Tryptophan-like and humic-like fluorophores are extracellular in groundwater: implications as real-time faecal indicators

Abstract

Fluorescent natural organic matter at tryptophan-like (TLF) and humic-like fluorescence (HLF) peaks is associated with the presence and enumeration of faecal indicator bacteria in groundwater. We hypothesise, however, that it is predominantly extracellular material that fluoresces at these wavelengths, not bacterial cells. We quantified total (unfiltered) and extracellular (filtered at < 0.22 µm) TLF and HLF in 140 groundwater sources across a range of urban population densities in Kenya, Malawi, Senegal, and Uganda. Where changes in fluorescence occurred following filtration they were correlated with potential controlling variables. A significant reduction in TLF following filtration (ΔTLF) was observed across the entire dataset, although the majority of the signal remained and thus considered extracellular (median 96.9%). ΔTLF was only significant in more urbanised study areas where TLF was greatest. Beneath Dakar, Senegal, ΔTLF was significantly correlated to total bacterial cells (ρs 0.51). No significant change in HLF following filtration across all data indicates these fluorophores are extracellular. Our results suggest that TLF and HLF are more mobile than faecal indicator bacteria and larger pathogens in groundwater, as the predominantly extracellular fluorophores are less prone to straining. Consequently, TLF/HLF are more precautionary indicators of microbial risks than faecal indicator bacteria in groundwater-derived drinking water.

Laboratory In-Situ Production of Autochthonous and Allochthonous Fluorescent Organic Matter by Freshwater Bacteria

“…In this study, for the microbial community cultures, Peak C was found to be predominantly associated with extracellular material. This is in agreement with the data associated with the bacterial isolates ( Table 3 ) and is also in line with recent environmental observations [ 31 ]. High Peak C intensities are observed within the microbial community culture samples ( Figure 1 ).…”

“…As with Peak B, the majority of Peak T fluorescence is observed in the resuspended and lysed cell fractions, indicating the cellular origin of Peak T fluorescence in this laboratory model. This finding challenges recent environmental groundwater research [ 31 ], which concludes that Peak T is predominantly associated with extracellular material [ 8 , 32 , 63 ]. The Peak T data associated with the environmental bacterial isolate monocultures ( Table 3 ) further support this observation.…”

“…This agrees with our previous work using bacterial reference strains, strongly suggesting that its origin and presence is due to microbial structural components and/or cellular constituents. However, this contradicts some recent groundwater research which demonstrated the majority (>90%) of Peak T fluorescence, in a natural groundwater, to be associated with extracellular material [ 31 ]. This demonstrates the need for more experimental work regarding impact of residence time on the release of cellular material into the water body, either via metabolic pathways or via cell lysis [ 69 ].…”

“…Classifying FOM in this way has enabled the study of the variable temporal and spatial detection of these fluorescent signatures within aquatic systems, as indicators of water quality [ 6 ]. Freshwater research has mainly focussed on the use of Peak T fluorescence as an in-situ indicator of the presence of microorganisms [ 27 , 28 ], and more recently how this can be used to infer microbial activity within freshwater systems [ 29 , 30 , 31 ]. Microbial activity has long been studied within marine sciences due to the importance of the microbial-carbon pump and long-term deep ocean carbon storage [ 11 , 32 , 33 , 34 ].…”

Stormwater Runoff and Tidal Flooding Transform Dissolved Organic Matter Composition and Increase Bioavailability in Urban Coastal Ecosystems

“…Protein-like fluorescence (C4, C5) was positively correlated with log-transformed E. coli concentrations in the wet season with highest bacterial loads at high tide (Figure 6). These findings support the notion that tryptophan-like fluorescence intensity may be used as a proxy for degraded water quality determination in wastewater-impacted streams (Baker & Inverarity, 2004;Gu et al, 2020;Mendoza et al, 2020;Sorenson et al, 2020). We found that likely diffuse sources of anthropogenic waste (e.g., septic release, pet waste), indicated by increased bacterial loads, including E. coli, and associated DOM composition, may result in increased microbial utilization of DOM and processing rates in the water column.…”