Patent Application
20230406742
This patent application claims the benefit and priority of Chinese Patent Application No. 202210571683.0, filed with the China National Intellectual Property Administration on May 24, 2022, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.
The present disclosure belongs to the technical field of environmental biology, and in particular relates to a method for promoting degradation of polychlorinated biphenyls (PCB s) by Pseudomonas JD37 with nanoscale zero-valent iron (nZVI) and use thereof.
Polychlorinated biphenyls (PCBs) are a class of synthetic organochlorine compounds with a general chemical formula C12H10-nCln. According to the number and position of chlorine atoms on a biphenyl ring, there are 209 homologues of PCBs. PCBs are typical persistent organic pollutants due to their extremely-stable chemical properties, and are difficult to degrade in the environment. Due to the potential hazards to fish, mammals, and human beings, the International Agency for Research on Cancer lists the PCBs as Class-I human carcinogens. The “Stockholm Convention” promulgated on May 22, 2001 listed PCBs as persistent organic pollutants.
In order to solve the PCBs contamination, relevant scholars have proposed many remediation schemes, including physical remediation, chemical remediation, and biological remediation. Physical remediation methods (including safe landfill and thermal desorption) and chemical remediation methods (including oxidation and reduction technologies have a simple process and strong operability, but may cause secondary pollution, seriously damaging the ecosystem function, which is not conducive to subsequent environmental restoration. The biological remediation is to remove environmental pollutants through the direct action of plants, animals, and microorganisms, which is a more economical-friendly technology. However, due to the physiological limitation of biodegradation or bioaccumulation, the biological remediation is low-efficiency and long-period. In general, it is highly important to protect the ecological function of the natural environment while shortening the restoration period of the polluted environment.
Plant growth promoting rhizobacteria (PGPR) are a type of beneficial bacteria that live freely in soil or attach in plant roots, which can promote plant growth and its absorption and utilization of mineral nutrients, and can inhibit harmful organisms. Pseudomonas is a typical representative of the PGPR. The biological control mechanism of Pseudomonas is to inhibit plant pathogenic bacteria and control plant pests and diseases by secreting disease-resistant substances (such as phenazines, pyrrolnitrin, and quinolones) to metabolize extracellular reactive oxygen species (ROS). Microbial metabolism produces extracellular ROS, which can accelerate the biodegradation of refractory organic matters. At present, the effect of this process on the remediation of organically-polluted environments has been less studied or reported. Moreover, iron is an essential nutrient element for organisms and participates in biochemical activities such as respiration, photosynthesis, anti-oxidative stress, hormones, and metabolic regulation of plants and microorganisms. Nanoscale zero-valent iron (nZVI) is a nanomaterial that has attracted much attention in environmental remediation. Compared with other zero-valent metals such as manganese (Mn) and copper (Cu), the nZVI has low mass production cost and environmental friendliness. The nZVI is a core-shell structure consisting of Fe(0) core and oxide shell, providing a unique reaction interface for the initial adsorption of pollutants and the subsequent reduction and/or oxidation processes on the particle surface. Meanwhile, nano-sized iron oxides have strong adsorption capacity and catalytic activity, which can directly remove pollutants in the environment. Therefore, it is necessary to understand the effect of iron-based nanomaterials and rhizosphere functional bacteria on a degradation process of PCB28.
Aiming at the deficiencies of the prior art, an objective of the present disclosure is to provide a method for promoting degradation of PCBs by Pseudomonas JD37 with nZVI and use thereof.
The objective of the present disclosure is achieved by the following technical solutions: in a first aspect, the present disclosure provides a method for promoting degradation of PCBs by Pseudomonas JD37 with nZVI, including the following steps: adding nZVI and Pseudomonas JD37 in an environment having PCBs, and promoting degradation of the PCBs by the Pseudomonas JD37 with the nZVI; where
Further, the nZVI has a size of 20 nm to 100 nm.
Further, the Pseudomonas JD37 is Pseudomonas JD37 in middle and late stages of a logarithmic growth phase, and is prepared by the following steps: inoculating a Pseudomonas JD37 strain into a liquid medium after sterilization, and conducting shaking culture until bacterial cells are in the middle and late stages of the logarithmic growth phase.
Further, the liquid medium has a pH value of 6.5 to 7.5, and includes 5 g/L to 10 g/L of tryptone, 2 g/L to 5 g/L of a yeast extract, 5 g/L to 10 g/L of sodium chloride, and water as a solvent; and
the shaking culture is conducted at 28° C. to 30° C. and 150 rpm to 200 rpm for 12 h to 18 h.
Further, when the environment is water, the nZVI is added in an amount of 10 mg/L to 100 mg/L;
Further, when the environment is soil, the nZVI is added in an amount of 10 mg/L to 1,000 mg/L.
In a second aspect, the present disclosure provides use of the method for promoting degradation of PCBs by Pseudomonas JD37 with nZVI in environmental remediation.
Compared with the prior art, the present disclosure has the following advantages and beneficial effects:
The research results show that nZVI has neither proliferation nor toxicity to Pseudomonas JD37 at low dose levels; meanwhile, the nZVI increases a yield of extracellular ROS of the Pseudomonas JD37, and significantly shortens a degradation half-life of the Pseudomonas JD37 in degrading PCBs, so as to rapidly degrade the PCBs.
To make the objective, technical solution, and advantages of the present disclosure clearer, the present disclosure will be further described in detail with reference to the drawings and examples. It should be understood that the specific examples described here are only used to explain the present disclosure, rather than all the examples. All other examples obtained by those of ordinary skill in the art based on the examples of the present disclosure without creative efforts should fall within the protection scope of the present disclosure.
In the following examples, the experimental methods in which specific conditions are not stated are generally conducted according to conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the following examples are all commercially available, unless otherwise specified.
(1) Shake-Flask Culture of Pseudomonas JD37
10 g of tryptone, 5 g of a yeast extract, and 10 g of sodium chloride were added into 1,000 mL of water, mixed well, adjusted to a pH value of 7.0, put into a 2,000 mL Erlenmeyer flask, autoclaved at 121° C. for 20 min, and cooled to a room temperature to obtain a liquid medium; a slant strain of Pseudomonas JD37 (Pseudomonas chlororaphis subsp. aurantiaca JD37, preserved in the CGMCC with a deposit number of CGMCC No. 1.10967) was inoculated into the liquid medium with an inoculation loop at an inoculum size of 2 loops; the Erlenmeyer flask was placed in a shaking incubator and incubated at 30° C. and 180 rpm for 18 h to obtain a shake-flask culture solution; and the shake-flask culture solution was centrifuged at 4,000 rpm and 4° C. for 10 min to obtain Pseudomonas JD37 cells.
(2) Degradation of PCBs by Pseudomonas JD37
In this example, the PCB was 2,4,4′-trichlorodiphenyl (PCB28).
1.0 mg of the PCB28 was dissolved in 10 mL of an acetone solution as a PCB stock solution used in the experiment, where the acetone was chromatographically-pure grade. 100 μL of the stock solution was added to a 200 mL Erlenmeyer flask, 10 mg of nZVI (nZVI100) with a size of 100 nm was added after the acetone volatilized, a mixture was added into 100 mL of a sterilized and cooled base salt medium, and then added with 0.4 g of glucose as a growth carbon source of the Pseudomonas JD37, to obtain a medium containing PCB28; the Pseudomonas JD37 cells prepared in step (1) were inoculated into the Erlenmeyer flask with the PCB28-containing medium, adjusted to OD600=0.1, the Erlenmeyer flask was put into a shaking incubator, and incubated by shaking at 30° C. and 180 rpm; a culture solution was extracted at 1 h, 4 h, 8 h, 18 h, 24 h, 48 h, and 72 h separately, and a concentration of PCB28 was measured in the culture solution; a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 8.1 h.
The base salt medium included: 2.8 g/L disodium hydrogen phosphate, 1.0 g/L potassium dihydrogen phosphate, 0.5 g/L ammonium sulfate, 0.1 g/L magnesium chloride hexahydrate, 0.05 g/L calcium salt tetrahydrate, 0.5 mg/L disodium EDTA, 0.01 mg/L zinc sulfate heptahydrate, mg/L manganese chloride tetrahydrate, 0.03 mg/L boric acid, 0.02 mg/L cobalt chloride hexahydrate, 0.001 mg/L copper chloride dihydrate, 0.002 mg/L nickel chloride hexahydrate, and mg/L sodium molybdate dihydrate.
(1) Shake-Flask Culture of Pseudomonas JD37
10 g of tryptone, 5 g of a yeast extract, and 10 g of sodium chloride were added into 1,000 mL of water, mixed well, adjusted to a pH value of 7.0, put into a 2,000 mL Erlenmeyer flask, autoclaved at 121° C. for 20 min, and cooled to a room temperature to obtain a liquid medium; a slant strain of Pseudomonas JD37 (Pseudomonas chlororaphis subsp. aurantiaca JD37, preserved in the CGMCC with a deposit number of CGMCC No. 1.10967) was inoculated into the liquid medium with an inoculation loop at an inoculum size of 2 loops; the Erlenmeyer flask was placed in a shaking incubator and incubated at 30° C. and 180 rpm for 18 h to obtain a shake-flask culture solution; and the shake-flask culture solution was centrifuged at 4,000 rpm and 4° C. for 10 min to obtain Pseudomonas JD37 cells.
(2) Degradation of PCBs by Pseudomonas JD37
In this example, the PCB was 2,4,4′-trichlorodiphenyl (PCB28).
1.0 mg of the PCB28 was dissolved in 10 mL of an acetone solution as a PCB stock solution used in the experiment, where the acetone was chromatographically-pure grade. 100 μL of the stock solution was added to a 200 mL Erlenmeyer flask, 10 mg of nZVI (nZVI20) with a size of 20 nm was added after the acetone volatilized, a mixture was added into 100 mL of a sterilized and cooled base salt medium, and then added with 0.4 g of glucose as a growth carbon source of the Pseudomonas JD37, to obtain a medium containing PCB28; the Pseudomonas JD37 cells prepared in step (1) were inoculated into the Erlenmeyer flask with the PCB28-containing medium, adjusted to OD600=0.1, the Erlenmeyer flask was put into a shaking incubator, and incubated by shaking at 30° C. and 180 rpm; a culture solution was extracted at 1 h, 4 h, 8 h, 18 h, 24 h, 48 h, and 72 h separately, and a concentration of PCB28 was measured in the culture solution; a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 13.2 h.
The base salt medium included: 2.8 g/L disodium hydrogen phosphate, 1.0 g/L potassium dihydrogen phosphate, 0.5 g/L ammonium sulfate, 0.1 g/L magnesium chloride hexahydrate, 0.05 g/L calcium salt tetrahydrate, 0.5 mg/L disodium EDTA, 0.01 mg/L zinc sulfate heptahydrate, mg/L manganese chloride tetrahydrate, 0.03 mg/L boric acid, 0.02 mg/L cobalt chloride hexahydrate, 0.001 mg/L copper chloride dihydrate, 0.002 mg/L nickel chloride hexahydrate, and mg/L sodium molybdate dihydrate.
Blank Group (CK Group)
(1) Shake-Flask Culture of Pseudomonas JD37
10 g of tryptone, 5 g of a yeast extract, and 10 g of sodium chloride were added into 1,000 mL of water, mixed well, adjusted to a pH value of 7.0, put into a 2,000 mL Erlenmeyer flask, autoclaved at 121° C. for 20 min, and cooled to a room temperature to obtain a liquid medium; a slant strain of Pseudomonas JD37 (Pseudomonas chlororaphis subsp. aurantiaca JD37, preserved in the CGMCC with a deposit number of CGMCC No. 1.10967) was inoculated into the liquid medium with an inoculation loop at an inoculum size of 2 loops; the Erlenmeyer flask was placed in a shaking incubator and incubated at 30° C. and 180 rpm for 18 h to obtain a shake-flask culture solution; and the shake-flask culture solution was centrifuged at 4,000 rpm and 4° C. for 10 min to obtain Pseudomonas JD37 cells.
(2) Degradation of PCBs by Pseudomonas JD37
In this example, the PCB was 2,4,4′-trichlorodiphenyl (PCB28).
1.0 mg of the PCB28 was dissolved in 10 mL of an acetone solution as a PCB stock solution used in the experiment, where the acetone was chromatographically-pure grade. 100 μL of the stock solution was added to a 200 mL Erlenmeyer flask, a resulting mixture was added to 100 mL of a sterilized and cooled base salt medium after the acetone volatilized, and then added with 0.4 g of glucose as a growth carbon source of the Pseudomonas JD37, to obtain a medium containing PCB28; the Pseudomonas JD37 cells prepared in step (1) were inoculated into the Erlenmeyer flask with the PCB28-containing medium, adjusted to OD600=0.1, the Erlenmeyer flask was put into a shaking incubator, and incubated by shaking at 30° C. and 180 rpm; a culture solution was extracted at 1 h, 4 h, 8 h, 18 h, 24 h, 48 h, and 72 h separately, and a concentration of PCB28 was measured in the culture solution; a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 16.5 h.
The base salt medium included: 2.8 g/L disodium hydrogen phosphate, 1.0 g/L potassium dihydrogen phosphate, 0.5 g/L ammonium sulfate, 0.1 g/L magnesium chloride hexahydrate, 0.05 g/L calcium salt tetrahydrate, 0.5 mg/L disodium EDTA, 0.01 mg/L zinc sulfate heptahydrate, mg/L manganese chloride tetrahydrate, 0.03 mg/L boric acid, 0.02 mg/L cobalt chloride hexahydrate, 0.001 mg/L copper chloride dihydrate, 0.002 mg/L nickel chloride hexahydrate, and mg/L sodium molybdate dihydrate.
(1) Shake-Flask Culture of Pseudomonas JD37
10 g of tryptone, 5 g of a yeast extract, and 10 g of sodium chloride were added into 1,000 mL of water, mixed well, adjusted to a pH value of 7.0, put into a 2,000 mL Erlenmeyer flask, autoclaved at 121° C. for 20 min, and cooled to a room temperature to obtain a liquid medium; a slant strain of Pseudomonas JD37 (Pseudomonas chlororaphis subsp. aurantiaca JD37, preserved in the CGMCC with a deposit number of CGMCC No. 1.10967) was inoculated into the liquid medium with an inoculation loop at an inoculum size of 2 loops; the Erlenmeyer flask was placed in a shaking incubator and incubated at 30° C. and 180 rpm for 18 h to obtain a shake-flask culture solution; and the shake-flask culture solution was centrifuged at 4,000 rpm and 4° C. for 10 min to obtain Pseudomonas JD37 cells.
(2) Degradation of PCBs by Pseudomonas JD37
In this example, the PCB was 2,4,4′-trichlorodiphenyl (PCB28).
1.0 mg of the PCB28 was dissolved in 10 mL of an acetone solution as a PCB stock solution used in the experiment, where the acetone was chromatographically-pure grade. 100 μL of the stock solution was added to a 200 mL Erlenmeyer flask, 10 mg of zero-valent iron (mZVI) with a size of 5 μm was added after the acetone volatilized, a mixture was added into 100 mL of a sterilized and cooled base salt medium, and then added with 0.4 g of glucose as a growth carbon source of the Pseudomonas JD37, to obtain a medium containing PCB28; the Pseudomonas JD37 cells prepared in step (1) were inoculated into the Erlenmeyer flask with the PCB28-containing medium, adjusted to OD600=0.1, the Erlenmeyer flask was put into a shaking incubator, and incubated by shaking at 30° C. and 180 rpm; a culture solution was extracted at 1 h, 4 h, 8 h, 18 h, 24 h, 48 h, and 72 h separately, and a concentration of PCB28 was measured in the culture solution; a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 16.1 h.
The base salt medium included: 2.8 g/L disodium hydrogen phosphate, 1.0 g/L potassium dihydrogen phosphate, 0.5 g/L ammonium sulfate, 0.1 g/L magnesium chloride hexahydrate, 0.05 g/L calcium salt tetrahydrate, 0.5 mg/L disodium EDTA, 0.01 mg/L zinc sulfate heptahydrate, mg/L manganese chloride tetrahydrate, 0.03 mg/L boric acid, 0.02 mg/L cobalt chloride hexahydrate, 0.001 mg/L copper chloride dihydrate, 0.002 mg/L nickel chloride hexahydrate, and mg/L sodium molybdate dihydrate.
Compared with Comparative Example 1, the difference was only in that hematite (α-Fe2O3) with a size of 20 nm was used to replace the zero-valent iron (mZVI) with a size of 5 μm, and a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 14.8 h.
Compared with Comparative Example 1, the difference was only in that maghemite (γ-Fe2O3) with a size of 20 nm was used to replace the zero-valent iron (mZVI) with a size of 5 μm, and a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 19.1 h.
Compared with Comparative Example 1, the difference was only in that magnetite (Fe3O4) with a size of 20 nm was used to replace the zero-valent iron (mZVI) with a size of 5 μm, and a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 16.9 h.
Compared with Comparative Example 1, the difference was only in that FeSO4 was used to replace the zero-valent iron (mZVI) with a size of 5 μm, and a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 15.2 h.
Compared with Comparative Example 1, the difference was only in that Fe2(SO4)3 was used to replace the zero-valent iron (mZVI) with a size of 5 μm, and a calculated half-life of PCB28 degradation by Pseudomonas JD37 was 18.3 h.
The contents of hydroxyl radicals during the degradation of PCBs by Pseudomonas JD37 in Example 1, Example 2, and the blank group were measured, respectively, and the measurement results were shown in
Through the degradation half-life of PCB28 in Comparative Example 1 to Comparative Example 6, it can be seen that other iron-containing materials cannot promote the degradation of PCBs by Pseudomonas JD37.
The above descriptions are merely preferred examples of the present disclosure, and are not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, and the like made within the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210571683.0 | May 2022 | CN | national |
