photographing an additional 10 random fields for each sam- ple and digitizing the ... phosphate substrate as described by Wetzel (41; values reported in that ...
Vol. 58, No. 1
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 150-156
0099-2240/92/010150-07$02.00O/0 Copyright © 1992, American Society for Microbiology
Effects of Nutrients on Specific Growth Rate of Bacterioplankton in Oligotrophic Lake Water Culturest MICHAEL F. COVENEYt* AND ROBERT G. WETZEL§ W. K. Kellogg Biological Station, Michigan State University, Hickory Corners, Michigan 49060 Received 22 July 1991/Accepted 29 October 1991
The effects of organic and inorganic nutrient additions on the specific growth rates of bacterioplankton in oligotrophic lake water cultures were investigated. Lake water was first passed through 0.8-p.m-pore-size filters (prescreening) to remove bacterivores and to minimize confounding effects of algae. Specific growth rates were calculated from changes in both bacterial cell numbers and biovolumes over 36 h. Gross specific growth rates in unmanipulated control samples were estimated through separate measurements of grazing losses by use of penicillin. The addition of mixed organic substrates alone to prescreened water did not significantly increase bacterioplankton specific growth rates. The addition of inorganic phosphorus alone significantly increased one or both specific growth rates in three of four experiments, and one experiment showed a secondary stimulation by organic substrates. The stimulatory effects of phosphorus addition were greatest concurrently with the highest alkaline phosphatase activity in the lake water. Because bacteria have been shown to dominate inorganic phosphorus uptake in other P-deficient systems, the demonstration that phosphorus, rather than organic carbon, can limit bacterioplankton growth suggests direct competition between phytoplankton and bacterioplankton for inorganic phosphorus.
Studies of the seasonal dynamics of bacterioplankton frequently show a relative constancy of cell densities over time (2, 28). It is commonly believed that densities of free planktonic bacteria are controlled by protozoan grazers but that growth rates are limited by the availability of reduced carbon substrates (2, 15, 16, 46). Organic carbon limitation is consistent with the fact that most natural waters constitute very dilute organic media and is supported by the positive relationship noted in cross-system studies between bacterial production and phytoplankton biomass (6, 44). Since bacterial production, which is the product of the two components specific growth rate (SGR) and cell density, could vary with changes in either component, an understanding of nutritional limitations for bacterioplankton is best achieved by consideration of SGRs. Direct evidence regarding controlling factors for bacterial SGRs in natural systems is rare. Temperature is clearly one environmental factor which can constrain bacterial growth (5, 18, 21, 30). A recent compilation of data from a variety of habitats showed a strong, positive relationship between bacterial SGRs and temperature (44). A seasonal shift from temperature regulation to probable organic substrate regulation of SGRs was demonstrated for Lake Michigan bacterioplankton (33). The requirement of aquatic bacteria for inorganic nutrients (especially N and P) is usually assumed to be met either through the utilization of organically bound fractions or through high-affinity uptake systems for inorganic forms. There is widespread support for the idea that Pi uptake in P-limited aquatic systems is dominated by bacteria because
their uptake systems have higher affinities than those of algae (11-13, 40). Nutrient bioassays have been used in a limited number of natural communities to compare the effects of organic and inorganic nutrient additions on bacterial growth. In the majority of these experiments, organic substrates, rather than inorganic nutrients, were concluded to limit bacterial growth (22, 23, 29). Recently, the addition of Pi was shown to stimulate the production of an inoculum of bacterioplankton diluted in filtered lake water (39). Our investigations of bacterial dynamics and pelagic carbon cycling in an oligotrophic lake provided evidence that bacterial production was not controlled solely by the availability of labile organic substrates. Bacterial metabolism of about one-half the labile extracellular organic carbon released by phytoplankton was slow (a first-order rate constant mode of 0.05 to 0.15 day-') (9), indicating the potential accumulation of a pool of labile substrate. Bacterial production in this lake was uncoupled from photosynthesis in the short term and varied little from day to night (7a), further evidence for sufficient organic carbon supply. To address the question of whether organic substrate availability limited bacterial growth rates in this ecosystem, we conducted a series of nutrient enrichment experiments with lake water batch cultures. Changes in bacterial numbers and biovolumes were used as direct measures of growth for the calculation of SGRs. We demonstrated that organic substrate enrichments alone did not increase bacterial growth and that the addition of inorganic phosphorus was
frequently stimulatory. MATERIALS AND METHODS
* Corresponding author. t Contribution no. 713 of the W. K. Kellogg Biological Station, Michigan State University, Hickory Corners, MI 49060. t Present address: Department of Surface Water Programs, St.
Experimental design. Nutrient addition experiments for examining bacterial growth were conducted with lake water batch cultures and were a subset of experiments for examining conversion factors for the [3H]thymidine assay for bacterial production (8). The lake water batch culture experiments were designed to test two a priori comparisons. First,
Johns River Water Management District, P.O. Box 1429, Palatka, FL 32078-1429. § Present address: Department of Biology, University of Alabama, Tuscaloosa, AL 35487-0344. 150
VOL. 58, 1992
the effects of nutrient additions on SGRs of bacteria in the absence of grazers were evaluated by nutrient amendments after filtering (prescreening). Bacterial growth with these nutrient additions was compared with that in prescreened water without nutrient additions. Prescreening served two functions: the removal of bacterivores to allow a bacterial growth response and the removal of most algae to ensure that bacteria responded directly to nutrient amendments and not indirectly to changes in algal metabolism. One requirement was that prescreening per se should have no direct effect on bacterial growth, so the second comparison examined possible effects of the prescreening process. In these experiments, SGRs in prescreened water were compared with those of controls corrected for grazing losses. Experimental procedures. Integrated epilimnetic (depth, 0 to 4 m) water samples were taken from August through October 1986 from Lawrence Lake, an oligotrophic, hardwater lake in southwestern Michigan. Lawrence Lake has a surface area of 5 ha and a mean depth of 5.9 m. Representative nutrient concentrations are as follows: soluble reactive P, 0.05). Bacterial growth was anomalous in most treatments on 1 October (Fig. 3 and Table 1). SGRs were negative after prescreening, evidence for nongrazing mortality. Both Pi and Pi plus organic additions to prescreened water significantly increased SGRs, which became positive. Mean bacterial cell sizes were low during this study, representative of conditions in Lawrence Lake and similar to those in other oligotrophic systems (33). Initial mean cell volumes ranged from 0.046 to 0.051 ,um3 for whole lake water (Fig. 4). Prescreening resulted in the selective removal of larger cells and decreased the initial mean cell volumes by an average of 7%. On the basis of mean cell volumes after experimental incubation, treatments were segregated into two groups for experiments on 6 August and 18 August: prescreening alone and prescreening plus organic substrates resulted in lower cell volumes, and prescreening plus Pi and
VOL. 58, 1992
NUTRIENT LIMITATION IN BACTERIOPLANKTON 0.50
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TREATMENT FIG. 3. SGRs in lake water cultures after prescreening only (F) or prescreening plus nutrient amendments (FO, organic substrates; FP, Pi; FPO, P1 plus organic substrates). Control (C) and penicillin-treated (PEN) cultures are shown for comparison. Asterisks denote significance levels in comparisons of SGRs after nutrient amendments with SGRs after prescreening only: *, marginally significant (P < 0.05); **, significant (P < 0.017). Error bars denote 1 standard error. NO, number; BIOVOL, biovolume.
the control treatment (whole water with no amendments) resulted in higher cell volumes. The large increase in the SGR after Pi plus organic additions on 18 August was accompanied by a severalfold increase in mean cell volume. Final cell volumes on 16 September showed no significant differences across treatments. With one exception, the ratios of number of cocci to number of rods were about 45:55 for control and treatment flasks in all experiments. In the single exception, this ratio changed to approximately 25:75 after Pi plus organic additions on 18 August.
TABLE 1. Gross SGRs estimated for unmanipulated control flasks and SGRs after prescreening. Prescreened values significantly different from those of controls are marked (1-way ANOVA and linear comparison; n = 2) measure
Growth
Gross SGR'
Prescreening SGR
Number Biovolume
0.258 0.530
0.222
18 August
Number Biovolume
0.155 0.213
0.126 0.207
16 September
Number Biovolume
0.197 0.387
0.135 0.281
1 October
Number Biovolume
0.145 0.416
-0.086b -0.069b
Expt
6 August
a See the text for details. bSignificant at P < 0.05.
0.061b
DISCUSSION In
no
experiment did the addition of mixed organic sub-
strates alone significantly increase the SGR of Lawrence Lake bacterioplankton (Fig. 3). These results are in contrast to those of other studies in which organic additions stimu-
lated bacterial growth (22, 23). The mixed organic amendments consisted of low-molecular-weight compounds which were shown to be readily utilized by oligotrophic bacterial strains (20). That the lack of response to organic additions did not result from the inability of the bacterioplankton to utilize the added compounds was demonstrated in the 18 August experiment: SGRs were much higher when Pi and organic substrates were added than when Pi was added alone (Fig. 3). These data do not support the common view that the growth of bacterioplankton in phosphorus-deficient lakes is limited by the availability of reduced carbon substrates (12). In contrast, the addition of Pi alone significantly increased one or both SGRs in three of the four experiments (Fig. 3). Our results indicate that low Pi availability can limit bacterioplankton growth in Lawrence Lake and are supported by a recent report of Pi stimulation of bacterial production in a meso-eutrophic lake (39). P1 limitations in the Lawrence Lake ecosystem also have been found for planktonic algae (41) and submersed macrophytes and epiphytic algae (27, 42a). The time period covered by these experiments was sufficient to demonstrate a temporal variability in bacterioplankton response to nutrient additions. P1 addition stimulated SGRs on both 6 August and 18 August, while the same experiment on 16 September resulted in no significant response (Fig. 3). The effects of Pi addition, significant in
APPL. ENVIRON. MICROBIOL.
COVENEY AND WETZEL
154
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TREATMENT FIG. 4. Initial and final cell volumes in control and treated lake water cultures. Initial values are shown for both whole water (blank columns) and prescreened water (cross-hatched columns), and final values are identified by corresponding columns. Error bars denote 1 standard error. Letter codes mark means which were not significantly different (P > 0.05), as determined by a one-way ANOVA and Tukey's studentized range procedure (34 [p. 234]). Treatments are as defined in the legend to Fig. 3.
and absent in September, changed in parallel with alkaline phosphatase activity assayed in the lake water (Fig. 1). Elevated alkaline phosphatase activity in Lawrence Lake has been shown to indicate phosphorus limitation of phyto-
August
plankton growth (41). The present experiments imply a contemporaneous Pi limitation of bacterioplankton growth as well. Differential centrifugation demonstrated that 40% of alkaline phosphatase activity was associated with nonalgal, presumably bacterial, particles on one occasion in this lake (35).
Bacterioplankton have been demonstrated to possess high-affinity uptake systems for Pi and to dominate P1 uptake in P-deficient lakes (4, 11-14, 31, 40). A model for the coexistence of bacteria and algae under dilute nutrient conditions has been proposed in which algae are P limited while bacteria obtain sufficient P through the efficient uptake
of Pi but are limited by the availability of organic substrates (12). The present experiments with Lawrence Lake water indicated the inverse for bacterioplankton-organic substrates did not increase growth, while Pi was stimulatory. Our data support an alternate hypothesis that for small P-limited lakes, allochthonous organic carbon supplies are large, and both bacteria and algae are P limited (37). Allochthonous and littoral sources contribute about 70% of annual organic carbon inputs to Lawrence Lake (42 [p. 699]), and an appreciable portion of this organic matter is likely transported to the pelagic zone. It is difficult to explain how phytoplankton with inferior Pi uptake systems survive in P-deficient environments. Estimates of P available to algae through phosphatase-mediated hydrolysis of phosphomonoesters either showed this to be an insufficient source (3, 7, 11, 17) or were inconclusive (11). Phytoplankton in Lake Michigan were shown to utilize primarily Pi, while bacterioplankton depended mainly on organic P (37). However, bacteria in Lake Michigan appear to be limited by organic substrates rather than by P1 (23). A recent analysis of multiple lake data sets did not support any of the current conceptual models for phosphorus-bacterialalgal interactions (10). Instead, a scheme was proposed in which bacterioplankton and phytoplankton both depend on P1 availability and some other factor (temperature, for example) and relate mutualistically to each other. However, this analysis also supported bacterial-algal competition for P at the low total phosphorus levels characteristic for Lawrence Lake (10). The demonstration in the present study that bacterioplankton, which are superior competitors for Pi, may periodically suffer P, rather than organic C, limitation raises the question as to what P sources are available to phytoplankton. One solution would depend on a heterogeneous P1 supply (patches) and a greater storage capacity for P in the larger algal cells. Lysing algae and feeding and excreting zooplankters are possible "point sources" that could create microscale patches of P1. Lehman and Scavia (25, 26) demonstrated that algal cells could exploit patches of Pi. Elevated P1 concentrations have been shown to favor uptake by algae in mixed algal-bacterial cultures (31) and by larger, presumably algal, particles in both freshwater and marine environments (24, 36). The experiment started on 1 October deviated from the other three experiments by the occurrence of nongrazing mortality (Fig. 3). At least two different but indistinguishable mechanisms could have caused these results. First, nongrazing mortality may have been present in all treatments. This would have increased the grazing estimate provided through penicillin treatment but would not have been prevented by prescreening. Second, the prescreening process may have induced nongrazing mortality. Regardless of the cause, the additional mortality was partially alleviated by P1 addition. The 1 October experiment followed a period of high rainfall, and epilimnetic bacterial numbers were unusually high (Fig. 1). This short-lived increase in bacterial density may have been stimulated by the heavy input to Lawrence Lake of allochthonous dissolved organic matter which accompanies heavy precipitation events (43). However, assays of in situ bacterial production ([3H]thymidine incorporation) conducted 1 week before and 1 week after 1 October revealed similar, moderate levels (data not shown). It is possible that high bacterial densities resulted from the input of allochthonous wetland soil bacteria rather than from autochthonous bacterial production. In this case, the nongrazing mortality evident in the 1 October experiment could
VOL. 58, 1992
NUTRIENT LIMITATION IN BACTERIOPLANKTON
have been evidence of acute nutrient limitation or other physiological stress of an aquatic environment. An allochthonous origin for the high densities of bacteria following this storm event is supported by observations in impoundments in which the heterotrophic activity of bacterioplankton increased sharply following storms but cell numbers remained unchanged (19, 32). Bacterial production was also measured as [3H]thymidine incorporation over the course of incubation of these lake water cultures, and the resulting data have been reported elsewhere (8). There was an effect of Pi addition on the relationship between [3H]thymidine incorporation and bacterial production (i.e., the conversion factor for the [3H]thymidine assay) (8). This sensitivity of the conversion factor to nutrient addition obviated the use of [3H]thymidine incorporation for measuring bacterial production in response to nutrient additions in the present experiments and necessitated direct cell counting and sizing techniques. SGRs were calculated for experimental flasks by use of an exponential growth model and initial and final cell numbers and biovolumes. Exponential growth has been shown to occur in the absence of bacterivores (1), and the use of SGR as the growth measure has several advantages. Under nutrient-limited conditions, SGR would respond directly to an increase in the concentration of the limiting nutrient. Also, SGR should be independent of small changes in initial cell density, allowing a direct comparison between whole-water and prescreening treatments. Finally, the calculation of the gross SGR is straightforward, as SGR for control flasks minus that for penicillin treatment flasks. Bacterioplankton growth in prefiltered batch cultures has sometimes been found to be linear or sigmoidal or to start after an initial lag phase (45). Sigmoidal or lag-phase patterns would result in an underestimation of the SGR by endpoint measurements. Although sigmoidal growth curves were not evident in the present experiments, initial growth lags or slight decreases in density were common (Fig. 2). To evaluate the importance of deviations from ideal exponential growth, we determined bacterial cell numbers and biomass at 12-h intervals for the 6 August experiment. An additional set of SGRs for 6 August were derived from these time course data by regression of the natural logarithm of cell density over time, with initial density omitted. As expected, endpoint (initial to final) SGRs were lower than those calculated from time courses and averaged 50 and 54% of the latter for SGRN and SGRB, respectively. Endpoint SGRs were regressed on time course SGRs, and the residuals were examined for treatment effects by a one-way ANOVA. Treatment effects were not significant (P > 0.05) for either SGRN or SGRB. Thus, although growth lags clearly resulted in low SGRs when calculation was limited to initial and final cell densities, there was no evidence for bias in the results of the present experiments.
2. Azam, F., T. Fenchel, J. G. Field, J. S. Gray, L. A. Meyer-Reil, and F. Thingstad. 1983. The ecological role of water-column microbes in the sea. Mar. Ecol. Prog. Ser. 10:257-263. 3. Boavida, M. J., and R. T. Heath. 1988. Is alkaline phosphatase always important in phosphate regeneration? Arch. Hydrobiol. 111:507-518. 4. Chr6st, R. J., and J. Overbeck. 1987. Kinetics of alkaline phosphatase activity and phosphorus availability for phytoplankton and bacterioplankton in Lake Plussee (north German eutrophic lake). Microb. Ecol. 13:229-248. 5. Coffin, R. B., and J. H. Sharp. 1987. Microbial trophodynamics in the Delaware Estuary. Mar. Ecol. Prog. Ser. 41:253-266. 6. Cole, J. J., S. Findlay, and M. L. Pace. 1988. Bacterial production in fresh and saltwater ecosystems: a cross-system overview. Mar. Ecol. Prog. Ser. 43:1-10. 7. Cotner, J. B., Jr., and R. T. Heath. 1988. Potential phosphate release from phosphomonoesters by acid phosphatase in a bog lake. Arch. Hydrobiol. 111:329-338. 7a.Coveney, M. F. Unpublished data. 8. Coveney, M. F., and R. G. Wetzel. 1988. Experimental evaluation of conversion factors for the [3H]thymidine incorporation assay of bacterial secondary productivity. Appl. Environ. Microbiol. 54:2018-2026. 9. Coveney, M. F., and R. G. Wetzel. 1989. Bacterial metabolism of algal extracellular carbon. Hydrobiologia 173:141-149. 10. Currie, D. J. 1990. Large-scale variability and interactions among phytoplankton, bacterioplankton, and phosphorus. Limnol. Oceanogr. 35:1437-1455. 11. Currie, D. J., E. Bentzen, and J. Kalff. 1986. Does algalbacterial phosphorus partitioning vary among lakes? A comparative study of orthophosphate uptake and alkaline phosphatase activity in freshwater. Can. J. Fish. Aquat. Sci. 43:311318. 12. Currie, D. J., and J. Kalff. 1984. A comparison of the abilities of freshwater algae and bacteria to acquire and retain phosphorus. Limnol. Oceanogr. 29:298-310. 13. Currie, D. J., and J. Kalff. 1984. The relative importance of bacterioplankton and phytoplankton in phosphorus uptake in freshwater. Limnol. Oceanogr. 29:311-321. 14. Currie, D. J., and J. Kalff. 1984. Can bacteria outcompete phytoplankton for phosphorus? A chemostat test. Microb. Ecol. 10:205-216. 15. Fenchel, T. 1982. Ecology of heterotrophic microflagellates. IV. Quantitative occurrence and importance as bacterial consumers. Mar. Ecol. Prog. Ser. 9:35-42. 16. Hadas, O., R. Pinkas, C. Albert-Diez, J. Bloem, T. Cappenberg, and T. Berman. 1990. The effect of detrital addition on the development of nanoflagellates and bacteria in Lake Kinneret. J. Plankton Res. 12:185-199. 17. Heath, R. T. 1986. Dissolved organic phosphorus compounds: do they satisfy planktonic phosphate demand in summer? Can. J. Fish. Aquat. Sci. 43:343-350. 18. Hobbie, J. E., and J. J. Cole. 1984. Response of a detrital foodweb to eutrophication. Bull. Mar. Sci. 35:357-363. 19. Hubbard, J. G., and T. H. Chrzanowski. 1986. Impact of storms on heterotrophic activity of epilimnetic bacteria in a southwestern reservoir. Appl. Environ. Microbiol. 51:1259-1263. 20. Ishida, Y., and H. Kadota. 1981. Growth patterns and substrate requirements of naturally occurring obligate oligotrophs. Microb. Ecol. 7:123-130. 21. Joint, I. R., and A. J. Pomroy. 1987. Activity of heterotrophic bacteria in the euphotic zone of the Celtic Sea. Mar. Ecol. Prog. Ser. 41:155-165. 22. Kirchman, D. L. 1990. Limitation of bacterial growth by dissolved organic matter in the subarctic Pacific. Mar. Ecol. Prog. Ser. 62:47-54. 23. Laird Pernie, G., D. Scavia, M. L. Pace, and H. J. Carrick. 1990. Micrograzer impact and substrate limitation of bacterioplankton in Lake Michigan. Can. J. Fish. Aquat. Sci. 47:1836-1841. 24. Lean, D. R. S., and E. White. 1983. Chemical and radiotracer measurements of phosphorus uptake by lake plankton. Can. J. Fish. Aquat. Sci. 40:147-155. 25. Lehman, J. T., and D. Scavia. 1982. Microscale nutrient patches
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation (grant BSR 8517039) and the Department of Energy (contracts DE-FG02-87ER60515 and COO-1599-342). N. L. Consolatti assisted with the experiments and with bacterial enumeration and size measurements. REFERENCES 1. Ammerman, J. W., J. A. Fuhrman, A. Hagstrom, and F. Azam. 1984. Bacterioplankton growth in seawater. I. Growth kinetics and cellular characteristics in seawater cultures. Mar. Ecol. Prog. Ser. 18:31-39.
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37. Tarapchak, S. J., and R. A. Moll. 1990. Phosphorus sources for phytoplankton and bacteria in Lake Michigan. J. Plankton Res. 12:743-758. 38. Taylor, W. D., and R. G. Wetzel. 1988. Phytoplankton community dynamics in Lawrence Lake of southwestern Michigan. Arch. Hydrobiol. Suppl. 81:491-532. 39. Toolan, T., J. D. Wehr, and S. Findlay. 1991. Inorganic phosphorus stimulation of bacterioplankton production in a mesoeutrophic lake. Appl. Environ. Microbiol. 57:2074-2078. 40. Vadstein, O., and Y. Olsen. 1989. Chemical composition and phosphate uptake kinetics of limnetic bacterial communities cultured in chemostats under phosphorus limitation. Limnol. Oceanogr. 34:939-946. 41. Wetzel, R. G. 1981. Longterm dissolved and particulate alkaline phosphatase activity in a hardwater lake in relation to lake stability and phosphorus enrichments. Verh. Int. Verein. Limnol. 21:369-381. 42. Wetzel, R. G. 1983. Limnology, 2nd ed. The W. B. Saunders Co., Philadelphia. 42a.Wetzel, R. G. Unpublished data. 43. Wetzel, R. G., and A. Otsuki. 1974. Allochthonous organic carbon of a marl lake. Arch. Hydrobiol. 73:31-56. 44. White, P. A., J. Kalif, J. B. Rasmussen, and J. M. Gasol. 1991. The effect of temperature and algal biomass on bacterial production and specific growth rate in freshwater and marine habitats. Microb. Ecol. 21:99-118. 45. Wright, R. T., and R. B. Coffin. 1984. Measuring microzooplankton grazing on planktonic marine bacteria by its impact on bacterial production. Microb. Ecol. 10:137-149. 46. Wright, R. T., R. B. Coffin, and M. E. Lebo. 1987. Dynamics of planktonic bacteria and heterotrophic microflagellates in the Parker Estuary, northern Massachusetts. Continental Shelf Res. 7:1383-1397.
