<p>This research explores the <em>in-situ</em> bacterial production of aquatic fluorescent organic matter (AFOM) under controlled laboratory conditions. Whilst fluorescence techniques have long been used to monitor AFOM distribution, origin and dynamics within aquatic systems, the extent to which AFOM characteristics are defined by microbial processing in surface freshwaters has largely been overlooked. Current convention champions the assumption that humic-like (Peak C) and protein-like (Peak T) fluorescence signatures are exclusively derived from terrestrial (allochthonous) or microbial (autochthonous) origins respectively, with Peak T having been directly correlated with microbial enumeration. Under intensifying anthropogenic perturbations and changing catchment characteristics, the complexities associated with bacterial-organic matter (OM) interactions in freshwater systems are increasing, challenging our understanding as to the origin and fate of aquatic OM. To what extent the observed AFOM in freshwater systems is defined by bacterial processing and how such processing may be influenced by nutrient availability are key knowledge gaps that need to be addressed. Previous research has observed the <em>in-situ</em> bacterial production of humic-like compounds in a laboratory model system with a high-nutrient and high-carbon content synthetic growth medium. This work describes a non-fluorescing, simulated freshwater matrix which is low in both nutrient and organic carbon concentrations. Using this model, growth curve incubation experiments have been undertaken over a 48-hour period with a monoculture laboratory strain of <em>Pseudomonas aeruginosa</em>. Microbiological and fluorescence analyses undertaken at regular time intervals demonstrate the bacterial production of humic-like OM (Peak C) under oligotrophic (after 8hrs) and simulated high-nutrient conditions (after 6hrs). These findings, albeit under laboratory conditions, are important as they show that this fluorescence region, currently viewed as allochthonous in origin, can also represent labile OM generated <em>in-situ</em> by bacteria and, furthermore, that this bacterial production increases as a function of nutrient loading. In addition, the data quantitatively demonstrates that fluorescence intensities increase independently of cell density. These results challenge the assumption that humic-like AFOM is exclusively terrestrial in origin and suggest that bacteria may “engineer” OM<em> in-situ</em> that gives rise to these fluorescence characteristics as a function of metabolism. Importantly, nutrient availability is a key driver of metabolic activity, outlining the potential for the use of fluorescence as a marker for stream metabolism as opposed to a measure of bacterial numbers. Further development of the laboratory model via the utilisation of environmentally-sourced bacterial communities is required. Ultimately, this laboratory model will inform field studies that look to improve our understanding of how microbial communities respond to catchment stressors, and how these responses influence AFOM fluorescence signatures and ultimately the origin and fate of OM in freshwater systems.</p>

Dissolved organic matter (DOM) is ubiquitous throughout aquatic systems. Fluorescence techniques can be used to characterize the fluorescing proportion of DOM, aquatic fluorescent organic matter (AFOM). AFOM is conventionally named in association with specific fluorescence “peaks,” which fluoresce in similar optical regions as microbially-derived proteinaceous material (Peak T), and terrestrially-derived humic-like compounds (Peaks C/C+), with Peak T previously being investigated as a tool for bacterial enumeration within freshwaters. The impact of anthropogenic nutrient loading on the processing of DOM by microbial communities is largely unknown. Previous laboratory studies utilizing environmental freshwater have employed growth media with complex background fluorescence, or very high nutrient concentrations, preventing the investigation of AFOM production under a range of more representative nutrient concentrations within a matrix exhibiting very low background fluorescence. We describe a laboratory-based model with Pseudomonas aeruginosa that incorporates a low fluorescence growth matrix consisting of a simulated freshwater (SFW), representative of low-hardness freshwater systems allowing controlled nutrient conditions to be studied. The effects of microbial processing of DOM as a function of available nitrogen, phosphorous, and dissolved organic carbon (DOC) in the form of glucose were investigated over 48 h at highly resolved time increments. The model system demonstrates the production of a range of complex AFOM peaks in the presence and absence of DOC, revealing no linear relationship between cell numbers and any of the peaks for the bacterial species studied, with AFOM peaks increasing with microbial cell number, ranging from 55.2 quinine sulfate units (QSU) per 106 cells to 155 QSU per 106 cells (p < 0.05) for Peak T during the exponential growth phase of P. aeruginosa under high nutrient conditions with 5 mg L−1 DOC. Nutrient and DOC concentration was found to cause differential production of autochthonous- or allochthonous-like AFOM, with lower DOC concentrations resulting in higher Peak T production relative to Peaks C/C+ upon the addition of nutrients, and high DOC concentrations resulting in higher Peak C/C+ production relative to Peak T. Our results show the production of allochthonous-like AFOM from a simple and non-fluorescent carbon source, and provide uncertainty in the use of Peak T as a reliable surrogate for specific bacterial enumeration, particularly in dynamic or nutrient-impacted environments, pointing toward the use of fluorescence as an indicator for microbial metabolism.