Characterization and evolution of natural aquatic biofilm communities exposed in vitro to herbicides.

River biofilms are assemblies of autotrophic and heterotrophic microorganisms that can be affected by pollutants such as those found in watersheds and wastewater treatment plants. In the laboratory, experimental biofilms were formed from river water, and their overall composition was investigated. Denaturing gradient gel electrophoresis (DGGE) and cytometry were used to assess the richness and diversity of these communities. The software Cytostack (available on request) was developed to treat and analyze the cytometric data. Measurements of chlorophyll-a and carotenoids were used to assess the global composition of the photoautotrophic community, whereas proteins, polysaccharides (PS) content, and esterase activities were used to assess overall changes in the mixed communities. We evaluated the effects that 3 weeks of treatment with the herbicides diuron and glyphosate (10 µg L(-1)) had on these biofilms. Exposed to diuron, bacterial communities adapted, changing their composition. Glyphosate inhibited growth of one autotrophic community but caused no chlorophyll deficit. As a whole, the biofilm acted as a micro-ecosystem, able to regulate and maintain a constant level of photosynthetic pigment through the structural adaptation of the autotrophic community. These results are one more proof that microbial diversity of aquatic biofilms is influenced by chemical stresses, potentially leading to disturbances within the ecosystems.

Dose-dependent effects of the herbicide mesotrione on soil cyanobacterial communities.

This study aimed to investigate the dose-response effects of an herbicide on soil photosynthetic microbial communities, particularly cyanobacteria, using a microcosm approach. Pure mesotrione (active ingredient), and Callisto (a commercial formulation of this triketone herbicide), were spread at different rates on soil microcosm surfaces. Soil Chlorophyll concentrations were quantified to assess the photosynthetic biomass, and the genetic structure and diversity of the cyanobacterial community were investigated by a group-specific polymerase chain reaction followed by denaturing gradient gel electrophoresis. Dose-dependent responses were evidenced for both functional and structural parameters. No effect was detected in soils treated with 1 × AR (1-fold recommended application rate) irrespective of the herbicide formulation. At 10 × AR (10-fold recommended application rate), only Callisto treatment induced significant decreases of photosynthetic biomass, whereas structural parameters were less affected. At the 100 × AR (100-fold recommended application rate), both pure mesotrione and Callisto had strong negative impacts on soil chlorophyll concentrations and cyanobacterial genetic structure and diversity. At both the 10 × AR and 100 × AR treatments, Callisto induced significant stronger effects than pure mesotrione. In addition, indicators of photosynthetic biomass, compared with structural parameters of cyanobacterial communities (within 14 days), responded (within 7 days) more quickly to herbicide stress. The results of this study underscore the relevance of soil photosynthetic microbial communities to develop indicators for herbicide risk assessment.

Glyphosate-degrading behavior of five bacterial strains isolated from stream biofilms.

The present study investigates the individual degrading behavior of bacterial strains isolated from glyphosate-degrading stream biofilms. In this aim, biofilms were subjected to enrichment experiments using glyphosate or its metabolite AMPA (aminomethyl phosphonic acid) as the sole phosphorus source. Five bacterial strains were isolated and taxonomically affiliated to Ensifer sp. CNII15, Acidovorax sp. CNI26, Agrobacterium tumefaciens CNI28, Novosphingobium sp. CNI35 and Ochrobactrum pituitosum CNI52. All strains were capable of completely dissipating glyphosate after 125-400 h and AMPA after 30-120 h, except for Ensifer sp. CNII15 that was not able to dissipate glyphosate but entirely dissipated AMPA after 200 h. AMPA dissipation was overall faster than glyphosate dissipation. The five strains degraded AMPA completely since formaldehyde and/or glycine accumulation was observed. During glyphosate degradation, the strain CNI26 used the C-P lyase degradation pathway since sarcosine was quantitatively produced, and C-P lyase gene expression was enhanced 30× compared to the control treatment. However, strains CNI28, CNI35 and CNI52 accumulated both formaldehyde and glycine after glyphosate transformation suggesting that both C-P lyase and/or glyphosate oxidase degradation pathways took place. Our study shows different and complementary glyphosate degradation pathways for bacteria co-existing in stream biofilms.

Effects of sulfonamide antibiotics on aquatic microbial community composition and functions.

Knowledge on interactions among microbial communities colonizing various streambed substrata (e.g. cobbles, sediment, leaf-litter etc.) is essential when investigating the functioning of stream ecosystems. However, these interactions are often forgotten when assessing the responses of aquatic microbial communities to chemical contamination. Using a stream microcosm approach, the respective impact of two sulfonamide antibiotics (sulfamethoxazole and sulfamethazine) on the ability of microbial heterotrophs to decompose alder leaves was investigated in the presence or absence of periphyton. Our hypothesis suggested that sulfonamides would negatively impair microbial litter decomposition and that periphyton could possibly alleviate this effect by stimulating microbial decomposer activity through a priming effect. Results showed that the presence of periphyton enriched water with oxygen and labile dissolved organic carbon forms. However, these labile organic carbon sources did not stimulate leaf-litter decomposition but mostly decoupled microbial decomposer activity from particulate organic matter to dissolved organic matter through negative priming. Also, the two sulfonamide molecules did not affect the leaf-litter decomposition process but significantly decreased bacterial biomass accrual on leaves. The reduction of bacteria was concomitant with an increase in biomass-specific ß-glucosidase activity and this was attributed to a stress response from bacteria to sulfonamides. Further research looking at microbial interactions would provide for better assessment of chemical contamination effects in communities and processes in stream ecosystems.

Stimulation or inhibition: Leaf microbial decomposition in streams subjected to complex chemical contamination.

Leaf litter decomposition is a key mechanism in headwater streams, allowing the transfer of nutrients and energy into the entire food web. However, chemical contamination resulting from human activity may exert a high pressure on the process, possibly threatening the structure of heterotrophic microbial communities and their decomposition abilities. In this study, the rates of microbial Alnus glutinosa (Alnus) leaf decay were assessed in six French watersheds displaying different land use (agricultural, urbanized, forested) and over four seasons (spring, summer, autumn, winter). In addition, for each watershed at each sampling time, both upstream (less-contaminated) and downstream (more-contaminated) sections were monitored. Toxicities (estimated as toxic units) predicted separately for pesticides and pharmaceuticals as well as environmental parameters (including nutrient levels) were related to microbial decay rates corrected for temperature and a range of fungal and bacterial community endpoints, including biomass, structure, and activity (extracellular ligninolytic and cellulolytic enzymatic activities). Results showed that agricultural and urbanized watersheds were more contaminated for nutrients and xenobiotics (higher pesticides and pharmaceuticals predicted toxicity) than forested watersheds. However, Alnus decay rates were higher in agricultural and urbanized watersheds, suggesting compensatory effects of nutrients over xenobiotics. Conversely, fungal biomass in leaves was 2-fold and 1.4-fold smaller in urbanized and agricultural watersheds than in the forested watersheds, respectively, which was mostly related to pesticide toxicity. However, no clear pattern was observed for extracellular enzymatic activities except that ß-glucosidase activity positively correlated with Alnus decay rates. Together, these results highlight microbial communities being more efficient for leaf decomposition in polluted watersheds than in less contaminated ones, which is probably explained by changes in microbial community structure. Overall, our study showed that realistic chemical contamination in stream ecosystems may affect the biomass of Alnus-associated microbial communities but that these communities can adapt themselves to xenobiotics and maintain ecosystem functions.

Biodegradation and toxicity of a maize herbicide mixture: mesotrione, nicosulfuron and S-metolachlor.

The prediction of chemical mixture toxicity is a major concern regarding unintentional mixture of pesticides from agricultural lands treated with various such compounds. We focused our work on a mixture of three herbicides commonly applied on maize crops within a fortnight, namely mesotrione (ß-triketone), nicosulfuron (sulfonylurea) and S-metolachlor (chloroacetanilide). The metabolic pathways of mesotrione and nicosulfuron were qualitatively and quantitatively determined with a bacterial strain (Bacillus megaterium Mes11). This strain was isolated from an agricultural soil and able to biotransform both these herbicides. Although these pathways were unaffected in the case of binary or ternary herbicide mixtures, kinetics of nicosulfuron disappearance and also of mesotrione and nicosulfuron metabolite formation was strongly modulated. The toxicity of the parent compounds and metabolites was evaluated for individual compounds and mixtures with the standardized Microtox® test. Synergistic interactions were evidenced for all the parent compound mixtures. Synergistic, antagonistic or additive toxicity was obtained depending on the metabolite mixture. Overall, these results emphasize the need to take into account the active ingredient and metabolites all together for the determination of environmental fate and toxicity of pesticide mixtures.

Epiplasmins and epiplasm in paramecium: the building of a submembraneous cytoskeleton.

In ciliates, basal bodies and associated appendages are bound to a submembrane cytoskeleton. In Paramecium, this cytoskeleton takes the form of a thin dense layer, the epiplasm, segmented into regular territories, the units where basal bodies are inserted. Epiplasmins, the main component of the epiplasm, constitute a large family of 51 proteins distributed in 5 phylogenetic groups, each characterized by a specific molecular design. By GFP-tagging, we analyzed their differential localisation and role in epiplasm building and demonstrated that: 1) The epiplasmins display a low turnover, in agreement with the maintenance of an epiplasm layer throughout the cell cycle; 2) Regionalisation of proteins from different groups allows us to define rim, core, ring and basal body epiplasmins in the interphase cell; 3) Their dynamics allows definition of early and late epiplasmins, detected early versus late in the duplication process of the units. Epiplasmins from each group exhibit a specific combination of properties. Core and rim epiplasmins are required to build a unit; ring and basal body epiplasmins seem more dispensable, suggesting that they are not required for basal body docking. We propose a model of epiplasm unit assembly highlighting its implication in structural heredity in agreement with the evolutionary history of epiplasmins.