海草根际沉积物好氧反硝化菌S22的筛选、脱氮特性及基因组分析

Screening, denitrification characterization, and genomic analysis of an aerobic denitrifying bacterium S22 from seagrass rhizosphere sediments

摘要:

近岸海域富营养化导致的过量氮输入是全球海草床退化的关键环境压力因素。目的 从海草床生态系统中筛选、鉴定高效好氧反硝化菌株，解析其脱氮特性与脱氮机制，为缓解氮负荷压力、修复富营养化海草床提供微生物资源。方法 采用富集驯化培养和溴百里酚蓝指示剂筛选法从烟台芝罘湾海草根际沉积物中分离筛选好氧反硝化菌株，并通过16S rRNA基因序列鉴定菌株的分类地位。结合脱氮性能测定选取一株高效好氧反硝化细菌，利用单因素实验和正交试验方法优化其脱氮条件，通过氮平衡实验与全基因组测序阐明其脱氮途径及关键功能基因。结果 从烟台芝罘湾海草床根际沉积物中分离获得34株反硝化菌株，经鉴定优势菌属为假单胞菌属(Pseudomonas)和不动杆菌属(Acinetobacter)。基于脱氮性能筛选获得一株高效好氧反硝化菌株Pseudomonas sp. S22，通过单因素实验与正交试验优化，确定其最适脱氮条件为以丁二酸钠为碳源、C/N=15、pH 9.0、盐度(salinity, S)=30‰、T=28 ℃。在此条件下，菌株对140 mg/L硝态氮的36 h去除率达99.99%，展现出高效脱氮能力。氮平衡实验结果表明约59.64%的硝态氮被转化为气态氮，证明反硝化为其主导脱氮途径。基因组测序进一步揭示菌株S22携带包括napA和nirS等好氧反硝化关键功能基因，从分子水平为其脱氮表型提供了遗传基础。结论 本研究系统分离鉴定了来源于我国北方海草床的好氧反硝化菌株，菌株S22具有优异的脱氮性能和环境适应性，氮平衡和基因组分析证实其以反硝化为主要脱氮途径，且携带好氧反硝化关键功能基因。菌株S22可作为海草床氮负荷削减的潜在微生物资源，为后续开发“微生物-海草”协同修复技术提供菌种资源和理论依据。

Abstract:

Excessive nitrogen input caused by eutrophication in nearshore waters is a major environmental stressor driving the global degradation of seagrass beds.Objective To screen and identify efficient aerobic denitrifying bacteria from seagrass bed ecosystems and elucidate their nitrogen removal performance and mechanisms, thus providing microbial resources for alleviating nitrogen loading and restoring eutrophic seagrass beds.Methods Aerobic denitrifying bacteria were isolated and screened from seagrass rhizosphere sediments in Zhifu Bay, Yantai by enrichment-domestication culture and bromothymol blue assay. The taxonomic status of the strains was determined by 16S rRNA gene sequencing. On the basis of nitrogen removal performance, an efficient aerobic denitrifying strain was selected. Single-factor and orthogonal experiments were conducted to optimize its denitrification conditions, and nitrogen balance experiments and whole-genome sequencing were employed to elucidate its nitrogen removal pathways and key functional genes.Results A total of 34 denitrifying strains were isolated from seagrass rhizosphere sediments in Zhifu Bay, Yantai. The dominant genera were Pseudomonas and Acinetobacter. A strain designated as Pseudomonas sp. S22 with high denitrification performance was selected. The denitrification conditions of this strain were optimized as follows: sodium succinate as the carbon source, C/N=15, pH 9.0, salinity (S)=30‰, and T=28 ℃. Under these conditions, the strain achieved a removal rate of 99.99% for 140 mg/L nitrate nitrogen within 36 h, demonstrating excellent nitrogen removal efficiency. Nitrogen balance analysis revealed that approximately 59.64% of the initial nitrate nitrogen was converted to gaseous nitrogen, confirming that denitrification was the dominant nitrogen removal pathway. Genomic sequencing revealed that strain S22 carried key functional genes for aerobic denitrification, including napA and nirS, providing a genetic basis for its denitrification phenotype at the molecular level.Conclusion This study systematically isolated and identified aerobic denitrifying bacteria from seagrass beds in northern China. Strain S22 exhibits outstanding nitrogen removal performance and environmental adaptability. Nitrogen balance and genomic analyses confirm that denitrification is its primary nitrogen removal pathway and the strain carries key functional genes for aerobic denitrification. Strain S22 can serve as a potential microbial resource for reducing nitrogen loading in seagrass beds. This study provides both a valuable strain and a theoretical basis for the future development of microbe-seagrass synergistic remediation technologies.