Preliminary Application Of Sandwich Plate Method In Bacterial Isolation From Marine Sediments And Polyphasic Taxonomy Of Two Novel Strains

The marine environment is complex and changeable,in which there are huge microbial resources.These marine microorganisms not only play a vital role in marine ecosystem and biogeochemical cycle,but also are important source of new bioactive substances and lead compounds.It is very important to isolate and identify marine microbial resources,especially the potential new species.Due to the limitation of isolation and culture conditions and technologies,many marine microorganisms are not unexploited,and pure culture strains are not obtained.Therefore,it is necessary to improve and optimize the existing isolation and culture conditions,develop new isolation and culture methods,and isolate and purify more previously uncultured microorganismsIn this thesis,the microbial diversity of Weihai offshore sediment samples was studied,using the sandwich plate method to separate the microorganisms in the marine sediment,and combined with the 16S rRNA gene high-throughput sequencing technology to analyze the flora of culturable bacteria.The dependent strains were screened out.Based on the analysis of genome and transcriptome of a representative strain,the mechanism of promoting growth of dependent and helper strains was explored.The marine sediment samples were divided into control group A01,experimental groups A08,A38,A71 and A72.The isolation and culture of microorganisms were carried out,and the changes of culturable bacterial flora were analyzed with 16S rRNA gene high-throughput sequencing.Four strains of bacteria were selected and 447 strains were isolated by sandwich plate method,which were distributed in 5 phylum,116 genera and 216 species,with 156 strains of potential new species.The pure culture strains isolated from the samples were mainly concentrated on Proteobacteria and Bacteroidetes,followed by Firmicutes,Actinobacteria and Deinococcus-Thermus.According to the 16S rRNA gene high-throughput sequencing results,the culturable bacterial flora in different treatment groups changed significantly.The species diversity of control group A01 was the highest,but the sample with the highest species richness was the experimental group A38,and there were also different bacterial groups in other experimental groups.Among them,the composition of bacterial groups of A38 and A71 in the experimental group was the most similar,but the proportions of dominant bacterial groups were different.The growth dependent strains were screened from the isolated strains,and the relationship between a dependent strain and helper was investigated by genome and transcriptome analysis.A total of 22 dependent strains were screened by the method of spot seeding,which were distributed in Proteobacteria and Bacteroidetes,and were mainly concentrated in Rhodobacteraceae and Flavobacteriaceae.There was a non-contact dependence between strain Gaetbulibacter sp.S0825 and Colwellia aestuarii KCTC 12480T.Preliminary analysis of the genome of strain Gaetbulibacter sp.S0825 revealed that some metabolic pathways were incomplete,which may result in its inability to grow alone.Transcriptome sequencing analysis was carried out on C.aestuarii KCTC 12480T in separate culture and mixed culture.There were 321 differentially expressed genes,184 up-regulated genes and 137 down regulated genes.In the GO enrichment analysis of differential genes,a total of 1184 GO terms were enriched,and 631 functional classifications of biological processes,171 functional classifications of cell components,and 382 functional classifications of molecular functions were annotated.The effect of C.aestuarii KCTC 12480T on the growth of strain Gaetbulibacter sp.S0825 was predicted by annotation and analysis of different genes.The polyphasic taxonomy of two novel strains was completed.By means of phenotypic characteristics,physiological and biochemical characteristics,molecular genetic and chemical classification characteristics,the taxonomic status of strains 2b14T and E84T was determined,and the taxonomic status of strains was identified.Strain 2b14 T represents a novel species within the genus Pontibacter,for which the name Pontibacter arcticus sp.nov.is proposed.The type strain is 2b14T(=KCTC 62596T=MCCC 1H00304T).Strain E84T represents a novel species of the genus Rhodosalinus,for which the name Rhodosalinus halophilus sp.nov.is proposed.The type strain is E84T(=KCTC 52697T=MCCC 1H00231T).