Viruses and flagellates sustain apparent richness and reduce biomass accumulation of bacterioplankton in coastal marine waters

To gain a better understanding of the interactions among bacteria, viruses and flagellates in coastal marine ecosystems, we investigated the effect of viral lysis and protistan bacterivory on bacterial abundance, production and diversity [determined by 16S rRNA gene polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)] in three coastal marine sites with different nutrient supplies in Hong Kong. Six experiments were set up using filtration and dilution methods to develop virus, flagellate and virus+flagellate treatments for natural bacterial populations. All three predation treatments had significant repressing effects on bacterial abundance. Bacterial production was significantly repressed by flagellates and both predators (flagellates and viruses). Bacterial apparent species richness (indicated as the number of DGGE bands) was always significantly higher in the presence of viruses, flagellates and both predators than in the predator-free control. Cluster analysis of the DGGE patterns showed that the effects of viruses and flagellates on bacterial community structure were relatively stochastic while the co-effects of predators caused consistent trends (DGGE always showed the most similar patterns when compared with those of in situ environments) and substantially increased the apparent richness. Overall, we found strong evidence that viral lysis and protist bacterivory act additively to reduce bacterial production and to sustain diversity. This first systematic attempt to study the interactive effects of viruses and flagellates on the diversity and production of bacterial communities in coastal waters suggests that a tight control of bacterioplankton dominants results in relatively stable bacterioplankton communities.     

Biomass, production and heterotrophic activity of bacterioplankton in the Great Astrolabe Reef lagoon (Fiji)

 Biomass, production and heterotrophic activity of bacterioplankton were determined for two weeks in the Great Astrolabe Reef lagoon, Fiji. Bacterial and Bacterial activities were distributed homogeneously throughout the water column (20 to 40 m deep) and varied little from site to site inside the lagoon. Bacterioplankton biomass and production also varied little over a diel period with coefficients of variation of 9 and 22%, respectively. On average, over the whole study, bacterial abundance was 0.77×10⁹ cells l^-1 and bacterial production averaged 0.36 μg-at. C l^-1 d^-1. Bacterial abundance and production were greater in the lagoon than in oceanic waters. Attachment to particles seems to provide an advantage for bacterioplankton growth because specific growth rates for attached bacterioplankton were, on average, significantly greater than that of the free community. Growth efficiency, determined by correlating the net increase of bacterial biomass and the net decrease of dissolved organic carbon (DOC) in dilution cultures, was very low (average 6.6%). Using carbon growth efficiency and bacterial production rates, heterotrophic activity was estimated to average 5.4 μg-at. C l^-1 d^-1. The turn-over rate of DOC (average 114 μg-at. C l^-1) due to bacterial consumption was estimated to be 0.048 d^-1 during the period of study. Accepted: 25 July 1998     

Fate of heterotrophic bacteria in Lake Tanganyika (East Africa)

Bacterial mortality was studied using two complementary methods between 2002 and 2004 in the two main basins (north and south) of Lake Tanganyika. The disappearance of radioactivity from the DNA of natural assemblages of bacteria previously labeled with tritiated thymidine was used to estimate the mortality due to grazing by predators (72%) and due to the cell lysis (28%). Measurements of ingestion rate of bacteria by protozoa using fluorescent micro-particles yielded protozoan grazing rates similar to those provided by the thymidine method, and showed that heterotrophic nano-flagellates were responsible for most of the grazing pressure on the bacterial community of the pelagic zone (92-99%). Bacterial cell lysis was the second process involved in bacterial mortality, ranking before ciliate grazing. Overall, bacterial mortality was balanced with bacterial production. With regard to the assessment of the trophic role of bacteria, it was estimated that c. 5-8% of the organic carbon taken up by bacteria was converted into protozoan biomass and was thus available for metazoans.     

Microbial food web in a large shallow lake (Lake Balaton, Hungary)

Seasonal variations of phyto-, bacterio- and colourless flagellate plankton were followed across a year in the large shallow Lake Balaton (Hungary). Yearly average chlorophyll-a concentration was 11 µg 1^–1, while the corresponding values of bacterioplankton and heterotrophic nanoflagellate (HNF) plankton biomass (fresh weight) were 0.24 mg 1^–1 and 0.35 mg 1^–1, respectively. About half of planktonic primary production was channelled through bacterioplankton on the yearly basis. However, there was no significant correlation between phytoplankton biomass and bacterial abundance. Bacterial specific growth rates were in the range of 0.009 and 0.09 h^–1, and ended to follow the seasonal changes in water temperature. In some periods of the year, predator-prey relationships between the HNF and bacterial abundance were obvious. The estimated HNF grazing on bacteria varied between 3% and 227% of the daily bacterial production. On an annual basis, 87% of bacterial cell production was grazed by HNF plankton.     

Heterotrophic bacterioplankton production and grazing mortality rates in an Ethiopian rift-valley lake (Awassa)

SUMMARY. 1. Heterotrophic bacterioplankton growth and production rates were estimated in a tropical lake by various methods. Mean growth rates, determined by tritiated thymidine incoporation into DNA, frequency of dividing cells and increase in cell density varied between 0.013 and 0.014 (with a range of 0.006–0.026) h⁻¹ corresponding to bacterial production of 1.16–1.22 (0.34–3.63) mg C m⁻³ h⁻¹.
2. Heterotrophic bacterial production estimated from oxygen and inorganic carbon consumption in the dark were compared with these values. The oxygen method gave similar results, while values from dark carbon uptake were as much as 2.5 times higher.
3. Although the different estimates of rates of bacterial production showed different patterns, the existence of spatial (vertical) and temporal (diel and seasonal) variation was demonstrated. Bacterial production was 13–41% of the net primary production and 10–30% of gross primary production.
4. Bacterial grazing mortality rate was estimated from size-fractionation and metablic inhibitor experiments. Average grazing rates were between 0.34 and 3.77 mg C m⁻³ h⁻¹ corresponding to 76–120% of the mean bacterial production rate. Organisms 1–12 μm in size, possibly mainly ciliates. were implicated as important bacterial grazers.     

Control of marine bacterioplankton populations: Measurement and significance of grazing

A variety of methods have been used to estimate the degree of control exercised upon marine bacterioplankton by grazing organisms. These include filtration or dilution of samples to reduce grazers, the use of specific inhibitors to prevent growth or grazing, and the use of artificial particles or radio-labelled bacteria as tracers for the natural bacterioplankton. Each of these techniques has drawbacks which may lead to under- or overestimates of grazing. In addition, they tell us little about which organisms are doing the grazing or the degree to which viruses or lytic bacteria compete with grazers for bacterial production. Because measurements of grazing and bacterioplankton growth rates are uncertain, exact comparisons are not presently possible. Thus measurements of bacterial and bacterivore abundance, concentrated on comparisons between seasons, on diel cycles and on spatial variations, have been used to evaluate mechanisms controlling bacterial populations. These give an idea of the degree of coupling between bacterial growth and bacterivore activity and of the time scales over which growth and grazing balance. Combined with laboratory studies of grazing, they currently provide the best insight into what controls populations of bacteria in the sea.     

Bacterioplankton growth, grazing mortality and quantitative relationship to primary production in a humic and a clearwater lake

Bacterial growth and grazing mortality were estimated from Mayto October in two south Swedish oligotrophic lakes, one beinga clearwater lake (water colour 5–10 mg Pt l^–1 DOC2.9–3.4 mg l^–1, Secchi disk depth 5.0–9.4m) and the other a humic, brownwater lake (water colour 105–165mg Pt l^–1, DOC 13.7–22.7mg l^–1, Secchi diskdepth 1.3–2.1 m). Specific rates of growth and grazingmortality were generally similar for both lakes. However, theabundance of bacteria was consistently 2–3 times higherin the water of the humic lake, suggesting that the total productionand consumption of bacterial cells were also higher than inthe dearwater lake. The ratio of bacterial secondary productionto primary production was higher in the humic lake than in theclearwater lake, indicating that the bacterioplankton of thehumic lake utilize allochthonous substrates, in addition tosubstrates originating from autochthonous primary production.Most of the bacterial loss in both lakes could be attributedto small protozoan grazers. This implies that allochthonousand autochthonous organic carbon fixed by bacterioplankton isless important in terms of carbon flow to higher trophic levelsthan would be expected if macrozooplankton were the dominantbacterivores, providing a more direct and efficient transferof carbon to larger organisms.     

Coupling Between Rates of Bacterial Production and the Abundance of Metabolically Active Bacteria in Lakes, Enumerated Using CTC Reduction and Flow Cytometry

Abstract In natural bacterioplankton assemblages, only a fraction of the total cell count is active, and, therefore, rates of bacterial production should be more strongly correlated to the number of active cells than to the total number of bacteria. However, this hypothesis has seldom been tested. Herein we explore the relationship between rates of bacterial production (measured as leucine uptake) and the number of active bacteria in 14 lakes in southern Québec. Active bacteria are defined as those cells capable of reducing the tetrazolium salt CTC to its fluorescent formazan; these cells were enumerated using flow cytometry. Bacterial production varied two orders of magnitude in the lakes studied, as did the number of active bacteria, whereas the total number of bacteria varied by only sixfold. The number and proportion of active bacteria were similar among lake strata, but rates of bacterial production were highest in the epilimnion and lowest in the hypolimnion. As expected, bacterial production was better correlated to the number of active cells, and bacterial growth rates calculated for active cells ranged from 0.7 to 1.8 day⁻¹, on average threefold higher than those calculated on the basis of total bacterial abundance. Growth rates scaled to active cells were, on average, similar among lake strata and did not show any pattern along a gradient of increasing chlorophyll concentration, so there was no systematic change of bacterial growth rates with lake productivity. In contrast, growth rates scaled to the entire bacterial assemblage were positively correlated to chlorophyll, were tenfold more variable among lakes than growth rates of active cells, and showed larger differences among lake strata. Scaling bacterial production to either the total number or the number of active cells thus results in very different patterns in bacterial growth rates among aquatic systems. Received: 12 July 1996; Accepted: 24 September 1996     

Size-selective grazing on bacteria by natural assemblages of estuarine flagellates and ciliates

The small average cell size of in situ bacterioplankton, relative to cultured cells, has been suggested to be at least partly a result of selection of larger-sized cells by bacterivorous protozoa. In this study, we determined the relative rates of uptake of fluorescence-labeled bacteria (FLB), of various cell sizes and cell types, by natural assemblages of flagellates and ciliates in estuarine water. Calculated clearance rates of bacterivorous flagellates had a highly significant, positive relationship with size of FLB, over a range of average biovolume of FLB of 0.03 to 0.08 microns3. Bacterial cell type or cell shape per se did not appear to affect flagellate clearance rates. The dominant size classes of flagellates which ingested all types of FLB were 3- to 4-microns cells. Ciliates also showed a general preference for larger-sized bacteria. However, ciliates ingested a gram-positive enteric bacterium and a marine bacterial isolate at higher rates than they did a similarly sized, gram-negative enteric bacterium or natural bacterioplankton, respectively. From the results of an experiment designed to test whether the addition of a preferentially grazed bacterial strain stimulated clearance rates of natural bacterioplankton FLB by the ciliates, we hypothesized that measured differences in rates of FLB uptake were due instead to differences in effective retention of bacteria by the ciliates. In general, clearance rates for different FLB varied by a factor of 2 to 4. Selective grazing by protozoa of larger bacterioplankton cells, which are generally the cells actively growing or dividing, may in part explain the small average cell size, low frequency of dividing cells, and low growth rates generally observed for assemblages of suspended bacteria.     

Bacterial Production in the Recently Flooded Sep Reservoir: Diel Changes in Relation to Dissolved Carbohydrates and Combined Amino Acids

The spatial distribution of bacterial abundance and production were measured every 4 h in a recently flooded oligo-mesotrophic reservoir (the Sep Reservoir, Puy-De-Dôme, France), in relation to concentrations of dissolved carbohydrates and combined amino acids. The concentration of dissolved organic matter (DOM) components in the recently flooded Sep Reservoir were higher than those measured in other lakes of similar trophic status. Short-term variations in the bacterial production in this new reservoir appeared cyclical and endogenous to bacterial communities. These results highlight the need for the evaluation of diel changes in bacterial production, if estimation of the daily production rate of bacteria is to be done accurately for a reliable model of carbon flow through bacterioplankton and ultimately through aquatic microbial food webs. Bacterial growth, measured over time and space, did not appear exclusively governed by DOM components from phytoplankton primary production.     

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. Offprint requests to: J. Iriberri.     

Estimates of bacterial production and mortality in the Ebro river (Spain)

In the terminal part of the Ebro river, bacterial production and mortality have been studied. Bacterial production was estimated by the thymidine incorporation method and from estimates of growth rate. Bacterial mortality and grazing were studied using differential filtration of a ³H-labelled population. Production was estimated as about 0.15 times 10⁹ bacteria/l/h and total mortality was about 0.016/h. Grazing accounted for about 56% of the total mortality processes.     

Relationship between bacteria, phytoplankton and heterotrophic nanoflagellates along the trophic gradient

Bacterial and heterotrophic nanoflagellates (HNF) abundance, as well as bacterial production and chlorophylla levels, were measured at five sites extending from the coastal zone toward the open Adriatic in the period from March to October 1995. The investigated areas were grouped into trophic categories according to concentrations of chlorophylla. All the biotic-para-meters increased along the trophic gradient, leading to eutrophy, but they did not increase at the same rate. The bacterial biomass: phytoplankton biomass (BB: chla) ratio decreased from about 10 in the very oligotrophic area to 0.8 at the eutrophic site. In contrast, the bacterial abundance: HNF abundance ratio (B: HNF) increased from 1000 bacteria per 1 flagellate in the oligotrophic system to 1700 bacteria flagellate⁴ in the eutrophic area. Decreasing BB: chla and increasing B: HNF ratios along the trophic gradient might reflect the different structures of the microbial food web. Relationships between bacterial abundance and production, and chla and HNF showed that bacterial abundance along the trophic gradient was regulated by the interplay between nutrient supply and grazing pressure. But in the oligotrophic system, bacterial abundance was more closely related to bacterial production and chla than in the eutrophic system, suggesting stronger control of bacterial abundance by substrate supply. On the other hand, the coupling between bacteria and HNF, and uncoupling between bacterial abundance and production in the eutrophic system, showed that the importance of bacteriovory increased in richer systems.     

Rate of Bacterial Mortality in Aquatic Environments

A method is proposed which provides a minimum estimate of the rate of bacterial mortality in growing natural populations of planktonic bacteria. This estimate is given by the rate of decrease of radioactivity from the DNA of a [³H]thymidine-labeled natural assemblage of bacteria after all added thymidine has been exhausted from the medium. Results obtained from river water, estuarine water, and seawater show overall bacterial mortality rates in the range 0.010 to 0.030 h⁻¹, in good agreement with the range of growth rates measured in the same environments. Use of selective filtration through Nuclepore filters (pore size, 2 μm) allowed us to determine the contribution of microzooplankton grazing to overall bacterial mortality. Grazing rates estimated by this method ranged from 0 to 0.02 h⁻¹.     

Bacterioplankton Growth Yield: Seasonal Variations and Coupling to Substrate Lability and beta-Glucosidase Activity

The seasonal variation in the carbon growth yield of pelagic bacteria in the eutrophic lake Frederiksborg Slotss? was studied. The growth yield was determined in dilution culture experiments, in which a substrate of dissolved organic carbon (DOC) from the lake was incubated with a natural bacterioplankton assemblage. Bacterial growth efficiency varied annually from 8 to 60% with an average (and standard deviation) of 41 +/- 11% (n = 29). Simultaneous measurements of growth yield, substrate lability (DOC(L)), chlorophyll and bacterial production, abundance, and extracellular enzymatic activity revealed new aspects of the regulation of bacterial DOC utilization. Growth yield correlated positively to DOC(L) and negatively to beta-d-glucosidase activity. These results indicated a close coupling between the substrate conditions and the physiological response of the bacteria. The large variations in yield within a few days and the close coupling to substrate availability showed that one single global carbon yield factor cannot be expected to apply in pelagic systems.     

Predation rates of flagellate and ciliated protozoa on bacterioplankton in a river

Abstract Predation rates of flagellate and ciliate protozoa on the bacterioplankton of Butrón River (Spain) were determined from FLB (fluorescently labelled bacteria) uptake rates. Bacterial and ciliate protozoa counts were higher when higher water temperature was recorded. Flagellate counts did not show this pattern, which suggested predation of flagellates by other organisms, or some other different nutritional mode besides phagotrophy. Average individual ciliate predation rates were up to 40-times higher than those of flagellates. These results were compared with similar data obtained from other authors in several aquatic systems. However, the population predation rates of flagellate protozoa were on average 6-times higher than that of ciliate protozoa, due to the low population numbers of the latter. Thus, flagellate protozoa can be considered as more important bacterial consumers than ciliates in this aquatic system.     

Culturability and Coexistence of Colony-Forming and Single-Cell Marine Bacterioplankton

Culturability and coexistence of bacterioplankton exhibiting different life strategies were investigated in the Baltic Sea and Skagerrak Sea. Bacterial numbers were estimated using a dilution-to-extinction culturing assay (DCA) and calculated as the most probable number, based on six different methods to detect bacterial growth in the DCA. Irrespective of the method used to detect growth, the fraction of multiplying cells never exceeded 10%, using the total count of 4′,6′-diamidino-2-phenylindole (DAPI)-stainable cells as a reference. Furthermore, the data also showed that non-colony-forming bacteria made up the majority of the viable cells, confirming molecular results showing dominance of non-colony-forming bacteria in clone libraries. The results obtained are in agreement with previous observations, indicating that bacterial assemblages in seawater are dominated by small, active subpopulations coexisting with a large group of inactive cells. The ratio of colony-forming to non-colony-forming bacteria was approximately 10 to 20 times higher in the brackish Baltic Sea than in the Skagerrak Sea. These two sea areas differ in (for example) their levels of bacterial production, dissolved organic carbon, and salinity. We suggest that the relative importance of colony-forming versus non-colony-forming bacterioplankton may be linked to environmental characteristics.     

Culturability and coexistence of colony-forming and single-cell marine bacterioplankton

Culturability and coexistence of bacterioplankton exhibiting different life strategies were investigated in the Baltic Sea and Skagerrak Sea. Bacterial numbers were estimated using a dilution-to-extinction culturing assay (DCA) and calculated as the most probable number, based on six different methods to detect bacterial growth in the DCA. Irrespective of the method used to detect growth, the fraction of multiplying cells never exceeded 10%, using the total count of 4',6'-diamidino-2-phenylindole (DAPI)-stainable cells as a reference. Furthermore, the data also showed that non-colony-forming bacteria made up the majority of the viable cells, confirming molecular results showing dominance of non-colony-forming bacteria in clone libraries. The results obtained are in agreement with previous observations, indicating that bacterial assemblages in seawater are dominated by small, active subpopulations coexisting with a large group of inactive cells. The ratio of colony-forming to non-colony-forming bacteria was approximately 10 to 20 times higher in the brackish Baltic Sea than in the Skagerrak Sea. These two sea areas differ in (for example) their levels of bacterial production, dissolved organic carbon, and salinity. We suggest that the relative importance of colony-forming versus non-colony-forming bacterioplankton may be linked to environmental characteristics.     

The impact of zooplankton feeding on the epilimnetic bacteria of a eutrophic lake

SUMMARY. The members of the zooplankion eat bacteria in natural systems, but the impact of this mechanism of removal of bacterial biomass on the bacterioplankton of freshwaters is unknown. Zooplankton abundance and biomass were followed for 2 years in Lake Mendota. Bacterial biomass and heterotrophic production were also assessed. Feeding of zooplankton on bacteria was measured by a cell counts method in 1979 and using radioactively labelled natural assemblages of bacteria in 1980. Total feeding was calculated and was found to account for l-60% of the bacterial biomass daily. Annually, it accounted for 1–10% of the bacterial heterotrophic production. Since bacterial biomass does not change significantly from year to year and yet bacterial production is very high compared to feeding by zooplankton, mechanisms other than feeding must exist which remove biomass from the epilimnetic bacteria in larger amounts.     

Effects of hydrophobic and electrostatic cell surface properties of bacteria on feeding rates of heterotrophic nanoflagellates

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.