Energy–Dependent Bacterivory in Ochromonas minima–A Strategy Promoting the Use of Substitutable Resources and Survival at Insufficient Light Supply

Energy–Dependent Bacterivory in Ochromonas minima–A Strategy Promoting the Use of Substitutable Resources and Survival at Insufficient Light Supply

ARTICLE IN PRESS Protist, Vol. 157, 291—302, July 2006 http://www.elsevier.de/protis Published online date 10 July 2006 ORIGINAL PAPER Energy—Depen...

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ARTICLE IN PRESS

Protist, Vol. 157, 291—302, July 2006 http://www.elsevier.de/protis Published online date 10 July 2006

ORIGINAL PAPER

Energy—Dependent Bacterivory in Ochromonas minima—A Strategy Promoting the Use of Substitutable Resources and Survival at Insufficient Light Supply Sabine Flo¨dera,1,2, Thomas Hansena, and Robert Ptacnikb a

IFM-Geomar, Leibniz-Institut fu¨r Meereswissenschaften, Du¨sternbrooker Weg 20, 24105 Kiel, Germany Norwegian Institute for Water Research NIVA, Postboks 173 Kjelsa˚s, N-0411 Oslo, Norway

b

Submitted December 23, 2005; Accepted May 11, 2006 Monitoring Editor: Michael Melkonian

Phagotrophy and competitive ability of the mixotrophic Ochromonas minima were investigated in a three-factorial experiment where light intensity (low: 1.0 lmol m2 s1 and high: 60 lmol m2 s1 PPFD), nutrient concentration (ambient: 7.0 lmol N l1, 0.11 lmol P l1 and enriched: 88 lmol N l1, 6.3 lmol P l1) and DOC supply (without and with enrichment, 250 lmol C l1) were manipulated. Ochromonas minima and bacterial abundance were monitored for 12 days. We found significant and interacting effects of light and nutrients on Ochromonas minima growth rate and abundance. At high light intensity, nutrient enrichment resulted in increased growth rates and population sizes. In contrast, reduced growth rates and population sizes were observed for nutrient enrichment when light intensity was low. Although, Ochromonas minima was able to ingest bacteria under both high and low light conditions, it grew only when light intensity was high. At high light intensity, Ochromonas minima grew exponentially under nutrient conditions that would have been limiting for photoautotrophic microalgae. In non-enriched low light treatments, Ochromonas minima populations survived, probably by using background DOC as an energy source, indicating that this ability can be of relevance for natural systems even when DOC concentrations are relatively low. When competing with photoautotrophic microalgae, the ability to grow under severe nutrient limitation and to survive under light limitation should be advantageous for Ochromonas minima. & 2006 Elsevier GmbH. All rights reserved. Key words: light intensity; mixotrophy; nutrient concentration; Ochromonas minima; phagotrophy; resource competition.

Introduction

1 Present address: Botanisches Institut, Universita¨t zu Ko¨ln, GyrhofstraXe 15, 50931 Ko¨ln, Germany 2 Corresponding author; fax +49 221 470 5181 e-mail [email protected] (S. Flo¨der).

& 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2006.05.002

Realizing to which extent mixotrophy occurs among aquatic microorganisms, provided new perspectives for resource limitation, competition, and coexistence. Since planktonic production is the basis for aquatic food webs, research on nutrition and competitive ability of mixotrophs

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affected the conception of aquatic ecosystem functioning. Mixotrophic protists combine the ability of photosynthetic primary production and heterotrophic modes of nutrition. Mixotrophs are common in the open ocean, in coastal waters, estuaries, and in inland waters (Stoecker 1998). Since many plankton algae are able to take up dissolved organic carbon (Lewitus and Kana 1995; Raven 1997; Schnepf and Elbra¨chter 1992), amino acids, or other organic nitrogen sources (Michaels 1991), the majority of phytoplankton species may be considered mixotrophic (Stoecker 1998) if osmotrophy (absorption of dissolved organic compounds) is included in the definition of mixotrophy (Jones 1997). Focusing on phagotrophy (ingestion of particles) as the heterotrophic mode of nutrition, mixotrophy has been found in six algal groups (haptophytes, chrysophytes, dinophytes, prasinophytes, euglenophytes, and cryptophytes) and in ciliates (Boraas et al. 1988; Graham and Wilcox 2000; Porter 1988; Sanders and Porter 1988). In freshwater systems (Bird and Kalff 1987, 1989; Hitchmann and Jones 2000) and in estuaries and coastal waters (Epstein and Shiaris 1992; Hall et al. 1993; Havskum and Riemann 1996), it has been shown that mixotrophic flagellates may contribute to more than 50% of total bacterivory. Maintaining metabolic pathways for both phototrophic and heterotrophic nutrition results in additional basic metabolic costs in mixotrophic organisms. Mixotrophs were found to reach lower reproductive rates than heterotrophs when growing phagotrophically. Similarly, purely phototrophic growth resulted in lower reproductive rates as compared to photoautotrophic organisms (Rothhaupt 1996a, b). Potential ecological advantages of phagotrophy in phototrophic microorganisms are still under discussion. In primarily phototrophic species, the additional carbon source has been discussed as an advantage of mixotrophy. Supplementing or substituting the energy supply from photosynthesis appears to be important especially under low light conditions (Bird and Kalff 1987, 1989). Another apparent advantage is the acquisition of macronutrients bound in particulate form, augmenting or replacing the uptake of these resources from the dissolved pool (Nygaard and Tobiesen 1993; Rothhaupt 1996b; Smalley et al. 2003; Urabe et al. 2000). Mixotrophs can also benefit from organic growth factors such as vitamins (Gu¨de 1989), B-vitamins (Aaronson and Baker 1959), or phospholipids (Kimura and Ishida 1985) which they obtain from ingested bacteria. Besides the

advantage of being provided with resources if the dissolved pool is depleted, ingestion of bacteria or other small planktonic organisms eliminates potential competitors for those resources (Thingstad et al. 1996). The proportion of primary production and heterotrophic nutrition in mixotrophs varies widely between species, ranging from primarily heterotrophic to primarily phototrophic (Jones 1994; Myers and Graham 1956; Sanders et al. 1990). Within species, factors such as prey abundance (Andersson et al. 1989; Sanders et al. 2001), light intensity (Caron et al. 1993; Keller et al. 1994), macronutrient concentration (Nygaard and Tobiesen 1993; Smalley et al. 2003), or the combination of light availability and nutrient concentration (Urabe et al. 2000), may influence the relative importance of primary production and phagotrophy. While some species seem to switch from one to the other extreme nutritional mode, depending on the presence or absence of prey or of certain resources, others use photosynthesis and phagotrophy simultaneously (Jones 1997; Stoecker 1998). There is growing evidence that mixotrophs combine phototrophic and phagotrophic nutrition for optimal foraging (Rothhaupt 1996a, b; Stibor and Sommer 2003; Tittel et al. 2003). The combined use of light and particulate prey allows mixotrophs to achieve positive growth rates at very low levels of prey and dissolved nutrients (Rothhaupt 1996a, b). This combined resource use enables them to compete simultaneously with obligate phototrophic and heterotrophic competitors (Ptacnik et al. 2004). In combination, the benefits of mixotrophic nutrition, thus, seem to compensate for the lower efficiency as compared to more specialized organisms. However, some mixotrophs have been reported not to survive in the dark (Caron et al. 1993; Jones 1997). While these organisms might be able to perform optimal foraging and to substitute mineral resources, they are not likely to substitute the source of energy in the dark. In this study, we investigated heterotrophic behavior and potential competitive abilities of Ochromonas minima (chrysophyta). Ochromonas minima ingests bacteria and other small planktonic organisms. Unlike other Ochromonas strains that are primarily phagotrophic and grow in the dark as well as in the light if particulate food is available (Andersson et al. 1989; Rothhaupt 1996b), O. minima is unable to grow in the dark (Ptacnik 2003) but cultures perform well at low light levels (see methods section) that are in the range of critical light intensities (Icrit) of photoautotrophic microalgae

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Energy—Dependent Bacterivory in Ochromonas minima

(Huisman et al. 1999). At this species-specific threshold level, population growth and losses are balanced. Consequently, populations do not survive at light levels below Icrit. The ability to grow or to survive at extremely low light intensities could be advantageous for O. minima when competing with photoautotrophic microalgae. To study O. minima’s obligately phototropic form of mixotrophy, we conducted a laboratory experiment with a three factorial design. The objective of this study was to investigate the growth and mode of nutrition of the mixotrophic O. minima in environments with alternative sources of energy (light, particulate prey, and DOC) at different levels of productivity.

Results and Discussion Bacteria Ingestion under Low Light Conditions Whether O. minima is able to ingest bacteria under low light conditions was tested experimentally using fluorescently labeled bacteria. Exposed to a light intensity of o1.0 mmol m2 s1 PPFD (photosynthetic photon flux density) after it had been kept in darkness for 2 days, fluroscently labeled bateria (FLB) uptake by O. minima showed a logarithmic increase with time (Fig. 1) that could be described by a power function (R2: 0.998, po0.01). After having ensured that our Ochromonas strain is able to ingest bacteria under low light conditions, we investigated its mixotrophic behavior experimentally for 12 days using non-axenic

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cultures. In a cross-classified factorial design, we manipulated light supply (low: 0.8—1.0 mmol m2 s1 PPFD; high: 6072 mmol m2 s1 PPFD), nutrients (ambient: 7.0 mmol N l1, 0.11 mmol P l1; enriched: 88 mmol N l1, 6.3 mmol P l1), and dissolved organic carbon (DOC) by adding glucose (G) (G levels: 0 mmol Cl1, 250 mmol Cl1).

Time Patterns of Ochromonas and Bacterial Abundance Ochromonas minima growth experienced a lag phase, before on Day 4 an increase in O. minima abundance could be detected in treatments with high light (+L) intensity (Fig. 2A—D). In low light (-L) treatments, O. minima abundance did not increase. However, O. minima was able to sustain a stable population at low numbers in low light treatments without nutrient enrichment (Fig. 2 C, D). When exposed to nutrient enrichment (N) and low light (Fig. 2 A, B), O. minima decreased in numbers. O. minima populations that received the high light intensity kept growing until the end of the experiment, at the same time reducing bacterial abundance. In contrast to the time pattern of O. minima abundance, bacterial abundance began increasing when the experiment was started and reached saturation plateaus in each treatment (Fig. 2 A—D) indicating that the bacterial community was resource limited. Average abundance at saturation (Days 4—12) was calculated for low light treatments (N-L: 1.770.29  107; NG-L: 2.470.39  107; L: 3.870.59  106; G-L 4.270.51  106 cells ml17SD). Except treatments with ambient nutrient concentration (L and GL), bacterial abundance at saturation differed significantly among treatments (Newman—Keuls test, po0.001).

Limiting Resources for Ochromonas and Bacterial Growth

Fig. 1. FLB ingestion at low light intensity (o1.0 mmol m2 s1 PPFD) as a function of time, flow cytometrical analysis of the increase in fluorescently labeled Ochromonas minima cells.

In nutrient-enriched treatments, nitrogen and phosphorus concentrations remained in the same range (Fig. 3 A, B) after bacterial abundance reached saturation level. Nitrogen (30—70 m mol l1) and SRP (3.5— 5.2 m mol l1) concentrations at the end of the experiment (Fig. 4 A) indicate that there was no limitation by these resources in nutrient-enriched treatments. However, the significant difference between high bacterial abundance in the low light treatment with nutrient and glucose addition

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Fig. 2. Time patterns of Ochromonas minima and bacterial abundance in the experimental treatments. A: Nutrient enriched; B: Nutrient enriched and glucose added; C: Ambient; D: Ambient but glucose added; L+: high light intensity; L: low light intensity (see methods section for nutrient, glucose, and light levels). Bars represent the standard error.

Fig. 3. Time course of nutrient concentration in the experimental treatments. A: Nutrient enriched; B: Nutrient enriched and glucose added; C: Ambient; D: Ambient but glucose added; L+: high light intensity; L: low light intensity (see methods section for nutrient, glucose, and light levels). Bars represent the standard error.

and lower bacterial abundance in the low light treatment with nutrient addition only (Fig. 4 B) indicates that these treatments were carbon limited.

Although no carbon measurements are available for this experiment, it is possible to estimate bacterial carbon uptake from literature data. Using X-ray microanalysis, Fagerbakke et al. (1996)

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Fig. 4. Nutrient concentration (A) and bacterial abundance (B) at the end (Day 12) of the experiment, and Ochromonas minima growth rate (C). N: nutrient addition; G: glucose addition; L+: high light intensity; L: low light intensity (see methods section for nutrient, glucose, and light levels). Significant differences among treatments (3-factorial ANOVA, see Table 1; Newman—Keuls test) are denoted by a different letter. Brackets signify marginally insignificant differences (0.1op40.05). Except for Ochromonas minima growth rates, logtransformed data were used for statistical analyses. Bars represent the standard error.

measured the elemental composition of natural marine bacteria and determined C:N ratios in the range of 3.85—5.88:1 in weight. C:N ratios of resource limited and exponentially growing bacteria were analyzed by Vrede et al. (2002) using the same technique. For carbon limited bacteria, the average C:N ratio in the study by Vrede et al. (2002) was 3.8:1. Nitrogen limited bacteria had an average C:N ratio of 7.5:1, whereas exponentially growing bacteria had a C:N ratio of on average 5.2:1 (ratios also given in weight). Assuming carbon in our experiment was incorporated

into bacterial biomass at the lowest ratio (3.8:1 wt ¼ 4.45:1 molar) given in the literature cited above, carbon content of the glucose and nutrient-enriched treatments would have been depleted with a nitrogen concentration of 32 mmol l1 remaining in the medium. The lowest nitrogen concentrations (NG+L: 3172.0 SE mmol l1, NG-L: 3773.0 SE mmol l1) measured at Day 5 of our experiment correspond quite well with this assumption. While carbon limitation of the bacterial community can be assumed for nutrient-enriched

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treatments, it was not likely in treatments without nutrient enrichment because glucose addition in ambient treatments did not result in different nutrient concentrations at the end of the experiment (Fig. 4 A). Nitrogen concentrations declined during the course of the experiment (Fig. 3 C, D) and at the end (Fig. 4A) consisted mostly of ammonia (0.3—0.7 mmol l1). Nitrate concentrations were below the detection level of 0.1 mmol l1. SRP concentrations were in the range of 0.03—0.17. N:P ratios of 7.0—8.4:1 at the end of the experiments were far below the Redfield (1958) ratio for unlimited phytoplankton growth (16:1) and the one Vrede et al. (2002) determined for exponential bacterial growth (15:1). This indicates that nitrogen concentrations were severely limiting for bacteria as well as phytoplankton in treatments without nutrient addition.

Final Bacterial Abundances and Ochromonas Growth Rates Bacterial abundance at the end of the experiment was affected by interactions between nutrient enrichment, glucose addition, and light intensity (significant 3-way interaction, Table 1). While glucose addition and nutrient enrichment had a positive effect on bacterial abundance, the effect of light intensity on bacterial abundance was negative. Consequently, O. minima growth at high light intensity resulted in a decreased bacterial abundance. Highest bacterial abundances were recorded in low light treatments with nutrient

addition (Fig. 4 B). Among these, bacterial density was significantly higher when additionally glucose was added. High light treatments with nutrient addition had an intermediate level of bacterial abundance with no significant difference between treatments with or without glucose addition. Treatments without nutrients added displayed comparably low bacterial abundances and neither the treatments with low and high light intensity nor those with or without glucose addition differed significantly from each other (Fig. 4 B). To calculate growth rates (m) for O. minima (Fig. 4 C), abundance data from the exponential growth phase were used. In high light treatments with nutrient addition, O. minima grew exponentially from Day 5 to 10; whereas in high light treatments without nutrient addition, an exponential growth phase was detected between Days 8 and 12 (Fig. 2). Since there was no exponential growth phase of O. minima in low light treatments, the time span (Day 5—12) covering the exponential growth phases of all high light treatments were used to calculate growth rates. Ochromonas minima growth rate was influenced by the interaction between light and nutrients (significant 2way interaction, Table 1). Under high light intensity, nutrient enrichment resulted in increased growth rates; whereas at low light intensity, nutrient enrichment was connected to reduced growth rates. O. minima grew with high rates in high light treatments with nutrient enrichment (Fig. 4 C) were the highest growth rate was recorded when glucose was added. However, growth rate

Table 1. Results of 3-factorial ANOVA: effects of light, nutrient, and glucose addition on Ochromonas minima growth rates (exponential growth phase, Days 4—12) and on Ochromonas minima and bacteria abundance on the final day (Day 12) of the experiment. See Fig. 4 for significant differences among treatments (Newman—Keuls test). Log-transformed data were used to analyze Ochromonas minima and bacterial abundance. However, Ochromonas minima abundance data failed to become homoscedastic and showed larger variances at low abundances.

Factor

df

Light (L) Nutrients (N) Glucose (G) LN LG NG LNG

1; 1; 1; 1; 1; 1; 1;

24 24 24 24 24 24 24

Adj. Whole mod. R2; p-level:

Ochromonas minima growth rate

Ochromonas minima abundance

Bacteria abundance

F

p-level

F

p-level

F

p-level

739.95 2.70 0.215 64.34 0.774 0.85 2.08

0.000 0.114 0.647 0.000 0.388 0.366 0.162

1455.5 59.9 0.32 148.2 0.17 0.01 2.69

0.000 0.000 0.577 0.000 0.687 0.927 0.114

212.6 1566.5 28.8 57.5 0.1 0.0 2.4

0.000 0.000 0.000 0.000 0.804 0.843 0.131

0.963; o0.0001

0.982; o0.0001

0.984; o0.0001

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difference between nutrient-enriched high light treatments with and without glucose addition was marginally insignificant (po0.076). In high light treatments without nutrient enrichment, O. minima growth rates (Fig. 4 C) were significantly lower than in treatments with enrichment. While O. minima populations died off in low light treatments with nutrient enrichment, growth rates were about zero (0.006—0.008 d1) in treatments without nutrient enrichment (Fig. 4 C). Thus, O. minima growth rates supported the observation that O. minima decreased in abundance when nutrients were added but was able to sustain a stable population at low numbers when light intensity and nutrient concentration were low (Fig. 2). Neither in low light treatments with nutrient addition nor in those with ambient nutrient concentrations glucose addition resulted in growth rate differences.

Factors Affecting Ochromonas Growth under Different Light Conditions Although O. minima in our experiments ingested bacteria under both low and high light conditions, it grew only when light supply was high. Under the high light condition, O. minima abundance stagnated during the first days of the experiment and started increasing after bacterial abundance was higher than 106 cells ml1 (Fig. 2). Thus, O. minima used phototropic and phagotrophic pathways simultaneously. Under nutrient enrichment, glucose addition promoted bacterial growth (Fig. 2 A, B) and the O. minima growth rate increased (Fig. 4 C), and therefore, appeared to depend on the number of prey available. Under nutrient limitation, bacteria ingestion enabled O. minima to grow exponentially (Fig. 2 C, D) under conditions that would have been extremely limiting for photoautotrophic microorganisms. This indicates that even a mainly phototrophic organism like O. minima can benefit in two ways when operating both metabolic pathways at the same time. Ochromonas minima utilizes energy from photosynthesis and nutrients from the bacteria ingested. Whether optimal foraging is as important in O. minima as it has been shown studying phosphorus uptake from dissolved and particulate pools in Chrysochromulina polylepis (Stibor and Sommer 2003), cannot be fully answered with our experimental design. However, regardless whether concentrations of dissolved nutrients were high or low, O. minima experienced a lagphase at low bacterial abundance (Fig. 3 A—D),

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illustrating that nutrition relied more on bacteria than on dissolved nutrients and that optimal foraging was of minor importance with regard to dissolved and particulate nutrient pools. In this study, we did not investigate whether O. minima is able to grow photoautotrophically in axenic culture. Therefore, it remains unclear whether O. minima can substitute particulate nutrient sources with dissolved ones. However, it is very likely that particulate nutrient resources are substitutable, as O. minima feeds not only on bacteria but also on other small planktonic organisms (Ptacnik 2003). In competition experiments using an Ochromonas strain with the ability of phototrophic growth in the light and heterotrophic growth in the dark, the use of substitutable resources has been shown to be a successful competitive strategy. It allowed Ochromonas to coexist with photoautotrophic Cryptomonas sp. in the light and with heterotrophic Spumella sp. in the dark (Rothhaupt 1996a, b). Since bacteria were the primary nutrient source for O. minima in our experiment, it is very likely that it can coexist with photoautotrophic microalgae if light supply is sufficient (Ptacnik 2003). Being able to substitute bacteria with other particulate nutrient sources should be advantageous for O. minima, when it competes with other mixotrophs that depend on bacteria as additional nutrient source. Under low light conditions, O. minima was able to sustain a stable population size under ambient nutrient concentrations (Fig. 3 C, D). With nutrient enrichment, when carbon limitation occurred, population size declined (Fig. 3 A, B). Since more dissolved nutrients as well as carbon, nitrogen, and phosphorus in the form of bacteria were abundant in nutrient-enriched treatments than in treatments without enrichment, we can exclude nutrient limitation as a reason for the observed decrease in O. minima’s population size. However, nutrient-enriched and ambient low light treatments differed in the availability of DOC. While nutrientenriched treatments became DOC limited, nitrogen was the limiting factor in ambient treatments. Although surrounded by nutrients in soluble and particulate form in nutrient-enriched treatments, O. minima seemed not to be able to use them. A possible mechanism is that phagotrophy in O. minima is energy dependent. Ochromonas minima was not able to take up and digest bacteria because energy from photosynthesis was lacking. Light dependency of phagotrophy has previously been discussed for some chrysophyte species. Compared to phagotrophy at high light supply (170 mmol m2 s1 PPFD), the marine

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Ochromonas clone CCMP 583 displayed increased phagotrophy when light intensity was low (17 mmol m2 s1 PPFD). In low light adapted cells, a reduced bacteria uptake was observed when these were exposed to darkness. This Ochromonas clone has been classified as an obligate phototroph that requires light for both photosynthesis and phagotrophy (Keller et al. 1994). Dinobryon cylindricum ceased growing at a light intensity as high as 30 mmol m2 s1 PPFD. The primary function of bacterivory in this species was thought to provide essential growth factors or major nutrients for photosynthetic growth (Caron et al. 1993). A study on the ingestion rate of the estuarine Gyrodinium galatheanum in dependence of light intensity provided more evidence for energy-dependent phagotrophy (Li et al. 2000). While this dinoflagellate did not show any phagotrophy in the dark, the ingestion rate increased with increasing light intensity and reached a saturation level at 60 mmol m2 s1 PPFD. Urabe et al. (2000) explained the observed decrease in the phagotrophic rate of a freshwater Cryptomonas sp. under low light conditions by high energetic costs of the phagotrophic mode of nutrition. If Cryptomonas was able to obtain significant amounts of N and P but not enough C from bacteria, this would explain why it ceased to ingest bacteria at night, when there is no C to gain from photosynthesis. However, light dependency and high energetic costs of phagotrophy alone are not sufficient to explain why O. minima in our experiment survived maintaining a low abundance in ambient low light treatments but declined in population numbers when nutrients were added. Since those treatments differed in the availability of DOC, O. minima could have used glucose in spiked ambient treatments and in ambient treatments without glucose addition background DOC to produce energy. If O. minima were able to grow on DOC, populations should have reached higher densities in treatments with glucose addition than in treatments without glucose addition where O. minima depended on background DOC alone. Since energy yield from photosynthesis is about 2.5 times higher than from glycolysis and pyruvate dehydrogenation (e.g. Lehninger et al. 2001), we would have expected O. minima to grow at lower rates on DOC as energy source than photosynthetically on CO2. However, in our experiment, O. minima did not grow on DOC. We therefore propose that O. minima is able to obtain energy from photosynthesis when light is available and from glycolysis and dehydrogenation of pyruvate

in the dark. Sustaining energy metabolism is sufficient to maintain a stable population at low light, but for population growth biosynthetic pathways need to be operational. If the biosynthetic metabolism lacked necessary enzymes, O. minima was not able to use C-molecules from organic material originating from DOC uptake or from bacteria digestion for growth. In other words—as an energy source, CO2 appears to be substitutable, but it seems to be essential for biosynthesis. This would explain why O. minima was able to maintain a stable population when DOC is available but did not grow heterotrophically on bacteria or DOC in the dark. Laboratory studies have shown that species belonging to the genus Ochromonas can subsist on dissolved organic substances when concentrations are high (Pringsheim 1952; Sanders et al. 2001). Humic lakes are an example of DOC-rich (750—1.600 mmol C l1) natural systems, where mixotrophic flagellates are abundant ( Bergstro¨m et al. 2003; Jansson et al. 2001; Jones 2000). The DOC concentration (1.500 mmol C l1) Sanders et al. (2001) used in their experiment is in the range that is characteristic for humic lakes. However, the concentration of organic substances is much lower in most aquatic systems. Therefore, Ochromonas’ ability to subsist on dissolved organic substances has been considered to be of little relevance for the natural situation (Capriulo 1990). In contrast to this assumption, our results show that O. minima is capable of sustaining a stable population at DOC concentrations (background DOC in sterile filtered water from Kattegat, Baltic Sea) that are relevant to natural systems. Background DOC concentrations determined in the Kattegat range from 100 to 250 mmol C l1 (Frette et al. 2004; Nagel 2003; Pinhassi et al. 2003).

Conclusion According to our results, O. minima was able to ingest bacteria at high (60 mmol m2 s1 PPFD) and at low light intensity (o1.0 mmol m2 s1 PPFD). Although O. minima used DOC as an energy source for phagocytosis and to maintain basic cell metabolism at the low light intensity, it did not grow. Bacterivory in O. minima is energy dependent and energy sources may be light or DOC. However, population growth depends on the availability of light. Ochromonas minima used phototrophic and phagotrophic pathways simultaneously. Growth rates depended on the number

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of prey available and population density tended to increase with bacterial abundance. Ochromonas minima is likely to acquire nutrients and possibly trace elements from particulate matter substituting or supplementing the dissolved pool. Under nutrient limitation, bacterivory enabled O. minima to grow exponentially under conditions that would have been limiting for photoautotrophic growth. Our results demonstrate that, although unable to grow without light, O. minima may have an advantage when competing for light and nutrients. Being able to utilize mineral resources from bacteria and to withstand long periods of extremely low light intensity, it can be expected to be competitively superior to strictly autotrophic forms of phytoplankton that usually have higher threshold levels for light and nutrients.

Methods Experimental design: The experiment consisted of 150 ml non-axenic Ochromonas minima cultures that were grown as batch cultures in 250 ml Erlenmeyer flasks for 12 days. Sterile filtered Baltic water originating from the Kattegat (25 psu) was used as growth medium. The Ochromonas minima culture that served as inoculum was grown at 6.4 mmol m2 s1 PPFD (photosynthetic photon flux density). A total of 2.5 ml of a dense culture (4  104 cells l1) were used to inoculate the experimental cultures. Depending on the experimental treatment, either ambient seawater was used or it was nutrient enriched (modified Stosch and Drebes (1964) medium) and spiked with NaHCO3 following Guillard (1975). The experimental temperature was 16 1C. All cultures experienced a day:night cycle of 16:8 h, they were swirled by hand, and randomly arranged in the light fields once a day. All culture work and sampling was done under sterile conditions and in dim light (1.1 mmol m2 s1 PPFD). In a three-factorial design, we manipulated the supply of light, nutrients, and dissolved organic carbon (glucose) to investigate how these factors may affect growth and mixotrophic behavior of Ochromonas minima. Experimental light intensities were low (0.8—1.0 mmol m2 s1 PPFD) and high (6072 mmol m2 s1 PPFD), the nutrient levels were ambient (nitrogen: 7.0 mmol l1, phosphorus: 0.11 mmol l1) and enphosphorus riched (nitrogen 88 mmol l1, 1 6.3 mmol l ), and glucose levels were without (0 mmol C l1) and with enrichment (250 mmol C l1). Light, nutrient, and glucose treatments were

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factorially crossed and replicated 4-fold, resulting in a 2  2  2  4 design. Samples for Ochromonas and bacteria counts and nutrient analyses were taken at the start of the experiment (Day 1) and on Days 3, 5, 8, 10, and 12. Additional sampling (Days 2 and 4) supplemented the analysis of Ochromonas minima and bacteria abundance. Grazing experiment: To test whether Ochromonas minima ingests bacteria at very low light intensities, a phosphorus-limited culture was kept in complete darkness for 2 days prior to the start of a grazing experiment. Light intensity in the laboratory was dimmed to o1.0 mmol m2 s1 PPFD when the culture was prepared for the grazing experiment. Light intensity experienced by the culture during the experiment was 0.02 mmol m2 s1 PPFD. After adding fluorescently labeled bacteria (FLB) to the Ochromonas minima culture, the number of cells that had ingested FLB in the course of 40 min was analyzed cytometrically. Labeled Ochromonas cells were counted at 0, 2, 10, 20, and 40 min. FLB production: A culture of Rhodomonas salina, grown on F/2 medium (20 PSU) was at stationary phase when harvested. The culture was filtered using a 0.8 mm membrane filter (Schleicher and Schuell). A total of 40 ml aquilots of the filtered solution were centrifuged at 13,500 rpm (10 1C) for 10 min. The supernatant was removed and the remaining residue (1-2 ml) collected. Thereby, 200 ml of the filtered solution were concentrated to a final volume of 18 ml. This concentrate was cleaned once more using a 0.8 mm membrane filter and then heated up to 80 1C for 5 min in a water bath. Afterwards, it was treated with 45 ml of SYBR-green (Comp. Molecular Probes; l 494/ 521) diluted 1:50, allowing the cells to stain for 10 min. Staining with SYBR-green produces an intensive DNA coloring (Marie et al. 1997). A cleaning procedure followed by centrifuging and washing with bacteria-free seawater two times. The residue was resuspended in 2 ml artificial seawater and frozen at 20 1C. Before use, the FLB samples were thawed under hot water and mixed afterwards for a few seconds using a vortex mixer. Flow cytometrical analysis: Ochromonas minima and bacterial abundances were analyzed cytometrically. Analyses were performed with a FACS CALIBUR 2 laser flow cytometer (Company Becton Dickinson, San Jose, California). A 488 nm laser was used for excitation. The fluorescence produced was collected as chlorophyll-a (FL3-H channel) using a 870 nm long pass filter and as

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SYBR green with a 530715 nm band pass filter (FL1-H channel). Relative size was determined as right angle front scatter (FSC-H) and right angle side scatter (SSC-H). To analyze Ochromonas minima abundance, living cultures were analyzed counting 5000 events at medium flow rate (68.5 ml min1, SD 2.08) per sample. The flow rate was calculated by ‘‘true count tubes’’ (tubes that contain a defined amount of fluorescent beads). Quality control was realized with AlignFlowTM beads in the size of 2.5 and 6.0 mm on excitation wavelengths 448 and 633 nm. Data were recorded in list mode and analyzed using Cellquest Pro (Becton Dickinson Software). Bacteria samples (2 ml) were fixed with 100 ml formaldehyde solution (37%) and stored at 4 1C over night. Before counting, bacteria samples were stained with 5 ml (stock dilution 1:50) SYBR Green I (Marie et al. 1997) for at least 10 min. Analyses were done at low flow rate (24.5 ml min1, SD 0.29) counting 100,000 events per sample. Bacterial abundances were counted as SYBR fluorescence using the right angle scatter. Nutrient analysis: Dissolved inorganic nitrogen (DIN) and soluble reactive phosphorus (SRP) were analyzed with a Scalar scanplus system autoanalyzer following standard methods as published in Grasshoff et al. (1999). Calculation of experimental results: Ochromonas minima growth rate (m), the change in population size per unit time, was calculated assuming exponential growth according to m¼

ðln N2  ln N1 Þ , ðt2  t1 Þ

where N1 and N2 are the population sizes at the times t1 and t2. Statistical analyses: To analyze light, nutrient, and glucose effects on Ochromonas minima growth rate and on Ochromonas minima and bacteria abundance, a 3-factorial ANOVA was performed. Newman—Keuls test was used to analyze between treatment differences of bacterial abundance, Ochromonas minima growth rates, and nutrient concentrations. FLB uptake of Ochromonas minima was characterized with a non-linear regression approach.

Acknowledgements We would like to thank Jahn Throndsen (University of Oslo, Norway) for the O. minima culture, Anja Fitter for inspiring discussions, and Helmut Hillebrand for helpful comments on the manuscript.

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