticky. When the bacterial strain grows on a solid basic salt culture medium which takes deltamethrin as a unique carbon source, a larger colony can grow.

5.2 physiological and Biochemical identification of the Strain

(1) Gram stain

Preparing a smear: under the aseptic condition, a clean glass slide is taken, a drop of bacteria liquid is dripped into the center (the smear is not too thick, the phenomenon that the bacterial dispersion does not affect the dyeing effect) and the glass slide is dried and fixed (the bacterium coating surface faces upwards during fixation, and the glass slide can not be overheated for 2-3 times through flame).

Primary dyeing: adding a drop of crystal violet, dyeing for 1min, washing with water gently, and removing water drops.

Coal dyeing: and (4) dropwise adding iodine solution, dyeing for 1min, washing with water, and removing water drops.

And (3) decoloring: decolorizing with 95% alcohol, shaking the slide slightly, washing with water until the flowing alcohol is not purple, and washing with water slightly after about 30 s.

Counterdyeing: adding safranin staining solution, and washing after 1 min.

Microscopic examination: after natural drying, the mixture was observed under a microscope.

The gram stain results are shown in FIG. 2, where the Paracoccus P-2 strain is gram-negative, coccoid.

(2) Starch hydrolysis test

Shaking the bacterial liquid to a growth logarithmic phase, dipping a small amount of bacterial liquid by using an inoculating loop, inoculating the bacterial liquid on a starch plate, and culturing for 24-48 h at 30 ℃. Iodine solution was then added dropwise to the vicinity of the inoculated colonies, and the whole colony was covered with iodine solution, and the result was immediately examined. If the reaction is positive (i.e., the starch is decomposed), the plate becomes blue as a whole, but the starch is decomposed around the colonies, and a white transparent ring appears.

(3) Experiment of liquefaction of gelatin

Shaking the bacterial liquid to the logarithmic phase of growth, taking 20 mu L of bacterial liquid by using a sterile syringe, penetrating through gelatin colloid, inoculating the bacterial liquid to the center of the culture medium, and slowly injecting to avoid breaking the gel. The culture was carried out at room temperature. From day 2 onwards, it was observed daily whether gelatin could be liquefied by bacteria, since some details could produce gelatinase, which hydrolyzes gelatin into polypeptides, and upon decomposition into amino acids, liquefaction occurred. Those that can cause this phenomenon are positive.

(4) Sugar fermentation experiments:

the bacterial solution was shaken to the logarithmic phase of growth, 20. mu.L of the bacterial solution was taken with a sterile syringe, inoculated into the central part of the medium, cultured at room temperature, and observed every day. If the bromocresol purple turns yellow, the test result is judged to be positive.

(5) Indole experiments

Shaking the bacterial liquid to a growth logarithmic phase, adding 20 mu L of the bacterial liquid into an indole culture medium, culturing at room temperature for 72h, adding an indicator, and judging the bacterial liquid to be positive if a red annular ring appears on the surface layer of the liquid surface. Care was taken not to shake the tube and add the indicator slowly dropwise along the tube wall.

(6) Methyl Red experiment (M. R experiment)

Shaking the bacterial liquid to a growth logarithmic phase, adding 20 mu L of the bacterial liquid into an M.R culture medium, culturing at a constant temperature of 30 ℃, taking out 1ml of the culture liquid after 24 hours, 48 hours, 72 hours and 96 hours respectively, adding a small amount of methyl red indicator (about 2 drops), and judging the culture liquid to be positive if the culture liquid is bright red.

(7) Acetylmethylmethanol experiment (V. P experiment)

And shaking the bacterial liquid to a growth logarithmic phase, adding 20 mu L of the bacterial liquid into a V.P culture medium, and culturing at the constant temperature of 37 ℃ for 1-2 days. Taking out the culture solution, shaking for 2min, adding 5-10 drops of 40% NaOH solution and 5% 1-naphthol solution with the same amount, shaking the test tube again, keeping the temperature at 37 ℃ for 30min, taking out the culture solution, observing, and judging as positive reaction if the culture solution is red.

(8) Catalase test

Dipping a small amount of bacteria on a new MCM plate culture medium by adopting a scribing method, picking fresh single bacterial colonies after the bacteria grow out, adding the single bacterial colonies into a sterile centrifuge tube, and dropwise adding 3% H₂O₂When 2mL of the solution was observed to have bubbles within 30 seconds, the solution was judged to be positive.

The results of the experiment are shown in table 3:

TABLE 3 physiological and biochemical characteristics of Strain P-2

Physiological and biochemical characteristics	Identification results	Standard bacterium	Physiological and biochemical characteristics	Identification results	Standard bacterium
						Starch hydrolysis	-	-	V.P experiment	-	-
Gelatin test	-	-	Catalase test	+	+
						Indole experiments	-	-	Glucose	-	-
M.R experiment	-	-	Sucrose	-	-

The physiological and biochemical characteristics show that the strain P-2 can not hydrolyze starch and liquefy gelatin, the V.P experiment and the M.R experiment are negative, and the catalase experiment is positive.

(9) Determination of strain species

The 16S rDNA sequence measured by the strain is compared and analyzed in a GenBank database by using BLAST, and the similarity of the sequence and the gene sequence of the strain such as Paracoccus sp is found to reach 99 percent. The phylogenetic tree, physiological, biochemical and morphological characteristics shown in FIG. 3 were combined to confirm that the strain P-2 is Paracoccus.

Example 2

1. Detection of deltamethrin content in culture solution

(1) Mixing 1mL culture solution, 2mL acetone and 2mL n-hexane, performing ultrasonic extraction for 20min, centrifuging at 6000r/min for 5min, and collecting the upper organic phase;

(2) repeating the step (1), and combining the obtained two extraction liquids;

(3)70℃N₂drying, and keeping the volume of normal hexane to 1 mL;

(4) pre-leaching the Florisil column by using normal hexane and acetone (9+1, V + V), and discarding the leaching solution;

(5) when the liquid level of the solvent reaches the upper surface in the column, immediately pouring the sample obtained in the step (3), and collecting leacheate;

(6) using N as the sample in (5)₂Drying, and keeping the volume of normal hexane to 1 mL;

(7) 100 mu L of the solution is diluted by n-hexane for 100 times to be detected.

The gas chromatographic conditions are represented as follows: the chromatographic column adopts a DB-1 nonpolar capillary chromatographic column 30m 0.25 μm;

temperature programming: maintaining at 80 deg.C for 1min, increasing to 230 deg.C at 15 deg.C/min, and maintaining for 1 min; raising the temperature to 270 ℃ at a speed of 40 ℃/min, and keeping the temperature for 10 min; sample inlet temperature: no shunt sampling at 240 ℃;

flow rate: 1.2mL/min, constant current; sample introduction amount: 1 mu L of the solution; a detector: ECD, 320 ℃; flow rate of carrier gas: nitrogen (N)₂) And the tail blowing flow rate is 60 mL/min.

Deltamethrin degradation rate (%) [ 1- (measured residue/control measured residue) ] × 00%

2. Deltamethrin standard recovery rate determination

(1) Adding deltamethrin into a sterilized basic salt liquid culture medium to make the final concentrations of the deltamethrin respectively 40, 80, 120, 160 and 320 mu g/L;

(2) taking 2mL of each concentration, adding 4, 4 and 3mL of n-hexane in sequence, fully oscillating for 2min, performing ultrasonic extraction for 20min, centrifuging at 6000r/min for 10min, and taking an upper organic phase;

(3) after extraction for three times, combining the extracts, filtering, adding a proper amount of anhydrous sodium sulfate to absorb water, using normal hexane to fix the volume to 10mL, and measuring by adopting a gas chromatography-mass spectrometry method. And calculating a regression equation according to the peak area, and calculating the concentration of the sample according to the equation.

The response value of deltamethrin is used as ordinate (Y) and the injection concentration is used as abscissa (X) to make a graph, and the standard curve of deltamethrin is obtained as shown in figure 4, and the linear regression equation is as follows: y 2474.324107 x, correlation coefficient 0.99088274. Therefore, the concentration and the response value of the deltamethrin have a good relationship, and the deltamethrin can be used for detecting the pyrethroid pesticides.

3. Effect of initial concentration on the degradation Rate of the Strain

Inoculating strain seed liquid into basic salt culture medium with final concentration of deltamethrin of 20, 50, 100, 150 and 200mg/L in an inoculation amount of 10%, culturing at 30 ℃ and 180r/min, setting three times for each group, and detecting deltamethrin residue after 5 d.

The results are shown in fig. 5, where represents significant differences (./p < 0.05); as the initial concentration of the pesticide increases, the degradation rate of paracoccus P-2 gradually decreases. When the concentration of the deltamethrin is 20mg/L, the 5d degradation rate is the highest and reaches 88.6%, the degradation effect is inhibited along with the increase of the substrate concentration, and when the concentration of the deltamethrin is 150mg/L, the 5d degradation rate is 83.2%, and is obviously reduced compared with that of the deltamethrin 20 mg/L. The overall degradation effect is obvious, and the degradation rate reaches more than 80 percent.

4. Influence of pH value on degradation rate of strain

In a basic salt culture medium with 100mg/L of deltamethrin, the pH is adjusted to be 2.0, 4.0, 6.0, 7.0 and 8.0. Inoculating strain seed liquid according to the inoculation amount of 10%, culturing at 30 ℃ for 5d at 180r/min, setting three times for each group, and detecting the residual amount of deltamethrin.

The results are shown in fig. 6, where p represents significant differences (× p <0.01, × p < 0.001); the pH value has a great influence on the degradation rate of the strain P-2. The result shows that the degradation effect is poor under the condition of strong acid or strong alkali. When the pH is 7, the degradation rate of 5d is 86.6% at most, and by taking the group as a control, when the pH is 2, 4 and 6, the degradation efficiency of 5d is 24.3%, 36.8% and 60.2% respectively, the degradation rate is obviously reduced, as can be seen from the trend in the figure, the degradation efficiency of the strain begins to show a reduction trend along with the alkaline environment, and when the pH is 8, the degradation efficiency is 74.4%.

5. Effect of inoculum size on degradation rate of strains

Inoculating fresh seed liquid of strains in a basic salt culture medium with 100mg/L of deltamethrin in an inoculation amount of 1%, 3%, 5%, 10% and 15%, culturing at 30 ℃, sampling after 180r/min and 5d, setting three times for each group, and determining the residual quantity of deltamethrin.

Results are shown in fig. 7, representing significant differences (./p < 0.01); the degradation efficiency and the inoculation amount of the strain show a positive correlation trend. The group with the inoculation amount of 15% is used as a control, when the inoculation amount is 1% and 3%, the degradation rate is obviously reduced, and when the inoculation amount reaches 5%, the difference is not obvious. The degradation rates at the inoculum levels of 10% and 15% were 80.1% and 82.5%, respectively.

6. Laboratory use of degrading strain P-2

Deltamethrin is prepared into mother liquor of 100mg/L, 10L of water is added into a culture box, deltamethrin is added to ensure that the final concentration is 3 mug/L, and the group is a control group. Adding deltamethrin with the same concentration into another culture box, and adding paracoccus P-2 to ensure that the concentration of the bacterial liquid in the water body is 6.0 multiplied by 10⁸CFU/L, this group is the experimental group.

After standing for 24h, 30 Chinese mitten crabs are respectively put into the two groups of breeding boxes. After 3, 6, 9, 12 and 15 days of cultivation, the two cultivation boxes respectively take five Chinese mitten crabs out of the five cultivation boxes, respectively take out livers and muscles, and put the livers and the muscles at the temperature of minus 80 ℃ for detecting the pesticide residue in each tissue. Taking the liver and muscle of normal Eriocheir sinensis as control.

Sample treatment:

(1) and adding 10mL of acetonitrile and 2g of NaCl into a centrifuge tube by taking 2g of the tissue homogenate sample, and fully whirling, vibrating and uniformly mixing. Centrifuging for 10min under 10000r/min, and taking the supernatant;

(2) repeating the above operations, and combining; clear liquid;

(3)80 ℃ water bath, N₂Drying;

(4) redissolving the residue with 200mL of n-hexane;

LC-Florisil solid phase extraction column

(5) Sequentially activating the small column by using 5mL of acetone and n-hexane (V + V is 1+9), and then adding 5mL of n-hexane;

(6) adding a sample solution, and collecting eluent;

(7) adding 5mL of acetone and n-hexane (V + V ═ 1+9) again for repeated elution, and collecting the eluent;

(8) at 50 deg.C，N₂And drying, and then adding n-hexane to a constant volume of 1mL for detection.

The residual amount of deltamethrin in the hepatopancreas of Eriocheir sinensis is shown in FIG. 8, wherein AS is a standard group; DM is deltamethrin group; DM + P-2 is deltamethrin + P-2; blank is normal hepatopancreas; represents significant differences (. about.. p.)<0.01，***p<0.001). The method is proved to be true and effective in detecting the residual quantity by taking no pesticide and degrading bacteria AS reference, the standard addition level (AS) is 4.0 mug/kg, and the detected deltamethrin content is 3.91 +/-0.26 mug/kg. And taking a group added with 3.0 mu g/L of deltamethrin as a control, the deltamethrin content in the hepatopancreas of the eriocheir sinensis shows a trend of increasing first and then decreasing, the deltamethrin content in the hepatopancreas reaches the maximum value of 3.28 +/-0.48 mu g/kg at the 6 th day, and then gradually decreases until the 12 th day, and the deltamethrin residue in the hepatopancreas is below the detection limit. The addition amount of deltamethrin is 3.0 mu g/L, and the added bacterial liquid is 6 multiplied by 10⁸In the CFU/L experimental group, the content of deltamethrin also shows a trend of increasing and then decreasing, at the 6 th day, the content of deltamethrin in the hepatopancreas reaches the maximum value of 1.22 +/-0.28 mu g/kg, and then gradually decreases, and after 12 days, deltamethrin residue cannot be detected in the hepatopancreas. Differential analysis shows that pesticide residue in the experimental group added with the paracoccus P-2 is obviously reduced compared with that in the control group. The degradation rate of the paracoccus P-2 to the deltamethrin is between 60 and 80 percent, and the degradation rate reaches the highest value at the 9 th day, namely 80 percent.

The residual amount of deltamethrin in the muscle of Eriocheir sinensis is shown in FIG. 9, wherein AS is a standard group; DM is deltamethrin group; DM + P-2 is deltamethrin + P-2; blank normal muscle. Note: represents significant differences (× p <0.05, × p <0.01, × p < 0.001). The normal eriocheir sinensis is used AS a reference, no deltamethrin residue is detected in the hepatopancreas of the normal eriocheir sinensis, the standard addition level (AS) is 4.0 mu g/kg, and the deltamethrin residue after the standard addition is detected to be 4.07 +/-0.39 mu g/kg, so that the method is proved to be true and effective in residue detection. The group with the addition of 3.0 mu g/L of deltamethrin is taken as a control group, and the detected trend that the content of deltamethrin in the muscle tissue of the eriocheir sinensis also shows a trend of increasing first and then decreasing is consistent with the detected trend of pesticide residue in the liver and pancreas. At 6d, the deltamethrin content in the muscle tissue reaches 3.93 plus or minus 0.47 mug/kg at the maximum, after which the deltamethrin content in the muscle tissue starts to decrease gradually until reaching the lowest value at 15d, which is 2.38 plus or minus 0.21 mug/kg. In the experimental group added with the pesticide and the paracoccus P-2, the content of deltamethrin in the muscle of the eriocheir sinensis also shows a trend of increasing firstly and then decreasing, and the difference analysis shows that the pesticide residue in the experimental group is obviously reduced compared with that in the control group after the degrading bacteria are added. At 12d, the degradation efficiency is highest and reaches 72%, the overall degradation efficiency in muscle tissues is lower than that of hepatopancreas, and the degradation rate is higher and reaches more than 60% at 3d, 12d and 15 d.

The paracoccus P-2 has a good degradation effect on deltamethrin, the residual quantity of the deltamethrin in hepatopancreas and muscle tissues of eriocheir sinensis can be remarkably reduced, the degradation rate of the deltamethrin in the hepatopancreas by the paracoccus P-2 is between 60% and 80%, and the degradation efficiency in muscles is over 60%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of Nanjing university

<120> Paracoccus P-2 for degrading deltamethrin and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1385

<212> DNA

<213> Paracoccus sp.

<400> 1

aacgaacgct ggcggcaggc ctaacacatg caagtcgagc gcacccttcg gggtgagcgg 60

cggacgggtg agtaacgcgt gggaatatgc cctttggtac ggaatagtcc tgggaaactg 120

ggggtaatac cgtatgcgcc cttcggggga aagatttatc gccaaaggat tagcccgcgt 180

tggattaggt agttggtggg gtaatggcct accaagccga cgatccatag ctggtttgag 240

aggatgatca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg 300

gggaatctta gacaatgggg gcaaccctga tctagccatg ccgcgtgagt gatgaaggcc 360

ctagggttgt aaagctcttt cagctgggaa gataatgacg gtaccagcag aagaagcccc 420

ggctaactcc gtgccagcag ccgcggtaat acggaggggg ctagcgttgt tcggaattac 480

tgggcgtaaa gcgcacgtag gcggaccgga aagttggggg tgaaatcccg gggctcaacc 540

ccggaactgc cttcaaaact atcggtctgg agttcgagag aggtgagtgg aattccgagt 600

gtagaggtga aattcgtaga tattcggagg aacaccagtg gcgaaggcgg ctcacaggct 660

cgatactgac gctgaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 720

ccacgccgta aacgatgaat gccagtcgtc gggcagcatg ctgttcggtg acacacctaa 780

cggattaagc attccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg 840

ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc 900

aacccttgac atcccaggac cggcccggag acgggtcttt cacttcggtg acctggagac 960

aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tcggttaagt ccggcaacga 1020

gcgcaaccca cactcttagt tgccagcatt tggttgggca ctctaagaga actgccgatg 1080

ataagtcgga ggaaggtgtg gatgacgtca agtcctcatg gcccttacgg gttgggctac 1140

acacgtgcta caatggtggt gacagtgggt taatccccaa aagccatctc agttcggatt 1200

ggggtccgca actcgacccc atgaagttgg aatcgctagt aatcgcggaa cagcatgccg 1260

cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggga gttgggtcta 1320

cccgacggcc gtgcgctaac cagcaatggg ggcagcggac cacggtaggc tcagcgactg 1380

gggtg 1385

Claims

1. A Paracoccus sp P-2 for degrading deltamethrin is characterized in that the preservation number of the Paracoccus P-2 is CCTCC M2019721.

2. The paracoccus P-2 of claim 1, wherein the 16S rDNA sequence of paracoccus P-2 is as set forth in SEQ ID NO: 1 is shown.

3. A deltamethrin degrading agent comprising paracoccus P-2 of claim 1 or 2.

4. Use of the Paracoccus P-2 according to claim 1 or 2 or the deltamethrin degrading agent according to claim 3 for reducing the content of deltamethrin in Eriocheir sinensis.

5. The application of claim 4, wherein the Paracoccus P-2 is added into the culture pond of the Eriocheir sinensis during the application, and the addition amount of the Paracoccus P-2 is 10⁸CFU/L。

6. The use according to claim 5, wherein the pH value of the water in the culture pond is 7-8.

7. The application of claim 6, wherein the concentration of deltamethrin in the culture pond is 20-150 mg/L.