CN115322942A - Haematerium, application and culture method thereof and method for degrading plastics - Google Patents
Haematerium, application and culture method thereof and method for degrading plastics Download PDFInfo
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- B09B3/60—Biochemical treatment, e.g. by using enzymes
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Abstract
The invention relates to the technical field of microorganism application, in particular to a marinobacter, an application and culture method thereof and a method for degrading plastics. The present invention provides a Haihacillus bacterium (Marinobacter salinexigens) The preservation number is CGMCC No.25242. The marinobacter provided by the invention has a plastic degradation function, can degrade plastics in seawater, and has high degradation efficiency. Experiments show that the invention successfully separates and screens the sea stems from the mangrove forest bottom mud near the mouth of the sea near the Yangtze riverThe polyethylene plastic film treated by the marinobacter MY04 for 4 weeks is degraded, and the degradation rate is 1.1 +/-0.12 g/(d.m) 3 ) And the polyethylene plastic film of the comparison group has no quality change.
Description
Technical Field
The invention relates to the technical field of microorganism application, in particular to a marinobacter, an application and culture method thereof and a method for degrading plastics.
Background
Since the invention of plastics in the early 20 th century, the plastics bring the change of covering the ground to the life of people, but the problems caused by the plastics are more obvious and urgently needed to be solved. Oceans account for approximately 71% of the surface area of the earth and have become serious areas of plastic contamination. More seriously, the plastic pollution damages the marine ecosystem, and the fragmented micro plastic is eaten by marine organisms and greatly harms the human health along with the food chain.
At present, an effective technical means is not yet available for the treatment problem of marine plastic pollution. After entering the sea, part of the plastic is deposited and buried in the mud and sand on the near shore or is deposited to the mud; plastics floating in seawater corrode into micro plastics over time, and are difficult to treat by methods of salvage and subsequent centralized disposal on land (such as accumulation, landfill, incineration, recycling and the like). The method for in-situ degradation and restoration of microorganisms has the characteristics of low cost, high benefit and environmental friendliness, but the quality resources of the plastic degradation strains which can adapt to the marine environment and are obtained at the present stage are very limited, so that the method has a great technical bottleneck in the aspect of in-situ restoration application of marine plastic pollution.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a marinobacter, an application and culture method thereof, and a method for degrading plastics.
The present invention provides a Haihacillus bacterium (Marinobacter salinexigens) In 2022, 7/6/month, the culture is preserved in China general microbiological culture Collection center at the address of: the preservation number of the institute of microbiology of the institute of sciences of China, no. 3 of Xilu No. 1 of Beijing, chaoyang, is CGMCC No.25242.
The Bacillus marinus and at least one of its cellular components, metabolites, derivatives of metabolites, and secretions are within the scope of the invention. It is understood that the cellular components include at least one of cells, a culture solution containing the cells, and various chemical components constituting the cells; the metabolites include at least one of intermediate metabolites and final metabolites in metabolism; the secretion includes at least one of a nucleic acid, an enzyme, an antibody, an exosome, and a hormone.
The invention provides application of the bacillus marinus in degrading plastics. The marinobacter provided by the invention can utilize plastics as a carbon source and an energy source, has the function of degrading plastics and is high in degradation efficiency. In certain embodiments of the present invention, the plastic comprises at least one of a polyethylene plastic, a polypropylene plastic, or a polystyrene plastic. In one embodiment, the plastic is plastic granules or a plastic film. In one embodiment, the plastic is a micro plastic.
The invention provides a method for degrading plastics, comprising: the plastics were degraded with the above-mentioned marinobacter. Specifically, the method for degrading plastics comprises the following steps: the above-mentioned marine bacillus can be contacted with plastic to make propagation and degradation. The method for degrading plastics can be carried out in natural environment or culture medium. In one embodiment, the above-mentioned sea bacillus is contacted with plastic, and propagated and degraded in seawater. In one embodiment, the above-mentioned Haibacillus is contacted with a plastic material, propagated in a culture medium, and degraded. The plastic of the invention is the same as the above and is not described in detail.
The invention provides a culture method of the bacillus marinus, which comprises the following steps: the above-mentioned marinobacter is inoculated into a culture medium containing plastic and cultured. Specifically, the present invention cultures the above-mentioned marine bacillus culture solution by inoculating it into a culture medium containing a plastic. More specifically, the present invention comprises inoculating the culture solution of the above-mentioned Bacillus marinus into an inorganic salt liquid medium or an inorganic salt solid medium containing a plastic, and culturing the culture solution. In some embodiments of the present invention, the present invention cultures the above mentioned marine bacillus culture solution on a plate containing plastic inorganic salt solid culture medium or a slant tube containing plastic inorganic salt solid culture medium. The plastic of the invention is the same as the above and is not described in detail. In the culture medium, the mass of the plastic in each liter of the culture medium is 1 g-3 g, and preferably 2 g.
In some embodiments of the present invention, the amount of the Bacillus marinus is 5% to 20%, preferably 5% to 10%, and more preferably 10%. In certain embodiments of the invention, the culturing is carried out at a pH of 6 to 8, preferably at a pH of 7. In some embodiments of the invention, the culturing is performed at 10 ℃ to 40 ℃ at 100 r/min to 150 r/min, preferably at 28 ℃ at 120 r/min. In certain embodiments of the invention, the culturing is for a period of 4 weeks or more.
The pH value of the culture medium is adjusted, so that the pH value in the culture process is adjusted. In one embodiment, the pH value of the culture medium is adjusted to 6 to 8, preferably 7 by using 1-3 mol/L NaOH.
In certain embodiments of the present invention, the inorganic salt liquid medium comprises, in parts by mass: 0.5 to 1 part of K 2 HPO 4 0.5 to 1 part of KH 2 PO 4 0.8 to 1.2 parts of NH 4 NO 3 0.08 to 0.12 part of CaCl 2 0.01 to 0.04 parts of FeSO 4 And 500-1500 parts of seawater; the inorganic salt solid medium comprises the following components: 0.5 to 1 part of K 2 HPO 4 0.5 to 1 part of KH 2 PO 4 0.8-1.2 parts of NH 4 NO 3 0.08 to 0.12 part of CaCl 2 0.01 to 0.04 parts of FeSO 4 10-20 parts of agar and 500-1500 parts of seawater. In one embodiment, the inorganic salt liquid medium comprises: 0.7 K of g 2 HPO 4 0.7 g KH 2 PO 4 1.0 g of NH 4 NO 3 0.1 g of CaCl 2 0.02g of FeSO 4 And 1L of seawater. In one embodiment, the inorganic salt solid medium comprises the following components: 0.7 K of g 2 HPO 4 0.7 g KH 2 PO 4 1.0 g of NH 4 NO 3 0.1 g of CaCl 2 0.02g of FeSO 4 15 g of agar and 1L of seawater.
In some embodiments of the present invention, the step of obtaining the culture solution of the above-mentioned marine bacillus comprises: domesticating, enriching, primarily screening and re-screening the marine bacillus in an environment sample to obtain the culture solution of the marine bacillus.
Firstly, the sea bacillus is domesticated in the environment. Specifically, the plastic is first placed in the environment and subjected to continuous in situ acclimation. In certain embodiments of the invention, plastic is placed in Hainan mangrove root mud, indigenous microorganisms are acclimated in situ continuously for more than 4 weeks, and the plastic and the root mud around the plastic are taken as environmental samples.
The sea bacillus is domesticated in the environment and then enriched. Specifically, the environmental sample obtained after acclimation is placed in an enrichment culture medium to perform enrichment culture on the marinobacter. More specifically, the environmental sample obtained after domestication is placed in an enrichment culture medium containing plastics, the Haibacterium is subjected to primary enrichment culture, and then continuous subculture enrichment culture is performed. In some embodiments of the present invention, the environmental sample obtained after acclimation is placed in an enrichment medium containing plastics, and the marine bacillus is subjected to primary enrichment culture for more than 4 weeks, preferably for 6 weeks; carrying out continuous subculture for 3 to 5 times, preferably 3 times; the time of enrichment culture of each continuous passage is 4 to 6 weeks. The plastic of the invention is the same as the above and is not described in detail. In the culture medium, the mass of the plastic in each liter of the culture medium is 1 g-3 g, and preferably 2 g. In one embodiment, the enrichment culture is performed at a pH of 6 to 8, preferably 7. In one embodiment, the enrichment culture is performed at 10 ℃ to 40 ℃ at 100 r/min to 150 r/min, preferably at 28 ℃ at 120 r/min. In one embodiment, the environmental sample is placed in the enrichment medium at an inoculum size of 5-10%.
The pH value in the enrichment process is adjusted by adjusting the pH value of the enrichment medium. In one embodiment, the pH value of the enrichment medium is adjusted to 6 to 8, preferably 7 by using 1-3 mol/L NaOH.
In certain embodiments of the invention, the enriched medium comprises, in parts by mass: 0.5 to 1 part of K 2 HPO 4 0.5 to 1 part of KH 2 PO 4 0.8 to 1.2 parts of NH 4 NO 3 0.08 to 0.12 part of CaCl 2 0.01-0.04 parts of FeSO 4 0.08-0.12 part of yeast extract powder and 500-1500 parts of seawater. In one embodiment, the composition of the enrichment medium comprises: 0.7 K of g 2 HPO 4 0.7 g of KH 2 PO 4 1.0 g of NH 4 NO 3 0.1 g of CaCl 2 0.02g of FeSO 4 0.1 g of yeast extract powder and 1L of seawater.
Domesticating and enriching the marine bacillus, and then carrying out primary screening and secondary screening to obtain a culture solution of the marine bacillus. Specifically, the enriched and cultured marinobacter is diluted and coated, and then is subjected to primary screening by adopting a plate-streaking method, and then is subjected to secondary screening culture in an inorganic salt liquid culture medium to obtain a culture solution of the marinobacter. In some embodiments of the invention, the marinobacter after enrichment culture is diluted, coated and transferred into an inorganic salt solid culture medium containing plastics for culture for 1 to 2 weeks, and then separated and purified by a plate-scribing method according to form, size and color to obtain the initially screened marinobacter; and then selecting a ring of the primarily screened sea bacillus to perform secondary screening culture in an inorganic salt liquid culture medium containing plastics for 1 to 2 weeks to obtain a culture solution of the sea bacillus. In the culture medium, the mass of the plastic in each liter of the culture medium is 1 g-3 g, and preferably 2 g. In certain embodiments of the invention, the dilution coating has a dilution gradient of 10 -1 、10 -2 、10 -3 . The culture conditions, the inorganic salt liquid culture medium, the inorganic salt solid culture medium and the plastic are independently the same as those described above, and thus detailed description thereof is omitted.
The invention provides a marinobacter, an application and culture method thereof and a method for degrading plastics. The present invention provides a bacterium (A) and (B)Marinobacter salinexigens) The preservation number is CGMCC No.25242. The marinobacter provided by the invention has a plastic degradation function, can degrade polyethylene in seawater, and has high degradation efficiency. Experiments show that the invention has a success component in the mangrove bottom sediment near the entrance of the Hainan Mitsui river to the HaikouThe surface of the polyethylene plastic film treated by the marinobacter MY04 for 4 weeks becomes rough and obviously cracks appear, the surface is degraded, and the degradation rate is 1.1 +/-0.12 g/(d.m) 3 ) And the surface of the polyethylene plastic film of the control group has no corrosion trace and no quality change, and infrared spectrum analysis further proves that the marine bacillus MY04 provided by the invention has a plastic degradation function by using plastic as an energy source.
Biological preservation Instructions
Biological material: MY04, classification name:Marinobacter salinexigensin 2022, 7/6/month, the culture is preserved in China general microbiological culture Collection center at the address of: the microbial research institute of China academy of sciences No. 3, xilu No. 1, beijing, chaoyang, with the collection number of CGMCC No.25242.
Drawings
FIG. 1 is a phylogenetic tree diagram of the strain obtained in example 1 constructed based on the 16S rRNA gene sequence;
FIG. 2 is a morphological diagram of the strain obtained in example 1 in a 2216E solid medium;
FIG. 3 is a graph showing the growth of the strain obtained in example 1;
FIG. 4 is a surface morphology of a polyethylene plastic film of a control group;
FIG. 5 is a surface morphology of polyethylene plastic films of the treatment group;
FIG. 6 is an infrared spectrum of the polyethylene plastic films of the treated group and the control group.
Detailed Description
The invention discloses a marinobacter, an application and culture method thereof and a method for degrading plastics. Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention is further illustrated with reference to the following examples:
example 1
Separating the polyethylene plastic marine degrading bacteria, which comprises the steps of in-situ domestication and enrichment culture, and specifically comprises the following steps:
1. in-situ domestication: burying polyethylene plastic film strips in the root mud of the shore mangrove near the entrance of the three-river of the city of Mitsui, hainan province in shallow layers, continuously domesticating indigenous microorganisms in situ, proliferating plastic degradation flora, and domesticating for 4 weeks. And then collecting the polyethylene plastic film and the root mud coated by the polyethylene plastic film as samples for next enrichment culture.
2. Enrichment culture: taking a sample obtained by in-situ domestication under the aseptic operation condition, namely taking the polyethylene plastic film and the coated root mud as an inoculum, wherein the polyethylene plastic film is cut into square blocks with the length and the width of 1 cm by using aseptic scissors, adding the square blocks into 100 mL of sterilized primary enrichment medium in a 300 mL conical flask according to the inoculum size of 10%, and culturing for 6 weeks by shaking at the temperature of 28 ℃ and at the speed of 120 r/min. The sterilized primary enrichment medium is sterilized by autoclaving at 121 ℃ for 20 minutes in advance, and after the primary enrichment medium is cooled to room temperature, a polyethylene plastic film which is 1 cm long and wide and is subjected to sterile treatment in advance is added according to 2 g/L, wherein the polyethylene plastic film is soaked in 2% SDS for 2 hours, then soaked in 75% alcohol for 2 hours, and washed in sterile water for 3 times. And continuously performing enrichment culture by using a primary enrichment culture medium without yeast extract powder components for directional enrichment, and continuously performing subculture enrichment for 3 times, 4 weeks each time. The primary enrichment liquid medium comprises the following formula (/ L): 0.7 K of g 2 HPO 4 0.7 g of KH 2 PO 4 1.0 g of NH 4 NO 3 0.1 g of CaCl 2 0.02g of FeSO 4 0.1 g of yeast extract powder and 1000mL of natural aged seawater, and adjusting the pH to 7.0.
Performing active screening on the enriched and cultured polyethylene plastic marine degrading bacteria, wherein the steps comprise primary screening and secondary screening, and specifically comprise the following steps:
1. primary screening: subjecting the above to enrichment cultureSeparating the bacterial liquid growing to logarithmic phase by plate dilution coating method with dilution gradient of 10 -1 、10 -2 、10 -3 . After the bacterial liquid grows on a culture dish flat plate for one week, colonies which are different in shape, size and color and grow out by coating are selected, and streak separation is carried out on a fresh inorganic salt solid culture medium flat plate for 2 to 3 times to obtain a pure bacterial strain. The formulation (/ L) of the inorganic salt solid medium was 0.7 g of K 2 HPO 4 0.7 g of KH 2 PO 4 1.0 g of NH 4 NO 3 0.1 g of CaCl 2 0.02g of FeSO 4 15 g of agar and 1000mL of natural seawater, 2.0 g of PE powder (polyethylene micro plastic) was added thereto, and the pH was adjusted to 7.0 with 1 mol/L of NaOH. Autoclaving at 121 deg.C for 20 min.
2. Rescreening (retesting): in order to prevent false positive result of agar in mixed flora and inorganic salt solid culture medium on active strain separation, primary screening is carried out on the obtained pure strains for secondary screening (retesting), a ring of grown pure strains are selected to 100 mL of inorganic salt liquid culture medium (namely the inorganic salt solid culture medium used for removing agar during primary screening), and the obtained pure strains are cultured for 1 week at 28 ℃ and 120 r/min to obtain the marinobacter culture solution. The culture solution of the marinobacter is inoculated as an inoculum onto a plate containing a solid medium, and the plate cultured with a growth product is used as an effective strain. The formula (/ L) of the inorganic salt liquid culture medium is as follows: 0.7 K of g 2 HPO 4 0.7 g KH 2 PO 4 1.0 g of NH 4 NO 3 0.1 g of CaCl 2 0.02g of FeSO 4 And 1L of natural seawater, to which 2.0 g of PE powder (polyethylene micro plastic) was added, and the pH was adjusted to 7.0 with 1 mol/L NaOH. Autoclaving at 121 deg.C for 20 min.
Example 2
The strains obtained in example 1 were classified and identified as follows:
the strain obtained in example 1 is inoculated to an ocean 2216E solid medium for growth, colonies on a plate are harvested for 2 to 3 days and used for extracting and sequencing bacterial nucleic acid, and the formula (/ L) of the 2216E solid medium is as follows: 5.0 The pH value of the feed additive is 7.6 +/-0.2 by weight of peptone, 1.0 g of yeast powder, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 5.98 g of magnesium chloride, 3.24 g of sodium sulfate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium carbonate, 0.08 g of potassium bromide, 0.034 g of strontium chloride, 0.022 g of boric acid, 0.004 g of sodium silicate, 0.0024 g of sodium fluoride, 0.0016 g of ammonium nitrate, 0.008 g of disodium hydrogen phosphate, 15 g of agar and 1L of deionized water. 16S rRNA gene sequencing and homology analysis were performed as follows:
the strain obtained in example 1 was subjected to DNA extraction using TSINGKE plant DNA extraction kit (general type) to obtain a DNA sample of the strain. The extracted DNA sample is diluted in a proper amount and used as a PCR template, and the PCR amplification is carried out by using a bacterial universal primer 27F (5 '-AGTTTGATCTMTGGCTCAG-3') and 1492R (5 '-GGTTACCTTGTTCGACTT-3'), wherein the length is 1500 bp. An amplification system: 45. mu L of 1 XTSE 101 gold primer mix,2 mu L of primer 27F,2 mu L of primer 1492R,1 mu L of DNA template. And (3) amplification procedure: pre-denaturing at 98 ℃ for 2 min; in the circulation stage, the temperature is 98 ℃ for 10 s, 56 ℃ for 10 s and 72 ℃ for 10 s, and the circulation is carried out for 35 times; extension phase 72 deg.C for 5 min, and storage phase 4 deg.C. And (3) carrying out agarose gel electrophoresis (2 muL sample +6 muL bromophenol blue) on the amplified PCR product, and obtaining an identification glue picture 12 min under the voltage of 300V. The prepared PCR product was sent to Guangzhou Ongchou sequencing department for one-generation sequencing. Splicing the obtained sequence by using contigExpress, removing the part with inaccurate two ends, comparing the spliced sequence in EzBioCloud, and finding that the spliced sequence is matched with the sequenceMarinobacter salinexigensSimilarity of 99.79%, a phylogenetic tree of the strain was constructed based on the 16S rRNA gene sequence, as shown in FIG. 1, FIG. 1 is a phylogenetic tree of the strain obtained in example 1 constructed based on the 16S rRNA gene sequence, and FIG. 1 shows the strain obtained in example 1 and the strain obtained in example 1Marinobacter salinexigensThe same branch was clustered in the clade tree and uploaded to NCBI for accession number ON878183.
The bacterial colony of the strain obtained in example 1 on the ocean 2216E solid medium is milky white, round in shape, smooth in surface and neat in edge, and as shown in figure 2, figure 2 is a morphological diagram of the strain obtained in example 1 in the 2216E solid medium. Then, the cells were inoculated into 2216E liquid medium, and aerobic culture was carried out at 28 ℃ and 120 r/min to determine the growth curve, and the results are shown in FIG. 3 and Table 1, in which FIG. 3 is a graph showing the growth curve of the strain obtained in example 1, and Table 1 is data showing the growth curve of the strain shown in FIG. 3. Wherein the formula (/ L) of the 2216E liquid culture medium is as follows: 5.0 The yeast extract comprises, by weight, peptone, yeast powder 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 5.98 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium carbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 0.034 g, boric acid 0.022 g, sodium silicate 0.004 g, sodium fluoride 0.0024 g, ammonium nitrate 0.0016 g, disodium hydrogen phosphate 0.008 g and deionized water 1000mL, and the pH value is 7.6 +/-0.2.
TABLE 1
| Time (h) | 0 | 2 | 4 | 6 | 8 | 10 | 12 | 14 | 18 | 22 | 26 | 30 | 34 |
| OD 600 | 0.027 | 0.0595 | 0.112 | 0.135 | 0.151 | 0.182 | 0.204 | 0.237 | 0.258 | 0.269 | 0.270 | 0.270 | 0.271 |
By comprehensively comparing the characteristics, the strain obtained in example 1 is preliminarily identifiedMarinobacter salinexigens MY04 strain, i.e. marinobacter MY04.
Example 3
The application of the marinobacter MY04 in the degradation of commercial polyethylene plastics is carried out by the following steps (the formula of the used culture medium is the same as that of the culture medium, and is not described again):
1. preparation of seed culture solution
There are two ways; the first method is that the hypsizygus marmoreus MY04 slant lawn of the invention is inoculated into an inorganic salt liquid culture medium, the pH is adjusted to 7.0 by 1 mol/L NaOH, no PE powder is added, and the culture medium is added with a polyethylene plastic film which is sterilized in advance and has the length and width of 1 cm according to the amount of 2 g/L (the processing method is the same as that of the embodiment 1); culturing at 28 deg.C and 120 r/min for 1 week to obtain bacterial suspension; secondly, the hypsizygus marmoreus MY04 slant lawn provided by the invention is inoculated into a 2216E liquid culture medium, and sterile polyethylene plastic films with the length and the width of 1 cm are added into the culture medium according to the amount of 2 g/L in advance (the processing method is the same as that described in example 1); culturing at 28 deg.C and 120 r/min for 24 hr to obtain bacterial suspension. The bacterial suspensions prepared by the two methods can be used as seed culture solutions, in the scheme, the bacterial suspension is prepared by the second method when the growth curve of the example 2 is measured, and the bacterial suspension is prepared by the first method when the polyethylene plastic is degraded and the surface morphology and the functional groups of the polyethylene plastic are measured by a scanning electron microscope.
2. And (3) carrying out expanded culture to degrade the polyethylene plastic.
Inoculating the seed culture solution into an inorganic salt liquid culture medium which is added with polyethylene plastic films with the length and the width of 1 cm in advance according to the inoculation amount of 10 percent, taking the inorganic salt liquid culture medium as a treatment group, taking the inorganic salt liquid culture medium which is not inoculated with bacteria and is added with the polyethylene plastic films in advance according to the amount of 2 g/L as a control group, and performing sterile treatment on the polyethylene films of the treatment group and the control group according to the method described in the embodiment 1 in advance and weighing the polyethylene films at constant weight to obtain the initial weight of the polyethylene films; each of the treated group and the control group is provided with 3 parallels, and after culturing for 4 weeks under the conditions of 28 ℃ and 120 r/min, the weight loss condition, the surface morphology change, the functional group change and the like of the polyethylene plastic film are measured.
3. Quantitative and qualitative characterization of polyethylene film degradation effect
1) The method for determining the degradation rate of the polyethylene film comprises the following steps: cleaning the polyethylene films in the treatment group and the control group by 5% SDS, oscillating and cleaning for 5 h, ultrasonically cleaning for 30 min, soaking for 2 h by 75% alcohol, finally washing by using sterile water for 3 times, placing in a 40 ℃ oven for drying for 24 h, and weighing constantly to obtain the weight of the polyethylene films after bacterial degradation. The degradation rate was calculated according to the following formula: degradation rate = (initial weight of polyethylene film-weight of polyethylene film after bacterial degradation)/(culture broth volume x degradation time).
After being treated by the marinobacter MY04 strain for 4 weeks, the plastic film is degraded, and the degradation rate is 1.1 +/-0.12 g/(d.m) 3 ) And the quality of the control group does not change, which indicates that the marinobacter MY04 can effectively degrade the polyethylene plastic.
2) Surface morphology observation of polyethylene film: the polyethylene plastic films of the treatment group and the comparison group are respectively cleaned and dried according to the same method in the step 1), then sputtering is carried out for 120 s to prepare an electron microscope observation sample, and the surface morphology of the polyethylene plastic film is observed by a field emission scanning electron microscope under the conditions of beam current 40 mA and acceleration voltage 5 kV, as shown in fig. 4 to 5, fig. 4 is a surface morphology diagram of the polyethylene plastic film of the comparison group, and fig. 5 is a surface morphology diagram of the polyethylene plastic film of the treatment group. As can be seen from fig. 4 to 5, the surface of the polyethylene plastic film of the treated group becomes rough, and obvious erosion holes and cracks appear, while the surface of the polyethylene plastic film of the control group which is not treated with bacteria has no erosion trace.
Polyethylene film functional group determination: respectively cleaning and drying the polyethylene plastic films of the treatment group and the control group according to the same method as 1), and measuring by using a Fourier infrared converter in an ATR mode, wherein the wave number is 4000 cm -1 ~400 cm -1 Scanning times of 32 times and resolution of 4 cm -1 . The measurement results are shown in fig. 6, and fig. 6 is an infrared spectrum of the polyethylene plastic films of the treatment group and the control group; the treated group was at 1260 cm compared to the control group -1 At position(s) where an ether bond (C-O-C extension) was introduced, was at 1650 cm -1 Introducing carbonyl bond (C = O) unstable polar functional group, and is at 2850 cm -1 Symmetric stretching vibration carbon-hydrogen bond (CH) 2 -) absorption peak is reduced, backbone CH 2 The structural formula is reduced, and the main chain has the possibility of being cut off, which indicates that the marine bacillus MY04 in the treatment group has chemical degradation effect on the polyethylene film.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (10)
1. Haibacterium (A), (B), (C)Marinobacter salinexigens) The preservation number is CGMCC No.25242.
2. Use of the marinobacter according to claim 1 for degrading plastics.
3. The use of claim 2, wherein the plastic comprises at least one of a polyethylene plastic, a polypropylene plastic, or a polystyrene plastic.
4. A method of degrading a plastic, comprising:
degradation of plastics by use of the marinobacter as claimed in claim 1.
5. The method for culturing a marine bacillus according to claim 1, comprising:
the method comprising inoculating the marinobacter sp.
6. The method of claim 5, wherein the amount of said Haemophilus is 5% to 20%.
7. The method according to claim 5, wherein the culturing is carried out at a pH of 6 to 8.
8. The method of claim 5, wherein the culturing is performed at 10 ℃ to 40 ℃ at 100 r/min to 150 r/min.
9. The method according to claim 5, wherein the composition of the medium comprises, in parts by mass:
0.5 to 1 part of K 2 HPO 4 、0.5-1 parts of KH 2 PO 4 0.8-1.2 parts of NH 4 NO 3 0.08 to 0.12 parts of CaCl 2 0.01 to 0.04 parts of FeSO 4 And 500-1500 parts of seawater;
alternatively, the first and second electrodes may be,
0.5 to 1 part of K 2 HPO 4 0.5 to 1 part of KH 2 PO 4 0.8-1.2 parts of NH 4 NO 3 0.08 to 0.12 part of CaCl 2 0.01 to 0.04 parts of FeSO 4 10-20 parts of agar and 500-1500 parts of seawater.
10. The method of claim 5, wherein said Haibacterium species may be cultured in enrichment medium prior to inoculation;
the enriched culture medium comprises the following components in parts by mass: 0.5 to 1 part of K 2 HPO 4 0.5 to 1 part of KH 2 PO 4 0.8 to 1.2 parts of NH 4 NO 3 0.08 to 0.12 parts of CaCl 2 0.01-0.04 parts of FeSO 4 0.08-0.12 part of yeast extract powder and 500-1500 parts of seawater.
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