Assessing the biodegradability of dissolved organic carbon in freshwater systems: A method evaluation study

Dissolved organic carbon (DOC) is an important contributor to both carbon cycling and other biogeochemical processes in aquatic ecosystems. The biodegradable fraction of DOC can be microbially degraded over time, producing carbon dioxide (CO₂), a greenhouse gas. In addition, microbial degradation-resistant DOC can accumulate in water bodies, causing chemical and physical changes to aquatic systems. Although biodegradable DOC (BDOC) is widely studied, there is no agreed-upon standard method for assessing its biodegradability. Here, we aimed to develop and evaluate a new method for determining BDOC in freshwater samples. Our method includes filtering water samples to below 0.22 µm, to remove existing microbial cells, prior to inoculating the samples with a concentrated microbial inoculum produced by stepwise isolation of microbial cells from a peat sample. In addition, we added solutions containing nitrogen and phosphorus (in the forms of NH₄NO₃ and K₂HPO₄, respectively) to ensure that the microbes were not nutrient-limited. The samples were then capped with foam stoppers and incubated in the dark at 25⁰C on a shaker for 28 days to allow constant aeration during BDOC degradation. When applied to five freshwater samples collected from rivers, stormwater ponds, and a lake, and a glucose control, we observed that the amount of BDOC in the natural samples ranged from 15% to 53% and was 90% in the glucose control. Rates of BDOC degradation were calculated from DOC measurements at six sampling time points between days 0 and 28. We found that the DOC trends with time were best explained by two successive phases for BDOC degradation in all of the samples: an initial, fast, phase of BDOC degradation followed by a second, slower, phase of BDOC degradation where the rate constant for the second phase was between 5.57 and 565 times slower than for the initial phase. Changes in chemical characteristics of DOC measured using absorbance and fluorescence parameters including specific ultraviolet absorbance at 254 nm (SUVA₂₅₄), humification index (HIX), and parallel factor analysis (PARAFAC) at each sampling time revealed that the initial, fast, phase of BDOC degradation often represents the utilization of small, non-aromatic compounds while the later, slower, phase of BDOC degradation often represents the utilization of more complex, aromatic compounds. The presented method provides a new approach to measure and characterize BDOC degradability and degradation kinetics that can be applied to future studies on biogeochemical processes in aquatic ecosystems.