CN112456649A - Method for enriching and fixing micro-plastics in water body by utilizing amoeba - Google Patents
Method for enriching and fixing micro-plastics in water body by utilizing amoeba Download PDFInfo
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- CN112456649A CN112456649A CN202011221509.0A CN202011221509A CN112456649A CN 112456649 A CN112456649 A CN 112456649A CN 202011221509 A CN202011221509 A CN 202011221509A CN 112456649 A CN112456649 A CN 112456649A
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- 239000004033 plastic Substances 0.000 title claims abstract description 59
- 229920003023 plastic Polymers 0.000 title claims abstract description 59
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 21
- 229920000426 Microplastic Polymers 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 9
- 239000002245 particle Substances 0.000 claims abstract description 8
- 230000009747 swallowing Effects 0.000 claims abstract description 8
- 230000002829 reductive effect Effects 0.000 claims abstract description 7
- 239000004005 microsphere Substances 0.000 claims description 40
- 239000004793 Polystyrene Substances 0.000 claims description 30
- 229920002223 polystyrene Polymers 0.000 claims description 30
- 239000006059 cover glass Substances 0.000 claims description 15
- 239000013535 sea water Substances 0.000 claims description 14
- 241000224422 Acanthamoeba Species 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 claims description 4
- 239000003761 preservation solution Substances 0.000 claims description 4
- 241000224421 Heterolobosea Species 0.000 claims description 3
- 240000007594 Oryza sativa Species 0.000 claims description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 210000003001 amoeba Anatomy 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- 210000003812 trophozoite Anatomy 0.000 claims description 3
- 206010006100 Bradykinesia Diseases 0.000 claims description 2
- 208000006083 Hypokinesia Diseases 0.000 claims description 2
- 238000004220 aggregation Methods 0.000 claims 1
- 230000002776 aggregation Effects 0.000 claims 1
- 238000009792 diffusion process Methods 0.000 abstract description 3
- 230000003834 intracellular effect Effects 0.000 abstract description 3
- 230000000152 swallowing effect Effects 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 3
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- 238000006731 degradation reaction Methods 0.000 description 3
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- 230000037406 food intake Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
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- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 241000461630 Naegleria sp. TH8 Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 241000254109 Tenebrio molitor Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 229920006248 expandable polystyrene Polymers 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 241000894007 species Species 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
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- Biotechnology (AREA)
- Botany (AREA)
- Biodiversity & Conservation Biology (AREA)
- Microbiology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of micro-plastic treatment, and discloses a method for enriching and fixing micro-plastic in water by utilizing amoeba, which is characterized in that a deep sea amoeba with micro-plastic swallowing capacity is obtained by screening; and putting the screened amoeba into the water body to enrich and fix the micro-plastics in the water body. The deep sea amoeba with the micro-plastic swallowing capacity is obtained by screening, has strong tolerance to the toxicity of the micro-plastic, can adhere intracellular digests to the water bottom after death, and reduces the diffusion level of the micro-plastic. The amoeba has good swallowing effect on micro plastic particles of 800 nm-2 um, and the initial concentration of the amoeba is more than 1 × 102cell/mL, at 5X 103The concentration of the micro-plastic in the water body can be obviously reduced under the concentration level of the micro-plastic per mL, and the micro-plastic control system can play a role in controlling the micro-plastic in the water body.
Description
Technical Field
The invention belongs to the technical field of micro-plastic treatment, and particularly relates to a method for enriching and fixing micro-plastic in water by utilizing amoeba.
Background
At present, Micro Plastics (MPs) are widely present in global marine environments as a new class of plastic pollutants, and have become a hot environmental problem of general concern in international society. The micro-plastics refer to plastic particles, fragments, fibers and the like with the particle size of less than 5um, MPs can be ingested by invertebrates (crustaceans, bivalve molluscs and hairy links), vertebrates (seabirds and sea fishes) and even mammals, and are transmitted from low nutrition level to high nutrition level through a food chain, so that adverse effects are generated on the ecological environment. The traditional plastic degradation method in the environment has been reported, all fungi, bacteria, degradation genes and enzymes can degrade polyurethane plastics, yellow mealworms and barley worms can gnaw foamed polystyrene and can effectively degrade polystyrene, insects and intestinal microorganisms thereof can efficiently degrade polyethylene and polystyrene, but the actual degradation rate is very low, and the method cannot be applied to practice.
The amoeba are benthos, have certain mobility in the water bottom, and are not easy to be preyed by other animals. Currently, researches on amoeba mainly focus on morphological and species identification, and the Naegleria sp.th8, which is reported in tang wei in 2012, reports about the fact that the amoeba can swallow pseudo-microalgae, but the researches are rarely reported about the study that the amoeba can swallow micro-plastics.
Through the above analysis, the problems and defects of the prior art are as follows: there is no precedent in the prior art regarding the ingestion of micro-plastics by amoebas.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for enriching and fixing micro-plastics in water by utilizing amoeba.
The invention is realized in such a way that the method for enriching and fixing the micro-plastics in the water body by utilizing the amoeba specifically comprises the following steps:
step one, obtaining a deep sea amoeba with the ability of swallowing micro-plastics by screening;
inoculating the amoeba preservation solution which is preserved and separated from seawater samples in different regions in a laboratory into a seawater culture dish in which a small amount of sterilized rice is put, and carrying out amplification culture at 25 ℃ for one week. Observation by microscopeWhen most of the amoebas were in the trophozoite stage of bradykinesia, the growth was good, and the culture broth was inoculated into 6-well plates and cultured at 25 ℃ until the density reached 1X 10 per well2For cell/mL, one cover glass was added to each well, 3 replicates of each amoeba were added with 20. mu.L of 800nm diameter polystyrene microspheres to a final concentration of 5X 104particle/mL. Another three samples without added microspheres were used as controls to which 20. mu.L of seawater was added. The samples were incubated for 1 day at 25 ℃ after addition of polystyrene microspheres. According to the characteristic that the amoeba are attached to the object, the cover glass at the bottom of the six-hole plate is taken out, the cover glass is slowly washed by seawater for 0.5-1min to remove the microspheres which are not attached to the cover glass, and then the cover glass is placed under a microscope for observation. In the experimental group with added polystyrene microspheres, a strain of amoeba acantha named as W138 shows that a plurality of microspheres are aggregated into clusters in vivo, and in the control group without added polystyrene microspheres, the microspheres do not exist in vivo, which indicates that the amoeba has the capability of swallowing micro-plastics.
And step two, throwing the screened amoeba into the water body to enrich and fix the micro-plastics in the water body.
Further, the initial concentration of the amoeba in the method for enriching and fixing the micro-plastics in the water body by utilizing the amoeba is more than 1 multiplied by 102cell/mL, at 5X 103And the concentration of the micro plastic in the water body is reduced under the concentration level of the micro plastic per mL.
By combining all the technical schemes, the invention has the advantages and positive effects that: the deep sea amoeba with the micro-plastic swallowing capacity is obtained by screening, has strong tolerance to the toxicity of the micro-plastic, can adhere intracellular digests to the water bottom after death, and reduces the diffusion level of the micro-plastic. The amoeba has good swallowing effect on micro plastic particles of 800 nm-2 um, and the initial concentration of the amoeba is more than 1 × 102cell/mL, at 5X 103The concentration of the micro-plastic in the water body can be obviously reduced under the concentration level of the micro-plastic per mL, and the micro-plastic control system can play a role in controlling the micro-plastic in the water body.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flow chart of a method for enriching and fixing micro-plastics in water by using amoebae according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of the ingestion of polystyrene microspheres by Acanthamoeba amoeba W138 provided in an embodiment of the present invention;
in the figure: a: a control group without polystyrene microspheres; b: experimental group with polystyrene microspheres added.
FIG. 3 is a schematic diagram showing the effect of different concentrations of polystyrene microspheres on the proliferation of Acanthamoeba amoeba W138 provided in the examples of the present invention; having the same letter superscript indicates no statistical difference; wherein p <0.05 is statistically different.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for enriching and fixing micro-plastics in water by utilizing amoeba, and the invention is described in detail below with reference to the attached drawings.
As shown in fig. 1, the method for enriching and fixing the micro-plastic in the water body by using amoeba provided by the invention specifically comprises the following steps:
s101, obtaining a deep sea amoeba with the micro-plastic swallowing capacity through screening.
S102, throwing the screened amoeba into the water body to enrich and fix the micro-plastics in the water body.
The method for enriching and fixing the micro-plastics in the water body by utilizing the amoeba provided by the invention can also be implemented by other steps by persons skilled in the art, and the method for enriching and fixing the micro-plastics in the water body by utilizing the amoeba provided by the invention in fig. 1 is only one specific embodiment.
According to the embodiment of the invention, the deep sea amoeba with the micro-plastic swallowing capacity is obtained by screening, the tolerance capacity to the toxicity of the micro-plastic is strong, and after death, intracellular digests can be adhered to the water bottom, so that the diffusion level of the micro-plastic is reduced. The amoeba has good swallowing effect on micro plastic particles of 800 nm-2 um, and the initial concentration of the amoeba is more than 1 × 102cell/mL, at 5X 103The concentration of the micro-plastic in the water body can be obviously reduced under the concentration level of the micro-plastic per mL, and the micro-plastic control system can play a role in controlling the micro-plastic in the water body.
The technical solution of the present invention is further described with reference to the following specific examples.
Example 1: screening amoeba with ability to swallow polystyrene plastic microspheres
Inoculating the amoeba preservation solution which is preserved and separated from seawater samples in different regions in a laboratory into a seawater culture dish in which a small amount of sterilized rice is put, and carrying out amplification culture at 25 ℃ for one week. Observing with microscope, when most amoeba are in slow-moving trophozoite stage, indicating that growth state is good, inoculating culture solution into 6-well plate, culturing at 25 deg.C until density reaches 1 × 10 per well2For cell/mL, one cover glass was added to each well, 3 replicates of each amoeba were added with 20. mu.L of 800nm diameter polystyrene microspheres to a final concentration of 5X 104particle/mL. Another three samples without added microspheres were used as controls to which 20. mu.L of seawater was added.
The samples were incubated for 1 day at 25 ℃ after addition of polystyrene microspheres. According to the characteristic that the amoeba are attached to the object, the cover glass at the bottom of the six-hole plate is taken out, the cover glass is slowly washed by seawater for 0.5-1min to remove the microspheres which are not attached to the cover glass, and then the cover glass is placed under a microscope for observation. In the experimental group with added polystyrene microspheres, a strain of amoeba acanthamoeba named as W138 was found to show that a plurality of microspheres are aggregated into clusters, while in the control group without added polystyrene microspheres, no microspheres are shown to exist in the amoeba (FIG. 2), indicating that the amoeba has the ability of micro-plastic ingestion.
Example 2 tolerance of acanthamoeba amoeba W138 to different concentrations of polystyrene microspheres
Polystyrene microspheres were added to a six-well plate of 4/mL Acanthamoeba amoeba W138 to final concentrations of 500, 5000, 50000/mL. The blank control group is set to be that the same amount of acanthamoeba amoeba W138 is added with the sterilized artificial seawater without the polystyrene microspheres. The experimental and control groups were set up in 3 replicates. Microscopic observations were made after 7 days of culture, and cell counts of protozoa were performed for each biological replicate in the different treatment groups. The results are shown in fig. 3, and it can be seen that the number of acanthamoeba amoeba W138 bodies is significantly increased at 500, 5000 and 50000 grains/mL, and the number of W138 at 50000 grains/mL is significantly lower than that of the control group and that at 500 and 5000 grains/mL, indicating that the high-concentration polystyrene microspheres have an inhibitory effect on the growth of W138.
Example 3 Elastasia W138 clearance of polystyrene microspheres of varying concentrations
The polystyrene microspheres with the concentrations of 500, 5000 and 50000 granules/mL are co-cultured with the same amount of acanthamoeba amoeba W138(4 granules/mL) at 25 ℃, the same amount of polystyrene microspheres are added into seawater without amoeba, and the concentration of the polystyrene microspheres is counted after 7 days, so that the concentrations of the 500, 5000 and 50000 granules/mL of the polystyrene microspheres are reduced by 83.2%, 26.4% and 0.32% respectively after 7 days of acanthamoeba amoeba addition, and the concentration of the polystyrene microspheres in a control group is not obviously changed, which indicates that the acanthamoeba amoeba W138 has an obvious removing effect on the polystyrene microspheres with the concentration of below 5000 granules/mL.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. The method for enriching the micro-plastics in the fixed water body by using the amoebas is characterized by comprising the following steps of:
step one, obtaining a deep sea amoeba with the ability of swallowing micro-plastics by screening;
and step two, throwing the screened amoeba into the water body to enrich and fix the micro-plastics in the water body.
2. The method for enriching the micro-plastics in the fixed water body by using the amoeba as claimed in claim 1, wherein the method for enriching the micro-plastics in the fixed water body by using the amoeba has an initial concentration of the amoeba of more than 1 x 102cell/mL, at 5X 103And the concentration of the micro plastic in the water body is reduced under the concentration level of the micro plastic per mL.
3. The method of claim 1, wherein the obtaining of a strain of deep sea amoeba with a capacity of micro-plastic feeding by screening further comprises: collecting the amoeba preservation solution which is preserved and separated from seawater samples in different regions, inoculating the amoeba preservation solution into a seawater culture dish in which a small amount of sterilized rice is put, and carrying out amplification culture at 25 ℃ for one week.
4. The method of claim 3, wherein the growth state is good when the majority of amoebae are in trophozoite stage of bradykinesia as observed by microscope, inoculating the culture solution into 6-well plate, culturing at 25 deg.C until the density reaches 1 × 10 per well2For cell/mL, one cover glass was added to each well, 3 replicates of each amoeba were added with 20. mu.L of 800nm diameter polystyrene microspheres to a final concentration of 5X 104particle/mL.
5. The method for enriching and fixing the micro-plastics in the water body by using the amoeba as claimed in claim 4, wherein another three samples without microspheres are taken as a control and 20 μ L of seawater is added. Adding polystyrene microspheres, and culturing the sample at 25 ℃ for 1 day; according to the characteristic that the amoeba are attached to the object, the cover glass at the bottom of the six-hole plate is taken out, the cover glass is slowly washed by seawater for 0.5-1min to remove the microspheres which are not attached to the cover glass, and then the cover glass is placed under a microscope for observation.
6. The method of claim 5, wherein the addition of polystyrene microspheres in an experimental group shows that a strain of Acanthamoeba named W138 shows aggregation of a plurality of microspheres in clusters, while the absence of polystyrene microspheres in a control group shows that the amoeba shows no microspheres, indicating that the amoeba has the ability to ingest the micro-plastics.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667624A (en) * | 2021-09-30 | 2021-11-19 | 闽江学院 | Bacillus siamensis and application thereof |
| CN114804367A (en) * | 2022-05-10 | 2022-07-29 | 天津农学院 | Method for removing marine micro-plastics by using protozoan broad bean worms |
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| US5051365A (en) * | 1987-04-27 | 1991-09-24 | The Regents Of The University Of California | Microorganisms and methods for degrading plant cell walls and complex hydrocarbons |
| CN103464426A (en) * | 2013-09-17 | 2013-12-25 | 杨永妹 | Microorganism cleaning method for waste plastic |
| US20150247018A1 (en) * | 2012-10-31 | 2015-09-03 | Jun Yang | Biodegradation of petroleum-based plastics |
| CN106645049A (en) * | 2016-09-30 | 2017-05-10 | 大连海洋大学 | Method for detecting plastic content of marine organism |
| CN107966393A (en) * | 2017-12-07 | 2018-04-27 | 厦门大学 | Micro- plastic content and organism absorb the assay method of micro- plastics in a kind of seawater |
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2020
- 2020-11-05 CN CN202011221509.0A patent/CN112456649A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US5051365A (en) * | 1987-04-27 | 1991-09-24 | The Regents Of The University Of California | Microorganisms and methods for degrading plant cell walls and complex hydrocarbons |
| US20150247018A1 (en) * | 2012-10-31 | 2015-09-03 | Jun Yang | Biodegradation of petroleum-based plastics |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667624A (en) * | 2021-09-30 | 2021-11-19 | 闽江学院 | Bacillus siamensis and application thereof |
| CN113667624B (en) * | 2021-09-30 | 2024-06-07 | 闽江学院 | Siamese bacillus and application thereof |
| CN114804367A (en) * | 2022-05-10 | 2022-07-29 | 天津农学院 | Method for removing marine micro-plastics by using protozoan broad bean worms |
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