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Keywords:

  • grazing on bacteria;
  • prey–predator interaction;
  • phenotypic adaptation;
  • filament;
  • microcolony

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Predation and competition are two main factors that determine the size and composition of aquatic bacterial populations. Using a simplified bacterial community, composed of three strains characterized by different responses to predation, a short-term laboratory experiment was performed to evaluate adaptations and relative success in communities with experimentally controlled levels of predation and nutrient availability. A strain with a short generation time (Pseudomonas putida), one with high plasticity in cell morphology (Flectobacillus sp. GC5), and one that develops microcolonies (Pseudomonas sp. CM10), were selected. The voracious flagellate Ochromonas sp. was chosen as a predator. To describe adaptations against grazing and starvation, abundance, biomass and relative heterogeneity of bacteria were measured. On the whole, the strains in the predation-free cultures exhibited unicellular growth, and P. putida represented the largest group. The presence of Ochromonas strongly reduced bacterial abundance, but not always the total biomass. The activity of grazers changed the morphological composition of the bacterial communities. Under grazing pressure the relative composition of the community depended on the substrate availability. In the presence of predators, P. putida abundance declined in both high and low nutrient treatments, and Pseudomonas CM10 developed colonies. Flectobacillus was only numerically codominant in the nutrient-rich environments.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The complexity of interrelations among trophic levels of the microbial food web, the heterogeneity of prey and predators, and the fast adaptation to change, result in a system with huge intricacy. The impact of flagellate grazing on bacteria (top-down) often results in the development of numerous resistance strategies, including morphological adaptations, toxicity, motility, cells communication, and exopolymer production (reviewed by Pernthaler, 2005; and Matz & Kjelleberg, 2005). Several studies have focused on the different morphological adaptations of bacteria against grazing, and the development of resistant morphotypes has been described for several strains (e.g. filamentous forms, Hahn et al., 1999; Corno & Jürgens, 2006; microcolonies, Matz et al., 2002).

The diversity of reactions to the presence of predators, based on the resistance of different bacterial strains, can be summarized by applying the concept of ‘prey heterogeneity’ to microbial communities (McCauley et al., 1988; Leibold, 1989, 1996; Abrams, 1993). Many strains can change their shape and size in order to become resistant to predation, whereas only a few bacterial strains resist predation by growing in an organized structure.

Bacterial community size and structure, however, may not only be determined by top-down factors. The availability of nutrients is also recognized as a basic factor controlling bacterial community characteristics (bottom-up control). Higher amounts of available nutrients normally result in higher total bacterial biomass and in a modification of the size-structure of the community (Samuelsson et al., 2002), in agreement with both theoretical and earlier empirical studies (Thingstad & Sakshaug, 1990; Kivi et al., 1996; Duarte et al., 2000). Due to higher biovolume-specific uptake rates, small cells are expected to out-compete larger forms in nutrient-poor conditions (Legendre & Rassoulzadegan, 1995). However, larger forms can establish when small forms are saturated or limited by predation (Thingstad & Sakshaug, 1990).

Bacteria also exhibit morphological changes in response to different nutrient conditions, as demonstrated in studies on single strains (Holmquist & Kjelleberg, 1993) and natural communities (Tuomi et al., 1995), where they can favour different survival mechanisms and adaptive responses of bacteria to the presence of protistan grazers (Jürgens & Matz, 2002; Matz & Jürgens, 2003).

When grazing-resistant strains were compared based on their competitiveness against strains unable to produce resistant forms (Matz et al., 2002; Matz & Jürgens, 2003), there was a clear advantage for the edible strains in the absence of predators, usually due to a smaller size and shorter generation time.

Despite huge scientific attention, there seems to be no general consensus about how ‘top-down’ and ‘bottom-up’ factors interact to control population dynamics (Pernthaler, 2005).

In order to evaluate capabilities and relative ecological success, three diverse bacterial strains (prey) were exposed to predation by a single heterotrophic nano-flagellate (HNF) strain in high and low productivity systems. The impact of grazing on similar populations, subjected to different substrate conditions, was tested.

The physiology and the morphological abilities of the selected strains were all very well described from previous studies and, because of this knowledge, it was possible to settle on a system that generates a true strategy competition. Pseudomonas sp. CM10 (described by Matz et al., 2002), usually lives in single small cells (cocci or rods with a maximum diameter of 2–3 μm, Fig. 1a), and becomes resistant to Ochromonas sp. grazing (the selected predator) by developing clusters (several tens of cells) in the presence of predators (Fig. 1d). The second strain, Flectobacillus sp. GC5 (described by Corno & Jürgens, 2006) can become resistant to grazing. Its usual shape is a C or an S composed of one or two rods of 3–5 μm (shown also in Fig 1b) but, in the presence of HNF, inedible filaments and chains of several cells (for a final length of 10–40 μm, Fig. 1e) appear. The third strain, Pseudomonas putida MM1 (used in Matz et al., 2002; Matz & Jürgens, 2005; Corno & Jürgens, 2006), is a completely edible strain. It is usually used as ideal food in laboratory systems for several HNF species. Its shape is a typical free-living coccus, it is small (2–3 μm, Fig. 1c and f), and has a very fast generation time.

image

Figure 1.  Photomicrographs of DAPI-stained cells of the three bacterial strains used in this study: Pseudomonas sp. CM10 (a, d), Flectobacillus sp. GC-5 (b, e), Pseudomonas putida (c, f). Scale bar=20 μm. The first column refers to strains grown free from predators. On the second column bacterial strains are under high grazing pressure by Ochromonas sp.

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These three strains are ‘masters’ in their respective abilities: making microcolonies, developing filaments, or simply growing extremely fast without making any morphological defences against protist grazing.

Another ‘specialist’ in the field was chosen as a predator: the ‘interception feeder’Ochromonas sp. (Salcher et al., 2005, following the description given by Fenchel, 1987), which is a voracious mixotrophic bacterivorous nanoflagellate (Caron, 1987; Sanders, 1991; Posch et al., 1999) is readily used as a predator in many studies on bacterial plasticity in laboratory experimental systems (summarized in Boenigk & Arndt, 2002), often feeding on the same bacterial strains used in this study (Hahn et al., 1999; Boenigk et al., 2001; Matz et al., 2002; Corno & Jürgens, 2006).

The relative competitiveness of these three bacterial strains was tested in short-term semi-continuous cultures in the laboratory, in the presence or absence of Ochromonas sp. in order to test the effectiveness of different grazing resistance strategies with different amounts of available substrate, and to describe the impact of the limiting factors on the composition of this extremely simplified bacterial community.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Flectobacillus sp. GC5 was isolated from a continuous culture system, enriched with HNF, and inoculated with a mixed bacterial community from Lake Shöhsee (Germany). A fragment of its 16S rRNA gene is 98% identical with Flectobacillus mayor, the type species of the genus Flectobacillus (Larkin & Borrall, 1984). The accession number of the nearly full-length 16S rRNA gene sequence is DQ145723 (Corno & Jürgens, 2006). Pseudomonas sp. CM10 was isolated from Lake Shöhsee by C. Matz; its 16S rRNA gene sequence is deposited in GenBank (AF380369 and AF380370, Matz et al., 2002). Flectobacillus sp. GC5 and Pseudomonas sp. CM10 are available in the bacterial strain collection of the CNR-Institute of Ecosystem Study (Italy). Pseudomonas putida MM1, originally derived from rhizosphere of barley (Christoffersen et al., 1997), is available in the bacterial strains collection of the Max Planck Institute for Limnology (Germany), and its 16S rRNA gene sequence is also deposited in GenBank (AY623928, Matz & Jürgens, 2005).

The strains were grown separately on agar plates and then transferred to WC liquid medium (Guillard & Lorenzen, 1972) enriched with 50 mg L−1 glucose. Clonal cultures of all the strains were obtained from single colonies. Axenic cultures of Ochromonas sp. isolated always from Lake Shöhsee were maintained on suspensions of heat-killed P. putida MM1 (Corno & Jürgens, 2006). From these cultures, batches and semi-continuous experimental treatments were inoculated.

Batch culture experiment

Batch culture experiments were performed in order to compare the specific growth rates of the three selected bacterial strains. A set of three replicates for each monoculture was performed. The flasks (100 mL) were carried out on WC medium supplemented with 25.0 mg glucose L−1 as additional carbon source. A sample of 10 μL from the precultures described above was inoculated in each replicate obtaining three monocultural series of three replicates each. All cultures were incubated in the dark for 6 days on a rotary shaker at 15°C and sampled every 12 h.

Fed batch culture experiment

The relative competitiveness of the three bacterial strains was tested in semi-continuous cultures free from predators (the flagellate Ochromonas sp.) in treatments called ‘−GRAZ’ and, at the same time, in other cultures under grazing pressure (treatments ‘+GRAZ’). The bottles (250 mL each) were carried out on WC medium enriched with 2.5 or 25.0 mg glucose L−1 (treatments ‘−GLC’ and ‘+GLC’ respectively). Twelve cultures, three for each possible combination of treatments, were established. Every bottle was inoculated at day 0 with all bacterial strains to obtain an initial population of a total 3 × 106 bacteria mL−1 (1 × 106 bacteria mL−1 for each strain) in all the treatments. Where present, Ochromonas sp. concentration at day 0 was 500 cells mL−1. The experimental duration was 9 days, during which time the microbial assemblages were incubated in the dark at 15°C and shaken for 15 min every 2 h. Substrate was continuously provided from a reservoir with a dilution rate of 0.02 h−1 to maintain a relatively constant volume and compensate for daily sampling (Matz et al., 2002).

The substrate availability for bacterial cells, and for units of bacterial biovolume (μm3) during the period between day 3 and day 9 of the run was calculated daily by dividing the amount of glucose supplied for the corresponding total number of bacteria and total bacterial biovolume (Table 1).

Table 1.   Comparison of bacterial populations during growth in high (+GLC) and low (−GLC) productivity semi-continuous cultures in the presence (+GRAZ) and in the absence (−GRAZ) of the bacterivorous flagellate Ochromonas sp
Bacterial community parameterTreatments
−GRAZ+GRAZ
−GLC+GLC−GLC+GLC
  • The period between days 3 and 9 of the run was analysed. Values, means of three replicates per treatment, are expressed as mean ± SD, or simple arithmetical mean, or relative proportion.

  • *

    Substrate availability for treatments +GRAZ does not take into account the eventual substrate released by Ochromonas sp. Grazing pressure is calculated as number of Ochromonas sp. cells per edible bacterial cell. Paired treatments (‘−GRAZ −GLC’ against ‘+GRAZ −GLC’ and ‘−GRAZ+GLC’ against ‘+GRAZ +GLC’) were statistically compared (

  • , P>0.05;

  • ••

    , P<0.01).

Total abundance (106 cells mL−1)4.98 ± 0.1122.21 ± 1.321.60 ± 0.29••12.45 ± 1.87••
Total biovolume (106 μm3 mL−1)2.58 ± 0.0212.87 ± 0.111.14 ± 0.41••14.20 ± 4.19
Mean cell biovolume (μm3)0.51 ± 0.100.58 ± 0.020.71 ± 0.151.14 ± 0.57••
Total biomass (108 μg C L−1)6.17 ± 0.8130.11 ± 2.222.60 ± 0.73••33.22 ± 9.82-
Proportion of inedible bacteria (%)1.39 ± 0.442.10 ± 0.5184.08 ± 5.4687.50 ± 7.87
Substrate availability (fg Glc bact−1)0.49 ± 0.031.13 ± 0.061.62 ± 0.32*2.21 ± 0.52*
Substrate availability (fg Glc μm−3)0.96 ± 0.121.97 ± 0.142.22 ± 0.57*1.76 ± 0.52*
Grazing pressure (HNF edible bact−1)0.0094 ± 0.00080.0072 ± 0.0005

Determination of bacterial and protistan abundance, biovolume and biomass

Bacterial and Ochromonas sp. abundances were determined daily using an epifluorescence microscope and staining formalin-fixed samples with DAPI (4′,6-diamidino-2-phenylindole, Porter & Feig, 1980). Cell size measurements were taken from DAPI-stained samples using an automated image analysis system (Image-Pro Plus 5.1, Media Cybernetics). Area and perimeter of 300–500 cells were measured for each sample according to the algorithms given in Massana et al. (1997). Accurate counting for filaments and microcolonies was done in order to reduce the risk of underestimation of abundance and biovolume due to the presence of extracellular polymeric substances (EPS), indistinct cell boundaries and 3D structure of these forms (Matz et al., 2002). For filamentous forms, measurements of cell dimensions were taken for at least 100 randomly selected units per filter with the help of an ocular grid (Corno & Jürgens, 2006). At least 30 microcolonies per sample were measured for size and abundance (Hahn et al., 2000).

Bacterial cell carbon content (in fg C cell−1) and bacterial biomass (in μg C L−1) were calculated following Loferer-Krößbacher et al. (1998).

Specific abundances of the three bacterial strains in competition were determined using strain-specific polyclonal antibodies produced from rabbits immunized with Flectobacillus sp. GC-5 and Pseudomonas sp. CM10 (Eurogentec, Herstal, Belgium). Staining with primary and secondary antibodies, and the assessment by epifluorescence microscopy, were done with a modification of the procedure described in Christoffersen et al. (1997). In order to properly evaluate specific abundances, and to avoid possible overlapping, two different filters for each sample were prepared, each one stained with a single specific antibody. Cross reactivity between the two strains and with P. putida MM1 was not observed.

Definitions

Suspended, floclike structures consisting of three or more bacterial cells were termed ‘microcolonies’ (see Fig. 1d; Hahn et al., 2000). In order to assess properly the impact of predation on the heterogeneous prey populations of bacteria, an appropriate definition of the edibility of the various bacterial morphologies was required. By selecting Ochromonas sp. as predator, it was possible to define a size limit of 7 μm, below which bacteria were considered edible (regardless of the strain). When bacteria developed filamentous forms, or chains of cells, longer than 7 μm, they appeared to be resistant to predation (Corno & Jürgens, 2006). A similar threshold was also identified for microcolonies: clusters of each strain composed of more than 10 cells were considered inedible for flagellates. The use of prey size limits for the identification of the degree of edibility was questioned by Wu et al. (2004), studying contradictory effects of the predation of an Ochromonas sp. strain on several filamentous bacteria; on the other hand, prey size and morphology was recognized as a decisive factor in determining the edibility of a bacterial prey for a class of predators in several studies. The results of this study will be discussed in the light of the problematic nature of the imposed size limit.

Statistical analyses

Bacterial and protistan dynamics and their relative proportions in all treatments were tested, between day 0 and day 9, for significance using two-way repeated measures anova, with Bonferroni corrected t-tests posthoc comparisons, except for the comparison of single-strain growth dynamics in batch cultures, which were tested with a paired t-test. Statistical analyses were performed with SigmaStat 3.0 packed with SigmaPlot 9.0 (Systat Software, Inc.).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Single-strain batch cultures

The specific growth dynamics for each of the three strains used in this study was considered in batch cultures, during the phase of exponential growth (Fig. 2). Pseudomonas putida reached the top of its phase of growth after 48 h, with 24.31±2.23 × 106 cells mL−1 and a total biovolume of 12.03±1.10 × 106 μm3 mL−1. Flectobacillus sp. GC5 grew up to 10.43±1.35 × 106 cells mL−1 (total biovolume of 14.84±1.91 × 106 μm3 mL−1), but the top was reached after 60 h. Pseudomonas sp. CM10 reached the highest abundance (21.77±2.56 × 106 cells mL−1) and total biovolume (13.81± 1.52 × 106 μm3 mL−1) after 72 h.

image

Figure 2.  Growth of Pseudomonas sp. CM10, Flectobacillus sp. GC-5, and Pseudomonas putida in batches, without predators. (a) Shows abundances, while (b) shows corresponding biovolumes (means of three replicates ±SD).

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The comparison of single-strain trends during the phase of exponential growth (between 12 and 48 h after the inoculum) showed that the number of cells for P. putida increased significantly faster than the other two strains (P=0.007 against Flectobacillus sp. GC5 and P=0.026 against Pseudomonas sp. CM10, respectively, Fig. 2a), whereas comparing Flectobacillus sp. GC5 and Pseudomonas sp. CM10 no significant advantage was noticed (P>0.05). Comparing population biovolumes (Fig. 2b) the situation was different: growth rates measured for the three strains, even during the exponential phase of growth, did not show any significant difference (P>0.05 for all possible combinations).

General dynamics of bacterial and protistan populations in semi-continuous cultures

Trends in the abundance and total biomass of bacterial and protistan populations in treatments were considered to start from day 3. This approach was required in order to skip the huge fluctuations measured in the first 3 days, when bacterial populations were exponentially growing and the added flagellates were breaking previous bacterial trends to equilibrium. Starting from day 3 it was possible to identify more stable dynamics in every treatment, and thus to discuss them and to compare abundances and tendencies. Bacterial abundances (Fig. 3a) were significantly positively correlated with glucose concentration, in absence (P<0.001) and presence of Ochromonas (P<0.01). Bacterial populations in treatments −GLC and −GRAZ reduced fluctuations in number by the second day, after which their growth rate was constant and the population abundance remained between 5.67 × 106 and 4.72 × 106 cells mL−1. The presence of Ochromonas sp. (2.35±0.30 × 103 cells mL−1, Fig. 3b) in treatments −GLC resulted in a reduction of bacterial abundance and a relative reduction in the stability of the population, which fluctuated between 2.01 × 106 and 1.20 × 106 cells mL−1. Bacterial abundances in bottles +GLC were higher than those from low productivity treatments in the absence and presence of predators: for treatments −GRAZ, a minimum of 20.99 × 106 cells mL−1 and a maximum of 24.34 × 106 cell mL−1 were counted from days 3 to 9, at which point these populations reached a clear equilibrium. In the presence of predators (11.03±1.09 × 103 cells mL−1, Fig. 3b) bacterial abundances reduced by almost one-half, ranged between 16.68 × 106 and 6.58 × 106 cells mL−1 and followed inconsistent trends. There were very high abundances in the first period of the experiment, followed by dramatic reductions and large subsequent oscillations.

image

Figure 3.  Bacterial and flagellate development between days 0–9. Bacterial abundances (a), in high (+GLC) and low (−GLC) productivity systems, in the presence (+GRAZ) or absence (−GRAZ) of predators. Ochromonas sp. abundances (b) are related to treatments +GRAZ. Values are expressed as means of three replicates ±SD.

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A comparison between the means of the period from days 3 to 9 (Table 1) shows that the biggest bacterial assemblage grew in the treatment +GLC and −GRAZ. Populations from treatments +GRAZ were always lower in number than the corresponding −GRAZ populations (P<0.01).

The mean values for total bacterial biovolume and biomass in +GLC and +GRAZ treatments were slightly higher (P>0.05, Table 1) than the populations with the same substrate concentration but without predators. This result is related to a development of larger cells for bacterial populations subjected to grazing pressure. It was not noticed for −GLC and +GRAZ treatments, where the difference in total biovolume and biomass between the two series was statistically significant (P<0.01) and comparable to the difference measured in abundances. In −GRAZ treatments the impact of the amount of substrate supplied influenced the average size of bacterial cells: smaller forms were noticed in −GLC treatments when compared with bacteria from +GLC treatments (average biovolume per cell of 0.51±0.10 vs. 0.58±0.02, P<0.01).

Assuming that bacterial populations from different treatments kept fairly constant growth rates during the period from days 3 to 9 (Fig. 2), a calculation of the substrate available for a single bacterial cell (and bacterial unit of biovolume) was proposed (Table 1). Bacterial populations in −GLC treatments, in the absence of predators, obtained less than 50% of the substrate available (both per cell and per unit of biovolume) compared to bacteria in treatments +GLC. In +GRAZ treatments the substrate available per cell was high, was unlikely to be limiting, and was in comparable amounts between treatments (P>0.05 for both measurements, per cell or per unit of biovolume).

Ochromonas sp. populations in +GLC treatments were subject to large fluctuations: the mean abundance during the period from days 3 to 9 was 1.10±0.87 × 104 cells mL−1 with a minimum of 0.45 × 104 cells mL−1 at day 5 and a maximum of 2.87 × 104 cells mL−1 at day 3. Grazers in −GLC treatments during the same period had a mean of 0.24±0.10 × 104 cells mL−1, a minimum of 0.12 × 104  cells mL−1 at day 7, and a maximum of 0.44 × 104 cells mL−1 at day 3 (Fig. 3b).

Effectiveness of different prey classes in different conditions

In order to obtain a clear description of developmental tendencies, and thus of the relative success of the diverse morphological classes, a description of class frequencies, instead of absolute values, was chosen (Fig. 4).

image

Figure 4.  Development of different bacterial morphologies (edible: bacterial cells shorter than 7 μm or clusters composed by fewer than 10 cells; and inedible), measured on bacterial abundances, in the absence (a, b) or presence (c, d) of Ochromonas sp. and during the period of days 0–9. (a) And (c) refer to treatments +GLC (enriched with 25.0 mg glucose L−1), (b) and (d) to −GLC (enriched with 2.5 mg glucose L−1).

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At day 0, the percentage of edible cells was 92% of the total number (87% of total biomass) for all treatments. Populations from −GRAZ, in +GLC or −GLC treatments, did not change their morphological composition (P>0.05): the relative proportion of the mean abundance of edible forms by day 3 for these treatments was 95% (Fig. 4a and b). The opposite situation was observed in the presence of grazers: the populations started with 95% edible cells, switched to a majority of inedible cells by day 3 in +GLC (74%, Fig. 4c) and in −GLC (63%, Fig. 4d). Following this trend, both treatments became dominated by inedible forms with percentages >90% after day 5 (see Table 1 for relative means).

Analysis of single-strain dynamics

By measuring the abundance of single strains and their relative success in diverse populations, it is possible to link them to the conditions of the treatment. The relative proportion of every strain at time 0 was 33.3% by number, in every bottle. In −GRAZ treatments, P. putida overtook the other two strains, independent of substrate supply. In −GLC treatments it reached 50% of the total population by day 3 and 90% by day 7 (Fig. 5b). In +GLC its success was even faster, and P. putida reached 70% after 24 h (Fig. 5a). In these treatments the other two strains were relegated to percentages usually lower than 10–15%, and it became impossible to determine which was better adapted for any specific substrate conditions. There was a constant increment in the relative proportion of the strain P. putida in both treatments, and this tendency held throughout the whole experiment.

image

Figure 5.  Relative abundance of the three bacterial strains, during the period of days 0–9. Treatments free from predators are represented in (a) and (b); (c) and (d) refer to populations subjected to Ochromonas sp. grazing. (a) and (c) refer to treatments +GLC (enriched with 25.0 mg glucose L−1), (b) and (d) to −GLC (enriched with 2.5 mg glucose L−1).

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Similar proportions were not seen in +GRAZ treatments, and the development of the bacterial communities was completely different. Pseudomonas putida seems to suffer particularly in the presence of grazers, and in both conditions it was reduced to less than 10% of the total bacterial population by day 4. Under conditions of nutrient limitation (−GLC, Fig. 5d) Pseudomonas sp. CM10 became dominant by day 4 (60% of the total population), and by the end of the experiment made up 90% of the total bacterial population. Pseudomonas sp. CM10 also grew well in +GLC (Fig. 5c), where it reached about 50% of the total population, but was unable to outcompete Flectobacillus sp. GC5, which grew in the very same proportion with long filaments and chains of rods.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The relative impact of predation by HNF (top-down control) and nutrient availability (bottom-up control) on abundance and biomass of natural and artificial bacterial communities was measured in several studies, but there are a few generally accepted concepts that govern microbial dynamics (summarized in Pernthaler, 2005). In this study, the presence of predators led to a reduction of bacterial abundance in both high (+GLC) and low (−GLC) productivity systems (Fig. 3), and the bacterial populations in +GLC were more abundant than in −GLC, regardless of the presence or absence of predators.

Moreover, when evaluating the shift in bacterial biomass due to the presence of predators, it was clear that grazing had contradictory effects on prey populations (Table 1): in −GLC grazers reduced total bacterial biomass, but in +GLC biomass of bacterial communities was unaffected by predation, even if the number of bacteria was half that in −GRAZ. This was not unexpected and can be explained by the additional source of organic carbon represented by Ochromonas sp. exudates (Pernthaler et al., 1997) that are directly available for bacteria, and by the resistance strategy (high phenotypic plasticity, used to develop inedible long filaments or chains of cells) of Flectobacillus sp. GC5 (further described in detail) which results in an increase of its single cell biomass under grazing pressure (Corno & Jürgens, 2006).

The small amount of substrate available for bacterial populations in −GLC treatments, drives the development of small forms: increasing the amount of substrate supplied (treatments +GLC) increased the average bacterial cell size significantly. On the other hand, species composition and relative abundances were not influenced by the amount of substrate supplied. In +GRAZ treatments the average cell size was bigger, and the impact of substrate supplied less evident, also because the amount of nutrients available per cell were much higher and probably has less impact on bacterial populations than predation.

Since Ochromonas sp. is considered an ‘interception feeder’, calculating the chance that an edible bacterial cell will meet the predator (Table 1) is a valid index of the grazing pressure on the different communities: the possibility for a flagellate to encounter an inedible filament, or a cluster of cells is not increasing the risk for the bacterial community, and thus is not considered. Grazing pressure was slightly higher (P<0.05) in −GLC treatments where this impact, coupled with the low amount of substrate supplied, resulted in a reduction of more than 50% of the total bacterial biomass.

Predation and nutrient availability both control the bacterial community, reciprocally increasing or reducing their respective impact. This confirms the speculations of Gasol (1994) and Pace & Cole (1994) that ‘top-down’ control by protistan grazing or ‘bottom-up’ control by the availability of organic carbon and nutrients might be related to overall ecosystem productivity both in marine and freshwater microbial food webs.

Moreover, these findings are consistent with comparative analyses (Gasol et al., 2002) and theoretical models (Thingstad & Lignell, 1997), which show that bacteria are more intensely controlled by protistan predation in nutrient-poor, oligotrophic systems than in more productive environments.

The morphological composition of the heterogeneous prey community was highly controlled by grazing (Fig. 4). By simply dividing the community into edible and inedible bacteria, it was possible to identify two contrasting patterns of development in the bacterial populations: (1) any increase, neither with higher population growth rates, of the proportion of costly resistant morphologies in −GRAZ treatments, and (2) fast shifts to inedible forms in +GRAZ treatments. The prey size limits used for the definition of the degree of edibility of bacterial cells (in this study simply ‘edible’ or ‘inedible’) were respected by the well known predilection of nanoflagellates as Ochromonas sp. for medium-sized bacteria (González et al., 1990; Šimek & Chrzanowski, 1992). It was shown that even filamentous bacteria are not absolutely resistant to HNF grazing but have a selective advantage due to a strongly reduced ingestion efficiency by bacterivorous flagellates (Wu et al., 2004). The results obtained confirm the validity of these thresholds, as already demonstrated (Matz et al., 2002; Corno & Jürgens, 2006) and point out a clear morphological advantage for cells exceeding the assigned limits. Nevertheless, in this study any direct observation of the strategy of predation was made and thus it is possible only indirectly to confirm that cell size and morphology represent a refuge against HNF grazing.

In any case, even if the evaluation of prey edibility is a useful tool (especially for HNFs), for an appropriate assessment of the effective success of the single bacterial strains it was necessary to observe their development in more detail. In this experiment it was possible to trace the bacterial strains throughout the duration of the experiment, and, finally, to confirm that, despite the fact that the three species selected were true ‘masters’ in their specialty, it was impossible to find a ‘winning strain’ that was able to out-compete the competitors independently of the environmental conditions.

For the first time in this study the exponential growth phases of the three well known bacterial strains used were compared. Simply considering the dynamics, measured on bacterial abundances (Fig. 2a), it was possible to confirm that P. putida is definitely the strain with faster generation time in our systems. Because of this ability, P. putida absolutely dominated the predator-free communities (Fig. 5) generally developing in small free-living cocci. Pseudomonas putida controlled those communities independently by the amount of substrate available: they did not seem to suffer at all the presence of other competitors growing with very similar abundances and dynamics in treatment +GLC with the other strains, and in the preliminary batch cultures as single strain. As a result, the other strains were relegated to a small minority of the total population. This ‘excluding trend’ imposed by P. putida may have resulted in the extinction of the other two strains in a longer experiment.

Pseudomonas putida was not able to survive grazing by Ochromonas sp. and in fact, the occurrence of predators ‘killed the potential winner’, as theorized by Thingstad (2000) for virus predation and by Beardsley et al. (2003) for protists. In the +GRAZ treatments the ecological niches left by P. putida were thus available for strains able to survive to the new limiting factor: predation by Ochromonas. With little substrate supply (−GLC), the competition was rapidly won by Pseudomonas sp. CM10. In +GLC treatments Flectobacillus sp. GC5 was able to compete with Pseudomonas sp. CM10, and, by the end of the experiment, its relative proportion was comparable with the CM10 strain. The different evolution of the bacterial communities observed in the presence of predators can be explained by the different strategies developed by the two strains. It is obvious that aggregation costs in terms of the effectiveness of nutrient uptake (Matz et al., 2002), but even a reduced uptake ability is enough to outcompete Flectobacillus sp. GC5. When subjected by high grazing pressure by HNF this strain develops long filaments and chain of cells: these morphologies are known not to be very competitive, in conditions of low or normal nutrient availability, and without grazing pressure. This is due to the huge cell size (filaments up to 40 μm long) and to the long intracellular distances that raising the nutrient demand and reducing the velocity of biological answers respectively, results in a largely disadvantaged morphology (Koch, 1996). The situation changes with high level of nutrient availability and with high grazing pressure by HNF: in this case, the strategy of Flectobacillus sp., filament formation, becomes efficient: these findings seem to limit its success to environments where HNF predation is coupled with high amounts of available nutrients.

Because of the reductionistic nature of this approach, and of the many simplifications introduced in the system, this study is only a simple model of real communities and the natural environment. For this reason future studies, using new tools [such as catalyzed reporter desposition-fluorescence in situ hybridization (CARD-FISH) coupled with microautoradiography and single cell sorting], are required to enlarge the number of tracked bacterial species, the diversity of predators and of their trophic levels.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The author is grateful to Carsten Matz and Klaus Jürgens for providing Pseudomonas sp. CM10. This work benefited from the insight and technical assistance of the staff of the microbial ecology laboratories of the Max Planck Institute for Limnology and of the CNR-Institute of Ecosystems Study. The author is also grateful to Peter Deines, Cristiana Callieri, Carsten Matz, Blake Matthews and to the anonymous reviewers for valuable comments and suggestions on the manuscript.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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