Diversity,Function And Metabolic Potentials Of Subsurface Crustal Microorganisms At North Pond,Western Flank Of The Mid-Atlantic Ridge

Deep-sea oceanic crust constitutes⁶5%of the earth's surface.Previous studies demonstrate that the upper 500 m of the oceanic crust is mainly composed of fractured basalts with high porosity.Bottom seawater could penetrate into the ocean crust through seawater circulation,and the volume of the crustal fluids make up 2%of the global seawater,which is the largest aquifer system on Earth.However,the biological process in the oceanic crust is less studied due to the lack of appropriate sampling and probing technologies.Recent studies have demonstrated the existence of active microorganisms in hot?65??and anaerobic oceanic crust,but the cool?5-25??and oxic crustal environments which represent a more common hydrologically active type of ocean crust is poorly known.This study investigated the microbial abundance,distribution,diversity,and metabolic potentials of the basaltic crustal samples collected during the Integrated Ocean Drilling Program?IODP?Expedition 336 at“North Pond”?NP?,western flank of the Mid-Atlantic Ridge.We seek to test the following hypotheses which is of great interest to researchers in the field of oceanic crustal biosphere:?1?a unique microbial community is hosted in the oceanic crust,and it is closely related to the geochemical and physical parameters;?2?nitrogen is a limiting factor for microbial growth in the oceanic crust;?3?microbial iron oxidation is an important energy producing metabolic pathway for supporting crustal biosphere,and contributes to oceanic crust weathering.This study enumerated the microbial cell densities of NP basalts in Hole U1383C by using the modified microbial cell extraction method,and showed that cell densities in the basalts are¹0⁴ cells cm^-3,with higher cell densities occurring between 115 and 145 m below seafloor.Enrichment experiments with different carbon?bicarbonate,acetate,methane?and nitrogen?nitrate and ammonium?sources revealed significant cell growth?one magnitude higher cell abundance?,higher intracellular DNA content and higher iron oxidation state only when nitrogen substrates were added,suggesting that nitrogen is a limiting factor for microbial growth in the oceanic crust.With the application of the high-throughput sequencing technology,we demonstrate that similar bacterial community structures,which were dominated by Marinobacter of the Gammaproteobacterial and Sediminibacterium of the Sphingobacterial species,were detected regardless of variations in sampling depth.Phylogenetic analysis of the 16S rRNA genes showed that some major and minor groups were closely related to cultured chemoautotrophic iron-oxidizing microorganisms,indicating that crustal microorganisms may participate in iron oxidation.Furthermore,the metabolic potentials of the crust microbiota were assayed by metagenomic analysis.Genes coding for energy metabolism involved in hydrocarbon degradation,dissimilatory nitrate reduction to ammonium,denitrification,hydrogen oxidation and iron oxidation were identified.Comparative metagenomics of the crustal biosphere of NP against metagenomes from other environments showed that NP was enriched in pathways for Fe³⁺uptake,siderophore synthesis and uptake,and Fe transport,suggesting that iron metabolism is an important energy production and conservation mechanism in this system.Notably,after two years of cultivation,this study isolated seven pure cultures of the neutrophilic iron-oxidizing bacteria from NP basalts by using the Gradient tube method.They are from the genera Halomonas,Idiomarina,Mameliella,Marinobacter?two strains?,Sulfitobacter and Thalassospira.Marinobacter sp.NP-4 and Marinobacter sp.NP-6,which are from the dominant bacterial group in the crustal microbial community,are proved to be mixtrophic,neutrophilic iron-oxidizing bacteria.High-pressure incubation of Marinobacter sp.NP-4 showed that the single-cell iron oxidation rate appeared to be higher at 20 MPa than that at 0.1 MPa,indicating its adaptation to high pressure and origin of the deep-sea environment.Whole genomic sequencing and comparative genomics of the two Marinobacter strains against three Marinobacter genomes from other environments showed that the genes for carbohydrate and amino acid metabolism were enriched,suggesting their adaptation strategies to the oligotrophic oceanic crust.In summary,this study is the first to demonstrate that unique and active microbial communities are hosted in the cool,oxic subsurface oceanic crust at NP.These microorganisms possibly participate in the iron-related weathering of the oceanic crust.Microbial growth could be stimulated by in vitro nitrogen addition,indicating that nitrogen is a limiting factor for microbial growth in the oceanic crust.We provide a comprehensive and systematic study of the microbial abundance,distribution,diversity,and metabolic potentials of the NP crustal biosphere.This is the first to study the cool,oxic ridge flank crustal system which represent a more common hydrologically active type of ocean crust,filling a prior gap in knowledge about the role of microorganisms in oceanic crust weathering and their contributions to global biogeochemical elements cycling.This study also guides future researches in the explorations of the oceanic crustal ecosystem.