A novel derivative synchronous fluorescence method for the rapid, non-destructive and intuitive differentiation of denitrifying bacteria

Abstract

It is challenging to differentiate bacteria residing in the same habitat by direct observation. This difficulty impedes the harvest, application and manipulation of functional bacteria in environmental engineering. In this study, we developed a novel method for rapid differentiation of living denitrifying bacteria based on derivative synchronous fluorescence spectroscopy, as exemplified by three heterotrophic nitrification-aerobic denitrification bacteria having the maximum nitrogen removal efficiencies greater than 90%. The intact bacteria and their living surroundings can be analyzed as an integrated target, which eliminates the need for the complex pre-processing of samples. Under the optimal synchronous scanning parameter (Δλ = 40 nm), each bacterium possesses a unique fluorescence spectral structure and the derivative synchronous fluorescence technique can significantly improve the spectral resolution compared to other conventional fluorescence methods, which enables the rapid differentiation of different bacteria through derivative synchronous fluorescence spectra as fast as 2 min per spectrum. Additionally, the derivative synchronous fluorescence technique can extract the spectral signals contributed by bacterial extracellular substances produced in the biological nitrogen removal process. Moreover, the results obtained from our method can reflect the real-time denitrification properties of bacteria in the biological nitrogen removal process of wastewater. All these merits highlight derivative synchronous fluorescence spectroscopy as a promising analytic method in the environmental field.