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Spatial heterogeneity along river networks is interrupted by dams, affecting the transport, processing, and storage of organic matter, as well as the distribution of biota. We here investigated the structure of planktonic (free-living, FL), particle-attached (PA) and sediment-associated (SD) bacterial and archaeal communities within a small reservoir. We combined targeted-amplicon sequencing of bacterial and archaeal 16S rRNA genes in the DNA and RNA community fractions from FL, PA and SD, followed by imputed functional metagenomics, in order to unveil differences in their potential metabolic capabilities within the reservoir (tail, mid, and dam sections) and lifestyles (FL, PA, SD). Both bacterial and archaeal communities were structured according to their life-style preferences rather than to their location in the reservoir. Bacterial communities were richer and more diverse when attached to particles or inhabiting the sediment, while Archaea showed an opposing trend. Differences between PA and FL bacterial communities were consistent at functional level, the PA community showing higher potential capacity to degrade complex carbohydrates, aromatic compounds, and proteinaceous materials. Our results stressed that particle-attached prokaryotes were phylogenetically and metabolically distinct from their free-living counterparts, and that performed as hotspots for organic matter processing within the small reservoir.

During summer drought, Mediterranean fluvial networks are transformed into highly heterogeneous landscapes characterized by different environments (i.e., running and impounded waters, isolated river pools and dry beds). This hydrological setting defines novel biogeochemically active areas that could potentially increase the rates of carbon emissions from the fluvial network to the atmosphere. Using chamber methods, we aimed to identify hot spots for carbon dioxide (CO₂) and methane (CH₄) emissions from two typical Mediterranean fluvial networks during summer drought. The CO₂ efflux from dry beds (mean ± SE = 209 ± 10 mmol CO₂m⁻²d⁻¹) was comparable to that from running waters (120 ± 33 mmol m⁻² d⁻¹) and significantly higher than from impounded waters (36.6 ± 8.5 mmol m⁻²d⁻¹) and isolated pools (17.2 ± 0.9 mmol m⁻² d⁻¹). In contrast, the CH₄ efflux did not significantly differ among environments, although the CH₄ efflux was notable in some impounded waters (13.9 ± 10.1 mmol CH₄ m⁻² d⁻¹) and almost negligible in the remaining environments (mean <0.3 mmol m⁻²d⁻¹). Diffusion was the only mechanism driving CO₂ efflux in all environments and was most likely responsible for CH₄ efflux in running waters, isolated pools and dry beds. In contrast, the CH₄ efflux in impounded waters was primarily ebullition-based. Using a simple heuristic approach to simulate potential changes in carbon emissions from Mediterranean fluvial networks under future hydrological scenarios, we show that an extreme drying out (i.e., a four-fold increase of the surface area of dry beds) would double the CO₂ efflux from the fluvial network. Correspondingly, an extreme transformation of running waters into impounded waters (i.e., a twofold increase of the surface area of impounded waters) would triple the CH₄ efflux. Thus, carbon emissions from dry beds and impounded waters should be explicitly considered in carbon assessments of fluvial networks, particularly under predicted global change scenarios, which are expected to increase the spatial and temporal extent of these environments.

Biofilms are a key component in the nutrient removal from the water column. However, nutrient uptake by biofilms may be hampered by the occurrence of pollutants or other stressors. This study aimed: (i) to investigate the biofilm phosphorus (P) uptake capacity as a relevant process for the maintenance of fluvial water quality and (ii) to explore the sensitivity of this process to different chemical and environmental stressors. We conducted chamber experiments to test for the relevance of biofilm P uptake capacity (PUC) as a tool to detect effects of pollutants on river self-depuration. PUC was calculated by measuring P temporal decay after performing controlled P-spikes in chambers with biofilm-colonized tiles. Four different experiments were conducted to evaluate the response of PUC to: (a) several river waters from increasing polluted sites; (b) the effect of the bactericide triclosan (TCS); (c) the combined effect of TCS and grazers; and (d) the effect of TCS after a drought episode that affected the biofilms. These experiments showed that biofilms decreased their PUC along the pollution gradient. The biofilm PUC was significantly reduced after receiving high TCS concentrations, though lower TCS concentrations also affected the biofilm when this was submitted to grazing pressure. PUC decrease was induced by flow interruption which further enhanced the TCS negative effects. Overall, PUC was sensitive to the effects of pollutants like TCS as well as to the action of biological (grazing) and environmental (drought) factors. The study also showed that multiple stressors enhance the negative effects of pollutants on the PUC of biofilms. Our study values the use of biofilms’ PUC as a sensitive ecological-based tool to assess the effects of chemicals on freshwater communities and their derived functioning in river ecosystems.

Abstract To evaluate the effects of hydrological variability on pesticide dissipation capacity by stream biofilms, we conducted a microcosm study. We exposed biofilms to short and frequent droughts (daily frequency), long and less frequent droughts (weekly frequency) and permanently immersed controls, prior to test their capacities to dissipate a cocktail of pesticides composed of tebuconazole, terbuthylazine, imidacloprid, glyphosate and its metabolite aminomethylphosphonic acid. A range of structural and functional descriptors of biofilms (algal and bacterial biomass, extracellular polymeric matrix (EPS) concentration, microbial respiration, phosphorus uptake and community-level physiological profiles) were measured to assess drought effects. In addition, various parameters were measured to characterise the dynamics of pesticide dissipation by biofilms in the different hydrological treatments (% dissipation, peak asymmetry, bioconcentration factor, among others). Results showed higher pesticide dissipation rates in biofilms exposed to short and frequent droughts, despite of their lower biomass and EPS concentration, compared to biofilms in immersed controls or exposed to long and less frequent droughts. High accumulation of hydrophobic pesticides (tebuconazole and terbuthylazine) was measured in biofilms despite the short exposure time (few minutes) in our open-flow microcosm approach. This research demonstrated the stream biofilms capacity to adsorb hydrophobic pesticides even in stressed drought environments. The capacity of stream biofilms to dissipate a cocktail of pesticides performed better in a high intermittent drought-stressed environment.

We investigated photoinhibition on natural communities of ammonia oxidizing (AO) archaea (AOA) and bacteria (AOB) embedded in complex stream biofilms, and its implications on nitrogen uptake at biofilm scale. Based on the strong photoinhibition previously exhibited by free living and cultured AOA and AOB, we expected AO activity to decrease in biofilms experimentally exposed to light, reducing the contribution of microbial nitrification to ammonium uptake. To test it, we conducted light manipulation experiments in mesocosms using biofilms naturally developed on stream cobbles sides both exposed to light (light-side) and facing the sediment (dark-side). We observed a strong AO photoinhibition in dark-side biofilms, accompanied by either biofilm-scale decreases in nitrification or increases in nitrogen uptake likely by heterotrophic activity. Conversely, in light-side biofilms photoinhibition was not observed suggesting that photoautotrophic layers may protect AO in situ by a sunshade effect. Experimental light and dark alternation cycles stimulated AO, enhancing both nitrification and ammonium uptake. These results support photoinhibition effects on natural AO communities, especially in biofilms developed under ambient dark conditions, whereas this effect seems to be buffered in biofilms developed under light conditions. Therefore, the contribution of nitrification to ammonium uptake at biofilm scale should consider not only the physiological study of AOA and AOB, but also the environmental conditions and community structure at the habitat microscale, since they may counterbalance, among others, the damaging light effects under natural conditions.

Stream microbial communities and associated processes are influenced by environmental fluctuations that may ultimately dictate nutrient export. Discharge fluctuations caused by intermittent stream flow are increasing worldwide in response to global change. We examined the impact of flow cessation and drying on in-stream nitrogen cycling. We determined archaeal (AOA) and bacterial ammonia oxidizer (AOB) abundance and ammonia oxidation activity in surface and deep sediments from different sites along the Fuirosos stream (Spain) subjected to contrasting hydrological conditions (i.e., running water, isolated pools, and dry streambeds). AOA were more abundant than AOB, with no major changes across hydrological conditions or sediment layers. However, ammonia oxidation activity and sediment nitrate content increased with the degree of stream drying, especially in surface sediments. Upscaling of our results shows that ammonia oxidation in dry streambeds can contribute considerably (~50%) to the high nitrate export typically observed in intermittent streams during first-flush events following flow reconnection. Our study illustrates how the dry channels of intermittent streams can be potential hotspots of ammonia oxidation. Consequently, shifts in the duration, spatial extent and severity of intermittent flow can play a decisive role in shaping nitrogen cycling and export along fluvial networks in response to global change.

Large‐scale factors associated with the environmental context of streams can explain a notable amount of variability in patterns of stream N cycling at the reach scale. However, when environmental factors fail to accurately predict stream responses at the reach level, focusing on emergent properties from small‐scale heterogeneity in N cycling rates may help understand observed patterns in stream N cycling. To address how small‐scale heterogeneity may contribute to shape patterns in whole‐reach N uptake, we examined the drivers and variation in microbial N uptake at small spatial scales in two stream reaches with different environmental constraints (i.e., riparian canopy). Our experimental design was based on two ¹⁵N additions combined with a hierarchical sampling design from reach to microhabitat scales. Regardless of the degree of canopy cover, small‐scale heterogeneity of microbial N uptake ranged by three orders of magnitude, and was characterized by a low abundance of highly active microhabitats (i.e., hot spots). The presence of those hot spots of N uptake resulted in a nonlinear spatial distribution of microbial N uptake rates within the streambed, especially in the case of epilithon assemblages. Small‐scale heterogeneity in N uptake and turnover rates at the microhabitat scale was primarily driven by power relationships between N cycling rates and stream water velocity. Overall, fine benthic organic matter (FBOM) assemblages responded clearly to changes in the degree of canopy cover, overwhelming small‐scale heterogeneity in its N uptake rates, and suggesting that FBOM contribution to whole‐reach N uptake was principally imposed by environmental constraints from larger scales. In contrast, N uptake rates by epilithon showed no significant response to different environmental influences, but identical local drivers and spatial variation in each study reach. Therefore, contribution of epilithon assemblages to whole‐reach N uptake was mainly associated with emerging properties from small‐scale heterogeneity at lower spatial scales.

Human‐induced salinization increasingly threatens inland waters; yet we know little about the multifaceted response of lake communities to salt contamination. By conducting a coordinated mesocosm experiment of lake salinization across 16 sites in North America and Europe, we quantified the response of zooplankton abundance and (taxonomic and functional) community structure to a broad gradient of environmentally relevant chloride concentrations, ranging from 4 to ca. 1400 mg Cl− L−1. We found that crustaceans were distinctly more sensitive to elevated chloride than rotifers; yet, rotifers did not show compensatory abundance increases in response to crustacean declines. For crustaceans, our among‐site comparisons indicate: (1) highly consistent decreases in abundance and taxon richness with salinity; (2) widespread chloride sensitivity across major taxonomic groups (Cladocera, Cyclopoida, and Calanoida); and (3) weaker loss of functional than taxonomic diversity. Overall, our study demonstrates that aggregate properties of zooplankton communities can be adversely affected at chloride concentrations relevant to anthropogenic salinization in lakes.

Nutrients and light are the most determinant factors for microbial benthic assemblages in oligotrophic forested streams. We investigated the importance of nutrients and light availability on the structure and the function of epilithic biofilms in a Mediterranean forested stream (Fuirosos, Spain). Biofilms grew on artificial substrata in both enriched and unenriched reaches where shade conditions were simulated. Four different treatments were generated: higher light unenriched, lower light unenriched, higher light enriched (HL-E) and lower light enriched. Chlorophyll a , bacterial density, extracellular polymeric substances (EPS), extracellular leucine aminopeptidase (LAmP) and alkaline phosphatase (APase) activities were analysed during the colonisation at days 4, 9, 16, 22 and 52. At day 52, confocal laser scanning microscopy (CLSM) was used to determine differences in biofilm architecture. CLSM evidenced differences in thickness and structural complexity of biofilms grown in different conditions. Biofilms in HL-E were the thickest and had the most complex structure. The CLSM highlighted that the EPS was agglomerated in the upper layer of enriched-grown biofilms, but evenly distributed through the biofilm in unenriched biofilms. CLSM 3D images suggested that cyanobacteria increased under higher nutrient conditions. Nutrient enrichment caused the decrease of APase activity. Interaction between the two factors affected LAmP activity. HL-E had the highest LAmP and the lowest APase activities, an indication that biofilm responses to nutrients mostly occurred with high-light availability. Our results revealed that the conjoint availability of light and nutrients caused the highest changes in biofilm spatial organisation, microbial structure and functioning in oligotrophic forested streams.

The salinization of freshwaters is a global threat to aquatic biodiversity. We quantified variation in chloride (Cl−) tolerance of 19 freshwater zooplankton species in four countries to answer three questions: (1) How much variation in Cl− tolerance is present among populations? (2) What factors predict intraspecific variation in Cl− tolerance? (3) Must we account for intraspecific variation to accurately predict community Cl− tolerance? We conducted field mesocosm experiments at 16 sites and compiled acute LC50s from published laboratory studies. We found high variation in LC50s for Cl− tolerance in multiple species, which, in the experiment, was only explained by zooplankton community composition. Variation in species‐LC50 was high enough that at 45% of lakes, community response was not predictable based on species tolerances measured at other sites. This suggests that water quality guidelines should be based on multiple populations and communities to account for large intraspecific variation in Cl− tolerance.