CN114891671A - Microorganism with polystyrene plastic degradation activity and method for degrading plastic by using microorganism - Google Patents
Microorganism with polystyrene plastic degradation activity and method for degrading plastic by using microorganism Download PDFInfo
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- C08J11/00—Recovery or working-up of waste materials
- C08J11/04—Recovery or working-up of waste materials of polymers
- C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
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- C08J2325/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Derivatives of such polymers
- C08J2325/02—Homopolymers or copolymers of hydrocarbons
- C08J2325/04—Homopolymers or copolymers of styrene
- C08J2325/06—Polystyrene
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Abstract
The invention provides a microorganism with polystyrene plastic degradation activity and a method for degrading plastic by using the microorganism. The microbial strain is identified as Bacillus cereus by sequence identification and is named Baci// us cereusCH 6. The strain has the characteristics of short generation time, high activity and quick substrate utilization. After 50 days of incubation, the amount of Bacillus// us cereus CH6 degraded by 0.50g of Bacillus cereus was 10.70%. In addition, the average degradation rate of the polystyrene reaches 0.97 mg/d. The surface of the plastic was observed to have cracks and pores by a scanning electron microscope. Fourier transform infrared spectroscopy and thermogravimetric analysis find that the plastic is degraded and then has chemical structure change. In addition, from the perspective of the change of biological indexes, it was found that the concentration of bacterial protein and esterase activity changed from low to high during the degradation of polystyrene particles. The invention is expected to provide a new idea for degrading the polystyrene.
Description
Technical Field
The invention discloses a microorganism with polystyrene plastic particle degradation activity and application thereof. More particularly, the present invention discloses a microorganism which is separated from lake bottom sediments and can partially or wholly degrade polystyrene plastics, and a method for degrading polystyrene plastics by using the microorganism.
Background
Plastic contamination has become a global problem because plastics have many properties, such as resistance to oxidation, fire degradation, and corrosion. In 2017, the global plastic cumulative yield reaches 83 hundred million tons. By 2050, this figure is expected to increase to 340 billion metric tons. In 2015 and 2017, global plastic production reached 6.3 and 8.3 million tons respectively, but only 9% of the plastic was recycled or incinerated, the remainder decomposed by weathering into fine pieces, and a large portion of the pieces had been converted to "microplastics" (diameter < 5 mm). Microplastics are consumed by a variety of organisms, leading to false satiety, pathological stress and reproductive complications. In short, the ultimate fate of plastics has become an increasing concern.
According to incomplete statistics, polystyrene accounts for about 6% of the world plastic yield in 2017 and is one of the most typical contributors to plastic pollution. However, polystyrene has a linear carbon backbone with alternating backbone atoms attached to the phenyl moiety. Enzymes secreted by microorganisms are more difficult to convert into monomers and degrade. In the course of exploring ways to degrade polystyrene, researchers have discovered that while polystyrene can persist in the environment and resist degradation, they can be degraded by insects, fungi, bacteria, and the like. In all degrading organisms, bacteria dominate. Because bacteria have the inherent ability to adapt to almost all environments and have the potential to degrade a variety of compounds, including polystyrene.
Some researchers have screened polystyrene degrading bacteria and successfully studied their degradation characteristics. For example, Chauhan et al reported that strains DR11 and DR14 can form strong biofilms on polystyrene surfaces and can also utilize polystyrene as a carbon source. The degradation rate of the two strains on polystyrene is about 4%. Yang et al investigated the role of the intestinal bacteria of mealworms in the degradation of polystyrene. Suspension cultures of strain YT2 were able to degrade 7.40 ± 0.40% polystyrene sheets within a 60 day incubation period. These results strongly demonstrate the ability of bacteria to degrade polystyrene. At the same time, no more functional microorganisms are isolated and the interaction between bacteria and plastics remains to be elucidated. There have been no reports of a large amount of information on physical and chemical analyses of polystyrene surfaces during degradation. Therefore, the physical, chemical and biological indicators in the degradation process of plastics need further research.
Therefore, a greater variety of microorganisms are required to decompose polystyrene more efficiently. In this patent, a bacillus cereus capable of degrading polystyrene particles is isolated. The growth characteristics of the strains and their ability to degrade polystyrene were examined. At the same time, changes in biodegradation were further confirmed using scanning electron microscopy and fourier transform infrared spectroscopy and thermogravimetric analysis. All results indicate that Bacillus cereus CH6 can colonize, degrade and utilize polystyrene. The research result can provide theoretical reference for the microbial remediation of the soil or water polluted by the micro-plastics. This may enrich the microbial strain pool and provide a new way for the degradation of polystyrene.
Disclosure of Invention
Aiming at the defects of the types of the existing microbial degradable plastics and the defects of the degradation method, the invention mainly aims to provide a functional strain capable of efficiently degrading the plastic-polystyrene, and the functional strain has the plastic degradation activity.
The second purpose of the invention is to provide a microorganism separation, purification and culture method of degradable plastic-polystyrene.
It is a third object of the present invention to provide a method for degrading a plastic, which is characterized by comprising the step of incubating a plastic sheet containing polystyrene with a microorganism having a degrading activity.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the microorganism separated from the lake bottom sediment is a pure culture with consistent appearance. The strain of the invention belongs to the genus Bacillus cereus. The diameter of the bacterial colony is about 2-4mm, the bacterial colony is milky, convex and opaque, the edge is regular, and the bacterial colony is viscous; gram staining positive. The optimal growth temperature is 25-30 ℃, and the pH is 6.0-8.0.
The bacillus cereus disclosed by the invention can tolerate poor nutrition conditions and has the characteristics of strong organic matter utilization capacity and high carbon source utilization rate; can convert and degrade polystyrene by taking the polystyrene as a sole carbon source under aerobic conditions. The bacillus cereus can be applied to the plastic degradation treatment in the actual environment.
The Bacillus cereus is named as Bacillus cereus CH6 in the application, is separated from lake bottom sediments, and the sequencing of strain DNA is completed by the biological company of Venezitongbao. A series of physiological and biochemical tests and process optimization tests carried out on the strain show that the Bacillus cereus CH6 provided by the invention has the performance of efficiently degrading polystyrene particles. The main advantages are as follows:
(1) the Bacillus Cereus CH6 can be used for growth and propagation by taking polystyrene plastic as a carbon source, and has the advantages of short generation period, fast growth and propagation, low culture cost and the like.
(2) Bacillus cereus CH6 has the ability to tolerate poor nutrient conditions, and has low requirements for nutrient conditions of the culture medium.
(3) The Bacillus cereus CH6 has the capability of efficiently degrading polystyrene plastics and has higher degradation rate.
(4) The Bacillus cereus CH6 is easy to expand and culture, has low cost, and can be used for treating soil polluted by polystyrene.
Due to the adoption of the scheme, the invention has the beneficial effects that:
the present invention provides a microorganism having polystyrene plastic particle degrading activity, which can degrade polystyrene particles and convert them into low molecular substances such as carbon dioxide, etc. when cultured under appropriate culture conditions. The microorganism with the degradation activity of the polystyrene plastic can utilize and convert the polystyrene particles by secreting different proteins, esterase and other substances with degradation effect, and can be suitable for degrading different types of polystyrene plastics or for regenerating or pretreating the polystyrene plastics.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
FIG. 1 is a phylogenetic analysis of Bacillus cereus CH 6;
FIG. 2 shows the growth curve and cell concentration of Bacillus cereus CH6 using polystyrene plastic as carbon source;
FIG. 3 is a graph showing the weight loss of Bacillus cereus CH6 in the degradation process of polystyrene plastic particles.
FIG. 4 is a SEM photograph of the degradation of polystyrene plastic particles by Bacillus cereus CH 6.
FIG. 5 is a graph showing the changes in protein content and esterase concentration in the degradation process of polystyrene plastic particles by Bacillus cereus CH 6.
Detailed Description
The present invention is further described below with reference to examples, but the embodiments of the present invention are not limited thereto.
First part of the sources of the microorganisms of the invention
The microorganism with the polystyrene plastic degradation activity separated by the invention is derived from sediments at the bottom of a lake of Tai lake of Suzhou, Jiangsu.
The method process obtained by separation comprises the following steps:
step 1, preparation of a degradation strain culture medium: firstly, preparing a nutrient medium capable of enriching the bacteria in the lake bottom sediment, and then preparing an inorganic salt medium required by enriching the bacteria to provide the required inorganic salt for the bacteria. Finally, adjusting the pH value of the culture medium to be neutral, and providing a proper growth condition for degrading bacteria;
Step 3, separating and purifying the degradation strains: the enriched bacterial liquid is subjected to gradient dilution by using cooled sterile distilled water. Then, the strain is subjected to static culture by adopting a plate coating method. And then selecting strains with regular appearance and uniform color in the flat plate, coating for multiple times, and scribing and purifying to finally obtain the pure strains for degrading the polystyrene plastic.
The strain can be obtained repeatedly by the above method.
The obtained strains are commonly present in lake bottom sediments and are not limited to lake bottom sediments of Taihu lake of Jiangsu Suzhou.
Characterization of the second part of the microorganisms isolated from the lake bottom sediment
Sequencing of DNA was performed by Competition Dalianbao Biometrics. DNA sequencing was carried out using Seq Forward, Seq Reverse, and Seq Internal as primers. The 16S rRNA amplification employed broad-spectrum primers F27(SEQ ID NO: 2-AGAGTTTGATCATGGCTCAG) and R1492(SEQ ID NO: 3-TACGGTTACCTTGTTACGACTT). According to the identification results, phylogenetic analysis is carried out on the degrading strain as shown in figure 1, and the strain Bacillus cereus CH6 is found to have homology with Bacillus aceticer strain CBD119, Bacillus sp.L28 and Bacillus sp.AK73, belong to the genus Bacillus and have the similarity of 97.5 percent with the Bacillus cereus ATCC 14579 strain.
The microbial strain is identified as Bacillus cereus by sequence identification and is named as Bacillus cereus CH 6.
Third part
The following are pure cultures, laboratory procedures. The microbial strain has the characteristics of short generation time, high activity and quick utilization of plastic degradation substrates. As described in detail below.
1. Experimental Material
The composition of the Bacillus cereus medium is as follows (g/L): (1) enrichment culture medium: beef extract: 5.0; peptone: 10.0; sodium chloride: 5.0. (2) inorganic salt culture medium: k 2 HPO 4 1.0；NH 4 NO 3 1.0；MgSO 4 7H 2 O 0.2；CaCl 2 2H 2 O0.1; KCl 0.15. (3) Cultivation of trace elementsBase: FeSO 4 6H 2 O 1.0；ZnSO 4 7H 2 O 1.0 and MnSO 4 1.0. The pH was adjusted to 7.0. Polystyrene plastic is required for degradation experiments.
Besides polystyrene, the carbon source can also directly utilize glucose, sodium acetate, sodium citrate and other common carbon sources.
The rest reagents are commercial analytical pure products.
2. Enrichment, separation and purification method of strain and plastic degradation experiment thereof
1) The method for obtaining the degraded strains-enrichment culture conditions and specific operations: inoculating 3mL of lake bottom sediment supernatant into a 250mL triangular flask containing 100mL of sterilized broth culture medium, shaking thoroughly, culturing at 25 deg.C and 160 r.min-1, and storing in refrigerator at 4 deg.C after 2 days. Then, the enriched microorganisms are inoculated into a 250mL triangular flask containing 100mL of sterilized polystyrene plastic culture medium according to the inoculation amount of 2 percent, subculture is carried out under the same conditions, and enrichment passage is carried out for 4-5 times.
2) The method for obtaining the degradation strain-separation and purification conditions and specific operation: diluting the enriched bacteria liquid with cooled sterile distilled water by 10 times to obtain 10-fold dilution -3 、10 -4 、10 -5 、10 -6 、10 -7 The diluent (2). The separated strain culture medium is cultured by a coating method. The plate was placed in a biochemical incubator and incubated at 30 ℃ for 2 days. And (4) carrying out multiple passages on the strains with good growth vigor, and purifying.
3) The degradation experiment research of the degradation strain on the plastic comprises the following steps: the identified bacteria are inoculated into beef extract peptone medium and cultured on a constant temperature shaking table at 25 ℃ and 120r/min until the culture reaches logarithmic phase. The initial cell density of the inoculum was adjusted to 7.5X 10 7 CFU/ml. 10% of the log phase culture was inoculated into Erlenmeyer flasks containing 30ml of pure medium and 0.5g of sterilized polystyrene plastic granules for plastic degradation experiments. As a control, the uninoculated medium and the polystyrene plastic particle samples were maintained under similar conditions. Optical Density (OD) was monitored every 5 days 600 ) And microbial cell concentration for 50 days. All experimentsPerformed in triplicate.
3. Identification of polystyrene degradation strain
The target fragment was amplified by PCR using QIAquick Genomic DNA Buffer Set. Mu.l of the DNA fragment was subjected to 3% agarose gel electrophoresis, and the desired fragment was recovered by using a cut gel to perform DNA sequencing. Sequencing of DNA was performed by Competition Dalianbao Biometrics. DNA sequencing was carried out using Seq Forward, Seq Reverse, and Seq Internal as primers. The 16S rRNA amplification employed broad-spectrum primers F27(SEQ ID NO: 2-AGAGTTTGATCATGGCTCAG) and R1492(SEQ ID NO: 3-TACGGTTACCTTGTTACGACTT). According to the identification results, phylogenetic analysis is carried out on the degrading strain as shown in figure 1, and the strain Bacillus cereus CH6 is found to have homology with Bacillus aceticer strain CBD119, Bacillus sp.L28 and Bacillus sp.AK73, belong to the genus Bacillus and have the similarity of 97.5 percent with the Bacillus cereus ATCC 14579 strain.
4. Detection method
After 50 days of incubation, the remaining polystyrene plastic particles were recovered from the culture medium by filtration. The plastic granules were then washed with a 70% ethanol solution and then dried overnight in a vacuum oven at 50 ℃. To determine the weight of the residual polystyrene plastic and investigate the degree of degradation. The initial weight of the uninoculated plastic sample was measured by the same sample handling method. And finally, evaluating the degradation effect of the polystyrene plastic according to the weight loss percentage W value, wherein the specific formula is as follows:
wherein W 0 Is the initial weight (g) of the polystyrene plastic particles, and W is the remaining weight (g) of the polystyrene plastic particles.
Application and implementation effect verification case:
EXAMPLE 1 measurement of growth Curve, cell concentration and degradation Rate of polystyrene Using polystyrene as a sole carbon Source for the Strain
Sterilizing 100mL polystyrene degradation culture medium in 250mL conical flask with high temperature steam at 121 deg.C for 30min, cooling, and transferring with liquid transfer device5mL of Bacillus cereus solution (OD) was added 600 nm of 0.50-1.00), sealing with sealing film, and placing in a shaker at 25 deg.C and 160 r.min -1 Culturing under the condition, and measuring OD of bacterial liquid every 10 days 600 Determining the growth condition of the thalli by the nm value, simultaneously determining the degradation rate of the polystyrene,from FIG. 2, it can be seen thatThe variation trend of the absorbance value of the strain and the concentration of the thallus is consistent. The strain showed significant growth in the medium inoculated with polystyrene plastic particles. From day 1 to day 15, the bacterial concentration increases exponentially, OD 600 Increasing from 0.2119 to 0.4433. Around 20d, the bacterial growth rate decreased, but the OD 600 Increasing from 0.4433 to 0.4592, the strain reached the maximum OD 600 . This indicates that the strain has finished the exponential growth phase and has just entered the stationary phase. OD 600 This increase coincides with an increase in the number of bacteria. The highest bacterial cell number (15.9X 10) was also recorded on day 20 of the experiment 7 CFU/ml)。
The initial mass of the polystyrene plastic particles in the medium was 0.50g, and after 50 days of degradation, 446.50mg of polystyrene plastic particles remained.As shown in fig. 3The weight loss of the polystyrene particles without microbial treatment was 1.03%. The weight loss of PS MP was 10.70% after 50 days of biodegradation evaluation with Bacillus cereus CH 6. The calculation shows that the degradation rate reaches 0.97 mg/d. Other strains are usually between 0.30 and 0.70 mg/d. This indicates that the isolated pure species had higher degradation activity and rate.
Example 2 surface topography Change of Bacillus cereus CH6 degradation polystyrene particle Plastic
100mL polystyrene degradation culture medium is taken in a 250mL conical flask, the polystyrene degradation culture medium is sterilized by high-temperature steam for 30min at the temperature of 121 ℃, and after cooling, a liquid transfer device is used for adding 5mL bacillus cereus liquid (OD) 600 nm is 0.50-1.00), sealing with sealing film, and placing in shaking table at 25 deg.C and 160r min -1 Culturing under the condition, and inspecting the shape change of the polystyrene surface by using SEM in the degradation process.As shown in fig. 4 After 50 days, the untreated (control) polystyrene particles had a smooth surface and no microorganisms on the surface of the polystyrene particlesAttachment of a substance (FIGS. 4a and 4c)。As shown in fig. 4b After 50 days of incubation, bacterial colonization was observed on the surface of the polystyrene particles. The microbially treated polystyrene particles exhibit increased adhesion and number. This indicates that Bacillus cereus CH6 can adapt to the living environment created by plastics and can grow normally. It was observed that various pores/pits and irregularities were formed on the surfaces of PS MPs exposed to the separator: (FIG. 4d) Simultaneously exhibit phenomena of surface erosion, fissures, folds and bacterial colonization (FIGS. 4e and 4f)。
Example 3 graph showing the change of protein content and esterase concentration in the degradation process of polystyrene plastic particles by Bacillus cereus CH 6.
100mL polystyrene degradation culture medium is taken in a 250mL conical flask, the polystyrene degradation culture medium is sterilized by high-temperature steam for 30min at the temperature of 121 ℃, and after cooling, a liquid transfer device is used for adding 5mL bacillus cereus liquid (OD) 600 nm is 0.50-1.00), sealing with sealing film, and placing in shaking table at 25 deg.C and 160r min -1 Culturing under the condition, and inspecting the shape change of the polystyrene surface by using SEM in the degradation process. Samples were taken every 10 days and the total protein content as well as the esterase activity in each sample was determined. The degradation of the strain was investigated from the molecular level.As shown in fig. 5The protein content of the isolate is about 50-106. mu.g/ml, which is highest at the stage of highest degradation rate. The esterase activity can be found to be in the range of 3-13U/ml through analysis. In the absence of any carbon source in the medium, changes in protein content and esterase activity of the bacterial species indicated that it used polystyrene plastic particles as a carbon source. In addition, the peak values of protein content and enzyme activity, 105ug/ml and 1350U/ml respectively, appeared at day 30 of fermentation, demonstrating that the strain is secreted in the logarithmic growth phase with vigorous metabolism.
The invention separates microbes with polystyrene plastic degradation activity from lake bottom sediments and a method for degrading plastics by using the microbes. The strain has the characteristics of short generation time, high activity and quick substrate utilization. After 50 days of incubation, the amount of Bacillus cereus CH6 degraded 0.50g of Bacillus cereus by 10.70%. In addition, the average degradation rate of the polystyrene reaches 0.97 mg/d. The surface of the plastic was observed to have cracks and pores by a scanning electron microscope. Fourier transform infrared spectroscopy and thermogravimetric analysis find that the plastic undergoes obvious chemical structure change after degradation.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Claims (6)
1. A microorganism, characterized in that the microbial species has been identified by the sequence number Bacillus cereus CH 6.
2. The microorganism according to claim 1, which is isolated from lake bottom sediments, has plastic degrading activity, and can perform growth and metabolism by using plastic particles as a sole carbon source; the plastic is polystyrene, and the type is polystyrene granules.
3. A method for degrading polystyrene plastics, which comprises the step of culturing particles containing polystyrene plastics together with microorganisms having a degrading activity thereof, wherein the microorganisms having a degrading activity of polystyrene are separated from lake sediment and can grow using the polystyrene particles as a sole carbon source.
4. The method of claim 3, wherein the degradation conditions are an inorganic salt medium supplemented with polystyrene particles and a trace element medium required by microbial species.
5. The method for degrading polystyrene particles according to claim 3, wherein one culture medium consists of an enrichment medium + a trace element medium + polystyrene particles.
6. The method for degrading polystyrene plastic of claim 3, wherein the microorganism is cultured at a temperature of 25 ℃ to 30 ℃ for 10 days to 50 days.
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| CN116949685B (en) * | 2023-09-18 | 2023-12-12 | 北京建工环境修复股份有限公司 | Microorganism composite fiber film and preparation method and application thereof |
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