Composition Analysis And Specific Primers Design Of Prokaryotic Microbial Community On The Surface Of Micro-Nano Antifouling Coating

The attachment of marine fouling organisms has brought serious negative impacts,which not only accelerated the corrosion of artificial facilities in seawater,but also brought great additional energy consumption,threatening the orderly development of the marine economy.The attachment organs and mechanisms of different fouling organisms on the surface sedimentation and attachment are different,forming a very complicated mechanism of action,but it is worth confirming that the fouling organisms will interact with certain specific bacterial flora on the surface biofilm.At present,there have been a small number of studies related to the role of microorganisms and fouling organisms,but the microbial information available at the genus and species levels is very limited.More experiments and research work are still needed to fill the gap.In this study,three carbon nanotube materials with good antifouling effect and one kind of nano-platinum material screened by the laboratory were selected as experimental objects,micro/nano composite antifouling coatings were prepared by adding 0.1wt% concentration to two common commercial antirust paint substrates:epoxy iron red antirust paint and M133 acrylic resin film,and conduct to the Marine experiments.The biofilm on each coating surface was sampled every 7 days,and the biofilm metagenome was extracted on day 28 for high-throughput determination of the full-length 16 S r RNA gene,and the main prokaryotic bacterial group structures and differences between the two primer substrates were analyzed.Based on the high-throughput test results,for the top 30 prokaryotic genus in biofilms,the design principle of Simple Allele-discriminating PCR primers was used to design and successfully obtain the excellent primers prokaryotic specificity of Neptuniibacter,Marinelicella,Fluviicola,Tenacibaculum.Studies have shown that Proteobacteria is the most dominant door in the biofilm on the coating surface,accounting for an average of up to 86%.The abundance difference of Rhodovulum,Neptuniibacter,Neptunomonas,Sulfitobacter in the two substrates is very obvious,especially Neptuniibacter.Its iron red matrix OTU(Operational Taxonomic Units)number is more than 100 times higher than acrylic resin matrix.The use of 16 S r RNA gene full-length high-throughput measurement has obtained a large number of species-level structural information,and found that the differences are mainly caused by Neptuniibacter?sp.?CAR-SF strains.Through principal component analysis,the main factor causing the difference in the structure of prokaryotic flora is the difference in the primer matrix,the difference can reach 92.7%,and the introduction of nano-materials has only a small effect on the structure of the flora.Scanning electron microscopy and static contact angle test characterization results were similar.No significant differences were found on the surface of the coating after the introduction of nanomaterials,or it played an anti-fouling role by changing the physical and chemical properties of the coating in the matrix.Through real-time observation and statistics of 7 kinds of fouling organisms,it was found that barnacles and Ciona intestinalis were the main fouling organisms in this experiment.The anti-fouling performance of the acrylic matrix coating was better than the iron red matrix coating on the whole.It was found that the attachment of Ciona intestinalis larvae was very different,which is consistent with the distribution of Neptuniibacter bacteria in high-throughput test results.In order to verify this rule,the specific primers of Neptuniibacter bacteria with good specificity were used to conduct QPCR experiments on the contents of the bacteria in each coating.The results showed that the bacteria content of iron red matrix coating was much higher than that of acrylic matrix coating.The total content of Neptuniibacter bacteria in iron red/platinum 2coating was up to 345202.6 copies/?l,which was the coating with the highest content of bacteria and the coating with the largest adhesion area of Ciona intestinalis.Through correlation analysis,it was found that there was a significant positive correlation between them(P<<0.01,r=0.967),which further verified that Neptuniibacter had a potential induction effect on the attachment of Ciona intestinalis.This study preliminarily established and verified the functional relationship between prokaryotes and fouling organisms,the relevant mechanism of action and in-depth marine verification experiments are worthy of further exploration.It may provide a new basis for solving the occurrence of fouling at the source.