Phytoplankton utilization of ammonium and nitrate in Lake Bonney: A ...
sumption during denitrification and possible mechanisms for its production. Deep-Sea Research, 25(6), 509-524. Glover, H.E. 1983. Measurement of chemoautotrophic CO2 assimilation in marine nitrifying bacteria: An enzymatic approach. Marine Biology, 74(3), 295-300. Goreau, T.J., W.A. Kaplan, S.C. Wofsy, M.B. McElroy, F.W. Valois, and S.W. Watson. 1980. Production of NO 2 and N20 by nitrifying bacteria at reduced concentrations of oxygen. Applied and Environmental Microbiology, 40(3), 526-532. Johnstone, B.H., and R.D. Jones. 1989. A study on the lack of [Methyl3H] thymidine uptake and incorporation by chemolithotrophic bacteria. Microbial Ecology, 18(1), 73-77. Priscu, J.C., B.B. Ward, and M.T. Downes. 1993. Water column transformations of nitrogen in Lake Bonney, a perennially ice-covered antarctic lake. Antarctic Journal of the U.S., 28(5). Ward, B.B., and A.F. Carlucci. 1985. Marine ammonium- and nitriteoxidizing bacteria: Serological diversity determined by immunofluorescence in culture and in the environment. Applied and Environonmental Microbiology, 50(2), 194-201. Ward, B.B., and A.R. Cockcroft. 1993. Immunofluorescence detection of the denitrifying bacterium, Pseudomonas perfectomarina, in seawater and intertidal sediment environments. Microbial Ecology, 25(3), 233-246. Ward, B.B., A.R. Cockcroft, and K.A. Kilpatrick. 1993. Antibody and DNA probes for detection of nitrite reductase in seawater. Journal of General Microbiology, 139(9), 2285-2293. Woolston, C., and J.C. Priscu. 1993. Phytoplankton utilization of ammonium and nitriate in Lake Bonney: A preliminary assessment. Antarctic Journal of the U.S., 28(5).
the west lobe in much higher numbers than in the east lobe at comparable depths in the oxygen depleted deep waters (figure 2). ELB17 shows a distribution pattern that is quite different from the bacterial population as a whole, albeit at much lower abundance levels. Before our next field season, we plan to analyze the approximately 100 DNA extracts we collected in 1992 (using tangential flow filtration to concentrate 4-liter samples). We are also planning to produce more polyclonal antisera to other Lake Bonney denitrifier isolates and to characterize these isolates as to their temperature and salinity ranges and optima. We have been unable so far to isolate any nitrifying strains from lake samples. Our nitrifier probes, however, are ready for application in next year's program. This research was supported by National Science Foundation grant OPP 91-17907.
References Bartlett, R., C. Woolston, and J.C. Priscu. 1993. Influence of high salinity levels on 15-nitrogen extraction efficiency in Lake Bonney, Antarctica. Antarctic Journal of the U.S., 28(5). Cohen, Y., and L.I. Gordon. 1978. Nitrous oxide in the oxygen minimum of the eastern tropical North Pacific: Evidence for its con-
Phytoplankton utilization of ammonium and nitrate in Lake Bonney: A preliminary assessment CHRISTOPHER D. WOOLSTON and JOHN C. PRISCU, Department of Biological Sciences, Montana State University, Bozeman, Montana 59717
ambient uptake rates of inorganic nitrogen and carbon. The results provide preliminary insight into the interactions of nutrients and microplankton; furthermore, they set the groundwork for future investigations of nutrient cycling and primary production in the lake. In this report, uptake refers to transport plus assimilation, two distinct pathways which could not be distinguished by our experimental designs. The Michaelis-Menten parameters Vm (the maximum specific uptake rate) and Km (the substrate concentration at which uptake is half of the maximal rate) were determined with substrate kinetics experiments. In these experiments, water samples from 4.5 meters (m) received varying levels of enrichment of nitrogen-15-labeled NH4 or NO 3- and were incubated for approximately 24 hours. The suuspended particulate matter in the water samples was collected on precombusted GF/C filters which were frozen until analysis. The accumulation of nitrogen-15 in the particulate matter was measured by atomic emission spectrometry following Dumas combustion of filters (Timperly and Priscu 1986). Uptake rates were calculated from the equations formulated by Dugdale and Goering (1967).
ake Bonney is characterized by strong gradients in nutriLents and algal concentrations (figure 1 and Priscu, Ward, and Downes, Antarctic Journal, in this issue). In this permanently ice-covered quiescent environment, nutrient transformations and microplanktonic activity are hypothesized to be tightly coupled. This should be particularly true for transformations of inorganic nitrogen, because nitrogen has been shown to limit phytoplankton photosynthesis in a number of antarctic lakes (Priddle et al. 1986; Priscu, Vincent, and Howard-Williams 1989; Sharp and Priscu 1990). Our current research on Lake Bonney is based on the hypothesis that regeneration of ammonium (NH 4 ) and nitrate (NO3i through heterotrophic remineralization and nitrification, respectively, support the bulk of productivity in the upper trophogenic zone of Lake Bonney (Priscu et al., Antarctic Journal, in this issue). This report focuses on a preliminary assessment of the physiological capacity of planktonic assemblages for inorganic nitrogen uptake in the trophogenic zone of the east lobe of Lake Bonney. Specifically, our study provides estimates for the effects of substrate concentration on uptake kinetics as well as
ANTARCTIC JOURNAL - REVIEW 1993
241
zooplankton capable of producing NH4 patches, the differential affinity can perhaps best be explained by the energy requirement of assimilation. Estimates of ambient uptake rates (figure 2) were computed using ambient nutrient concentrations in conjunction with Michaelis- Menten parameters for uptake. These ambient rates indicate that NH4 is the predominant source of nitrogen utilized throughout the trophogenic zone. Furthermore, the relative contribution of NH4 to nitrogenous nutrition increases with depth from 6 to 12 m. This supports the hypothesis that phytoplankton near the first chemical gradient (at approximately I m) are supported by the upward dif fusion of NH4 (Priscu et al., Antarctic Journal, this issue). There is reason, however, to interpret these data with caution. First, because nutrient distributions and physiological response may change with time, any estimate of uptake based on single measurements of nutrient concentration and Michaelis- Menten parameters will reflect instantaneous conditions only (Goldman and Glibert 1983). Second, nitrogen-15 in the substrate during incubation could be significantly diluted by heterotrophic regeneration of inorganic nitrogen. Because such an effect could lead to underestimation of inorganic nitrogen-14 uptake, rates of isotope dilution in Lake Bonney remain tentative until further investigations are made.
CHLOROPHYLL 0.0 2.0 4.0 6.0 0
5
10 H 15
20
25
NITROGEN UPTAKE
0.0
2.0 4.0 6.0
0.0000 0
PARTICULATE NITROGEN Figure 1. Chlorophyll-a (micrograms per liter) and particulate nitrogen (micromolar) in the east lobe of Lake Bonney. Measurements were made on 12 December 1989 and 24 November 1989, respectively.
0.0005 0.0010
5
The uptake rates were fitted to the Michaelis -Menten model with a nonlinear curve fitting program utilizing Marquardt's algorithm (Dodds, Priscu, and Ellis 1991). From data collected on 29 November 1990, Vm (plus standard deviation) and Km (plus standard deviation) for NH 4 at 4.5 in 0.00083 per hour + 2.81x10- 14 and 1.000 micromoles per liter + 1.65x10- 10 , respectively. Vmax and Km for NO3- at 4.5 m, calculated from data collected on 18 November 1990, were 0.00039 per hour + 6.87x10- 15 and 4.83 micromoles per liter + 3.36x 10-10, respectively. The high Vmax and low Km for NH4 , relative to NO3-, indicate a high affinity for this nutrient at both saturating and limiting levels (Goldman and Glibert 1983). Two potential explanations for this affinity are, first, that NH 4 requires less energy for assimilation than does NO 3-, and second, that increased capacity for NH4 uptake may represent an adaptive response for utilization of temporary micropatches of NH4 produced through regeneration. In Lake Bonney, both regenerated NO3- and regenerated NH4 are hypothesized to support primary production in the euphotic zone (Priscu et al., Antarctic Journal, this issue). Because of the virtual lack of
10 H 15
20
25 Figure 2. Estimated ambient specific nitrogen uptake (per hour) of ammonium and nitrate in the east lobe of Lake Bonney.
ANTARCTIC JOURNAL - REVIEW 1993 242
inorganic nitrogen uptake (figure 3) indicate that nitrogen limitation, if present, is most pronounced above the layer of intense chemical stratification. That nitrogen uptake exceeds carbon uptake below 15 meters may represent a dominance of heterotropl'iic nitrogen utilization below this depth. In conclusion, our preliminary study suggests that NH4 is the primary nitrogenous nutrient supporting microplankton production above 20 in Lake Bonney. Substrate kinetics experiments suggest a high affinity for this nutrient, and nutrient profiles indicate that it is more abundant than NO3 and nitrite (NO 2 i combined. Our future investigations of heterotrophic regeneration combined with direct measurement of inorganic nitrogen uptake in the water column will clarify the exact relationship between nutrient transformations and planktonic activity. We thank T. Sharp for conducting many of the field experiments. This work was supported in part by National Science Foundation grant OPP 91-17907 to John C. Priscu.
VOLUMETRIC UPTAKE 0.00 0.04 0.08 0 I I
5
10
1L::
References Dodds, W.K., J.C. Priscu, and B.K. Ellis. 1991. Seasonal uptake and regeneration of inorganic nitrogen and phosphorus in a large oligotrophic lake: Size fractionation and antibiotic treatment. Journal of Plankton Research, 13(6), 1339-1358. Dugdale, R.C., and J.J. Goering. 1967. Uptake of new and regenerated forms of nitrogen in primary productivity. Limnology and Oceanography, 12(2),196-206. Goldman, J.C., and P.M. Glibert. 1983. Inorganic nitrogen uptake in phytoplankton. In E.J. Carpenter and D.G. Capone (Eds.), Nitrogen and the marine environment. New York: Academic Press. Priddle, J., I. Hawes, J.C. Ellis-Evans, and T.J. Smith. 1986. Antarctic aquatic ecosystems as habitats for phytoplankton. Biology Review, 61,199-238. Priscu, J.C., W.F. Vincent, and C. Howard-Williams. 1989. Inorganic nitrogen uptake and regeneration in Lakes Fryxell and Vanda, Antarctica. Journal of Plankton Research, 11(2), 335-351. Priscu, J.C., B.B. Ward, and M.T. Downes. 1993. Water column transformations of nitrogen in Lake Bonney, a perennially ice-covered antarctic lake. Antarctic Journal of the U.S., 28(5). Sharp, T.R., and J.C. Priscu. 1990. Ambient nutrient levels and the effects of nutrient enrichment on primary productivity in Lake Bonney. Antarctic Journal of the U.S., 25(5), 226-227. Timperly, M.H., and J.C. Priscu. 1986. Determination of nitrogen-15 by emission spectrometry using an atomic absorption spectrometer. Analyst, 111, 23-28.
20
25 Figure 3. Volumetric uptake of inorganic carbon (micromoles carbon per liter per hour) and volumetric uptake of inorganic nitrogen (micromoles nitrogen per liter per hour) in the east lobe of Lake Bonney. Addition of carbon- 14-bicarbonate to water samples and subsequent incubation allowed for measurements of inorganic carbon uptake (primary production) in units of micromoles per liter per hour. To achieve similar units and to compute total inorganic nitrogen uptake, the specific uptake rates of NH 4 and NO 3 - (V) were added and then multiplied by the concentration of particulate nitrogen at the depths where rates were to be obtained. Ratio of inorganic carbon uptake to
ANTARCTIC JOURNAL - REVIEW 1993 243
the west lobe in much higher numbers than in the east lobe at comparable depths in the oxygen depleted deep waters (figure 2). ELB17 shows a distribution pattern that is quite different from the bacterial population as a whole, albeit at much lower abundance levels. Before our next field season, we plan to analyze the approximately 100 DNA extracts we collected in 1992 (using tangential flow filtration to concentrate 4-liter samples). We are also planning to produce more polyclonal antisera to other Lake Bonney denitrifier isolates and to characterize these isolates as to their temperature and salinity ranges and optima. We have been unable so far to isolate any nitrifying strains from lake samples. Our nitrifier probes, however, are ready for application in next year's program. This research was supported by National Science Foundation grant OPP 91-17907.
References Bartlett, R., C. Woolston, and J.C. Priscu. 1993. Influence of high salinity levels on 15-nitrogen extraction efficiency in Lake Bonney, Antarctica. Antarctic Journal of the U.S., 28(5). Cohen, Y., and L.I. Gordon. 1978. Nitrous oxide in the oxygen minimum of the eastern tropical North Pacific: Evidence for its con-
Phytoplankton utilization of ammonium and nitrate in Lake Bonney: A preliminary assessment CHRISTOPHER D. WOOLSTON and JOHN C. PRISCU, Department of Biological Sciences, Montana State University, Bozeman, Montana 59717
ambient uptake rates of inorganic nitrogen and carbon. The results provide preliminary insight into the interactions of nutrients and microplankton; furthermore, they set the groundwork for future investigations of nutrient cycling and primary production in the lake. In this report, uptake refers to transport plus assimilation, two distinct pathways which could not be distinguished by our experimental designs. The Michaelis-Menten parameters Vm (the maximum specific uptake rate) and Km (the substrate concentration at which uptake is half of the maximal rate) were determined with substrate kinetics experiments. In these experiments, water samples from 4.5 meters (m) received varying levels of enrichment of nitrogen-15-labeled NH4 or NO 3- and were incubated for approximately 24 hours. The suuspended particulate matter in the water samples was collected on precombusted GF/C filters which were frozen until analysis. The accumulation of nitrogen-15 in the particulate matter was measured by atomic emission spectrometry following Dumas combustion of filters (Timperly and Priscu 1986). Uptake rates were calculated from the equations formulated by Dugdale and Goering (1967).
ake Bonney is characterized by strong gradients in nutriLents and algal concentrations (figure 1 and Priscu, Ward, and Downes, Antarctic Journal, in this issue). In this permanently ice-covered quiescent environment, nutrient transformations and microplanktonic activity are hypothesized to be tightly coupled. This should be particularly true for transformations of inorganic nitrogen, because nitrogen has been shown to limit phytoplankton photosynthesis in a number of antarctic lakes (Priddle et al. 1986; Priscu, Vincent, and Howard-Williams 1989; Sharp and Priscu 1990). Our current research on Lake Bonney is based on the hypothesis that regeneration of ammonium (NH 4 ) and nitrate (NO3i through heterotrophic remineralization and nitrification, respectively, support the bulk of productivity in the upper trophogenic zone of Lake Bonney (Priscu et al., Antarctic Journal, in this issue). This report focuses on a preliminary assessment of the physiological capacity of planktonic assemblages for inorganic nitrogen uptake in the trophogenic zone of the east lobe of Lake Bonney. Specifically, our study provides estimates for the effects of substrate concentration on uptake kinetics as well as
ANTARCTIC JOURNAL - REVIEW 1993
241
zooplankton capable of producing NH4 patches, the differential affinity can perhaps best be explained by the energy requirement of assimilation. Estimates of ambient uptake rates (figure 2) were computed using ambient nutrient concentrations in conjunction with Michaelis- Menten parameters for uptake. These ambient rates indicate that NH4 is the predominant source of nitrogen utilized throughout the trophogenic zone. Furthermore, the relative contribution of NH4 to nitrogenous nutrition increases with depth from 6 to 12 m. This supports the hypothesis that phytoplankton near the first chemical gradient (at approximately I m) are supported by the upward dif fusion of NH4 (Priscu et al., Antarctic Journal, this issue). There is reason, however, to interpret these data with caution. First, because nutrient distributions and physiological response may change with time, any estimate of uptake based on single measurements of nutrient concentration and Michaelis- Menten parameters will reflect instantaneous conditions only (Goldman and Glibert 1983). Second, nitrogen-15 in the substrate during incubation could be significantly diluted by heterotrophic regeneration of inorganic nitrogen. Because such an effect could lead to underestimation of inorganic nitrogen-14 uptake, rates of isotope dilution in Lake Bonney remain tentative until further investigations are made.
CHLOROPHYLL 0.0 2.0 4.0 6.0 0
5
10 H 15
20
25
NITROGEN UPTAKE
0.0
2.0 4.0 6.0
0.0000 0
PARTICULATE NITROGEN Figure 1. Chlorophyll-a (micrograms per liter) and particulate nitrogen (micromolar) in the east lobe of Lake Bonney. Measurements were made on 12 December 1989 and 24 November 1989, respectively.
0.0005 0.0010
5
The uptake rates were fitted to the Michaelis -Menten model with a nonlinear curve fitting program utilizing Marquardt's algorithm (Dodds, Priscu, and Ellis 1991). From data collected on 29 November 1990, Vm (plus standard deviation) and Km (plus standard deviation) for NH 4 at 4.5 in 0.00083 per hour + 2.81x10- 14 and 1.000 micromoles per liter + 1.65x10- 10 , respectively. Vmax and Km for NO3- at 4.5 m, calculated from data collected on 18 November 1990, were 0.00039 per hour + 6.87x10- 15 and 4.83 micromoles per liter + 3.36x 10-10, respectively. The high Vmax and low Km for NH4 , relative to NO3-, indicate a high affinity for this nutrient at both saturating and limiting levels (Goldman and Glibert 1983). Two potential explanations for this affinity are, first, that NH 4 requires less energy for assimilation than does NO 3-, and second, that increased capacity for NH4 uptake may represent an adaptive response for utilization of temporary micropatches of NH4 produced through regeneration. In Lake Bonney, both regenerated NO3- and regenerated NH4 are hypothesized to support primary production in the euphotic zone (Priscu et al., Antarctic Journal, this issue). Because of the virtual lack of
10 H 15
20
25 Figure 2. Estimated ambient specific nitrogen uptake (per hour) of ammonium and nitrate in the east lobe of Lake Bonney.
ANTARCTIC JOURNAL - REVIEW 1993 242
inorganic nitrogen uptake (figure 3) indicate that nitrogen limitation, if present, is most pronounced above the layer of intense chemical stratification. That nitrogen uptake exceeds carbon uptake below 15 meters may represent a dominance of heterotropl'iic nitrogen utilization below this depth. In conclusion, our preliminary study suggests that NH4 is the primary nitrogenous nutrient supporting microplankton production above 20 in Lake Bonney. Substrate kinetics experiments suggest a high affinity for this nutrient, and nutrient profiles indicate that it is more abundant than NO3 and nitrite (NO 2 i combined. Our future investigations of heterotrophic regeneration combined with direct measurement of inorganic nitrogen uptake in the water column will clarify the exact relationship between nutrient transformations and planktonic activity. We thank T. Sharp for conducting many of the field experiments. This work was supported in part by National Science Foundation grant OPP 91-17907 to John C. Priscu.
VOLUMETRIC UPTAKE 0.00 0.04 0.08 0 I I
5
10
1L::
References Dodds, W.K., J.C. Priscu, and B.K. Ellis. 1991. Seasonal uptake and regeneration of inorganic nitrogen and phosphorus in a large oligotrophic lake: Size fractionation and antibiotic treatment. Journal of Plankton Research, 13(6), 1339-1358. Dugdale, R.C., and J.J. Goering. 1967. Uptake of new and regenerated forms of nitrogen in primary productivity. Limnology and Oceanography, 12(2),196-206. Goldman, J.C., and P.M. Glibert. 1983. Inorganic nitrogen uptake in phytoplankton. In E.J. Carpenter and D.G. Capone (Eds.), Nitrogen and the marine environment. New York: Academic Press. Priddle, J., I. Hawes, J.C. Ellis-Evans, and T.J. Smith. 1986. Antarctic aquatic ecosystems as habitats for phytoplankton. Biology Review, 61,199-238. Priscu, J.C., W.F. Vincent, and C. Howard-Williams. 1989. Inorganic nitrogen uptake and regeneration in Lakes Fryxell and Vanda, Antarctica. Journal of Plankton Research, 11(2), 335-351. Priscu, J.C., B.B. Ward, and M.T. Downes. 1993. Water column transformations of nitrogen in Lake Bonney, a perennially ice-covered antarctic lake. Antarctic Journal of the U.S., 28(5). Sharp, T.R., and J.C. Priscu. 1990. Ambient nutrient levels and the effects of nutrient enrichment on primary productivity in Lake Bonney. Antarctic Journal of the U.S., 25(5), 226-227. Timperly, M.H., and J.C. Priscu. 1986. Determination of nitrogen-15 by emission spectrometry using an atomic absorption spectrometer. Analyst, 111, 23-28.
20
25 Figure 3. Volumetric uptake of inorganic carbon (micromoles carbon per liter per hour) and volumetric uptake of inorganic nitrogen (micromoles nitrogen per liter per hour) in the east lobe of Lake Bonney. Addition of carbon- 14-bicarbonate to water samples and subsequent incubation allowed for measurements of inorganic carbon uptake (primary production) in units of micromoles per liter per hour. To achieve similar units and to compute total inorganic nitrogen uptake, the specific uptake rates of NH 4 and NO 3 - (V) were added and then multiplied by the concentration of particulate nitrogen at the depths where rates were to be obtained. Ratio of inorganic carbon uptake to
ANTARCTIC JOURNAL - REVIEW 1993 243
