CN114107092A

CN114107092A - Plant endophyte Gordonia L191 for degrading phthalate and application thereof

Info

Publication number: CN114107092A
Application number: CN202111289654.7A
Authority: CN
Inventors: 刘丽辉; 蔡全英; 张佳研; 莫测辉; 李彦文
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2022-03-01
Anticipated expiration: 2041-11-02
Also published as: CN114107092B

Abstract

The invention discloses a plant endophytic bacterium Gordonia (Gordonia polyisoprenivorans) L191 for degrading phthalate and application thereof, wherein the strain is preserved in Guangdong province microorganism strain preservation center at 24 months 9 in 2021 and has a preservation number of GDMCC NO: 61949. according to the invention, a Gordoniella L191 with good degradation performance on PAEs is screened and separated from wild rice, and researches show that the strain can carry out aerobic degradation by taking DBP and DEHP as unique carbon sources, and after the strain is cultured for 5 days in an inorganic salt liquid culture medium taking DBP and DEHP (200mg/L) as unique carbon sources, the degradation rate of DBP and DEHP reaches 93.3% and 62.5%; and the DBP in the rice tissue fluid can be efficiently degraded, and the degradation rate of the DBP in the rice tissue fluid is up to 99.61% on the 5 th day. The invention provides excellent strains for degrading phthalic acid ester, and has good application value in degrading phthalic acid ester and protecting environment.

Description

Plant endophyte Gordonia L191 for degrading phthalate and application thereof

Technical Field

The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to a plant endophytic bacterium Gordonia L191 for degrading phthalate and application thereof.

Background

Phthalate esters (PAEs) are used as a main plasticizer, are widely applied to plastic products and are distributed in various environmental media along with the production, use and treatment of the products, at present, the PAEs are detected in soil, rivers, lakes, marine sediments, drinking water, garbage sites and even foods, and the PAEs become one of the most common pollutants in the world. Particularly in agricultural production, due to the wide application of plastic films and plastic greenhouses, PAEs remained in agricultural soil are easy to be enriched in human bodies through food chains. PAEs and their metabolites can cause teratogenicity, mutagenicity and carcinogenicity to the body, so the Environmental Protection Agency (EPA) and the national environmental monitoring center (CNEMC) have both listed them as the priority pollutants. The hydrolysis and photolysis rate of PAEs in the environment is very slow, and the microbial degradation is considered as the main process of completely mineralizing PAEs in the natural environment. In recent years, a great deal of research has been carried out on PAEs (PAEs) degraded by bacteria, a great number of strains for efficiently degrading PAEs have been separated from various environments such as mangrove, soil, ocean, river and activated sludge of wastewater treatment plants, and the first problem to be solved in the microbial remediation technology of PAEs-polluted soil is to screen out strains capable of efficiently degrading PAEs.

At present, in the research of PAEs degrading bacteria at home and abroad, more than 80 PAEs degrading bacteria have been screened, including Bacillus, Rhodococcus, Gordonia, Agromyces, Pseudomonas and the like. Most PAEs degrading bacteria are separated and screened from soil or water, for example, patent CN110283755A discloses a Gordoniella terricola RL-JC02 separated from farmland soil, and the degrading rate of polycyclic aromatic hydrocarbon substances or phthalate ester substances in the polluted soil can reach as high as 99.6 percent within 7 days in the aspect of degrading organic pollutants. Although the fertilizer has strong degradation capability, the fertilizer can only be applied to soil or water body pollution, but the survival rate of the fertilizer in soil is low, and the fertilizer and plants are difficult to mutually benefit and symbiotic.

The endophyte can utilize organic pollutants as raw materials required by the growth of the endophyte, can also improve the tolerance of plants to the organic pollutants, and is better suitable for the polluted environment. In addition, the endophyte can also regulate and control the expression of degradation genes of self-degraded pollutants of plants, stimulate the activity of antioxidant enzyme and promote the degradation of the pollutants by the plants. However, there are few reports of plant endophytes capable of degrading PAEs at present, and no PAEs degrading bacteria applied to plants are available; the germplasm resources of the plant endophyte which can degrade PAEs are deficient. Therefore, a new plant endophyte needs to be screened out to be applied to the degradation of PAEs in crops and soil, can well degrade phthalic acid esters in plants or natural environment, and can also promote the mutual beneficial symbiosis of the endophyte and the plants.

Disclosure of Invention

The invention aims to provide a novel plant endophyte capable of degrading phthalate, and provides more efficient strain choices for degrading pollutant phthalate in the environment.

The second purpose of the invention is to provide the application of the Gordonia L191 strain.

The third purpose of the invention is to provide a preparation for degrading phthalate.

The above object of the present invention is achieved by the following technical solutions:

gordonia (Gordonia polyisoprenivorans) L191 strain, which is deposited at 24 months 9 in 2021 in the Guangdong province culture Collection (GDMCC), and the strain number is GDMCC NO: 61949.

the Gordoniella L191 strain is obtained by separating wild rice tissues through a dilution plate coating and scribing method, the bacterial colony of the Gordoniella L191 is pink, the bacterial colony is rod-shaped under a scanning electron microscope, the bacterial colony is identified as a gram-positive bacterium through gram staining, the growth temperature is suitable for 20-40 ℃, and the growth pH is suitable for 5-8. The 16S rRNA sequence of the strain L191 is compared with the 16S rRNA sequences of other registered bacterial strains through a BLAST program of an NCBI official website, and the result shows that the strain has the highest similarity with Gordonia polysopropaniforans and the homology rate reaches 99%.

The research of the invention finds that the strain can carry out aerobic degradation by taking DBP and DEHP as unique carbon sources, and the degradation rates of DBP and DEHP after the strain is cultured for 5 days in an inorganic salt liquid culture medium taking DBP and DEHP (with the concentration of 200mg/L) as unique carbon sources reach 93.3% and 62.5%; and the DBP in the rice tissue fluid can be efficiently degraded, and the degradation rate of the DBP in the rice tissue fluid is up to 99.61% on the 5 th day.

The invention provides application of Gordoniella L191 strain in repairing an environment polluted by organic pollutants.

The invention provides application of Gordonia L191 strain in degrading phthalate in plant tissues.

The invention provides application of Gordonia L191 strain in degrading phthalate.

Preferably, the strain is used for degrading di-n-butyl phthalate (DBP) and/or di (2-ethylhexyl) phthalate (DEHP).

The invention also provides application of the L191 strain in preparation of phthalate degradation products.

Preferably, the strain is applied to the preparation of products for degrading di-n-butyl phthalate (DBP) and/or di (2-ethylhexyl) phthalate (DEHP).

A phthalate-degrading agent comprising the Gordonia L191 strain of claim 1 or a bacterial solution thereof.

Preferably, the concentration of the bacterial liquid is 1 × 10⁸～1×10¹⁰cfu/mL。

The invention provides application of the preparation in degrading and degrading phthalate.

The invention also provides application of the preparation in repairing organic pollutant polluted environment.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a novel plant endophytic bacterium Gordonia (Gordonia polyisoprenivorans) L191 capable of degrading phthalate, and the strain has high activity, simple culture method and strong adaptability; meanwhile, the strain can grow and propagate by using DBP and DEHP as unique carbon sources, and has stronger degradation capability of DBP and DEHP; the DBP in the rice tissue fluid can be efficiently degraded, and the degradation rate of the DBP in the rice tissue fluid is up to 99.61% on day 5; the strain L191 can survive in the rice tissue fluid and can well grow by utilizing the nutrient substances in the rice tissue fluid. Greatly supplements the database of PAEs degrading bacteria. The L191 strain is used as a novel plant endophyte for degrading phthalate and has good application value.

Drawings

FIG. 1 shows the growth pattern of strain L191 cultured on LB medium for 3 days.

FIG. 2 is a 16S rRNA phylogenetic tree of strain L191.

FIG. 3 is a scanning electron micrograph of strain L191.

FIG. 4 shows the effect of strain L191 on the degradation of DBP and DEHP.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 isolation and identification of endophytes

(1) Preparation of culture Medium

Inorganic salt medium (MSM, g/L): k₂HPO₄ 5.8g，KH₂PO₄ 4.5g，(NH₄)₂SO₄ 2.0g，MgCl₂0.16g，CaCl₂ 0.02g，Na₂MoO₄·2H₂O 0.0024g，KNO₃ 0.0012g，FeCl₃ 0.0018g，MnCl₂·2H₂O0.0015 g, and adjusting the pH value of the culture medium to 7.0;

beef extract peptone medium (LB, g/L): 5.0g of yeast powder, 10.0g of peptone and 10.0g of NaCl, adding ultrapure water to 1L, and adjusting the pH to 7.0;

the culture medium is sterilized at 121 deg.C for 20min before use.

(2) Treatment of host plants

Cleaning collected wild rice, processing in a clean bench, cutting root, stem and leaf of host plant into small segments of about 4cm, soaking in 75% alcohol for 5min, and stirring with sterile forceps; then soaking the mixture for 3 minutes by using 0.1 percent mercuric chloride, and stirring the mixture by using a pair of tweezers; after the mercuric chloride was poured out, the mixture was washed 7 times with sterile water for 5 minutes each, and the distilled water from the last washing was left to stand. Then, cutting off two ends of the root, the stem and the leaf with the surfaces being disinfected by about 1cm respectively by using sterile scissors, then cutting the root, the stem and the leaf into pieces in a sterile mortar, and fully grinding the pieces; the milled liquid was inoculated into MSM liquid cultures (200mg/L concentration) using dibutyl phthalate DBP and phthalic acid (22 ethyl hexyl ester) DEHP as sole carbon sources, respectively. And (4) coating the distilled water washed for the last time on an LB solid culture medium, and observing whether microorganisms grow or not to determine whether the surfaces of the host plants are disinfected completely or not.

(3) Separation and purification of bacterial strains

The strain culture conditions are as follows: culturing for 1-3 days at the constant temperature of 28 ℃ and 120r/min in a shaking table. After bacteria grow out, respectively inoculating the bacterial liquid to a fresh MSM solid culture plate, and growing in an incubator at 28 ℃ for 1-3 days. The strain is separated and purified by a dilution coating method and a plate marking method.

The strain is subjected to streak culture on an LB solid culture medium for 7 days, and a colony is pink red, round, opaque, regular in edge and glossy, and is shown in figure 1. The strain morphology and physiological and biochemical characteristic determination indexes comprise the following items: gram staining, detection of appropriate growth temperature and pH range, anaerobic growth test, methyl red test, acetyl methyl methanol (V-P) test, gelatin liquefaction, catalase, indole test, starch hydrolysis, nitrate reduction, ammonia production test, urease and cellulase. The indexes for measuring the physiological and biochemical characteristics of the strains are detailed in table 1.

TABLE 1 measurement of physiological and biochemical characteristics of the strains

Note that: "+" indicates positive; "-" indicates negative

(4) 16S rDNA molecular identification and phylogenetic tree analysis of strains

Transferring the strain to LB liquid culture medium for amplification culture, centrifuging to collect thallus, and extracting total thallus DNA according to the method provided by E.Z.N.A bacteria DNA extraction kit of OMEGA. The genome of the bacterium is subjected to PCR amplification by using a universal primer of the 16S rRNA gene of the bacterium.

After the PCR product is sequenced, the sequencing result is compared with 16S rRNA gene sequence homology reported in GenBank, and a plurality of strains are selected for evolutionary tree analysis, and the result is shown in figure 2, the 16S rRNA gene sequence of the strain L191 obtained by separation and purification of the invention has the highest homology with Gordonia polyspirovorans W7924T (NR 117829). Therefore, the strain obtained by the screening of the present invention was identified as Gordonia polyisoprenivorans (Gordonia polyisoprenivorans) named Gordonia polyisoprenivorans L191.

(5) And (3) observing and identifying by a scanning electron microscope:

the purified strain L191 was inoculated into LB liquid medium containing 10mL overnight for activation. And (3) sucking 800 mu L of bacterial liquid, centrifuging at 8000rpm for 3-5 min, removing supernatant, and adding 500 mu L of PBS for washing bacteria for 3 times. To the harvested cell pellet, 1mL of 2.5% (v/v) glutaraldehyde was added and mixed well, followed by standing at 4 ℃ overnight. Then centrifuging at 8000rpm for 3-5 min, removing supernatant, and adding 500 μ L PBS to wash bacteria for 3 times. The cells were then dehydrated 2 times in 30%, 50%, 70%, 85%, 90% and 100% ethanol gradients, each gradient approximately 15min, then centrifuged at 8000rpm to remove the supernatant, and finally replaced 2 times with isoamyl acetate for 20min, in the same manner as above for ethanol dehydration.

By CO₂After drying, the sheet was observed, and as shown in FIG. 3, the morphology of the sheet was characterized by a rod shape, a length of about 2.0 to 5.0 μm and a width of about 0.3 to 0.7. mu.m, as observed by a scanning electron microscope.

The strain is deposited in the Guangdong province microorganism culture collection center at 24/9/2021, and the deposit number is GDMCC NO: 61949, the preservation address is No. 100 Michelia furiosa of Guangzhou, Guangdong province.

Example 2 analysis of the degradation Effect of Strain L191 on DBP and DEHP

(1) Preparation of a suspension of the Strain

Inoculating the purified strain L191 into LB liquid culture medium containing 10mL for overnight activation culture to logarithmic phase, centrifuging at 5000rpm for 10min to collect the strain, washing with 0.05mol/LPBS buffer solution of pH7.0 for 3 times, resuspending, and adjusting OD₆₀₀And nm is 1.0, and the strain suspension is obtained.

(2) Determination of the degradation Performance of Strain L191

The sterilized MSM medium was dispensed into 250mL glass flasks at 50mL per flask for use, and DBP and DEHP were added to adjust the concentration to 200mg/L, to obtain MSM medium containing DBP or DEHP as the sole carbon source. MSM medium was inoculated with 1mL of the suspension, while a control group (CK) was treated with the non-inoculated strain, three replicates per group. The shake flask was incubated in a constant temperature shaker at 30 ℃ and 150rpm/min in the dark for 5 days. Samples were taken and extracted at 5d and the concentrations of sample species DBP and DEHP were determined by GC/MS.

(3) Extraction method

Placing the culture sample in a triangular flask, adding 20mL of chromatographic grade dichloromethane, oscillating for 10min, pouring into a separating funnel, and taking a lower layer organic phase to transfer into a heart-shaped flask after the sample is layered and stabilized; pouring the upper water phase back to the original triangular flask and adding dichloromethane for repeated extraction twice, combining the organic phase dichloromethane, and passing through 15cm of anhydrous Na₂SO₄Column, will passThe filtrate was transferred to a heart flask, rotary evaporated to near dryness, the concentrate was transferred to a 10mL volumetric flask, and the heart flask was washed with chromatographically pure methanol and then brought to a volume of 10 mL. Filtering 1mL of the solution through a 0.45um microporous membrane, collecting the filtrate in a sample injection bottle, and performing GC-MS analysis.

(4) Chromatographic conditions

A GC/MS tandem mass spectrometer of model QP2010 Plus from Shimadzu was used. The chromatographic column is Agilent HP-5 column (0.25 μm × 0.25mm × 30 μm), the sample introduction temperature is 250 deg.C, the ion source (EI) temperature is 220 deg.C, non-split sample introduction volume is 1 μ L, and the carrier gas is high purity helium. The temperature rising procedure is as follows: the initial temperature was 100 deg.C, the gradient was increased to 280 deg.C at 30 deg.C/min, and held for 6 min.

(5) Quality control

And (5) preparing a standard curve by adopting an external standard method and a six-point calibration standard substance. The standard addition average recovery rate of the six PAEs mixed standard matrix is 89.3-105.5%, the relative deviation is lower than 10.8%, and the detection limit of an instrument is 0.13-0.45 ug/g. The method meets the requirement of quantitative analysis of trace organic matters; the OD value was measured by using Shimadzu UV-2450 type ultraviolet-visible spectrophotometer.

The degradation effect of the strain L191 on DBP and DEHP is shown in figure 4, the strain has a remarkable degradation effect on DBP and DEHP under the condition of 5-day shaking culture, the strain L191 shows a strong DBP degradation capability, and the degradation efficiency is as high as about 93.3%. The DEHP side chain is longer, the degradation rate is lower than DBP, and the degradation efficiency of the strain L191 to DEHP can reach about 62.5%. And during the culture of strain L191 for degrading DBP and DEHP, in the culture after 24h, the degradation intermediate products are detected by a GC/MS tandem mass spectrometer: phthalic acid monoester and phthalic acid, confirming the degradation of DBP and DEHP by strain L191.

Example 3 analysis of the degradation Effect of Strain L191 on DBP in Rice tissue fluid

(1) Preparation of a suspension of the Strain

Inoculating the purified strain L191 into LB liquid culture medium containing 10mL for overnight activation culture to logarithmic phase, centrifuging at 5000rpm for 10min to collect the strain, washing with 0.05mol/LPBS buffer solution with pH of 7.0 for 3 times, resuspending, and adjusting OD₆₀₀1.0 nm asThe strain suspension is obtained.

(2) Preparation of Rice tissue fluid

Cleaning collected rice plants in the tillering stage, then placing the rice plants in an ultra-clean workbench for treatment, respectively cutting roots, stems and leaves of tissue organs of the rice into small segments of about 4cm, soaking the small segments in 75% alcohol for 5 minutes, and stirring the small segments by using sterile forceps; then soaking the mixture for 3 minutes by using 0.1 percent mercuric chloride, and stirring the mixture by using a pair of tweezers; after the mercuric chloride was poured out, the mixture was washed 7 times with sterile water for 5 minutes each, and the distilled water from the last washing was left to stand. And (4) coating the distilled water washed for the last time on an LB solid culture medium, and observing whether microorganisms grow or not to determine whether the surfaces of the host plants are disinfected completely or not. A tissue sample (100g) of surface sterilized rice was slurried in an ice bath and diluted with 250ml of sterilized distilled water. Then 25ml of diluted rice tissue fluid is subpackaged in a 100ml triangular flask, sterilized at the high temperature of 121 ℃ for 20min, and cooled for standby.

(3) Strain L191 degrades DBP in rice tissue fluid

Adding DBP stock solution with final concentration of 30mg/L into sterilized rice tissue solution, inoculating 0.5ml bacterial suspension into the rice tissue solution, and using uninoculated rice tissue solution as control. All treatments were repeated 3 times and incubated at 140r/min and 30 ℃ in an incubator protected from light for 5 days. Samples were taken and extracted at 5d and the DBP concentration in the samples was determined by GC/MS.

(4) Method for extracting DBP (DBP) from rice tissue fluid

Freeze-drying the rice tissue fluid sample for 2-3 days, adding 20mL of dichloromethane, wrapping a bottle opening with tinfoil, performing ultrasonic extraction for 10min, performing refrigerated centrifugal separation at 8000r/min, collecting supernatant, and repeating for 3 times. Combining the three supernatants, and purifying with anhydrous sodium sulfate-silica gel-alumina column. Elution was repeated 3 times by adding 10mL of dichloromethane. Concentrating by a rotary evaporator at the rotating speed of 50r/min, and metering to 30 mL. Filtering 1mL of the solution through a 0.45um microporous membrane, collecting the filtrate in a sample injection bottle, and performing GC-MS analysis.

(5) Chromatographic conditions

(6) Quality control

The strain L191 can degrade DBP in the rice tissue fluid, and the degradation rate of the strain on DBP in the rice tissue fluid is up to 99.61% (DBP residue is 0.75 +/-0.12 mg/L) under the shaking culture for 5 days. The reduction rate of DBP in the tissue fluid of the rice which is not inoculated with the strain is only 3.78% (DBP residual quantity is 28.87 +/-1.67 mg/L). The bacterial strain L191 can survive in the rice tissue fluid and can well grow by utilizing the nutrient substances in the rice tissue fluid. And the strain L191 can efficiently degrade DBP in the tissue fluid of the rice, and compared with the culture medium, the degradation effect is better, so that the strain L191 and the rice can synergistically degrade the DBP. The invention provides strong evidence for degrading PAEs in plants by endophytes.

In conclusion, the screened plant endophytic phthalate degrading bacterium Gordonia polyisoprenvorans L191 has the advantages of high vitality, simple culture method, strong adaptability and capability of degrading phthalate by about 93 percent; the strain L191 can survive in the rice tissue fluid, the degradation rate of DBP in the rice tissue fluid is up to 99.61% on day 5, and DBP in the rice tissue fluid can be efficiently degraded. The invention provides excellent plant endophytic bacteria for producing degradable phthalate, and has good application value in phthalate and environmental protection.

The above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and it is obvious to those skilled in the art that other variations or modifications can be made based on the above description and ideas, and all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A plant endophytic bacterium Gordonia (Gordonia polyisoprenivorans) L191 strain for degrading phthalate is characterized in that the strain is preserved in Guangdong province microbial culture Collection (GDMCC) at 24 months 9 and 2021, and the strain is deposited as GDMCC NO: 61949.

2. the use of the Gordonia L191 strain of claim 1 for remediating an environment contaminated with organic pollutants.

3. Use of the Gordonia L191 strain of claim 1 for the degradation of phthalates.

4. Use of the Gordonia L191 strain of claim 1 for degrading phthalates in plant tissue.

5. Use of the Gordonia L191 strain of claim 1 for the preparation of a phthalate ester degrading product.

6. Use according to any one of claims 3 to 5, wherein the phthalate is di-n-butyl phthalate and/or di (2-ethylhexyl) phthalate.

7. A phthalate degradation preparation comprising the Gordonia L191 strain of claim 1 or a bacterial solution thereof.

8. The preparation of claim 7, wherein the bacterial fluid concentration is 1 x 10⁸～1×10¹⁰cfu/mL。

9. Use of a formulation according to claim 7 or 8 for the degradation of phthalates.

10. Use of the formulation of claim 7 or 8 for remediation of an environment contaminated with organic contaminants.