Annotation and assembly of the contigs.

The intensification of shrimp farming contributes to the accumulation of toxic nitrogen compounds, which in turn affect productivity and complicates water quality management, especially under conditions of reduced salinity. This study evaluated the effects of 25% (CO, T2) and 50% (T1) water exchange, in combination with a bioaugmentation treatment using native Bacillus (T2), on nitrogen compound concentrations and bacterial community structure in Litopenaeus vannamei culture under reduced salinity conditions (4ppt). The results demonstrated that treatment with native Bacillus megaterium and Bacillus paralicheniformis (T2) leads to a significant reduction in nitrite and nitrate concentrations, reaching nearly 0 mg/L from day 4. In contrast, T1 and CO treatments showed markedly higher concentration, reaching up to 5 mg/mL and 160 mg/L, respectively. The full-length 16S rRNA gene used for the metataxonomic analysis revealed changes in bacterial composition towards species with nitrifying and probiotic potential, with native Bacillus strains detected exclusively in T2. In addition, a reduction in bacterial diversity was detected, and significant differences were observed between the bacterial communities of T2 and those of T1 and CO (p = 0.001, R2 = 0,328). The shotgun analysis further revealed a higher abundance of enzymes related to nitrification and dissimilatory nitrate reduction to ammonium in T2 treatment. The results highlighted the active involvement of Gram positive – Bacillus and Gram-negative bacteria such as Shewanella and Psychrobacter, and suggesting heterotrophic nitrification and aerobic denitrification. Overall, native B. megaterium and B. paralicheniformis provided an effective bioaugmentation strategy for the managing nitrate and nitrite in low-salinity shrimp farming, providing an eco-friendly alternative that may enhance productivity and reduce the industry´s water footprint.虾类养殖集约化程度的提升，会导致有毒含氮化合物的累积，进而降低养殖生产力并加剧水质管理难度，在盐度降低的养殖环境中这一问题尤为突出。本研究针对盐度降至4ppt的凡纳滨对虾（Litopenaeus vannamei）养殖体系，评估了25%换水量（对照组CO、实验组T2）与50%换水量（实验组T1），结合本地芽孢杆菌属（Bacillus）生物强化（bioaugmentation）处理（仅T2组采用）对养殖水体含氮化合物浓度及细菌群落结构的影响。研究结果显示，采用本地巨大芽孢杆菌（Bacillus megaterium）与副地衣芽孢杆菌（Bacillus paralicheniformis）处理的T2组，可显著降低亚硝酸盐与硝酸盐浓度，自第4日起浓度即可降至近0 mg/L。与之相比，T1组与CO组的含氮化合物浓度显著更高，最高分别可达5 mg/mL与160 mg/L。用于宏分类组学分析（metataxonomic analysis）的全长16S核糖体RNA基因（16S rRNA gene）测序结果显示，细菌群落组成向具备硝化功能与益生潜力的物种转变，且本地芽孢杆菌菌株仅在T2组中被检测到。此外，T2组的细菌多样性有所降低，且T2组与T1、CO组的细菌群落结构存在显著差异（p=0.001，R²=0.328）。鸟枪宏基因组测序（shotgun analysis）进一步显示，T2组中与硝化作用及异化硝酸盐还原产铵（dissimilatory nitrate reduction to ammonium, DNRA）相关的酶类丰度更高。研究结果表明，革兰氏阳性菌（芽孢杆菌属）与希瓦氏菌属（Shewanella）、冷杆菌属（Psychrobacter）等革兰氏阴性菌均发挥了积极作用，提示该体系通过异养硝化与好氧反硝化途径实现氮去除。综上，本地巨大芽孢杆菌与副地衣芽孢杆菌可为低盐度虾类养殖中的硝酸盐与亚硝酸盐管控提供有效的生物强化策略，是一种可提升养殖生产力并降低水产养殖业水足迹（water footprint）的环保型替代方案。