克雷伯氏菌属(Klebsiella sp.) WH-E的筛选及脱氮机制

Screening and denitrification mechanisms of Klebsiella sp. WH-E

摘要:

目的 畜禽养殖污水排放量激增，环境污染问题日益凸显。筛选高效异养硝化好氧反硝化菌株并探究其脱氮机制，对解决养殖污水氮素污染问题具有重要的理论与实践意义。方法 从猪场活性污泥中筛选一株异养硝化-好氧反硝化(heterotrophic nitrification-aerobic denitrification, HN-AD)能力优异的菌株，采用响应面法优化其培养条件；通过测定菌株对单一和混合氮源的利用效率，借助氮平衡分析方法，评估其对无机氮的转化能力；结合气相色谱法测定N₂O、N₂以验证反硝化过程的完整性；结合全基因组分析解析菌株WH-E的脱氮途径及机制。结果 成功分离出一株克雷伯氏菌属(Klebsiella sp.) WH-E，该菌株具有优异的异养硝化好氧反硝化性能。经优化试验确定其最适生长参数为：碳源为柠檬酸钠，温度为34.18 ℃，初始pH为7.1，C/N为14.53，摇床转速为159.59 r/min。菌株以氨氮、硝氮、亚硝氮为单一氮源培养时氮素去除率分别为99.80%、81.54%、80.00%；以氨氮为唯一氮源时，35.84%和35.91%的氮分别被转化为细胞氮和气态氮。以氨氮和硝氮为混合氮源培养时氮素去除率均为100.00%；以氨氮和亚硝氮为混合氮源培养时，氨氮去除率为100.00%，亚硝氮去除率为91.97%。全基因组测序分析发现glnB、norVWR、narGHI、nasBC、nirBD等多个与氮代谢相关的功能基因。结论 Klebsiella sp. WH-E有3种氮代谢途径，包括氨同化途径、硝化反硝化途径和硝酸盐同化与异化途径。本研究证实了HN-AD菌株在实际养殖废水脱氮处理中的应用潜力，并为其工程化实施提供了理论支撑。

Abstract:

Objective The rapid increase in wastewater discharge from animal husbandry has caused severe environmental pollution. Identifying efficient heterotrophic nitrifying-aerobic denitrifying bacteria and investigating their denitrification mechanisms are of great theoretical and practical importance for mitigating nitrogen pollution in the wastewater.Methods A strain exceling in heterotrophic nitrification-aerobic denitrification (HN-AD) was isolated and from activated sludge in pig farms. Culture conditions were optimized by response surface methodology. We evaluated the inorganic nitrogen-transforming capacity of the strain by assessing its utilization efficiency of single and mixed nitrogen sources and through nitrogen balance analysis. The completeness of the denitrification process was confirmed via gas chromatographic measurements of N₂ and N₂O. Finally, the nitrogen removal pathways and underlying mechanisms were elucidated through whole-genome analysis.Results The successfully isolated strain Klebsiella sp. WH-E exhibited excellent HN-AD capabilities. The growth conditions of the strain were optimized as follows: sodium citrate as the carbon source, 34.18 °C, initial pH 7.1, a C/N ratio of 14.53, and a shaking speed of 159.59 r/min. When the strain was cultured with ammonium, nitrate, or nitrite as the sole nitrogen source, the nitrogen removal rates were 99.80%, 81.54%, and 80.00%, respectively. Furthermore, when ammonium was the sole nitrogen source, 35.84% and 35.91% of nitrogen were converted into cellular nitrogen and gaseous nitrogen, respectively. When ammonia nitrogen was combined with nitrate nitrogen as mixed nitrogen sources, the nitrogen removal rate was 100.00%; When ammonia nitrogen was combined with nitrite nitrogen as mixed nitrogen sources, the ammonia nitrogen removal rate was 100.00%, and the nitrite nitrogen removal rate was 91.97%, respectively. Whole-genome sequencing identified several nitrogen metabolism-related functional genes, including glnB, norVWR, narGHI, nasBC, and nirBD.Conclusion Klebsiella sp. WH-E possesses three nitrogen metabolism pathways: ammonium assimilation, nitrification-denitrification, and nitrate assimilation and dissimilation. This study confirms the applicability of Klebsiella sp. WH-E for nitrogen removal from full-scale piggery wastewater and establishes a solid theoretical foundation for its engineering applications.