Begoña Ayo

In this study, we hypothesized that shifts in the kinetic parameters of extracellular hydrolytic enzymes may occur as a consequence of seasonal environmental disturbances and would reflect the level of adaptation of the bacterial community to the organic matter of the ecosystem. We measured the activities of enzymes that play a key role in the bacterial growth (leucine aminopeptidase, β‐ and α‐glucosidases) in surface coastal waters of the Eastern Cantabrian Sea and determined their kinetic parameters by computing kinetic models of distinct complexity. Our results revealed the existence of two clearly distinct enzymatic systems operating at different substrate concentrations: a high‐affinity system prevailing at low substrate concentrations and a low‐affinity system characteristic of high substrate concentrations. These findings could be the result of distinct functional bacterial assemblages growing concurrently under sharp gradients of high‐molecular‐weight compounds. We constructed an ecological network based on contemporaneous and time‐delayed correlations to explore the associations between the kinetic parameters and the environmental variables. The analysis revealed that the recurring phytoplankton blooms registered throughout the seasonal cycle trigger the wax and wane of those members of the bacterial community able to synthesize and secrete specific enzymes.

Summary: 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 (T _A ) and September (T _S ). 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 T _A and the bloom of prokaryotic phytoplankton before T _S . 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.

Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO₂ sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature dependence has not yet been evaluated. Here, we report a global assessment of the temperature‐sensitivity, as represented by the activation energies (E_a), of extracellular β‐glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen, and phosphorus, respectively. These E_a were calculated from two different approaches, temperature experimental manipulations and a space‐for‐time substitution approach, which generated congruent results. The three activities showed contrasting E_a in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication.

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 M editerranean 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 SAR 11 (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 .

This study investigated the generation of dissolved free amino acids (DFAA) by the bacterivorous flagellate Rhynchomonas nasuta when feeding on abundant prey. Specifically, it examined whether this flagellate protist exhibits a chemosensory response towards those amino acids. The concentrations of glycine and the L- and D-enantiomers of glutamate, serine, threonine, alanine, and leucine were determined in co-cultures of the flagellate and bacteria. Glycine, L- and D-alanine, and L-serine were found to accumulate under these conditions in amounts that correlated positively with flagellate abundance, suggesting that protists are involved in their generation. Investigations of the chemotactic response of young and old foraging protists to the same amino acids, offered in concentrations similar to those previously generated, showed that glycine elicited the strongest attraction in both age groups. Young protists were strongly attracted to all the assayed amino acids, whereas older protists maintained a high level of attraction only for glycine. These results suggest that glycine generated by protists actively grazing in bacterially enriched patches functions as an infochemical, signaling to foraging protists the presence of available prey in the aquatic environment.

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.

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 differential 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 kinetics of glucose and leucine uptake in attached and free-living prokaryotes in two types of microcosms with different nutrient qualities were compared. Microcosm type M1, derived from unaltered seawater, and microcosm type M2, from phytoplankton cultures, clearly expressed different kinetic parameters (Vmax/cell and K' m). In aggregates with low cell densities (M1 microcosm), the attached prokaryotes benefited from attachment as reflected in the higher potential uptake rates, while in aggregates with high cell densities (M2 microcosm) differences in the potential uptake rates of attached and free-living prokaryotes were not evident. The aging process and the chemical changes in aggregates of M2 microcosms were followed for 15-20 days. The results showed that as the aggregates aged and prokaryotic abundance increased, attached prokaryotes decreased their potential uptake rate and their K' m for substrate. This suggests an adaptive response by attached prokaryotes when aggregates undergo quantitative and qualitative impoverishment.

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, α-glucosidase and β-glucosidase were measured in the ambient water and the aggregates in the two types of microcosms. Bacteria attached to aggregates expressed enzymes with K_m 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 V_max/K_m, 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.