Multi-wavelength Fluorescence Monitoring of Faecal Contamination in Waters: A Laboratory-based Quantification

Near real-time monitoring of faecal indicator bacteria (FIB) in waters is currently not feasible, and current monitoring methods require field sampling and laboratory testing that inhibits decision-making within a relevant timeframe. While recent studies identified the potential of using specific fluorescence regions for FIB monitoring, sufficient accuracy often requires site-specific calibration due to minor variations in fluorescence peak locations. In this study, a series of lab experiments were completed to address some of the selectivity issues. Specifically, the study explored correlations between wavelength-specific fluorescence signals acquired through fluorescence excitation-emission matrices (EEM) and the amount of E. coli K-12 ( E. coli ) and E. faecalis (enterococci) in exponential and stationary phase broth cultures. Subsequently, the experiments quantified how the addition of known concentrations of L-tryptophan amplifies an indole pulse, specifically its concentration and the corresponding fluorescence properties. Results show unique peak excitation/emission (λex/λem) wavelengths (± 5 nm) in EEMs for E. coli cell pellet and in M9 broth (~ 280/ ~ 327 nm), enterococci cell pellet (~ 276/ ~ 324 nm), L-tryptophan (~ 278/ ~ 343 nm and ~ 298/ ~ 344 nm), and indole (~ 232/ ~ 321 nm). The findings demonstrate that L-tryptophan concentrations in E. coli broth were reduced. At the same time, the indole content increased throughout the initiation phase to the stationary phase of the bacteria growth curve, with the peak indole pulse occurring approximately at the time of transition from the exponential to stationary phase. Such unique fluorescence signatures for not only FIB but also indole (whose pulse can be triggered by L-tryptophan) provide foundations for developing reliable and near real-time in situ FIB sensors. Highlights Fluorescence regions specific to common E. coli and enterococci strains, tryptophan and indole are identified. High accuracy between concentrations and fluorescence intensity are observed. E. coli broth with 6 mM L-tryptophan produces 5 mM indole to effectively infer FIB.