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The microbial response to environmental changes in coastal waters of the eastern Cantabrian Sea was explored for four years by analysing a broad set of environmental variables along with bacterial community metabolism and composition. A recurrent seasonal cycle emerged, consisting of two stable periods, characterized by low bacterial metabolic activity (winter) from October to March, and high bacterial metabolic activity (summer) from May to August. These two contrasting periods were linked by short transition periods in April (TA ) and September (TS ). The phylogenetic groups Alphaproteobacteria and Bacteroidetes were dominant during winter and summer respectively, and their recurrent alternation was mainly driven by the bloom of eukaryotic phytoplankton before TA and the bloom of prokaryotic phytoplankton before TS . Bacterial growth efficiency remained high and stable during the winter and summer periods but dropped during the two short transition periods. Our results suggest that bacterial growth efficiency should be considered a very resilient property that reflects different stages in the adaptation of the bacterial community composition to the environmental changes occurring throughout the seasonal cycle in this coastal ecosystem., SI

Summary We explored how marine dissolved organic matter (DOM) altered by bacterial growth and protozoan grazing modify the metabolism of Southeastern Cantabrian Sea (CS) and NW Mediterranean Sea (MS) coastal bacterial communities. Major metabolic features were measured in treatments with half of the natural water replaced by water with different DOM quality, characterized by fluorescent DOM analysis and collected from key times of the predator–prey curve. In both ecosystems, protozoan-altered DOM led to similar increases in bacterial carbon demand (238% and 213%) and decreases in bacterial growth efficiency (BGE: 56% for the CS and 46% for the MS). These low BGEs were caused by similar bacterial production but much higher bacterial respiration rates, which in turn were positively related to aminopeptidase activity. However, in the CS bacterial community dominated by Bacteroidetes (41%), the enhanced hydrolytic activity was produced at a lower metabolic cost than in the MS, dominated by SAR11 (47%), which suggests a better adaptation of Bacteroidetes to the DOM altered during protozoan grazing. These results highlight protozoan grazing as a relevant factor influencing BGE in coastal ecosystems, and relate bacterial community composition to the major metabolic processes that result after a change in the quality of marine DOM.

We analysed changes in the abundance, biomass, activity and composition of coastal marine prokaryotic communities after the addition of organic substrates, such as glucose, leucine and yeast extract, and the effect of grazing pressure exerted by nanoflagellates. The addition of a carbon source (i.e. glucose) promoted the growth of Gammaproteobacteria, while a combined source of C and N (i.e. leucine) favoured the development of Alphaproteobacteria. The addition of yeast extract, a complex substrate rich in N and growth factors, promoted the proliferation of Alphaproteobacteria and Gammaproteobacteria. Grazing pressure exerted by nanoflagellates produced marked differences on the size structure of the prokaryotic biomass. A pronounced tendency to filamentation and aggregation was observed in the glucose treatment, while in the case of yeast extract, small and mainly freely dispersed prokaryotes were maintained throughout the incubations. Thus, the final community in the yeast extract treatment showed a high percentage of edible biomass, while an important fraction of potentially grazing-resistant prokaryotes (more than 50% of total prokaryotic biomass) was detected in the microcosms enriched with glucose. These results suggest a marked effect of DOM sources on the development of grazing-resistant prokaryotes.

The influence of the quality of organic matter on the hydrolysis of polymers by marine bacteria was investigated in microcosms containing aggregates created in rolling tanks. Two types of microcosms were analysed: microcosms type M1 from unaltered seawater and microcosms type M2 from phytoplankton cultures. Kinetics of aminopeptidase, �-glucosidase and �-glucosidase were measured in the ambient water and the aggregates in the two types of microcosms. Bacteria attached to aggregates expressed enzymes with Km values higher than those of the bacteria in the ambient water for the three hydrolytic activities analysed in both types of microcosms. Attached bacteria showed higher rates of polymer hydrolysis than free-living bacteria only in microcosms M2 created from freshly produced phytoplanktonic material, while free-living bacteria were more active than attached bacteria in the microcosms M1 containing unaltered seawater. The ratio Vmax/Km, which describes the ability of enzymes to compete at low substrate concentration, shows that free-living bacteria are more efficient at dealing with low substrate concentrations in microcosms derived from natural seawater, where the liquid phase may be depleted of utilizable dissolved organic matter, than in the microcosms derived from phytoplankton cultures. Our data suggest that the hydrolytic activities of both attached and free-living bacteria are significantly influenced by the quality of the aggregates and the consequences of this influence are discussed.

Bacterivory on aggregates and bulk seawater by three nanoflagellates and a microciliate was investigated. A new approach to measure bacterivory on the attached bacterial community is proposed. Macroaggregates containing attached fluorescently labeled bacteria (FLB) were obtained by adding labeled planktonic bacteria to rolling tanks filled with natural seawater, and they were used to measure protistan grazing rates on the bacteria attached to aggregates. Protistan grazing on free-suspended bacteria was measured according to the monodispersed FLB uptake technique. The four protists showed low grazing rates when feeding on free-suspended bacteria at the low densities usually found in seawater. Surprisingly, the analyzed protists also showed low grazing rates when feeding on aggregates despite the very high bacterial densities inhabiting them. The analysis of the influence of prey density on bacterial grazing rates suggests that feeding on bacteria attached to aggregates involves a relevant additional effort when compared to feeding on free-suspended bacteria. The paradox between these results and the very high abundance of protistan communities usually found in aggregates is discussed attending to the view of aggregates as biological reactors. Therefore, marine macroaggregates can be considered as microniches able to support the maintenance of a protistan community without food limitation, as well as to attract protistan colonizers by generating a water layer enriched in organic matter and bacteria.

Kinetics of glucose and amino acid uptake by attached and free-living bacteria were compared in the upper 70 m of the oligotrophic north-western Mediterranean Sea. Potential uptake rates of amino acids were higher than those of glucose in all the samples analysed. Cell-specific potential uptake rates of attached bacteria were up to two orders of magnitude higher than those of total bacteria, both for amino acids and glucose (0.72–153 amol amino acids cell−1 h−1 and 0.05–58.42 amol glucose cell−1 h−1 for attached bacteria and 0.34–1.37 amol amino acids cell−1 h−1 and 0.07–0.22 amol glucose cell−1 h−1 for total bacteria). The apparent Km values were also higher in attached bacteria than in total bacteria, both for amino acids and glucose. These results would reflect the presence of different uptake systems in attached and free-living bacteria, which is in accordance with the different nutrient characteristics of their microenvironments, ambient water and particles. Attached bacteria show transport systems with low affinity, which characterise a bacterial community adapted to high concentration of substrates.

Colonization and succession over time by bacterivorous protists on laboratory-ma de marine snow were analysed in five assays during 1994. Marine snow was made from natural seawater using rolling tanks. In all experiments, the macroaggregates were stable in size and consistency after the fourth day, and the colonization and succession processes were similar. Newly formed macro- aggregates became colonized by heterotrophic nanoflagellates on the fourth day, most of them kineto- plastids (Bodo designis and Rhynchomonas nasuta) and bicosoecids (Pseudobodo tremulans and Bicosoeca sp.). Sarcodines and ciliates appeared 1 day later. Among the former, the most abundant genus was Vannella sp., while scuticociliates (Uronema marinum) and hypotrichs (Euplotes vannus and Aspidisca sleini) were the most abundant ciliates. Most of the species observed in the study were more common to benthic habitats than to pelagic ones. The planktonic existence of the genera Bodo, Rhynchomonas, Bicosoeca, Euplotes and Aspidisca depends on the presence of surfaces because they are poor swimmers or immotile, and Pseudobodo and Vannella need attachment for feeding. The only pelagic protist observed was Uronema, probably because its opportunistic behaviour leads it to exploit enriched environments such as marine snow. Flagellate and ciliate abundances in laboratory-made macroaggregates were much higher than in surrounding water, which indicates that marine snow rep- resents an enhanced habitat for protist growth.

SUMMARY 1. The influences of bacterial density and water temperature on the grazing activity of the ciliates Uronema sp. and Colpoda inflata were studied. The conditions assayed were two prey densities (106 and 4 × 107 bacteria ml−1) and three water temperatures (10, 15 and 22 °C). 2. The response of the ciliates was measured from changes in protistan biovolumes and specific clearance rates. At high prey density, both ciliates showed lower biovolumes as water temperature increased, while at low prey density this tendency was minimized. 3. At the intermediate temperature of 15 °C both ciliates filtered ten times more body volume when bacteria were scarce; however, the ingested bacteria were fewer than at high prey density. At low prey density, a decrease from 15 to 10 °C evidenced different strategies of the two ciliates, which led to a similar ingestion of bacteria: C. inflata reduced its specific clearance rates and increased its biovolume, while Uronema sp. did not show changes. At high prey density, an increase from 15 to 22 °C caused lower biovolumes and a noticeable increase in specific clearance rates in both ciliates, indicating opportunist behaviour.

To enhance our understanding of the factors affecting feeding selectivity of bacterivorous protists in aquatic systems, we examined the preference responses of the freshwater ciliate Uronema nigricans towards three bacterial prey taxa, Pseudomonas luteola, Serratia rubidaea, and Aeromonas hydrophila. Potential factors influencing the predator-prey contact rate included the previous feeding history of the ciliate and physiological state of bacteria. Preference indexes were obtained from multiple-choice mazes in which ciliates moved preferentially towards alternative bacteria or the prey species on which they had been feeding. Uronema nigricans showed differ- ential attraction towards the offered prey types, and these preferences varied as a function of the ciliate feeding history: U. nigricans growing on P. luteola showed lower preference responses towards the offered bacteria than U. nigricans growing on S. rubidaea. The bacteria in stationary phase elicited a higher degree of attraction than bacteria in exponential phase, probably due to a higher concentration of carbohydrates in the former. Therefore, this protist will preferentially swim towards bacteria in stationary growth phase, although the degree of this response will be affected by the recent feeding history of the ciliate.

The disappearance of bacteria through grazing was studied. Five allochthonous and eight autochthonous bacterial strains, and the bacterioplankton of the Butron River were tested. There were differences in the susceptibility of different strains. These differences seemed to be related to intrinsic characteristics of the bacteria rather than to their origin.

Aims: The permanence in aquatic systems of allochthonous bacteria coming from sewage effluents is a risk for public health. This work aimed to analyse the elimination of the bacteria Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli and Klebsiella pneumoniae by a riverine ciliate community. Methods and Results: The ciliates were characterized and identified on the basis of morphological and behavioural features and grouped in nine morphotypes. Differential grazing by each morphotype on the four allochthonous bacteria was carried out by adding fluorescently labelled bacteria to the water samples, and measuring their uptake along time. Conclusions: The nine morphotypes were present in all the samples but in different proportions, being the most abundant the small scuticociliates and hipotrichs. The smallest morphotypes showed grazing rates lower than 20 FLB ciliate–1 h–1, with a preference towards K. pneumoniae. The larger morphotypes showed in general the highest grazing rates, but the selectivity was hardly attributable to bacterial size or type of cell wall. Significance and Impact of the Study: The elimination of allochthonous bacteria in an aquatic system may be highly different depending on the diversity of the ciliated protistan community in the system and on the nature of the bacterial prey.

Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from [ methyl - 3 H]thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 � 10 11 and 8.678 � 10 11 μg of C mol −1 for free-living and attached bacteria in the freshwater system, and 1.276 � 10 11 and 1.354 � 10 11 μg of C mol −1 for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different trophic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria.

The spatial distribution of marine protistan communities in the presence of organic macroaggregates, formed from natural seawater, was studied in several microcosm experiments. The presence of macroaggregates had two main effects. First, the size of the communities of bacteria, flagellates and ciliates increased, as these communities were three orders of magnitude higher in the aggregates than in the microcosm water. In addition, it brought the diversification on the niches accessible to planktonic microorganisms, as three phases were formed: water, aggregates and aggregate-water interphase. Some of the detected protistan taxa were only found in the water, and therefore they can be considered as truly free-swimming protists. Others quickly colonised the aggregates, and finally, some of them showed a preference for the aggregate-water interphase. We discuss this spatial structuring of the protistan community on the basis of their feeding strategies and structural and behavioural characteristics.

Protozoan predation on bacteria and bacterioplankton secondary production were simultaneously determined in La Salvaje Beach water during 1990. Protozoan grazing on bacterioplankton was measured from fluorescently labeled bacterium uptake rates; estimates of bacterial secondary production were obtained from [ 3 H]thymidine incorporation rates. Two different conversion factors were used to transform thymidine incorporation rates into bacterial production rates; both of them were specific for La Salvaje Beach and were calculated by using empirical and semitheoretical approaches. The average flagellate predation rate was 14.0 bacteria flagellate -1 h -1 ; the average population predation rate was 7.35 x 10 6 bacteria liter -1 h -1 . The estimates of bacterial production differed greatly depending on the conversion factor used, and so did the percentages of bacterial production consumed by flagellated protozoa (4.6% when the empirical conversion factor for La Salvaje Beach was used and 113% when the semitheoretical conversion factor specific for this system was used). The ecological implications of each of these values are discussed.

Summary A flow cytometric protocol to detect and enumerate heterotrophic nanoflagellates (HNF) in enriched waters is reported. At present, the cytometric protocols that allow accurate quantification of bacterioplankton cannot be used to quantify protozoa for the following reasons: i) the background produced by the bacterial acquisitions does not allow the discrimination of protozoa at low abundance, ii) since the final protozoan fluorescence is much higher than the bacterioplankton fluorescence (more than 35 fold) the protozoa acquisitions lie outside the range. With an increase in the fluorescence threshold and a reduction of the fluorescence detector voltage, low fluorescence particles (bacteria) are beneath the detection limits and only higher fluorescence particles (most of them heterotrophic nanoflagellates) are detected. The main limitation for the application of the cytometric protocol developed is that a ratio of bacteria/HNF below 1000 is needed. At higher ratios, the background of larger cells of bacterioplankton makes it difficult to discriminate protozoa. The proposed protocol has been validated by epifluorescence microscopy analyzing both a mixed community and two single species of HFN: Rhynchomonas nasuta and Jakoba libera. Taking into account the required bacteria/HNF ratio cited above, the results provide evidence that the flow cytometric protocol reported here is valid for counting mixed communities of HNF in enriched seawater and in experimental micro or mesocosms. In the case of single species of HNF previous knowledge of the biological characteristics of the protist and how they can affect the effectiveness of the flow cytometric count is necessary.