CN109666612B

CN109666612B - Bacillus subtilis and application thereof in degradation of dibutyl phthalate

Info

Publication number: CN109666612B
Application number: CN201910079682.2A
Authority: CN
Inventors: 万群; 徐文君; 李易芯; 冯发运; 余向阳
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2019-01-28
Filing date: 2019-01-28
Publication date: 2022-06-21
Anticipated expiration: 2039-01-28
Also published as: CN109666612A

Abstract

The invention relates to a Bacillus subtilis with the preservation number of CGMCC No.16233 and application thereof in degrading dibutyl phthalate; the bacillus subtilis can be prepared into a microbial inoculum, is applied to the restoration of residual pollution of a soil source plasticizer in a leaf vegetable planting system, and reduces the absorption and transportation of the soil source plasticizer to an edible part of the leaf vegetable, belongs to common probiotics in soil, can be planted in vegetable plants for a long time, efficiently reduces the plasticizer residue on the premise of not influencing the vegetable quality, and realizes green and safe production.

Description

Bacillus subtilis and application thereof in degradation of dibutyl phthalate

Technical Field

The invention relates to the fields of microbiology and food safety, in particular to a Bacillus subtilis strain and application thereof in degrading dibutyl phthalate.

Technical Field

Plasticizers (plasticizers), also known as plasticizers, are colorless oily synthetic compounds and are widely used in the production of various plastic products, such as paints, plastic packaging boxes or films, medical pipes, retractable toys, pesticides, polyvinyl chloride materials, and the like. Dibutyl phthalate (DBP), a representative plasticizer, is produced in high yields of millions of tons per year, and is also very easily released into the environment, contaminating the ecosphere and the food chain.

Because of the volatility of DBP, and its weak chemical bond with plastic products, food products come into contact with plastic products during packaging and shipping to contaminate the plasticizer, but the most harmful is the plasticizer residue absorbed into the crop during production. Residual accumulated DBP in leafy vegetables is a major source of human DBP exposure (Zhang, y., Tao, y., Zhang, h., Wang, l., Sun, g., Sun, x., eringle, k., Feng, c., Song, q., Li, m.,2015.Effect of di-n-butyl phthalate on root physiology and rhizosphere microbial community of cucumber seeds.j. hazard. mater.289,9-17.) long term contact accumulation is carcinogenic, interferes with the endocrine system and reduces male fertility. Thus, dibutyl phthalate has been classified as a major contaminant by the United States Environmental Protection Agency (USEPA). The currently reported soil remediation agents mainly focus on the remediation and remediation of soil heavy metal pollution and utilize beneficial microbial groups to improve the fertility of soil, and the remediation agents specially aiming at the remediation of the soil pollution caused by plasticizers, particularly dibutyl phthalate, are not reported.

The document "screening identification of dibutyl phthalate degrading strain and research on degradation characteristics thereof" (Lijianlong, et al, modern food science and technology, 2014) discloses a bacillus subtilis with DBP degradation function screened from soil, but the document only discloses an in vitro degradation experiment (under the condition of a culture medium), and does not disclose the in vivo degradation experiment effect of soil and plants.

With the improvement of living standard of people, the demand of fresh vegetables is also larger. The greenhouse plastic greenhouse and the use of chemical pesticides are the most efficient means for guaranteeing the supply of leaf vegetables at present, and therefore, the pollution of plasticizers in planting soil is inevitably caused. The rapid growth characteristics of leaf vegetables determine that it will absorb most of the water and nutrients from the soil through the root tissue, which is accompanied by plasticizer residues entering the edible parts of the leaf vegetables, which are eventually eaten by humans and endanger human health. In view of this, exploring a method for effectively repairing the residual soil plasticizer and blocking the plasticizer from entering the leaf vegetables from the planting soil is very important for ecological environment protection, green agricultural production and human sustainable development.

Disclosure of Invention

Aiming at the problems, the invention provides a plant endophyte Bacillus subtilis screened and separated from plants, which can degrade main soil plasticizer dibutyl phthalate (DBP) and can be further prepared into a repairing agent, so that plasticizer pollution remained in planting soil can be effectively degraded, absorption and transfer of the strain to leafy vegetables are reduced, normal growth of the leafy vegetable plants is not influenced, and other pollution sources are not introduced.

Firstly, the invention provides a Bacillus subtilis with the preservation number of CGMCC NO:16233, which is named HB-T2 by the applicant and belongs to the genus Bacillus; the strain is separated from a perennial herb sorrel body, is rod-shaped, can produce spores, and is mostly harmless to animals and plants.

The invention also provides application of the bacillus subtilis with the preservation number of CGMCC NO:16233 in degrading dibutyl phthalate, in particular application in degrading dibutyl phthalate pollution in soil or leafy vegetable plants.

Further, the application of the bacillus subtilis with the preservation number of CGMCC NO:16233 in degrading dibutyl phthalate pollution in leafy vegetable plants means that the bacillus subtilis with the preservation number of CGMCC NO:16233 is prepared into the bacillus subtilis with the bacterium content of 10^7-8And (3) soaking the roots of the leaf vegetable plants for 24 hours by using CFU/ml (OD value of 1) bacterial liquid to degrade the residual dibutyl phthalate in the leaf vegetable plants.

Further, the application of the Bacillus subtilis with the preservation number of CGMCC NO:16233 in degrading dibutyl phthalate pollution in soil means that the Bacillus subtilis with the preservation number of CGMCC NO:16233 is prepared into the bacillus subtilis with the bacterium content of 10^7- ⁸Spraying a bacterial solution of CFU/ml (OD value of 1) on the soil polluted by dibutyl phthalate, wherein the added bacterial solution accounts for 5% of the soil by mass and is used for degrading the residual dibutyl phthalate in the soil; the concentration of the dibutyl phthalate remaining in the soil is preferably 127ppm or less.

Drawings

FIG. 1 is a colony map, microscope and scanning electron microscope image of Bacillus subtilis HB-T2.

FIG. 2 is a schematic diagram showing the result of functional verification of the degradation of dibutyl phthalate by Bacillus subtilis HB-T2.

FIG. 3 shows the repairing function of the microbial agent on the residual dibutyl phthalate in soil.

FIG. 4 is a graph showing the effect of the microbial agent on reducing plasticizer contamination in leaf vegetables.

FIG. 5 is a schematic diagram of the effect of the biological agent on enhancing the capability of an antioxidant protection system in a leaf vegetable body.

Detailed Description

Culture media referred to in the examples:

LB medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and pH 7.0;

inorganic salt liquid culture medium MgSO₄·7H₂O(0.4g),FeSO₄·7H₂O(0.2g),K₂HPO₄(0.2g),(NH4)2SO₄(0.2g),and CaSO₄(0.08g), 1L of deionized water, and the pH value is 7.0-7.2;

seed culture medium: k₂HPO₄(4.8g),KH₂PO4(3.5g),(NH₄)₂SO₄(2g),MgCl₂(0.16g),CaCl₂(0.02g),NaMoO₄.2H₂O(0.0024g),FeCl₃(0.0018g),MnCl₂.2H₂O (0.0015g), PH 7.0, water to 1L.

Inorganic salt solid medium: adding 20g/L agar into an inorganic salt liquid culture medium;

the strain activation culture medium comprises 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and pH 7.0;

inorganic salt liquid culture medium MgSO4 & 7H2O (0.4g), FeSO4 & 7H2O (0.2g), K2HPO4(0.2g), (NH4)2SO4(0.2g), and CaSO4 (0.08g), deionized water 1L, pH 7.0-7.2;

the reagents referred to in the following examples, unless otherwise specified, were purchased commercially.

Example 1 isolation and identification of Bacillus subtilis HB-T2 and verification of degradation function

1. Strain Bacillus subtilis sp.HB-T2 separation and identification

The applicant collects wild weed garden sorrel growing in a plastic greenhouse in an experimental field of agricultural academy of sciences of Jiangsu province in 2016, then carries out surface disinfection on garden sorrel plants, grinds the garden sorrel plants into juice, coats the juice on an inorganic salt solid culture medium containing DBP as a unique carbon and nitrogen source, and screens the juice to obtain a plant endophyte with dibutyl phthalate degradation characteristic, and the applicant obtains the plant endophyte with the name HB-T2, and a colony photo, a microscopic picture and a scanning electron microscope picture of the plant endophyte are sequentially shown in a figure 1A, a figure 1B and a figure 1C, and the plant endophyte is in a rod shape and has endophyte spores as shown in the figure 1.

The strain HB-T2 is identified as Bacillus subtilis by comparing physiological and biochemical characteristics with a 16SrDNA conserved sequence, and the applicant is preserved in 8.8.8.2018 to China general microbiological culture Collection center (CGMCC), and the address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101, the preservation number is CGMCC No.16233, and the classification name is Bacillus subtilis.

2. Functional verification of DBP degradation function of Bacillus subtilis sp.HB-T2 strain

The pure HB-T2 strain obtained by screening in step 1 was cultured overnight in LB medium, the cells were centrifuged (5000rpm, 15min,4 ℃) and washed three times with sterile phosphate buffer (PBS, pH 7.0), and then resuspended in PBS to obtain HB-T2 suspension (OD)₆₀₀About 1.0); adding the HB-T2 bacterial suspension into inorganic salt liquid culture medium containing DBP as unique carbon nitrogen source with different concentrations (DBP concentration is 5 mug/mL, 10 mug/mL and 20 mug/mL in sequence) according to the inoculation amount of 1% of the volume ratio for culturing for 1, 3, 6, 9, 12, 24, 48 and 120 hours; and finally detecting the DBP residual content in the inorganic salt liquid culture medium by using High Performance Liquid Chromatography (HPLC), comparing a control without inoculation (adding 1% PBS with the same volume), and calculating the degradation rate of the HB-T2 strain:

DBP degradation rate ═ (control DBP content-experimental DBP content)/control DBP content 100%;

finally determining the DBP degradation performance of the Bacillus subtilis sp.HB-T2.

For the above High Performance Liquid Chromatography (HPLC), see literature: chess 172, 418. chess 428, Gao, M., Dong, Y., Zhang, Z., Song, W., Qi, Y.,2017.Growth and antioxidant feedback responses of leather seeds to di-n-butyl phthalate and di (2-ethylhexyl) phthalate stress.

As shown in FIG. 2, the 1% volume of HB-T2 bacterial suspension was able to completely degrade the bacterial suspension at concentrations of 5. mu.g/mL, 10. mu.g/mL, and 20. mu.g/mL for 9h, 24h, and 120h, respectively. The efficient degradation characteristic of Bacillus subtilis sp.HB-T2 on dibutyl phthalate is fully verified. According to the report of the literature 'separation and screening of plant endophytes degrading phthalate and pollution remediation application research' (doctor academic paper, 2017, of river-south university, of von nebrodensis), the highest degradation rate of endophytic bacillus subtilis degrading DBP is 82%, so HB-T2 is considered as the endophytic bacillus subtilis with the highest DBP degrading capability at present.

Example 2 preparation of DBP inoculum

The preparation method of the HB-T2 microbial inoculum comprises the following specific steps:

A) inoculating the bacillus subtilis HB-T2 obtained by screening in the example 1 to an LB culture medium, streaking at 30 ℃, selecting a single colony for culturing twice, selecting the single colony to a strain activation culture medium, and performing shake culture for 12-24h at 30 ℃, 150-one-rotation-rate (220 rpm) in a shaking table to obtain an activated strain;

B) inoculating activated strain into a fermentation tank filled with a seed culture medium, wherein the inoculation amount is 1% of the volume of the seed culture medium, the inoculation amount is 25-38 ℃, and introducing air for culturing for 16-24h to obtain liquid seeds;

C) inoculating liquid seeds into a fermentation tank filled with a seed culture medium, wherein the inoculation amount is 1% of the volume of the seed culture medium, and culturing the liquid seeds to a logarithmic phase at 30-35 ℃ and 200rpm in a dark place to obtain a viable organism culture;

D) centrifuging 50ml of viable bacteria culture at 4 deg.C and 5000rpm for 20min, washing the precipitate with sterile normal saline for 3 times, and adjusting to bacteria concentration of 10¹⁰cfu/mL, thus obtaining the HB-T2 microbial inoculum.

Example 3 DBP contamination experiment in soil remediation by HB-T2 microbial inoculum

The natural soil of a farmland is sampled, the natural soil is exposed to the sun and dried in the air, and then the natural soil is sieved by a 30-mesh sieve to remove large stone particles, wherein the soil composition property is pH 5.97, the organic matter content is 66.8g/kg of dry soil, the total nitrogen content is 0.36%, the total carbon content is 49.61%, the total hydrogen content is 5.7174%, the total sulfur content is 0.01%, the oxygen content is 44.37%, the clay content is 2.33%, the sludge content is 16.1%, and the sand content is 81.5%.

Spreading the soil sample in a stainless steel plate, spraying DBP mother liquor (dissolving DBP in acetonitrile), standing for 24 hours to volatilize an organic solvent, then filling the soil sample into a glass container, placing the soil sample in a shaking table, uniformly mixing the soil sample overnight to uniformly disperse DBP, setting the DBP residual quantity of the experimental soil to be 400 milligrams per kilogram of soil, and after naturally standing for one month, determining the DBP residual quantity of the soil to be 127 milligrams per kilogram.

Adding HB-T2 microbial inoculum diluent (diluted with sterile normal saline to bacterial inoculum concentration of 10) obtained in example 2 into experimental soil according to the addition amount of c^7-8cfu/mL), the shaker is tumbled overnight and mixed. The glass container is placed in a dark room, and meanwhile, experimental soil without the microbial inoculum is used as a control. And (3) periodically sampling and determining the degradation dynamic of DBP in the experimental soil by using a high performance liquid chromatography, and determining the repairing effect of the HB-T2 microbial inoculum on the DBP pollution of the plasticizer.

The experimental result is shown in FIG. 3, compared with the DBP contaminated soil of the control group which is not treated, after the HB-T2 microbial inoculum is added (experimental group), the degradation rate constant and the degradation half-life period of the DBP in the contaminated soil are from 0.260d^-1And 2.669d increased to 1.835d^-1and 0.377 d. After the microbial inoculum is added, the DBP concentration of the soil can be rapidly reduced from 127mg/kg to 36 mg/kg within 12 hours. Even if DBP is difficult to degrade by HB-T2 strain and other degradation media due to combination with other adsorbed substances in soil in a combined state at the later period of remediation (after 7 days), so that the degradation speed of the DBP at the later period becomes slow, the microbial inoculum still reduces the DBP content of the soil by more than 10 percent within an experimental period of 21 days.

Example 4 HB-T2 microbial inoculum experiment for repairing DBP (Dictyophora Indusiata) contaminated soil by leaf vegetables

Experimental groups: the roots of the first-heart-triloba herb Shanghai Qingn are all soaked in the HB-T2 microbial inoculum diluent (diluted by sterile normal saline to the bacterial liquid concentration of 10) obtained in the example 2^7-8cfu/mL) for 24 hours, and then transplanted into the soil added with DBP in example 3 (same as the natural soil of farmland in example 3);

soaking the control group in normal saline;

after 3h, 6h, 12h, 1d, 3d, 7d, 14d and 21d, edible parts of the leaf vegetables are taken, and the DBP residue content of the edible parts of the leaf vegetables is detected by gas chromatography-mass spectrometry (GC-MS) (the detection method is disclosed in the publications of Feng, N., Yu, J., Mo, C., ZHao, H., Li, Y., Wu, B., Cai, Q., Li, H., ZHou, D., Wong, M.,2017.Biodegradation of di-n-butyl phthalate (DBP) by a non-end photosynthetic bacterium strain YJB3.Sci.Total Environ.616-617, 117. one drugs).

The experimental result is shown in fig. 4 and 5, and the microbial inoculum can remarkably reduce the DBP residue content of edible parts of the leafy vegetables. Besides, experiments show that the microbial inoculum can reduce the toxicity of high-concentration DBP (127mg/Kg soil) on the growth of leaf vegetables and has the effect of promoting the growth of plants. DBP residue in aerial parts of leaf vegetables is reduced by 30-60% compared with the control, the residue is degraded to below 0.4mg/L after 12 hours of inoculation, and the residue can be naturally degraded to below 0.4mg/L after 14 days of cultivation of the control.

Meanwhile, the concentrations of various enzymes of the antioxidant system of the inoculated plants are found to be changed, namely MDA, POD, SOD and H₂O₂The concentration is reduced, and the change of the indexes indicates that DBP pollution stress suffered by the leaf vegetable plant is relieved, possibly because the bacterial strain HB-T2 reduces the oxidative stress of the phytotoxicity to the plant host by degrading DBP on one hand, and on the other hand, the endophyte HB-T2 can regulate the ROS level in plant tissues, further regulate and control a plant antioxidant system, regulate the activities of SOD and POD enzymes and protect the plant host from oxidative stress damage.

Claims

1. Bacillus subtilis with preservation number of CGMCC NO 16233Bacillus subtilis）。

2. The use of the bacillus subtilis according to claim 1 for degrading dibutyl phthalate.

3. The use of claim 2, wherein the use is of the bacillus subtilis for degrading dibutyl phthalate in soil or of the bacillus subtilis for degrading dibutyl phthalate in leafy vegetable plants.

4. The use according to claim 3, wherein the use for degrading dibutyl phthalate in soil is prepared by preparing the bacillus subtilis to have a bacterial content of 10^7-8And spraying the bacterial liquid of the CFU/ml to the soil polluted by the dibutyl phthalate to degrade the dibutyl phthalate remained in the soil.

5. The use of claim 3, wherein the use of dibutyl phthalate in degrading leafy vegetable plants is to prepare the Bacillus subtilis to have a bacterial content of 10^7-8And (3) soaking roots of the leaf vegetable plants by using the CFU/ml bacterial solution to degrade the residual dibutyl phthalate in the leaf vegetable plants.