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Seasonal patterns of ammonium regeneration from size-fractionated microheterotrophs
Continental Shelf Research 19 (1999) 1755}1770
Seasonal patterns of ammonium regeneration from size-fractionated microheterotrophs Jean-Franc7 ois Maguer*, SteH phane L'Helguen, Christian Madec, Pierre Le Corre Laboratoire de Chimie Marine, Universite& de Bretagne Occidentale et U.P.R. 9042 C.N.R.S. Roscow, Place Nicolas Copernic, TechnopoL le Brest-Iroise, 29280 Plouzane, France Received 12 August 1997; received in revised form 11 June 1998; accepted 13 April 1999
Abstract Ammonium regeneration by size-fractionated plankton was measured for 1 year at a coastal station in the shallow well-mixed waters of the western English Channel. Rates of ammonium regeneration in the (200 lm fraction varied from 0.6 to 27 nmol N l\ h\. On the seasonal scale, these rates were relatively low ((7 nmol N l\ h\) in autumn and winter, increased steadily from March to attain a maximum (27 nmol N l\ h\) at the end of May and thereafter decreased steadily to the seasonal minimum in December. This pattern is distinctly di!erent from that observed in deep well-mixed waters where the peak ammonium regeneration occurs in summer (Le Corre et al., 1996, Journal of Plankton Research 18, 355}370). Total ammonium regenerated in a year by the microheterotrophs was 15 g N m\, equivalent to about 60% of the total nitrogen uptake. Microplankton (200}15 lm) accounted for about 50% of the regeneration measured between early spring and late summer. Percent contribution of nanoplankton to total ammonium regeneration varied considerably between the seasons, from very high (83}88%) levels in winter to very low (2}13%) levels in summer. Contribution by picoplankton ((1 lm) was high (20}45%) in summer but was less than 20% in other seasons. Ammonium regeneration in micro- and nanoplankton fractions was mainly associated with ciliates and in the picoplankton fraction with bacteria. Macrozooplankton dynamics appears to regulate ammonium regeneration by ciliates and bacteria. Low macrozooplankton biomass in spring may favour a high growth of ciliates and an associated high in ammonium regeneration. In summer, the increase in macrozooplankton may exert a grazing pressure on ciliates. This, coupled with the fact that most of the #agellates are autotrophs, would, in turn, lower the grazing pressure on the bacteria, thus favouring their development and increasing the importance of their role in ammonium regeneration. This situation, where the macrozooplankton dynamics apparently regulates ammonium regeneration in nano- and picoplankton fractions,
* Corresponding author. Tel.: 0033-2-9849-8778. E-mail address: [email protected] (J.-F. Maguer) 0278-4343/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 7 8 - 4 3 4 3 ( 9 9 ) 0 0 0 3 7 - 0
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appears to be di!erent from that in deep well-mixed waters. Here, the relative contribution of ciliates and bacteria to ammonium regeneration shows little variation with an increase in macrozooplankton biomass. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Nitrogen; Ammonium regeneration; Size fractionation; Seasonal cycle; English Channel
1. Introduction The permanently well-mixed waters of the western English Channel constitute a unique temperate coastal ecosystem. The strong tidal action maintains a homogeneous water column (down to &100 m) throughout the year (Pingree, 1978) and the euphotic zone seldom becomes exhausted of nutrients (Wafar et al., 1983). The primary production cycle is synchronous with the seasonal increase in the depth of the euphotic zone and thus, in terms of carbon assimilation, tends to a broad summer maximum (Boalch et al., 1978; Wafar et al., 1983). Seasonal changes of the uptake of nitrogen compounds also re#ect the characteristic of the summer maximum (L'Helguen et al., 1996). Ammonium uptake alone accounted for more than half of the nitrogen taken up in the annual cycle even though the winter nitrate reserve was large and, in the absence of a physical constraint, could be replenished continuously with the in#ux of new nitrogen into the euphotic zone. The winter reserve of ammonium is generally an order of two magnitude lower than that of nitrate and the allochthonous input is negligible (Wafar et al., 1983; L'Helguen et al., 1993). Hence, the in situ regeneration of ammonium is the main mechanism of supply of nitrogen for uptake, with a close balance between its production and consumption (Le Corre et al., 1996). In all these studies, the location of the stations was such that the depth of the euphotic zone was no more than half of that of the entire water column, even in summer. Understandably, therefore, these patterns in the biological processes rely on a seasonal increase in the depth of the euphotic zone and by consequence, of the residence time of the phytoplankton cells within. In the sectors of the well-mixed waters, where the depth of the euphotic zone and that of the water column are more or less the same, the phytoplankton cells can reside for relatively longer periods within the euphotic zone. Hence the phytoplankton cells receive relatively more light energy than the cells from the deeper sectors. Under these conditions, the peak phytoplankton growth in the shallow well-mixed waters occurs much earlier in spring. Consequently, the uptake of nitrogen, especially that of ammonium, attains a peak in spring and remains relatively high through the summer (Maguer et al., 1996b). As ammonium was the major nitrogen source at the time and the allochthonous supply was negligible, it remains to be seen whether in situ regeneration of ammonium, as in deep well-mixed waters, is substantial enough to support this high uptake rate. This assessment of the relative importance of di!erent groups of planktonic microheterotrophs ((200 lm) to ammonium regeneration was prompted by the relatively
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low abundance of such data in the literature. Planktonic microheterotrophs contribute signi"cantly to ammonium regeneration (Harrison, 1992) but the importance of the size classes varies in some instances. The 1}10 lm size range was considered to be responsible for most of the ammonium production (Glibert, 1982; Probyn, 1987; Miller et al., 1995; Le Corre et al., 1996). Elsewhere, either the '10 lm (Paasche and Kristiansen, 1982; Hanson and Robertson, 1988) or the (1 lm (Harrison et al., 1983) fraction was more important. Also, the contribution of planktonic microheterotrophs to ammonium production may vary as a function of the season (Glibert, 1982; Glibert et al., 1992; Selmer et al., 1993). This, in particular, may be closely related to the trophic relationships between the organisms of the microbial loop and their predators (Glibert et al., 1992; Miller et al., 1995). The present study was thus intended to describe the seasonal cycle of ammonium regeneration in the shallow well-mixed waters of the western English Channel and to quantify the contribution of di!erent microheterotroph size classes ((200, (15 and (1 lm) and their taxonomic groups to this process.
2. Material and methods 2.1. Collection of samples Between March 1992 and March 1993, 14 "eld trips, each coinciding with a neap tide, were made to the sampling site (Fig. 1). In each instance, two sets of data collection were made at around 0800 h. In the "rst, water samples were obtained from three standard depths (0, 10 and 20 m) and used for the analyses of nutrients. In the second, samples were obtained from 50% optical depth, fractionated to three size classes ((200,(15 and (1 lm) and used for measurements of chlorophyll a (Chl a), particulate organic nitrogen (PON) and uptake and regeneration of ammonium. The (200 and (15 lm fractions were obtained by gravity "ltration through bolting silk nets and the (1 lm fraction by gentle suction ((100 mmHg) across a 1 lm Nuclepore "lter. The e$ciency of size fractionation was veri"ed by microheterotroph counts. In addition, measurements of uptake and regeneration of ammonium on unfractionated samples ((200 lm) collected from "ve optical depths (100, 50, 22.5, 8 and 3.5% of surface-incident light) were made in April, May, July, September, November, 1992 and January, 1993. On each "eld trip, macrozooplankton samples were collected in vertical hauls with a WP2 plankton net (mesh size 200 lm). In this paper, the 200}15 lm fraction is referred to as microplankton, the 15}1 lm fraction, as nanoplankton and the (1 lm fraction, as picoplankton. Uptake and regeneration rates were obtained directly for the picoplankton fraction and indirectly (by di!erence), for the other two fractions. 2.2. Analytical methods Temperature was recorded with reversing thermometers mounted on water samplers. Nitrate concentrations were measured following the method of Wood et al.
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Fig. 1. Position of the sampling station.
(1967), as adapted by Treguer and Le Corre (1975) for automated analyses with a Technicon II Auto-analyser. Ammonium concentrations were measured manually by the indo-phenol blue method (Korole!, 1970). Chl a content was measured #uorometrically (Yentsch and Menzel, 1963). Particulate matter for PON measurements was collected on pre-ignited GF/F "lter pads and analysed in a Perkin-Elmer model 240 elemental analyser. Analytical precisions in these measurements were: 0.05 lmol N l\ at 10 lmol N l\ level for nitrate, 0.02 lmol N l\ at 0.5 lmol N l\ level for ammonium, 0.05 lg l\ at 1 lg l\ level for Chl a and 0.1 lg N l\ at 2 lmol l\ level for PON. The biomass of macrozooplankton was estimated as dry weight. Samples designated for microzooplankton ((200 lm) enumeration were "xed in Lugol-formalin and counted in an inverted microscope. Samples for enumeration of bacterial numbers were "xed with formalin, stained with acridin orange, "ltered onto 0.2 lm Nuclepore "lters and counted by epi#uorescence in a BH2 Olympus microscope. 2.3. Uptake and regeneration experiments Ammonium uptake and regeneration measurements were made from single incubation experiments using N tracer. Ammonium was added to the samples in the form of NH Cl (97.5 at %, CEA, France) at 10}20% of the ambient concentrations. Half of the sample was immediately "ltered through pre-ignited GF/F "lter pads so as to obtain zero-time enrichment with N of the particulate and dissolved pools. The
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remaining fraction was incubated in 2.5 l polycarbonate bottles under simulated in situ conditions for 4 h around local noon. Incident light on the incubation bottles was attenuated to 50% by enveloping them with pre-calibrated nickel screens. The ambient temperature was maintained constant with a continuous #ow of seawater. The incubations were terminated by "ltering the samples onto pre-ignited GF/F "lter pads. The "lters were dried at low temperature (&403C) and stored dry until analysis for isotopic ratios. A part of the "ltrate was used to measure the ammonium concentration (in triplicate) and the rest was stored frozen at !203C. Ammonium contained in the "ltrate was later extracted by di!usion in basic pH (Kristiansen and Paasche, 1989). The N : N isotopic ratios of the particulate matter and of the ammonium from the "ltrate were measured in a SOPRA GS1 model emission spectrometer following the methods described by Guiraud and Fardeau (1980). Ammonium uptake rates were calculated using the equation of Dugdale and Wilkerson (1986) where the PON content is the one measured at the beginning of the incubation. Ammonium uptake rates were corrected for isotope dilution (Glibert et al., 1982). Ammonium regeneration rates were calculated either by using the equation of Glibert et al. (1982) when ammonium concentration did not change measurably during the incubation, or by that of Laws (1984) when the reverse was true. The coe$cients of variation of the measurements of uptake and regeneration were respectively 4 and 6% at the level of 10 nmol N l\ h\. Total ammonium regeneration in a year was computed from a summation of the day-time regeneration pro"les of each month and a night/day ratio of 0.52 for regeneration rates (Maguer et al., 1996a).
3. Results 3.1. Ambient and biological parameters Consistent with the main characteristic of the permanently well-mixed waters (Wafar et al., 1983), water temperatures were vertically uniform and varied from 9.5 to 16.03C during the seasonal cycle. Nitrate was the major component of the winter stock of dissolved inorganic nitrogen (DIN) (Fig. 2). Coincident with the spring phytoplankton development, nitrate concentrations decreased rapidly through April and May to very low values in June ((0.5 lmol N l\) that persisted until the end of July. Ammonium concentrations varied between 0.15 and 0.55 lmol N l\ (Fig. 2). From very low values in autumn and winter, ammonium concentrations began to increase in spring, in parallel with the phytoplankton growth, to reach maximum values ('0.30 lmol N l\) in summer, with a peak value (0.55 lmol N l\) in July. Chl a concentrations ranged from 0.2 to 2.8 lg l\ (Fig. 3). It was noteworthy that in the seasonal changes of Chl a the peak value (2.8 lg l\) occurred in May and there was a persistence of high values (&1.5 lg l\) in summer (June}August). At other times of the year, Chl a concentrations were generally (0.5 lg l\. Microplankton contributed to between 8 and 46% (average: 30%) of the Chl a, with the maximum
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Fig. 2. Concentrations of ammonium (solid line) and nitrate (dashed line) at the study station during 1992}1993. Vertical distribution of nutrients was homogeneous and the data are averages from three depths.
Fig. 3. Concentrations of Chlorophyll a in micro-, nano-, and picoplankton fractions at the study site during 1992}1993.
percentages in April (42%) and September (46%). Nanoplankton accounted for a higher proportion almost throughout the year (30}73%, average: 54%), except at the beginning of spring ((35% in March}April). The contribution of picoplankton was less (12}27%, average: 16%) throughout the year, except at the beginning of spring (March}April).
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Fig. 4. Density of #agellates in the (200 lm fraction at the study site during 1992}1993.
Fig. 5. Densities of ciliates (solid line), bacteria (dashed line) and macrozooplankton (discontinuous line) at the study site during 1992}1993.
Flagellate counts varied from 0.19;10 to 2.43;10 cells l\ over the year, with a major peak in April followed by two other peaks in June}July and October (Fig. 4). The major part of the #agellates counted (62%) were in the 15}1 lm fraction, 32% were in the 200}15 lm fraction and only 6% were in the (1 lm fraction. Ciliate counts varied from nil to 3.6;10 cells l\ and were generally less than 500 cells l\ (Fig. 5), except in spring when they formed a major peak in May. On average, about 65% of the ciliates counted were in the 200}15 lm fraction and the rest, in the
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15}1 lm fraction. Bacterial numbers ranged from 12;10 to 190;10 cells l\, with 89% of them recorded in the (1 lm fraction. Seasonal changes of bacteria in this size class (Fig. 5) show a broad maximum extending from late May to early August. Macrozooplankton biomass (Fig. 5) varied from 2.9 to 16.5 mg m\ in the seasonal cycle, with a peak in June. 3.2. Ammonium regeneration by microheterotrophs 3.2.1. Total community Rates of ammonium regeneration in the (200 lm fraction varied from 0.6 to 27 nmol N l\ h\. On a seasonal scale (Fig. 6), they were relatively low ((7 nmol N l\ h\) in autumn and winter, increased steadily from March to attain a maximum (27 nmol N l\ h\) at the end of May and thereafter decreased steadily to the seasonal minimum in December. The increase in regeneration occurred in parallel with those of phytoplankton growth and ammonium uptake (Fig. 7). Regeneration rates related linearly with ammonium uptake rates (r"0.83; P(0.01; regeneration/uptake"0.42) and with the total nitrogen uptake (r"0.91; P(0.01). Pro"les of ammonium regeneration (Fig. 8) showed a surface/subsurface maximum, followed by a progressive decrease with depth. Similar patterns were observed also in uptake pro"les. Vertical and temporal integration of the pro"les gave a total ammonium regeneration by microheterotrophs of 15 g N m\ yr\. 3.2.2. Size fractions Patterns of ammonium regeneration by microplankton (0.1}13.5 nmol l\ h\) and unfractionated microheterotrophs were similar (Fig. 6). Percent contribution by microplankton to total ammonium production, however, varied seasonally ranging
Fig. 6. Ammonium regeneration rates in micro-, nano- and picoplankton fractions at the study site during 1992}1993.
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Fig. 7. Uptake (dashed line) and regeneration (solid line) of ammonium by unfractionated plankton ((200 lm) at the study site during 1992}1993.
between 32% (June) and 55% (April) in spring, high and nearly constant in summer (52}56%) and autumn (46}58%) but was consistently low (9}17%) in winter. Nanoplankton regenerated ammonium at rates ranging between 0.2 and 10.4 nmol N l\ h\ (Fig. 6), with the maximum rates in May. From June, regeneration in this fraction became less important and remained at less than 2.5 nmol N l\ h\ until December, when it ranged between 0.5 and 4.7 nmol N l\ h\ throughout winter. Contribution by nanoplankton to total ammonium regeneration, similar to that of microplankton, varied markedly between the seasons. It was between 23 and 31% in spring but decreased substantially in summer (2}13%) before increasing again through autumn (29}44%) to account for nearly all of the ammonium regenerated (83}88%) in winter. Ammonium regeneration rates of picoplankton ranged from below limits of detection to 6 nmol N l\ h\ (Fig. 6). On a seasonal scale, the rates were maximum (4}6 nmol N l\ h\) and occurred as a single broad peak in April}July but in other months they were generally less than 1 nmol N l\ h\. Contribution of picoplankton to total ammonium regeneration was substantial in summer (20}45%) but was relatively unimportant in other seasons ((20% in spring, (17% in autumn and (4% in winter).
4. Discussion 4.1. Regeneration of ammonium by microheterotrophs In coastal ecosystems that are relatively undisturbed by anthropogenic input of nitrogen, ammonium regeneration by microheterotrophs varies seasonally from
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undetectable levels to 100 nmol N l\ h\ (Paasche and Kristiansen, 1982; Cochlan, 1986; Owens et al., 1986; Selmer, 1988; Le Corre et al., 1996). The rates measured in the present study (0}27 nmol N l\ h\) fall in this range but towards the lower end. In the shallow permanently well-mixed waters, ammonium regeneration proceeds at appreciable rates in the entire water column for several months of the year (Figs. 6 and 8). As shown by the regeneration/uptake ratio of 0.42 (Fig. 7), this rate is adequate to provide for more than 40% of the ammonium assimilated by the phytoplankton at the 50% light depth. Integrating the water column regeneration rates (Fig. 8) and raising them to an annual scale gave a still greater importance to the role of microheterotrophs in the regeneration of nitrogen that can sustain phytoplankton uptake. The total ammonium production was 15 g N m\ yr\, equivalent to 60% of the needs in ammonium (24 g N m\ yr\) and 33% of the needs in nitrogen (45 g N m\ yr\) (Maguer et al., 1996a). This is of the same order (respectively 64 and
Fig. 8. Vertical pro"les of ammonium uptake (dashed line) and regeneration (solid line) by the unfractionated plankton in (a) April, (b) May, (c) July, (d) September, (e) November, (f ) January. Note scale di!erence.
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34%) as that found in the deep well-mixed waters (L'Helguen, 1991). Our results not only agree well with a number of uptake and regeneration measurements on discrete samples (e.g. Harrison, 1992) but also demonstrate that ammonium regeneration by microheterotrophs can be substantially important to nitrogen #ux through plankton on annual time-scales. Such an importance of ammonium regeneration in supporting phytoplankton production can perhaps be related to the strong tidal mixing and the permanent homogeneity of the water column. In the absence of a thermocline, the whole water column behaves as a single layer throughout the year and hence the organic matter produced remains within. Decomposition of this organic matter would, therefore, occur in situ and its regeneration products (principally as ammonium) almost immediately become available for uptake by phytoplankton. By favouring a rapid return of nitrogen, the strong mixing thus maintains a high nitrogen uptake throughout the year (Maguer et al., 1996b) and, in turn, also a high level of ammonium regeneration. This would also explain the close coupling between ammonium regeneration and total nitrogen uptake, observed both in the shallow (r"0.91; P(0.01) and deep (r"0.95; P(0.01*L'Helguen, 1991) well-mixed waters, with the only di!erence between the two systems being the pattern of the seasonal changes. 4.2. Size fraction variability in ammonium regeneration during spring and summer In spring, micro- and nanoplankton were responsible for a major proportion (between 73 and 92%) of ammonium regeneration. The absence of metazoans (copepod nauplii, rotifers) and the low abundance of bacteria in these two size classes suggest that the protozoans (ciliates and #agellates) would have been responsible for the bulk of ammonium regenerated at this time. While the importance of protozoans to the regeneration of ammonium has already been demonstrated in a number of marine ecosystems (Glibert, 1982; Wheeler and Kirchman, 1986; Probyn, 1987; Ferrier and Rassoulzadegan, 1991), the relative importance of ciliates and #agellates to ammonium regeneration is less known. In the shallow well-mixed waters of the western English Channel, the patterns of ammonium regeneration rates and the number of ciliates in the 200}15 and 15}1 lm fractions were similar and related statistically (r"0.89, n"15, p(0.01 and r"0.82, n"15, p(0.01 respectively) (Fig. 9). These results suggest that the ciliates could play an important role in ammonium regeneration. Taking into consideration the rates published for ammonium excretion by ciliates (Verity, 1985; Ferrier-Pages and Rassoulzadegan, 1994), at the densities observed in our study, ciliates can produce ammonium at between 8.3 (June) and 18 nmol N l\ h\ (May). This rate accounts for, on average, 72% of the ammonium regenerated by microheterotrophs in spring. A similar importance for ciliates in ammonium regeneration has also been shown in the deep well-mixed waters of the western English Channel (Le Corre et al., 1996) and in the Mediterranean coastal waters (Selmer et al., 1993). The relatively high biomass of #agellates in the microheterotroph fraction at any one time (Goldman et al., 1985), however, has led several authors (Paasche and Kristiansen, 1982; Probyn, 1987) to attribute a much more important role to them in
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Fig. 9. Ammonium regeneration rates (solid lines) and number of ciliates (dashed line) in (a) micro- and (b) nanoplankton at the study site during 1992}1993.
ammonium production. We did not detect any signi"cant relationship between the number of #agellates and ammonium regeneration rates. The high proportion of #agellates (62%) and of Chl a (56%) in the nanoplankton fraction on one hand, and the high ammonium uptake (&60% of total uptake*Maguer, 1995) measured on the other, suggest instead that a large fraction of #agellates are probably autotrophs. In that event, even if their densities were high, their contribution to ammonium production could become relatively less important than that of ciliates. A large fraction of the ammonium regeneration in summer was associated with the organisms (1 lm in size (Fig. 6) and this follows the decline of the diatom bloom
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Fig. 10. Ammonium regeneration rates (solid lines), number of bacteria in picoplankton (dashed line) and ratio between regeneration and bacteria in picoplankton (discontinuous line) at the study site during 1992}1993.
(Maguer et al., 1996b). Similar observations have been reported from the Middle Atlantic Bight (Harrison et al., 1983) and the coastal waters of the Vineyard Sound (Glibert, 1982). The similarity in the changes of regeneration rates and bacterial cell counts (Fig. 10) suggest that the increase in ammonium regeneration in this size fraction in summer could be related to an increase in the density of bacteria. Nevertheless, the ratio between the ammonium production rate and the number of bacteria (Fig. 10) does not remain constant but follows a cyclic change, ranging from 0.6;10\ in autumn to 4.0;10\ nmol N cell\ h\ in summer. This would suggest that it is not only the increase in bacterial biomass but an increase in their activity as well, which is responsible for the seasonal increase in ammonium production in this size fraction. The ratio of ammonium production to bacterial numbers was, in particular, nearly twice as high (4.0;10\ nmol N cell\ h\) in summer than at the beginning of spring (2.5;10\ nmol N cell\ h\). This increased activity could have been stimulated by an increase of nitrogen substrates following phytoplankton development in spring: Wafar et al. (1984) observed a high accumulation of DON in these waters after the spring phytoplankton growth. It may also result from an increase in water temperature, as has been suggested by Solic and Krstulovic (1994) in the marine environment (Adriatic Sea) and by Haga et al. (1995) in freshwater (Kisaki Lake, Japan). 4.3. Impact of trophic interactions on nitrogen regeneration The in#uence of macrozooplankton on the structure of the microbial food chain and ammonium #ux can be deduced from the temporal di!erences in the abundance
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of macrozooplankton and microheterotrophs (Fig. 5) and in the ammonium #ux through nano- and picoplankton (Fig. 6) in the spring}summer period. The peak abundance of #agellates in April is followed by that of ciliates in May suggesting that the ciliates develop at the expense of the #agellates. The low abundance of macrozooplankton at this time, and hence the low grazing pressure, would also help in the proliferation of the ciliates. This would explain the greater role deduced for ciliates in ammonium regeneration in spring. The rapid decline in ciliate density from May and the concomitant increase in macrozooplankton abundance reaching a peak in June also suggest that the latter occurs most likely at the expense of the former. This, coupled with the possibility that most of the #agellates are autotrophs (see above), could reduce substantially the grazing pressure on the bacteria in summer, thus favouring an enhanced role for them in ammonium regeneration at that time. This regulatory e!ect, as shown in the present study, of macrozooplankton dynamics on the structure of microheterotrophic communities, in#uencing considerably the ammonium #ux in the process, has also been shown recently in Chesapeake Bay (Glibert et al., 1992; Miller et al., 1995). Such an e!ect becomes more manifest if the phytoplankton community is dominated by smaller autotrophs (Van Wambeke et al., 1996). This was the case in the shallow well-mixed waters where the abundance of diatoms decreased by 40}60% between spring and summer (Maguer et al., 1996b). In contrast, in the deep well-mixed waters of the western English Channel, where the abundance of diatoms increased by 60}80% in the same period, the relative contributions of ciliates and bacteria to ammonium regeneration were more or less constant (Le Corre et al., 1996) when the biomass of macrozooplankton increased.
5. Conclusion The seasonal cycle of ammonium regeneration by microheterotrophs in the shallow well-mixed waters of the western English Channel is characterised by a spring maximum. On an annual scale, this source of regeneration accounts for about 60% of the ammonium taken up by phytoplankton. Such a high and important contribution can be ascribed to the permanent mixing which enables a rapid turnover of N in this ecosystem. Microplankton accounts for more than 50% of the ammonium regenerated from spring to the end of summer. The contribution by nanoplankton is high only in winter and that of picoplankton in summer. Macrozooplankton dynamics appears to have a signi"cant in#uence on the structure of the microbial food chain and regeneration of ammonium by microheterotrophs. The low biomass of macrozooplankton in spring would favour the proliferation of ciliates and a high in ammonium regeneration by them. In summer, the increase of macrozooplankton biomass would exert a strong grazing pressure on the ciliates, thus favouring the development of bacteria and enhancing their importance in the regeneration of ammonium.
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References Boalch, G.T., Harbour, D.S., Butler, E.I., 1978. Seasonal phytoplankton production in the Western English Channel 1964}1974. Journal of the Marine Biological Association of the United Kingdom 58, 943}953. Cochlan, W.P., 1986. Seasonal study of uptake and regeneration of nitrogen on the Scotian Shelf. Continental Shelf Research 5, 555}577. Dugdale, R.C., Wilkerson, F.P., 1986. The use of N to measure nitrogen uptake in eutrophic oceans; experimental considerations. Limnology and Oceanography 31, 673}689. Ferrier, C., Rassoulzadegan, F., 1991. Density-dependant e!ects of protozoans on speci"c growth rates in pico- and nanoplanktonic assemblages. Limnology and Oceanography 36, 657}669. Ferrier-Pages, C., Rassoulzadegan, F., 1994. N remineralisation in planktonic protozoa. Limnology and Oceanography 39, 411}419. Glibert, P.M., 1982. Regional studies of daily, seasonal and size fraction variability in ammonium remineralization. Marine Biology 70, 209}222. Glibert, P.M., Lipchultz, F., McCarthy, J.J., Altabet, M.A., 1982. Isotope dilution models of uptake and remineralization of ammonium by marine plankton. Limnology and Oceanography 27, 639}650. Glibert, P.M., Miller, C.A., Garside, C., Roman, M.R., McManus, G.B., 1992. NH> regeneration and grazing: interdependent processes in size-fractionated NH> experiments. Marine Ecology Progress Series 82, 65}74. Goldman, J.C., Caron, D.A., Anderson, O.K., Dennett, M.R., 1985. Nutrient cycling in a micro#agellate food chain: I Nitrogen dynamics. Marine Ecology Progress Series 24, 231}242. Guiraud, G., Fardeau, J.C., 1980. DeH termination isotopique par spectromeH trie optique de composeH s faiblement enrichis en azote-15. Analusis 8, 148}152. Haga, H., Nagata, T., Sakamoto, M., 1995. Size-fractionated NH> regeneration in the pelagic environments of two mesotrophic lakes. Limnology and Oceanography 40, 1091}1099. Hanson, R.B., Robertson, C.Y., 1988. Spring recycling rates of ammonium in turbid continental shelf waters o! the Southeastern United States. Continental Shelf Research 8, 49}68. Harrison, W.G., 1992. Regeneration of nutrients. In: Falkowski, P.G., Woodhead, A.D. (Eds.), Primary Productivity and Biogeochemical Cycles in the Sea. Plenum Press, New York, pp. 385}407. Harrison, W.G., Douglas, D., Falkowski, P., Rowe, G., Vidal, J., 1983. Summer nutrient dynamics of the Middle Atlantic Bight: nitrogen uptake and regeneration. Journal of Plankton Research 5, 539}556. Korole!, F., 1970. Direct determination of ammonia in natural waters as indophenol blue. Information on techniques and methods for seawater analysis. Conseil International pour l'Exploration de la Mer, Interlaboratory Report 3, 19}22. Kristiansen, S., Paasche, E., 1989. An improved method for determining relative N abundance in ammonium regeneration studies by direct di!usion. Marine Ecology Progress Series 54, 203}207. Laws, E.A., 1984. Isotope dilution models and the mystery of the vanishing N. Limnology and Oceanography 29, 379}386. Le Corre, P., Wafar, M., L'Helguen, S., Maguer, J.F., 1996. Ammonium assimilation and regeneration by fractionated plankton in permanently well-mixed temperate waters. Journal of Plankton Research 18, 355}370. L'Helguen, S., 1991. Absorption et reH geH neH ration de l'azote dans les eH cosyste`mes peH lagiques du plateau continental de la Manche occidentale. Relations avec le reH gime de meH lange vertical des masses d'eaux; cas du front thermique de Ouessant. The`se d' Doctorat de SpeH cialiteH , UniversiteH de Bretagne Occidentale, Brest, 212pp. L'Helguen, S., Madec, C., Le Corre, P., 1993. Nutrition azoteH e du phytoplancton dans les eaux brasseH es de la Manche occidentale. Oceanologica Acta 16, 653}659. L'Helguen, S., Madec, C., Le Corre, P., 1996. Nitrogen uptake in permanently well-mixed temperate waters. Estuarine Coastal and Shelf Science 42, 803}818. Maguer, J.F., 1995. Absorption et reH geH neH ration de l'azote dans les eH cosyste`mes co( tiers. Relations avec le reH gime de meH lange vertical des masses d'eaux; cas du domaine homoge`ne peu profond de la Manche occidentale. The`se de Doctorat de SpeH cialiteH , UniversiteH de Bretagne Occidentale, Brest, 194pp.
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Maguer, J.F., Le Corre, P., L'Helguen, S., 1996a. Sources d'azote pour le phytoplancton dans le syste`me brasseH peu profond de la Manche occidentale. Importance de la production reH geH neH reH e: bilan annuel. Journal de Recherche OceH anographique 21, 103}108. Maguer, J.F., Le Corre, P., Madec, C., 1996b. Variations saisonnie`res de l'absorption de l'azote dans un syste`me brasseH peu profond (Manche occidentale). Oceanologica Acta 19, 529}537. Miller, C.A., Penry, D.L., Glibert, P.M., 1995. The impact of trophic interactions on rates of nitrogen regeneration and grazing in Chesapeake Bay. Limnology and Oceanography 40, 1005}1011. Owens, N.J.P., Mantoura, R.C.F., Howland, R.J.M., Pomroy, A.J., Woodward, E.M.S., 1986. Nutrient cycling in Camarthen Bay: phytoplankton production, nitrogen assimilation and regeneration. Marine Biology 93, 329}342. Paasche, E., Kristiansen, S., 1982. Ammonium regeneration by microzooplankton in the Oslofjord. Marine Biology 69, 55}63. Pingree, R.D., 1978. Mixing and stabilization of phytoplankton distributions on the northwest European continental shelf. In: Steele, J.H. (Ed.), Spatial Patterns in Plankton Communities. Plenum Press, New York, pp. 181}220. Probyn, T.A., 1987. Ammonium regeneration by microplankton in an upwelling environment. Marine Ecology Progress Series 37, 53}64. Selmer, J.S., 1988. Ammonium regeneration in eutrophicated coastal waters of Sweden. Marine Ecology Progress Series 44, 265}273. Selmer, J.S., Ferrier-Pages, C., Cellario, C., Rassoulzadegan, F., 1993. New and regenerated production in relation to the microbial loop in the NW Mediterranean Sea. Marine Ecology Progress Series 100, 71}83. Solic, M., Krstulovic, N., 1994. Role of predation in controlling bacterial and heterotrophic nano#agellate standing stocks in the coastal Adriatic Sea: seasonal patterns. Marine Ecology Progress Series 114, 219}235. Treguer, P., Le Corre, P., 1975. Manuel d'analyse des sels nutritifs dans l'eau de mer (utilisation de l'autoanalyseur II Technicon), 2e`me ED dition. UniversiteH de Bretagne Occidentale, 110pp. Van Wambeke, F., Christaki, U., Gaudy, R., 1996. Carbon #uxes from the microbial food web to mesozooplankton. An approach in the surface layer of a pelagic area (NW Mediterranean Sea). Oceanologica Acta 19, 57}66. Verity, P.G., 1985. Grazing, respiration, excretion and growth rates of tintinids. Limnology and Oceanography 30, 1268}1282. Wafar, M.V.M., Le Corre, P., Birrien, J.L., 1983. Nutrients and primary production in permanently well-mixed temperate coastal waters. Estuarine Coastal and Shelf Science 17, 431}446. Wafar, M.V.M., Le Corre, P., Birrien, J.L., 1984. Seasonal changes of dissolved organic matter (C, N, P) in permanently well-mixed temperate waters. Limnology and Oceanography 29, 1127}1132. Wheeler, P.A., Kirchman, D.L., 1986. Utilization of inorganic and organic nitrogen by bacteria in marine systems. Limnology and Oceanography 31, 998}1009. Wood, E.D., Armstrong, F.A.J., Richards, F.A., 1967. Determination of nitrate in sea water by cadmium}copper reduction of nitrite. Journal Marine Biology Association UK 47, 23}31. Yentsch, C.S., Menzel, D.W., 1963. A method for the determination of phytoplankton chlorophyll and phaeophytin by #uorescence. Deep-Sea Research 10, 221}231.
Seasonal patterns of ammonium regeneration from size-fractionated microheterotrophs Jean-Franc7 ois Maguer*, SteH phane L'Helguen, Christian Madec, Pierre Le Corre Laboratoire de Chimie Marine, Universite& de Bretagne Occidentale et U.P.R. 9042 C.N.R.S. Roscow, Place Nicolas Copernic, TechnopoL le Brest-Iroise, 29280 Plouzane, France Received 12 August 1997; received in revised form 11 June 1998; accepted 13 April 1999
Abstract Ammonium regeneration by size-fractionated plankton was measured for 1 year at a coastal station in the shallow well-mixed waters of the western English Channel. Rates of ammonium regeneration in the (200 lm fraction varied from 0.6 to 27 nmol N l\ h\. On the seasonal scale, these rates were relatively low ((7 nmol N l\ h\) in autumn and winter, increased steadily from March to attain a maximum (27 nmol N l\ h\) at the end of May and thereafter decreased steadily to the seasonal minimum in December. This pattern is distinctly di!erent from that observed in deep well-mixed waters where the peak ammonium regeneration occurs in summer (Le Corre et al., 1996, Journal of Plankton Research 18, 355}370). Total ammonium regenerated in a year by the microheterotrophs was 15 g N m\, equivalent to about 60% of the total nitrogen uptake. Microplankton (200}15 lm) accounted for about 50% of the regeneration measured between early spring and late summer. Percent contribution of nanoplankton to total ammonium regeneration varied considerably between the seasons, from very high (83}88%) levels in winter to very low (2}13%) levels in summer. Contribution by picoplankton ((1 lm) was high (20}45%) in summer but was less than 20% in other seasons. Ammonium regeneration in micro- and nanoplankton fractions was mainly associated with ciliates and in the picoplankton fraction with bacteria. Macrozooplankton dynamics appears to regulate ammonium regeneration by ciliates and bacteria. Low macrozooplankton biomass in spring may favour a high growth of ciliates and an associated high in ammonium regeneration. In summer, the increase in macrozooplankton may exert a grazing pressure on ciliates. This, coupled with the fact that most of the #agellates are autotrophs, would, in turn, lower the grazing pressure on the bacteria, thus favouring their development and increasing the importance of their role in ammonium regeneration. This situation, where the macrozooplankton dynamics apparently regulates ammonium regeneration in nano- and picoplankton fractions,
* Corresponding author. Tel.: 0033-2-9849-8778. E-mail address: [email protected] (J.-F. Maguer) 0278-4343/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 7 8 - 4 3 4 3 ( 9 9 ) 0 0 0 3 7 - 0
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appears to be di!erent from that in deep well-mixed waters. Here, the relative contribution of ciliates and bacteria to ammonium regeneration shows little variation with an increase in macrozooplankton biomass. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Nitrogen; Ammonium regeneration; Size fractionation; Seasonal cycle; English Channel
1. Introduction The permanently well-mixed waters of the western English Channel constitute a unique temperate coastal ecosystem. The strong tidal action maintains a homogeneous water column (down to &100 m) throughout the year (Pingree, 1978) and the euphotic zone seldom becomes exhausted of nutrients (Wafar et al., 1983). The primary production cycle is synchronous with the seasonal increase in the depth of the euphotic zone and thus, in terms of carbon assimilation, tends to a broad summer maximum (Boalch et al., 1978; Wafar et al., 1983). Seasonal changes of the uptake of nitrogen compounds also re#ect the characteristic of the summer maximum (L'Helguen et al., 1996). Ammonium uptake alone accounted for more than half of the nitrogen taken up in the annual cycle even though the winter nitrate reserve was large and, in the absence of a physical constraint, could be replenished continuously with the in#ux of new nitrogen into the euphotic zone. The winter reserve of ammonium is generally an order of two magnitude lower than that of nitrate and the allochthonous input is negligible (Wafar et al., 1983; L'Helguen et al., 1993). Hence, the in situ regeneration of ammonium is the main mechanism of supply of nitrogen for uptake, with a close balance between its production and consumption (Le Corre et al., 1996). In all these studies, the location of the stations was such that the depth of the euphotic zone was no more than half of that of the entire water column, even in summer. Understandably, therefore, these patterns in the biological processes rely on a seasonal increase in the depth of the euphotic zone and by consequence, of the residence time of the phytoplankton cells within. In the sectors of the well-mixed waters, where the depth of the euphotic zone and that of the water column are more or less the same, the phytoplankton cells can reside for relatively longer periods within the euphotic zone. Hence the phytoplankton cells receive relatively more light energy than the cells from the deeper sectors. Under these conditions, the peak phytoplankton growth in the shallow well-mixed waters occurs much earlier in spring. Consequently, the uptake of nitrogen, especially that of ammonium, attains a peak in spring and remains relatively high through the summer (Maguer et al., 1996b). As ammonium was the major nitrogen source at the time and the allochthonous supply was negligible, it remains to be seen whether in situ regeneration of ammonium, as in deep well-mixed waters, is substantial enough to support this high uptake rate. This assessment of the relative importance of di!erent groups of planktonic microheterotrophs ((200 lm) to ammonium regeneration was prompted by the relatively
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low abundance of such data in the literature. Planktonic microheterotrophs contribute signi"cantly to ammonium regeneration (Harrison, 1992) but the importance of the size classes varies in some instances. The 1}10 lm size range was considered to be responsible for most of the ammonium production (Glibert, 1982; Probyn, 1987; Miller et al., 1995; Le Corre et al., 1996). Elsewhere, either the '10 lm (Paasche and Kristiansen, 1982; Hanson and Robertson, 1988) or the (1 lm (Harrison et al., 1983) fraction was more important. Also, the contribution of planktonic microheterotrophs to ammonium production may vary as a function of the season (Glibert, 1982; Glibert et al., 1992; Selmer et al., 1993). This, in particular, may be closely related to the trophic relationships between the organisms of the microbial loop and their predators (Glibert et al., 1992; Miller et al., 1995). The present study was thus intended to describe the seasonal cycle of ammonium regeneration in the shallow well-mixed waters of the western English Channel and to quantify the contribution of di!erent microheterotroph size classes ((200, (15 and (1 lm) and their taxonomic groups to this process.
2. Material and methods 2.1. Collection of samples Between March 1992 and March 1993, 14 "eld trips, each coinciding with a neap tide, were made to the sampling site (Fig. 1). In each instance, two sets of data collection were made at around 0800 h. In the "rst, water samples were obtained from three standard depths (0, 10 and 20 m) and used for the analyses of nutrients. In the second, samples were obtained from 50% optical depth, fractionated to three size classes ((200,(15 and (1 lm) and used for measurements of chlorophyll a (Chl a), particulate organic nitrogen (PON) and uptake and regeneration of ammonium. The (200 and (15 lm fractions were obtained by gravity "ltration through bolting silk nets and the (1 lm fraction by gentle suction ((100 mmHg) across a 1 lm Nuclepore "lter. The e$ciency of size fractionation was veri"ed by microheterotroph counts. In addition, measurements of uptake and regeneration of ammonium on unfractionated samples ((200 lm) collected from "ve optical depths (100, 50, 22.5, 8 and 3.5% of surface-incident light) were made in April, May, July, September, November, 1992 and January, 1993. On each "eld trip, macrozooplankton samples were collected in vertical hauls with a WP2 plankton net (mesh size 200 lm). In this paper, the 200}15 lm fraction is referred to as microplankton, the 15}1 lm fraction, as nanoplankton and the (1 lm fraction, as picoplankton. Uptake and regeneration rates were obtained directly for the picoplankton fraction and indirectly (by di!erence), for the other two fractions. 2.2. Analytical methods Temperature was recorded with reversing thermometers mounted on water samplers. Nitrate concentrations were measured following the method of Wood et al.
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Fig. 1. Position of the sampling station.
(1967), as adapted by Treguer and Le Corre (1975) for automated analyses with a Technicon II Auto-analyser. Ammonium concentrations were measured manually by the indo-phenol blue method (Korole!, 1970). Chl a content was measured #uorometrically (Yentsch and Menzel, 1963). Particulate matter for PON measurements was collected on pre-ignited GF/F "lter pads and analysed in a Perkin-Elmer model 240 elemental analyser. Analytical precisions in these measurements were: 0.05 lmol N l\ at 10 lmol N l\ level for nitrate, 0.02 lmol N l\ at 0.5 lmol N l\ level for ammonium, 0.05 lg l\ at 1 lg l\ level for Chl a and 0.1 lg N l\ at 2 lmol l\ level for PON. The biomass of macrozooplankton was estimated as dry weight. Samples designated for microzooplankton ((200 lm) enumeration were "xed in Lugol-formalin and counted in an inverted microscope. Samples for enumeration of bacterial numbers were "xed with formalin, stained with acridin orange, "ltered onto 0.2 lm Nuclepore "lters and counted by epi#uorescence in a BH2 Olympus microscope. 2.3. Uptake and regeneration experiments Ammonium uptake and regeneration measurements were made from single incubation experiments using N tracer. Ammonium was added to the samples in the form of NH Cl (97.5 at %, CEA, France) at 10}20% of the ambient concentrations. Half of the sample was immediately "ltered through pre-ignited GF/F "lter pads so as to obtain zero-time enrichment with N of the particulate and dissolved pools. The
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remaining fraction was incubated in 2.5 l polycarbonate bottles under simulated in situ conditions for 4 h around local noon. Incident light on the incubation bottles was attenuated to 50% by enveloping them with pre-calibrated nickel screens. The ambient temperature was maintained constant with a continuous #ow of seawater. The incubations were terminated by "ltering the samples onto pre-ignited GF/F "lter pads. The "lters were dried at low temperature (&403C) and stored dry until analysis for isotopic ratios. A part of the "ltrate was used to measure the ammonium concentration (in triplicate) and the rest was stored frozen at !203C. Ammonium contained in the "ltrate was later extracted by di!usion in basic pH (Kristiansen and Paasche, 1989). The N : N isotopic ratios of the particulate matter and of the ammonium from the "ltrate were measured in a SOPRA GS1 model emission spectrometer following the methods described by Guiraud and Fardeau (1980). Ammonium uptake rates were calculated using the equation of Dugdale and Wilkerson (1986) where the PON content is the one measured at the beginning of the incubation. Ammonium uptake rates were corrected for isotope dilution (Glibert et al., 1982). Ammonium regeneration rates were calculated either by using the equation of Glibert et al. (1982) when ammonium concentration did not change measurably during the incubation, or by that of Laws (1984) when the reverse was true. The coe$cients of variation of the measurements of uptake and regeneration were respectively 4 and 6% at the level of 10 nmol N l\ h\. Total ammonium regeneration in a year was computed from a summation of the day-time regeneration pro"les of each month and a night/day ratio of 0.52 for regeneration rates (Maguer et al., 1996a).
3. Results 3.1. Ambient and biological parameters Consistent with the main characteristic of the permanently well-mixed waters (Wafar et al., 1983), water temperatures were vertically uniform and varied from 9.5 to 16.03C during the seasonal cycle. Nitrate was the major component of the winter stock of dissolved inorganic nitrogen (DIN) (Fig. 2). Coincident with the spring phytoplankton development, nitrate concentrations decreased rapidly through April and May to very low values in June ((0.5 lmol N l\) that persisted until the end of July. Ammonium concentrations varied between 0.15 and 0.55 lmol N l\ (Fig. 2). From very low values in autumn and winter, ammonium concentrations began to increase in spring, in parallel with the phytoplankton growth, to reach maximum values ('0.30 lmol N l\) in summer, with a peak value (0.55 lmol N l\) in July. Chl a concentrations ranged from 0.2 to 2.8 lg l\ (Fig. 3). It was noteworthy that in the seasonal changes of Chl a the peak value (2.8 lg l\) occurred in May and there was a persistence of high values (&1.5 lg l\) in summer (June}August). At other times of the year, Chl a concentrations were generally (0.5 lg l\. Microplankton contributed to between 8 and 46% (average: 30%) of the Chl a, with the maximum
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Fig. 2. Concentrations of ammonium (solid line) and nitrate (dashed line) at the study station during 1992}1993. Vertical distribution of nutrients was homogeneous and the data are averages from three depths.
Fig. 3. Concentrations of Chlorophyll a in micro-, nano-, and picoplankton fractions at the study site during 1992}1993.
percentages in April (42%) and September (46%). Nanoplankton accounted for a higher proportion almost throughout the year (30}73%, average: 54%), except at the beginning of spring ((35% in March}April). The contribution of picoplankton was less (12}27%, average: 16%) throughout the year, except at the beginning of spring (March}April).
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Fig. 4. Density of #agellates in the (200 lm fraction at the study site during 1992}1993.
Fig. 5. Densities of ciliates (solid line), bacteria (dashed line) and macrozooplankton (discontinuous line) at the study site during 1992}1993.
Flagellate counts varied from 0.19;10 to 2.43;10 cells l\ over the year, with a major peak in April followed by two other peaks in June}July and October (Fig. 4). The major part of the #agellates counted (62%) were in the 15}1 lm fraction, 32% were in the 200}15 lm fraction and only 6% were in the (1 lm fraction. Ciliate counts varied from nil to 3.6;10 cells l\ and were generally less than 500 cells l\ (Fig. 5), except in spring when they formed a major peak in May. On average, about 65% of the ciliates counted were in the 200}15 lm fraction and the rest, in the
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15}1 lm fraction. Bacterial numbers ranged from 12;10 to 190;10 cells l\, with 89% of them recorded in the (1 lm fraction. Seasonal changes of bacteria in this size class (Fig. 5) show a broad maximum extending from late May to early August. Macrozooplankton biomass (Fig. 5) varied from 2.9 to 16.5 mg m\ in the seasonal cycle, with a peak in June. 3.2. Ammonium regeneration by microheterotrophs 3.2.1. Total community Rates of ammonium regeneration in the (200 lm fraction varied from 0.6 to 27 nmol N l\ h\. On a seasonal scale (Fig. 6), they were relatively low ((7 nmol N l\ h\) in autumn and winter, increased steadily from March to attain a maximum (27 nmol N l\ h\) at the end of May and thereafter decreased steadily to the seasonal minimum in December. The increase in regeneration occurred in parallel with those of phytoplankton growth and ammonium uptake (Fig. 7). Regeneration rates related linearly with ammonium uptake rates (r"0.83; P(0.01; regeneration/uptake"0.42) and with the total nitrogen uptake (r"0.91; P(0.01). Pro"les of ammonium regeneration (Fig. 8) showed a surface/subsurface maximum, followed by a progressive decrease with depth. Similar patterns were observed also in uptake pro"les. Vertical and temporal integration of the pro"les gave a total ammonium regeneration by microheterotrophs of 15 g N m\ yr\. 3.2.2. Size fractions Patterns of ammonium regeneration by microplankton (0.1}13.5 nmol l\ h\) and unfractionated microheterotrophs were similar (Fig. 6). Percent contribution by microplankton to total ammonium production, however, varied seasonally ranging
Fig. 6. Ammonium regeneration rates in micro-, nano- and picoplankton fractions at the study site during 1992}1993.
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Fig. 7. Uptake (dashed line) and regeneration (solid line) of ammonium by unfractionated plankton ((200 lm) at the study site during 1992}1993.
between 32% (June) and 55% (April) in spring, high and nearly constant in summer (52}56%) and autumn (46}58%) but was consistently low (9}17%) in winter. Nanoplankton regenerated ammonium at rates ranging between 0.2 and 10.4 nmol N l\ h\ (Fig. 6), with the maximum rates in May. From June, regeneration in this fraction became less important and remained at less than 2.5 nmol N l\ h\ until December, when it ranged between 0.5 and 4.7 nmol N l\ h\ throughout winter. Contribution by nanoplankton to total ammonium regeneration, similar to that of microplankton, varied markedly between the seasons. It was between 23 and 31% in spring but decreased substantially in summer (2}13%) before increasing again through autumn (29}44%) to account for nearly all of the ammonium regenerated (83}88%) in winter. Ammonium regeneration rates of picoplankton ranged from below limits of detection to 6 nmol N l\ h\ (Fig. 6). On a seasonal scale, the rates were maximum (4}6 nmol N l\ h\) and occurred as a single broad peak in April}July but in other months they were generally less than 1 nmol N l\ h\. Contribution of picoplankton to total ammonium regeneration was substantial in summer (20}45%) but was relatively unimportant in other seasons ((20% in spring, (17% in autumn and (4% in winter).
4. Discussion 4.1. Regeneration of ammonium by microheterotrophs In coastal ecosystems that are relatively undisturbed by anthropogenic input of nitrogen, ammonium regeneration by microheterotrophs varies seasonally from
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undetectable levels to 100 nmol N l\ h\ (Paasche and Kristiansen, 1982; Cochlan, 1986; Owens et al., 1986; Selmer, 1988; Le Corre et al., 1996). The rates measured in the present study (0}27 nmol N l\ h\) fall in this range but towards the lower end. In the shallow permanently well-mixed waters, ammonium regeneration proceeds at appreciable rates in the entire water column for several months of the year (Figs. 6 and 8). As shown by the regeneration/uptake ratio of 0.42 (Fig. 7), this rate is adequate to provide for more than 40% of the ammonium assimilated by the phytoplankton at the 50% light depth. Integrating the water column regeneration rates (Fig. 8) and raising them to an annual scale gave a still greater importance to the role of microheterotrophs in the regeneration of nitrogen that can sustain phytoplankton uptake. The total ammonium production was 15 g N m\ yr\, equivalent to 60% of the needs in ammonium (24 g N m\ yr\) and 33% of the needs in nitrogen (45 g N m\ yr\) (Maguer et al., 1996a). This is of the same order (respectively 64 and
Fig. 8. Vertical pro"les of ammonium uptake (dashed line) and regeneration (solid line) by the unfractionated plankton in (a) April, (b) May, (c) July, (d) September, (e) November, (f ) January. Note scale di!erence.
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34%) as that found in the deep well-mixed waters (L'Helguen, 1991). Our results not only agree well with a number of uptake and regeneration measurements on discrete samples (e.g. Harrison, 1992) but also demonstrate that ammonium regeneration by microheterotrophs can be substantially important to nitrogen #ux through plankton on annual time-scales. Such an importance of ammonium regeneration in supporting phytoplankton production can perhaps be related to the strong tidal mixing and the permanent homogeneity of the water column. In the absence of a thermocline, the whole water column behaves as a single layer throughout the year and hence the organic matter produced remains within. Decomposition of this organic matter would, therefore, occur in situ and its regeneration products (principally as ammonium) almost immediately become available for uptake by phytoplankton. By favouring a rapid return of nitrogen, the strong mixing thus maintains a high nitrogen uptake throughout the year (Maguer et al., 1996b) and, in turn, also a high level of ammonium regeneration. This would also explain the close coupling between ammonium regeneration and total nitrogen uptake, observed both in the shallow (r"0.91; P(0.01) and deep (r"0.95; P(0.01*L'Helguen, 1991) well-mixed waters, with the only di!erence between the two systems being the pattern of the seasonal changes. 4.2. Size fraction variability in ammonium regeneration during spring and summer In spring, micro- and nanoplankton were responsible for a major proportion (between 73 and 92%) of ammonium regeneration. The absence of metazoans (copepod nauplii, rotifers) and the low abundance of bacteria in these two size classes suggest that the protozoans (ciliates and #agellates) would have been responsible for the bulk of ammonium regenerated at this time. While the importance of protozoans to the regeneration of ammonium has already been demonstrated in a number of marine ecosystems (Glibert, 1982; Wheeler and Kirchman, 1986; Probyn, 1987; Ferrier and Rassoulzadegan, 1991), the relative importance of ciliates and #agellates to ammonium regeneration is less known. In the shallow well-mixed waters of the western English Channel, the patterns of ammonium regeneration rates and the number of ciliates in the 200}15 and 15}1 lm fractions were similar and related statistically (r"0.89, n"15, p(0.01 and r"0.82, n"15, p(0.01 respectively) (Fig. 9). These results suggest that the ciliates could play an important role in ammonium regeneration. Taking into consideration the rates published for ammonium excretion by ciliates (Verity, 1985; Ferrier-Pages and Rassoulzadegan, 1994), at the densities observed in our study, ciliates can produce ammonium at between 8.3 (June) and 18 nmol N l\ h\ (May). This rate accounts for, on average, 72% of the ammonium regenerated by microheterotrophs in spring. A similar importance for ciliates in ammonium regeneration has also been shown in the deep well-mixed waters of the western English Channel (Le Corre et al., 1996) and in the Mediterranean coastal waters (Selmer et al., 1993). The relatively high biomass of #agellates in the microheterotroph fraction at any one time (Goldman et al., 1985), however, has led several authors (Paasche and Kristiansen, 1982; Probyn, 1987) to attribute a much more important role to them in
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Fig. 9. Ammonium regeneration rates (solid lines) and number of ciliates (dashed line) in (a) micro- and (b) nanoplankton at the study site during 1992}1993.
ammonium production. We did not detect any signi"cant relationship between the number of #agellates and ammonium regeneration rates. The high proportion of #agellates (62%) and of Chl a (56%) in the nanoplankton fraction on one hand, and the high ammonium uptake (&60% of total uptake*Maguer, 1995) measured on the other, suggest instead that a large fraction of #agellates are probably autotrophs. In that event, even if their densities were high, their contribution to ammonium production could become relatively less important than that of ciliates. A large fraction of the ammonium regeneration in summer was associated with the organisms (1 lm in size (Fig. 6) and this follows the decline of the diatom bloom
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Fig. 10. Ammonium regeneration rates (solid lines), number of bacteria in picoplankton (dashed line) and ratio between regeneration and bacteria in picoplankton (discontinuous line) at the study site during 1992}1993.
(Maguer et al., 1996b). Similar observations have been reported from the Middle Atlantic Bight (Harrison et al., 1983) and the coastal waters of the Vineyard Sound (Glibert, 1982). The similarity in the changes of regeneration rates and bacterial cell counts (Fig. 10) suggest that the increase in ammonium regeneration in this size fraction in summer could be related to an increase in the density of bacteria. Nevertheless, the ratio between the ammonium production rate and the number of bacteria (Fig. 10) does not remain constant but follows a cyclic change, ranging from 0.6;10\ in autumn to 4.0;10\ nmol N cell\ h\ in summer. This would suggest that it is not only the increase in bacterial biomass but an increase in their activity as well, which is responsible for the seasonal increase in ammonium production in this size fraction. The ratio of ammonium production to bacterial numbers was, in particular, nearly twice as high (4.0;10\ nmol N cell\ h\) in summer than at the beginning of spring (2.5;10\ nmol N cell\ h\). This increased activity could have been stimulated by an increase of nitrogen substrates following phytoplankton development in spring: Wafar et al. (1984) observed a high accumulation of DON in these waters after the spring phytoplankton growth. It may also result from an increase in water temperature, as has been suggested by Solic and Krstulovic (1994) in the marine environment (Adriatic Sea) and by Haga et al. (1995) in freshwater (Kisaki Lake, Japan). 4.3. Impact of trophic interactions on nitrogen regeneration The in#uence of macrozooplankton on the structure of the microbial food chain and ammonium #ux can be deduced from the temporal di!erences in the abundance
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of macrozooplankton and microheterotrophs (Fig. 5) and in the ammonium #ux through nano- and picoplankton (Fig. 6) in the spring}summer period. The peak abundance of #agellates in April is followed by that of ciliates in May suggesting that the ciliates develop at the expense of the #agellates. The low abundance of macrozooplankton at this time, and hence the low grazing pressure, would also help in the proliferation of the ciliates. This would explain the greater role deduced for ciliates in ammonium regeneration in spring. The rapid decline in ciliate density from May and the concomitant increase in macrozooplankton abundance reaching a peak in June also suggest that the latter occurs most likely at the expense of the former. This, coupled with the possibility that most of the #agellates are autotrophs (see above), could reduce substantially the grazing pressure on the bacteria in summer, thus favouring an enhanced role for them in ammonium regeneration at that time. This regulatory e!ect, as shown in the present study, of macrozooplankton dynamics on the structure of microheterotrophic communities, in#uencing considerably the ammonium #ux in the process, has also been shown recently in Chesapeake Bay (Glibert et al., 1992; Miller et al., 1995). Such an e!ect becomes more manifest if the phytoplankton community is dominated by smaller autotrophs (Van Wambeke et al., 1996). This was the case in the shallow well-mixed waters where the abundance of diatoms decreased by 40}60% between spring and summer (Maguer et al., 1996b). In contrast, in the deep well-mixed waters of the western English Channel, where the abundance of diatoms increased by 60}80% in the same period, the relative contributions of ciliates and bacteria to ammonium regeneration were more or less constant (Le Corre et al., 1996) when the biomass of macrozooplankton increased.
5. Conclusion The seasonal cycle of ammonium regeneration by microheterotrophs in the shallow well-mixed waters of the western English Channel is characterised by a spring maximum. On an annual scale, this source of regeneration accounts for about 60% of the ammonium taken up by phytoplankton. Such a high and important contribution can be ascribed to the permanent mixing which enables a rapid turnover of N in this ecosystem. Microplankton accounts for more than 50% of the ammonium regenerated from spring to the end of summer. The contribution by nanoplankton is high only in winter and that of picoplankton in summer. Macrozooplankton dynamics appears to have a signi"cant in#uence on the structure of the microbial food chain and regeneration of ammonium by microheterotrophs. The low biomass of macrozooplankton in spring would favour the proliferation of ciliates and a high in ammonium regeneration by them. In summer, the increase of macrozooplankton biomass would exert a strong grazing pressure on the ciliates, thus favouring the development of bacteria and enhancing their importance in the regeneration of ammonium.
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