Cobalt effects on prokaryotic communities of river biofilms: Impact on their colonization kinetics, structure and functions.

Although cobalt (Co) plays a significant role in the transition to low-carbon technologies, its environmental impact remains largely unknown. This study examines Co impacts on the prokaryotic communities within river biofilms to evaluate their potential use as bioindicators of Co contamination. To this end, biofilms were cultivated in artificial streams enriched with different environmental Co concentrations (0.1, 0.5, and 1 µM Co) over 28 days and examined for prokaryotic abundance and diversity via quantitative PCR and DNA-metabarcoding every 7 days. The prokaryotic community's resilience was further investigated after an additional 35 days without Co contamination. The prokaryotic communities were affected by 0.5 and 1 µM Co from the onset of biofilm colonization. The biofilm biomass was comparable between treatments, but the community composition differed. Control biofilms were dominated by Cyanobacteria and Planctomycetes, whereas Bacteroidetes dominated the Co-contaminated biofilms. Potential functional redundancy was observed through the implementation of carbon fixation alternatives by non-photosynthetic prokaryotes in biofilms exposed to high Co concentrations. No structural resilience was observed in the biofilms after 35 days without Co contamination. Measuring the prokaryotic community structural response using molecular approaches appears to be a promising method for assessing shifts in water quality owing to Co contamination.

New sensitive tools to characterize meta-metabolome response to short- and long-term cobalt exposure in dynamic river biofilm communities.

Untargeted metabolomics is a non-a priori analysis of biomolecules that characterizes the metabolome variations induced by short- and long-term exposures to stressors. Even if the metabolite annotation remains lacunar due to database gaps, the global metabolomic fingerprint allows for trend analyses of dose-response curves for hundreds of cellular metabolites. Analysis of dose/time-response curve trends (biphasic or monotonic) of untargeted metabolomic features would thus allow the use of all the chemical signals obtained in order to determine stress levels (defense or damage) in organisms. To develop this approach in a context of time-dependent microbial community changes, mature river biofilms were exposed for 1 month to four cobalt (Co) concentrations (from background concentration to 1 × 10-6 M) in an open system of artificial streams. The meta-metabolomic response of biofilms was compared against a multitude of biological parameters (including bioaccumulation, biomass, chlorophyll a content, composition and structure of prokaryotic and eukaryotic communities) monitored at set exposure times (from 1 h to 28 d). Cobalt exposure induced extremely rapid responses of the meta-metabolome, with time range inducing defense responses (TRIDeR) of around 10 s, and time range inducing damage responses (TRIDaR) of several hours. Even in biofilms whose structure had been altered by Co bioaccumulation (reduced biomass, chlorophyll a contents and changes in the composition and diversity of prokaryotic and eukaryotic communities), concentration range inducing defense responses (CRIDeR) with similar initiation thresholds (1.41 ± 0.77 × 10-10 M Co2+ added in the exposure medium) were set up at the meta-metabolome level at every time point. In contrast, the concentration range inducing damage responses (CRIDaR) initiation thresholds increased by 10 times in long-term Co exposed biofilms. The present study demonstrates that defense and damage responses of biofilm meta-metabolome exposed to Co are rapidly and sustainably impacted, even within tolerant and resistant microbial communities.

Meta-metabolomic responses of river biofilms to cobalt exposure and use of dose-response model trends as an indicator of effects.

The response of the meta-metabolome is rarely used to characterize the effects of contaminants on a whole community. Here, the meta-metabolomic fingerprints of biofilms were examined after 1, 3 and 7 days of exposure to five concentrations of cobalt (from background concentration to 1 × 10-5 M) in aquatic microcosms. The untargeted metabolomic data were processed using the DRomics tool to build dose-response models and to calculate benchmark-doses. This approach made it possible to use 100% of the chemical signal instead of being limited to the very few annotated metabolites (7%). These benchmark-doses were further aggregated into an empirical cumulative density function. A trend analysis of the untargeted meta-metabolomic feature dose-response curves after 7 days of exposure suggested the presence of a concentration range inducing defense responses between 1.7 × 10-9 and 2.7 × 10-6 M, and of a concentration range inducing damage responses from 2.7 × 10-6 M and above. This distinction was in good agreement with changes in the other biological parameters studied (biomass and chlorophyll content). This study demonstrated that the molecular defense and damage responses can be related to contaminant concentrations and represents a promising approach for environmental risk assessment of metals.