Community Structure Characteristics Of Bacteria Attached On Microplastics In Sanggou Bay Mariculture Area

Marine microplastic pollution has become a hot environmental issue worldwide.Microplastics have the characteristics,including small particles,strong hydrophobicity,large specific surface area,low density and long degradation cycle.Studies have shown that marine microplastics can be colonized rapidly by microorganisms(including potentially pathogenic bacteria),and together constitute a marine "Plastisphere" micro-ecology system.Studies also found that pathogenic microorganisms distributed in the "Plastisphere" would pose a huge potential threat to the ocean,especially the mariculture area.At present,the research on the community structure characteristics of bacteria attached on microplastics is mainly concentrated in estuaries and freshwater areas,and there is less research on the community structure characteristics of bacteria attached on the microplastics in mariculture areas.The mariculture area of Sanggou Bay has a long history.The research found that the abundance of microplastics in water and sediment is much higher than those in open seas.It will cause a serious threat to the mariculture organisms if the microplastics with pathogenic bacteria were ingested by the farmed organisms.Therefore,it is imminent to explore the the community structure characteristics of the bacteria attached on the microplastics in mariculture area.Based on this,this research used an in-situ experiment to study the characteristics of bacteria communities on different substates for different exposure time in coastal areas and mariculture areas of the north of China(Sanggou Bay,Shandong Province),in August 2018.Mariculture related microplastics(fishing net,foam,float)and natural organic particles(feather)with a particle size of 1 to 4 mm were selected as research materials.Six experimental groups and two control groups were set up in mariculture and nearshore areas respectively,with a total of 24 in-situ experiment devices.The samples were collected at 3 h,1 d,7 d,and 21 d.Firstly,the biofilm formation process on the microplastics in nearshore area and mariculture area in different time periods was studied.Then,the high-throughput sequencing technology was used to study the dynamic changes of the community structure of bacteria attached on microplastics.Finally,the strains attached on the microplastics were obtained by screening,separation,purification and identification techniques.The main research results are as follows:(1)The microplastics were obtained by processing of commercial mariculture related plastics,and Fourier infrared spectrometer was used to characterize them.The polymer type was identified by comparing the infrared spectrum with the standard spectrum of the database and analyzing the characteristic peaks.The polymer type of the fishing net,foam and foams are linear low density polyethylene,polystyrene and polyvinyl chloride,respectively.(2)Scanning electron microscope(SEM)was used to observe and analyze the in-situ experiment microplastic samples.The SEM results of the microplastic surface at different periods showed that micron-sized particles attached on the surface of microplastics at the initial stage of the in-situ experiment(3 hours and 1 day),indicating that organic and inorganic particles such as bacteria can rapidly colonize on the microplastics in a short time.After the 7 and 21 days,the surface attachments of most microplastics and feather particles increased significantly.After 21 days,obvious biofilm formation characteristics could be observed on the microplastics and feather particles.The attached bacteria were rod-shaped and granular mainly.(3)The community structure of bacteria attached on the surface of microplastics was identified by DNA extraction and high-throughput sequencing analysis of microplastics,seawater and sediment samples collected in in-situ experiment,to determine the community structure composition of bacteria on microplastics,and analyze the diversity of bacteria.The results showed that the bacteria attached on microplastics mainly came from seawater,but the contribution of sediments cannot be ignored.The microplastic polymer type had no significant effect on the community structure characteristics(diversity and abundance)of the bacteria attached on microplastics,and the trends of relative abundance of different bacteria on different material particles are inconsistent.The community structure composition of the bacteria on microplastics mainly includes Vibrio,Pseudoalteromonas,and Alteromonas,and the potential pathogenic Vibrio is distributed in the genus Vibrio.This result is consistent with the analysis of the KEGG pathways,which explains disease and metabolism in pathway,may pose a potential threat to farmed organisms.(4)At the same time,based on the analysis of bacterial community diversity,the alpha diversity of bacterial communities on microplastics is lower than that in seawater environment.The alpha diversity of bacterial communities on microplastics in mariculture area is lower than that in nearshore area,and their relative abundance of the main species is higher than the surrounding seawater and sediments.This shows that the attachment process of bacteria on microplastics is selective.The resultsof ? diversity analysis of the bacterial community on microplastics showed that the similarity between the bacterial communities on substrates in the mariculture area and seawater was becoming lower with time.(5)For the dominant bacterial genus of microplastics,7 strains were isolated and purified from microplastic samples of in-situ experiment by plate separation method.Partial gene sequences of the strains were obtained by 16 S rRNA gene sequencing technology,and the strains were identified by comparing the sequences with GenBank data.Finally,7 single strains were obtained,6 of them were Vibrio,including Vibriofortis,Vibriorotiferianus,Vibrioalginolyticus,Vibrioowensii,Vibriocyclitrophicus and Vibriochagasii,1 strain was Shewanellawaksmanii,the similarity rate is higher than 99.30%,and the isolated strains have been preserved.In summary,bacteria can quickly attach to microplastic and form the biofilm in Sanggou Bay,and the bacterial community structure on the surface of microplastics is significantly different from the bacterial community in the seawater environment.Potentially pathogenic bacteria have a relatively higher abundance on microplastics.Many Vibrio strains were detected on the surface of microplastics,and some of them are pathogenic Vibrio spp.Therefore,the presence of microplastics in the mariculture environment increases the threat of pathogenic bacteria to the cultured organisms.