Stefano Amalfitano

Responses of Benthic Bacteria to Experimental Drying in Sediments from Mediterranean Temporary Rivers

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.

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.

Bacterial uptake of DOM released from P-limited phytoplankton

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 gamma-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.

Aquatic invasive species: challenges for the future

Humans have effectively transported thousands of species around the globe and, with accelerated trade; the rate of introductions has increased over time. Aquatic ecosystems seem at particular risk from invasive species because of threats to biodiversity and human needs for water resources. Here, we review some known aspects of aquatic invasive species (AIS) and explore several new questions. We describe impacts of AIS, factors limiting their dispersal, and the role that humans play in transporting AIS. We also review the characteristics of species that should be the greatest threat for future invasions, including those that pave the way for invasions by other species (“invasional meltdown”). Susceptible aquatic communities, such as reservoirs, may serve as stepping stones for invasions of new landscapes. Some microbes disperse long distance, infect new hosts and grow in the external aquatic medium, a process that has consequences for human health. We also discuss the interaction between species invasions and other human impacts (climate change, landscape conversion), as well as the possible connection of invasions with regime shifts in lakes. Since many invaders become permanent features of the environment, we discuss how humans live with invasive species, and conclude with questions for future research.

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.

A microbial perspective on biological invasions in aquatic ecosystems

Microorganisms are essential components of all aquatic ecosystems and are primarily responsible for biogeochemical cycles and key environmental processes. Despite their potential to influence the ecological functioning of biological communities and ecosystems at a global scale, the mechanisms of microbial invasions still lack specific and generalizable theories. Here, we review some of the ecological mechanisms, among those relevant under the global change scenario, which may facilitate the spread of microbial invaders, with a specific focus on aquatic prokaryotes (i.e. Bacteria). We summarize a selection of ecological features of vulnerable microbial communities (functional and structural stability, diversity, invasibility, and invasiveness) and environmental stressors for invasions (temperature increase, pollution, nutrients and resources fluctuations, and food web alterations). Owing to the rapid microbial adaptation to laboratory conditions, straightforward experimental approaches appear as suitable and informative tools to explore the invasion mechanisms in artificially assembled communities. We conclude by delineating future research steps required for a better understanding of the potential consequences of microbial invasions in the aquatic environment.

CARD–FISH and confocal laser scanner microscopy to assess successional changes of the bacterial community in freshwater biofilms

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.

Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification

Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.

Flow cytometric analysis of benthic prokaryotes attached to sediment particles.

Appropriate detachment treatments are required to analyze prokaryotes associated with streambed sediments by flow cytometry. Using our previously optimized protocol, two groups of cells exhibiting different nucleic acid contents were easily detectable. However, the Nucleic Acid Double Staining assay proved that detachment procedures negatively affect the cell membrane integrity.

Grazing-induced Synechococcus microcolony formation: experimental insights from two freshwater phylotypes

Freshwater cyanobacteria of the genus Synechococcus are ubiquitous and organized either as single cells of diverse morphology or as microcolonies of different size. We studied the formation of microcolonies induced by the mixotrophic nanoflagellate Poterioochromonas sp. grazing on two Synechococcus strains belonging to phylotypes with different content of phycobiliproteins (PE: phycoerythrin-rich cells, L.Albano Group A; PC: phycocyanin-rich cells, MW101C3 Group I). The quantitative variations in cell abundance, morphological and physiological conditions were assessed on short-term incubations in semi-continuous cultures, single culture (PE, PC) and co-culture (PE+PC), with and without predators, by flow cytometry, and PhytoPAM. Under grazing pressure, we observed that (i) the abundance of PE single cells decreased over time with a concomitant formation of PE microcolonies; (ii) in PC single cultures, no significant variation in single cells was found and microcolonies did not form; (iii) both PE and PC formed monoclonal microcolonies in co-culture; (iv) PC cells increased the photosynthetic efficiency of the PSII (higher Fv/Fm) in co-culture. In the aftermath of microcolony formation as a predation-induced adaptation, our findings indicated a different response of Synechococcus phylotypes potentially co-existing in natural environment and the importance of their interaction.