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Notes 433 phate uptake: Are larger cells better competitors for pulses of phosphate than smaller cells? Oecologia 74: 57 l-576. TURPIN, D. H., AND P. J. HARRISON. 1980. Cell size manipulation in natural marine, planktonic, dia318: 55-58. tom communities. Can. J. Fish. Aquat. Sci. 37: SAKSHALJG, E., AND 0. HOLM-HANSEN. 1977. Chem1193-I 195. ical composition of Skeletonemu cost&urn (Grev.) WHEELER, P. A., AND D. L. KIRCHMAN. 1986. UtiCleve and Pavlova (Monochrysis) lutheri (Droop) lization of inorganic and organic nitrogen by bacGreen as a function of nitrate-, phosphate-, and teria in marine systems. Limnol. Ckeanogr. 31: iron-deficiency. J. Exp. Mar. Biol. Ecol. 29: l-34. 998-1009. SUTTLE,C. A., J. G. STOCIUVER, AND P. J. HARRISON. 1987. Effects of nutrient pulses on community structure and cell size of a freshwater phytoplankton assemblage in culture. Can. J. Fish. Aquat. Submitted: 7 June 1989 Sci. 44: 1768-1774. Accepted: 4 October 1989 -, -, K. S. SHORTREED, AND P. J. HARRISON. 1988. Time-courses of size-fractionated phosRevised: 4 December 1989 et al. [eds.], Tidal mixing and plankton dynamics. Springer. PLATT. T.. AND W. G. HARRISON. 1985. Biogenic fluxes’of carbon and oxygen in the ocean. Nature Limnd. Oceanogr.. 35(Z), 1990,433-443 Q 1990, by the American Society of Limaolcgy and Ocean@raphy, Inc. Carbon, nitrogen, and phosphorus budgets for the mixotrophic phytoflagellate Poterioochromonasmalhamensis(Chrysophyceae) during bacterial ingestion Abstract-Growth, grazing, C and nutrient incorporation by the mixotrophic phytoflagellate Poterioochromonas malhamensis were examined under various nutrient and light regimes in the presence of heat-killed bacteria. Poterioochromonas malhamensis readily ingested bacteria in all culture treatments containing heat-killed bacteria, and growth rates of the protist were much greater for heterotrophic (bacterivorous) growth than for phototrophic growth. C incorporation efficiencies by the phytoflagellate were virtually identical to P incorporation efficiencies during heterotrophic growth, but N incorporation efficiencies were somewhat lower in nearly all of the treatments. Algal growth in cultures with heatkilled bacteria was similar in continuous darkness and in continuous light. Bacterial organic C was the primary source of protist cellular C for P. malhamensis during heterotrophic growth. Based on our observations, we conclude that P. malhamensis is primarily heterotrophic and phagotrophic and that it is highly competent in this mode of nutrition. Acknowledgments We are grateful to Kay Ho, Harold King, Douglas Leeper, and Ee Lin Lim for technical assistance. The research was supported by NSF grants BSR 8620441 (to KG.P.) and BSR 86-20443 (to D.A.C.). Contribution 7273 from Woods Hole Oceanographic Institution. Contribution 46 from Lake Oglethorpe Limnological Association. Phagotrophic phytoflagellates occur within a number of algal orders. Although algal phagotrophy has been known for many years (see Sanders and Porter 1988) only recently have the ecological implications of this behavior been recognized. There are at least two important implications of phagotrophy by photosynthetic protists. This behavior may constitute an important source of predation on bacteria and other protists in aquatic ecosystems. It is now well established that flagellated heterotrophic protists are important consumers of bacterial and algal picoplankton in marine and freshwater communities (e.g. Azam et al. 1983; Sherr et al. 1983; Davis and Sieburth 1984; Caron et al. 1985; Sanders et al. 1989). More recently, mixotrophic chrysophytes, exhibiting both photosynthetic and phagotrophic activity, have been identified as potentially important bacterivores in some marine and freshwater ecosystems (e.g. Estep et al. 1984; Porter et al. 1985; Bird and Kalff 1986; Borass et al. 1988; Porter 1988; Salonen and Jokinen 1988). Based on the results of these studies, there is a growing realization that the magnitude of predation by mixotrophic algae mav have been overlooked in previous 434 Notes studies. The potential importance of this behavior in most natural communities remains largely unknown. Phagotrophy also may constitute an important nutritional strategy for mixotrophic algae. The ingestion of particulate organic material may be a mechanism for the acquisition of organic C, major nutrients (N and P) or other factors (e.g. vitamins, essential amino acids) required for growth. The ingestion of particulate organic C contributes significantly to the total C budgets of some mixotrophs (Bird and Kalff 1987, 1989; Andersson et al. 1989). For autotrophic flagellates, however, supplementing the uptake of inorganic N or P by grazing is potentially more important than organic C nutrition because these organisms are capable of producing organic C photosynthetically. Whether particle ingestion could be an important source of these elements in environments with low concentrations of dissolved nutrients and whether the nutrients contained in food particles could be utilized more efficiently by these algae than by heterotrophic protists have not yet been examined. We examined these latter questions by comparing the C, N, and P budgets of the phagotrophic phytoflagellate Poterioochromonas malhamensis (Pringsheim) Peterfi growing under various culture conditions. Poterioochromonas malhamensis is an 81O-pm spherical, flagellated unicellular chrysophyte. It was obtained from the University of Texas algal collection (stock number UTEX L 1297). Stock cultures were maintained in the light at 20°C on f/2-enriched distilled water (Stein 1973) or on organically enriched medium consisting of 1.O g liter-’ each of glucose, tryptone, yeast extract, and liver extract (Starr 1978). Heat-killed bacteria were used in all treatments involving bacteria in this study in order to circumvent the complications of a live bacterial population releasing or assimilating nutrients during incubations. In this way, all C and nutrient cycling in the cultures was attributable to the protist. Bacterial activity is a potentially important consideration for experiments designed to examine C and nutrient cycling by photo- synthetic and phagotrophic protists (Giide 1985; Caron et al. 1988). The uptake of nutrients by live algae also can cause problems in nutrient cycling experiments where algae are used as prey, even if the grazing phase of these experiments is carried out in the dark (Goldman et al. 1987). An axenic freshwater bacterial isolate, Pasteurella sp. (size, -0.647 x 1.5-2.0 pm), was grown at 20°C on a shaker table in 0.2% yeast extract in distilled water. Three-day-old, stationary growth-phase cultures of the bacterium were heat killed at 70°C for 30 min. The effectiveness of heat killing was verified by trial and error at several temperatures and for several incubation times by testing the viability of the resulting bacterial suspension in 0.5% yeast extract broth and on nutrient agar plates. Heatkilled bacteria were centrifuged in sterile, 250-ml polycarbonate centrifuge flasks with caps in place at 12,000 rpm for 15 min at 5°C. The bacteria were resuspended in sterile distilled water, centrifuged, and resuspended again a total of three times. Heat-killing Pasteurella sp. resulted in some loss of bacterial biomass from the cell suspension (Table 1). Relative to the live culture, equivalent volumes of the cell suspension were -20% lower in particulate C, N, and P immediately after the procedure. After rinsing and resuspension, however, the particulate and dissolved constituents in the cultures remained virtually unchanged for 7 d (length of the experimental incubations with P. malhamensis). Also, the C: N and C : P ratios of the heat-killed bacterial cells were not substantially different than these parameters for the live culture and were consistent with the ranges of these values for live bacteria from natural assemblages (Bratbak 1985; Nagata 1986). The concentrations of NH,+ and soluble reactive phosphorus (SRP) were high in this test treatment because the bacteria were rinsed only once. Bacteria used in the experimental treatments were rinsed three times, and the residual concentrations of these dissolved constituents were therefore much lower in the stock suspension of dead bacteria. Bacterial density and the concentrations of particulate and dissolved constituents were vir- Notes 435 Table 1. Changes in the particulate and dissolved constituents during heat killing of the freshwater bacterial isolate Pasteurellu sp. The C and nutrient constituents measured were soluble reactive P (SRP, rg liter-‘), total dissolved P (TDP, mg liter-‘), particulate P (PP, mg liter-‘), ammonium (NH,+, pg liter-‘), particulate N (PN, mg liter-‘), and particulate C (PC, mg liter-‘). PP, PN, and PC values were much lower after rinsing because of dilution and filtration through a LO-Nrn filter. Sampling time (d) Before killing After killing, before rinsing After killing and 1 rinse (T = 0) 1 d after rinse 4 d after rinse 7 d after rinse Particulate C:N:Pratio @Y w PP 11,100 36.8 7.43 7,500 45.9 182 - 10,900 37.1 6.13 7,300 36.9 143 - 25~6.2: 1 23:6.0: 1 1.44 1.41 1.40 1.86 440 450 500 460 13.0 13.3 12.1 12.9 1.17x10~~0.57 2.42 x 10%0.44 2.30x 10*+0.25 37 : 9.0 : 1 40 : 9.4 : 1 35:8.6:1 29 : 6.9 : 1 38.1 16.8 21.1 21.6 0.14 0.10 0.10 0.11 PN Bacterial density (No. ml-l) TDP SRP NH,* tually unchanged over the length of the experiment in a control culture of bacteria not inoculated with the alga. The heat-killed bacteria were resuspended in a basal salts culture medium (Sanders et al. 1990) after the final rinse, filtered aseptically through a sterile 5-pm Nuclepore filter with a sterile filter assembly to remove clumped cells, and diluted to a final concentration in those treatments containing bacteria. Sterility tests (culture in 0.5% yeast extract in distilled water) were performed on the stock suspension of heat-killed bacteria initially and on all cultures during sampling and at the end of the experiment to ensure that no viable bacteria were present in any of the cultures. Light intensity, inorganic nutrient concentration, and the availability of bacteria were tested for their effects on the nutritional mode (heterotrophy vs. phototrophy) of P. malhamensis. Five basic experimental treatments were used, consisting of various nutrient and bacteria additions to an N-free and P-free inorganic basal medium (see Sanders et al. 1990). This basal medium was enriched with bacteria only (no inorganic N or P added), bacteria and NH,+ (no inorganic P added), bacteria and POd3- (no organic N added), bacteria and NH,+ and POd3-, or NH,+ and POd3- (no bacteria added). Control cultures consisted of heatkilled bacteria in basal medium (not inoculated with P. malhamensis), an N-free and P-free algal culture, an algal culture with NH4+, POd3-, and vitamins but without bac- PC 53.0 56.0 48.4 53.1 teria, and an algal culture in organically enriched medium (see above) without bacteria. Concentrations of NH,’ and POd3- were 200 PM and 20 PM after they were added to the culture medium. For all treatments, inocula from a phototrophically growing culture of P. malhamensis in bacteria-free f/2-enriched distilled water were transferred to 900-ml tissue culture flasks containing 800 ml of culture medium. Flasks of each of the five experimental treatments were incubated at 20°C with gentle shaking in continuous low or high light intensity (4-10 or 400-500 rmol quanta rnpz ss’) and in continuous darkness (a total of 15 experimental and 3 control flasks). Samples incubated in continuous darkness were wrapped in foil and sampled in almost total darkness. Samples for bacterial and algal cell counts and chemical analyses were removed aseptically at t = 0 and at intervals of several hours for a period of -7 d in each experimental treatment. Cell densities of the bacterium and the microalga were determined with epifluorescence microscopy on acridine orange- or DAPI-stained samples preserved with 1.O% glutaraldehyde (Porter and Feig 1980; Davis and Sieburth 1982). Growth rates of the alga in the various treatments were determined from regressions of the linear portion of the increase in the natural Iog of cell density during exponential growth of the alga in each treatment. Particulate C (PC) and particulate N (PN) were analyzed with a Perkin Elmer 240 el- 436 Time (h) 0 50 100 0 150 Time (h) 100 150 Time (h) Fig. 1. Representative changes in the population densities, SRP, and DOP in cultures of Poterioochromonas malhamensis medium for this treatment was the basal salts medium with were incubated at high light intensity. A. Changes in bacterial in PN and NH,‘. D. Changes in SRP, PP, and DOP. emental analyzer on samples retained on precombusted Gelman GF/F glass-fiber filters. NH,+ concentration was measured in the filtrate from the PC/PN samples with standard calorimetric procedures (Parsons et al. 1984). SRP, total dissolved P (TDP), and particulate P (PP) were determined with procedures outlined by Parsons et al. (1984), with modifications for TDP and PP according to Menzel and Cot-win (1965). SRP was determined on unfiltered samples. PP was determined as SRP after persulfate oxidation of samples filtered onto Gelman GA-8 Supor filters (0.2~pm pore size). TDP was determined as SRP (after persulfate oxidation) in the filtrate of the latter samples. The 50 and in the concentrations of PC, PN, PP, NH,‘, containing heat-killed Pasteure[la sp. The culture no inorganic nutrients (N or P) added. Cultures and algal density. B. Changes in PC. C. Changes concentration of dissolved organic P (DOP) was calculated as TDP - SRP. Two complete sets of samples from the 1.5 experimental flasks also were analyzed for SRP after persulfate oxidation of unfiltered samples. The resulting “total” P concentrations were highly consistent with the sum of the SRP, TDP, and PP measurements for these same samples determined by the procedure described above. Phagotrophy was the dominant nutritional mode of P. malhamensis under all experimental conditions when bacteria were present at densities >, lo6 ml-’ (Fig. 1). Poterioochromonas malhamensis grew rapidly only in the presence of bacteria or in or- Notes 437 Table 2. Specific growth rates (d-l) of Poterioochromonas malhamensis cultured under 17 different light and nutrient conditions and in the presence and absence of heat-killed bacteria. Numbers in parentheses are averages for all cultures with bacteria at each light intensity. Light regime culture treatment Bacteria only Bacteria + NH,+ Bacteria + POZBacteria + NH,+ + PO,>NH,+ + PO,‘(bacteria-free) NH,+ + PO,‘+ vitamins (bacteria-free) Organically enriched medium (bacteria-free) Avg of cultures with bacteria m 0 100 50 150 Time (hl Fig. 2. Summary of the changes in algal and bacterial density in 12 cultures ofPoten’oochromonns maihamtwsis with heat-killed Pusteurelfa sp. Data from the algal cultures with heat-killed bacteria from all four nutrient regimes and all three light regimes are included. ganically enriched medium (Table 2). Furthermore, algal growth rates were virtually identical in all treatments containing bacteria (Fig. 2). Bacterivorous growth of the alga was more than an order of magnitude more rapid than growth in inorganic medium without bacteria. Rapid, exponential growth ceased as the density of bacteria in the cultures decreased to 0.5-l .O x lo6 ml-’ (Figs. 1A, 2A). Bacterivorous growth in P. malhamensis was rapid, and chloroplast autofluorescence was just barely visible by epifluorescence light microscopy. When bacteria were reduced below - lo6 ml-‘, however, rapid algal growth ceased, giving the appearance of a stationary growth phase (Fig. 2A). Algae cultured in the light became light green dur- Hi% Low Dark 1.8 1.5 1.8 1.6 1.6 1.6 1.5 1.5 1.5 1.7 1.5 1.5 CO.1 KO.1 (1.6) (1.5) co.1 CO.1 1.5 (1.7) ing this growth phase, chloroplast autofluorescence became readily apparent by epifluorescence microscopy, and clumping of the cells was visible macroscopically. Algal cells in the inorganic medium without bacteria remained autofluorescent and viable in the light, but changes in population density were negligible throughout the experiment. Bacterivory by P. malhamensis was the primary mode of nutrition in all experimental treatments containing bacteria. Light had virtually no effect on the growth rate of P. malhamensis with heat-killed bacteria present in the medium (Table 2). The average algal growth rates in the bacterized cultures were 1.7, 1.6, and 1.5 d-’ for cultures incubated at high light intensity, low light intensity, and in continuous darkness (four treatments in each light regime). These small differences in average growth rate are within the variability associated with the method used to calculate growth rates. The growth rate of the alga in culture vessels with bacteria also was unaffected by the concentrations of inorganic N (NH,‘), inorganic P (POd3-), or the addition of vitamins. The similarity of the growth curves of the alga in the bacterized cultures, and of the losses of bacteria from these cultures, in- Notes 438 Table 3. Maximal ingestion (bacteria alga’ h-l) and clearance (nl alga-’ h-l) rates for P. malhamensis on heat-killed bacteria (Pasteurella sp.) in the presence of various nutrient enrichments. (Nutrient concentrations given in text.) T~~t~Wlt High light Bacteria alone Bacteria + NH.,+ Bacteria + PO,+ Bacteria + NH,+ + PO,)Low light Bacteria alone Bacteria + NH.,+ Bacteria + POa3Bacteria + NH,+ + PO,‘Continuous darkness Bacteria alone Bacteria + NH,+ Bacteria + POa3Bacteria + NH.,+ + POd3- Ingestion Clearance 103 77 77 49 0.8 0.9 0.8 0.9 64 94 47 70 0.6 0.9 0.9 0.5 72 52 57 83 0.9 0.6 0.8 0.6 dicated the ineffectiveness of inorganic nutrient concentrations or light in altering the bacterivorous nature of P. malhamensis (Fig. 2). Growth curves for the alga were virtually identical in all treatments containing heatkilled bacteria. The patterns of disappearance of the bacteria in these cultures also were identical, indicating that grazing rates were similar in all treatments. “Maximal” ingestion (bacteria consumed alga-’ h-l) and clearance rates (nl of water filtered alga-’ h-l) were calculated according to the equations of Heinbokel (1978) for each experimental treatment. These rates were based on changes in the cell densities of the bacteria and algae between consecutive samples. The highest rates calculated for each parameter were taken as maximal. In all cases, maximal ingestion rates occurred during the first sampling interval during which the bacterial density decreased significantly. Maximal clearance rates occurred over the last sampling interval during which the bacterial density decreased significantly. The maximal rates of bacterivory by P. malharnensis were high in all cultures containing bacteria (Table 3). The presence or absence of light, inorganic N, inorganic P, or vitamins had no consistent effect on bacterivory by P. malhamensix The overall average for maximal inges- tion rate was 70 bacteria alga-i h-i for cultures with bacteria. No significant changes in PC, PN, PP, NH4+, SRP, or DOP occurred during the experimental period in the vessels with heatkilled bacteria but not inoculated with P. malhamensis. In contrast, the concentrations of PC, PN, and PP in the cultures of P. malhamensis with bacteria decreased rapidly during exponential growth of the alga (e.g. Fig. lB,C,D). Concentrations of NH4+, SRP, and DOP increased concomitantly. Most of the bacterial N and P released by the alga during bacterivory was eventually released as NH,+ and SRP (Fig. lC,D). In addition, however, some of the C, N, and P in the consumed bacterial biomass was released by the alga as dissolved organic material. Dissolved organic C and N were not determined, but small amounts of DOP (= TDP - SRP) amounting to lo-20% of the PP concentrations were measured in all algal cultures with heat-killed bacteria (Fig. 1D). We assume that a similar proportion of DON was released because, on average, the amount of NH,’ appearing in the cultures was lO-25% less than the amount of PN disappearing from the cultures (e.g. in Fig. lC, the loss of PN x 1.40 - 0.35 = 1.05 mM, the increase ofNH,+ z 0.70 mM, 1.05-0.70=0.35,and0.35+1.40=25%). It is possible that some dissolved organic material released by the alga was reassimilated and remineralized by the alga during the experimental period. It is also possible, however, that some of the observed dissolved organic material in these cultures may have been due to cell breakage during filtration rather than to algal excretion. It is difficult, therefore, to estimate the exact importance of dissolved organic material in these cultures. Nevertheless, the overall outcome is that most of ,the bacterial biomass released as dissolved material is eventually released in remineralized form. Carbon and nutrient incorporation efficiencies of P. malhamensis (expressed as percent) feeding phagotrophically on bacteria were calculated from the concentration of PC, PN, and PP in the algal cultures at the end of exponential growth of the algae (when algal biomass highly dominated the particulate fraction) divided by these con- Notes centrations at the beginning of the experiment (when bacterial biomass constituted all of the particulate fraction). It was assumed in these calculations that the contribution of bacterial biomass and defecated particulate material in the cultures at the end of the exponential growth phase of the alga was negligible. Although the former was certainly true (bacterial biomass remaining in the cultures at the end of the exponential growth phase was calculated to be less than a few percent of the particulate material), significant amounts of defecated particulate material may have been present. Therefore, incorporation efficiencies cannot be truly equated with growth efficiencies in these cultures. We estimate that our incorporation efficiencies may have overestimated the true growth efficiencies by not more than 10%. Relatively large percentages of the prey C, N, and P were incorporated into algal biomass during bacterivorous growth of P. malhamensis. Overall, the incorporation efficiencies of the alga for bacterial N, C, and P were 33, 43, and 45% at the end of bacterivorous growth (Fig. 3). The average incorporation efficiencies for C and P were not significantly different from one another, but these latter efficiencies were significantly different from the average N incorporation efficiency. The N incorporation efficiency was less than the C incorporation efficiency in all treatments with bacteria and less than the P incorporation efficiency in all of these treatments except one. There were no apparent correlations between the incorporation efficiencies of P. malhamensis in bacterized cultures and the type of experimental treatment (i.e. with light regime or nutrient additions). Average N, C, or P incorporation efficiencies were comparable for the four experimental treatments with bacteria in each of the three light regimes (Table 4). Likewise, incorporation efficiencies averaged for all bacterized treatments in each of the three light regimes were similar for the four types of bacterized treatments (Table 4). Thus, neither light nor the presence of dissolved inorganic nutrients significantly affected the growth efficiency of the alga when it was consuming bacteria. Poterioochromonas malhamensis contin- 439 60 q Nitrogen P 50 B0 40 El ig 30 3 t 2o 24 10 n Exponential Algal Growth stationary Phase Fig. 3. Average incorporation efficiencies of Poterioochromonas malhamensis fed heat-killed Pasteurella sp. in 12 cultures at the time of maximal algal density (“exponential” phase) and 2 d after the depletion of bacterial prey (“stationary” phase). Error bars are f 1 SD. ued to remineralize significant amounts of N, P, and C even after rapid population growth stopped (Fig. 3). The C, N, and P incorporation efficiencies of the alga decreased an average of 13% for all bacterized cultures between the time of maximal algal density (the end of bacterivorous growth) and 2 d after the onset of the stationary growth phase. The losses of C or nutrients during this phase were not related to the light regime and presumably were due to cannibalism or the metabolism of cellular reserves by the alga. Strictly speaking, these losses during this growth phase no longer represented incorporation efficiencies of the alga because the bacterial prey already had been exhausted. We hypothesized that P. maIhamensis might incorporate a larger percentage of the N or P from the bacterial biomass when these nutrients were not added to the culture medium. Alternatively, we hypothesized that the presence of high concentrations of inorganic nutrients might depress the rate of bacterial ingestion by the alga because these nutrients would be readily available as dissolved constituents. Neither of these possibilities occurred. These results for P. malhamensis contradict a report of enhanced predation in the absence of growth- Notes 440 Table 4. Summary of C, N, and P incorporation efficiencies (mean % + 1 SD) of Poterioochromonas malhamensis during bacterivorous growth in three different light regimes and four nutrient conditions (total of 12 treatments). Four treatments were performed in each light regime (the four nutrient conditions) and three light regimes were used for each nutrient condition (high light, 400-500 pmol quanta me2 s-l; low light, 4-10 rmol quanta rndz s-l; and continuous darkness). Light regime Nutrient High Low Carbon Nitrogen Phosphorus 44+7 38+6 41k8 42-t3 31k4 44+4 limiting, dissolved inorganic nutrients closely related species of Ochromonas Nutrient continuous darkness 43k2 3Ok2 49*7 by a (Keller et al. 1988), but they agree with the observations of Andersson et al. (1989) for another Ochromonas species in which heterotrophy was the primary mode of nutrition and in which bacterivory was not important in nutrient acquisition for photosynthetic growth. Based on the large percentage of the bacterial C that was incorporated into the alga, it is improbable that P. malhamensis consumed bacteria solely as a source of major nutrients or solely as a source of some specific growth factor. Instead, P. malhamensis apparently switched nearly completely from phototrophic growth to heterotrophic growth when sufficient bacteria were present in the culture medium. The processes involved in switching between phototrophy and heterotrophy in this species are examined elsewhere (Sanders et al. 1990). Phototrophy contributed negligibly to the growth of P. malhamensis in this study. Algal densities in cultures containing inorganic nutrients and vitamins did not change significantly during the experiment. Previous studies and our own work have demonstrated that P. malhamensis is capable of limited phototrophic growth (Handa et al. 198 1). The phototrophic growth rate of P. malhamensis was slightly greater in an acidic medium (Sanders et al. 1990) but bacterivory was the main source of C for the alga regardless of the pH of the medium or the light regime. In this study, phototrophic nutrition of the alga was important only for survival in the absence of bacterial biomass that was sufficient for heterotrophic growth. Bacteria only 41k2 31*5 48k4 Bacteria + N 45*4 33*9 49k6 condition Bacteria + P 42k5 32k4 43*11 Bacteria +N+P 45*4 35+7 40+5 Overall, the behavior of P. malhamensis in bacterized cultures was highly analogous to results that have been obtained with purely heterotrophic nanoflagellates. Maximal ingestion rates and clearance rates of P. malhamensis observed in this study are within the range of rates published for some heterotrophic flagellates of similar size, although they are certainly not the highest rates ever observed (e.g. Fenchel 1986; Davis and Sieburth 1984; Geider and Leadbeater 1988). The C, N, and P incorporation efficiencies of the alga during bacterivory also were comparable to the incorporation efficiencies of some obligately heterotrophic nanoflagellates (e.g. Kopylov et al. 1980; Fenchel 1982a; Sherr et al. 1983; Geider and Leadbeater 1988). In conceiving this experiment, we hypothesized that the concentration of dissolved inorganic nutrients in the bacterized cultures without NH,+ or POd3- might remain low if P. malhamensis was an efficient mixotroph: that is, if the nutrients contained in the ingested bacterial biomass were efficiently retained by the alga and converted into algal biomass by using photosynthesis to supplement the C budget of the cell. This result was not observed. The retention of N in the particulate fraction during grazing by P. malhamensis on average was less than the retention of C (or P) and was not affected by NH,+ availability (Fig.3; Table 4). This result is consistent with expectations for a purely heterotrophic nanoflagellate based on the relatively low C : N ratio of bacterial biomass (Table 1; see Nagata 1986; Lee and Fuhrman 1987) the percentage of C incorporated by P. malhamen- Notes sis, and the expected C : N ratio of protist biomass (Goldman et al. 1987). It is also consistent with the observation that heterotrophic nanoflagellates apparently catabolize protein-rich compounds more readily than other cellular components (Fenchel 1982b; Caron et al. 1985; Goldman et al. 1985; Andersen et al. 1986). In general, the cycling of C and nutrients by the chrysophyte alga P. malhamensis was very similar to previous studies on nutrient and C cycling by the omnivorous (but obligately heterotrophic) marine nanoflagellate, Paraphysomonas imperjbrata (Goldman et al. 1985, 1987; Caron et al. 1985; Andersen et al. 1986). The relative proportions of N, P, and C incorporated and remineralized by the alga were very similar to patterns observed in C and nutrient cycling by P. imperforata (Fig. 3). The proportions of particulate C, N, and P retained in the particulate fraction during algal phagotrophy indicate an inability by the alga to retain “excess” nutrients contained in its prey for subsequent use during phototrophic growth. The release of remineralized N and P from the particulate fraction of the P. malhamensis cultures in the light even after the bacterial population was depleted and autofluorescence increased is particularly striking (Fig. 3). This reduction may indicate the “autophagy” of cellular components during the stationary growth phase of the species, similar to results obtained for Ochromonas (Fenchel 1982a). Alternatively, this result may indicate a shift to a cannibalistic mode of nutrition once bacterial prey were depleted. This latter alternative is highly probable because we frequently observed cannibalism by this alga, and the high density of algal cells in these cultures at the end of the experimental period increases the probability of encounters between the cells. The tendency of the alga to cannibalize other algal cells rather than rely on photosynthesis for growth further indicates the importance of phagotrophy for the nutrition of this species. The information that we have obtained on the mixotrophic chrysophyte P. malhamensis indicates that this alga strongly favors phagotrophy as its primary mode of 441 nutrition. Under all conditions tested, P. malhamensis rapidly ingested bacteria when they were present in the culture medium at densities > 1O6 ml-*. The alga is a highly competent bacterivore and should be able to compete successfully with other bacterivorous protists in planktonic communities. Under conditions that will support phagotrophy of P. malhamensis, it supplies organic C and major nutrients for growth. In these situations, C and nutrient incorporation and remineralization are analogous to C and nutrient cycling by truly heterotrophic protists. Poterioochromonas malhamensis developed visible autofluorescence (i.e. increased its chlorophyll content) only when bacteria were not present in sufficient densities, and population growth of the alga under our experimental conditions occurred only during baterivory. On the basis of the present study, we conclude that phototrophy is of minor importance for the growth of this species. Presumably the ecological significance of mixotrophy for P. malhamensis involves a long-term survival strategy (phototrophy) for times when bacterial density is insufficient to support its primary mode of nutrition (phagotrophy). Moreover, major nutrients (N and P) obtained during bacterivory are not retained efficiently by the alga for subsequent use during phototrophic growth. For the mixotrophic P. malhamensis, phagotrophy and phototrophy seem to be nearly mutually exclusive but in series promote the growth and survival of the organism. David A. Caron Biology Department Woods Hole Oceanographic Institution Woods Hole, Massachusetts 02543 Karen G. Porter Zoology Department University of Georgia Athens 30602 Robert W. Sanders Division of Environmental Research Philadelphia Academy of Natural Sciences 19th and the Parkway Philadelphia, Pennsylvania 19 103 References ANDE~SEN, 0. K., J. C. GOLDMAN, D. A. CARON, AND M. R. DENNIZTT. 1986. Nutrient cycling in a microflagellate food chain: 3. Phosphorus dynamics. Mar. Ecol. Prog. Ser. 31: 47-55. ANDERSSON,A., S. FALK, AND G. SAMUEISSON. 1989. Nutritional characteristics of a mixotrophic nanoflagellate, Ochromonas sp. Microb. Ecol. 17: 252262. 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MAITA, AND C. M. LALLI. 1984. A manual of chemical and biological methods for seawater analysis. Pergamon. PORTER, K. G. 1988. Phagotrophic phytoflagellates in microbial food webs. Hydrobiologia 159: 8997. -, AND Y. S. FEIG. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948. -, E. B. SHERR, B. F. SHERR, M. PACE, AND R. W. SANDERS. 1985. Protozoa in planktonic food webs. J. Protozool. 32: 409-415. SALONEN, K., AND S. JOK~NEN. 1988. Flagellate grazing on bacteria in a small dystrophic lake. Hydrobiologia 161: 203-209. SANDERS,R. W., AND K. G. PORTER. 1988. Phagotrophic phytoflagellates. Adv. Microb. Ecol. 10: 167-192. -, -, S. J. BENNETT, AND A. E. DEBIASE. 1989. Seasonal patterns of bacterivory by flagellates, ciliates, rotifers and cladocerans in a freshwater planktonic community. Limnol. Oceanogr. 34: 673-687. -, -, AND D. A. CARON. 1990. Relationship between phototrophy and phagotrophy in the mixotrophic chysophyte Poterioochromonas malhamensis. Microb. Ecol. 19: 97-109. Notes B. F., E. B. SHERR, AND T. BERMAN. 1983. Grazing, growth, and ammonium excretion rates of a heterotrophic microflagellate fed with four species of bacteria. Appl. Environ. Microbial. 45: 1196-1201. STARR, R. C. 1978. The culture collection of algae at the University of Texas at Austin. J. Phycol. 14: 47-100. SHERR, Limnol. Oceanogr.. 3X2), Q 1990, by the American 1990,443-447 Society of Limnology and Oceanography, STEIN, 443 J. R. [ED.]. 1973. Handbook methods. Cambridge. of phycological Submitted: 12 June 1989 Accepted: 16 August 1989 Revised: 18 December 1989 Inc. Distribution of labile dissolved organic carbon in Lake Michigan Abstract -Bioassay-measured, labile dissolved organic carbon (LDOC) concentrations were compared in near-bottom and near-surface Lake Michigan water between April and October 1986. In five of seven experiments, the LDOC concentration was higher in near-bottom water. LDOC reached 40.2% of the total DOC pool in the near-bottom water in late May and 13.8% in the near-surface water in early July. Concentration in near-bottom water was highest during early stratification; concentration in surface water varied less but was highest in early July. The data suggest that an allochthonous source of labile organic C may be important. Identifying primary sources of labile dissolved organic carbon (LDOC) in pelagic systems has taken on new interest in light of recent evidence that a major portion of the C fixed via autotrophic production passes through heterotrophic bacteria (Love11 and Konopka 1985; Scavia et al. 1986; Scavia and Laird 1987; Nagata 1987) and that much of the bacterial production is grazed (Scavia and Laird 1987; Nagata 1987). This transfer of LDOC to primary consumers captures C that might otherwise be lost. Recently, we reported that in Lake Michigan a disequilibrium between phytoplankton organic C production (supply) and bacterial C use (demand) on seasonal and spatial scales may exist (Scavia and Laird 1987). Acknowledgments We thank M. T. Babbitt for technical assistance and W. S. Gardner and G. L. Fahnenstiel for their suggestions and improvements to the manuscript. GLERL Contribution 66 1. Although annual bacterial C demand (236 g C m-2 yr-I) appeared to be balanced by annual phytoplankton net C production (225 g C me2 yr-I), summer bacterial C demand (1,087 mg C mm2 d-‘) could not be satisfied by net summer phytoplankton C production (627 mg C mm2 d-l), indicating that a large discrepancy exists between DOC demand and supply. This discrepancy was even more dramatic when restricted to the summer epilimnion. We suggested that seasonal and spatial disequilibrium between organic C supply and demand could explain the apparent deficit. The hypothesis suggested that temporal disequilibrium is driven when bacterial production is low (winter or early spring) and LDOC accumulates, exceeding bacterial demand. Also, spatial disequilibrium is driven by accumulation of organic matter in cold, deep regions through sedimentation of phytoplankton, detritus, and other decaying material. These supplies of LDOC would then become available throughout the water column during winter mixing. Strayer (1988) generalized the issue of secondary production in ecosystems and questioned the need for our disequilibrium hypothesis. He based his analysis on model calculations demonstrating that because organic C is recycled, total consumer C demand may exceed organic C inputs to ecosystems. Extension of that model analysis (Scavia 1988) corroborated that consumer demand can be met by autotrophic production on an annual water-column basis in Lake Michigan; it also suggested, however,