Ecological stoichiometry in the microbial food web: a test of the light ...

AQUATIC MICROBIAL ECOLOGY Aquat Microb Ecol

Vol. 31: 49–65, 2003

Published February 13

Ecological stoichiometry in the microbial food web: a test of the light:nutrient hypothesis J. J. Elser1,*, M. Kyle1, W. Makino2, T. Yoshida2, 3, J. Urabe 2 1 Department of Biology, Arizona State University, Tempe, Arizona 85287-1501, USA Center for Ecological Research, Kyoto University, Kamitanakami Hirano, Otsu 520-2113, Japan

2 3

Present address: Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New York 14853, USA

ABSTRACT: The ‘light:nutrient hypothesis’ (LNH) states that the importance of the microbial food web relative to grazing impacts by macrozooplankton and the nature of the relationship between algae and bacteria (competition or commensalism) are affected by the balance of light and nutrients experienced by phytoplankton. We tested this hypothesis in field enclosures by manipulating irradiance and nutrient supply in a P-limited lake in Ontario, Canada. Shading and P-enrichment had little effect on standing biomass of small suspended particles ( 85% of the added P. Response variables. Abundance and stoichiometry of plankton constituents and dissolved nutrients: Enclosures were mixed every day by pulling a 0.3 m perforated PVC disk several times through the length of the enclosure. Monitoring for a variety of parameters occurred at 5 d intervals. Methods of monitoring of macrozooplankton are described by Urabe et al. (2002b). Water samples for quantification of heterotrophic nanoflagellates (HNF, as an index of overall

microzooplankton abundance), algae, bacteria, seston, and dissolved nutrient analysis were taken after each enclosure was mixed. HNF samples were preserved with 4% glutaraldehyde and kept refrigerated until enumeration by epifluorescent microscopy after staining with primulin (Caron 1983). Algal samples were fixed with Lugol’s solution for later microscopic enumeration. Algal biomass was estimated by combining cell counts with taxon-specific cell-volume estimates following Findlay & Kling (1998). Biovolume for algal taxa with variable morphology or colony sizes was estimated independently for each sample while single constant values were used for taxa that did not exhibit substantial variation in size and shape. Bacteria samples were preserved with 2% filtered formaldehyde and kept refrigerated until enumeration by epifluorescent microscopy using DAPI (4’6-diamidino-2phenylindole) staining following Porter & Feig (1980). Thus our data pertain to responses of the entire microbial assemblage to experimental conditions. As previous studies have shown that manipulations of nutrients or grazing pressure can induce physiological and taxonomic shifts within the overall microbial assemblage (e.g. Jürgens et al. 1999, Larsen et al. 2001), this means that our results pertaining to microbial responses to light and nutrient manipulations will be essentially conservative; some potentially important responses may therefore have been missed. Water samples for seston analysis were size-fractionated using a 1 µm polycarbonate filter after which suspended particles in whole-water and 0.10). Occurrence of significant (p < 0.05) light × Penrichment interaction terms are indicated under L × P. Other significant interaction terms: A, Light lowered seston C:P in controls but increased C:P in +P; effect was modest. B, Effect of P was slightly stronger when incubated in lake. C, New C:P decreased slightly with light in controls but increased in +P bottles. D, Effect of P was (marginally) stronger when bottles were incubated in the lake. E, Effect of P was stronger when bottles were incubated in the lake. F, Effect of light was (marginally) stronger in controls relative to +P bottles. G, Effect of P was negative in enclosure but positive in the lake. H, Effect of light was (marginally) larger in controls than in +P bottles. I: Effect of P was (marginally) stronger when bottles were incubated in the lake P treatment

Light treatment

+ 0 µM + 0.024 µM + 0.048 µM + 0.097 µM + 0.19 µM + 0.39 µM + 0 µM + 0.024 µM + 0.048 µM + 0.097 µM + 0.19 µM + 0.39 µM

Unshaded Unshaded Unshaded Unshaded Unshaded Unshaded Shadeded Shadeded Shadeded Shadeded Shadeded Shadeded

L

Growth P

L×P

L

(+) ns ns ns – ns ns ns (replicate lost; no statistics possible) ns ns ns ns + ns ns ns (+) + B + + + (D) + + + E + + + ns + + ns ns ns + (+) G + + + (I) +

should vary negatively with the degree of algal light limitation. To examine this, we plotted initial seston C:P in each bioassay versus LL assessed for that bioassay, expecting a negative correlation. As anticipated, seston C:P in the enclosures was negatively associated with the degree of apparent light limitation of phytoplankton growth (Fig. 6; p < 0.003, R2 = 0.67, after natural log-transformation of LL). Above an LL value of ~0.1, seston C:P reached values approximating the Redfield ratio. In addition, the relationship appeared to be non-linear, with steep increases in seston C:P at low levels of light limitation (LL < 0.1). Thus, it appears that considerable alleviation of light limitation is required before seston C:P increases substantially. For shaded enclosures, increasing enclosure P-fertilization was associated with a shift from left to right in this figure but there was no indication in unshaded enclosures that nutrient enrichment per se influenced the position of different enclosures on this plot. Because we also measured seston C and P in each bioassay bottle at the end of the incubations, we were also able to estimate the C:P of new seston produced (C:Pnew) during the bioassay. Bioassay P enrichment inevitably lowered C:Pnew (Table 1) to values of ~30 to 75; this response is not surprising given the high per capita P-supply in the P-enriched dilution bottles. In contrast, raising light (LC vs EC, LP vs EP) generally led to increased C:Pnew (Table 1). This effect was significant in 5

Final seston C:P P L×P

New seston C:P L P L×P

– –

ns A

(+) (–)

– –

ns ns

– – – – – – – – –

ns ns ns ns ns (F) ns (H) ns

ns + ns + + + + + +

– – – – – – – – –

ns ns C ns ns ns ns ns ns

of 6 experiments for shaded enclosures but only for 1 of 5 experiments involving unshaded enclosures. To consider the effect of enclosure irradiance and bioassay irradiance on the stoichiometry of algal production, we calculated mean C:Pnew for each unenriched bioassay treatment combination for each enclosure (that is, the mean values of C:Pnew for LC and EC treatments). We pooled all enclosure data for various enclosure enrichment levels for each irradiance treatment and performed a 2-way ANOVA (C:Pnew was natural log-transformed to stabilize the variance) to evaluate the effects of enclosure irradiance (shaded vs unshaded) and bioassay irradiance (in enclosure vs in lake) manipulations on C:Pnew. Data for C:Pnew for bottles incubated inside the enclosure clearly show that algae in fully illuminated enclosures were forming new biomass at a higher C:P ratio relative to algae growing in shaded enclosures (Fig. 7). Not surprisingly, C:Pnew in unshaded enclosures did not respond very strongly to the modest increase in irradiance when enclosure plankton were incubated at 0.5 m depth in L239. However, C:Pnew for shaded enclosures increased dramatically when enclosure plankton were incubated in L239 (Fig. 7), reaching values even higher than those seen for unshaded enclosures. Thus, in the 2-way ANOVA for C:Pnew, enclosure treatment (unshaded vs shaded) and enclosure-bioassay treatment interactions were statistically significant (p < 0.05). Examination of the data for individual

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Fig. 6. Correlation between initial seston C:P ratio in bioassay bottles and the degree of light limitation (LL) exhibited during the bioassay. LL calculated as average increase in growth rate of algal seston (caught on GF/C filters) in elevated vs ambient irradiance treatments. P-fertilization treatment for each data point is shown. (----) Results of regression analysis on lntransformed data for initial seston C:P (p < 0.003, R2 = 0.61)

Fig. 5. Shifts in algal growth responses to (A) light enrichment (LL; incubation at 0.5 m depth in Lake 239 relative to incubation at 1 m depth in their respective enclosure) and (B) to P enrichment (LP; + 2 µM PO4) at the end of the enclosure experiment. LL calculated as average difference in algal growth when bottles incubated at relatively high light in lake vs in enclosure; average involved responses in unenriched bottles (µLC – µEC) and responses in P-enriched bottles (µLP – µEP). LP for each enclosure was calculated as average difference in algal growth for P-enriched vs unenriched bottles; average involved data from bottles incubated at relatively high light in lake (µLP – µLC) and from bottles incubated in enclosure (µEP – µEC). Results of statistical tests of algal responses to light or P-enrichment are given in Table 1. (----) Results of regression analysis of LL for shaded enclosures and LP for unshaded enclosures, the only relationships that were statistically significant (enrichment treatment was ln + 1 transformed to normalize the variance)

enclosures suggested that the responsiveness of C:Pnew to incubation location (enclosure vs L239) for shaded enclosures increased with enclosure P-enrichment (p = 0.09, R2 = 0.55). Thus, it appeared that algae growing in enclosures with the greatest likelihood of light limitation (shaded, high P-enrichment) developed a carbon fixation overcapacity that resulted in stoichiometrically unbalanced production of new biomass when irradiance was increased. In summary, these responses clearly show that the C:P stoichiometry of production of new biomass in L239 is sensitive to both P supply and irradiance experienced by the phytoplankton.

Fig. 7. C:P ratio of ‘new’ seston produced in bioassay bottles incubated under different conditions of illumination using sample water from unshaded and shaded enclosures. Data for all P-enrichment treatments were pooled (there was no statistically significant effect of P enrichment on C:Pnew in either irradiance treatment). Error bars indicate ±1 SE. Two-way ANOVA indicated significant effect of bioassay incubation treatment (incubation in enclosure vs in lake), no effect of enclosure treatment (unshaded vs shaded), but significant bioassay incubation × enclosure treatment interaction. Letters on each bar reflect results of Scheffe’s multiple comparison test following the ANOVA; different letters indicate means significantly different from each other (p < 0.05)

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Table 2. Statistical analyses of bacterial growth responses in enrichment bioassays performed at the end of the enclosure experiment. Responses to DOC addition (unamended control or enriched with 250 µM C as glucose and acetate) are indicated under DOC, while responses to bioassay P-enrichment (unamended control or enriched with 2 µM PO4) are indicated under P. Significant treatment effects (p < 0.05) are indicated by ‘+’ when the response parameter increased in response to the manipulation of that factor or by ‘–’ when the response parameter decreased in response to manipulation of that factor. Marginally significant responses and interaction terms (0.05 ≤ p < 0.10) are indicated by parentheses. ns = non-significant (p > 0.10). Occurrence of significant (p < 0.05) DOC × P-enrichment interaction terms are indicated under DOC × P. Other significant interaction terms: A, DOC stimulatory with P but inhibitory without P. B, P inhibitory without DOC but stimulatory with DOC P treatment

Light treatment

DOC

+ 0 µM + 0.024 µM + 0.048 µM + 0.097 µM + 0.19 µM + 0.39 µM + 0 µM + 0.024 µM + 0.048 µM + 0.097 µM + 0.19 µM + 0.39 µM

Unshaded Unshaded Unshaded Unshaded Unshaded Unshaded Shadeded Shadeded Shadeded Shadeded Shadeded Shadeded

ns ns ns (+) ns + ns (+) ns + (+) +

Growth P DOC × P ns + ns ns + (-) + + ns + ns +

A ns ns ns ns (B) ns ns ns ns ns ns

Bioassay responses of bacteria Bacteria in the enclosures responded to addition of both DOC and PO4 in various enclosures but there were few significant DOC × PO4 interactions in the experiments (Table 2). Following the approach for algal bioassay response, for each bioassay an index of bacterial DOC limitation (LDOC) was calculated as the average of µDOC – µC and µDOC+P – µP while a similar index of bacterial P-limitation (LP) was calculated as the average of µP – µC and µDOC+P – µDOC. In shaded enclosures, addition of DOC had a significant or marginally significant effect on bacterial growth rate in 4 of the 6 bioassays but DOC effects were observed in only 1 of the 5 bioassays in the fully illuminated enclosures (Table 2). However, a paired t-test comparing LDOC for shaded and unshaded enclosures was not statistically significant (p > 0.85), largely due to high variation in LDOC among the unshaded enclosures. Statistically significant stimulation of bacterial growth by added P occurred in 4 of the 6 experiments in the shaded enclosures (Table 2) but in only 2 of the 6 experiments in the unshaded enclosures (there was also a significant decrease in growth to P-enriched bottles in the + 0.30 µM P enclosure). There was no

clear tendency of bacterial P-limitation to change as a function of P-fertilization level (regression analyses of LP for bacteria against P-fertilization were non-significant, p > 0.20) nor was there any significant effect of irradiance on bacterial P response (paired t-test, p > 0.65). Visual examination indicated no consistent effect of bioassay treatments on bacterial cell size; thus, treatment effects on growth rates determined by cell counts probably reflected changes in overall bacterial production.

DISCUSSION The main results of our study are summarized in a diagram that semi-quantitatively depicts the status of major planktonic pools and regulatory interactions under contrasting conditions of light and P input imposed in our enclosures (Fig. 8). Here we summarize the 4 scenarios illustrated there: (1) Under high light and low P supply (i.e. unshaded enclosures receiving no experimental nutrient input), a pelagic community with high C:P ratio in overall seston as well as in bacteria-sized particles occurred (Fig. 8A). Algal biomass was high relative to bacterial biomass and neither algae nor bacteria responded significantly to bioassay enrichments of nutrients or energy (light, DOC). Macrozooplankton biomass was low and representation of high P taxa such as Daphnia was diminished (Urabe et al. 2002b); therefore, C:P ratio of zooplankton biomass was high. Macrozooplankton growth was constrained by stoichiometric food quality with low growth efficiency in terms of C (Urabe et al. 2002b). Hence, macrozooplankton grazing was weak, microzooplankton (as indexed by HNF in our study) probably experienced modest grazing losses, and thus had relatively high biomass despite potentially poorer quality (high C:P) of bacterial prey. As a result, a dominant trophic pathway in this system probably involved DOC transfer from algae to bacteria and subsequent consumption of bacteria by microzooplankton with strong dissipation of excess C by microzooplankton due to stoichiometric imbalance with their bacteria prey. (2) Under low P but low light (Fig. 8B), shifts in both stoichiometric aspects and biomass pools were observed. Algal biomass was decreased somewhat but was also more P-rich. Signs of algal light limitation were observed, as expected. However, somewhat paradoxically, growth rates of both algae and bacteria responded to P-enrichment, perhaps reflecting a decrease of overall availability of P to phytoplankton and bacteria due to the large increase in the biomass and P-content of the macrozooplankton community (Urabe et al. 2002b). Stoichiometric limitation of

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Fig. 8. Changes in major components of the planktonic food web under contrasting conditions of light and nutrient balance. Scenarios summarize data from this study and for whole seston and macrozooplankton from Urabe et al. (2002b). (A–D) Sizes of boxes indicate approximate magnitude of biomass of particular functional groups; absolute sizes of boxes for different functional groups (e.g. macrozooplankton vs macrozooplankton under high light, low P) are not meant to be compared directly, as we do not have direct measurements of the absolute biomass levels of all of the functional groups depicted. Dark shading within each box indicates relative P-content of that pool. A grey box is used to indicate microzooplankton because we do not have data on their P-content. Indications of likely relative magnitude of particular fluxes and of efficiency of trophic transfers (in terms of gross growth efficiency, or the fraction of ingested C that is transformed to new biomass production) are included. Also, indication is given of primary limiting factors for algae (light, phosphorus) and bacteria (DOC, phosphorus). Z = macrozooplankton; M = microzooplankton; A = algae; B = bacteria; P = PO4

macrozooplankton production was relieved by shading, resulting in greater zooplankton production and growth efficiency in terms of C (Urabe et al. 2002b). Despite higher macrozooplankton biomass that probably imposed high loss rates, microzooplankton biomass was unchanged (relative to unshaded enclosures), perhaps due to more efficient growth in response to increased P-content of bacterial prey. Important pathways of trophic flow involved both direct links and

indirect links (i.e. via bacteria and microzooplankton) to macrozooplankton. (3) At high rates of P-input and ambient light (Fig. 8C), strong shifts in both biomass and stoichiometry were also observed relative to ambient light and nutrient conditions. Inorganic P levels, as indicated by SRP, were higher than at low P-input. The absolute and relative abundances of algae and bacteria were largely unchanged compared to unenriched conditions

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but seston C:P ratios, both overall and in bacteria-sized particles, were significantly decreased. While algal growth surprisingly responded only to P-enrichment despite elevated P-input levels, bacterial growth rates were not DOC-limited, as expected. Again, perhaps the increased sink of P in massively larger biomass of P-rich macrozooplankton (Urabe et al. 2002b) was responsible for continued P-limited algal growth rate despite increased P-inputs. Large increases in macrozooplankton due to relief of stoichiometric constraints and increased growth efficiency were accompanied by strongly decreased abundance of microzooplankton. Thus, in this condition the dominant trophic links primarily involved direct macrozooplankton consumption of algal and bacterial production. (4) To a large degree these responses to increased P input were even more pronounced at low light input (Fig. 8D). Here, algal biomass was decreased due to shading but seston C:P ratios were quite low. Algae experienced strong light limitation (along with a weaker response to P-enrichment) while bacteria responded to both DOC and P-amendment. Zooplankton biomass was somewhat lower than in the fertilized, unshaded enclosures, perhaps due to lower overall food abundance. Microzooplankton abundance was also low relative to low enrichment enclosures. Thus, dominant trophic pathways probably involved direct links to macrozooplankton under these conditions. Our study represents the first large-scale field experiment to test microbial aspects of the LNH of Sterner et al. (1997) and has produced the following primary findings consistent with these aspects: (1) The relative abundance of macrozooplankton over microzooplankton was significantly affected by both irradiance and nutrient supply. In particular, fertilization led to increases in macrozooplankton abundance (Urabe et al. 2002b), decreases in HNF abundance (Fig. 4A), and thus to a major increase in the relative abundance of macrozooplankton versus HNF as P-enrichment increased (Fig. 4B). It appears that relieving stoichiometric constraints on macrozooplankton production in L239 induced a massive shift in the pathways of organic matter processing between the grazing chain and the microbial food web, implying that in L239 and similar unproductive lakes, light:nutrient balance affects microzooplankton primarily through ‘top down’ effects. P-enrichment did not lead to increased abundances of heterotrophic nanoflagellates in the enclosures. On the contrary, abundance of these microzooplankton declined dramatically in high P enclosures, in concert with large increases in macrozooplankton induced by the improved stoichiometric food quality due to P-fertilization (Urabe et al. 2002b). This suggests that microzooplankton abundance in L239 is strongly determined by

the proliferation of macrozooplankton and that the effects of light:nutrient balance on microzooplankton are indirect and occur mainly via the impacts of light:nutrient balance on food quality for larger grazers. However, there is some suggestion that HNF success was also influenced by stoichiometric food quality, as has been suggested in laboratory studies (Nakano 1994, Rothaupt 1996). That is, in low P (+ 0 µM P, + 0.024 µM P) enclosures, HNF abundances in shaded and unshaded enclosures were nearly identical (Fig. 4A), despite the fact that macrozooplankton biomass was 2 to 4 times higher in shaded enclosures (Urabe et al. 2002b) and that HNF in those enclosures probably experienced higher loss rates. Thus, HNF growth rates were probably higher in shaded enclosures to compensate for those losses, perhaps in response to a more suitable food quality (lower C:P in overall seston and in bacteria-sized 0.50 in regression of µm vs P-enrichment treatment). (3) Irradiance and P-fertilization affected bacteria mostly by altering their physiological state (as indexed by the relative frequency of bioassay responses and by