CN115109717B - Gordonia strain for efficiently degrading polystyrene plastic - Google Patents

Abstract

Gordonia strain for efficiently degrading polystyrene plastic, and relates to plastic pollution treatment and waste recycling. The high-efficiency polystyrene plastic degrading bacteria are used for harmless treatment of polystyrene plastic garbage and recycling of resources. Providing the nucleotide sequences of 16S rRNA genes of 5 strains of polystyrene plastic degrading bacteria, wherein the preservation number of the preservation center is CGMCCNO: 22163-22167; the application of the 5 polystyrene plastic degradation strains Gordonia spp in the harmless treatment, recycling and environmental restoration of polystyrene plastic resources is provided. The Gordonia spp. Strain can be applied to biodegradable polystyrene. The polystyrene with the average initial quantity of 0.05 to 0.07g can be degraded, and the degradation rate is 2.66 to 7.73 percent.

Description

Gordonia strain for efficiently degrading polystyrene plastic

Technical Field

The invention relates to plastic pollution control and waste recycling, in particular to a high-efficiency polystyrene plastic degrading bacterium Gordonia spp and application thereof.

Background

Synthetic plastics are great inventions in recent centuries, bring great convenience to the life of people and accelerate the pace of human society development, but bring a series of environmental pollution problems. Synthetic plastics which account for 80% of the global plastic usage are Polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyurethane (PU), polystyrene (PS), polyethylene terephthalate (PET), respectively. Plastic landfill can pollute soil and groundwater, and completely degrade the landfill waste, and the time required for the landfill waste is as short as decades to hundreds of years, so that the soil hardening is unfavorable for agricultural production; plastic waste discarded into the natural environment can be a threat to the life safety of humans and animals. It is reported that large plastic blocks in nature become microplastic with smaller particles (particle size is less than 5 mm) after physical and chemical actions or animal misfeeding, and the microplastic is widely distributed in natural environment, and can float in the air or be deposited on the seabed. Microplastic, also known as "PM 2.5" in the ocean, is a significant global environmental problem in parallel with global climate change, ozone depletion and ocean acidification. In marine environments scientists find that worldwide marine environments are contaminated with varying levels of marine microplastic, from the ocean surface to deep sea sediments, from the offshore area to the ocean, from the equatorial area to polar regions (a GK, K A, M H, K S, g.review on plastic wastes in marine environment-Biodegradation and biotechnological solutions.mar poll bull.2020; 150:110733). Scientists recently found that microplastic was found in snowfall from alps to arctic (Melanie Bergmann SM, sebastian Primpke, mine B Tekman, jurg Trachsel, gunar gerds.white and wonderfulMicroplastics prevail in snow from the Alps to the arctic.science Advances.2019;

5). Recently, scientists have found plastic particles in both Ma Liya nanometer sea ditch sediment and bodies of water of more than 10,000 meters, wherein the content of plastic particles in the sediment is much higher than the content in the same volume of water (Peng X, chen M, chen S, dasgupeta S, xu H, ta K, et al microplasties contaminate the deepest part of the world' S ocean. Geochem Perspect Let.2018; 1-5).

The bulk plastic floating on the sea water surface is sunk to the sea bottom after solar irradiation, physical chemical mechanical degradation, or adhesion by organisms, and improper feeding of marine organisms. Researchers generally believe that deep sea is not available for light and ultraviolet rays, so that the degradation of deep sea plastics can only be accomplished by living things. At present, few reports on plastic degrading microorganisms have been focused mainly on land organisms (such as yellow meal worm, wax moth, snail, etc.) and intestinal microorganisms thereof (Exiguobacterium sp., bacillus sp., serratia sp.), and on degradation microorganisms isolated from landfill (Mohan AJ, sekhar VC, bhaskar T, nampoothiri KM. Microbial assisted High Impact Polystyrene (HIPS) degradation. Bioresource technology.2016; 213:204-07; yang Y, yang J, wu WM, zhao J, song Y, gao L, et al Biodegradation and Mineralization of Polystyrene by Plastic-Eating Mealworms: part1.chemical and Physical Characterization and Isotopic Tests. Environ Sci technology.2015; 49:12080-6;Mor R,Sivan A.Biofilm formation and partial biodegradation of polystyrene by the actinomycete Rhodococcus ruber:biodegradation of polystyrene.Biodegradation.2008;19:851-8).

Polystyrene (polystyrene) plastics are polymers synthesized from styrene monomers by free radical addition polymerization, are the most commonly used and produced plastics in the world today, and are mainly used for food and disposable materials (2017 PtF, plastics-the pictures 2017: an analysis of European plastics production, demand and waste data, in Publisher for the polymer reduction.2017, publisher for the polymer industry:Germany), including ordinary polystyrene, expanded Polystyrene (EPS), high Impact Polystyrene (HIPS) and Syndiotactic Polystyrene (SPS). The polystyrene plastic, especially the foaming type plastic, has small mass, low residual value and difficult recycling, so the recycling rate is extremely low; other types of polystyrene, because of their strong inertness, are difficult to get into the bio-geochemical cycle via photodecomposition and biodegradation, thus imposing a great burden on the environmental ecosystem.

At present, the research on the degradation of polystyrene plastics from marine environment is mainly focused on the research on the diversity of microorganisms attached to the surfaces of plastics in the environment, and functional strains or organisms with degradation capability are very few. Scientists, when studying the polystyrene surface microbiota in the ocean, found by high throughput sequencing analysis that the common species colonized on the polystyrene surface were Flavobacteria, hyphomonadaceae, rhodobaceae, sphingomonaseae, alcamaracaceae, ocenospirillales, vibrionaceae (Wright RJ, langille MGI, walker TR. Food or just a free ride A meta-analysis reveals the global diversity of the Plastisphere. ISME J.2021; 15:789-806), but the degradability of these species was to be further experimentally verified.

In 2008, a first strain (Rhodococcus ruber) having polystyrene degrading ability was found (Mor R, sivan A.Biofilm formation and partial biodegradation of polystyrene by the actinomycete Rhodococcus ruber: biodegradation of polystyrene.Biodegradation.2008; 19:851-8), wherein the strain had a degradation rate of 0.8% within 8 weeks, and no further study on polystyrene degrading genes and enzymes has been made so far. Research on the biodegradation mechanism of polystyrene is helpful to find key degrading enzymes, so as to optimize degradation paths; secondly, the treatment efficiency of the functional enzyme in the recycling treatment of polystyrene plastic can be improved by carrying out corresponding genetic engineering on the discovered functional enzyme.

Disclosure of Invention

The first aim of the invention is to provide a high-efficiency polystyrene plastic degrading bacterium which is used for harmless treatment of polystyrene plastic garbage and recycling of resources.

A second object of the present invention is to provide nucleotide sequences of 16S rRNA genes of 5 strains (Gordonia spp.) of the polystyrene plastic degrading bacteria, with the strain numbers of MCCC1A04216, MCCC1A14786, MCCC1A 17966 (ZN 14-R1), MCCC1A 17970 (ZN 15-R9), and MCCC1A17979 (ZN 17-RX).

The third purpose of the invention is to provide the application of the 5 polystyrene plastic degradation strains (Gordonia spp.) in the harmless treatment, recycling and environmental remediation of polystyrene plastic resources.

In order to achieve the above object, the present invention provides a polystyrene high-efficiency plastic degrading strain identified as Gordonia sp; the Gordonia (Gordonia spp.) strains include Gordonia amicalis a04216, gordonia didemni 1a14786, gordonia bronchialis ZN17-RX, gordonia sihwensis ZN-R1, gordonia mangrovi ZN-R9, which have been deposited at the chinese classical-R1 culture deposit of the chinese general microbiological bacterial deposit, month 4, 12, 2021, deposit address: the institute of microbiology, academy of sciences, post code: at 100101, the process of the present invention, the preservation number of the preservation center is CGMCC NO: 22163-22167.

For clarity of description, the species "MCCC 1a04216, MCCC1a14786, MCCC1a 17966 (ZN 14-R1), MCCC1a 17970 (ZN 15-R9), MCCC1a17979 (ZN 17-RX)" are given. "and description of sequence Listing," the arrangement of the corresponding information is as follows:

MCCC1a04216 corresponds to CGMCC number 22163, corresponds to sequence listing SEQ ID NO:1

MCCC1a14786 corresponds to CGMCC number 22164, corresponds to sequence listing SEQ ID NO:2

MCCC1a 17966 corresponds to ZN14-R1, to CGMCC number 22166, to sequence listing SEQ ID NO:3

MCCC1a 17970 corresponds to ZN15-R9, to CGMCC number 22167, to sequence listing SEQ ID NO:4

MCCC1a17979 corresponds to ZN17-RX, to CGMCC number 22165, to sequence listing SEQ ID NO:5 to 7

Gordonia bronchialis ZN17-RX, gordonia sihwensis ZN-R1, gordonia mangrovi ZN-R9 in Gordonia spp strains are separated from the red woodland styrofoam plastic refuse pile in Zhangzhou purple mud; to further verify the degradation capability of the bacteria of the genus to polystyrene, the degradation activity verification is carried out on the bacteria of the genus with unknown function in the reservoir, and 2 strains with degradation capability are found to be respectively separated from south China sea and offshore in Shandong Weifang, and the reservoir numbers of the China marine microorganism strain collection center are MCC 1A04216,MCCC1A14786, namely Gordonia amicalis A04216 and Gordonia didemni 1A14786.

The main biological characteristics of the Gordonia (Gordonia spp.) strain of the invention are: in the R2A solid culture medium, the flat plate is inverted and is cultured in a constant temperature incubator for 72 hours at 28 ℃, the colony size is 2-3mm, the colony is round, orange or pink or milky, the surface is opaque, smooth and moist, the surface is regular, no halo and bulge exist, and part of bacterial strains have pigment production. The degradation strain can grow by using polystyrene plastic as a unique carbon source and energy source, amplifying and sequencing the strain 16S rRNA gene sequence, and performing BLAST analysis on the obtained sequence and the sequence existing in NCBI database, wherein the results show that the above 5 species are MCCC1A04216 (Gordonia amicalis, 100%), MCCC1A14786 (Gordonia didemni, 98.9%), MCCC1A 17966 (Gordonia sihwensis, 100%), MCCC1A 17970 (Gordonia mangrovi, 98.7%), and MCCC1A17979 (Gordonia bronchialis, 99.3%), respectively.

The Gordonia (Gordonia spp.) strain, wherein MCCC1A04216,MCCC1A14786 is from the chinese marine microorganism collection; the obtaining of three other MCCCs 1A17966,MCCC 1A17970,MCCC1A17979 comprises the following steps:

collecting and bringing back to a laboratory a waste plastic foam sample from a natural protection area of a red forest in purple mud of Dragon sea in Zhangzhou, and carrying out 3-cycle inoculation enrichment culture on the foam sample in the laboratory to obtain a culturable strain with degraded styrofoam (polystyrene); the samples taken were waste foam with a weathered trace on the surface and a small amount of residual sludge after tidal passage.

The screening method of the Gordonia (Gordonia spp.) strain MCCC 1A17966,MCCC 1A17970,MCCC1A17979 comprises the following steps:

1) Sample collection: the laboratory goes to the edge of the natural protection area of the purple mud mangrove in Zhanghai city in 10 months and 23 days in 2019, and a large amount of waste foam samples positioned in the protection area are collected in a concentrated way on the premise of not damaging the local protection area, are packaged in a sterile sample collection bag and are stored in an incubator at the temperature of 4 ℃. The waste foam accumulation area is an area which can be immersed after tide water rises and tides, so that a small amount of sludge is arranged on the surface of a foam sample;

2) Laboratory enrichment: the collected foam sample is subjected to enrichment transfer by taking foam as a unique carbon source in a laboratory, and the specific operation steps are as follows:

a, taking a 300mL glass conical flask, cleaning, adding 150mL sterile artificial seawater culture medium, sealing by a sealing film, sterilizing at high temperature and high pressure of 121 ℃ for 20min, and supplementing trace element solution and collected waste foam plastic on an ultra-clean workbench after the system is cooled to room temperature;

the composition (MMC) of the artificial seawater medium: potassium chloride 0.7g/L, ammonium chloride 1g/L, sodium nitrate 1g/L, sodium chloride 20g/L, monopotassium phosphate 2g/L, disodium phosphate 3g/L, magnesium sulfate heptahydrate 3.5g/L, and magnesium sulfate heptahydrate is independently sterilized, and the pH is adjusted to 7.2-7.4. The microelements comprise the following components: 2.0mg/L of ferrous sulfate hexahydrate, 2.0mg/L of zinc sulfate heptahydrate and 2.0mg/L of manganese sulfate;

culturing the inoculated foam enrichment sample at 150rpm/min and 28 ℃ for 2 months, transferring 2% enrichment culture solution into a new artificial seawater culture medium, adding new sterile polystyrene foam, and culturing for 2 months under the same conditions;

and c, repeating the same transfer culture for 1 time, taking out the foam after microorganism treatment in a 50mL sterile centrifuge tube after the third transfer, adding a proper amount of sterile phosphate buffer salt solution, oscillating at a low speed by an oscillator, and combining and centrifuging for multiple times to obtain thalli on the surface of the foam plastic. The obtained thalli are used for DNA extraction and bacterial strain separation and identification;

and d, after the obtained microorganisms on the surface of the foam are diluted in a gradient manner, the foam is coated on an R2A solid culture medium, is cultured in a constant temperature incubator at 28 ℃ for 1 week, and then the strains with different morphology and size are selected, purified and sequenced, and then the strains are stored in 20% glycerol and are stored in a refrigerator at-80 ℃.

The R2A solid culture medium is R2A agar of BD company in America, and the composition of the R2A solid culture medium is 18.2g/L of the R2A agar product.

e, the obtained culturable strain is subjected to the sequence elimination after the completion of the sequence measurement. The residual strains are respectively inoculated in a sterile artificial seawater culture medium, and degradation capacity verification is carried out by taking polystyrene chemical pure flake plastics (GF 94795701, sigma, USA) as the only carbon source and energy source; in the process of functional verification, 3 biological repetitions are set for each strain of degrading bacteria, and a control without adding polystyrene is set, so that the inoculated experimental group culture solution is observed to be obviously turbid, and the functional strain with the polystyrene degrading capability is determined.

The other 2 strains obtained in the present invention, after being obtained from the marine microorganism deposit management center, are simultaneously subjected to step e in the same screening method as mangrove strains, and are as follows:

the 5 Gordonia obtained by the invention are high-efficiency polystyrene plastic degrading bacteria obtained by the experiment, genomic DNA thereof is extracted, a bacterial universal primer 27F/1492R is adopted to amplify a 16S rRNA gene segment by taking the genome DNA as a template, high similarity sequences are selected from EzBiocloud and NCBI, and the systematic evolution distance of the sequences is calculated by using MEGA-7 to construct a phylogenetic tree.

Activating the 5 strains of single bacteria by using an R2A liquid culture medium, culturing for 2 days at 28 ℃ in a dark place at 150rpm/min, centrifugally collecting the bacteria at 6000rpm/10min, washing the bacteria for 2 times by using a sterile MMC culture medium, respectively inoculating the bacteria into a 100mL MMC liquid culture medium of a system, setting three biological repetitions by taking polystyrene with initial weighing as a unique carbon source, culturing in a shaking table at the constant temperature of 28 ℃ in a dark place at the rotating speed of 150rpm/min, taking out the polystyrene, washing a surface biological film by using 2% SDS, airing and weighing; wherein the control group is not inoculated with bacteria, and the other treatments are consistent with the inoculated group.

The nucleotide sequences of the 16S rRNA genes of the class of highly efficient plastic degrading bacteria (Gordonia spp.) are respectively as follows:

the nucleotide sequence of the 16S rRNA of Gordonia amicalis A04216 is as follows:

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAAGGCCCGCTTGCGGGTACTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTTACATCGCATGGTGTTTGGTGGAAAGCTTTTGCGGTTCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCAGGGACGAAGCGCAAGTGACGGTACCTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATTCTGCAACTCAATTGTAGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGGCTCATTTCACGAGTTCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCAGAAAGCTATAGAGATATAGCCCCCCTTGTGGTTGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTATTGCCAGCGGGTTATGCCGGGGACTTGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCTGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCAGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCT。

the 16S rRNA nucleotide sequence of Gordonia didemni 1A14786 is: AGGTTGGGGTGCTACCTGCAAGTCGACGGAAAGGCCCAGCTTGCTGGGTACTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCAACTGTCGCATGGTGGTTGGTGGAAAGCTTTTGCGGTGTGGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGGAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCAGGGACGAAGCGTGAGTGACGGTACCTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATTCTGCAACTCAATTGCAGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTCCTTTTCACGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCAGACGCGGCTAGAGATAGTCGTTCCCTTGTGGTTGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTATTGCCAGCGGGTTATGCCGGGGACTTGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCTTCGAAGGGGATCGGGT.

The nucleotide sequence of the 16S rRNA of Gordonia sihwensis A17966 is as follows: ACGGAAAGGCCCAGCTTGCTGGGTACTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATAGGACCACTGATTGCATGGTTGGTGGTGGAAAGCTTTTGCGGTTCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCGCTAGGGACGAAGCGTAAGTGACGGTACCTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATTCTGCAACTCAATTGCAGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGGCTCATTTCACGAGTTCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCAGAAAGCCGTAGAGATACGGCCCCCCTTGTGGTTGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTATTGCCAGCGGGTTATGCCGGGGACTTGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCGCGTACAGAGGGCTGCGAGACCGTGAGGTGGAGCGAATCCCTTAAAGCGCGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGG.

The nucleotide sequence of the 16S rRNA of Gordonia mangrovi A17970 is as follows: GCAAGTCGAACGGAAGGCCCAGCTTGCTGGGTGCTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCCTGACTTTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCAGTTGGTGCATGCCTTCTGGTGGAAAGCCTTGTGCGGTTGGGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCAGGGACGAAGCTTTTGTGACGGTACCTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATTCTGCAGCTTAACTGCAGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGGCTCATTTCACGAGTTCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCAGATGCGGGTAGAGATAGTCGTTCCCTTGTGGTTGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTATTGCCAGCGGGTTATGCCGGGGACTTGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGG.

The nucleotide sequence of the 16S rRNA of Gordonia bronchialis A17979 is as follows:

16S-1:

GATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAAGGCCCAGCTTGCTGGGTGCTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCCTGACTTTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTTCCCTCGCATGGGGGTTGGTGGAAAGCCTTGTGCGGTTGGGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCAGGGACGAAGCGCAAGTGACGGTACCTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATTCTGCAGCTTAACTGCAGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGGCTCATTTCACGAGTTCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCAGACGCGGCTAGAGATAGTCGTTCCCTTGTGGTTGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTATTGCCAGCGGGTTATGCCGGGGACTTGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCCGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGAGCAA

16S-2：

TGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAAGGCCCAGCTTGCTGGGTGCTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCCTGACTTTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTTCCCTCGCATGGGGGTTGGTGGAAAGCTTTTGCGGTTGGGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCAGGGACGAAGCTTTTGTGACGGTACCTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATTCTGCAGCTTAACTGCAGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGGCTCATTTCACGAGTTCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCAGACGCGGCTAGAGATAGTCGTTCCCTTGTGGTTGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTATTGCCAGCGGGTTATGCCGGGGACTTGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCTACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCCGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGAGCAT

16S-3:

CGATAGGTTTTCATGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAAGGCCCAGCTTGCTGGGTGCTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCCTGACTTTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTTACGTTGCATGACGTTTGGTGGAAAGCCTTGTGCGGTTGGGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCAGGGACGAAGCGCAAGTGACGGTACCTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATTCTGCAGCTTAACTGCAGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGGCTCATTTCACGAGTTCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCAGACGCGGCTAGAGATAGTCGTTCCCTTGTGGTTGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTATTGCCAGCGGGTTATGCCGGGGACTTGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCCGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGA。

the Gordonia (Gordonia spp.) strain may be used in biodegradable polystyrene.

The Gordonia (Gordonia spp.) strain was inoculated in a sterile artificial seawater medium and degradation capacity was verified using polystyrene chemically pure sheet plastic (GF 94795701, sigma, USA) as the sole carbon and energy source. During functional verification, 3 biological replicates per strain were set, and a control without polystyrene addition was set, and significant turbidity was observed in the post-inoculation experimental group broth, confirming that the Gordonia (Gordonia spp.) strain was a functional strain with polystyrene degrading ability.

The Gordonia (Gordonia spp.) strain may be used in the aerobic degradation of organic compounds. The organic compound was polystyrene flake plastic (GF 94795701, sigma, USA) purchased from Sigma company. The organic compound is polystyrene plastic.

The high-efficiency polystyrene degrading bacteria (Gordonia spp.) are identified to degrade about 0.05-0.07 g of polystyrene with an average initial quantity after being inoculated for 30 days in a shaking table with the rotating speed of 150rpm/min at 28 ℃ and the degradation rate of 2.66-7.73%.

The specific method of the application can be as follows: activating the Gordonia (Gordonia spp.) strain with an R2A liquid culture medium, centrifuging to collect 2mL of thalli, preparing the thalli into a bacterial suspension again by using MMC artificial seawater, washing for 2-3 times, inoculating the bacterial suspension to 50mL of MMC culture medium with polystyrene as a unique carbon source and energy source in an inoculum size of 4%, culturing the plastic sheet with polystyrene average initial size of about 0.05-0.07 g in a shaking table at a speed of 150rpm/min in a dark place at 28 ℃, washing the surface of the polystyrene plastic sheet with 2% SDS after inoculating for 30 days, completely removing the thalli, airing at room temperature after washing the plastic again with deionized water, weighing the degraded plastic sheet with a precision balance, and calculating the degradation rate of the Gordonia to the polystyrene to be 2.66-7.73%.

The application of the high-efficiency polystyrene degrading bacteria (Gordonia spp.) and genes and enzymes thereof in the depolymerization and degradation of polystyrene engineering applications; the degradation rate can reach 2.66% -7.73%.

The degradation bacteria are suitable for degrading organic compounds, especially polystyrene, are few strains for degrading polystyrene, and have obvious degradation effect from the degradation rate and the observation and analysis of a scanning electron microscope, thereby providing reliable basis for further excavation research of genes and enzymes of the degradation bacteria. Is suitable for innocent treatment, cyclic utilization and environmental restoration of polystyrene plastic resources.

Drawings

FIG. 1 is a macroscopic morphology of colonies of the polystyrene plastic degradation strain (Gordonia spp.) according to the invention;

FIG. 2 is a measurement of polystyrene degradation rate of a polystyrene plastic degradation strain (Gordonia spp.) according to the present invention;

FIG. 3 is a graph showing the morphological characteristics of a biological film formed on the surface of a polystyrene plastic sheet by a strain after 15 days of degradation of the polystyrene plastic degradation strain under a Scanning Electron Microscope (SEM);

FIG. 4 is a phylogenetic analysis tree of the polystyrene plastic degradation strain (Gordonia spp.) according to the present invention.

Detailed Description

The invention will be further illustrated by the following examples in conjunction with the accompanying drawings.

The present invention provides:

1) Has a preservation number of CGMCC No.22163 or comprises SEQ ID NO:1 and having all the identifying characteristics of gordonia Gordonia amicalis a 04216;

2) Has a preservation number of CGMCC No.22164 or comprises SEQ ID NO:2 and having all the identifying characteristics of Gordonia didemni 1a 14786;

3) Has a preservation number of CGMCC No.22166 or comprises SEQ ID NO:3 and having all the identifying characteristics of gordonia Gordonia sihwensis ZN-R1;

4) Has a preservation number of CGMCC No.22167 or comprises SEQ ID NO:4 and having all the identifying characteristics of gordonia Gordonia mangrovi ZN-R9;

5) Has a preservation number of CGMCC No.22165 or comprises SEQ ID NO: 5-7 and having all the identifying characteristics of Gordonia Gordonia bronchialis ZN-RX.

The gordonia strain comprises all the identifying characteristics selected from the group consisting of:

1) Pink or white on the R2A culture medium, circular colony, wet surface and regular edge;

2) The 16s RNA gene has a single copy or multiple copies;

3) Has polystyrene degradation capability.

The Gordonia strain is an isolated Gordonia strain.

Example 1: morphological characteristics of strains

The single colony three-area line is inoculated into an R2A solid culture medium, the plate is inverted in a constant temperature incubator and is cultured for 72 hours at 28 ℃, the colony size is 2-3mm, the colony is round, orange or pink or milky white, the surface is opaque and smooth and moist, the colony is regular, no halo exists, the bulge exists, and part of the colony is generated, and the colony is shown in figure 1.

Example 2: screening and identification of strains

The screening method of the high-efficiency polystyrene degrading bacteria (Gordonia spp.) MCC 1A17966,MCCC 1A17970,MCCC1A17979 comprises the following steps:

1) Sample collection: the applicant goes to the edge of the natural protection area of the red forest in purple mud town of Dragon sea city in Zhangzhou in 10 months 23 days of 2019, and a large amount of waste foam samples positioned in the protection area are collected in a concentrated manner on the premise of not damaging the local protection area, are packaged in a sterile sample collection bag, and are stored in an incubator at 4 ℃. The waste foam accumulation area is an area which can be immersed after tide water rises and tides, so that a small amount of sludge exists on the surface of a foam sample.

2) Laboratory enrichment: the collected foam sample is subjected to enrichment transfer by taking foam as a unique carbon source in a laboratory, and the specific operation steps are as follows:

a, taking a 300mL glass conical flask, cleaning, adding 150mL sterile artificial seawater culture medium, sealing by a sealing film, sterilizing at high temperature and high pressure at 121 ℃ for 20min, and supplementing trace element solution and collected waste foam plastic on an ultra-clean workbench after the system is cooled to room temperature.

The artificial seawater culture medium comprises the components (MMC) of 0.7g/L potassium chloride, 1g/L ammonium chloride, 1g/L sodium nitrate, 20g/L sodium chloride, 2g/L potassium dihydrogen phosphate, 3g/L disodium hydrogen phosphate, 2g/L magnesium sulfate heptahydrate and 2g/L magnesium sulfate heptahydrate, wherein the pH is adjusted to 7.2-7.4 by independent sterilization. The microelements comprise the following components: ferrous sulfate hexahydrate 2.0mg/L, zinc sulfate heptahydrate 2.0mg/L, manganese sulfate 2.0mg/L.

Culturing the inoculated foam enrichment sample at 150rpm/min and 28 ℃ for 2 months, transferring 2% enrichment culture solution into a new artificial seawater culture medium, adding new sterile polystyrene foam, and culturing for 2 months under the same conditions;

and c, repeating the same transfer culture for 1 time, taking out the foam after microorganism treatment in a 50ml sterile centrifuge tube after the third transfer, adding a proper amount of sterile phosphate buffer salt solution, oscillating at a low speed by an oscillator, and combining and centrifuging for multiple times to obtain the thalli on the surface of the foam plastic. The obtained thalli are used for DNA extraction and bacterial strain separation and identification.

And d, after the obtained microorganisms on the surface of the foam are diluted in a gradient manner, the foam is coated on an R2A solid culture medium, is cultured in a constant temperature incubator at 28 ℃ for 1 week, and then the strains with different morphology and size are selected, purified and sequenced, and then the strains are stored in 20% glycerol and are stored in a refrigerator at-80 ℃.

The R2A solid culture medium is R2A agar of BD company in America, and the composition of the R2A solid culture medium is 18.2g/L of the R2A agar product.

e, the obtained culturable strain is kicked off the strain of the 16S rDNA repetitive sequence after the sequencing is completed. The remaining strains were inoculated in sterile artificial seawater medium, respectively, and degradation capacity was verified using polystyrene chemically pure sheet plastic (GF 94795701, sigma, USA) as the sole carbon source and energy source. In the process of functional verification, 3 biological replicates are arranged for each strain, and a control without adding polystyrene is arranged, so that the inoculated experimental group culture solution is observed to be obviously turbid, and the functional strain with polystyrene degradation capability is determined.

The other 2 strains obtained in the present invention, after being obtained from the marine microorganism deposit management center, are subjected to the same verification steps as mangrove strains, as described in step e above and as follows:

the invention obtains 5 single strains with polystyrene degradation capability, extracts genome DNA thereof, uses the genome DNA as a template to amplify 16S rDNA fragments by adopting a bacterial universal primer 27F/1492R, carries out BLAST sequence analysis on EzBiocloud and NCBI, selects high similarity sequences, and constructs a phylogenetic tree (NJ) by using MEGA-7 software, see figure 4.

Example 3: determination of polystyrene degradation Capacity of Strain

1) The preparation of artificial seawater liquid culture is based on a glass conical flask, the culture system is 100ml, the thallus activated by an R2A liquid culture medium is centrifuged with 2% of inoculation amount, the thallus is washed 3 times by sterile artificial seawater, and the residual nutrient medium components are removed. Then re-suspending the thalli by using a sterile artificial seawater culture medium, inoculating the thalli into the 100ml sterile artificial seawater culture medium, and adding quantitative weighing polystyrene plastic as a unique carbon source;

2) Quantitatively weighing polystyrene plastic, respectively setting three biological repetitions, culturing for 30 days, taking out the plastic in a 50ml centrifuge tube, immersing the plastic in 2% SDS, placing in a 50 ℃ oven for incubation for 5 hours, and shaking and mixing uniformly every 1 hour to ensure that the SDS and the biological film on the surface of the plastic have full effect; taking out the plastic, flushing the plastic with sterile water for multiple times, placing the plastic in a sterile culture dish, drying the plastic in a baking oven at 50 ℃, weighing, wherein the average weight is a precision balance of ten thousandth;

3) Mass decay rate (%) = (polystyrene initial mass-mass after degradation)/initial mass of polystyrene plastic film × 100%. 30 days after inoculation, the MCCC1A 17966 can degrade the polystyrene with the average initial quantity of 0.0476g and the degradation rate of 4.69 percent; MCCC1a 17970 was able to degrade polystyrene with an average initial amount of 0.0475g and a degradation rate of 7.72%; MCCC1a17979 was able to degrade polystyrene with an average initial amount of 0.0451g, with a degradation rate of 6.69%; MCCC1a04216 was able to degrade polystyrene with an average initial amount of 0.0576g, with a degradation rate of 2.66%; MCCC1a14786 was able to degrade polystyrene with an average initial amount of 0.0702g, degradation rate of 3.37%; whereas the control treatment was unchanged, indicating that polystyrene was degraded by this strain (Gordonia spp.), and the specific degradation rate measurement results are shown in fig. 2.

Example 4: scanning electron microscope observation of biological film on polystyrene plastic surface

After the polystyrene degradation strain is cultured on a culture medium with polystyrene as a unique carbon source for 15 days, a piece of plastic is randomly taken out from the culture system, the culture medium on the surface is washed out by sterile seawater, and after the culture medium is fixed and tabletted, the formation of a biological film on the surface is observed under a Scanning Electron Microscope (SEM), and the biological film is shown in figure 3. As can be seen from fig. 3, the PS plastic film before biodegradation had a smooth surface without particularly significant defects. After 30 days incubation, the PS film surface became rough, and a large number of pores and grooves were present, which demonstrated that the film was degraded by the action of microbial PS plastic degrading enzymes. Surface adhesion and colonization of the cells on the PET plastic film are also clearly visible in SEM images.

Sequence listing

<110> institute of natural resources third sea

<120> Gordonia strain for efficiently degrading polystyrene Plastic

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1509

<212> DNA

<213> Gordonia amicalis 1A04216

<400> 1

agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60

ggaaaggccc gcttgcgggt actcgagtgg cgaacgggtg agtaacacgt gggtgatctg 120

ccctggactc tgggataagc ctgggaaact gggtctaata ccggatatga ccttacatcg 180

catggtgttt ggtggaaagc ttttgcggtt caggatgggc ccgcggccta tcagcttgtt 240

ggtggggtaa tggcctacca aggcgacgac gggtagccga cctgagaggg tgatcggcca 300

cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga atattgcaca 360

atgggcgcaa gcctgatgca gcgacgccgc gtgagggatg acggccttcg ggttgtaaac 420

ctctttcacc agggacgaag cgcaagtgac ggtacctgga gaagaagcac cggccaacta 480

cgtgccagca gccgcggtaa tacgtagggt gcgagcgttg tccggaatta ctgggcgtaa 540

agagctcgta ggcggtttgt cgcgtcgtct gtgaaattct gcaactcaat tgtaggcgtg 600

caggcgatac gggcagactt gagtactaca ggggagactg gaattcctgg tgtagcggtg 660

aaatgcgcag atatcaggag gaacaccggt ggcgaaggcg ggtctctggg tagtaactga 720

cgctgaggag cgaaagcgtg ggtagcgaac aggattagat accctggtag tccacgccgt 780

aaacggtggg tactaggtgt ggggctcatt tcacgagttc cgtgccgtag ctaacgcatt 840

aagtaccccg cctggggagt acggccgcaa ggctaaaact caaaggaatt gacgggggcc 900

cgcacaagcg gcggagcatg tggattaatt cgatgcaacg cgaagaacct tacctgggtt 960

tgacatacac cagaaagcta tagagatata gccccccttg tggttggtgt acaggtggtg 1020

catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080

cttgtcctgt attgccagcg ggttatgccg gggacttgca ggagactgcc ggggtcaact 1140

cggaggaagg tggggatgac gtcaagtcat catgcccctt atgtccaggg cttcacacat 1200

gctacaatgg ctggtacaga gggctgcgat accgtgaggt ggagcgaatc ccttaaagcc 1260

agtctcagtt cggattgggg tctgcaactc gaccccatga agtcggagtc gctagtaatc 1320

gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacgt 1380

catgaaagtc ggtaacaccc gaagccggtg gcctaacccc ttgtgggagg gagctgtcga 1440

aggtgggatc ggcgattggg acgaagtcgt aacaaggtag ccgtaccgga aggtgcggct 1500

ggatcacct 1509

<210> 2

<211> 1418

<212> DNA

<213> Gordonia didemni 1A14786

<400> 2

aggttggggt gctacctgca agtcgacgga aaggcccagc ttgctgggta ctcgagtggc 60

gaacgggtga gtaacacgtg ggtgatctgc cctgcacttt gggataagcc tgggaaactg 120

ggtctaatac cggatatgac caactgtcgc atggtggttg gtggaaagct tttgcggtgt 180

gggatgggcc cgcggcctat cagcttgttg gtggggtaat ggcctaccaa ggcgacgacg 240

ggtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 300

cgggaggcag cagtggggaa tattgcacaa tgggcggaag cctgatgcag cgacgccgcg 360

tgagggatga cggccttcgg gttgtaaacc tctttcacca gggacgaagc gtgagtgacg 420

gtacctggag aagaagcacc ggccaactac gtgccagcag ccgcggtaat acgtagggtg 480

cgagcgttgt ccggaattac tgggcgtaaa gagctcgtag gcggtttgtc gcgtcgtctg 540

tgaaattctg caactcaatt gcaggcgtgc aggcgatacg ggcagacttg agtactacag 600

gggagactgg aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccggtg 660

gcgaaggcgg gtctctgggt agtaactgac gctgaggagc gaaagcgtgg gtagcgaaca 720

ggattagata ccctggtagt ccacgccgta aacggtgggt actaggtgtg ggttcctttt 780

cacgggatcc gtgccgtagc taacgcatta agtaccccgc ctggggagta cggccgcaag 840

gctaaaactc aaaggaattg acgggggccc gcacaagcgg cggagcatgt ggattaattc 900

gatgcaacgc gaagaacctt acctgggttt gacatacacc agacgcggct agagatagtc 960

gttcccttgt ggttggtgta caggtggtgc atggctgtcg tcagctcgtg tcgtgagatg 1020

ttgggttaag tcccgcaacg agcgcaaccc ttgtcctgta ttgccagcgg gttatgccgg 1080

ggacttgcag gagactgccg gggtcaactc ggaggaaggt ggggatgacg tcaagtcatc 1140

atgcccctta tgtccagggc ttcacacatg ctacaatggc cggtacagag ggctgcgata 1200

ccgtgaggtg gagcgaatcc cttaaagccg gtctcagttc ggatcggggt ctgcaactcg 1260

accccgtgaa gtcggagtcg ctagtaatcg cagatcagca acgctgcggt gaatacgttc 1320

ccgggccttg tacacaccgc ccgtcacgtc atgaaagtcg gtaacacccg aagccggtgg 1380

cctaaccctt gtggagggag cttcgaaggg gatcgggt 1418

<210> 3

<211> 1369

<212> DNA

<213> Gordonia sihwensis 1A17966

<400> 3

acggaaaggc ccagcttgct gggtactcga gtggcgaacg ggtgagtaac acgtgggtga 60

tctgccctgg actctgggat aagcctggga aactgggtct aataccggat aggaccactg 120

attgcatggt tggtggtgga aagcttttgc ggttcaggat gggcccgcgg cctatcagct 180

tgttggtggg gtaatggcct accaaggcga cgacgggtag ccgacctgag agggtgatcg 240

gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg 300

cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatgacggcc ttcgggttgt 360

aaacctcttt cgctagggac gaagcgtaag tgacggtacc tggagaagaa gcaccggcca 420

actacgtgcc agcagccgcg gtaatacgta gggtgcgagc gttgtccgga attactgggc 480

gtaaagagct cgtaggcggt ttgtcgcgtc gtctgtgaaa ttctgcaact caattgcagg 540

cgtgcaggcg atacgggcag acttgagtac tacaggggag actggaattc ctggtgtagc 600

ggtgaaatgc gcagatatca ggaggaacac cggtggcgaa ggcgggtctc tgggtagtaa 660

ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 720

ccgtaaacgg tgggtactag gtgtggggct catttcacga gttccgtgcc gtagctaacg 780

cattaagtac cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg 840

ggcccgcaca agcggcggag catgtggatt aattcgatgc aacgcgaaga accttacctg 900

ggtttgacat acaccagaaa gccgtagaga tacggccccc cttgtggttg gtgtacaggt 960

ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1020

aacccttgtc ctgtattgcc agcgggttat gccggggact tgcaggagac tgccggggtc 1080

aactcggagg aaggtgggga tgacgtcaag tcatcatgcc ccttatgtcc agggcttcac 1140

acatgctaca atggcgcgta cagagggctg cgagaccgtg aggtggagcg aatcccttaa 1200

agcgcgtctc agttcggatt ggggtctgca actcgacccc atgaagtcgg agtcgctagt 1260

aatcgcagat cagcaacgct gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1320

acgtcatgaa agtcggtaac acccgaagcc ggtggcctaa cccttgtgg 1369

<210> 4

<211> 1378

<212> DNA

<213> Gordonia mangrovi 1A17970 ZN15-R9

<400> 4

gcaagtcgaa cggaaggccc agcttgctgg gtgctcgagt ggcgaacggg tgagtaacac 60

gtgggtgatc tgcccctgac tttgggataa gcctgggaaa ctgggtctaa taccggatat 120

gaccagttgg tgcatgcctt ctggtggaaa gccttgtgcg gttggggatg ggcccgcggc 180

ctatcagctt gttggtgggg taatggccta ccaaggcgac gacgggtagc cgacctgaga 240

gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag gcagcagtgg 300

ggaatattgc acaatgggcg caagcctgat gcagcgacgc cgcgtgaggg atgacggcct 360

tcgggttgta aacctctttc accagggacg aagcttttgt gacggtacct ggagaagaag 420

caccggccaa ctacgtgcca gcagccgcgg taatacgtag ggtgcgagcg ttgtccggaa 480

ttactgggcg taaagagctc gtaggcggtt tgtcgcgtcg tctgtgaaat tctgcagctt 540

aactgcaggc gtgcaggcga tacgggcaga cttgagtact acaggggaga ctggaattcc 600

tggtgtagcg gtgaaatgcg cagatatcag gaggaacacc ggtggcgaag gcgggtctct 660

gggtagtaac tgacgctgag gagcgaaagc gtgggtagcg aacaggatta gataccctgg 720

tagtccacgc cgtaaacggt gggtactagg tgtggggctc atttcacgag ttccgtgccg 780

tagctaacgc attaagtacc ccgcctgggg agtacggccg caaggctaaa actcaaagga 840

attgacgggg gcccgcacaa gcggcggagc atgtggatta attcgatgca acgcgaagaa 900

ccttacctgg gtttgacata caccagatgc gggtagagat agtcgttccc ttgtggttgg 960

tgtacaggtg gtgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1020

aacgagcgca acccttgtcc tgtattgcca gcgggttatg ccggggactt gcaggagact 1080

gccggggtca actcggagga aggtggggat gacgtcaagt catcatgccc cttatgtcca 1140

gggcttcaca catgctacaa tggccggtac agagggctgc gataccgtga ggtggagcga 1200

atcccttaaa gccggtctca gttcggatcg gggtctgcaa ctcgaccccg tgaagtcgga 1260

gtcgctagta atcgcagatc agcaacgctg cggtgaatac gttcccgggc cttgtacaca 1320

ccgcccgtca cgtcatgaaa gtcggtaaca cccgaagccg gtggcctaac ccttgtgg 1378

<210> 5

<211> 1521

<212> DNA

<213> Gordonia bronchialis 1A17979

<400> 5

gatcctggct caggacgaac gctggcggcg tgcttaacac atgcaagtcg aacggaaagg 60

cccagcttgc tgggtgctcg agtggcgaac gggtgagtaa cacgtgggtg atctgcccct 120

gactttggga taagcctggg aaactgggtc taataccgga tatgaccttc cctcgcatgg 180

gggttggtgg aaagccttgt gcggttgggg atgggcccgc ggcctatcag cttgttggtg 240

gggtaatggc ctaccaaggc gacgacgggt agccgacctg agagggtgat cggccacact 300

gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat tgcacaatgg 360

gcgcaagcct gatgcagcga cgccgcgtga gggatgacgg ccttcgggtt gtaaacctct 420

ttcaccaggg acgaagcgca agtgacggta cctggagaag aagcaccggc caactacgtg 480

ccagcagccg cggtaatacg tagggtgcga gcgttgtccg gaattactgg gcgtaaagag 540

ctcgtaggcg gtttgtcgcg tcgtctgtga aattctgcag cttaactgca ggcgtgcagg 600

cgatacgggc agacttgagt actacagggg agactggaat tcctggtgta gcggtgaaat 660

gcgcagatat caggaggaac accggtggcg aaggcgggtc tctgggtagt aactgacgct 720

gaggagcgaa agcgtgggta gcgaacagga ttagataccc tggtagtcca cgccgtaaac 780

ggtgggtact aggtgtgggg ctcatttcac gagttccgtg ccgtagctaa cgcattaagt 840

accccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg ggggcccgca 900

caagcggcgg agcatgtgga ttaattcgat gcaacgcgaa gaaccttacc tgggtttgac 960

atacaccaga cgcggctaga gatagtcgtt cccttgtggt tggtgtacag gtggtgcatg 1020

gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080

tcctgtattg ccagcgggtt atgccgggga cttgcaggag actgccgggg tcaactcgga 1140

ggaaggtggg gatgacgtca agtcatcatg ccccttatgt ccagggcttc acacatgcta 1200

caatggccgg tacagagggc tgcgataccg tgaggtggag cgaatccctt aaagccggtc 1260

tcagttcgga tcggggtctg caactcgacc ccgtgaagtc ggagtcgcta gtaatcgcag 1320

atcagcaacg ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacgtcatg 1380

aaagtcggta acacccgaag ccggtggcct aaccccttgt gggagggagc cgtcgaaggt 1440

gggatcggcg attgggacga agtcgtaaca aggtagccgt accggaaggt gcggctggat 1500

cacctccttt ctaaggagca a 1521

<210> 6

<211> 1521

<212> DNA

<213> Gordonia bronchialis 1A17979

<400> 6

tgatcctggc tcaggacgaa cgctggcggc gtgcttaaca catgcaagtc gaacggaaag 60

gcccagcttg ctgggtgctc gagtggcgaa cgggtgagta acacgtgggt gatctgcccc 120

tgactttggg ataagcctgg gaaactgggt ctaataccgg atatgacctt ccctcgcatg 180

ggggttggtg gaaagctttt gcggttgggg atgggcccgc ggcctatcag cttgttggtg 240

gggtaatggc ctaccaaggc gacgacgggt agccgacctg agagggtgat cggccacact 300

gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat tgcacaatgg 360

gcgcaagcct gatgcagcga cgccgcgtga gggatgacgg ccttcgggtt gtaaacctct 420

ttcaccaggg acgaagcttt tgtgacggta cctggagaag aagcaccggc caactacgtg 480

ccagcagccg cggtaatacg tagggtgcga gcgttgtccg gaattactgg gcgtaaagag 540

ctcgtaggcg gtttgtcgcg tcgtctgtga aattctgcag cttaactgca ggcgtgcagg 600

cgatacgggc agacttgagt actacagggg agactggaat tcctggtgta gcggtgaaat 660

gcgcagatat caggaggaac accggtggcg aaggcgggtc tctgggtagt aactgacgct 720

gaggagcgaa agcgtgggta gcgaacagga ttagataccc tggtagtcca cgccgtaaac 780

ggtgggtact aggtgtgggg ctcatttcac gagttccgtg ccgtagctaa cgcattaagt 840

accccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg ggggcccgca 900

caagcggcgg agcatgtgga ttaattcgat gcaacgcgaa gaaccttacc tgggtttgac 960

atacaccaga cgcggctaga gatagtcgtt cccttgtggt tggtgtacag gtggtgcatg 1020

gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080

tcctgtattg ccagcgggtt atgccgggga cttgcaggag actgccgggg tcaactcgga 1140

ggaaggtggg gatgacgtca agtcatcatg ccccttatgt ccagggcttc acacatgcta 1200

caatggccgg tacagagggc tgcgataccg tgaggtggag cgaatccctt aaagccggtc 1260

tcagttcgga tcggggtctg ctactcgacc ccgtgaagtc ggagtcgcta gtaatcgcag 1320

atcagcaacg ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacgtcatg 1380

aaagtcggta acacccgaag ccggtggcct aaccccttgt gggagggagc cgtcgaaggt 1440

gggatcggcg attgggacga agtcgtaaca aggtagccgt accggaaggt gcggctggat 1500

cacctccttt ctaaggagca t 1521

<210> 7

<211> 1521

<212> DNA

<213> Gordonia bronchialis 1A17979

<400> 7

cgataggttt tcatggagag tttgatcctg gctcaggacg aacgctggcg gcgtgcttaa 60

cacatgcaag tcgaacggaa aggcccagct tgctgggtgc tcgagtggcg aacgggtgag 120

taacacgtgg gtgatctgcc cctgactttg ggataagcct gggaaactgg gtctaatacc 180

ggatatgacc ttacgttgca tgacgtttgg tggaaagcct tgtgcggttg gggatgggcc 240

cgcggcctat cagcttgttg gtggggtaat ggcctaccaa ggcgacgacg ggtagccgac 300

ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag 360

cagtggggaa tattgcacaa tgggcgcaag cctgatgcag cgacgccgcg tgagggatga 420

cggccttcgg gttgtaaacc tctttcacca gggacgaagc gcaagtgacg gtacctggag 480

aagaagcacc ggccaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttgt 540

ccggaattac tgggcgtaaa gagctcgtag gcggtttgtc gcgtcgtctg tgaaattctg 600

cagcttaact gcaggcgtgc aggcgatacg ggcagacttg agtactacag gggagactgg 660

aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg 720

gtctctgggt agtaactgac gctgaggagc gaaagcgtgg gtagcgaaca ggattagata 780

ccctggtagt ccacgccgta aacggtgggt actaggtgtg gggctcattt cacgagttcc 840

gtgccgtagc taacgcatta agtaccccgc ctggggagta cggccgcaag gctaaaactc 900

aaaggaattg acggggcccg cacaagcggc ggagcatgtg gattaattcg atgcaacgcg 960

aagaacctta cctgggtttg acatacacca gacgcggcta gagatagtcg ttcccttgtg 1020

gttggtgtac aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080

cccgcaacga gcgcaaccct tgtcctgtat tgccagcggg ttatgccggg gacttgcagg 1140

agactgccgg ggtcaactcg gaggaaggtg gggatgacgt caagtcatca tgccccttat 1200

gtccagggct tcacacatgc tacaatggcc ggtacagagg gctgcgatac cgtgaggtgg 1260

agcgaatccc ttaaagccgg tctcagttcg gatcggggtc tgcaactcga ccccgtgaag 1320

tcggagtcgc tagtaatcgc agatcagcaa cgctgcggtg aatacgttcc cgggccttgt 1380

acacaccgcc cgtcacgtca tgaaagtcgg taacacccga agccggtggc ctaacccctt 1440

gtgggaggga gccgtcgaag gtgggatcgg cgattgggac gaagtcgtaa caaggtagcc 1500

gtaccggaag gtgcggctgg a 1521

Claims (7)

1. Gordonia genusGordonia spp.) strain, characterized in that it is gordoniaGordonia sihwensisZN14-R1 with preservation number CGMCC No.22166.

2. A genus Gordonia as claimed in claim 1Gordonia spp.), characterized in that the gordonia strain comprises all the identifying characteristics selected from the group consisting of:

1) Pink or white on the R2A culture medium, circular colony, wet surface and regular edge;

2) The 16s RNA gene has a single copy or multiple copies;

3) The strain has polystyrene degrading ability.

3. The Gordonia genus as claimed in claim 1Gordonia spp.), characterized in that the gordonia strain is an isolated gordonia strain.

4. The Gordonia genus as claimed in claim 1Gordonia spp.) strain in the biodegradation of polystyrene.

5. The use as claimed in claim 4, characterized in that the genus GordoniaGordonia spp.) strain degrades polystyrene under aerobic conditions.

6. The Gordonia genus as claimed in claim 1Gordonia spp.) strain is applied to innocent treatment, recycling and environmental remediation of polystyrene plastic resources.

7. A process comprising the step of preparing a strain of Gordonia as defined in claim 1Gordonia spp.) microbial inoculant for the strain.

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