Tryptophan-like fluorescence as a high-level screening tool for detecting microbial contamination in drinking water.

Regular monitoring of drinking water quality is vital to identify contamination of potable water supplies. Testing for microbial contamination is important to prevent transmission of waterborne disease, but establishing and maintaining a water quality monitoring programme requires sustained labour, consumables and resources. In low resource settings such as developing countries, this can prove difficult, but measuring microbial contamination is listed as a requirement of reaching the UN's Sustainable Development Goal 6 for water and sanitation. A nine-month water quality monitoring programme was conducted in rural Malawi to assess the suitability of tryptophan-like fluorescence (TLF), an emerging method for rapidly detecting microbial contamination, as a drinking water quality monitoring tool. TLF data was compared with thermotolerant coliforms (TTCs, E. coli) and inorganic hydrochemical parameters. A large (n = 235) temporal dataset was collected from five groundwater drinking water sources, with samples collected once or twice weekly depending on the season. The results show that TLF can indicate a broader contamination risk but is not as sensitive to short term variability when compared to other faecal indicators. This is likely due to a broad association of TLF with elevated DOC concentrations from a range of different sources. Elevated TLF may indicate preferential conditions for the persistence of TTCs and/or E. coli, but not necessarily a public health risk from microbial contamination. TLF is therefore a more precautionary risk indicator than microbial culturing techniques and could prove useful as a high-level screening tool for initial risk assessment. For widespread use of TLF to be successful, standardisation of TLF values associated with different levels of risk is required, however, this study highlights the difficulties of equating TLF thresholds to TTCs or E. coli data because of the influence of DOC/HLF on the TLF signal.

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

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.

Large-scale survey of seasonal drinking water quality in Malawi using in situ tryptophan-like fluorescence and conventional water quality indicators.

Faecally-contaminated drinking water is a risk to human health, with the greatest risks to those living in developing countries. UN Sustainable Development Goal 6 aims to address this issue. Tryptophan-like fluorescence (TLF) shows potential as a rapid method for detecting microbial contamination in drinking water, which could reduce the spread of waterborne diseases. This study is the first to investigate the effectiveness of TLF for a large-scale survey using a randomised, spot-sampling approach. The large-scale survey took place in Malawi, sub-Saharan Africa, in the dry season (n = 183). A subset of sources were revisited at the end of the following wet season (n = 41). The effectiveness of TLF was assessed by comparing TLF results to thermotolerant coliforms (TTC), humic-like fluorescence (HLF), inorganic hydrochemical data and sanitary risk scores. The most prominent differences in microbial water quality were observed between source types, with little variation between districts and seasons. TLF, TTCs, turbidity and sanitary risk scores were all elevated at alternative sources (shallow wells and tap stands) compared to hand-pumped boreholes. In the dry season, 18% of hand-pumped boreholes showed TTC contamination, which increase to 21% in the wet season. Groundwater recharge processes are likely responsible for seasonal variability of inorganic hydrochemistry at hand-pumped boreholes. TLF was able to distinguish no and low WHO risk classes (TTC 0-9 cfu/100 mL) from medium, high and very high risk classes (TTC 10 - >1000 cfu/100 mL). TLF failed to distinguish between no and low risk classes, which limits the use of TLF for assessing water quality to drinking water standards. This dataset indicates that HLF may raise baseline TLF for samples with low TLF values, increasing false positives. Therefore, TLF is better suited as a rapid high-level water quality screening tool to assess moderate and high levels of faecal contamination.

In-situ fluorescence spectroscopy indicates total bacterial abundance and dissolved organic carbon.

We explore in-situ fluorescence spectroscopy as an instantaneous indicator of total bacterial abundance and faecal contamination in drinking water. Eighty-four samples were collected outside of the recharge season from groundwater-derived water sources in Dakar, Senegal. Samples were analysed for tryptophan-like (TLF) and humic-like (HLF) fluorescence in-situ, total bacterial cells by flow cytometry, and potential indicators of faecal contamination such as thermotolerant coliforms (TTCs), nitrate, and in a subset of 22 samples, dissolved organic carbon (DOC). Significant single-predictor linear regression models demonstrated that total bacterial cells were the most effective predictor of TLF, followed by on-site sanitation density; TTCs were not a significant predictor. An optimum multiple-predictor model of TLF incorporated total bacterial cells, nitrate, nitrite, on-site sanitation density, and sulphate (r2 0.68). HLF was similarly related to the same parameters as TLF, with total bacterial cells being the best correlated (ρs 0.64). In the subset of 22 sources, DOC clustered with TLF, HLF, and total bacterial cells, and a linear regression model demonstrated HLF was the best predictor of DOC (r2 0.84). The intergranular nature of the aquifer, timing of the study, and/or non-uniqueness of the signal to TTCs can explain the significant associations between TLF/HLF and indicators of faecal contamination such as on-site sanitation density and nutrients but not TTCs. The bacterial population that relates to TLF/HLF is likely to be a subsurface community that develops in-situ based on the availability of organic matter originating from faecal sources. In-situ fluorescence spectroscopy instantly indicates a drinking water source is impacted by faecal contamination but it remains unclear how that relates specifically to microbial risk in this setting.

Using boreholes as windows into groundwater ecosystems.

Groundwater ecosystems remain poorly understood yet may provide ecosystem services, make a unique contribution to biodiversity and contain useful bio-indicators of water quality. Little is known about ecosystem variability, the distribution of invertebrates within aquifers, or how representative boreholes are of aquifers. We addressed these issues using borehole imaging and single borehole dilution tests to identify three potential aquifer habitats (fractures, fissures or conduits) intercepted by two Chalk boreholes at different depths beneath the surface (34 to 98 m). These habitats were characterised by sampling the invertebrates, microbiology and hydrochemistry using a packer system to isolate them. Samples were taken with progressively increasing pumped volume to assess differences between borehole and aquifer communities. The study provides a new conceptual framework to infer the origin of water, invertebrates and microbes sampled from boreholes. It demonstrates that pumping 5 m(3) at 0.4-1.8 l/sec was sufficient to entrain invertebrates from five to tens of metres into the aquifer during these packer tests. Invertebrates and bacteria were more abundant in the boreholes than in the aquifer, with associated water chemistry variations indicating that boreholes act as sites of enhanced biogeochemical cycling. There was some variability in invertebrate abundance and bacterial community structure between habitats, indicating ecological heterogeneity within the aquifer. However, invertebrates were captured in all aquifer samples, and bacterial abundance, major ion chemistry and dissolved oxygen remained similar. Therefore the study demonstrates that in the Chalk, ecosystems comprising bacteria and invertebrates extend from around the water table to 70 m below it. Hydrogeological techniques provide excellent scope for tackling outstanding questions in groundwater ecology, provided an appropriate conceptual hydrogeological understanding is applied.