(2000). Picocyanobacteria single forms, aggregates and microcolonies: survival strategy or species succession? SIL Proceedings, 1922-2010: Vol. 27, No. 4, pp. 1879-1883.
Picocyanobacteria (Pcy) single-cells and microcolonies are common in lakes throughout the world, and abundant across a wide spectrum of trophic conditions. The single-celled Pcy populations tend to be predominant in large, deep oligo-mesotrophic lakes, while the microcolonies find optimal conditions in warmer, shallower and more nutrient rich lakes. Microcolonies of different size (from 5 to 50 cells) constitute a gradient without a net separation from single-celled types. Considering microcolonies as transitional forms from single-cells to colonial morphotypes it is conceivable to propose a common ecology where local communities are not isolated but linked by dispersal of multiple, potentially interactive, species. In this review abiotic forcing and biotic regulation of Pcy community structure and dynamics are examined to offer an updated view of Pcy ecology.
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.
The interest for microorganisms inhabiting the hypolimnion and for their role in biogeochemical cycles of lakes is considerable, but knowledge is far from complete. The presence of chemolithoautotrophic Bacteria and mesophilic Archaea (e.g., Thaumarchaeota) assimilating inorganic carbon in the deep hypolimnion of lakes has been ascertained. We measured, for the first time at 350 m in Lake Maggiore (Northern Italy), the prokaryotic in situ dark [14C]HCO3 incorporation with a new custom-made apparatus, which takes samples and adds tracers in situ. Thereby stress factors affecting prokaryotes during sample recovery from the depth were avoided. We tested the new instrument at different depths and conditions, performing parallel conventional on board incubations. We found that dark [14C]HCO3 incorporations had lower standard deviation in in situ incubations with respect to the on board ones, but their means were not statistically different. At 350 m we estimated an uptake of 187.7±15 μg C m–3 d–1, which is in line with the published uptake rates in aquatic systems. By inhibiting the bacterial metabolism, we found that Archaea were responsible for 28% of the total CO2 uptake. At the same depth, Thaumarchaeota, on average, constituted 11% of total DAPI counts. Dark [14C]HCO3 incorporation integrated along the aphotic water column was 65.8±5.2 mg C m–2 d–1 which corresponds to 87% of picophytoplanktonic autotrophic fixation in the euphotic layer. This study provides the first evidence of Bacteria and Archaea dark CO2 fixation in the deep hypolimnion of a subalpine lake and indicates a potentially significant prokaryotic CO2 sink.
The study of the hydrodynamics of the hypolimnion of a deep holo-oligomictic lake (Lake Maggiore, Northern Italy, z max = 370 m) during the last 28 years showed that hypolimnetic waters remained isolated, not exchanging with the mixing zone even in winter when the full overturn conditions are most likely. The thickness of the isolated layer can range from 100 to 300 m. Thus, water masses of variable size reside in the lake for many years, and their physical and chemical conditions remain relatively unaffected by seasonal variability and epilimnetic imputs. In the hypolimnetic waters prokaryote abundance is three times lower than in the mixing layer but cell size is significantly higher. In addition, the relative abundance of Archaea and Crenarchaeota increases with depth in respect to that of Bacteria. The heterogeneous distribution of the two domains within the habitat can be attributed to the existence in the same environment of isolated water masses.
The application of flow cytometry is well-established for the characterization of aquatic microbial communities in natural systems 1-3. These microbial assemblages are of fundamental importance for the so called “Earth's Critical Zone” (the planet surface including rivers, lakes, and oceans) as they are the main drivers of all biochemical cycles in waters and are responsible for more than half of the global production of oxygen. In waters limited by nutrients (e.g., glaciers, pristine freshwaters, and open oceans), they control energy fluxes and the transport of organic compounds from the surface to the deeper layers, allowing life even in extreme conditions. Finally, they represent the largest part of the biodiversity of marine and freshwaters, having >99% of the aquatic species comprised between bacteria and microscopic organisms 4. The manuscript by Amalfitano et al. (this issue, page 194; DOI: cyto.a.23304) presents an attractive approach to dredge the cytometric fingerprinting of planktonic microorganisms. Though there are studies providing algorithms to differentiate more cytometric groups 5, the novel deconvolution model proposed by Amalfitano et al. (this issue) allows sorting out the recurrent cell subgroups within a complex microbial community, without a priori knowledge of the event nature. Deconvolution models have been mainly proposed for clinical applications 6 and only sporadically in environmental microbiology 7. The computational workflow by Amalfitano et al. presents novel aspects and can be applied to analyze the dynamics of microbial community structures in space (e.g., along a river) and in time (e.g., experimental time-series), with the potential to fill a gap in modern microbial ecology. Major novelties in cytogram deconvolution consist in: taking into account (i) local maxima (i.e., the different density peaks) as well as local minima, (ii) a data-driven selection of the number and localization of recurrent peaks (i.e., the BIC approach), and (iii) the Voronoi tessellation for a data-based gate design. Therefore, this model is not fundamentally affected by skewness and distribution of plotted events subgroups, which could represent an issue when comparing cytometric fingerprinting between single cytograms from different samples 8. Extraordinarily different morphotypes are concomitantly present in waters, and their identification and quantification is of striking importance for a correct understanding of microbial dynamics 1. In particular, there is a call for rapid detection of phenotypically diverse aquatic microbes, comprising small protists, hetero and autotrophic prokaryotes, and viruses, all showing a vast range of different sizes and morphologies (Fig. 1). The ecological interactions among microorganisms (e.g., predation, competition for resources, mutualism, and parasitism) and their metabolic performances may modulate their growth at very short space and time scales. Aquatic microbes can also form colonies, aggregates and flocs by reacting very fast to local inputs and stressors 9-11. Consequently, the cytometric fingerprinting of microbial communities from complex natural settings (e.g., marine and freshwaters, underground waters, wastewaters, marine aerosols, irrigational channels, and ponds) can be far more puzzled than those comprising well-dispersed single cells. In general, the problem is overcome by a strong simplification of the system, considering each detected particle as a single event, independently by its size and actual composition. This can lead to a dramatic underestimation of the true microbial cell number in complex environmental settings (e.g., waters rich in particulate matter and microbial aggregates), bringing to misleading understanding of the system composition 12, 13. In other cases, complex gating supported by epifluorescence microscopy was implemented to recognize specific morphologies like microcolonies or single cells 7, and the number of clustered cells (at least for size classes of aggregates) 13-15. For example, a recent study of simplified aquatic communities composed by three species forming complex clusters as response to ecological factors (predation, cooperation) was conducted by defining, through very precise gating, a number of different size classes for each detected event. By applying epifluorescence and confocal microscopy a defined number of cells was assigned to each gate, and thus to the events comprised in it 15. This approach works very well on simplified communities, where the number of potential morphologies is controlled, and where it can support a precise quantification of the final cell number. Still, also in these cases, its application is laborious and it is limited to the study and the samples for which gates are designed. This is because the correct identification of the gates, as well as the correct enumeration of microbial cells in aggregates is strictly dependent from the conditions of each sample, and these are generally very different in different environments, or in different time of the year. In detail, a correct evaluation of peaks overlapping, and the selection of the correspondent morphologies, cannot be generalized. The application of this approach thus requires a constant validation and recalculation, reducing reproducibility and drastically incrementing the time needed for each analysis. The presented bioinformatics tool (this issue, page 194) is likely suitable for automation and could considerably increase the potential of flow cytometry for microbiological studies in environment, as the time of analysis will be shortened and cohesive microbial populations will be detected through an operator-independent procedure. For this reason, when broadly applied by microbial ecologists, the deconvolution model proposed by Amalfitano and coworkers (this issue) could represent a fundamental step toward a correct flow-cytometric assessment of microbial numbers and morphological distribution, promoting a significant advance in deeper understanding the microbial dynamics of aquatic systems. Phenotypic diversity in a drop of lake water. The left panel is an epifluorescence microphotography of the microbial community of lake Zurich (Switzerland) sampled in 2011 (the sample was DAPI stained and collected on a black Nucleopore filter, Millipore). It is possible to distinguish single bacterial cells of different shape (rods, cocci, filaments), microbial cell clusters (composed by one or more bacterial strains), and heterotrophic protists (bacterial predators). On the right panel, the cytogram of the same drop of water is presented. The different morphologies as depicted in the microphotography in epifluorescence microscopy are partially overlapping in the cytogram, with serious problems when it comes to the quantification of small and large cell clusters, and on the identification of protists. These problems are compromising an accurate quantification of the overall bacterial number in the drop, reducing the power of the cytometric gating and screening when simple events gating is applied.
Two approaches may be utilized to explain the predominance of picocyanobacteria (Pcy) in oligotrophic lakes: the analysis of their interannual evolution in one single lake and their relative importance in different lakes along a trophic gradient. Here we discuss results from field data on picocyanobacteria over several seasons from a deep oligotrophic subalpine lake - Lago Maggiore, and variables influencing their abundance. Comparing data from lakes along a trophic gradient, no simple relationship emerges between lake's trophic state and picocyanobacteria abundance and contribution to total phytoplanktonic biomass. That is, trophic state alone cannot explain the success/absence of picocyanobacteria that appear to be favored under P limitation, but seem more sensitive to grazing pressure and light. In some oligotrophic lakes, if light climate, grazing, and competition are favorable, picocyanobacteria can grow rapidly, out-compete competitors and become very abundant, but there are a host of factors that can influence the outcome of this competition, and ultimately influence Pcy success in lakes of all trophic types.
The horizontal heterogeneity of seston, dissolved (DOC) and particulate (POC) organic matter, Chlorophyll-a (Chl) and of autotrophic (APP) and heterotrophic (HPP) picoplankton was evaluated seasonally during a two-year period in Lago Maggiore, a large deep oligotrophic lake in Northern Italy. The dissolved fraction resulted homogeneously distributed in the epilimnion during the whole sampling period. The particulate fraction showed the higher heterogeneity in winter 2000 and 2001. For those years, POC and Chl heterogeneity was, respectively, 3-5 and 5-7 times higher than DOC. A similar trend was evident for Chl and for APP. The spatial heterogeneity of HPP resulted always smaller than the analytical variability, thus preventing any consideration about the spatial distribution of HPP. POC and Chl concentration exhibited a recurrent pattern of their spatial heterogeneity, possibly in relation with the location of the part of the drainage basin conveying higher amounts of nutrients to the lake.
Abstract The Black Sea is the largest meromictic sea with a reservoir of anoxic water extending from 100 to 1000 m depth. These deeper layers are characterised by a poorly understood fluorescence signal called “deep red fluorescence”, a chlorophyll a- (Chl a) like signal found in deep dark oceanic waters. In two cruises, we repeatedly found up to 103 cells ml−1 of picocyanobacteria at 750 m depth in these waters and isolated two phycoerythrin-rich Synechococcus sp. strains (BS55D and BS56D). Tests on BS56D revealed its high adaptability, involving the accumulation of Chl a in anoxic/dark conditions and its capacity to photosynthesise when re-exposed to light. Whole-genome sequencing of the two strains showed the presence of genes that confirms the putative ability of our strains to survive in harsh mesopelagic environments. This discovery provides new evidence to support early speculations associating the “deep red fluorescence” signal to viable picocyanobacteria populations in the deep oxygen-depleted oceans, suggesting a reconsideration of the ecological role of a viable stock of Synechococcus in dark deep waters.
