Classification,identification And Aerobic Denitrification Mechanisms Of Marine Halomonas Bacteria

Denitrification is considered as one of the main biological process,which causes nitrogen loss in the ocean,including oxygen minimum zone,offshore and continental shelf sediments,deep sea sediments,and so on.A growing number of studies have shown that in addition to anaerobic denitrification,aerobic denitrification also contributes greatly to the nitrogen loss in the offshore and deep sea environments.There are abundant resources of aerobic denitrifying bacteria in marine environment,however,majority of them are still waiting to be explored and recognized.In this study,the isolation and screening of marine aerobic denitrifying bacteria were carried out,in order to provide resources for biological treatment of high-salt wastewater.Moreover,their performance and mechanisms of aerobic denitrification were also studied,to provide a theoretical basis for engineering application.Meanwhile,their ecological distribution and metabolic pathways were investigated,to deepen the understanding of their ecological role in the marine nitrogen cycle.The Isolation and screening of marine aerobic denitrifying bacteria and polyphasic identification of seven novel species were performed.At least 86 aerobic denitrifiers were isolated from various habitats in the ocean.The genus Halomonas dominated in these aerobic denitrifiers,among which,15 Halomonas strains exhibited excellent nitrate removal capacity,as represented by strain MCCC 1A14433.Phylogenetic analyses based on 16S r RNA genes,concatenation of housekeeping genes and genomes sequences,allocated the 15 strains in this study into seven potential novel species of the genus Halomonas,and intriguingly they formed a unique group with other tweleve species designated as Halomonas desiderata group.Ecological survey showed that H.desiderata group was widely distributed in the ocean,but mainly in the coastal environment,and abundant in surface seawater and sediments.Based on the phenotypic,genotypic,and chemotaxonomic analyses,seven novel species belonging to H.desiderata group are established in this study,for which the names Halomonas zhangzhouensis,Halomonas aerodenitrificans,Halomonas sulfidoxydans,Halomonas ethanolica,Halomonas diversa,Halomonas sulfidivorans,and Halomonas tianxiuensis are proposed.The establishment of these species provides important resources for understanding the functional profile and ecological roles of H.desiderata group.The nitrogen metabolism pathways and performance of aerobic denitrification of representative strain MCCC 1A14433^Twere analyzed.Mass balance calculation of nitrogen and determination of N₂O and N₂showed that,when ammonia as the sole nitrogen source,strain MCCC 1A14433^Tutilized ammonia completely through assimilation process,but not heterotrophic nitrification;when nitrate or nitrite as the sole nitrogen source,strain MCCC 1A14433^Tcould perform assimilation and aerobic denitrification simultaneously.By contrast,when using ammonia and nitrate（nitrite）as mixed nitrogen sources,nitrate（nitrite）was reduced via aerobic denitrification;the presence of ammonia could enhance the reduction rate of nitrate and nitrite,which greatly promoted aerobic denitrification;besides,the removal rate of nitrate and nitrite was significantly higher than that of ammonia.The optimal aerobic denitrification conditions for strain MCCC 1A14433^Twere as follows:sodium citrate or sodium succinate as electron donors,C/N of 7,p H of 8,salinity of 1%-6%,and culture temperature of 37°C.Strain MCCC 1A14433^Texhibited excellent nitrogen removal ability,with average nitrate reduction rate within 16 h was 17.5 mg L^-1h^-1.It could perform complete aerobic denitrification pathway,with N₂and intermediate N₂O as the gas products.The molecular mechanisms of aerobic denitrification of strain MCCC 1A14433^Twere investigated through genomic and transcriptome analysis.Strain MCCC 1A14433^Tharbored complete denitrification pathway,which contained nar GHJI,nap CBADFE,nir SFCDLGHJEN,nor ECBQD and nos RZDFYL,encoding membrane-bound nitrate reductase,periplasmic nitrate reductase,cytochrome cd₁-type nitrite reductase,cytochrome c nitric oxide reductase and nitrous oxide reductase,respectively.Transcriptome analysis showed that nar G,nir S,nor B and nos Z were the main functional genes involved in denitrification under aerobic conditions for strain MCCC1A14433^T,however,nap A seemed not played the major role.dnr1 gene may positively regulate the nitrite reduction process.nar K genes are mainly responsible for the transport of nitrate and nitrite during assimilation and denitrification.When ammonia and nitrate as the mixed nitrogen source,the presence of ammonia significantly increased the transcriptional level of related genes involved in denitrification,transport and regulation,however,it inhibited nitrate assimilation process which was conducted by nas A-nir BD genes.Response to nitrosation stress,various nitrosation/oxidative stresses and nitrogen starvation related genes were induced by nitrate and nitrite,and showed similiar expression pattern to that of denitrification genes.The aerobic denitrification and heterotrophic sulfur oxidation pathways of“Halomonas desiderata group”were explored based on comparative genomic analysis.Complete denitrification pathways were confirmed in the H.desiderata group in which nap A,nar G,nir S,nor B,and nos Z genes co-exist.Their nitrite reductase Nir S formed a unique and novel evolutionary lineage,distinguished from other denitrifiers in Halomonas.Besides,dnr1 gene encoding FNR-like regulator was almost exclusively present in the H.desiderata group strains,showing a significant positive correlation with nir S1.In addition,multiple nitrite reductase genes co-existed in at least seven strains in the H.desiderata group.Phylogenies of denitrification functional genes including nir S,nap A,nor B,and nos Z in H.desiderata group showed diverse evolutionary clades in the genus Halomonas,incongruence with 16S r RNA phylogeny,which suggested they may have undergone different evolutionary processes.Besides,diverse occurrence patterns of denitrification genes were observed in different Halomonas,suggesting the metabolic diversity of this genus in denitrification.H.desiderata group were capable of heterotrophic sulfide oxidation,accompanied with the production of thiosulfate,with the co-occurrence of sqr/fcc AB and pdo genes in their genomes.Besides,four strains of H.desiderata group which harbored tsd A genes,could oxidize thiosulfate rapidly,producing tetrathionate.The widespread presence of aerobic denitrification and heterotrophic sulfide oxidation genes within H.desiderata group,implies their unneglectable ecological contribution in the nitrogen and sulfur cycle in the marine environment.These insights help to explain their wide distribution in marine environments and highlight their application potential for the removal of nitrogen and sulfide.