Aquatic dissolved organic matter (DOM) plays an essential role in biogeochemical cycling and transport of organic matter throughout the hydrological continuum. To characterise microbially-derived organic matter (OM) from common environmental microorganisms (Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa), excitation-emission matrix (EEM) fluorescence spectroscopy was employed. This work shows that bacterial organisms can produce fluorescent organic matter (FOM) in situ and, furthermore, that the production of FOM differs at a bacterial species level. This production can be attributed to structural biological compounds, specific functional proteins (e.g. pyoverdine production by P. aeruginosa), and/or metabolic by-products. Bacterial growth curve data demonstrates that the production of FOM is fundamentally related to microbial metabolism. For example, the majority of Peak T fluorescence (> 75%) is shown to be intracellular in origin, as a result of the building of proteins for growth and metabolism. This underpins the use of Peak T as a measure of microbial activity, as opposed to bacterial enumeration as has been previously suggested. This study shows that different bacterial species produce a range of FOM that has historically been attributed to high molecular weight allochthonous material or the degradation of terrestrial FOM. We provide definitive evidence that, in fact, it can be produced by microbes within a model system (autochthonous), providing new insights into the possible origin of allochthonous and autochthonous organic material present in aquatic systems.

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.

Organic matter (OM) has an essential biogeochemical influence along the hydrological continuum and within aquatic ecosystems. Organic matter derived via microbial processes was investigated within a range of model freshwater samples over a 10-day period. For this, excitation-emission matrix (EEM) fluorescence spectroscopy in combination with parallel factor (PARAFAC) analysis was employed. This research shows the origin and processing of both protein-like and humic-like fluorescence within environmental and synthetic samples over the sampling period. The microbial origin of Peak T fluorescence is demonstrated within both synthetic samples and in environmental samples. Using a range of incubation temperatures provides evidence for the microbial metabolic origin of Peak T fluorescence. From temporally resolved experiments, evidence is provided that Peak T fluorescence is an indication of metabolic activity at the microbial community level and not a proxy for bacterial enumeration. This data also reveals that humic-like fluorescence can be microbially derived in situ and is not solely of terrestrial origin, likely to result from the upregulation of cellular processes prior to cell multiplication. This work provides evidence that freshwater microbes can engineer fluorescent OM, demonstrating that microbial communities not only process, but also transform, fluorescent organic matter.

The Ganga River (known internationally as the Ganges) is one of the world's most prominent rivers, running from the Himalayas to the Bay of Bengal and supporting the livelihoods of > 40 % of India's 1.4 billion population. The Ganga River is regionally and globally important, supporting agriculture and industry, yet faces potentially detrimental water quality challenges arising from runoff and discharge from increasing urbanization, industry and agriculture. A ∼ 2700 km longitudinal survey of the nutrient and microbial water quality, including phytoplankton composition, of the Ganga River was undertaken in November 2019. The aim was to investigate if and how anthropogenic activities (e.g. urbanisation, industry, and agriculture) and tributary convergence (potentially reflecting both human activity and flow influences) affect and shift physicochemical, nutrient, and microbial water quality parameters along the river continuum. Segmented regression identified four zones of distinct nutrient/microbial characteristics along the Ganga River, with breakpoints located near Kanpur, Varanasi and downstream of the Farakka Barage, at distances of ∼ 1020, ∼ 1500 and ∼ 2350 km downstream from the Himalayan Ganga source. Population density, land use and urban cover were associated with selected water quality parameters in parts of the catchment, with elevated nutrient, microbial and chemical concentrations likely associated with agriculture, industry, and sewage inputs. Some urban areas (e.g. Kanpur and Varanasi), converging tributaries (e.g. Yamuna and Varuna) and barrages (e.g. Farakka) were associated with changes in nutrient availability, microbial activity/abundance and modelled discharge, likely driving apparent water quality changes in the relevant locations. Downstream shifts in nutrient and microbial water quality parameters were observed throughout the ∼ 2700 km Ganga River continuum. This information can help prioritize locations for targeted monitoring and/or remediation interventions and has illustrated an approach to quantify impacts of anthropogenic inputs on major river systems, such as the Ganga River.

This work investigates the origin and range of fluorescent organic matter (FOM) produced in-situ by environmentally sourced freshwater bacteria. Aquatic FOM is an essential component in global carbon cycling and is generally classified as either autochthonous, produced in-situ via microbial processes, or allochthonous, transported into aquatic systems from external sources. We have demonstrated that, within laboratory model systems, environmentally sourced mixed microbial communities and bacterial isolates can produce and/or export FOM associated with both autochthonous and allochthonous material. This study focuses on fluorescence peak B, T, M, C and C+, exploring (1) the cellular nature of FOM produced, (2) FOM exported as extracellular material into the water column and (3) the impact of physical cell lysis on FOM signature. For the laboratory model systems studied, Peak T fluorescence is retained within bacterial cells (>68%), while Peak C fluorescence is mainly observed as extracellular material (>80%). Peak M is identified as both cellular and extracellular FOM, produced by all isolated freshwater microorganisms investigated. The origin of Peak C+ is postulated to originate from functional metabolites associated with specific microorganisms, seen specifically within the Pseudomonas sp. monoculture here. This work challenges the binary classification of FOM as either allochthonous or autochthonous, suggesting that FOM processing and production occurs along a dynamic continuum. Within this study, fluorescence intensity data for the environmental bacteria isolate monocultures are presented as enumeration corrected data, for the first time providing quantitative fluorescence data per bacterial colony forming unit (cfu). From this, we are able to assess the relative contribution of different bacteria to the autochthonous FOM pool and if this material is cellular or extracellular.

With increasing pressures on water resources due to population, industrialization, agriculture, urbanization and climatic changes, improved temporal and spatial understanding of water quality is required. The development of new monitoring parameters, along with new monitoring technologies, are needed to provide real-time insight into the biogeochemical processes that underpin aquatic ecosystem health. Aquatic fluorescent organic matter (AFOM) has recently been explored for its potential to measure underpinning microbial activity within aquatic systems, which are essential in maintaining ecosystem health and function, with specific focus on the utilisation of tryptophan-like fluorescence (TLF or Peak T). In situ real-time portable fluorimeters have been extensively used for the identification and measurement of anthropogenic pollutants, such as polycyclic aromatic hydrocarbons (PAH) and optical brighteners. More recently, this portable fluorescence technology has been adapted for the monitoring and sensing of biological contamination, using microbially derived fluorescence signals (TLF).  The principal aim of this research was to deploy, for the first time in the field, the VLux TPro sensor (Chelsea Technologies Ltd., UK) and to assess the ability of this novel fluorescence-based sensor to detect the presence of biological contamination and elevation of microbial activity. This sensor has been developed to correct in situ real-time sensing data for optical interferences (caused by high turbidity and absorbance), as well as to provide quantitative fluorescence data by reporting in standardised quinine sulphate units (QSU). The urban surface waters within the city of Kolkata provide an interesting challenge for water quality sensors, allowing exploration of sensor performance in a range of water bodies ranging from turbid river waters to open sewer canals.  The sensor data collected demonstrates the ability of the VLux to identify waters with high bacterial loads using Peak T fluorescence. Moderate and weak positive correlations are seen for Peak T and E. coli or total coliform counts, R2 = 0.55 and 0.38 respectively. However, a strong significant correlation is identified between Peak T and the total bacterial cell counts (R2 = 0.75). This demonstrates that Peak T should not be used as a species-specific enumerator in complex surface water matrices. It does, however, demonstrate the ability of the VLux to successfully measure optically corrected and quantitative Peak T fluorescence in QSU. Therefore, data regarding the activity and fast-acting dynamics of freshwater bacterial communities, in response to pollution events, can now be reliably sensed and collected. This was demonstrated by the elevated Peak T fluorescence intensity observed when biologically contaminated water entered the main river channel, enabling identification of contamination hotspots. Sensing data has been further validated by laboratory analysis of spot samples confirming the significant correlations between Peak T and bacteria and nutrient concentrations. Further field-based research is required to determine the feasibility of long-term catchment scale sensor deployment as part of a sensing network, for the monitoring of biological activity and pollution events in freshwaters.     Acknowledgements: This research was supported by the NERC-DST Indo-UK Water Quality Programme NE/R003106/1 and DST/TM/INDO-UK/2K17/30. We would also like to acknowledge Chelsea Technologies Ltd. for ongoing VLux TPro sensor support.