Flow cytometric analysis of bacteria- and virus-like particles in lake sediments

Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (×1 / 20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or − 22 °C. For FCM analysis, the samples were diluted in Tris–EDTA buffer (pH 8) and heated for 10 min at 75 °C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology

Phosphorus Limitation, Uptake, and Turnover in Benthic Stream Algae

In this chapter, three different aspects of P utilization by benthic algae will be covered: (1) assessment of P limitation, (2) measurement of P uptake rates, and (3) determination of the release rate of P (expressed as the turnover rate). We note two caveats regarding this chapter. First, we focus exclusively on inorganic P; it is likely that dissolved organic phosphorus plays an important, albeit relatively undefined role, in the nutrient dynamics of freshwater algae (cf. Hwang et al., 1998; Pant et al., 2002), especially in P-limited environments (Karl and Björkman, 2015). However, treatment of this topic is beyond the scope of this chapter (but see Chapter 24). Second, although we use the term benthic algae throughout the chapter, it should be noted that the benthic algae attached to submerged substrata in streams usually exist as part of a complex assemblage variously referred to as periphyton, aufwuchs, or biofilm. This assemblage usually consists of algae, bacteria, fungi, and meiofauna (see Chapters 9e11, 14) that exist within a mucilaginous, poly- saccharide matrix (Lock et al., 1984), and each biotic group has different affinities for P. Indeed, even within a group, P uptake and cycling may be influenced by the abundance of different species and growth forms (cf. Steinman et al., 1992; Davies and Bothwell, 2012)

Variable phosphorus uptake rates and allocation across microbial groups in the oligotrophic Gulf of Mexico

Microbial uptake of dissolved phosphorus (P) is an important lever in controlling both microbial production and the fate and cycling of marine P. We investigated the relative role of heterotrophic bacteria and phytoplankton in P cycling by measuring the P uptake rates of individual microbial groups (heterotrophic bacteria and the phytoplankton groups Synechococcus, Prochlorococcus, and picoeukaryotic phytoplankton) in the P-depleted Gulf of Mexico. Phosphorus uptake rates were measured using incubations with radiolabeled phosphate and adenosine triphosphate coupled with cell sorting flow cytometry. We found that heterotrophic bacteria were the dominant consumers of P on both a biomass basis and a population basis. Biovolume normalized heterotrophic bacteria P uptake rate per cell (amol P μm-3 hr-1) was roughly an order of magnitude greater than phytoplankton uptake rates, and heterotrophic bacteria were responsible for generally greater than 50% of total picoplankton P uptake. We hypothesized that this variation in uptake rates reflects variation in cellular P allocation strategies, and found that, indeed, the fraction of cellular P uptake utilized for phospholipid production was significantly higher in heterotrophic bacteria compared to cyanobacterial phytoplankton. These findings indicate that heterotrophic bacteria have a uniquely P-oriented physiology and play a dominant role in cycling dissolved P

Effects of water level fluctuation and sediment–water nutrient exchange on phosphorus biogeochemistry in two coastal wetlands

Freshwater coastal wetlands are sensitive to drying and wetting events (DWE), associated with water level fluctuations. Prior studies have shown that DWE influence sediment–water nutrient exchange, but the fate of these nutrients has received much less attention. To address this information gap, we investigated how microbial (i.e., bacteria and phytoplankton) structure and function respond to DWE-induced release of nutrients from the sediment of mesotrophic and eutrophic coastal wetlands. Our approach was three-fold: (1) sediment from each wetland was subjected to a desiccation (2 month) and re-wetting (2 days) period in the laboratory; (2) the overlying water column from the DWE was measured for nutrient concentration; and (3) we conducted a microbial response experiment using the water from either DWE experiment (containing the nutrients released during the DWE) or with added nutrients obtained from commercially available sources (but at similar concentrations to those released from the sediments), to test the effect of nutrient concentration and source on microbes inhabiting the water column and their role in the fate of the released nutrients, in particular phosphate (Pi). In both coastal wetlands, the microbial community structure in the water column (absolute and relative abundance of different microbial groups) was modified by bacteria resuspended from the sediment after re-wetting. However, results from the microbial response experiments showed that the microbial communities did not grow following inputs of commercially available nutrients or release of sediment nutrients. In Pi amended treatments, Pi uptake rates increased but not enough to reduce Pi turnover time to values measured in the control. As a result, Pi concentration and turnover remained high. In coastal wetlands with limited hydrologic exchange, these elevated nutrients will accumulate and lead to further eutrophication. However, in open coastal wetlands, with substantial hydrologic exchange, advected nutrients may influence biological activity in the nearshore zone

Alkaline phosphatase activity and regulation in the North Pacific Subtropical Gyre

Alkaline phosphatase (AP) activity (APA) was measured at several stations in the North Pacific Subtropical Gyre in July 2008, and in a series of nutrient addition experiments: nitrate plus ammonium (+N) or phosphate (+P), to study APA regulation and to evaluate the capacity of picoplankton organisms (i.e., in the 0.2–2‐µm size range) to access the AP‐hydrolyzable fraction of dissolved organic phosphorus (DOP). The data indicated a primary limitation of the biomass by nitrogen. Both total (measured with a soluble DOP analog) and cell‐specific (measured with the enzyme‐labeled fluorescence [ELF] phosphate cell labeling method) APA were enhanced in the +N samples and reduced in the +P samples, suggesting that DOP is an important resource for picoplankton nutrition. Cell‐free APA represented > 65% of the APA in all samples, but its contribution to total APA significantly decreased in the +N treatment as microbial biomass increased. In the +N treatment, < 5% and up to 96% of the cells in the heterotrophic bacteria‐enriched and picophytoplankton‐enriched fractions, espectively, were ELF‐alcohol‐labeled after 5 d. Following N enrichment, the microbial assemblage shifted from cell‐free phosphatase dominated under N limitation and P stress (i.e., physiological response) to picophytoplankton‐based phosphatase dominated under P limitation (i.e., production or growth rate limitation). If, as predicted, the ocean evolves towards P limitation, DOP availability would become of major importance to sustain productivity

Copepods promote bacterial community changes in surrounding seawater through farming and nutrient enrichment

Bacteria living in the oligotrophic open ocean have various ways to survive under the pressure of nutrient limitation. Copepods, an abundant portion of the mesozooplankton, release nutrients through excretion and sloppy feeding that can support growth of surrounding bacteria. We conducted incubation experiments in the North Atlantic Subtropical Gyre to investigate the response of bacterial communities in the presence of copepods. Bacterial community composition and abundance measurements indicate that copepods have the potential to influence the microbial communities surrounding and associating with them – their ‘zoosphere’, in two ways. First, copepods may attract and support the growth of copiotrophic bacteria including representatives of Vibrionaceae, Oceanospirillales and Rhodobacteraceae in waters surrounding them. Second, copepods appear to grow specific groups of bacteria in or on the copepod body, particularly Flavobacteriaceae and Pseudoalteromonadaceae, effectively ‘farming’ them and subsequently releasing them. These distinct mechanisms provide a new view into how copepods may shape microbial communities in the open ocean. Microbial processes in the copepod zoosphere may influence estimates of oceanic bacterial biomass and in part control bacterial community composition and distribution in seawater

Detection of Extracellular Phosphatase Activity of Heterotrophic Prokaryotes at the Single‐Cell Level by Flow Cytometry

Monitoring cell activity using substrates, which turn fluorescent due to biological activity, allows observing the presence and dynamics of sub‐populations, and provides a very valuable insight in ecological studies. The phosphatase substrate ELF97 phosphate (ELF‐P) is a useful tool to detect and quantify phosphatase activity (PA) of microorganisms at the single‐cell level. Most of the studies dealing with PA at the single‐cell level focus on autotrophic cells and only few concern heterotrophic prokaryotes (referred as bacteria in the text). While flow cytometry is a promising tool to assess the single‐cell analysis, only microscopy tools have been used until now to measure the ELF labeling associated with bacteria expressing PA. Therefore, we have developed a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF‐P hydrolysis, making possible the specific identification of bacteria showing PA using flow cytometry

Analysis of Phosphatase Activity from Aquatic Heterotrophic Bacteria at the Single Cell Level by Flow Cytometry: Example of a Development Achieved in the Regional Flow Cytometry Platform for Microbiology (Precym) Hosted by the Oceanology Center of Marseille

Marine microbes play a very important role in biogeochemical cycles. Because of their ­biodiversity, the diversity of their metabolisms, and their ­physiological heterogeneity (live/dead, active/inactive cells), it is necessary to study them at the single cell level. Flow cytometry has become a technique of choice to reach this goal, providing a fast, reliable, and multiparametric analysis of individual cells. However, the cost of such a technique makes flow cytometry out of reach for small institutes or laboratory units. It also requires a qualified and well-trained operator. To make this technique available to all the microbiologists of the “Provence Alpes Côte d’Azur” Region, a Regional Flow Cytometry Platform (PRECYM), hosted by the Oceanology Centre of Marseille, was created by a consortium of six laboratories. Its goal is to provide researchers with the possibility to consider new approaches and to guarantee optimal technical, economic, and scientific exploitations of these instruments. An example of such a technical development is described in this chapter. The ELF97 phosphatase substrate has been shown to be a performing tool to assess the phosphorus status of phytoplankton populations. Recently, this technique has been successfully applied to marine heterotrophic bacteria in culture samples in which ELF-labeling was observed by microscopy. In this study, we describe a new protocol for ­ELF-labeling of heterotrophic bacteria that allow detection by flow cytometry. ELF-labeled samples were stored in liquid nitrogen for up to 4 months before analysis without any significant loss of total or ELF-labeled cell abundance and of relative ELF ­fluorescence intensity. This method enables studying ectoenzymatic alkaline phosphatase activity of heterotrophic bacteria at the single cell level in environments even with low cell abundance. It has been successfully applied in both fresh and marine samples. This approach is set to improve our understanding of the physiological response of heterotrophic bacteria to phosphorus limitation

Small pigmented eukaryotes play a major role in carbon cycling in the P‐depleted western subtropical North Atlantic, which may be supported by mixotrophy

We found that in the phosphate (PO4)‐depleted western subtropical North Atlantic Ocean, small‐sized pigmented eukaryotes (P‐Euk; < 5 μm) play a central role in the carbon (C) cycling. Although P‐Euk were only ~ 5% of the microbial phytoplankton cell abundance, they represented at least two thirds of the microbial phytoplankton C biomass and fixed more CO2 than picocyanobacteria, accounting for roughly half of the volumetric CO2 fixation by the microbial phytoplankton, or a third of the total primary production. Cell‐specific PO4 assimilation rates of P‐Euk and nonpigmented eukaryotes (NP‐Euk; < 5 μm) were generally higher than of picocyanobacteria. However, when normalized to biovolumes, picocyanobacteria assimilated roughly four times more PO4 than small eukaryotes, indicating different strategies to cope with PO4 limitation. Our results underline an imbalance in the CO2 : PO4 uptake rate ratios, which may be explained by phagotrophic predation providing mixotrophic protists with their largest source of PO4. 18S rDNA amplicon sequence analyses suggested that P‐Euk was dominated by members of green algae and dinoflagellates, the latter group commonly mixotrophic, whereas marine alveolates were the dominant NP‐Euk. Bacterivory by P‐Euk (0.9 ± 0.3 bacteria P‐Euk−1 h−1) was comparable to values previously measured in the central North Atlantic, indicating that small mixotrophic eukaryotes likely exhibit similar predatory pressure on bacteria. Interestingly, bacterivory rates were reduced when PO4 was added during experimental incubations, indicating that feeding rate by P‐Euk is regulated by PO4 availability. This may be in response to the higher cost associated with assimilating PO4 by phagocytosis compared to osmotrophy

Dissolved organic matter uptake by <i>Trichodesmium </i>in the Southwest Pacific

International audienceThe globally distributed diazotroph Trichodesmium contributes importantly to nitrogen inputs in the oligotrophic oceans. Sites of dissolved organic matter (DOM) accumulation could promote the mixotrophic nutrition of Trichodesmium when inorganic nutrients are scarce. Nano-scale secondary ion mass spectrometry (nanoSIMS) analyses of individual trichomes sampled in the South Pacific Ocean, showed significant 13 C-enrichments after incubation with either 13 C-labeled carbohydrates or amino acids. These results suggest that DOM could be directly taken up by Trichodesmium or primarily consumed by heterotrophic epibiont bacteria that ultimately transfer reduced DOM compounds to their host trichomes. Although the addition of carbohydrates or amino acids did not significantly affect bulk N 2 fixation rates, N 2 fixation was enhanced by amino acids in individual colonies of Trichodesmium. We discuss the ecological advantages of DOM use by Trichodesmium as an alternative to autotrophic nutrition in oligotrophic open ocean waters. Nitrogen is recognized as the proximate limiting nutrient for primary production in the oceans 1. The oceanic nitrogen reservoir is controlled by a balance between fixed nitrogen gains (via dinitrogen-N 2-fixation) and losses (denitrification) 2. While global nitrogen budget estimations determine that denitrification exceeds N 2 fixation considerably 3 , recent improvements in the 15 N 2 isotope tracer method used to measure biological N 2 fixation have evidenced that formerly published rates could be underestimated by a factor of ~2 to 6 4–8 , and thus could be high enough to balance denitrification on a global basis. However, the variability among N 2 fixation rates obtained when using the two different methods (adding 15 N 2 as a bubble or pre-dissolved in seawater) 4,9 can be high 7 and at times not significant 10–12. A mechanistic understanding of which factors determine the degree of discrepancy between the two 15 N 2 methods is currently lacking. Moreover, marine pelagic N 2 fixation had been long attributed to the tropical and subtropical latitudinal bands of the ocean, e.g. 13 , while other ecological niches such as high latitude waters, oxygen minimum zones and the vast dark realm of the ocean (below the euphotic zone) are now recognized as active N 2 fixation sites 14–16. It is likely that the inclusion of these previously unaccounted for active N 2 fixation sites will be more important in equilibrating denitrification and N 2 fixation rates than the underestimation of rates due to discrepancies between isotopic tracer methods. In chronically stratified open ocean regions such as the vast subtropical gyres, primary production depends largely on external fixed nitrogen inputs provided by N 2 fixation performed by prokaryotes termed 'diazotrophs'. Diazotrophic cyanobacteria are photosynthetic prokaryotes (with the exception of the photoheterotrophic Candidatus Atelocyanobacterium thalassa which cannot photosynthesize) 17 that thrive in oligotrophic tropical and subtropical waters of the oceans where they provide an important source of fixed nitrogen for other phyto-plankton 13. Despite being classically regarded as photoautotrophs, some unicellular diazotrophic cyanobacteria like Cyanothece are able to take up dissolved organic matter (DOM) molecules photoheterotrophically 18. As well, various filamentous diazotrophic cyanobacteria such as Anabaena bear genes for amino acids transport, which may be used to incorporate amino acids from the in situ DOM pool, or to assimilate amino acids self-produced during diazotrophic growth 19