Browning (DOC concentration) was a strong predictor of the stoichiometry of the total nutrient pools across all sampling dates, and warming did not alter these relationships along the DOC gradient (Fig. 1; Table 1). In linear mixed-effects models, the estimate (Est.) indicates the direction and magnitude of the relationship, i.e., the expected change in the response for a one-unit increase in the predictor. While increased cDOM and DOC concentrations increase the absolute amount of nutrients (open data), DOC strongly increased DOC:TN (Est.=0.72, p<0.001) and DOC:TP (Est.=0.72, p<0.001), indicating progressive carbon enrichment relative to N and P with browning (Fig. 1A and B). This pattern is uniform across browning systems across the northern hemisphere (Creed et al., 2018; Isles et al., 2020). Although declines in TN:TP along the DOC gradient suggested increasing nutrient imbalance and stronger N limitation, this relationship was weak (Est.=-0.12, p=0.06; Fig. 1C). Nonetheless, this effect may be underestimated, as DIN:TP are shown to be better estimators for phytoplankton nutrient limitation (Bergström, 2010).

In the dissolved nutrient pools, DOC was also an important predictor of stoichiometry (Table 1). DOC marginally decreased DIC:DIN (Est.=-0.34, p=0.04), strongly decreased DIC:PO43- (Est.=-2.32, p=<0.001) and DIN:PO43- (Est.=-1.09, p=0.0002), indicating decreasing inorganic carbon enrichment relative to dissolved nutrients along the DOC gradient (Fig. 1D–F). The increasing importance of carbon in the total nutrient pools (DOC:TN and DOC:TP), but decreasing importance of carbon in the inorganic pools (DIC:DIN and DIC:PO43-), reflects the increasing importance of organic nutrients along the DOC gradient (Andersen et al., 2025; Corman et al., 2018; Stetler et al., 2021). The ternary plots with Redfield normalized C:N:P ratios, confirm that ponds are consistently carbon-rich, and therefore N and P-poor relative to Redfield proportions in both dissolved (DIC–DIN–PO43-) and total (DOC–TN–TP) nutrient pools throughout the sampling season (Fig. 2B and C; Fig. B1 in Appendix B). Most observations fell within the P-depleted and co N&P-depleted, and occasionally N-depleted regions of conceptual stoichiometric space (Fig. 2A). Stoichiometric imbalances were strongest in the dissolved fraction, which showed greater carbon dominance and more pronounced seasonal variability than total pools. Most importantly, browning increased the C:N and C:P ratios in the dissolved carbon and total nutrient pools, indicating lower TN and TP relative to DOC, while decreasing these ratios in the dissolved fractions, indicating lower DIC relative to DIN and PO43- (Fig. 1A–B, D–E). This contrasting pattern suggests that browning enhances the availability of carbon substrates for heterotrophs while reducing the relative carbon resources available to photoautotrophs.

As the ponds are consistently enriched in carbon relative to N and P (Fig. 2), it is particularly interesting to assess the N and P pools. The stoichiometric optimum that approximates the Redfield ratio (N:P = 16:1, molar) corresponds to a log⁡10-transformed value of 1.2 (10log⁡ (16:1)) for our molar concentrations (Fig. 1). Ratios around this value indicate likely nitrogen–phosphorus co-limitation, whereas higher values suggest increasing P limitation and lower values indicate N limitation (Redfield, 1958). In our experiment, N:P ratios spanned this critical range, from just below 1.0 to >1.5, thereby capturing conditions from N limitation to P limitation. Moreover, DOC quality is held constant as all ponds are treated with the same DOC source, but in natural systems DOC composition varies and strongly shapes its bioavailability and the resulting nutrient limitation of microbial production (Berggren et al., 2014, 2025; Soares et al., 2017). The mixed-effects model results indicate that in our experiment the stoichiometric optimum of 1.2 occurs at a DOC concentration of 7.2 mgL-1 for total pools, and a DOC concentration of 2.5 mgL-1 for dissolved pools (not accounting for the temperature treatments). These values are way below the proposed turning point of ∼11mgL-1 DOC, below which primary producers are primarily nutrient-limited and above which light limitation becomes more important (Hanson et al., 2003; Jones and Lennon, 2015). Accordingly, the magnitude of the algal production peaks near this transition (∼11mgL-1 DOC) is additionally driven by N:P nutrient stoichiometry (Kelly et al., 2018; Puts et al., 2023a). In our ponds, the optimal N:P ratio occurs well below 11 mgL-1 DOC, indicating that algal production is constrained by suboptimal nutrient ratios rather than by light availability. This explains the observed decline in PP, as light limitation in our system is unlikely (Sect. 2.4).

For both total and dissolved nutrient pools, browning clearly dominated over warming, and warming did not modify the DOC effect (Table 1). Interaction models provided no evidence that warming altered DOC effects on total nutrient stoichiometry (with ΔAICInt–Base ranging from +0.03 to +1.45; Table 1). A notable exception was DIN:PO43-, which was significantly affected by warming (Est.=0.22, p=0.038). Seasonal effects were similarly strong in both total and dissolved nutrient pools, with all pools showing pronounced temporal variation. This strong seasonal signal across both pools suggests that stoichiometric dynamics is not solely driven by the DOC loading within a waterbody but are also regulated by seasonal (moth-to-month) biogeochemical cycling (Kolzau et al., 2014). The ternary plots (Fig. 2) indicate P limitation early in the season, with N becoming relatively more limiting later. Seasonal differences in stoichiometry were limited in total pools, whereas dissolved pools showed greater variability, particularly in May when the relative contribution of N was higher than later in the season. Seasonal variability likely reflects synchronized changes in biological uptake, microbial processing, and hydrological inputs (e.g., spring run-off), which jointly restructure both total and dissolved nutrient pools.

Together, these results suggest that organic carbon enrichment was the primary driver of stoichiometric shifts in both pools, and 2 °C warming did not modify these effects except for DIN:PO43-. However, seasonal effects, where temperature is compounded, are observed. The observed effect of warming on DIN:PO43- suggests that temperature mainly influences fast-cycling nitrogen processes. Other stoichiometric ratios from both the total and dissolved pools did not respond to warming because they may be buffered by slower turnover of total nutrient pools (Sinsabaugh and Follstad Shah, 2012) or influenced by variation in microbial and phytoplankton community composition that can dampen the direct effects of temperature on nutrient cycling.

The nutrient limitation assay shows consistent N–P co-limitation across all DOC treatments, with chl a responses strongest when N and P were added together (Fig. 3A). Responses to P in the highest DOC treatment increased significantly compared to the control with low DOC. Responses to N and P alone were low to moderate, with slight increases at higher DOC concentrations. Our results are in line with large spatial studies across Sweden showing phytoplankton in southern lakes are co-limited by N and P, while phytoplankton in carbon-poor northern lakes were mainly N-limited (Bergström, 2010; Bergström et al., 2008). DOC enrichment (browning) therefore shifts the system toward stronger nutrient stress for phytoplanktonic algal (chl a) production, confirming that browning leads to less favorable nutritional requirements for algal production (Sect. 2.1).

Temperature had a minimal and insignificant effect (Fig. 3B). Nonetheless, the highest RRchl a values were observed under Ambient temperature conditions, and warming generally reduced response, particularly in Mid and High DOC treatments, although this response was not significant. Overall, higher DOC enhanced nutrient-driven responses, particularly under NP additions, while warming tended to dampen these effects, consistent with patterns observed for PP (Sect. 2.4), although these effects were not statistically significant.

Microbial production responded differently to browning and warming. Both BP and PP (p<0.001 and p<0.01, respectively), as well as PP:BP (p=0.022) varied significantly across sampling times, indicating strong seasonal influence on microbial responses. BP increased strongly with DOC (Est.=0.43, p=0.0003; Fig. 1H) and was unaffected by warming or DOC-warming interactions (Fig. 1H; Table 1). In contrast to primary producers, bacterial (heterotrophic) producers are well known to assimilate organic carbon efficiently, have a more flexible stoichiometry (Cotner et al., 2010) and are a substantial part of carbon cycling and the microbial food web via the “microbial loop” (Fenchel, 2008). Browning increased C:N and C:P ratios in the DOC versus total nutrient pools, but decreased them in the dissolved fractions (Fig. 1A–B, D–E), suggesting greater carbon availability for heterotrophs and reduced carbon availability for photoautotrophs. This pattern is consistent with increasing BP and unaffected (though a trend of declining) PP and along the cDOM gradient (Fig. 1E and G).

Indeed, while PP showed no significant response to DOC or warming (in the linear mixed-effects models), it showed a trend (linear regression) of higher values at low DOC that declined more sharply with DOC under warming, resulting in lower PP at high DOC in warmed treatments (Table 1; Fig. 1G). Accordingly, warming amplified DOC-related declines in the PP:BP ratio (interaction: Est.=-0.73, p=0.047) indicating that the ponds shifted toward stronger heterotrophic dominance, although net heterotrophy (PP:BP<1) was not reached. Net heterotrophy was likely not reached because measurements were taken at a single, well-lit depth (0.6 m) rather than integrated over the full water column (Klaus et al., 2022), and because shallow lakes and ponds (≤4m) typically exhibit high rates of both primary and heterotrophic production and large variability in net ecosystem production (Rabaey et al., 2024).

This pattern, indicating interactive effects of DOC and warming on metabolic balance (PP:BP), can only be disentangled in an experimental setting, as in natural lakes temperature variations are confounded by other factors that govern microbial production, such as nutrients, light, variability in DOC quality and quantity. Our finding of inhibited PP due to warming contrasts with observations from Swedish Arctic lakes, where PP is typically temperature-limited (Bergström et al., 2013; Faithfull et al., 2011; Puts et al., 2023a). Instead, our results controlled the confounded factors, and show that browning promotes net heterotrophy, but that this shift is not driven solely by increased BP, it can also arise from warming-induced declines in PP, so-called “thermal compensation” (Liu et al., 2022). Indeed, temperatures during this experiment were unusually high (see Appendix Sect. A1), meaning the additional +2°C warming may have pushed the system beyond the thermal optimum of primary producers, whose growth declines sharply at temperatures higher than their thermal optimum (Kontopoulos et al., 2020). Moreover, we showed that along the DOC gradient inorganic nutrient availability declines (Sect. 2.1), which would impact production negatively. In less warm years, where primary producers are not passing optimal growth temperatures, the effect of temperature may not be negative, still, our results show that with browning PP will decrease. Our results indicate that BP is thriving under 2 °C warming and browning, whereas primary producers struggle, likely due to (1) thermal compensation (2) reduced inorganic nutrients, and (3) suboptimal stoichiometry.

With the increasing occurrence of heatwaves (Perkins-Kirkpatrick and Lewis, 2020) and intensified browning and warming of surface waters (Pilla et al., 2018; Puts et al., 2023a), the conditions observed in our ponds are likely to become more common. Altogether, our results indicate that in ponds and natural lakes, net ecosystem respiration and heterotrophy would be likely under browning and warming, particularly as warming intensifies epilimnetic temperatures (Bogard and del Giorgio, 2016; Duarte and Prairie, 2005; Sobek et al., 2007).

As with the majority of ecosystem-scale experiments, our study simplifies natural shallow water systems. Although the EXEF ponds contain sediments, microbial communities, primary producers, consumers, and a natural shoreline, natural lakes have more variation in size and their catchments. Moreover, natural lakes have varying cDOM sources, with varying quality (Berggren et al., 2018). Thus, the experiment was designed to isolate the effects of browning and warming rather than replicate the full complexity of freshwater ecosystems. While DOC quality was constant across treatments (not seasons), natural lakes vary in cDOM composition, nutrient inputs, catchment influences, and mixing regimes, all of which can affect water physico-chemistry and productivity. Nevertheless, the observed patterns are consistent with large-scale studies of browning lakes. Our study does not capture the full spectrum of DOC concentrations found in lakes: the control lake had relatively high DOC values already, and thus pristine mountain lakes are not captured in this gradient. Nonetheless, because our conclusions are still based on responses across a broader DOC gradient, we expect the direction of the responses to be robust, although their magnitude may vary among lake types and climatic conditions. The unusually warm summer during our study may also have amplified temperature effects. Accordingly, our findings should be viewed as evidence for the mechanisms underlying browning-warming interactions rather than as quantitative predictions for all boreal lakes.