Experiments on nutrient limitation in bottles

Transcription

1 Journal of Plankton Research Vol.20 no.8 pp , 1998 Experiments on nutrient limitation in bottles D.H.Cushing and J.W.Horwood Yarmouth Road, Lowestoft, Suffolk NR32 4AB and * Centre for Environment, Fisheries and Aquaculture Science, Pakefield Road, Lowestoft, Suffolk NR32 OHT, UK Abstract. A simple model is used to illustrate the character of production, within an incubation bottle subject to addition of a potentially limiting nutrient, over time periods long relative to the algal dynamics (1-10 days). Grazing by microheterotrophs significantly affects algal and nutrient dynamics, and production reflects the integration over time of grazing and nutrient-regulated growth. Greater production is found with increased nutrient, but this increase continues into the region of luxuriant nutrient. Increased production per se cannot be used to determine whether a nutrient is limiting. Introduction Production in the sea has for a long time been estimated with bottle experiments. For example, Nielsen's (1952) radiocarbon measurements were based on the assumption that the grazing copepods were excluded by filtration from a population of algal cells, which we would now classify as relatively large ones. The cyanobacteria of the picoplankton were discovered in 1979 (Johnson and Sieburth, 1979; Waterbury et al, 1979). They are eaten by the protozoa, possibly the most abundant grazers in the ocean. Nielsen's experiments lasted for 3 h in an apparently simple system. Today, some experiments last as long as 10 days and the ecosystem in the bottle is now recognized as a complex one. Smith et al. (1984) and Jackson (1983) noted that, in such bottle experiments, protozoan herbivores must play a considerable part in carbon cycling and, indeed, that the radiocarbon on the filter paper included much grazed material. The 'grow out' experiments (Martin et al., 1988,1989), with iron enrichment, lasted from 4 to 6.5 days; with high division rates and high grazing rates, the bottle ecosystem may change rapidly and radically, not only in numbers, but also in structure, as indeed Martin et al. (1989) pointed out. In both papers, Martin et al. believed that nutrient limitation could be shown by adding the nutrient in question to sea water in a bottle which was incubated for some days, and by monitoring the response of algae and nutrients. The division rate of the algae, u, will decline with falling nutrients by the Michaelis-Menten relationship (Dugdale and Goering, 1967; as displayed in Figure 1). At a high nutrient level (A), u is very close to p, the maximal division rate; then the increment in algae with nutrient added, AP n, is the same as AP C, the control increment with no added nutrient. At B, at a low nutrient level, AP n > AP C, so the added nutrient was the limiting one. Banse (1990) expressed the results of Martin et al. (1989) as (log chlorophyll on time) as an estimate of the division rate of the algae in the exponential phase. In two sets of observations, the estimated division rates did not differ from the control; in the third set, a difference was established, but only after a delay. Martin et al. (1990) countered the argument in using the regressions of the decline in nutrient and of Oxford University Press 1527

2 D.H.Cushing and J.WJionvood \i on nutrient Nutrient Fig. L The Michaelis-Menten relationship [u(/v) = jini(k + N), where \L is the division rate of the algae, ji is the maximal division rate. A' is the nutrient in the water and K is the half-saturation coefficient]: the division rate, u, is plotted on nutrient in umol I" 1. (A) is at high nutrient and (B) at low nutrient. the increase in chlorophyll during exponential phases; however, only in one case out of six was a regression significantly different from the control (at P = 0.05). The difference between Banse and Martin et al. was really whether AP n differed from AP C. The argument requires that the bottle contains only algae. However, the protozoan grazers must also have been present. With grazers present (g is the instantaneous mortality due to grazing), nutrient limitation can be masked, e.g. if (u - g) = 0. Further, u - g could be negative and (assuming no grazing) a negative division rate is impossible. Hence, we should examine the role of grazing in the system. Model The following brief model represents an attempt to caricature the effects of grazing and regeneration; the units are in p.mol N0 3 -N. We have used nitrate because it is believed that it limits production in the sea; it is also the prime nutrient of the Redfield series (which, of course, includes iron). The conclusions drawn should apply to any long-term enrichment experiment. About one-third of grazed nitrogen is regenerated [Butler et al. (1969), based on estimates of gross growth efficiency; Suzuki et al. (1996) used 15 N]. Fasham et al (1990) showed that the bacterial regeneration was about half that from the zooplankton (and presumably from the quantity grazed), so the total regeneration is modelled as QI2 or as g/2- where P is umol N in the algal cells, representing algal stock at time t, u is the 1528

3 Experiments on nutrient limitation in bottles instantaneous nutrient-dependent division rate of the algal cells and g is the instantaneous rate of algal mortality due to grazing. and dnidt = (-u(a0 + (1/2) g) P u(a/)= where p is the maximal instantaneous algal division rate and K is the halfsaturation coefficient of the Michaelis-Menten equation (which here is fixed at 0.5 umol N I" 1 )- This can be solved numerically using simple forward finite difference equations: P, where Q, is the cumulative quantity grazed from time 0 to t and AP is P, + & - P,. where Pr, is the production from time 0 to t. The initial concentration of NO3-N in the water was initially taken as 10 umol I" 1, which represents the maximal winter value in the North East Atlantic, i.e. nutrient replete. The initial quantity of algae is 5 umol I" 1, a typically high observed quantity (Elmgren, 1984; Fransz and Gieskes, 1984; Mommaerts et al., 1984) in the North Sea and in the Baltic. The quantities are high in order to reveal the mechanisms. The results are expressed in P,, u, N,, Q, and Pr, for 10 days and the time step At is one-hundredth of a day. A range of values was used of (p - g), where p and g are each varied from 0.1 to 1.0, with the other parameters fixed. Results Figure 2 shows the time course of a typical sequence of P, and N, for a period of 10 days. P, runs to a peak and then declines, after which N, is very low. Recall that u declines by the Michaelis-Menten equation. The nitrogen in the water, TV, quickly converges to where dmdf = 0, at N = , generally at N* = V 2 gk/(\i - Figure 3 displays the dynamics for a range of parameter values. For p constant (= 0.5), and for g = (p > g) and (p < g), the following three figures display the changes. Figure 3a shows the changes in P, with time (with the highest values associated with lowest g). Figure 3b illustrates changes in j with time; u 1529

4 DJLCushing and J.WJMorwood declines by the Michaelis-Menten equation and the decrease is moderated by regeneration. Figure 3c displays the changes in N t, nitrogen in the water. In Figure 4 are shown production, Pr, and Q, the cumulative quantity grazed on days to day t. Figure 4a display curves of production on time; p = 0.5 and g = (p > g) and (ji < g). Figure 4b shows curves of Q, the quantity 16.0 r P t, N t on days Days Fig. 2. Time course of a typical sequence (ji = 1; g = 0.1) of the algal stock. P,, in umol NO 3 N I' 1, rises to a peak and then declines as nutrients are reduced, u falls to near zero and grazing predominates. Nutrients in the water, N, (in the same units), decline to a trough as they are taken up by the algae. P, on days

5 Experiments on nutrient limitation in bottles (b) Division rate on days \ \ \ 1 \ \. % * \ A ( ) Days (c) 10 a N, on days Fig. 3. The sequence of algal N, P,, division rate, u, and nutrient in the water. A', (in umol NO 3 N I' 1 ) in time. The panel at the right of the figure shows the values of g for the indicated lines, (a) The change in time of P,, the algal N, on days; p = 0.5 and g = (p > g) and g = (ji < g). (b) The change in time of division rate, u; p = 0.5, initially, and g = (u >g) and g = (u < g) The algal division rate declines by the Michaelis-Menten equation and the rate of decline is moderated by regeneration, (c) The change in time of <V, nitrogen in the water (in umol NO3 I" 1 ); p = 0.5, and for g = (u > g) and (u < g). The division rate declines to an equilibrium at V 2 g, as it is taken up by the algae. grazed; g = 0.5 and p = ( > g) and (fi < g). Production and the quantity grazed were defined in terms of AP, or P l -P 0 = P 0 [exp(u -g)t-1]. There are two important controls, u/(u - g) [or g/(u - g)] and exp(u - g). In Figure 4a, the curves cross over because with increasing g, u/(u -g) increases when u >g and 1531

6 DJLCnshing and J.W-Horwood (a) 20 P r on days ^» //*-»* ^2^^ ^,^ft// ^ ^ J I f 1 1 I Days 10 (b) Q, on days 12 r Q, o.8o Fig. 4. Production and the quantity grazed (both in urool NOj N1*'). The panels to the right of the figure show the values of for the indicated lines, (a) Cumulated production for ji = 0.5 and g = (p > g) and (ji < g). (b) Cumulated quantity grazed, Q,, for p = 0.5 and g = (p > g) and (ji<s). exp((j - g) weakens. In Figure 4b (when ji > g), as g increases, g/(ji - g) increases and exp(u - g) increases and there are no cross-overs. In Figure 5 are shown the relationships of P, (a) and A*, (b) on time at different initial values of P t, the initial value of N, remaining the same, at 10 umol I" 1 ; 1532

7 Experiments on nutrient limitation in bottles (b) Fig. 5. The dependence of P, and N, (both in units of NO3 N I" 1 ) on days. The panels to the right of the figure show the values of g for the indicated lines, (a) P, at different initial values of algal nitrogen, 0.1-S umol I" 1, with the initial value of N, constant at 10 umol I" 1, (b) N, at different initial values of P, of ; the initial value of N, is always 10 umol h 1. P o is varied from 0.1 to 5.0. The patterns in Figure 5 should be compared with those in Figure 3. In Figure 6 is displayed the algal stock, P t, for 10 days as a function of the initial quantity of nutrient with no grazing; the greater the initial nutrient, the greater the stock of algae and then later the asymptote. Figure 7 illustrates the 1533

8 DJLCnshing and J.WJJorwood P t on days at different initial nutrients X Days Fig. 6. Changes in P, (in imol NO 3 NH) with no grazing. P, is plotted on days with different initial levels of nutrient (indicated on the panel to the right of the figure); u = r Pt on days at different initial nutrients Days Fig. 7. Changes in P, (in umol NO] N I" 1 ) with grazing (g = 0.1) on days at different initial quantities of nutrient (indicated on the panel to the right of the figure); u = 1.0. dependence of stock on the initial nutrient in the water (N, = ) by days with grazing mortality (g - 0.1); stock declines after the peak as it is eaten. Figure 8 displays the relationship between stock, P t, at 10 days on the initial nutrient added with g varying. In Figure 8a, at low g it is approximately linear, but at high g a curvilinear relationship appears; in Figure 8b, where g is varied 1534

9 Experiments on nutrient limitation in bottles (a) 25 P, at ten days on initial nutrients 20 D15 I ^ Initial nutrient 25 (b) P t at ten days on initial nutrients to Initial nutrient Fig. 8. P, (in umol NO 3 N I" 1 ) at 10 days on initial quantities of nutrient. The panels on the right of the figure indicate the initial levels of nutrient (a) g = 0.1-; (b) g = from 0.7 to 1.05, a more complex relationship emerges. The essential point is that with added nutrient, increased production is expected. Understanding of the shapes of the relationships between P(10) and the initial level of nutrients [N(0)] can be obtained by inspection of the plots of the dynamics of P(0 and N(t), e.g. Figure 2. For low levels of grazing (0.1 < g < 0.6), the increment in P(10) with N(0) is approximately linear. At low g, not only is P(10) higher, but so too is the rate of increase with N(0). Low g allows a rapid growth of algae, little regeneration, and a rapid exhaustion of nutrients. A maximum for the algae is reached near the point of nutrient exhaustion (Figure 2). 1535

10 DJLCushing and J.WHomood The level of the peak, above the initial algae, is almost identical to the level of added nutrients There is then a slow decline governed by g. Hence, the level of P(10) is more or less linearly related to the level of initial nutrients. As g increases to about g = 0.6, a similar relationship is maintained and the peak algal concentration is not much affected by grazing. At higher levels of g, the relationship becomes more non-linear. The peak of the algae is severely constrained by grazing, and importantly there is more variation in the time of the peak. For g = 0.8, the peak density is still approximately linear with N(0), but the time of the peak is at 3, 5 and 6 days for N(0) of 10, 20 and 30, respectively. There is an exponential decay after the peak and the difference in duration of the decay phase gives an approximately exponential increase for the P(l0) versus N(0) relationship. As grazing matches maximum growth (g = 1.0), a sigmoid relationship appears. At low N(0), algae and nutrients are depleted by day 10. For higher levels [N(0) > 20], the dynamics are very slow (g = u) and although N(t) is continuously in decline, the absolute level is still well above K and the algal density remains more or less constant, to give the asymptotic values. Discussion The design of the experiments of Martin et at (1988,1989) presumed the presence of algae only; if grazers were present, the test against the control is not unambiguous. The picoplankton and their predators, the protozoa, live in all oceans and most lakes so it is very unlikely that the protozoan grazers were absent from the bottles of Martin et al. The result of the model shows that when a nutrient is added, an increment in production is always expected, but the size depends strongly on the grazing rate. Hence, the 'grow out' experiments of Martin et al. must yield the expected production and the comparison with a control is misleading. There is a direct relationship between the initial quantity of nutrient and algal production. At any level of nutrient, an addition yields more production. This is true at those levels of nutrient normally regarded as limiting, i.e. in the North East Atlantic, at nitrate concentrations of <10 umol I" 1. An interesting point is that the production with grazing is the same as (or slightly greater than) that without. The results are generally applicable to any nutrient subject to Michaelis Menten kinetics. The problem has been formulated in nitrate because it represents most fully the nutrients in the Redfield series. Each nutrient obviously has a different function; indeed, the N/P ratio shows that more nitrogen is used, but phosphorus is equally essential; each represents a different part of the physiological needs of the ecosystem. Production in the ecosystem is driven by irradiance and restrained by nutrient lack. Hence, at any time, the quantity of nutrient represents a residuum from the processes of production. If a nutrient is added (as in a bottle), it need not surprise us that there is more material at the end of the experiment. The biological processes that lead to this result may, however, be complex, as we have shown. The study of nutrients in the sea must be both chemical and biological. 1536

11 Experiments on nutrient limitation in bottles When Dugdale and Goering (1967) formalized the nature of nutrient limitation, the question seemed to have been resolved. The difficulty was to estimate u/ji. Now Falkowski and his colleagues have shown that they can estimate this quantity in the sea (Bielefeld et al, 1996) and so nutrient limitation can be properly assessed. Liebig's (1840) Law of the minimum stated that the 'growth of a plant is dependent on the minimum amount of the foodstuff presented'. Cullen (1991) distinguished Blackman (1905) limitation to the rate of production from Liebig's limitation to the quantity produced. We have shown that Liebig limitation follows from Blackman limitation, i.e. if the limiting rate of production is integrated over time, the quantity produced is limited. There is, of course, permanent nutrient limitation in the Michaelis-Menten relationship (and in the Droop equation; Droop 1968), for \a is only found at infinite N. However, the next question is whether \i(n) is much diminished. The results of this study apply, at this stage, to the experiments in bottles that last up to 10 days in which the vital rates are very high. More generally, however, we have clarified the nature of nutrient limitation and have linked the Blackman and Liebig forms. The simplest marine ecosystem is that in the centre of the subtropical gyres where numbers of algae remain in a quasi steady state for long periods, where nitrate is absent from the euphotic layer, where most cells do not sink, and where the ecosystem is fed with ammonia excreted from the whole animal ecosystem. The balance is probably maintained between u(n) and g. Acknowledgements J.W.H. was partly supported by the Ministry of Agriculture, Fisheries and Food (MF 420). We are grateful for comments from Dr Trevor Platt, Professor John Shepherd, Professor Brian Rothschild, Dr Kevin Flynn and Professor A.N.Watson. References Banse.K. (1990) Does iron really limit phytoplankton in the offshore subarctic Pacific? Limnol. Oceanogr., 35, Bielefeld,M., BalcAJ., Kolber.ZS., AikenJ. and Falkowski.P.G. (1996) Confirmation of iron limitation of phytoplankton photosynthesis in the Equatorial Pacific Ocean. Nature, 383, Blackman,F.F. (1905) Optima and limiting factors. Ann. Bot., 19, Butlerf.1., Corner,E.D.S. and Marshall.S.M. (1969) On the nutrition and metabolism of zooplankton VI: Feeding efficiency of Calanus in terms of nitrogen and phosphorus./ Mar. BioL Assoc. N.S., 49, Cullenj. (1991) Hypotheses to explain high nutrient conditions in the open sea. Limnol Oceanogr., 36, Droop,M.R. (1968) Vitamin B and marine ecology IV. The kinetics of uptake, growth and inhibition in Monochrysis lutheri J. Mar. BioL Assoc. UK, 48, Dugdale,R.C. and GoeringJJ. (1967) Uptake and regenerated forms of nitrogen in primary production. Limnol Oceanogr., 12, Elmgren.R. (1984) Trophic dynamics in the enclosed brackish Baltic Sea. Rapp. P.-V. Cons. Int Explor. Mer, 183, Fasham>IJ.R., Ducklow,H.W. and McKelvie.S.M. (1990) A nitrogen based model of plankton dynamics in the oceanic mixed layer. / Mar. Res., 48,

12 D.H.Cushing and J.W.Horwood Fransz.H.G. and Gieskes.W.W.C. (1984) The imbalance of phytoplankton and copepods in the North Sea. Rapp. P.-V. Com. InL Explor. Mer, 183, Jackson.G.A. (1983) Zooplankton grazing effects on I4 C based phytoplankton production measurements: a theoretical study. /. Plankton Res., S, Johnson,P.W. and SieburthJ.McN. (1979) Chroococcoid cyanobacteria in the sea: a ubiquitous and diverse phototrophic biomass. UmnoL Oceanogr., 24, LJebigJ. (1840) Chemistry: Its Application to Agriculture and Physiology, 4th edn. Taylor and Watson, London, p Martin J.H. and Fitzwater.S.E. (1988) Iron deficiency limits growth in the North east Pacific subarctic. Nature, 331, MartinJ.H., Gordon.R.M., Fitzwater,S.E. and Broenkow.W.W. (1989) Vertex phytopiankton iron studies in the Gulf of Alaska. Deep-Sea Res., 36, MartirU.H., Broenkow.W.W., Fitzwater.S.E. and Gordon.R.M. (1990) Yes, it does. A reply to the comment by Banse. LimnoL Oceanogr., 35, MommaertsJ.P., Pichot.G., OzerJ., Adam.Y. and Baegens.W. (1984) Nitrogen cycling and budget in Belgian coastal waters: North Sea areas with and without river inputs. Rapp. P.-V. Cons. InL Explor. Mer, 183, Nielsen.E.S. (1952) The use of radioactive carbon for measuring organic production in the sea. 1. Cons. InL Explor. Mer, 18, Smith.R.E.H., Geider.R.E. and Platt.T. (1984) Phytoplankton productivity in the oligotrophic ocean. Nature, 311, Suzuki.M., SherrJE.B. and Sherr.B.F. (1996) Estimates of regeneration efficiencies associated with bacterivory in pelagic food webs via a 15 N tracer method. J. Plankton Res., 18, WaterburyJ.B., Watson,S.W., Guillard.R.R.L. and Brand,L.E. (1979) Widespread occurrence of a unicellular marine planktonic cyanobacterium. Nature, 277, Accepted on April 4,