Study On The Bacterial Community Structure Of Arctic Ocean Sediments And Extracellular Hydrolytic Enzymes Of Cultivable Bacteria

There is a unique microbial ecosystem due to the special environmental factors of low temperatures,oligotrophicity and high UV–radiations in the Arctic.Polar microbes can adapt the extreme environments by the adaptation of metabolic functions and the synthesis of cold-active enzymes.The bacterial community structure of 14 marine sediment samples collected during the 7th Chinese National Arctic Scientific Expedition was investigated by high-throughput sequencing technology,and the relationship between physicochemical factors and community structure was also analyzed.Cultivable bacteria from 37 marine sediments and 24overlying water samples were isolated and purified by serially dilution and spreading on modified marine ZoBell 2216E plate,and nine extracellular hydrolytic enzyme activity of some isolates was further analyzed.A total of 1,416,738 trimed sequences were obtained,resulting in 1,283 OTUs at the 97%sequence identity by high-throughput sequencing of 14 marine sediments on the basis of 16S rRNA V3+V4 variable region.At the phylum level,Proteobacteria showed the highest relative sequence abundance?29.79%–62.86%?;meanwhile,Cyanobacteria also showed high relative sequence abundance with remarkable difference between the samples investigated?0.03%–38.64%?.At the class level,the abundances of Gammaproteobacteria and Deltaproteobacteria were relatively high?6.82%–39.47%and 6.98%–35.22%?;and the relative abundance of Alphaproteobacteria varied significantly among the different samples?0.48%–34.29%?.At the genus level,the dominant genus was distinctive,in general,Desulfococcus,Plesiomonas and Lutimonas were the dominant genera.Principal coordinates analysis?PCoA?and principal component analysis?PCA?revealed that the bacterial community structure was closely related with the sampling depth,the relative content of cyanobacteria is negatively correlated with the sampling station depth significantly.Canonical correspondence analysis?CCA?revealed that there was a significant correlation between the physicochemical factors and the bacterial community structure,and the depth of sampling station,NO₂^--N and NO₃^--N contnent were the most significant factors that correlated with the bacterial community composition.A total of 428 strains were isolated from 37 sediments and 24 overlying seawater;Molecular identification and phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates belonged to 4 phyla,6 classes,12 orders,24families,51 genera and 101 species.The isolates from sediments belonged to 4 phyla,6 classes,12 orders,22 families,44 genera and 80 species.The isolates from overlying water belonged to 4 phyla,5 classes,11 orders,18 families,30 genera and49 species.?–Proteobacteria was the most abundant in terms of number and species,accounting for 50.00%of the total strains and 53.47%of total species.33 and 3strains isolated from sediments and overlying waters respectively,which had 16S rRNA gene similarity less than 97%with the closest type strain,might be four and two potential novel species.These results implied that there were cultivable bacteria with abundant diversity and novelty in Arctic ocean sediments and overlying water,which might have great potential application values.Based on the screening medium plate,the preliminary screening of 9 kinds of extracellular hydrolase such as protease,lipase and amylase of 352 isolates was performed.The results showed that most of strains?81.53%?produced more than one type of extracellular hydrolase,and 10.51%could produce more than 7 type of extracellular hydrolase tested.The number of gelatinase-producing strains was the largest,up to 64.49%,followed by beta-galactosidase-producing strains?54.55%?.The number of phospholipase-producing strains was the least,and it only accounted fot 10.51%of the total strains tested.The supernatant enzymatic activity of caseinase,carrageenase,agarase and lipase of some strains showing high activity at the screening medium plate were further analyzed.The results showed that the activity of each strain was very different.Enzymatic inhibition test and zymogram analysis of some protease-producing strains showed that the protease produced by each strain was significantly inhibited by at least two kinds of enzymatic inhibitors,and was able to produce at least three kinds of proteases with different molecular weights.In conclusion,this study enriches the resources of polar extracellular hydrolase isolates,and is helpful to the development and utilization of microbial resources in Arctic.