Detection of N-Acyl-homoserine Lactones Signal Molecules of Quorum Sensing Secreted by Denitrification Flora in Microaerobic Nitrogen Removal Processes by Ultra-performance Liquid Chromatography Tandem Mass Spectrometry

Gram-negative (G−) bacteria, such as denitrifying bacteria and anaerobic ammonia oxidation bacteria, are highly social organisms capable of sophisticated cooperative behavior mediated via quorum sensing. As the signal molecules of chemical communication, N-acyl-homoserine lactones (AHLs) can mediate the quorum sensing of the functional microbial population and regulate the population density. To understand the growth of functional microbial population and the mechanism for biological nitrogen removal in upflow microaerobic sludge reactors (UMSRs) treating organic wastewater with low ratio of chemical oxygen demand to total nitrogen, a method was established to simultaneously detect AHLs in the microaerobic processes. Water-sludge mixtures sampled from the UMSRs were pretreated in sequence by liquid-liquid extraction using ethyl acetate, rotary evaporation, constant volume with methanol, and separation by C18 column. Gradient elution was carried out using 5 mM ammonium acetate (containing 0.1% formic acid) and methanol as mobile phases. On the base of multiple reaction monitoring analysis, a triple quadrupole mass spectrometer with an electrospray ionization was applied to detect the target compounds. 9 kinds of AHLs were used to evaluate the established method and the results showed that the detection limits were 0.01−0.5 μg L−1 and all of the AHLs presented excellent linearity with the concentration ranged from 0.5 μg L−1 to 100 μg L−1. The recovery and relative standard deviation ranged from 62.5% to 118.1% and 2.9% to 12.1%, respectively. The analysis was completed within 6.5 min. The rapid, accurate and precise method for detecting AHLs provides a new insight into the growth and metabolic activity of functional microbial population in the activated sludge processes to understand the mechanism of biological nitrogen removal, suggesting a good application in regulation and operation of wastewater biological treatment processes.

Graphical abstractA rapid and accurate method was developed for detecting signal molecules AHLs in a microaerobic nitrogen removal system, via ultra performance liquid chromatography tandem mass spectrometry, providing an approach to understanding the biological mechanism of denitrification and optimizing the control of the microaerobic process.Figure optionsDownload full-size imageDownload as PowerPoint slide