Overview and Challenges of Large-Scale Cultivation of Photosynthetic Microalgae and Cyanobacteria.

Microalgae and cyanobacteria are diverse groups of organisms with great potential to benefit societies across the world. These organisms are currently used in food, feed, pharmaceutical and cosmetic industries. In addition, a variety of novel compounds are being isolated. Commercial production of photosynthetic microalgae and cyanobacteria requires cultivation on a large scale with high throughput. However, scaling up production from lab-based systems to large-scale systems is a complex and potentially costly endeavor. In this review, we summarise all aspects of large-scale cultivation, including aims of cultivation, species selection, types of cultivation (ponds, photobioreactors, and biofilms), water and nutrient sources, temperature, light and mixing, monitoring, contamination, harvesting strategies, and potential environmental risks. Importantly, we also present practical recommendations and discuss challenges of profitable large-scale systems associated with economical design, effective operation and maintenance, automation, and shortage of experienced phycologists.

Deconvolution model to resolve cytometric microbial community patterns in flowing waters.

Flow cytometry is suitable to discriminate and quantify aquatic microbial cells within a spectrum of fluorescence and light scatter signals. Using fixed gating and operational settings, we developed a finite distribution mixture model, followed by the Voronoi tessellation, to resolve bivariate cytometric profiles into cohesive subgroups of events. 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 subgroups within each of the commonly retrieved populations of cells with Low and High content of Nucleic Acids (namely, LNA, and HNA cells). Moreover, the advanced analysis allowed assessing changes of community patterns perturbed by a wastewater feed. Our approach for cytometric data deconvolution confirmed that flow cytometry could represent a prime candidate technology for assessing microbial community patterns in flowing waters. © 2017 International Society for Advancement of Cytometry.

Chlamydial seasonal dynamics and isolation of 'Candidatus†Neptunochlamydia vexilliferae' from a Tyrrhenian coastal lake.

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 Arsenite Oxidation Potential of Native Microbial Communities from Arsenic-Rich Freshwaters.

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.

Magnetite particles triggering a faster and more robust syntrophic pathway of methanogenic propionate degradation.

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.

Anaerobic treatment of groundwater co-contaminated by toluene and copper in a single chamber bioelectrochemical system.

Addressing the simultaneous removal of multiple coexisting groundwater contaminants poses a significant challenge, primarily because of their different physicochemical properties. Indeed, different chemical compounds may necessitate establishing distinct, and sometimes conflicting, (bio)degradation and/or removal pathways. In this work, we investigated the concomitant anaerobic treatment of toluene and copper in a single-chamber bioelectrochemical cell with a potential difference of 1 V applied between the anode and the cathode. As a result, the electric current generated by the bioelectrocatalytic oxidation of toluene at the anode caused the abiotic reduction and precipitation of copper at the cathode, until the complete removal of both contaminants was achieved. Open circuit potential (OCP) experiments confirmed that the removal of copper and toluene was primarily associated with polarization. Analogously, abiotic experiments, at an applied potential of 1 V, confirmed that neither toluene was oxidized nor copper was reduced in the absence of microbial activity. At the end of each experiment, both electrodes were characterized by means of a comprehensive suite of chemical and microbiological analyses, evidencing a highly selected microbial community competent in the biodegradation of toluene in the anodic biofilm, and a uniform electrodeposition of spherical Cu2O nanoparticles over the cathode surface.

Carbonate precipitation and phosphate trapping by microbialite isolates from an alkaline insular lake (Bagno dell'Acqua, Pantelleria Island, Italy).

The Bagno dell'Acqua lake is characterized by CO2 emissions, alkaline waters (pH = 9) and Eh values which indicate strongly oxidizing conditions. A typical feature of the lake is the presence of actively growing microbialites rich in calcium carbonates and silica precipitates. Mineralogy, petrography and morphology analyses of the microbialites were coupled with the analysis of the microbial community, combining molecular and cultivation approaches. The DNA sequencing revealed distinct patterns of microbial diversity, showing pronounced differences between emerged and submerged microbialite, with the upper layer of emerged samples exhibiting the most distinctive composition, both in terms of prokaryotes and eukaryotes. In particular, the most representative phyla in the microbial community were Proteobacteria, Actinobacteriota, and Bacteroidota, while Cyanobacteria were present only with an average of 5%, with the highest concentration in the submerged intermediate layer (12%). The role of microorganisms in carbonate mineral formation was clearly demonstrated as most of the isolates were able to precipitate calcium carbonate and five of them were characterized at molecular level. Interestingly, when microbial isolates were cultivated only in filtered water, the precipitation of hazenite was observed (up to 85%), opening new prospective in P (phosphate) recovery from P depleted environments.

Structural properties and microbial diversity of the biofilm colonizing plastic substrates in Terra Nova Bay (Antarctica).

Microbial colonization on plastic polymers has been extensively explored, however the temporal dynamics of biofilm community in Antarctic environments are almost unknown. As a contribute to fill this knowledge gap, the structural characteristics and microbial diversity of the biofilm associated with polyvinyl chloride (PVC) and polyethylene (PE) panels submerged at 5 m of depth and collected after 3, 9 and 12 months were investigated in four coastal sites of the Ross Sea. Additional panels placed at 5 and 20 m were retrieved after 12 months. Chemical characterization was performed by FTIR-ATR and Raman (through Surface-Enhanced Raman Scattering, SERS) spectroscopy. Bacterial community composition was quantified at a single cell level by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) and Confocal Laser Scanning Microscopy (CLSM); microbial diversity was assessed by 16S rRNA gene sequencing. This multidisciplinary approach has provided new insights into microbial community dynamics during biofouling process, shedding light on the biofilm diversity and temporal succession on plastic substrates in the Ross Sea. Significant differences between free-living and microbial biofilm communities were found, with a more consolidated and structured community composition on PVC compared to PE. Spectral features ascribable to tyrosine, polysaccharides, nucleic acids and lipids characterized the PVC-associated biofilms. Pseudomonadota (among Gamma-proteobacteria) and Alpha-proteobacteria dominated the microbial biofilm community. Interestingly, in Road Bay, close to the Italian "Mario Zucchelli" research station, the biofilm growth - already observed during summer season, after 3 months of submersion - continued afterwards leading to a massive microbial abundance at the end of winter (after 12 months). After 3 months, higher percentages of Gamma-proteobacteria in Road Bay than in the not-impacted site were found. These observations lead us to hypothesize that in this site microbial fouling developed during the first 3 months could serve as a starter pioneering community stimulating the successive growth during winter.

Geochemical and microbiological profiles in hydrothermal extreme acidic environments (Pisciarelli Spring, Campi Flegrei, Italy).

Although terrestrial hydrothermal systems are considered among the most fascinating environments, how their unique and extreme conditions can affect microorganisms selection and the role in biogeochemical cycles has not yet been well elucidated. A combined geochemical and microbiological exploration in waters and sediments from 10 sampling points along a sharp temperature gradient (15-90°C) within an extremely acidic hydrothermal system (Pisciarelli Spring, Campi Flegrei area, southern Italy) displayed how hydrothermal fluids influence the microbial dynamics. This area was characterized by high levels of reduced gaseous species (e.g. H2S, H2, CH4, CO) and very low pH values (<2.3). Thermodynamic calculations revealed a high microbial catabolic potential in oxidation/reduction reactions of N-, S- and Fe-bearing species. Overall, an increase of the archaeal/bacterial abundance ratio was observed by decreasing temperature and pH values. In particular, Archaea and Bacteria were present in almost equal cell abundance (up to 1.1 × 109 and 9.3 × 108 cell/g, respectively) in the <70°C sampling points (average pH = 2.09); on the contrary, the highest temperature waters (85-90°C; average pH = 2.26) were characterized by a low abundance of archaeal cells. The high-throughput sequencing of the 16S rRNA genes indicated strong differences in archaeal and bacterial communities composition along the temperature gradient. However, the microbiome in this extreme environment was mainly constituted by chemoautotrophic microorganisms that were likely involved in N-, S- and Fe-bearing species transformations (e.g. Acidianus infernus, Ferroplasma acidarmanus, Acidithiobacillus,Sulfobacillus,Thaumarchaeota), in agreement with thermodynamic calculations.

Interplay between abiotic and microbial biofilm-mediated processes for travertine formation: Insights from a thermal spring (Piscine Carletti, Viterbo, Italy).

Active hydrothermal travertine systems are ideal environments to investigate how abiotic and biotic processes affect mineralization mechanisms and mineral fabric formation. In this study, a biogeochemical characterization of waters, dissolved gases, and microbial mats was performed together with a mineralogical investigation on travertine encrustations occurring at the outflow channel of a thermal spring. The comprehensive model, compiled by means of TOUGHREACT computational tool from measured parameters, revealed that mineral phases were differently influenced by either abiotic conditions or microbially driven processes. Microbial mats are shaped by light availability and temperature gradient of waters flowing along the channel. Mineralogical features were homogeneous throughout the system, with euhedral calcite crystals, related to inorganic precipitation induced by CO2 degassing, and calcite shrubs associated with organomineralization processes, thus indicating an indirect microbial participation to the mineral deposition (microbially influenced calcite). The microbial activity played a role in driving calcite redissolution processes, resulting in circular pits on calcite crystal surfaces possibly related to the metabolic activity of sulfur-oxidizing bacteria found at a high relative abundance within the biofilm community. Sulfur oxidation might also explain the occurrence of gypsum crystals embedded in microbial mats, since gypsum precipitation could be induced by a local increase in sulfate concentration mediated by S-oxidizing bacteria, regardless of the overall undersaturated environmental conditions. Moreover, the absence of gypsum dissolution suggested the capability of microbial biofilm in modulating the mobility of chemical species by providing a protective envelope on gypsum crystals.

Two different approaches of microbial community structure characterization in riverine epilithic biofilms under multiple stressors conditions: Developing molecular indicators.

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.

Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters.

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

High concentrations of dissolved biogenic methane associated with cyanobacterial blooms in East African lake surface water.

The contribution of oxic methane production to greenhouse gas emissions from lakes is globally relevant, yet uncertainties remain about the levels up to which methanogenesis can counterbalance methanotrophy by leading to CH4 oversaturation in productive surface waters. Here, we explored the biogeochemical and microbial community variation patterns in a meromictic soda lake, in the East African Rift Valley (Kenya), showing an extraordinarily high concentration of methane in oxic waters (up to 156 µmol L-1). Vertical profiles of dissolved gases and their isotopic signature indicated a biogenic origin of CH4. A bloom of Oxyphotobacteria co-occurred with abundant hydrogenotrophic and acetoclastic methanogens, mostly found within suspended aggregates promoting the interactions between Bacteria, Cyanobacteria, and Archaea. Moreover, aggregate sedimentation appeared critical in connecting the lake compartments through biomass and organic matter transfer. Our findings provide insights into understanding how hydrogeochemical features of a meromictic soda lake, the origin of carbon sources, and the microbial community profiles, could promote methane oversaturation and production up to exceptionally high rates.

Investigating the influences of quorum quenching and nutrient conditions on activated sludge flocs at a short-time scale.

Quorum sensing signals regulate various functions within activated sludge processes such as formation of microbial aggregates. Disturbance of this signaling system, known as quorum quenching (QQ), provides opportunities for eliminating some problems related to biological wastewater treatment (e.g., biofouling and excess sludge production). However, it is poorly understood how and to what extent QQ systems can affect the microbial aggregation processes and the following floc formation. In particular, an in-depth structural characterization at the scale of microbial aggregate while considering nutrient conditions in the reactor is still largely disregarded. Here, we evaluated the QQ effects at the short-term time scale (i.e., after 4 h for the exogenous period and 19 h for exogenous/endogenous period), by combining advanced techniques for microbial characterization (flow cytometry, CARD-FISH, and confocal laser scanning microscopy) and conventional physical-chemical assessments. The results indicated that by implementing QQ agents (immobilized Acylase I enzyme in porous alginate beads) the abundance of single cells and suspended microbial aggregates in the supernatant did not show significant changes during the exogenous period. Conversely, at the end of the exogenous/endogenous period a significant increase of single prokaryotic cells, small and large microbial aggregates favored the growth of grazers, including free-living nanoflagellates and ciliates. Flocs became looser and thinner than those in the control reactor, thus affecting the sludge settling behavior. Inability of microbial community in degradation of soluble protein during the endogenous period confirmed that the QQ agents are likely to inhibit the secretion of protease enzyme within microbial communities of activated sludge.

Biological As(III) oxidation in biofilters by using native groundwater microorganisms.

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.

Microbiomes in Soils Exposed to Naturally High Concentrations of CO2 (Bossoleto Mofette Tuscany, Italy).

Direct and indirect effects of extremely high geogenic CO2 levels, commonly occurring in volcanic and hydrothermal environments, on biogeochemical processes in soil are poorly understood. This study investigated a sinkhole in Italy where long-term emissions of thermometamorphic-derived CO2 are associated with accumulation of carbon in the topsoil and removal of inorganic carbon in low pH environments at the bottom of the sinkhole. The comparison between interstitial soil gasses and those collected in an adjacent bubbling pool and the analysis of the carbon isotopic composition of CO2 and CH4 clearly indicated the occurrence of CH4 oxidation and negligible methanogenesis in soils at the bottom of the sinkhole. Extremely high CO2 concentrations resulted in higher microbial abundance (up to 4 × 109 cell g-1 DW) and a lower microbial diversity by favoring bacteria already reported to be involved in acetogenesis in mofette soils (i.e., Firmicutes, Chloroflexi, and Acidobacteria). Laboratory incubations to test the acetogenic and methanogenic potential clearly showed that all the mofette soil supplied with hydrogen gas displayed a remarkable CO2 fixation potential, primarily due to the activity of acetogenic microorganisms. By contrast, negligible production of acetate occurred in control tests incubated with the same soils, under identical conditions, without the addition of hydrogen. In this study, we report how changes in diversity and functions of the soil microbial community - induced by high CO2 concentration - create peculiar biogeochemical profile. CO2 emission affects carbon cycling through: (i) inhibition of the decomposition of the organic carbon and (ii) promotion of CO2-fixation via the acetyl-CoA pathway. Sites naturally exposed to extremely high CO2 levels could potentially represent an untapped source of microorganisms with unique capabilities to catalytically convert CO2 into valuable organic chemicals and fuels.

Colonization of overlaying water by bacteria from dry river sediments.

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.

Recovery and quantification of bacterial cells associated with streambed sediments.

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 (3)H-Leucine incorporation rates, rising uncertainties on the possibility to apply potential metabolic assays after Nycodenz purification.

Improved quantification of Dehalococcoides species by fluorescence in situ hybridization and catalyzed reporter deposition.

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.

The biogeochemical vertical structure renders a meromictic volcanic lake a trap for geogenic CO2 (Lake Averno, Italy).

Volcanic lakes are characterized by physicochemical favorable conditions for the development of reservoirs of C-bearing greenhouse gases that can be dispersed to air during occasional rollover events. By combining a microbiological and geochemical approach, we showed that the chemistry of the CO2- and CH4-rich gas reservoir hosted within the meromictic Lake Averno (Campi Flegrei, southern Italy) are related to the microbial niche differentiation along the vertical water column. The simultaneous occurrence of diverse functional groups of microbes operating under different conditions suggests that these habitats harbor complex microbial consortia that impact on the production and consumption of greenhouse gases. In the epilimnion, the activity of aerobic methanotrophic bacteria and photosynthetic biota, together with CO2 dissolution at relatively high pH, enhanced CO2- and CH4 consumption, which also occurred in the hypolimnion. Moreover, results from computations carried out to evaluate the dependence of the lake stability on the CO2/CH4 ratios, suggested that the water density vertical gradient was mainly controlled by salinity and temperature, whereas the effect of dissolved gases was minor, excepting if extremely high increases of CH4 are admitted. Therefore, biological processes, controlling the composition of CO2 and CH4, contributed to stabilize the lake stratification of the lake. Overall, Lake Averno, and supposedly the numerous worldwide distributed volcanic lakes having similar features (namely bio-activity lakes), acts as a sink for the CO2 supplied from the hydrothermal/magmatic system, displaying a significant influence on the local carbon budget.