Marian Unanue

Temporal variability of attached and free-living bacteria in coastal waters

The temporal variability of the abundance and the incorporation of 3H-thymidine and 14C-glucose by attached and free-living bacteria, as well as their relation with environmental factors, were analyzed in a coastal marine ecosystem during a year. Both communities were quantitatively very different. Attached bacteria represented only 6.8% of the total bacterial abundance, whereas free-living bacteria represented 93.2%. The environmental factors most closely linked to the abundance and activity of free-living bacteria were temperature and the concentration of dissolved nutrients. Moreover, the free-living community showed similar temporal variations in abundance and in activity, with lower values in the cold months (from October to May). The attached community did not present the same pattern of variation as the free-living one. The abundance of the attached bacteria was mainly correlated to the concentration of particulate material, whereas their activity was correlated to temperature. We did not find a significant correlation between the abundance and the activity of the attached community. On the other hand, the activity per cell of the two communities did not present a clear temporal variation. Attached bacteria were more active than free-living ones in the incorporation of radiolabeled substrates on a per cell basis (five times more in the case of glucose incorporation and twice as active in thymidine incorporation). However, both communities showed similar specific growth rates. The results suggest that the two aquatic bacterial communities must not be considered as being independent of each other. There appears to be a dynamic equilibrium between the two communities, regulated by the concentrations of particulate matter and nutrients and by other environmental factors.

Ectoenzymatic Activity and Uptake of Monomers in Marine Bacterioplankton Described by a Biphasic Kinetic Model.

A bstractThe kinetics of bacterial hydrolytic ectoenzymatic activity and the uptake of monomeric compounds were investigated in the Northwestern Mediterranean Sea. Aminopeptidase and α- and β-glucosidase activities were analyzed by using fluorogenic substrates at 15–22 concentrations ranging from 1 nM to 500 μM. Radiolabeled glucose and a mixture of amino acids were chosen as representatives of monomeric compounds, and the bacterial uptake rates (assimilation plus respiration) were determined over a wide range of substrate concentrations (from 0.2 nM to 3 μM). We found biphasic kinetics both for hydrolytic enzymes and uptake systems: high affinity enzymes at low concentrations of substrates (Km values ranged from 48 nM to 2.7 μM for ectoenzymes and from 1.4 nM to 42 nM for uptake systems), and low affinity enzymes at high concentrations of substrates (Km values ranged from 18 μM to 142 μM for ectoenzymes and from 0.1 μM to 1.3 μM for uptake systems). Transition between high and low affinity enzymes was observed at 10 μM for aminopeptidase and from 1 μM to 25 μM for glucosidases, and it was more variable and less pronounced for the uptake of glucose (40 nM–0.28 μM) and amino acids (10 nM–0.16 μM). Results showed that the potential rates of hydrolysis and uptake are tightly coupled only if the high affinity hydrolytic ectoenzymes and the low affinity uptake systems are operating simultaneously.

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles: Cleavage of Peptides and Uptake of Amino Acids

A bstractPhytoplankton-derived model particles were created in laboratory from a mixture of autoclaved diatom cultures. These particles were colonized by a marine bacterial community and incubated in rolling tanks in order to examine the relationship between aminopeptidase activity and leucine uptake. Bacteria inhabiting particles and ambient water were characterized for abundance, biovolume, aminopeptidase activity, leucine uptake, and growth rate. Particles were a less favorable habitat than ambient water for bacterial growth since growth rates of particle-attached bacteria were similar or even lower than those of free-living bacteria. During the first ∼100 h of the particle decomposition process, there were not statistically significant differences in the aminopeptidase activity:leucine uptake ratio between attached and free-living bacteria. From ∼100 h to ∼200 h, this ratio was higher for attached bacteria than for free-living bacteria. This indicates an uncoupling of aminopeptidase activity and leucine uptake. During this period, attached and free-living bacteria showed similar hydrolytic activities on a cell-specific basis. In the free-living bacterial community, variations in aminopeptidase activity per cell were associated with variations in leucine uptake per cell and growth rates. However, in the attached bacterial community, when leucine uptake and growth rates decreased, aminopeptidase activity remained constant. Thus, after ∼100 h, particle-attached bacteria were not taking advantage of their high aminopeptidase activity; consequently the hydrolysed amino acids were released into the ambient water, supporting the growth of free-living bacteria. These results demonstrate that over the particle decomposition process, the relationship between hydrolysis and uptake of the protein fraction shows different patterns of variation for attached and free-living bacterial communities. However, in our experiments, this uncoupling was not based on a hyperproduction of enzymes by attached bacteria, but on lower uptake rates when compared to the free-living bacteria.

Channeling of bacterioplanktonic production toward phagotrophic flagellates and ciliates under different seasonal conditions in a river

The objective of this study was to analyze the flux of biomass through the communities of bacteria and phagotrophic protists in the cold and warm conditions occurring seasonally in Butrón River. Bacterial and heterotrophic protistan (flagellate and ciliate) abundance was determined by epifluorescence direct counts; protistan grazing on planktonic bacteria was measured from fluorescently labeled bacteria uptake rates; and the estimate of bacterial secondary production was obtained from [3H]thymidine incorporation rates. The abundance of bacterial, flagellate, and ciliate communities was similar during cold and warm situations. However, we observed that estimates of dynamic parameters, i.e., secondary bacterial production and protistan grazing, in both situations were noticeably different. In the warm situation, grazing rates of flagellates and ciliates (bacteria per protist per hour) were, respectively, 7 times and 18 times higher than those determined in the cold situation, and the grazing rates of the protistan communities (bacteria per protists present in 1 ml of water per hour) increased up to 5 times in the case of flagellates and 42 times in the case of ciliates. Estimates of bacterial secondary production were also higher during the warm situation, showing a ninefold increase. The percentage of bacterial production preyed upon by flagellates or ciliates was not significantly different between the two conditions. These results showed that in the different conditions of a system, the flux of biomass between the trophic levels may be quite different although this process may not be reflected in the abundance of each community of bacteria, flagellates, and ciliates.

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles. Part II. Cleavage and Uptake of Carbohydrates

A bstractThe bacterial colonization and development of the ectoenzymatic glucosidase activity and glucose uptake were followed together with bacterial growth (measured as thymidine incorporation) in laboratory experiments, using phytoplankton-derived particles incubated in rolling tanks. Bacterial colonization of the particles was rapid. In the particles, bacterial turnover rates (production/biomass) were low (0.02 to 0.14 d−1). In the ambient water, turnover rates increased from 0.1 d−1 to 23.3 d−1, until the end of the experiment. In the control, lacking any particles, turnover of bacteria ranged from 0.3 to 7.6 d−1. Similarly, glucose uptake rates, per bacterium, were 1 to 2 orders of magnitude lower for particle-attached bacteria than for their free-living counterparts. Generally, Km values for glucosidase activity declined, over the incubation period, in particles and free-living bacteria until 168 h, and slightly increased, thereafter, to values of approximately 0.1 μM. Particle-attached bacteria exhibited significantly lower uptake rates of both thymidine and glucose, per bacterium, throughout the incubation. The per-cell ectoenzymatic activity was similar in particle-associated and free-living bacteria during the initial phase of the experiment, but was significantly higher after ≈200 h. Dissolved total (TCHO), as well as monomeric carbohydrates (MCHO), declined continuously in both particles and ambient water; they remained constant in the control; TCHO comprised about 50% of the dissolved organic carbon (DOC) in the particles. In ambient water TCHO contribution to DOC varied, with only one exception, between 25 and 45%; and in the control, between 20 and 50%. The shift detectable in the relation between ectoenzymatic activity and uptake of glucose between free-living and attached bacteria over the incubation period may reflect changes in the physiological status of the bacteria.

Attached and free-liviing dividing bacteria in two aquatic systems

The percentage of dividing biomass was calculated for attached and free‐living bacteria, in a coastal marine and a freshwater system. In the marine system with low concentrations of total and dissolved organic carbon (TOC and DOC) the percentage of dividing biomass was higher for attached (41.4 ± 13.9) than for the free‐living bacteria (22.0 ± 11.7). However, in the freshwater system, which had a higher concentration of TOC and DOC, the percentage of dividing biomass was similar for both communities‐attached (53.4 ± 26.5) and free‐living (78.4 ± 21.9). Thus the attachment to particulate material is not necessarily an advantage in waters where dissolved organic nutrients are readily available.

Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities

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 (Ea ), 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 Ea 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 Ea 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.