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Effect of nitrogen and silicate enrichment on photosynthate allocation by ice algae from Resolute Passage, Canadian Arctic
arine Systems
11( 1997) 53-6 1
a, *
b 9
a Hokkaido National Fisheries Research institute, Kushiro, Japan b Biology Department, University of Watevloo, Waterloo, Ont., Canada Received 15 August 1994; accepted 6 October 1995
Abstract Enrichment experiments with ammonium, nitrate and silicate were conducted to determine their effects on the photosynthate allocation by ice algae collected during the spring of 1992 from Resolute Passage, Canadian Arctic. Ammonium concentrations of 100 pJ4 inhibited the synthesis of protein, which was the largest fraction in the end products for the ammonium and nitrate enrichment experiments. No inhibition was detected for up to 200 @4 of added nitrate. Silicate enrichment of > 5 pA4 combined with 50 PM ammonium increased photosynthate allocation to protein to more than 45%. However, protein synthesis showed no regular pattern of increase or decrease with enrichments of varying concentration of silicate plus 50 p,M of nitrate. The dominant fraction in the silicate plus nitrate experiment was polysaccharide and nucleic acids, which accounted for more than 35% of the total photosynthesis. The total lipid fraction was only slightly affected by nitrogen or silicate, but neutral lipids decreased while membrane-associated polar lipids increased with silicate plus ammonium enrichment. Our findings indicate that ice algae in Resolute Passage were likely limited by ammonium and silicate during the declining period of the algal bloom in 1992. Keysoords: ammonium: nitrate; silicate; limitation; photosynthate;relative growth rate
1. Introduction Ice algae are often associated with brine channels within the skeletal ice, where concentrations of nutrients are assumed to be much higher than in the underlying water column. Consequently, ice algae are not generally considered to be nutrient Limited (Meguro et al., 1967; Bunt and Lee, 1970; Grainger, 1977; Homer and Schrader, 1982; Clarke and Ackley, 1984). However, Demers et al. (1986) and Cota
* Corresponding author. Present address: Lab. of Biological Oceanography, Dep. of Bioengineering, Soka University Hachioji, Tokyo 192, Japan. Phone/fax: 81 426 91 8002. E-mail: [email protected].
et al. (1987) estimated that ice algal standing stocks were higher than could be accounted for by nutrient accumulation in the brine channels. The close associ. . . -m f~r\fl nhntncvnatlon between nutnent concentratiolxa Lcl.UYIaV.VUJ thetic efficiency of ice algae at the botto observed by Gosselin et al. (1985) may suggest that ice algal cells are indeed nutrient limited. This observation has been confirmed by relating fortnightly tidal mixing to variations in (1) carbon allocation to protein (Smith et al., 1987), (2) photosynthetic performance (Cota and Home, 1989), and (3) uptake of anic nitrogen by ice algae (Demers et al. (1990) also demonstrated a tween chlorophyll and nitrate concentrations at the ice-water interface. Indepen-
0924-7963/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PU SO924-7963(96)00027-9
54
S. Tagrrclri,R.E. H. Sm?h / Jorrrnai of Marine System I I f 1997) 5.3-61
bioassays conducted by Maestrini ct al. (1986) showed that ice algae responded to nitrogen enrichment in a way that indicated nitrogen limitation in an estuarine area of Hudson Bay. However, using stable isotope bioassays, Harrison et al. (1990) concluded that ice algal assemblages never appeared to be nitrogen-limited in fully marine waters. These contrasting reports may be due to regional differences and the stage of algal accumulation. GOSselin et al. (1990) suggested that ice algal growth was light limited at the beginning of the growth season and became silicon limited later on, when in situ light and the accumulated algal biomass were high and the tidally driven nutrient supply was not adequate to satisfy algal nutrient requirements. Although the effect of siiicon was not determined in their study, Harrison et al. (1990) showed a systcmatic shift from predominantly nitrate metabolism during the early growth season to predominantly ammonium metabolism during the declining period of algal growth. Furthermore, Harrison et al. (1990) suggested that the conceptual model of Dugdale and Goering (1967) for the cycling of nitrogen in the pelagic ocean and its relation to primary production was also valid for ice algal communities. Ice algal growth in relation to nutrients can be studied by several methods: (1) changes in the algal biochemical composition; (2) differential enrichment bioassays; (3) photosynthate allocation; and (4) source of nitrogen nutrition (i.e., nitrate or ammonium). All four approaches were used to study ice algal communities in Resolute Passage, Northwest Territories, Canada, during the Saroma-Resolute Study @ARES) program. The present study is concerned mainly with photosynthate allocation. Protein synthesis of algae is directly related to growth (Morris, 1981; DiTullio and Laws, 1986). Determination of r4C incorporation into the protein fraction through photosynthate allocation is a crude but rapid and convenient way to study the protein synthesis of algae (Morris, 1981; Hitchcock, 1983). Nutrient limitation causes low protein synthesis and subsequently high production of storage products such as polysaccharide and lipids (Shifrin and Chisholm, 198 1; Taguchi et al., 1987). Determination of phoosynthate allocation permits the study of physiological change in response to nutrients. Early dent enrichment
studies (McConville, 1985; Nichols et al., 1986) drew inferences on ice algal photosynthate allocation based on experiments with non-ice algae at much higher light and temperature conditions. Photosynthate allocation in ice algae has been studied in the Antarctic (McConville et al., 1985; Palmisano and Sullivan, 1985) and more recently in the high Arctic (Smith et al., 1987, 1989; Smith and Herman, 1992). McConville ( 1985) and Nichols et al. ( 1986) observed a relatively large share (31-59%) of photosynthate in lipid and a small share (20-24%) in the protein fraction of ice algae. These studies also indicated that a substantially higher allocation to protein (up to 40%) was directly related to an increase in the nutrient supply caused by tidal mixing. Allocation of carbon to protein seems well suited for tes&ing the i~SpOnse of ice algae to varying nutrient enrichments at low light and low temperature. The purpose of the present study was to characterize the response of photosynthate allocation by arctic ice algae as a function of changes in nitrogen and silicon enrichment. We had three hypotheses: that the photosynthate allocation to protein would (1) exhibit an acclimation similar to those induced by the tidally driven nutrient supply (Smith et al., 1987) (2) have silicon-limited growth at high algal biomass (Gosselin et al., 1990), and (3) have ammonium-dependent growth during the declining period of the algal bloom (Harrison et al., 1990). The results confirmed the latter two hypotheses, indicating a combined control of ice algal growth by the availability of silicon and ammonium.
2. Materials and methods Samples were taken on 15 and 21 May 1992 at a station on land-fast ice in Resolute Passage, Northwest Territories, Canada (74’41.19’N, 95”15.59’W), about 2 km south of Cornwallis Island. Water pumped from just below the bottom of the ice was transferred to a dark bottle and then filtered immediately through a Nuclepore filter (0.2 pm pore size). Although a detailed sampling protocol has been provided by Smith et al. (1995), a brief description is given here. The first-year ice was sampled with a SIPRE coring device (Smith et al., 1987). Ice cores were collected for the nitrate enrichment experiment (NO,) and the
Table 1 Mean (*standard deviation) of photosynthesis [mg C (mg CHLa)- ’d- ‘1 of ice algal communities during 24 were significant ( p < 0.05) hxperiment
Treatment
IPR~~u~y,,ilr&
Number of
observations nitrate ammonium NH4 SiO, + NO, silicate + nitrate SiO, + NH, silicate + ammonium NO3
0.22 zk0.05 0.40 * 0.09 0.31 kO.07 0.72 + 0.16
6 5 7 7
ammonium enrichment experiment (N 1992 and for the silicate enrichme (SD, + NO, and SiO, + N J) on 21 May 1992. The loosely consolidated ice crystals from the botn*tn-.q’ me*P ~~~~~~11~.1 tom ‘ luycr WuIb bUIb.U,J scraped inntofiltered ceawater and allowed to melt slowly in a dark bottle (Garrison and Buck, 1986). The resulting suspension was a.bout 10% melted ice and 90% seawater with a salinity of about 30 psu. Chlorophyll a concentrations were determined and were about 1 mg per liter of suspension. Subsamples were taken from the suspension to measure chlorophyll pigments and dissolved ammonium, nitrate, nitrite, phosphate and silicate. Subsamples for nutrient analysis were passed through a membrane filter (type HV) and all but those for ammonium were stored at - 20°C until analysis; ammonium analyses were made on fresh samples within 12 hr of collection using the method of Solorzano (1969). All other nutrients were analyzed using the methods of Strickland and Parsons (1972). Chlorophyll a and pheopigments were determined by the method of Holm-Hansen et al. (1965). Subsamples were taken for the measurement of photosynthetic rate by the ice algal communities in the d;lmtm P.IFnPnP;AnP .#p& 14C_&]& gj&~, t;,(-g-_ IIULC, JU3pmrll”‘ IJ bonate. Incubations were carried out in acid-cleaned plastic bottles for 24 h in blue-filtered, artificial light incubators (Smith et al., 1987) maintained at ambient temperature ( - 1.7”C) and an ambient irradiance level of 20 p,Ern-* s -I. No controls with zero addition were employed in the nitrate and ammonium enrichment experiments. Nitrate (0.125, 0.25, 1, 10, 50 and 200 PM) was added to the first series of incubation bottles (NO,). The first three nitrate enrichments were not significantly different from the
the measurement of total organic carbon fixation and triplicate samples for the dete~ination of photosynmate allocation. End products of photosynthesis were determined by the method described by Smith et al. ( 1987). By convention, the four resulting fractions are referred to as follows: methanol-water soluble =
NITRATE (PM) Fig. 1. Rate of ice algal photosynthesis per mg chlorophyll a in the ammonium (upper) and nitrate (lower) enrichment experiments. Vertical bar indicates two standard deviations.
3. I. Nitrate and ammoniclm enrichment
low molecular weight compounds (LMWC); chloroform-soluble = lipid (LIP); TCA-soluble = polysaccharide with nucleic acids (PNA); and TCA-insoluble = protein (PRO). The samples of the lipid fraction were further analyzed to distinguish four lipid classes: triacylglycerols (TG), free fatty acids (FFA), acetone-mobile polar lipids (AMPL) and phospholipids (PL) by the method described by Smith and D’Souza ( 1993).
Enrichment with ammonium (except at 100 FM) stimulated higher photosynthetic rates [in mgC (mg CHL a)-’ d- ‘1 than enrichments with nitrate (Fig. 1). Ammonium enrichment with 0.1 PM doubled the ambient concentration of ammonium. This enrichment is often sufficient to enhance the total photosynthesis per chlorophyll a. However, no systematic effect was observed (Fig. 1). Ice algae showed little response to nitrate enrichments even at concentrations above 10 @Z. Ammonium enrichment of 100 PM inhibited the synthesis of protein, which was the predominant fraction in the photosynthetic end products (Fig. 2). This minimum value of 40 + 3% was 73% of the maximum value (55 _I 6%) found in the incubation with 0.1 PM ammonium added. No protein inhibition was detected for up to 200 FM of added nitrate (Fig. 2). The maximum value was 53 f 6% at 50 and 200 (-LMof added nitrate.
3. Results The study period encompassed the stage in the ice-algal growth cycle with declining chlorophyll biomass (Smith et al., 1997-this issue). The background concentrations of nutrients in the incubation bottles were l-2 PM of phosphate, 7 ~JJV of silicate, 8 PM of nitrate and 0.1 pM of ammonium. The ice algal community actively assimilated carbon during the incubations (Table 1). Photosynthetic rates were significantly different between experiments ( p C 0.05; Student’s z-test) except between NH, and SiO, + NO,.
O.lpM
0.2pM
3.2. Silicate enrichment Total photosynthesis per unit chlorophyll a was enhanced more in enrichments with SiO, + NH, than
1OpM
1W
50pM
1OOpM
AMMONIUMENRICHED 60 50 40 30 20 10 0 i
.J
0.125pM
0.25j.iM
1PM
10pM
50pM
200pM
NITRATEENRICHED Fig. 2. Change in relative abundance of photosynthetic end products in relation to different concentrations of ammonium or nitrate. Maxirmim coefficients of variance were 16% for LMWC, 12% for PNA, 9% for PRO and 14% for LIP.
e2 Lipid crass allocation of ice algal
synthesis was 30 f 5% while percent incorpo of 14C into polysaccharide plus nucleic acids ( was always highest, with a mean of photosynthetic end products. Although allocation to fractions other than lipid
munities as percent of total
Treatment:nitrate (50 j_kLM) 0 18 6.3
13
2.5 IO 50
3.8 5.6 3.9
14 14 14
62 53 64 55
TreQtntent: amtnnnium(50 C_L 0 29 2.5 19 a.3 10 14 4.8 50 15 5.0
15 14 15 16
49 58 67 64
30 17 27
TG = triacylglycerols (neutral lipids); FFA = free fatty acids; AMPL = acetone-mobile polar lipids; PL = phospholipids. NO duplicate observations
1.0
CO
were made.
~ +50/.&lAMMONIUM
varied with the enrichment, the allocation to lipid stayed relatively low and stable (7.3 + 0.6% for ammonium and 7.5 + 1% for nitrate; Fig. 4). Further analysis of lipid classes revealed allocation predomiohpi& (PI j in nantly to membrane-associated pho 4 experiments both the SiO, + NO, and SiO, + (Table 2). However, in the SiO, + NH, enrichments, neutral lipid (TG) tended to decrease while BL tended to increase with increasing silicate enrichments.
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Fig. 3. Rate of ice algal photosynthesis per mg chlorophyll a in the silicate enrichment experiments with 50 piI4 ammonium (upper) and 50 PM nitrate (lower). Vertical bar indicates two standard deviations.
Effects of “C tracer disequilibrium among different fractions and their precursors must be considered in drawing conclusions from incubation experiments for photosynthate allocation of carbon by microalgae (Li and Harrison, 1982; Smith a.&c:Geider, 1985). This is particularly the case for slowly growing cells such as ice algae, whose generation time can be longer than 72 h (Maestrini et al., 1986). Smith et al. (1987) showed that the labeling time courses of photosynthate allocation in arctic ice algae were predominantly linear even up to 70 h. Although the percent distribution of allocation changed s!owly with increasing incubation time, the distribution was quite similar to that at 24 h even after 70 h. While our standard 24 h incubations should have been able to minimize the effects of the 14Ctracer disequilibrium, short-term incubations may be of considerable value
S. Taguchi, R.E. H. Stnith / Jomml
58
of Marirw Systerrrs I1 f 1997) 53-6 I
for understanding the kinetics of biochemical synthesis by the ice algae. The physiological response to nutrient enrichment may differ depending on the nutrient, as shown in the present study. Nitrate enrichment probably had no effect on photosynthesis at 0.125, 0.25 and 1 PM because the ambient nitrate concentration was 8 ~.LM (Fig. 1 and Fig. 2). The highest protein allocation (53 f: 6%) was observed at 50 PM. A similar phenomenon was observed for the SiO, + NH, enrichments (Fig. 41, where carbon allocation to protein increased to 46 + 7%; again, this is probably because the ambient silicate concentration was 7 PM. In contrast to nitrate and silicate enrichment, 0.1 C_LM ammonium enrichment was already significant compared to the ambient concentration (0.1 h M). Therefore, quantifying the responses of the ice algal community to enrichment in an ammonium-depleted environment can be complicated. The extraction procedure we used was the same one previously applied to measure photosynthate allocation in arctic ice algae (Smith et al., 1989). However, the allocation to low molecular weight compounds (LMWC) was about 15 + 4% of the total
in the SiO, + NO, enrichment experiments (Fig. 4), which is less than half the values previously reported for arctic ice algae in the same geographic area (Smith et al., 1987, 1989). We currently have no explanation for this discrepancy and plan to address it in future studies. The minimum value of 14C incorporation into protein was 22 f 4% in the SiO, + NO, enrichment experiment (Fig. 4), which is similar to the value of 20-24% obtained by Smith et al. (1989). Both enrichment experiments with nitrate and ammonium revealed that the maxima of photosynthate allocation to protein were 51 _t 4 and 48 + 5%, respectively (Fig. 2). The degree of enhancement in photosynthate allocation to protein can be estimated by the relative growth rate since algal protein synthesis is directly related to growth (Morris, 198 1). A direct relationship has been observed between the percent protein allocation and the relative growth rate (l.~&~x) (DiTullio and Laws, 1986) and is described by the equation: %proteinC = 15 + 3S-
(1)
I-CMAX
+50pM AMMONIUM 24hr 50 40 30 g
20 10
8
0
iii
CJaoa EZ
g$45 J
8
5”
ggg fQ +50pM NITRATE 24hr
0
uaok 5””
OrcM
3
“a&g
SC&” W
!jgis~
f””
A
2.5pM
5PM slucAlE
10pM
25pM
J 50pM
ENfwliED
Fig. 4. Change in relative abundance of photosynthetic end products in relation to different concentrations of silicate + 50 p M ammonium or silicate + 50 p, M nitrate. Maximum coefficients of variance were 18% for LMWC, 13% for PNA, 9% for PRO and 9% for LIP.
Table 3 Mean (k standard deviation) of % protein allocation and estimated relative growth rate of ice algal communities based on triplicate observations. SiO, + NO3 and SiOl, + N cantly different ( p < 0.01) Experiment
Treatment
Protein allocation (96)
Wkk4x)
NO, NH, SiO, +NO, SiO, +NH,
nitrate ammonium silicate + nitrate silicate + ammonium
51 L-3.7 48kS.3 3Ok5.0 40 + 8.1
1.o + 0.07 0.94 + 0. I 0.43 + 0.07 0.71+0.1
Relative growth rate
where % protein C is the percentage of photoassimilated carbon allocated to protein after a 24-h incubation, p is the growth rate and l.~~*x is the maximum growth rate -when % protein C is 50% or higher. The high protein values in the NO, and N may suggest a high relative growth rate (l.~/p,~~x) of 1.O-0.94 (DiTullio and Laws, 1986). Ice algae had the highest relative growth rate with the nitrate and ammonium enrichments (Table 3). When algae are perturbed, as they were in the present experiments, protein synthesis may not be so simply related to growth rate. In fact, addition of the limiting nutrient(s) may produce a temporary suppression of photosynthesis and/or protein synthesis as cellular reserves are devoted to nutrient assimilation; this effect is particularly clear for nitrogen (Turpin et al., 198P). In our study, the response to ammonium would suggest that the benefits of improved nitrogen nutrition outweighed the energetic costs of assimilation (except at very high additions) so far as total photosynthesis is concerned, Protein synthesis, however, was still somewhat depressed after 24 hr at intermediate ammonium additions. In the case of SiS, + NH, additions, the cells were evidently able to exploit the nutrients for both photosynthesis and protein synthesis within 24 h. The results from the ammonium and nitrate enrichment experiments may suggest the simultaneous enhancement of allocation to protein by ammonium and nitrate (Table 3). The simultaneous utilization of ammonium and nitrate has been observed in benthic algae including ice algae (Maestrini et al., 1982; Price et al., 1985; Queguiner et al., 1986; Demers et al., 1989). Harrison et al. (1990), in contrast, observed a systematic shift from predominantly nitrate
1988; Lizotte and Sullivan, 1992; Smith et al., 1993). enrichment experiment showed a er allocation of carbon to protein than the SiO, + NO, experiment (Table 3). The relative growth rate of SiO, + NH, &/pMAX = 0.71 + 0. I) was also significantly hig relative growth of SiO, + NO, (p/p ned suppressive 0.07). This may indicate a co effect of silicate with ammonium. owever, it is not clear why the allocation pattern and total photosynthesis might be different between NO, and SiO, + NO, (Tables 1 and 3). Alternative explanations for the different allocation pattern reported here might be because physiologically different ice algal populations were used in the bioassajs. When the combined effects of various nutrients are considered, ammonium appears to be controlling ice algal growth with limitation by silicate during the ice algal population decline in the high Arctic (Table 3). Regenerated ammonium might be mediated by prokaryotic microorganisms (Carey, 1985; Kuosa et al., 1992) and silicon-limitation might be caused by the massive growth of ice algae but alleviated by hydrodynamic processes that occur in the underlying water column (Cota and Home, 1989).
Acknowledgements The authors appreciate the financial support from the Federal Department of Fisheries and Oceans in Canada and the Monbusho International Science Research Program (#03044146) and the Ministry of Education in Japan. Logistical support was provided
S. Tnguclri. R.E.H. Shth / Journnl of A&wineSwtetns11 ( 1997) 53-61
60
by the Polar Continental Shelf Project in Canada. M. and M. Levasseur coordinated the field t&&in sampling and sample analysis. Contribution (B-579) from Hokkaido National Fisheries Research Institute.
References Bunt, J.S. and Lee, C.C., 1970. Seasonal primary production in Antarctic sea ice at McMurdo Sound in 1967. J. Mar. Res., 28:
304-320. Carey Jr., A.G., 1985. Marine ice fauna: Arctic. In: R. Homer (Editor), Sea Ice Biota. CRC Press. Boca Raton. FL, pp. 173- 190. Clarke, D.B. and Ackley, S.F., 1984. Sea ice structure and biological activity in the Antarctic marginal ice zone. J. Geophys. Res., 89: 2087-2095. Conover. R.J., Cota, G.F., Harrison, W.G.. Horne, E.P.W. and Smith, R.E.H., 1990. Ice-water interactions and their effect on biological oceanography in the Arctic Archipelago. In: C.R. Harington (Editor), Canada’s Missing Dimension: Science and History in the Canadian Arctic Islands. Can. Natl. Mus. Nat. Sci., 1: 204-228. Cota, G.F. and Horne, E.P.W., 1989. Physical control of arctic ice algal production. Mar. Ecol. Prog. Ser., 52: 11 I- 121. Cota, G.F., Prisenberg, S.J., Nennett, E.G., Loder, J.W., Lewis, M.R., Anning, J.L., Watson, N.H.F. and Harris, L.R., 1987. Nutrient flux during extended blooms of Arctic ice algae. 1. Geophys. Res., 92: 1951- 1962. Demers, S., Legendre, L., Therriault, J.-C. and Ingram, R.G., 1986, Biological production at the ice-water ergocline. In: J.C.J. Nihoul (Editor), Marine Interfaces Ecohydrodynamics. Elseviet, Amsterdam, pp. 3 l-54. Demers, S., Legendre, L., Maestrini, S.Y., Rochet. M. and Ingram,R,G.,1989.Nitrogenous nutri&?n of sea-ic.c algae. Polar Biol,, 9: 377-383, DiTullio, G.R. and Laws, E.A., 1986. L:el periodicily of nitrogen and carbon assimilation in five species of marine phytoplankton. Accuracy of methodology for predicting N-assimilation rates and N/C composition ratios. Mar, Ecol. Prog. Ser., 32: 123-132. Dugdale, R.C. and Goering, J,J.. 1367. Uptake of D..W and regenerated forms of nitrogen in primary production. LimnoI. Oceanogr., 12: 196-206. Garrison, D.L.and Buck, K.R., 1986. Q-;Anism losses during ice melting: a serious bias in sea ice cummunity stl!dies. Polar Biol., 6: 237-239. Gosselin, M., Legendre, L., Demers, S. and Ingram, R.G., 1985. Responses of sea-ice microalgae to climate and fortnightly tidal energy inputs (Manitounuk Sound, Hudson Bay). Can. J. Fish.Aquat.Sci., 42: 999-1006. GosseIin, M.. Legendre, L., Therriault. J.-C. and Demers, S., 1990. Light and nutrient limitation of sea-ice microalgae (Hudson Bay, Canadian Arctic). J. Phycol., 26: 220-232. Grainger, E.H.. 1977. The annual nutrient cycle in sea-ice. In:
M.J. Dunbar (Editor) Polar Oceans. Arctic Inst. North America, Calgary, Alta., pp. 285-299. Harrison, W.G., Cota, G.F. and Smith, R.E.H., 1990. Nitrogen utilization in ice algal communities of Barrow Strait, Northwest Territories, Canada. Mar. Ecol. Proo. Ser., 67: 275-283. Hitchcock, G.L., 1983. Photosynthate partitioning in cultured marine phytoplankton. I. Dinoflagellates. J. Exp. Mar. Biol. Ecol., 69: 2 l-36. Helm-Hansen, O., Lorenzen, C.J., Holmes, R.W. and Strickland. J.D.H., 1965. Fluorometric determination of chlorophyll. J. Cons. Int. Explor. Mer, 30: 3-15. Horner, R.A. and Schrader, G.C., 1982. Relative contribution of ice algae, phytoplankton and benthic microalgae to primary production in near-shore regions of the Beaufort Sea. Arctic, 35: 485-503. Kuosa, H., Norman, B., Kivi. K. and Brandini. F., 1992. Effects of Antarctic sea ice biota on seeding as studied in aquarium experiments. Polar Biol., 12: 333-339. Li. W.K.W. and Harrison, W.G., 1982. Carbon flow into the end-products of photosynthesis in short and long incubations of a natural phytqlankton population. Mar. Biol., 72: 175182. Lizotte, M.P. and Sullivan, C.W., 1992. Biochemical composition and photosynthate distribution in sea ice microalgae of McMurdo Sound, Antarctica: evidence for nutrient stress during the spring bloom. Antarct. Sci., 4: 23-30. Maestrini, S.Y., Robert, Y.M. and Truquet, I., 1982. Simulta,.zous uptake of eq;monium and nitrate by oyster-pond algae. Mar. Biol. Lett., 3: 143-153. Ma.estrini, S.Y.. Rochet, M., Legendre, L. and Demers, S., 1986. Nutrient limitation of the bottom-ice microalgal biomass (southeastern Hudson Bay, Canadian Arctic). Limnol. Oceanogr., 3 1: 969-982. McConville, M.J.. 1985. Chemical composition and biochemistry of sea ice microalgae. In: R.A. Horner (Editor), Sea Ice Biota. CRC Press, Boca Raton, FL, pp. 105- 157. McConville, M.J., Mitchell, C. and Wetherbee, R., 1985. Patterns of carbon a&qimilation in a microalgal community from annual sea ice, East Antarctica. Polar Biol., 4: 135-141. Meguro, H., Ito, K. and Fukuqhima, H., 1967. Ice flora (bottom type): a mechanism of primary production in polar seas and the growth of diatom in sea-ice. Arctic, 20: 114-133. Morris, I., 1981. Phasosynthesis products, physiological state and phytoplankton growth. In: T. Platt (Editor) Physiological Bases of Phytoplankton Ecology. Can. Bull. Fish. Aquat. Sci., 210: 83- 102. Nichols, P.D., Palmisano, AC., Smith, GA. and White, D.C., 1986. Lipids of the antarctic sea ice diatom Nitzschiclcylindrus. Phytochemistry, 25: 1649-1653. Palmisano, AC. and Sullivan, C.W., 1985. Pathways of photosynthetic carbon assimilation in sea-ice microalgae from McMurdo Sound, Antarctica. Limnol. Oceanogr., 30: 674-678. Palmisano, AC., Lizotte, M.P., Smith, G.A., Nichols, P.D., White, D.C. and Suliivan, C.W., 1988. Changes in photosynthetic carbon assimilation in Antarctic sea-ice diatoms during spring bloom: variation in synthesis of lipid classes. J. Exp. Mar. Biol. Ecol., 116: I-13.
Price. N.M., Cochlan, W. of uptake of inorganic in the strait of Georgia: communities. Mar. Ecol. Queguiner, B.. Hafsanoui, M. and Treguer. P., 1986. Simultaneous uptake of ammonium and nitrate by phytoplankton in coastal ecosystems. Est. Coastal Shelf Sci., 23: 751-757. Shifrin, N.S. and Chisholm, SW., 1981. Phytoplankton lipids: interspecific differences and effects on nitrate, silicate and light-dark cycles. J. Phycol., 17: 374-384. Smith, R.E.H. and D’Souza. F.D., 1993. Macromolecular labelling patterns and inorganic nutrient limitation of a North Atlantic spring bloom. Mar. Ecol. Prog. Ser., 92: 111-I 18. Smith, R.E.H. and Geider. R.J., 1985. Kinetics of intracellular carbon allocation in a marine diatom. J. Exp. Mar. Biol. Ecol.. 93: 191-210. Smith, R.E.H. and Herman, A.W., 1992. In situ patterns of Intracellular photosynthate allocation by sea ice algae in the Canadian High Arctic. Polar Biol., !2: 545-55 I. Smith, R.E.H., Clement, P., Cota, G.F. and Li, W.K.W.. 1987. Intracellular photasynthate allocation and the control of Arctic marine ice algal production. J. Phycol., 23: l24- 132. Smith, R.E.H., Clement, P. and Head, P., 1989. Biosynthesis and photosynthate allocation patterns of arctic ice algae. Limnol. Oceanogr., 34: 59 I-605.
Kudoh, S., Legendre, L., B., Suzuki, S., Takahashi, 1995. Biological and chemical investigations of the SaromaResolute Project in ice-covered Resolute Passage, 1992. Can. Data Rep. Hydrogr. Ocean Sci. 137, 19 pp. Smith, R.E.H., Gosselin, M.. Kudoh, S., Robineau, B. and Taguchi, S.. 1997. DOC and its relationship to algae in bottom ice communities. J. Mar. Syst., I 1: 71-80. Solorzano, L., 1969. Determination of ammonia in natural waters by the phenolhypochloride method. Limnol. Oceanogr., 14: 799-80 I. Strickland, J.D.H. and Parsons, T.R., 1972. A practical handbook of seawater analysis. Bull. Fish. Res. Bd. Can., 167: l-31 1. Taguchi, S., Hirata, J.A. and Laws, E.A.. 1987. Silicate deficiency and lipid synthesis of marine diatoms. J. Phy Turpin, D.H.. Elrifi, J.R., Birch. D.G., Weger, J.J.. 1988. Interactions between photosynthesis, respiration and nitrogen assimilation. Can. J. Bot., 66: 2083-2097.
11( 1997) 53-6 1
a, *
b 9
a Hokkaido National Fisheries Research institute, Kushiro, Japan b Biology Department, University of Watevloo, Waterloo, Ont., Canada Received 15 August 1994; accepted 6 October 1995
Abstract Enrichment experiments with ammonium, nitrate and silicate were conducted to determine their effects on the photosynthate allocation by ice algae collected during the spring of 1992 from Resolute Passage, Canadian Arctic. Ammonium concentrations of 100 pJ4 inhibited the synthesis of protein, which was the largest fraction in the end products for the ammonium and nitrate enrichment experiments. No inhibition was detected for up to 200 @4 of added nitrate. Silicate enrichment of > 5 pA4 combined with 50 PM ammonium increased photosynthate allocation to protein to more than 45%. However, protein synthesis showed no regular pattern of increase or decrease with enrichments of varying concentration of silicate plus 50 p,M of nitrate. The dominant fraction in the silicate plus nitrate experiment was polysaccharide and nucleic acids, which accounted for more than 35% of the total photosynthesis. The total lipid fraction was only slightly affected by nitrogen or silicate, but neutral lipids decreased while membrane-associated polar lipids increased with silicate plus ammonium enrichment. Our findings indicate that ice algae in Resolute Passage were likely limited by ammonium and silicate during the declining period of the algal bloom in 1992. Keysoords: ammonium: nitrate; silicate; limitation; photosynthate;relative growth rate
1. Introduction Ice algae are often associated with brine channels within the skeletal ice, where concentrations of nutrients are assumed to be much higher than in the underlying water column. Consequently, ice algae are not generally considered to be nutrient Limited (Meguro et al., 1967; Bunt and Lee, 1970; Grainger, 1977; Homer and Schrader, 1982; Clarke and Ackley, 1984). However, Demers et al. (1986) and Cota
* Corresponding author. Present address: Lab. of Biological Oceanography, Dep. of Bioengineering, Soka University Hachioji, Tokyo 192, Japan. Phone/fax: 81 426 91 8002. E-mail: [email protected].
et al. (1987) estimated that ice algal standing stocks were higher than could be accounted for by nutrient accumulation in the brine channels. The close associ. . . -m f~r\fl nhntncvnatlon between nutnent concentratiolxa Lcl.UYIaV.VUJ thetic efficiency of ice algae at the botto observed by Gosselin et al. (1985) may suggest that ice algal cells are indeed nutrient limited. This observation has been confirmed by relating fortnightly tidal mixing to variations in (1) carbon allocation to protein (Smith et al., 1987), (2) photosynthetic performance (Cota and Home, 1989), and (3) uptake of anic nitrogen by ice algae (Demers et al. (1990) also demonstrated a tween chlorophyll and nitrate concentrations at the ice-water interface. Indepen-
0924-7963/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PU SO924-7963(96)00027-9
54
S. Tagrrclri,R.E. H. Sm?h / Jorrrnai of Marine System I I f 1997) 5.3-61
bioassays conducted by Maestrini ct al. (1986) showed that ice algae responded to nitrogen enrichment in a way that indicated nitrogen limitation in an estuarine area of Hudson Bay. However, using stable isotope bioassays, Harrison et al. (1990) concluded that ice algal assemblages never appeared to be nitrogen-limited in fully marine waters. These contrasting reports may be due to regional differences and the stage of algal accumulation. GOSselin et al. (1990) suggested that ice algal growth was light limited at the beginning of the growth season and became silicon limited later on, when in situ light and the accumulated algal biomass were high and the tidally driven nutrient supply was not adequate to satisfy algal nutrient requirements. Although the effect of siiicon was not determined in their study, Harrison et al. (1990) showed a systcmatic shift from predominantly nitrate metabolism during the early growth season to predominantly ammonium metabolism during the declining period of algal growth. Furthermore, Harrison et al. (1990) suggested that the conceptual model of Dugdale and Goering (1967) for the cycling of nitrogen in the pelagic ocean and its relation to primary production was also valid for ice algal communities. Ice algal growth in relation to nutrients can be studied by several methods: (1) changes in the algal biochemical composition; (2) differential enrichment bioassays; (3) photosynthate allocation; and (4) source of nitrogen nutrition (i.e., nitrate or ammonium). All four approaches were used to study ice algal communities in Resolute Passage, Northwest Territories, Canada, during the Saroma-Resolute Study @ARES) program. The present study is concerned mainly with photosynthate allocation. Protein synthesis of algae is directly related to growth (Morris, 1981; DiTullio and Laws, 1986). Determination of r4C incorporation into the protein fraction through photosynthate allocation is a crude but rapid and convenient way to study the protein synthesis of algae (Morris, 1981; Hitchcock, 1983). Nutrient limitation causes low protein synthesis and subsequently high production of storage products such as polysaccharide and lipids (Shifrin and Chisholm, 198 1; Taguchi et al., 1987). Determination of phoosynthate allocation permits the study of physiological change in response to nutrients. Early dent enrichment
studies (McConville, 1985; Nichols et al., 1986) drew inferences on ice algal photosynthate allocation based on experiments with non-ice algae at much higher light and temperature conditions. Photosynthate allocation in ice algae has been studied in the Antarctic (McConville et al., 1985; Palmisano and Sullivan, 1985) and more recently in the high Arctic (Smith et al., 1987, 1989; Smith and Herman, 1992). McConville ( 1985) and Nichols et al. ( 1986) observed a relatively large share (31-59%) of photosynthate in lipid and a small share (20-24%) in the protein fraction of ice algae. These studies also indicated that a substantially higher allocation to protein (up to 40%) was directly related to an increase in the nutrient supply caused by tidal mixing. Allocation of carbon to protein seems well suited for tes&ing the i~SpOnse of ice algae to varying nutrient enrichments at low light and low temperature. The purpose of the present study was to characterize the response of photosynthate allocation by arctic ice algae as a function of changes in nitrogen and silicon enrichment. We had three hypotheses: that the photosynthate allocation to protein would (1) exhibit an acclimation similar to those induced by the tidally driven nutrient supply (Smith et al., 1987) (2) have silicon-limited growth at high algal biomass (Gosselin et al., 1990), and (3) have ammonium-dependent growth during the declining period of the algal bloom (Harrison et al., 1990). The results confirmed the latter two hypotheses, indicating a combined control of ice algal growth by the availability of silicon and ammonium.
2. Materials and methods Samples were taken on 15 and 21 May 1992 at a station on land-fast ice in Resolute Passage, Northwest Territories, Canada (74’41.19’N, 95”15.59’W), about 2 km south of Cornwallis Island. Water pumped from just below the bottom of the ice was transferred to a dark bottle and then filtered immediately through a Nuclepore filter (0.2 pm pore size). Although a detailed sampling protocol has been provided by Smith et al. (1995), a brief description is given here. The first-year ice was sampled with a SIPRE coring device (Smith et al., 1987). Ice cores were collected for the nitrate enrichment experiment (NO,) and the
Table 1 Mean (*standard deviation) of photosynthesis [mg C (mg CHLa)- ’d- ‘1 of ice algal communities during 24 were significant ( p < 0.05) hxperiment
Treatment
IPR~~u~y,,ilr&
Number of
observations nitrate ammonium NH4 SiO, + NO, silicate + nitrate SiO, + NH, silicate + ammonium NO3
0.22 zk0.05 0.40 * 0.09 0.31 kO.07 0.72 + 0.16
6 5 7 7
ammonium enrichment experiment (N 1992 and for the silicate enrichme (SD, + NO, and SiO, + N J) on 21 May 1992. The loosely consolidated ice crystals from the botn*tn-.q’ me*P ~~~~~~11~.1 tom ‘ luycr WuIb bUIb.U,J scraped inntofiltered ceawater and allowed to melt slowly in a dark bottle (Garrison and Buck, 1986). The resulting suspension was a.bout 10% melted ice and 90% seawater with a salinity of about 30 psu. Chlorophyll a concentrations were determined and were about 1 mg per liter of suspension. Subsamples were taken from the suspension to measure chlorophyll pigments and dissolved ammonium, nitrate, nitrite, phosphate and silicate. Subsamples for nutrient analysis were passed through a membrane filter (type HV) and all but those for ammonium were stored at - 20°C until analysis; ammonium analyses were made on fresh samples within 12 hr of collection using the method of Solorzano (1969). All other nutrients were analyzed using the methods of Strickland and Parsons (1972). Chlorophyll a and pheopigments were determined by the method of Holm-Hansen et al. (1965). Subsamples were taken for the measurement of photosynthetic rate by the ice algal communities in the d;lmtm P.IFnPnP;AnP .#p& 14C_&]& gj&~, t;,(-g-_ IIULC, JU3pmrll”‘ IJ bonate. Incubations were carried out in acid-cleaned plastic bottles for 24 h in blue-filtered, artificial light incubators (Smith et al., 1987) maintained at ambient temperature ( - 1.7”C) and an ambient irradiance level of 20 p,Ern-* s -I. No controls with zero addition were employed in the nitrate and ammonium enrichment experiments. Nitrate (0.125, 0.25, 1, 10, 50 and 200 PM) was added to the first series of incubation bottles (NO,). The first three nitrate enrichments were not significantly different from the
the measurement of total organic carbon fixation and triplicate samples for the dete~ination of photosynmate allocation. End products of photosynthesis were determined by the method described by Smith et al. ( 1987). By convention, the four resulting fractions are referred to as follows: methanol-water soluble =
NITRATE (PM) Fig. 1. Rate of ice algal photosynthesis per mg chlorophyll a in the ammonium (upper) and nitrate (lower) enrichment experiments. Vertical bar indicates two standard deviations.
3. I. Nitrate and ammoniclm enrichment
low molecular weight compounds (LMWC); chloroform-soluble = lipid (LIP); TCA-soluble = polysaccharide with nucleic acids (PNA); and TCA-insoluble = protein (PRO). The samples of the lipid fraction were further analyzed to distinguish four lipid classes: triacylglycerols (TG), free fatty acids (FFA), acetone-mobile polar lipids (AMPL) and phospholipids (PL) by the method described by Smith and D’Souza ( 1993).
Enrichment with ammonium (except at 100 FM) stimulated higher photosynthetic rates [in mgC (mg CHL a)-’ d- ‘1 than enrichments with nitrate (Fig. 1). Ammonium enrichment with 0.1 PM doubled the ambient concentration of ammonium. This enrichment is often sufficient to enhance the total photosynthesis per chlorophyll a. However, no systematic effect was observed (Fig. 1). Ice algae showed little response to nitrate enrichments even at concentrations above 10 @Z. Ammonium enrichment of 100 PM inhibited the synthesis of protein, which was the predominant fraction in the photosynthetic end products (Fig. 2). This minimum value of 40 + 3% was 73% of the maximum value (55 _I 6%) found in the incubation with 0.1 PM ammonium added. No protein inhibition was detected for up to 200 FM of added nitrate (Fig. 2). The maximum value was 53 f 6% at 50 and 200 (-LMof added nitrate.
3. Results The study period encompassed the stage in the ice-algal growth cycle with declining chlorophyll biomass (Smith et al., 1997-this issue). The background concentrations of nutrients in the incubation bottles were l-2 PM of phosphate, 7 ~JJV of silicate, 8 PM of nitrate and 0.1 pM of ammonium. The ice algal community actively assimilated carbon during the incubations (Table 1). Photosynthetic rates were significantly different between experiments ( p C 0.05; Student’s z-test) except between NH, and SiO, + NO,.
O.lpM
0.2pM
3.2. Silicate enrichment Total photosynthesis per unit chlorophyll a was enhanced more in enrichments with SiO, + NH, than
1OpM
1W
50pM
1OOpM
AMMONIUMENRICHED 60 50 40 30 20 10 0 i
.J
0.125pM
0.25j.iM
1PM
10pM
50pM
200pM
NITRATEENRICHED Fig. 2. Change in relative abundance of photosynthetic end products in relation to different concentrations of ammonium or nitrate. Maxirmim coefficients of variance were 16% for LMWC, 12% for PNA, 9% for PRO and 14% for LIP.
e2 Lipid crass allocation of ice algal
synthesis was 30 f 5% while percent incorpo of 14C into polysaccharide plus nucleic acids ( was always highest, with a mean of photosynthetic end products. Although allocation to fractions other than lipid
munities as percent of total
Treatment:nitrate (50 j_kLM) 0 18 6.3
13
2.5 IO 50
3.8 5.6 3.9
14 14 14
62 53 64 55
TreQtntent: amtnnnium(50 C_L 0 29 2.5 19 a.3 10 14 4.8 50 15 5.0
15 14 15 16
49 58 67 64
30 17 27
TG = triacylglycerols (neutral lipids); FFA = free fatty acids; AMPL = acetone-mobile polar lipids; PL = phospholipids. NO duplicate observations
1.0
CO
were made.
~ +50/.&lAMMONIUM
varied with the enrichment, the allocation to lipid stayed relatively low and stable (7.3 + 0.6% for ammonium and 7.5 + 1% for nitrate; Fig. 4). Further analysis of lipid classes revealed allocation predomiohpi& (PI j in nantly to membrane-associated pho 4 experiments both the SiO, + NO, and SiO, + (Table 2). However, in the SiO, + NH, enrichments, neutral lipid (TG) tended to decrease while BL tended to increase with increasing silicate enrichments.
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Fig. 3. Rate of ice algal photosynthesis per mg chlorophyll a in the silicate enrichment experiments with 50 piI4 ammonium (upper) and 50 PM nitrate (lower). Vertical bar indicates two standard deviations.
Effects of “C tracer disequilibrium among different fractions and their precursors must be considered in drawing conclusions from incubation experiments for photosynthate allocation of carbon by microalgae (Li and Harrison, 1982; Smith a.&c:Geider, 1985). This is particularly the case for slowly growing cells such as ice algae, whose generation time can be longer than 72 h (Maestrini et al., 1986). Smith et al. (1987) showed that the labeling time courses of photosynthate allocation in arctic ice algae were predominantly linear even up to 70 h. Although the percent distribution of allocation changed s!owly with increasing incubation time, the distribution was quite similar to that at 24 h even after 70 h. While our standard 24 h incubations should have been able to minimize the effects of the 14Ctracer disequilibrium, short-term incubations may be of considerable value
S. Taguchi, R.E. H. Stnith / Jomml
58
of Marirw Systerrrs I1 f 1997) 53-6 I
for understanding the kinetics of biochemical synthesis by the ice algae. The physiological response to nutrient enrichment may differ depending on the nutrient, as shown in the present study. Nitrate enrichment probably had no effect on photosynthesis at 0.125, 0.25 and 1 PM because the ambient nitrate concentration was 8 ~.LM (Fig. 1 and Fig. 2). The highest protein allocation (53 f: 6%) was observed at 50 PM. A similar phenomenon was observed for the SiO, + NH, enrichments (Fig. 41, where carbon allocation to protein increased to 46 + 7%; again, this is probably because the ambient silicate concentration was 7 PM. In contrast to nitrate and silicate enrichment, 0.1 C_LM ammonium enrichment was already significant compared to the ambient concentration (0.1 h M). Therefore, quantifying the responses of the ice algal community to enrichment in an ammonium-depleted environment can be complicated. The extraction procedure we used was the same one previously applied to measure photosynthate allocation in arctic ice algae (Smith et al., 1989). However, the allocation to low molecular weight compounds (LMWC) was about 15 + 4% of the total
in the SiO, + NO, enrichment experiments (Fig. 4), which is less than half the values previously reported for arctic ice algae in the same geographic area (Smith et al., 1987, 1989). We currently have no explanation for this discrepancy and plan to address it in future studies. The minimum value of 14C incorporation into protein was 22 f 4% in the SiO, + NO, enrichment experiment (Fig. 4), which is similar to the value of 20-24% obtained by Smith et al. (1989). Both enrichment experiments with nitrate and ammonium revealed that the maxima of photosynthate allocation to protein were 51 _t 4 and 48 + 5%, respectively (Fig. 2). The degree of enhancement in photosynthate allocation to protein can be estimated by the relative growth rate since algal protein synthesis is directly related to growth (Morris, 198 1). A direct relationship has been observed between the percent protein allocation and the relative growth rate (l.~&~x) (DiTullio and Laws, 1986) and is described by the equation: %proteinC = 15 + 3S-
(1)
I-CMAX
+50pM AMMONIUM 24hr 50 40 30 g
20 10
8
0
iii
CJaoa EZ
g$45 J
8
5”
ggg fQ +50pM NITRATE 24hr
0
uaok 5””
OrcM
3
“a&g
SC&” W
!jgis~
f””
A
2.5pM
5PM slucAlE
10pM
25pM
J 50pM
ENfwliED
Fig. 4. Change in relative abundance of photosynthetic end products in relation to different concentrations of silicate + 50 p M ammonium or silicate + 50 p, M nitrate. Maximum coefficients of variance were 18% for LMWC, 13% for PNA, 9% for PRO and 9% for LIP.
Table 3 Mean (k standard deviation) of % protein allocation and estimated relative growth rate of ice algal communities based on triplicate observations. SiO, + NO3 and SiOl, + N cantly different ( p < 0.01) Experiment
Treatment
Protein allocation (96)
Wkk4x)
NO, NH, SiO, +NO, SiO, +NH,
nitrate ammonium silicate + nitrate silicate + ammonium
51 L-3.7 48kS.3 3Ok5.0 40 + 8.1
1.o + 0.07 0.94 + 0. I 0.43 + 0.07 0.71+0.1
Relative growth rate
where % protein C is the percentage of photoassimilated carbon allocated to protein after a 24-h incubation, p is the growth rate and l.~~*x is the maximum growth rate -when % protein C is 50% or higher. The high protein values in the NO, and N may suggest a high relative growth rate (l.~/p,~~x) of 1.O-0.94 (DiTullio and Laws, 1986). Ice algae had the highest relative growth rate with the nitrate and ammonium enrichments (Table 3). When algae are perturbed, as they were in the present experiments, protein synthesis may not be so simply related to growth rate. In fact, addition of the limiting nutrient(s) may produce a temporary suppression of photosynthesis and/or protein synthesis as cellular reserves are devoted to nutrient assimilation; this effect is particularly clear for nitrogen (Turpin et al., 198P). In our study, the response to ammonium would suggest that the benefits of improved nitrogen nutrition outweighed the energetic costs of assimilation (except at very high additions) so far as total photosynthesis is concerned, Protein synthesis, however, was still somewhat depressed after 24 hr at intermediate ammonium additions. In the case of SiS, + NH, additions, the cells were evidently able to exploit the nutrients for both photosynthesis and protein synthesis within 24 h. The results from the ammonium and nitrate enrichment experiments may suggest the simultaneous enhancement of allocation to protein by ammonium and nitrate (Table 3). The simultaneous utilization of ammonium and nitrate has been observed in benthic algae including ice algae (Maestrini et al., 1982; Price et al., 1985; Queguiner et al., 1986; Demers et al., 1989). Harrison et al. (1990), in contrast, observed a systematic shift from predominantly nitrate
1988; Lizotte and Sullivan, 1992; Smith et al., 1993). enrichment experiment showed a er allocation of carbon to protein than the SiO, + NO, experiment (Table 3). The relative growth rate of SiO, + NH, &/pMAX = 0.71 + 0. I) was also significantly hig relative growth of SiO, + NO, (p/p ned suppressive 0.07). This may indicate a co effect of silicate with ammonium. owever, it is not clear why the allocation pattern and total photosynthesis might be different between NO, and SiO, + NO, (Tables 1 and 3). Alternative explanations for the different allocation pattern reported here might be because physiologically different ice algal populations were used in the bioassajs. When the combined effects of various nutrients are considered, ammonium appears to be controlling ice algal growth with limitation by silicate during the ice algal population decline in the high Arctic (Table 3). Regenerated ammonium might be mediated by prokaryotic microorganisms (Carey, 1985; Kuosa et al., 1992) and silicon-limitation might be caused by the massive growth of ice algae but alleviated by hydrodynamic processes that occur in the underlying water column (Cota and Home, 1989).
Acknowledgements The authors appreciate the financial support from the Federal Department of Fisheries and Oceans in Canada and the Monbusho International Science Research Program (#03044146) and the Ministry of Education in Japan. Logistical support was provided
S. Tnguclri. R.E.H. Shth / Journnl of A&wineSwtetns11 ( 1997) 53-61
60
by the Polar Continental Shelf Project in Canada. M. and M. Levasseur coordinated the field t&&in sampling and sample analysis. Contribution (B-579) from Hokkaido National Fisheries Research Institute.
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