Isolation And Cultivation Of Deep-sea Difficult-to-cultivate Microorganisms And Mechanism Studies On Their Driving Element Cycling

Biogeochemical cycle is one of the core research directions of earth system science.It is important to describe,trace and predict the cycling processes of carbon,nitrogen,phosphorus,sulfur and heavy metals in the earth’s sphere.Among the various life forms on the earth,microorganisms play a key driving role in the elemental biogeochemical cycle because of their diverse types,wide distribution and abundant metabolisms.To reveal the genetic and metabolic diversity of microorganisms,the biogeochemical cycles,coupling mechanisms and driving modes of key elements will help to clarify the driving mechanisms of microorganisms in the biogeochemical cycling of important elements on earth.Deep-sea microorganisms(bacteria,archaea and eukaryotic organisms,etc.)have abundant genetic and metabolic diversity,but due to the limitation of sampling and culture conditions,so far,more than 99% of the deep-sea microbial has not yet been established to develop technology.And the fundamental role of deep-sea difficult-to-cultivate microorganisms in the cycle of geochemical elements is still poorly understood.Thus,these different deep-sea environments(such as abyss,hydrothermal,cold spring,seamounts,etc.)of microbial pure culture and their metabolic pathway mediated substances drive element cycling mechanism could help us to clarify for deep-sea microbes in the ocean and the whole earth scope element cycling contribution and achieve the depth of deep-sea microbial resources mining.In this study,sediment samples obtained during the deep-sea cold spring and hydrothermal habitat operation of the research vessel “Kexue” in 2017 and 2018 were used to break through the culture technology bottleneck of the deep-sea difficult-to-cultivation microorganisms based on the substance metabolism-driven separation strategy.In the end,28 potential new bacteria were successfully purified from deep sea sediments,which belonged to 7 different phyla(Chloroflexi,Planctomycetes,Tenericutes,Bacteroidetes,Firmicutes,Proteobacteria and Spirochaetes),showing good biodiversity.According to the similarity of 16 S r RNA gene sequences,the taxonomic status of these new bacteria was tentatively classified into 4 new classes,1 new order,6 new families,9 new genera and 8 new species.And we selected four of these strains(Tenericutes bacterium,Chloroflexi bacterium,Bacteroidetes bacterium and Sulfate-reducing bacterium)for further study and revealed the mechanism by which they mediated metabolic pathways to drive the cycling of elements(phosphorus,sulfur,carbon and heavy metals).Tenericutes are a group of bacteria without cell wall and with small genome,which have special research value in environmental adaptation mechanism and evolution,but most of them have not obtained pure culture.Previous metagenomic studies have shown that this group has the potential to degrade nucleic acids for energy metabolism.Based on this,we enriched and purified 7 Tenericutes bacteria by the novel method of adding Escherichia coli genomic DNA in basal culture medium,of which 4 were potential new classes.One of the strains,Xianfuyuplasma coldseepsis ZRK13,belonging to the class Izemoplasma,was selected for further study.Based on transcriptomics,the mechanism of energy conversion between metabolized organic matter and sodium thiosulfate was revealed.Based on biochemistry and other methods,the outstanding ability of degrading DNA was confirmed for the first time,and the deep-sea in situ experiments were carried out with the advanced equipment of the scientific research ship “Kexue”,which verified that strain zrk13 also had the ability to degrade DNA and participated in energy synthesis in natural habitats.The unique life process plays an important driving role in the biogeochemical cycle process and material cycle of life elements such as phosphorus,carbon and nitrogen in the deep-sea habitat.Chloroflexi bacteria are widely distributed in deep-sea environment.However,due to the limitation of culture conditions,there are very few Chloroflexi bacteria that can be isolated and cultured.Based on previous reports that Chloroflexi can tolerate rifampine antibiotics,we successfully isolated two new strains from deep-sea cold seep sediments by using the isolation strategy of adding rifampicin antibiotic to basal culture medium.One of the strains,Chloroflexus methaneseepsis ZRK33(a potential new order),was selected for in-depth study.First,16 S r RNA gene sequencing based on deep sea sediments revealed that Chloroflexi bacteria occupy a high abundance in deep sea sediments.Based on the similarity of 16 S r RNA gene sequence and phylogenetic analysis of the genome,it was indicated that strain ZRK33 was a representative bacterium of Chloroflexi,belonging to the new family(Sulfochloroflexaceae)and the new order(Sulfochloroflexales).Based on the analysis of genome and biochemistry,the assimilative sulfate reduction pathway of strain ZRK33 was revealed,and high concentrations of sulfate and thiosulfate significantly promoted the growth and morphology of strain ZRK33.Based on proteomic results,it was revealed that sulfate or thiosulfate could significantly promote the transport and degradation of macromolecular carbohydrates by strain ZRK33,thus stimulating the production of energy to promote its growth.Based on metagenomic analysis,the key genes related to assimilation and dissimilation of sulfate reduction are widely distributed in the genome of Chloroflexi bacteria,suggesting that Chloroflexi may play an important role in driving the biogeochemical cycle of sulfur in the deep sea.Bacteroides are considered to be effective degraders of marine polysaccharides and major contributors to the marine carbon cycle.However,compared with the extensive studies on the mechanism of polysaccharide degradation by gut Bacteroides,there has been few researches on the degradation of polysaccharides by marine Bacteroides(especially deep-sea Bacteroides),mainly due to the lack of pure cultures of deep-sea Bacteroides.We have successfully isolated four new Bacteroides bacteria from deep-sea cold seep sediments by using different polysaccharides added to the basal medium.One of them,Maribellus comscasis WC007,was selected for further study.Analysis based on amplicon and metagenomic sequencing showed that Bacteroides were one of the dominant communities in deep-sea surface sediments and had more genes encoding carbohydrate enzymes responsible for polysaccharide degradation than other bacteria.Based on genomic analysis,we found that strain WC007 had 82 Polysaccharide Utilization Locus(PULs),including 634 carbohydrate enzymes(CAZymes),82 pairs of channel forming transporters(SusC)/substrate binding proteins(SusD)and 58 pairs of σ-70 factors/anti-σ factors.Based on the analysis of growth test and transcriptomics,it was confirmed that strain WC007 could degrade and utilize different polysaccharides(cellulose,pectin,fucoidan,mannan,xylan and starch).Combined transcriptome and metabolome analysis revealed the potential mechanism of cellulose degradation and utilization by strain WC007.In conclusion,we firstly reveal the high abundance of deep-sea Bacteroides for significant degradation of polysaccharides,and the mechanism of the polysaccharide metabolism pathway mediated by Bacteroides can drive carbon cycling.Sulfate-reducing bacteria(SRB)exist widely in nature,but due to the difficulty of sampling and the limitation of culture conditions,there are few sulfate-reducing bacteria isolated from the deep sea.Using the separation strategy of sulfate and heavy metal ions added to the basal medium,we have successfully isolated two new sulfate-reducing bacteria from the deep-sea cold seep sediments.And one of them,Pseudodesulfovibrio cashew SRB007(a potential new species),has been selected for further study.Based on genomics and biochemistry methods,revealing that strain SRB007 can generate sulfur ions by reducing sulfate,and then form insoluble minerals with heavy metal ions such as cadmium,cobalt and mercury in the environment.While removing heavy metal stress,it also drives the biogeochemical cycle of sulfur and heavy metal elements in the deep sea.In summary,our separation strategy based on material metabolism has broken through the technical bottleneck of many difficult-to-cultivation microorganisms in deep-sea sediments.Many difficult-to-cultivation microorganisms have been isolated from deep-sea sediments,and we reveal the coupling relationship between metabolism of deep-sea microbial matter and biogeochemical cycles of key elements,which helps to clarify the driving mechanism of microorganisms on the biogeochemical cycle of important elements(carbon,nitrogen,sulfur,phosphorus,heavy metals,etc.)on the earth.This study provides an important theoretical basis for breaking through the culture bottleneck of difficult-to-cultivation microorganisms in deep sea,and providing an in-depth understanding of the environmental adaptation mechanism of rare microorganisms in deep sea,and expanding the cognition of the special life process of microorganisms in deep sea.