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Microbial communities are major players in the biogeochemical processes and ecosystem functioning of river networks. Despite their importance in the ecosystem, biomonitoring tools relying on prokaryotes are still lacking. Only a few studies have employed both metabarcoding and quantitative techniques such as catalysed reported deposition fluorescence in situ hybridization (CARD-FISH) to analyse prokaryotic communities of epilithic biofilms in river ecosystems. We intended to investigate the efficacy of both techniques in detecting changes in microbial community structure associated with environmental drivers. We report a significant correlation between the prokaryotic community composition and pH in rivers from two different geographical areas in Norway. Both CARD-FISH and metabarcoding data were following the pattern of the environmental variables, but the main feature distinguishing the community composition was the regional difference itself. Beta-dispersion analyses on both CARD-FISH abundance and metabarcoding data revealed higher accuracy of metabarcoding to differentiate regions and river systems. The CARD-FISH results showed high variability, even for samples within the same river, probably due to some unmeasured microscale ecological variability which we could not resolve. We also present a statistical method, which uses variation coefficient and overall prevalence of taxonomic groups, to detect possible biological indicators among prokaryotes using metabarcoding data. The development of new prokaryotic bioindicators would benefit from both techniques used in this study, but metabarcoding seems to be faster and more reliable than CARD-FISH for large scale bio-assessment.

International audience; Bioindication has become an indispensable part of water quality monitoring in most countries of the world, with the presence and abundance of bioindicator taxa, mostly multicellular eukaryotes, used for biotic indices. In contrast, microbes (bacteria, archaea and protists) are seldom used as bioindicators in routine assessments, although they have been recognized for their importance in environmental processes. Recently, the use of molecular methods has revealed unexpected diversity within known functional groups and novel metabolic pathways that are particularly important in energy and nutrient cycling. In various habitats, microbial communities respond to eutrophication, metals, and natural or anthropogenic organic pollutants through changes in diversity and function. In this review, we evaluated the common trends in these changes, documenting that they have value as bioindicators and can be used not only for monitoring but also for improving our understanding of the major processes in lotic and lentic environments. Current knowledge provides a solid foundation for exploiting microbial taxa, community structures and diversity, as well as functional genes, in novel monitoring programs. These microbial community measures can also be combined into biotic indices, improving the resolution of individual bioindicators. Here, we assess particular molecular approaches complemented by advanced bioinformatic analysis, as these are the most promising with respect to detailed bioindication value. We conclude that microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems.

Microorganisms play an important role in speciation and mobility of arsenic in the environment, by mediating redox transformations of both inorganic and organic species. Since arsenite [As(III)] is more toxic than arsenate [As(V)] to the biota, the microbial driven processes of As(V) reduction and As(III) oxidation may play a prominent role in mediating the environmental impact of arsenic contamination. However, little is known about the ecology and dynamics of As(III)-oxidizing populations within native microbial communities exposed to natural high levels of As. In this study, two techniques for single cell quantification (i.e., flow cytometry, CARD-FISH) were used to analyze the structure of aquatic microbial communities across a gradient of arsenic (As) contamination in different freshwater environments (i.e., groundwaters, surface and thermal waters). Moreover, we followed the structural evolution of these communities and their capacity to oxidize arsenite, when experimentally exposed to high As(III) concentrations in experimental microcosms. Betaproteobacteria and Deltaproteobacteria were the main groups retrieved in groundwaters and surface waters, while Beta and Gammaproteobacteria dominated the bacteria community in thermal waters. At the end of microcosm incubations, the communities were able to oxidize up to 95 % of arsenite, with an increase of Alphaproteobacteria in most of the experimental conditions. Finally, heterotrophic As(III)-oxidizing strains (one Alphaproteobacteria and two Gammaproteobacteria) were isolated from As rich waters. Our findings underlined that native microbial communities from different arsenic-contaminated freshwaters can efficiently perform arsenite oxidation, thus contributing to reduce the overall As toxicity to the aquatic biota.

The influence of the freshwater runoff on marine prokaryotes has been largelly studied in the pelagic environment, little is known about the effects on the prokaryotes in coastal sediments. We investigated the distribution of benthic prokaryotes in the northern Adriatic Sea, when an extreme Po River flood event determined high heterogeneity along the estuarine salinity gradient (from mainly freshwater to marine). The microbial diversity was assessed by 16S rRNA gene Illumina sequencing, the quantification of the dominant taxa of Bacteria and Archaea by microscopy techniques (CARD-FISH). Our results showed dominance of Bacteria (eight-fold higher) over Archaea, and highlighted Proteobacteria as the most represented phylum, followed by Bacteroidetes. Prokaryotic abundance, quantified by DAPI staining, was mainly affected by sediment characteristics (Total Organic C and Total N contents, and silt fraction). Abundance reached the highest values in sites directly affected by the plume (range 7.3 × 108 - 2.8 × 109 cell g−1). A considerable difference in prokaryotes composition was observed along the salinity gradient, with an increasing presence of freshwater taxa at stations more influenced by the river discharge. CARD-FISH analyses corroborated these findings, showing higher abundance of beta-Proteobacteria and Planctomycetes at stations closest to the river mouths. Nevertheless, a core microbiome (about 10% of the total OTUs) was present across all the study areas, indicating the ability of this assemblage to survive in rather diverse environmental conditions, differently impacted by the river plume. Our results show that the river plume can affect the diversity and distribution patterns of benthic prokaryotes far into the pro-delta, although further investigations are needed in order to understand how the mixing of bacterial communities of different origin might reflect on ecosystem functioning.

Glyphosate-based herbicides are widely used for several crops, such as transgenic soybean and forestry. The aim of this study was to determine the effect of glyphosate on the community structure of riverine bacterioplankton and to evaluate the potential of bacterioplankton to degrade the herbicide. River water to which 13C-labelled glyphosate (10, 100µgL–1) was added or not (control) was incubated for 6 days at the temperature measured in situ (20°C). Significant differences in bacterioplankton community composition, as assessed by microfluidics-based automated ribosomal intergenic spacer analysis, were found among treatments, with differences in the presence of 100µgL–1 of glyphosate being more pronounced, namely significant decreases in bacterial richness and diversity. The glyphosate degradation product aminomethylphosphonic acid (AMPA) was detected, accounting for 1.2% of glyphosate conversion in water with 100µgL–1 of 13C-labelled glyphosate, together with a significant enrichment of 13C in the bacterial biomass. These findings suggest that glyphosate had a direct detrimental effect on most bacterioplankton taxa, but enriched those that were able to degrade the herbicide. Together, the results indicate that glyphosate degradation in the river assessed would be a slow process (months–years), taking place through the AMPA degradation pathway and meaning glyphosate accumulate in the ecosystem.

An integrated geochemical and microbiological investigation of soils from the Solfatara Crater (Campi Flegrei, southern Italy) demonstrated that interstitial soil gases dominated by CO2 and other typical hydrothermal gaseous species (e.g. H2S, CH4, ethane, benzene, alkenes and S-bearing organic compounds) influenced the composition of microbial communities. The relatively high concentrations of hydrothermal fluids permeating the soil produced acidic conditions and whitish deposits that characterize the Solfatara Crater floor. Archaea and Bacteria showed almost equal cell abundance (up to 3.2 x 10(7) and 4.2 x 10(7) cell/g, respectively) with relatively low levels of biodiversity and equitability in sites characterized by elevated temperatures (up to 70 degrees C), very low pH values (up to 2.2) and reducing conditions. In these sites, high-throughput sequencing showed the marked selection of microorganisms, mainly affiliated with the genera Thermoplasma, Ferroplasma and Acidithiobacillus. A relatively high biodiversity and concomitant distinctive structure of the microbial community were observed in soils poorly affected by fumarolic emissions that were oxic and rich in organic matter.

Arsenic (As) contamination in drinking water represents a worldwide threat to human health. During last decades, the exploitation of microbial As-transformations has been proposed for bioremediation applications. Among biological methods for As-contaminated water treatment, microbial As(III)-oxidation is one of the most promising approaches since it can be coupled to commonly used adsorption removal technologies, without requiring the addition of chemicals and producing toxic by-products. Despite the As(III) oxidation capability has been described in several bacterial pure or enrichment cultures, very little is known about the real potentialities of this process when mixed microbial communities, naturally occurring in As contaminated waters, are used. This study highlighted the contribution of native groundwater bacteria to As(III)-oxidation in biofilters, under conditions suitable for a household-scale treatment system. This work elucidated the influence of a variety of experimental conditions (i.e., various filling materials, flow rates, As(III) inflow concentration, As(III):As(V) ratio, filter volumes) on the microbially-mediated As(III)-oxidation process in terms of oxidation efficiency and rate. The highest oxidation efficiencies (up to 90% in 3 h) were found on coarse sand biofilters treating total initial As concentration of 100 μg L−1. The detailed microbial characterization of the As(III) oxidizing biofilms revealed the occurrence of several OTUs affiliated with families known to oxidize As(III) (e.g., Burkholderiaceae, Comamonadaceae, Rhodobacteraceae, Xanthomonadaceae). Furthermore, As-related functional genes increased in biofilter systems in line with the observed oxidative performances.

Microbial electrochemical systems (MES) represent an innovative reagent-free technology for in situ remediation of groundwater contaminated by petroleum hydrocarbons. Here we describe the long-term (>160 days) anaerobic treatment of synthetic groundwater containing toluene (25 mg L−1) in a novel laboratory-scale, continuous-flow bioelectrochemical reactor, termed the “bioelectric well”. Under optimal operating conditions (i.e., anode potential potentiostatically controlled at +0.2 V vs. SHE and recycle flow-rate set at 75 mL min−1), the observed electrogenic toluene oxidation rate was 67.2 ± 5.7 mg L d−1, a value which is among the highest reported in the literature for laboratory-scale anaerobic treatment systems. Correspondingly, electric current was 5.1 ± 0.1 mA and the Coulombic efficiency (i.e., the yield of toluene conversion into electric current) was 79 ± 7%. Electrogenic toluene oxidation was most likely catalyzed by Geobacter species, which were found to dominate the surface of the graphite anode. The combined application of GC-MS (for detection and identification of metabolites) and qPCR (for quantification of functional genes) revealed that toluene degradation was initiated by fumarate addition, an activation pathway that is widely distributed among anaerobic hydrocarbon-degrading microorganisms. Collectively, the results of this study indicate that the bioelectric well holds remarkable potential for in situ treatment of groundwater contaminated by petroleum hydrocarbons.

The Chlamydiae are a phylum of obligate intracellular bacteria comprising important human and animal pathogens, yet their occurrence in the environment, their phylogenetic diversity and their host range has been largely underestimated. We investigated the seasonality of environmental chlamydiae in a Tyrrhenian coastal lake. By catalysed reporter deposition fluorescence in situ hybridization, we quantified the small planktonic cells and detected a peak in the abundance of environmental chlamydiae in early autumn with up to 5.9 × 10(4) cells ml(-1) . Super-resolution microscopy improved the visualization and quantification of these bacteria and enabled the detection of pleomorphic chlamydial cells in their protist host directly in an environmental sample. To isolate environmental chlamydiae together with their host, we applied a high-throughput limited dilution approach and successfully recovered a Vexillifera sp., strain harbouring chlamydiae (93% 16S rRNA sequence identity to Simkania negevensis), tentatively named 'Candidatus Neptunochlamydia vexilliferae'. Transmission electron microscopy in combination with fluorescence in situ hybridization was used to prove the intracellular location of these bacteria representing the first strain of marine chlamydiae stably maintained alongside with their host in a laboratory culture. Taken together, this study contributes to a better understanding of the distribution and diversity of environmental chlamydiae in previously neglected marine environments.

The presence of arsenic (As) in aquatic environments is often attributable to geogenic processes occurring within aquifers. Arsenic exists in two prevalent oxidation states, with the trivalent arsenite [As(III)] exerting stronger toxicity effects on the aquatic biota than the pentavalent arsenate [As(V)]. The review evaluates the literature available on the main arsenic removal technologies and the application of combined chemical and biological treatments. We provide a synthetic outlook on the potential strategies of biological As(III) oxidation to As(V) by means of cell-detoxifying mechanisms or metabolic processes with the aim to enhance As removal efficiency. Furthermore, the role of microorganisms in the mobility of arsenic in natural systems as well as the distribution of As-resistant bacteria, potentially suitable for arsenic removal, is discussed in the context of a case study carried out in Latium region (Italy), which is known for arsenic contamination of waters.

Arsenic (As) is a toxic element released in aquatic environments by geogenic processes or anthropic activities. To counteract its toxicity, several microorganisms have developed mechanisms to tolerate and utilize it for respiratory metabolism. However, still little is known about identity and physiological properties of microorganisms exposed to natural high levels of As and the role they play in As transformation and mobilization processes. This work aims to explore the phylogenetic composition and functional properties of aquatic microbial communities in As-rich freshwater environments of geothermal origin and to elucidate the key microbial functional groups that directly or indirectly may influence As-transformations across a natural range of geogenic arsenic contamination. Distinct bacterial communities in terms of composition and metabolisms were found. Members of Proteobacteria, affiliated to Alpha- and Betaproteobacteria were mainly retrieved in groundwaters and surface waters, whereas Gammaproteobacteria were the main component in thermal waters. Most of the OTUs from thermal waters were only distantly related to 16S rRNA gene sequences of known taxa, indicating the occurrence of bacterial biodiversity so far unexplored. Nitrate and sulphate reduction and heterotrophic As(III)-oxidization were found as main metabolic traits of the microbial cultivable fraction in such environments. No growth of autotrophic As(III)-oxidizers, autotrophic and heterotrophic As(V)-reducers, Fe-reducers and oxidizers, Mn-reducers and sulfide oxidizers was observed. The ars genes, involved in As(V) detoxifying reduction, were found in all samples whereas aioA (As(III) oxidase) and arrA genes (As(V) respiratory reductase) were not found. Overall, we found that As detoxification processes prevailed over As metabolic processes, concomitantly with the intriguing occurrence of novel thermophiles able to tolerate high levels of As.

Antibiotics are emerging contaminants, which wing to their bioactivity, may lead to short-term and long-term alterations of natural microbial communities in aquatic environment. We investigated the effects of antibiotics on biofilm bacterial communities in the Llobregat River (Northeast Spain). Three sampling sites were selected: two less polluted sites and one hotspot. River water was collected from each site and used both as inoculum and medium for growing biofilms in independent mesocosms. After 25 d of biofilm colonization, we exposed the colonized biofilms to river waters from the downstream sites (progressively contaminated by antibiotics). A control from each site was maintained where the growing biofilm was always exposed to water from the same site. The bacterial community composition, bacterial live/dead ratio and extracellular enzyme activities of the biofilms were measured before and 9 d after exposing the biofilms to increasing contaminated waters. Sixteen antibiotic compounds were detected in the water from the three sampling sites. At each site, the antibiotics present in the highest concentrations were sulfonamides, followed by quinolones and macrolides. Bacterial communities of biofilms grown with the three river waters differed markedly in their structure, but less so in terms of functional descriptors. After switching the medium water to increasing pollution, biofilms exhibited increased levels of actinobacteria (HGC), a trend that was associated to the higher antibiotic concentrations in the water. These biofilms also showed increased bacterial mortality, and decreased extracellular leucine-aminopeptidase and alkaline phosphatase. There was a significant correlation between antibiotic concentrations and biofilm responses. Our results indicate that the continuous entrance of antibiotics in running waters cause significant structural and functional changes in microbial attached communities.

Flow cytometry is suitable to discriminate and quantify aquatic microbial cells within a spectrum of fluorescence and light scatter signals. Using fixed operational and gating settings, a mixture model, coupled to Laplacian operator and Nelder-Mead optimization algorithm, allowed deconvolving bivariate cytometric profiles into single cell subgroups. This procedure was applied to outline recurrent patterns and quantitative changes of the aquatic microbial community along a river hydrologic continuum. We found five major persistent subgroups within each of the commonly retrieved populations of cells with Low and High content of Nucleic Acids (namely, LNA and HNA cells). Moreover, we assessed changes of the cytometric community profile over-imposed by water inputs from a wastewater treatment plant. Our approach for multiparametric data deconvolution confirmed that flow cytometry could represent a prime candidate technology for assessing microbial community patterns in flowing waters.

Among the bacterivorous protists, heterotrophic nanoflagellates (HNFs) are considered to be the main grazers of bacteria in freshwaters due to their size-selective grazing. In this work, we assessed the change of a riverine bacterial community in controlled incubations, where HNFs' predation pressure was initially released through filtration. Filtration did not prevent the passage of cysts, which grew in the enrichments afterwards. Data on the composition of the bacterial community were gathered by Catalyzed Reporter Deposition Fluorescent In situ Hybridization (CARD-FISH) using 16S probes targeting phylogenetic groups. Bacterial cell size was also examined using image analysis. Overall, the initial filtration directly (through release of predation pressure) or indirectly (through competition among bacterial groups) affected the bacterial community composition. When nanoflagellate abundance rose, a reduction of bacterial abundance and changes in cell size distribution were observed. Gamma-Proteobacteria and Actinobacteria were the groups showing the greatest reduction in abundance. Beta-Proteobacteria showed a reduction of cell size and were found in aggregates. Alpha-Proteobacteria and Actinobacteria developed two distinct filamentous morphotypes: short, segmented rods and long chains of rods. Our results showed that the release of the predation pressure and the successive rise of the nanoflagellates changed the bacterial community in terms of composition at large phylogenetic scale. HNF grazing is highly group-specific and seems to reconstruct the community based on cell size, and thus, not only drastically changing the bacterial community composition, but also increasing its functional diversity.

The Po River runoff strongly affects the oceanographic and ecological characteristics of the Northern Adriatic Sea. Catalysed reported deposition - fluorescence in situ hybridization (CARD-FISH) analysis was employed to assess how the composition of the coastal bacterioplankton community is influenced by the river runoff in two different seasons (spring and autumn). Samples were collected from the water column along a coastal–offshore transect in the Northern Adriatic Sea at different depths. Four clone libraries were then constructed from coastal (0 m) and offshore waters (−65 m). Higher abundances of bacteria were recorded in coastal waters as compared with the offshore samples. This result was mainly due to the trophic state of the water column, and it was related to salinity. Particularly, Actinobacteria and Gammaproteobacteria were affected by the riverine inputs, whereas Bacteroidetes and Alphaproteobacteria showed only minor responses. This was particularly clear in the autumn sample, in which a clear difference between the coastal and the offshore samples was found due to a strong influence of the less saline river water with high nutrient concentrations. Analysis of 205 partial length 16S rRNA gene sequences indicated a high diversity with the dominance of Alphaproteobacteria, Bacteroidetes and Gammaproteobacteria mainly affiliated to coastal and marine bacterioplankton clades. Actinobacteria were also detected and, together with Gammaproteobacteria, strongly follow the freshwater intrusion. Overall, our results indicate that the seasonal difference in the Po River discharge greatly affects the bacterioplankton community. In spring we observed a smooth transition from coastal to open-sea conditions owing to the more superficial freshwater plume. In autumn the deeper freshwater intrusion in the coastal site and thermal stratification offshore resulted in a marked difference between the coastal and offshore microbial communities.

River and stream biofilms in mediterranean fluvial ecosystems face both extreme seasonality as well as arrhythmic fluctuations. The hydrological extremes (droughts and floods) impose direct changes in water availability but also in the quantity and quality of organic matter and nutrients that sustain the microbial growth. This review analyzes how these ecological pulses might determine unique properties of biofilms developing in mediterranean streams. The paper brings together data from heterotrophic and autotrophic community structure, and extracellular enzyme activities in biofilms in mediterranean streams. Mediterranean stream biofilms show higher use of peptides during the favorable period for epilithic algae development (spring), and preferential use of cellulose and hemicellulose in autumn as a response to allochthonous input. The drying process causes the reduction in bacterial production and chlorophyll biomass, but the rapid recovery of both autotrophs and heterotrophs with rewetting indicates their adaptability to fluctuations. Bacteria surviving the drought are mainly associated with sediment and leaf litter which serve as "humid refuges". Some algae and cyanobacteria show resistant strategies to cope with the drought stress. The resistance to these fluctuations is strongly linked to the streambed characteristics (e.g., sediment grain size, organic matter accumulation, nutrient content) This study was funded by the projects CGL2011-30151-C02-01, and SCARCE (Consolider-Ingenio 2010 CSD2009-00065) of the Spanish Ministry of Economy and Competitiveness; the Italy-Spain Exchange Project CNR-CSIC 2006IT0010; and the EU project MIRAGE (FP7-ENV-2007-1 n.211732)

Summary For a long time the bacterial phylum of Chlamydiae exclusively consisted of one family of obligate intracellular bacteria, the Chlamydiaceae, which encompassed causative agents of severe diseases. In the 1990s, environmental chlamydiae were discovered as symbionts of free-living amoebae and other eukaryotic hosts. During a sampling campaign in September 2008, while monitoring Planctomycetes, we retrieved 20 almost full-length 16S rRNA gene sequences affiliated with Chlamydiales from a lake at the Tyrrhenian coast of central Italy (Lago di Paola, Latium). Two main clusters were identified. The nine sequences within the tight cluster I shared ∼98% identity, just like the six sequences of cluster II. The 16S rRNA sequence identity between the two novel groups was with 88% higher than with all known families of the order Chlamydiales. Four types of less frequent chlamydial 16S rRNA sequences were also detected. Two oligonucleotide probes were designed, and optimized. Chl282 targets the cluster I and almost all other Chlamydiales, while Chl282bis targets the cluster II and few other sequences. By catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH), we identified in the Lago di Paola picoplankton abundant tiny cells with dot-shaped morphology and, interestingly, rarely also protists with intracellular pleomorphic chlamydiae. Abundances of the novel chlamydial clusters were up to 5 × 104 cells per millilitre. The two clusters were also detected in similar numbers during a second sampling in October 2010. This confirmed the relevance of the two newly described clusters of chlamydiae in Lago di Paola, not only enlarging the knowledge on the biodiversity of environmental chlamydiae in aquatic habitats, but also raising sanitary issues that should be addressed in the future.

Efficient detachment and purification of bacterial cells associated with streambed sediments are required in order to quantify cell abundance and to assess community composition through the application of epifluorescence microscopy techniques. We applied chemical (i.e., sodium pyrophosphate and polysorbate) and physical treatments (i.e., shaking and sonication), followed by Nycodenz density gradient centrifugation to efficiently recover benthic bacteria. This procedure resulted in a highly purified cell suspension allowing for a precise cell quantification through the application of fluorescent dyes. About 93% of total cells were recovered from the original sediment, with higher recovery from the finer grain-size class (90%) in comparison to the coarse fraction (69%). The potential damaging effects of the applied procedures on cell integrity were assessed on planktonic bacteria in a pre-filtered water control. As a consequence of the high purity of the extracted bacteria, flow cytometry was successfully applied as counting method for sediment cell suspension. However, a significant decrease of protein synthesis in purified samples was measured by estimating the 3H-Leucine incorporation rates, rising uncertainties on the possibility to apply potential metabolic assays after Nycodenz purification.

We studied the diversity, community composition and activity of the primary microbial colonizers of the water above freshly re-wetted sediments from a temporary river. Dried sediments, collected from Mulargia River (Sardinia, Italy), were covered with sterile freshwater in triplicate microcosms, and changes of the planktonic microbial assemblage were monitored over a 48 h period. During the first 9 h bacterial abundance was low (1.5 x 10(4) cells ml(-1)); it increased to 3.4 x 10(6) cells ml(-1) after 28 h and did not change thereafter. Approximately 20% of bacteria exhibited DNA de novo synthesis already after 9 h of incubation. Changes of the ratios of (3)H-leucine to (3)H-thymidine incorporation rates indicated a shift of growth patterns during the experiment. Extracellular enzyme activity showed a maximum at 48 h with aminopeptidase activity (430.8 +/- 22.6 nmol MCA l(-1) h(-1)) significantly higher than alkaline phosphatase (98.6 +/- 4.3 nmol MUF l(-1) h(-1)). The primary microbial colonizers of the overlaying water - as determined by 16S rRNA gene sequence analysis - were related to at least six different phylogenetic lineages of Bacilli and to Alphaproteobacteria (Brevundimonas spp. and Caulobacter spp.). Large bacterial cells affiliated to one clade of Bacillus sp. were rare in the dried sediments, but constituted the majority of the planktonic microbial assemblage and of cells with detectable DNA-synthesis until 28 h after re-wetting. Their community contribution decreased in parallel with a rise of flagellated and ciliated protists. Estimates based on cell production rates suggested that the rapidly enriched Bacillus sp. suffered disproportionally high loss rates from selective predation, thus favouring the establishment of a more heterogenic assemblage of microbes (consisting of Proteobacteria, Actinobacteria and Cytophaga-Flavobacteria). Our results suggest that the primary microbial colonizers of the water above dried sediments are passively released into the plankton and that their high growth potential is counteracted by the activity of bacterivorous protists.

We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased from dry to wet sediments, showing a positive correlation with the community activity measured via incorporation of 3H leucine. FISH and signal amplification by catalyzed reporter deposition (CARD-FISH) could significantly better detect cells with low activity in dried sediments. Through the application of an optimized cell permeabilization protocol, the percentage of hybridized cells by CARD-FISH showed comparable values in dry and wet conditions. This approach was unrelated to 3H leucine incorporation rates. Moreover, the optimized protocol allowed a significantly better visualization of Gram-positive Actinobacteria in the studied samples. CARD-FISH is, therefore, proposed as an effective technique to compare bacterial communities residing in sediments with contrasting water content, irrespective of differences in the activity state of target cells. Considering the increasing frequencies of flood and drought cycles in European temporary rivers, our approach may help to better understand the dynamics of microbial communities in such systems.

In the semiarid Mediterranean regions, water scarcity represents a common physiological stress for microbial communities residing in river sediments. However, the effect of drying has not yet adequately been evaluated when analyzing riverine microbiological processes. The bacterial community structure (abundance, biomass, composition) and functioning (carbon production, live cell percentage) were assessed during experimental desiccation in microcosms with sediments from different Mediterranean temporary rivers (Tagliamento, Krathis, Mulargia, Pardiela). Our results showed that the overall responses to drying of the bacterial community were independent from sediment origin and strictly related to water content. During desiccation, a prompt decline (up to 100%) of the initial bacterial carbon production was followed by a slower decrease in abundance and biomass, with an overall reduction of 74% and 78%, respectively. By the end of the experiment, live cells were still abundant but depressed in their main metabolic functions, thus resulting in a drastic increase in the community turnover time. Only 14% of the initial live cell biomass was available in dry sediments to immediately start the reactivation of the aquatic microbial food web after the arrival of new water. Community composition analysis showed a relative increase in alpha- and beta-Proteobacteria, when passing from wet to dry conditions. Our results suggest that the occurrence of drought events could affect carbon cycling through the freshwater microbial compartment, by temporarily limiting microbial mineralization and altering bacterial community structure.

Interspecies electron transfer mechanisms between Bacteria and Archaea play a pivotal role during methanogenic degradation of organic matter in natural and engineered anaerobic ecosystems. Growing evidence suggests that in syntrophic communities electron transfer does not rely exclusively on the exchange of diffusible molecules and energy carriers such as hydrogen or formate, rather microorganisms have the capability to exchange metabolic electrons in a more direct manner. Here, we show that supplementation of micrometer-size magnetite (Fe3O4) particles to a methanogenic sludge enhanced (up to 33%) the methane production rate from propionate, a key intermediate in the anaerobic digestion of organic matter and a model substrate to study energy-limited syntrophic communities. The stimulatory effect most probably resulted from the establishment of a direct interspecies electron transfer (DIET), based on magnetite particles serving as electron conduits between propionate-oxidizing acetogens and carbon dioxide-reducing methanogens. Theoretical calculations revealed that DIET allows electrons to be transferred among syntrophic partners at rates which are substantially higher than those attainable via interspecies H2 transfer. Besides the remarkable potential for improving anaerobic digestion, which is a proven biological strategy for renewable energy production, the herein described conduction-based DIET could also have a role in natural methane emissions from magnetite-rich soils and sediments.

The effect of macroinvertebrate detritivore density on the mass loss rates of leaf litter of Alnus glutinosa (alder) was assessed. Experimental freshwater macrocosms, with increasing densities of four species of macroinvertebrate detritivores belonging to two functional groups (shredders and scrapers), were set up outdoors. The litter bag technique was used to assess decomposition rates of alder leaves. Indirect effects of increasing density of macroinvertebrates on phytoplankton standing crop in the water column were investigated by analysing Chlorophyll a concentration. Decomposition rate increased as animal density increased, although a continuous increase in detritivores density resulted in a discrete, step-wise increase of the decomposition rates. Animal colonisation followed an exponential pattern in low-medium density treatments versus a typical `bell-shape' curve in high density treatments; animals started to leave the consumed patches when about 60% of the initial leaf mass was lost (35th day in high-density treatments). Diversity (Hs) of the simplified detritivore community decreased as decomposition proceeded, with a dominance of shredders during the last phase of decomposition. Faster decomposition rate of detritus in the benthic compartment lead to a higher microalgae standing crop in the water column emphasising the role of allochthonous detritus as a source of nutrients for algae primary production in coastal freshwater ecotones.

10 páginas, 6 figuras, 1 tabla., In Mediterranean intermittent streams, the hydrological fragmentation in summer and the successive water flow reconvergence in autumn allow exploring how local processes shape the microbial community within the same habitat. The objectives of this study were to determine how bacterial community composition responded to hydrological fragmentation in summer, and to evaluate whether the seasonal shifts in community composition predominate over the effects of episodic habitat fragmentation. The bacterial community was assessed along the intermittent stream Fuirosos (Spain), at different levels of phylogenetic resolution by in situ hybridization, fingerprinting, and 16S rRNA gene sequencing. The hydrological fragmentation of the stream network strongly altered the biogeochemical conditions with the depletion of oxidized solutes and caused changes in dissolved organic carbon characteristics. In the isolated ponds, beta-Proteobacteria and Actinobacteria increased their abundance with a gradual reduction of the alpha-diversity as pond isolation time increased. Moreover, fingerprinting analysis clearly showed a shift in community composition between summer and autumn. In the context of a seasonal shift, the temporary stream fragmentation simultaneously reduced the microbial dispersion and affected local environmental conditions (shift in redox regime and quality of the dissolved organic matter) tightly shaping the bacterioplankton community composition., Participation of AB and EV was supported by Project CGL2011-30151-C02-02/BTE, funded by Ministerio de Economı`a y Competitividad. AB, EOC and EV are members of the Graccie Consortium.

24 páginas, 2 figuras, 1 tabla., The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EUWater Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel ecogenomic indices for routine application in biodiversity assessments of European fresh- and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments.

Bacterial community composition was assessed during riverine biofilm development by the Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) in combination with Confocal Laser Scanning Microscopy. Using artificial substrates, it was possible to follow the dynamics of specific bacterial clusters, while maintaining the unaltered structure and architecture of the biofilm.

International audience; The phylum Planctomycetes is involved in important processes, such as the mineralization of algal biomass and the removal of nitrogen. Using a combination of 16S rRNA sequence analysis and in situ hybridization, we analyzed the diversity and dynamics of Planctomycetes in a shallow meso-eutrophic lake, Lago di Paola, Italy. Planctomycetes detected by the probe PLA46 accounted for 1 to 5% of prokaryotic picoplankton. Abundances were higher in the coastal lake than in the adjacent marine waters. In the surface waters of the lake, the numbers of Planctomycetes fluctuated greatly, reaching a maximum of 1.5 x 10(6) cells ml(-1) in July. The hypoxic bottom waters had less variable cell abundances. The Planctomycetes counts were positively correlated with chlorophyll a concentrations, confirming the role of this phylum in the degradation of algal biomass. We obtained 70 almost full-length 16S rRNA gene sequences of Planctomycetes from 2 libraries. Four distinct clades could be identified. The Pirellula-related group F and the uncultured Planctomycetes group B both had the highest identity with sequences retrieved from marine habitats, whereas the Pirellula-related group E was affiliated with sequences known from freshwater and brackish water environments. The Planctomyces-related group A seems to have a wide habitat range. Catalyzed reported deposition fluorescence in situ hybridization with newly developed probes revealed abundances of the 4 clades in surface and bottom waters ranging from 1 x 10(3) to 5 x 10(4) cells ml(-1). Together, the 4 clade-specific probes identified only about a quarter of the Planctomycetes detected by probe PLA46. This indicates that the diversity of Planctomycetes has not yet been fully explored.

1. Knowledge about processes and dynamics underlying organic matter accumulation in transitional waters is crucial for the protection of these ecologically important coastal habitats. This study investigated the relationship between large particle accumulation and decomposition in a coastal lake included in a Site of Community Interest (SCI) using sediment trap and litterbag techniques. 2. Two sets of sediment traps were deployed at five sites along the longitudinal axis of the lake. One set was emptied once a month for 12 months, and the other twice a year. The contents of the monthly and the 6-monthly traps and the superficial sediments were then compared to estimate the organic matter accumulation and loss. To determine the mass loss rate of the three major allochthonous sources of detritus (the reed Phragmites australis, the cordgrass Spartina juncea, and the seagrass Posidonia oceanica), litterbags were placed near the sediment traps at three out of the five sites at the beginning of each 6-month period and retrieved monthly. 3. The amount of annual particulate organic matter (POM) deposition in the traps was 1320.2±58.5 g m−2 y−1 and consisted of 25% large particles (CPOM). Allochthonous litter comprised an important fraction of CPOM, and its breakdown rate changed with the plant species, site and season. Loss rate of CPOM accumulated at the lake bottom was significantly related with the mass loss rate of reed and cordgrass litter, but not with that of seagrass. 4. Due to slow litter breakdown and tidal landward advection, the lake acted as a sediment trap for allochthonous matter, especially from Posidonia. The important role of marine-derived litter in organic matter sedimentation suggests addressing conservation strategies of the lake functioning towards the selective control of allochthonous CPOM inputs, in particular at the mouth where the incoming tide brings suspended material and salinity decelerates matter flow to the sea and decomposition. This study supports the hypothesis that sedimentation and decomposition dynamics are important factors for coastal lake evolution, and shows litterbag and sediment trap techniques as simple useful investigation tools in management strategies aiming at conserving transitional waters. Copyright © 2008 John Wiley & Sons, Ltd.

Chlorinated ethenes contamination of soil and groundwater is a widespread problem in most industrialized countries. To date, there is a general consensus in the literature that members of the genus Dehalococcoides are required for complete dechlorination of these compounds. The availability of specific identification tools to track their distribution in the field is therefore a topic of particular relevance in environmental studies. These microorganisms have been successfully visualized by fluorescence in situ hybridization (FISH) in highly active dechlorinating cultures. However, FISH detection of Dehalococcoides under low activity conditions can be strongly hampered by their small cell size and low ribosome content. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative detection method. In a trichloroethene (TCE) dechlorinating enrichment culture, CARD-FISH, using proteinase K as a permeabilization pre-treatment, was found to be significantly superior to conventional FISH in terms of both microscopic visualization and quantification efficiency (about 30%). An application of this method on contaminated aquifer samples is also presented and discussed.

Bacterial degradation of mucilaginous aggregates (creamy layers, stringers and macroflocs) collected during two summer events (2001-2002) was tested. The objective was to describe the temporal trend of the bacterial activity, abundance and composition in the aggregated and dissolved organic matter under different trophic conditions. In the native aggregates proteins and organic phosphorous were actively hydrolyzed as aminopeptidase and alkaline phosphatase activities represented up to 87% and 25% of total activity, respectively; polysaccharides were less hydrolyzed and the highest activities were observed for beta-glucosidase (5% of the total). This hydrolysation pattern tends to a progressive accumulation of long persistent polysaccharides. During short term incubations nutrient addition (P, N and Glucose) differently stimulated bacterial growth in the seawater: P played the main role in stimulating bacterial production from 3 to 6 folds higher than in the control, whereas a secondary C-limitation was observed only for bacteria growing on seawater from macroflocs. This scarce dissolved organic carbon (DOC) bioavailability was confirmed by the lower DOC removal (13% macroflocs, 36% stringers). The total amount of carbon incorporated by bacteria living on aggregates was similar (0.58 mg C L(-1)) both in the control and under P enrichments showing a more balanced condition with respect to the seawater. Hence the well-known P limitation in the Northern Adriatic Sea affects only dissolved organic carbon uptake without influencing the uptake of aggregated organic matter. Organic matter limitation was observed only on stringers--total C incorporated raised to 0.96 mg C L(-1) after PNG addition. Macroflocs release of refractory compounds leads to DOC accumulation (73 microM DOC) contributing to inflate the pool of refractory DOC in the surrounding waters. Several evidences, including different monosaccharide composition of stringers and macroflocs (glucose 15% and 56% on stringers and macroflocs, respectively), bring to the conclusion that stringers are in an older stage in comparison with macroflocs. Community composition described by fluorescence in situ hybridization did not show significant differences between free-living and attached bacteria but it was modified by the different enrichment conditions: Cytophaga-Flavobacteria increased after inorganic nutrients enrichments while organics advantaged gamma-Proteobacteria.

Summary Bacterial communities associated with a variety of benthic detritus types were studied in three streams in the context of the chemical characteristics of the sediment material and the stream water. A cell purification assay was developed for a quantitative microscopic evaluation of bacterial community structure in detritus samples by fluorescence in situ hybridization (FISH). The efficiency of FISH with fluorescently monolabelled probes was compared with FISH with signal amplification by catalysed reporter deposition (CARD-FISH). In detritus types poor in organic carbon and nitrogen, the numbers of prokaryotes were related to the chemical characteristics of the stream water column, whereas no such relationship was found for detritus types rich in organic carbon and nitrogen. These results might help to provide criteria for the selection of detritus types for river ecosystem assessment and monitoring. The percentage of bacteria detected by FISH with monolabelled probes was correlated with the detritus total organic matter (OM). This is likely attributed to a higher ribosome content of microbial cells on substrates rich in OM. Cell detection by CARD-FISH did not show any correlation with OM content, indicating that this technique renders the results more independent from the activity state of cells. Fluorescence in situ hybridization with four group-specific probes suggested a relationship between substrate quality and the composition of the microbial assemblages on the various types of detritus. The improved protocol for cell purification and CARD-FISH may facilitate future investigations on the relationship between the riverine benthic detritus quality and microbial community composition.

The growth and the structure of a coastal bacterioplankton community were monitored in short-term bottle experiments in order to investigate the bacterial uptake of extracellular organic carbon released by the diatom Cylindrotheca closterium grown under P-balanced and P-depleted conditions. Bacterial specific growth rates and carbon demand were significantly lower in the exudates from P-depleted algae (24% and 30% reduction, respectively). The origin of the extracellular carbon appeared also to affect the taxonomic composition of the bacterioplankton assemblage, mainly reducing the development of ?-Proteobacteria. This pattern of bacterial carbon uptake could contribute to a longer persistence of the exudates released in P-depleted conditions affecting the dynamics of the carbon cycle in marine environments. © 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.