Christian Amblard

The Microbial Loop at the Surface of a Peatland:Structure, Function, and Impact of Nutrient Input

A bstractThe aim of this study was to assess the structure and function of the microbial loop in a peatland of the French Massif central, and the impact of fertilization on the different microbial communities.In terms of biomass, testate Amoeba (48% of the total microbial biomass), heterotrophic bacteria (15%), cyanobacteria (14%) and Bacillariophyceae (13%) were the dominant groups of microorganisms. Other microalgae (7%), ciliates (2%) and heterotrophic flagellates (1%) accounted for only a low proportion of total microbial biomass. The relative importance of heterotrophic microorganisms was higher than in marine or lacustrine environments. In addition, ciliates and heterotrophic flagellates only constituted a small proportion of the total protozoan biomass, which was heavily dominated by testate amoeba. Thus, the structure of the protist community in the subaquatic peatland was completely different from that reported for lakes or marine environments.In other aspects, the supply of nutrients (PKCa and NPKCa) resulted in increases of the relative biomasses of heterotrophic bacteria, Bacillariophyceae, and ciliates and by a decrease in the relative proportion of testate amoeba and of other microalgae.

The molecular diversity of freshwater picoeukaryotes reveals high occurrence of putative parasitoids in the plankton

Eukaryotic microorganisms have been undersampled in biodiversity studies in freshwater environments. We present an original 18S rDNA survey of freshwater picoeukaryotes sampled during spring/summer 2005, complementing an earlier study conducted in autumn 2004 in Lake Pavin (France). These studies were designed to detect the small unidentified heterotrophic flagellates (HF, 0.6–5 µm) which are considered the main bacterivores in aquatic systems. Alveolates, Fungi and Stramenopiles represented 65% of the total diversity and differed from the dominant groups known from microscopic studies. Fungi and Telonemia taxa were restricted to the oxic zone which displayed two fold more operational taxonomic units (OTUs) than the oxycline. Temporal forcing also appeared as a driving force in the diversification within targeted organisms. Several sequences were not similar to those in databases and were considered as new or unsampled taxa, some of which may be typical of freshwater environments. Two taxa known from marine systems, the genera Telonema and Amoebophrya, were retrieved for the first time in our freshwater study. The analysis of potential trophic strategies displayed among the targeted HF highlighted the dominance of parasites and saprotrophs, and provided indications that these organisms have probably been wrongfully regarded as bacterivores in previous studies. A theoretical exercise based on a new ‘parasite/saprotroph-dominated HF hypothesis’ demonstrates that the inclusion of parasites and saprotrophs may increase the functional role of the microbial loop as a link for carbon flows in pelagic ecosystems. New interesting perspectives in aquatic microbial ecology are thus opened.

Viral activity in two contrasting lake ecosystems

ABSTRACT For aquatic systems, especially freshwaters, there is little data on the long-term (i.e., >6-month period) and depth-related variability of viruses. In this study, we examined virus-induced mortality of heterotrophic bacteria over a 10-month period and throughout the water column in two lakes of the French Massif Central, the oligomesotrophic Lake Pavin and the eutrophic Lake Aydat. Concurrently, we estimated nonviral mortality through heterotrophic nanoflagellate and ciliate bacterivory. Overall, viral infection parameters were much less variable than bacterial production. We found that the frequency of visibly infected cells (FVIC), estimated using transmission electron microscopy, peaked in both lakes at the end of spring (May to June) and in early autumn (September to October). FVIC values were significantly higher in Lake Pavin (mean [M] = 1.6%) than in Lake Aydat (M = 1.1%), whereas the opposite trend was observed for burst sizes, which averaged 25.7 and 30.2 virus particles bacterium−1, respectively. We detected no significant depth-related differences in FVIC or burst size. We found that in both lakes the removal of bacterial production by flagellate grazing (MPavin = 37.7%, MAydat = 18.5%) was nearly always more than the production removed by viral lysis (MPavin = 16.2%, MAydat = 19%) or ciliate grazing (MPavin = 2.7%, MAydat = 8.8%). However, at specific times and locations, viral lysis prevailed over protistan grazing, for example, in the anoxic hypolimnion of Lake Aydat. In addition, viral mortality represented a relatively constant mortality source in a bacterial community showing large variations in growth rate and subject to large variations in loss rates from grazers. Finally, although viruses did not represent the main agent of bacterial mortality, our data seem to show that their relative importance was higher in the less productive system.

Structure of microbial communities in Sphagnum peatlands and effect of atmospheric carbon dioxide enrichment

In laboratory experiments, bacterioplankton were incubated under different nutrient conditions, and the percentage of bacteria exhibiting a polysaccharidic capsule (capsulated bacteria) and that of CTC (cyanotetrazolium chloride)-positive and therefore metabolically highly active bacteria were determined. In these seawater cultures amended with nutrients more than 95% of the CTC-positive cells exhibited a capsule. During two cruises, one to the North Atlantic and one to the North Sea, we investigated the distribution of capsulated bacteria throughout the water column. Capsulated bacteria were generally more abundant in eutrophic surface waters than in deeper layers or more oligotrophic regions. In the upper 100 m of the North Atlantic, about 6–14% of the total bacterioplankton community was capsulated, while in the layers below 100 m depth, 97% of the bacteria lacked a visible capsule. The percentage of capsulated bacteria correlated with bacterial abundance and production, and chlorophyll a concentration. Also, the bioavailability of DOC (dissolved organic carbon), estimated by the ratio between bacterial production and DOC concentration, significantly correlated with the percentage of capsulated bacteria. In the North Sea, the contribution of capsulated bacteria to the total number of bacteria decreased from the surface (3 m depth) to the near-bottom (25–35 m) layers from 20% to 14% capsulated bacteria. In the nearshore area of the North Sea, about 27% of the bacteria exhibited a capsule. Overall, a pronounced decrease in the contribution of capsulated bacteria to the total bacterial abundance was detectable from the eutrophic coastal environment to the open North Atlantic. Using this epifluorescence-based technique to enumerate capsulated bacterioplankton thus allowed us to routinely assess the number of capsulated bacteria even in the oceanic water column. Based on the data obtained in this study we conclude that almost all metabolically highly active bacteria exhibit a capsule, but also some of the metabolically less active cells express a polysaccharide capsule detectable with this method.

A comparison of methods for counting viruses in aquatic systems.

ABSTRACT In this study, we compared different methods—including transmission electron microscopy—and various nucleic acid labeling methods in which we used the fluorochromes 4′,6′-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylmethyledene]-1-(3′-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>108 particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.

Protistan Bacterivory in an Oligomesotrophic Lake: Importance of Attached Ciliates and Flagellates

Seasonal and depth variations of the abundance, biomass, and bacterivory of protozoa (heterotrophic and mixotrophic flagellates and ciliates) were determined during thermal stratification in an oligomesotrophic lake (Lake Pavin, France). Maximal densities of heterotrophic flagellates (1.9 × 103 cells ml−1) and ciliates (6.1 cells ml−1) were found in the metalimnion. Pigmented flagellates dominated the flagellate biomass in the euphotic zone. Community composition of ciliated protists varied greatly with depth, and both the abundance and biomass of ciliates was dominated by oligotrichs. Heterotrophic flagellates dominated grazing, accounting for 84% of total protistan bacterivory. Maximal grazing impact of heterotrophic flagellates was 18.9 × 106 bacteria 1−1h−1. On average, 62% of nonpigmented flagellates were found to ingest particles. Ciliates and mixotrophic flagellates averaged 13% and 3% of protistan bacterivory, respectively. Attached protozoa (ciliates and flagellates) were found to colonize the diatom Asterionella formosa. Attached bacterivores had higher ingestion rates than free bacterivorous protozoa and may account for 66% of total protozoa bacterivory. Our results indicated that even in low numbers, epibiotic protozoa may have a major grazing impact on free bacteria.

Short-term effect of nitrogen enrichment on the microbial communities of a peatland

The aim of this study was to assess the structure of the microbial loop in a Sphagnum fallax —Carex rostrata fen of the French Massif central,and the impact of nitrogen supplies on the different microbial communities. Microalgae (46% of the total microbial biomass),Protozoa (26%) and heterotrophic Bacteria (17%) were the dominant microorganisms.Rotifera (5%),Cyanobacteria (3%),Fungi (2%) and Nematoda (< 1%) were also present.Testate Amoebae were well represented in Sphagnum peatland (14% of total microbial biomass).Thus,the structure of the protist community in the surface of fen peatlands is notably different from that recorded in other environments.The input of nitrogen led to a steady increase in microbial biomasses,but only changed the structure of microbial communities significantly when the input was 50 kg ha-1.At high inputs,nitrogen supply increased the relative importance of Cyanobacteria, Euglenophyceae, Bacillariophyceae and Ciliates, and decreased the relative importance of heterotrophic Bacteria, other microalgae and testate Amoebae. The increase in the values of photosynthetic assimilation and heterotrophic activities could also reflect changes in community functioning.

Viral Lysis, Flagellate Grazing Potential, and Bacterial Production in Lake Pavin

Abundances of different compartments of the microbial loop (i.e., viruses, heterotrophic bacteria, nonpigmented nanoflagellates, and pigmented nanoflagellates), bacterial heterotrophic production (BHP), viral lysis, and potential flagellate grazing impacts on the bacterial assemblages were estimated during a short-term study (24 h) conducted in June 1998 in the epilimnion (5 m) and metalimnion (10 m) of a moderate-altitude oligomesotrophic lake (Lake Pavin, France). Viral and bacterial abundances were higher in the metalimnion than in the epilimnion, whereas pigmented and nonpigmented nanoflagellates were more numerous in the epilimnion. The control of the BHP due to viral lysis (determined by examination of viral-containing bacteria using a transmission electron microscope) was significantly higher in the meta- (range = 6.0–33.7%, mean = 15.6%) than in the epilimnion (3.5–10.3%, 6.4%). The same was for the losses of BHP from the potential predation by nanoflagellates which ranged from 0.5 to 115.4% (mean = 38.7%) in the epilimnion, and from 0.7 to 97.5% (mean = 66.7%) in the metalimnion. Finally, estimated viral mediated mortality rates from the percentage of visibly infected cells and potential nanoflagellate grazing rates based on assumed clearance rates suggest that flagellates consumed a larger proportion of bacterial production than was lost to viral lysis.

Short-term variations in specific biovolumes of different bacterial forms in aquatic ecosystems.

Short-term and spatial fluctuations in specific biovolumes (volume x cell−1) of different morphological categories of planktonic bacteria were estimated microscopically. Samples were taken from two lakes occurring in two different climatic systems: Lake Aydat (France) and Lake Cromwell (Canada). The study was done in summer, using 24-hour cycles of sampling.Due to their large size, the specific volume of filamentous bacteria constituted, on average, the major part (>70%) of the total specific volume of all bacterial forms considered. Greatest variations in specific biovolumes were recorded for filamentous bacteria (coefficients of variation ranged from 16 to 109%). These variations were more pronounced in the oxygenated and microaerophilic strata (DOC ≈1.5 mg liter−1). Fluctuations in cell volume were high (coefficients of variation =12–80%) for coccal bacteria, whereas no marked fluctuations were found for the rod and vibrio bacteria (coefficients of variation =4–10%).Evidence of diel patterns of cell volume of filamentous bacteria is provided. These cells displayed their maximum size during the day until early night, indicating cell division was occurring at night. Homogeneous circadian patterns were not provided by specific volume variations of coccal, rod, and vibrio bacteria.Statistical relationships between bacterial specific biovolumes and the biotic and abiotic parameters considered are discussed.

The microbial loop in a humic lake: seasonal and vertical variations in the structure of the different communities

Seasonal and vertical variations of the main microbial communities (heterotrophic bacteria, autotrophic picoplank- ton, auto- and heterotrophic nanoflagellates, ciliated protozoa and microalgae) and auto- and heterotrophic activities were estimated in a brown-colored humic and moderately acid lake in central France, the lake of Vassiviere. The results demonstrated the dominant role of light in the vertical distribution of autotrophic and mixotrophic microor- ganisms which are confined to the 0–5 m layer during thermal stratification. The bacterial biomass was high throughout the water column probably because of the great availability of dissolved organic matter. Consequently, the predatory microzooplankton and particularly the various trophic groups of ciliated protozoa, were distributed in the water column according to the vertical distribution of the particular food resources (detritus, bacteria, algae). However, despite the great abundance of algae and bacteria, biomass of flagellated and ciliated protozoa was relatively weak. Most of the phytoplanktonic biomass was filamentous (Diatoms) or colonial (Cyanobacteria) and therefore almost probably difficult to ingest for algivorous microzooplankton. Regarding the low abundance of bacterivorous protozoa, the relation with the special physicochemical properties of this lake is discussed.