Isolation And Cultivation Of Deep-sea Anaerobic Microorganisms And Study Of The Polysaccharide Metabolism Mechanism Of A Spherical Viscospor

The ocean accounts for more than 71%of the total surface area of the earth,and75%of the sea belong to the deep sea（>1,000 m）,including extreme habitats such as cold-seep and hydrothermal.Due to the special deep-sea environment,the difficult collection of deep-sea samples and the limitation of isolation and culture technology in lab,only 1%of deep-sea microorganisms have been pure cultured,which are called"difficult-to-culture microorganisms".At present,there is little research on the physiological and biochemical characteristics and metabolic pathways of the deep-sea microorganisms.Therefore,breaking the bottleneck of isolation and culture technology and extending the resources of uncultured microorganisms can not only strengthen the understanding of the characteristics and metabolic mechanisms of deep-sea microorganisms,but also expand the cognition of deep-sea microbial diversity and the ecological processes they may participate in.In this study,sediment samples obtained during the deep sea operation of the research vessel“Kexue”in 2018 were used to isolate and culture 50 anaerobic bacteria based on the substance metabolism-driven strategy,including 4 potential new species:two potential new species belonging to Actinobacteria and Proteobacteria respectively;a representative of Firmicutes bacteria;a representative of Lentisphaerae bacteria.At the same time,the Lentisphaerae bacteria mentioned above（strain zth2）was used as the research object.Through molecular biology methods,transmission electron microscope（TEM）observation and transcriptomic approach,its physiological and biochemical characteristics were systematically analyzed and the mechanism of polysaccharide metabolism was preliminarily revealed.In this study,we successfully cultured a novel Lentisphaerae bacteria（strain zth2）from deep-sea cold-seep samples by using a polysaccharide-degradation approach.And it was identified as a representative of Lentisphaerae by taxonomic method.We found that porphyra polysaccharide,carrageenan,ulva,starch and laminaria can promote the growth of strain zth2,especially laminaria and starch,which indicates that strain zth2may have a strong ability to metabolize polysaccharides.To further validate the mechanism of polysaccharides degradation mediated by strain zth2,we performed transcriptomic analysis.Indeed,the expression of many genes encoding glycan degradation（amylase,glycosyl hydrolases,glycosyl transferases and glucanases）was evidently upregulated when starch or laminaria was added in the medium.Meanwhile,many genes associated with energy production were also significantly upregulated,which showed that strain zth2 can exactly obtain the energy for growth through metabolism and utilization of polysaccharides,so as to accelerate the growth rate.Moreover,the supplement of starch or laminaria could also dramatically upregulate the expression of many genes responsible for phosphorus and iron metabolisms including phosphoadenosine phosphosulfate reductase,Pho U,Fe-S binding protein and so on,which indicated that the utilization of polysaccharide by strain zth2 can promote the metabolism of phosphorus and iron.To verify this ratiocination,we analyzed the growth status of strain zth2 in a basic medium supplemented with phosphate（Na₃PO₄or Ca₃（PO₄）₂）and Fe²⁺（Fe SO₄）.The results showed that strain zth2 can grow best in the presence of both Na₃PO₄ and Fe SO₄.Through the transcriptomic analysis,compared the supplement of both Na₃PO₄ and Fe SO₄ with only supplement of Fe SO₄ could upregulate the expression of genes responsible for energy production and the expression of genes associated with iron metabolism and polysaccharide degradation were also evidently upregulated,suggesting the presence of soluble phosphate could facilitate iron metabolism and polysaccharide degradation.On the other hand,the absence of Fe SO₄ in the medium indeed weakened the metabolism of phosphorus,iron and polysaccharide and finally affected the generation of energy.The above results show that the utilization of polysaccharide,phosphate and iron by strain zth2 is correlative and mutually promoted,which also reminds us to pay attention to the potential driving effect of Lentisphaerae bacteria on the biogeochemical cycle of carbon,phosphorus and iron in the deep sea.In addition,we mimicked the marine natural environment in the laboratory and the operational taxonomic units（OTUs）about marine microorganisms were analyzed.The results showed that strain zth2 could enrich the diversity of surrounding microorganisms by metabolizing polysaccharides or other substances,which provided more data support for the study of the ecological function of Lentisphaerae bacteria.To sum up,we used the substance metabolism-driven strategy to enrich and culture from deep-sea and successfully obtained some pure cultures,including a kind of difficult-to-culture bacteria（Lentisphaerae bacteria）.Mechanism of polysaccharide metabolism by Lentisphaerae bacteria is reported for the first time,and the metabolism of polysaccharide is coupled with the metabolism of phosphorus and iron.Our study provides research materials and a unique perspective for the study of resource acquisition and substance metabolism mechanism of deep-sea microorganisms.