重水标记-单细胞拉曼光谱表征反硝化菌代谢活性与电子传递

Heavy water-labeled single-cell Raman spectroscopy characterizes the metabolic activity and electron transport of denitrifying bacteria

细菌内部电子传递快慢及其代谢水平是其外在活性的决定因素之一.然而,目前对于细菌活性、电子传递及代谢水平三者之间的关系鲜有报道.本研究针对氮循环的经典过程—反硝化过程,以施氏假单胞菌为模式反硝化菌,系统探究了不同电子供体条件(不同C/N比)下电子传递对反硝化脱氮效率的影响,并通过重水标记(DIP)-拉曼光谱在单细胞水平上分析了代谢活性与电子传递、脱氮相关因素的潜在联系.结果表明,在反硝化前期(0~9 h),NADH脱氢酶活力均处于较高水平,导致电子传递系统活性(ETSA)迅速升高,加速了反硝化脱氮效率,NADH脱氢酶平均活力相对于C/N为5时分别增加50.97%(C/N=10)和83.40%(C/N=20),同时NADH/NAD+比率分别增加43.98%(C/N=10)和65.27%(C/N=20).在反硝化后期(9~24 h),碳源的消耗降低了ETSA,导致脱氮活性降低.同时,在反硝化过程中,C—D键的拉曼特征峰变化表明微生物代谢活性与NO_(3)^(-)-N还原量呈负向的线性关系,揭示了反硝化菌代谢和脱氮活动的不同步性.

The rapidity of electron transfer within bacteria and their metabolic level are among the determinants of their extrinsic activity.However,little has been reported on the relationship between bacterial activity,electron transfer and metabolic level.In this study,we systematically investigated the effect of electron transfer on denitrification efficiency under different electron donor conditions(different C/N ratios)for a classical process of nitrogen cycle,denitrification,and analyzed the potential link between metabolic activity and electron transfer and denitrification-related factors at the single-cell level by heavy water labeling(DIP)-Raman spectroscopy.The results showed that during the pre-denitrification period(0~9 h),NADH dehydrogenase activity was all at a high level,leading to a rapid increase in electron transfer system activity(ETSA),which accelerated denitrification efficiency,and the average NADH dehydrogenase activity increased by 50.97%(C/N=10)and 83.40%(C/N=20),respectively,relative to C/N of 5,while the NADH/NAD+ratio increased by 43.98%(C/N=10)and 65.27%(C/N=20),respectively.In the late stage of denitrification(9~24 h),the consumption of carbon source reduced ETSA,leading to a decrease in denitrification activity.Meanwhile,the changes in the Raman characteristic peak of the C—D bond during denitrification indicated that the microbial metabolic activity showed a negative linear correlation with NO_(3)^(-)-N reduction,revealing the asynchronous nature of denitrifying bacteria metabolism and denitrification activities.