Study On The Composition And Distribution Of DNRA Fungi And The DNRA Characteristics Of Aspergillus Sydowii 29R-4-F02 In ?2km Deep Sediment Environment Below Seafloor

The ocean occupies about 71%of the earth's surface area and plays a vital role in the earth's material and energy cycle.From the first global scientific expedition of the“Challenger”(1872-1876)and the continuous implementation of the Ocean Drilling Program(ODP)and Integrated Ocean Drilling Program(IODP),it has been proven that the deep sea and submarine sediment environment has extremely rich biodiversity and plays an important role in driving the geochemical cycle of elements.However,the research on the deep biosphere of the ocean floor in the past mainly focused on the study of prokaryotic microbial groups,distribution limits,genetic evolutionary laws and ecological functions,while the study of fungal groups belonging to eukaryotes was scarce.This study was focus on the role of sub-seafloor fungi,isolated from coal associated sediments at?2 km deep below the sea floor(mbsf)during IODP Expedition 337,in driving the nitrogen cycle in the ocean floor sedimentary environment,especially to determine whether these fungi possess the DNRA ability of dissimilation nitrate reduction to ammonia.Similarly,the key environmental factors that determine the geological distribution of DNRA fungi,its genetic basis and environmental factors effecting the DNRA ability of the fungi especially 29R-4-F02 strain,were also explored in detail.Our findings might provide a scientific basis for understanding the survival mechanism of fungi in the deep ocean biosphere and role they play in driving the nitrogen cycle.The main results are as follows:1.Most sub-seafloor isolated fungi showed DNRA ability and among them Aspergillus and Penicillium were the dominant DNRA fungi;however,this DNRA ability was different in different species.In order to study the difference in DNRA ability of fungi in different geological layers,18 factors of the environment of the fungus were selected for principal component analysis,and dimensionality reduction was processed to the two main components of PC1 and PC2.Among them,water,porosity,Ca²⁺and NH₄⁺have high positive contribution to PC1;TOC,TC,Salt,and TN have high positive contributions to PC2.The fungal DNRA ability of the ocean floor sediment environment is positively correlated with the pH,alkalinity,Ca²⁺and NH₄⁺concentration of the sediment environment,and negatively correlated with temperature,pressure,CaCO₃ and inorganic carbon.2.Environmental factors such as carbon source,C/N,substrate nitrogen source(NO₃^--N)concentration,oxygen,temperature and pH have an effect on DNRA ability of 29R-4-F02.C/N source greatly affected the DNRA ability of 29R-4-F02,compared to carbon source.Similarly,nitrogen source(NO₃^--N),its concentration(1-10m M),pH(5-9)and temperature(20-45?)were positively correlated with DNRA ability of the fungus.In addition,the fungus showed significantly improved DNRA ability under aerobic conditions,compared to anaerobic conditions.3.The results of genome sequencing and phylogenetic analysis showed that the genome size of Aspergillus sydowii 29R-4-F02 was 37.19 Mb,with a total of 12 contigs,with an average length of 3,381,064.8 bp,an N50 of 4,575,881 bp,a GC base content of 50.05%,and annotated protein.There are 12488 coding genes.The total length of all coding genes is19,572,123 bp,and the average length is 1,567 bp.The total length of the coding region accounts for 52.62%of the whole genome.It is closely related to Aspergillus versicolor.Collinearity analysis showed that the colinear genes of the ocean floor-derived Aspergillus sydowii 29R-4-F02 and the terrestrial-derived Aspergillus sydowii CBS563.65 accounted for86.10%,indicating that the ocean floor and terrestrial-derived Aspergillus sydowii were relatively conservative in evolution.Metabolic pathway analysis revealed that the bacterium possesses related enzyme genes of the DNRA pathway——nitrate reductase gene nirA and nitrite reductase gene nrfA,among which nrfA is the key enzyme gene of DNRA pathway.Phylogenetic tree analysis of domains and genes showed that the nrfA of Aspergillus sydowii29R-4-F02 was closely related to the nrfA genes of other Aspergillus genus.Due to this huge differences,it is therefore,speculated that the metabolic DNRA pathways of Aspergillus sydowii 29R-4-F02 may be different from terrestrial Aspergillus.Based on the above experiments,we found that fungi in the deep ocean bottom biosphere generally possess the DNRA pathway,and their DNRA ability is comprehensively regulated by geological environmental factors and genetic basis.The results of the study have an important role in understanding the survival mechanism of ocean floor fungi and in driving elemental geochemical processes.