基于DNA/RNA平行测序的海水人工湿地系统根际活跃微生物群落演替特征OA

The treatment of saline aquaculture effluent with high nutrient loads poses a significant challenge for coastal environmental protection.While constructed wetlands offer a sustainable solution,their performance often declines under high-salinity stress due to the intolerance of conventional biological components.To clarify the microbial mechanisms that underpin the stable operation of halophyte-based constructed wetlands,this study investigated the dynamic succession and in situ metabolic activity of the rhizosphere microbiome associated with the halophyte Suaeda salsa.Three vertical subsurface flow constructed wetlands were established and operated continuously for 90 days to treat real effluent from Litopenaeus vannamei aquaculture.The operational period was divided into three phases:initial(P1,days 1‒30),middle(P2,days 31‒60),and late(P3,days 61‒90),corresponding to both the maturation trajectory of the constructed wetland system and the developmental stages of the plants,during which treatment performance progressively improved.Rhizosphere and bulk substrate samples were collected at the end of each phase.A parallel DNA/RNA sequencing strategy was employed to distinguish the total and metabolically active communities.Specifically,total genomic DNA and cDNA(reverse-transcribed from community RNA)were subjected to high-throughput 16S rRNA gene sequencing.Bacterial community structures were analyzed based on the sequencing profiles.Furthermore,the RNA/DNA index was calculated for key genera to indicate their potential in situ metabolic activity,focusing on microorganisms involved in nitrogen metabolism.The results showed that the wetland system demonstrated steady improvement in treatment performance over time.The removal efficiencies for total nitrogen and chemical oxygen demand were not only maintained but also significantly improved during P3,reaching 34.67%and 42.60%,respectively,despite a significant increase in the influent load.The active microbial community exhibited a distinct successional dynamic compared to the relatively stable total community.Specifically,the α-diversity of the active rhizosphere community increased continuously,peaking in P3 and remaining significantly higher than in the bulk substrate.Non-metric multidimensional scaling(NMDS)analysis revealed greater sample dispersion within the active community and a more pronounced separation between rhizosphere and bulk soil niches,indicating its higher sensitivity to environmental changes.Moreover,the rhizosphere active community underwent substantial temporal restructuring.At the phylum level,Desulfobacterota and Cyanobacteria were significantly enriched during P2 and P3,respectively(P<0.05).At the genus level,the dominant active taxon shifted from unclassified_PB19 in the early two phases to Phormidium(a cyanobacterium)in P3.Furthermore,co-occurrence network analysis showed that the active community formed a more complex and interconnected microbial network than that of the total community,exhibiting a higher average degree,clustering coefficient,and proportion of positive correlations.Assessment of potential metabolic activity via the RNA/DNA index revealed a clear functional shift in nitrogen-metabolizing pathways.The denitrifier Halomonas increased in DNA-based relative abundance to 0.80%by P3,while its RNA/DNA index surged above 1.00 in P2 and remaining elevated at 1.08 in P3.In contrast,although the nitrifiers Nitrosomonas and Nitrospira accumulated steadily(reaching 0.39%and 0.38%by P3,respectively),their metabolic activities were highest initially and became significantly suppressed later.Notably,Marinobacter,which performs dissimilatory nitrate reduction to ammonium(DNRA),exhibited the most pronounced metabolic response;despite its lowest DNA-based abundance(0.11%)in P2,its RNA/DNA index rose continuously from 0.96 in P1 to 3.89 in P3,indicating a high per-cell metabolic potential.Collectively,these results confirm that the Suaeda salsa rhizosphere fosters a highly active and synergistic microbial consortium,which undergoes distinct functional succession driven by the metabolically active population.Fundamentally,these findings underscore the limitation of DNA-based surveys,which may obscure functionally critical microbial populations.Integrating RNA-based sequencing is therefore essential for accurately identifying the active drivers of biogeochemical processes.These insights provide a robust microbiological framework for optimizing halophyte selection and operational strategies to steer microbial communities toward desired functions,thereby enhancing both treatment performance and long-term stability of constructed wetlands for saline aquaculture effluent treatment.