ICES mar. Sei. Symp., 197: 149-158. 1993

Nutrient recycling in production experiments

W. Glen Harrison

Harrison, W. G. 1993. Nutrient recycling in production experiments. - ICES mar. Sei. Symp., 197: 149-158.

Regenerated nutrients support most of the primary production in the world’s oceans. The link between nutrient regeneration and phytoplankton growth cycles was recog­nized decades ago but elaboration of the quantitative role of regenerated nutrients in primary productivity was not established until the introduction of isotope tracers in the early 1960s. The first direct measurements of nutrient regeneration were made about a decade later, employing substrate “isotopc-dilution” principles. Techniques have been described for measuring regeneration of the principal reduced N-forms (am­monium, urea, amino acids), phosphorus, and silicon. Measurements to date have shown a strong co-variance between nutrient regeneration and utilization rates and primary productivity in a wide range of environments. Size fractionation studies show that most of the regeneration can be attributed to protozoans and their prey; however, the reliability of current experimental approaches has been questioned. The preva­lence and magnitude of nutrient regeneration in productivity experiments suggest that depletion of the major nutrients in standard in vitro incubations should not be of great concern. Productivity based on conventional estimates of nutrient utilization, on the other hand, may be in error (underestimated) as a consequence of substrate isotope dilution. Historical values of ammonium utilization, for example, may be low by a factor of two or more, particularly in productive coastal waters. These potential errors have implications for current conceptual models of the cycling of nutrients in the upper ocean and their role in regulating primary production.

W. G. Harrison: Department o f Fisheries and Oceans, Bedford Institute o f Oceano­graphy, Box 1006, Dartmouth, Nova Scotia, Canada, B2Y 4A2.

Introduction

The link between nutrients and algal growth cycles in nature was established well before the development of traditional methods for quantifying primary production (Harvey, 1926). The implications of these early studies were instrumental later in the development of views that nutrient resupply to the surface ocean could largely explain the major features of phytoplankton distribution globally (Sverdrup, 1955); contemporary analyses of biomass and productivity have tended to support this concept (Berger et al. , 1989; Lewis, 1989).

Of the major nutrients present in sea water that are essential for phytoplankton growth and metabolism, nitrogen has most often been the subject of study be­cause of its central role in algal photosynthesis and respiration (McCarthy, 1980; Turpin, 1991) and because it is considered one of the major “limiting nutrients" in the environment (Dugdale, 1967).

The contemporary conceptual model of nutrient lim­ited phytoplankton growth in the ocean describes two principal modes of nitrogen supply to the euphotic zone,

one largely physical and the other biological (Dugdale and Goering, 1967). New nitrogen, mainly in the form of nitrate, is brought to the sea surface by oceanic mixing processes. Regenerated nitrogen (ammonium, urea, and other dissolved organics), on the other hand, is locally produced as metabolic by-products (excretion, decom­position) of the resident plankton communities. Although both nutrient sources contribute to the pro­ductivity of phytoplankton, only the so-called new pro­duction contributes to net community growth; the re­generated production is viewed as that portion that meets and maintains the basic metabolic demands of the community (Platt et al., 1989). Despite the importance of new production in food web dynamics, regenerated nutrients fuel most of the global oceanic primary pro­duction (Eppley and Peterson, 1979; Fig. 1). It is this latter component that will be the focus of this paper.

The process of nutrient regeneration can be con­sidered on a spectrum of space/time scales from the organism level with characteristic time scales of hours to days to the global level with time scales of years to decades and longer (Harrison, 1992). In the context of

150 W. G. Harrison IC E S m ar . Sei. S y m p . . 197 (1993)

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Figure 1. Distribution of primary productivity by ocean surface area and its fraction supported by regenerated nutrients. Productivity data from Berger et al. (1989), fractional regenerated production from Eppley and Peterson (1979). Vertical line indicates that 25% of the global ocean primary production occurs in waters (principally coastal) representing <10% of the ocean surface. Primary productivity, over the rest of the ocean surface, is fueled predominantly (>75% ) by regenerated nutrients (redrawn from Harrison, 1992).

this Symposium, however, emphasis will be placed on the inherent scales characterized by the contemporary in

vitro productivity measurement, i.e., hours to seasonal, local to regional.

It is well known that the in vitro bottle containment experiment may compromise important natural loss (grazing, sinking) and supply (nutrient regeneration) processes (McCarthy, 1980). This paper will attempt to evaluate the important nutrient supply processes in the context of the in vitro experiment. How is nutrient regeneration measured? What is the magnitude of nutri­ent regeneration in productivity experiments? Which organisms are responsible? What are the effects of containment on nutrient regeneration? What are the effects of nutrient regeneration (or its absence) on primary productivity?

Measurement methodologies

Recognition of the importance of regenerated nutrients in sustaining primary production dates to the earliest studies of natural nutrient cycles (Harvey, 1926; Harvey et a l ., 1935; Cooper 1937), however, methods had not yet been devised to quantify the processes involved. Among the first in vitro measurements designed specifi­cally to study nutrient regeneration were those of von Brand and colleagues in the 1930s and 1940s (von Brand et a l., 1937). Although these studies were significant in elaborating qualita tive aspects of the nitrogen decompo­

sition and remineralization pathways, they did not mimic the natural environment sufficiently well to be of much quantita tive value. More quantitative approaches were introduced in the 1950s and 1960s combining in

vitro measurements (light/dark oxygen changes) and in

situ data (nutrient concentrations) to evaluate absolute biological transformations, i.e., nutrient uptake and regeneration (Riley, 1956; Ketchum and Corwin, 1965). By these methods, nutrient regeneration estimates were derived indirectly and thus subject to the uncertainties of the assumptions relating nutrient transformations to their "proxies” (oxygen changes).

Harris (1959) was one of the first directly to measure nutrient regeneration (excretion) by large zooplankton and to relate these measurements to nutrient require­ments for primary production. Experimentally, this approach proved simple and relatively unambiguous; because of their relatively large size, these nutrient “producers” could easily be separated from the “con­sumers” (phytoplankton) and their nutrient regener­ation thus measured by concentration difference before and after incubation. This approach, however, has proved to be of limited utility, as it relates to total community nutrient regeneration: (1) questions have arisen about the reliability of the estimates because of manipulation artifacts (Mullin et a l ., 1975), (2) sub­sequent studies have shown that important nutrient regenerators are of a similar size to the nutrient con­sumers and cannot therefore be physically separated for experimentation, moreover, (3) these smaller forms

IC E S m ar . Sei. S y m p . . 197 (1993) Nutrient recycling in production experiments 151

appear to carry out most of the nutrient regeneration in the plankton, i.e., the easily manipulated mesozoo- plankton have a relatively minor role (Harrison, 1980; Bidigare, 1983). Since mesozooplankton are usually absent in standard productivity measurements, either because of their low densities in surface waters or because they are intentionally excluded, their impact on nutrient regeneration has not been assessed using more contemporary experimental approaches.

Similar to the impact of radioisotopically labeled carbon on the study of primary productivity, the intro­duction of isotopically labeled nitrogen, 15N (Dugdale and Goering, 1967), phosphorus, 32P (Watt and Hayes, 1963), and silicon, 3()Si (Nelson and Goering, 1977a) compounds about a decade later provided for the first time means for direct nutrient flux measurements in

vitro. The conventional (and most extensive) appli­cation of the 15N tracer method has been to determine plankton utilization rates (U) by following the isotopic enrichment of particulate matter during incubation (Dugdale and Goering, 1967). Concurrent knowledge of the substrate concentration changes with time permit calculation of nitrogen regeneration rates (R) by mass balance (Fisher et a l . , 1981):

R = d(S)/dt + U

where R is regeneration rate, d(S)/dt is the change in substrate concentration during the incubation and U is the measured utilization rate. The reliability of R using this method depends on accurate estimates of substrate concentration and U, both of which may be difficult to achieve under certain circumstances (McCarthy, 1980; Glibert et a l., 1982; Dugdale and Wilkerson, 1986).

A more reliable yet laborious method, introduced in the 1960s (Dugdale and Goering, 1967) and imple­mented in the 1970s (Harrison, 1978; Caperon et a l .,

1979), allowed the determination directly of regener­ation of ammonium by monitoring the “isotope dilu­tion” of the substrate, resulting from the biological regeneration of unlabeled ammonium during incu­bation. This method permitted for the first time direct determination of nutrient regeneration by the commu­nity of microorganisms normally comprising the produc­tivity experiment, including the nutrient consumers. The analytical “breakthrough” in this procedure was the isolation of the substrate, in this case ammonium, for isotope analysis. A number of procedures have been introduced and refined for recovering ammonium from sea water in isotope dilution experiments, including the conventional distillation procedure (Harrison, 1978; Caperon et a l., 1979; Glibert et a l . , 1982), direct dif­fusion (Kristiansen and Paasche, 1989), mercury pre­cipitation (Fisher and Morrissey, 1985), and solvent extraction procedures (Dudck et a t ., 1986; Selmer and

Sorensson, 1986). Procedures have also been described for the isolation and isotope dilution analysis of other regenerated N-forms including urea (Slawyk et a l .,

1990), amino acids (Fuhrman, 1987), phosphorus (Har­rison, 1983a), and silicon (Nelson and Goering, 1977b). Hansell and Goering (1989) describe a novel approach for determining urea regeneration by combining l5N and l4C-labeled urea with the isotope dilution methods. 15N urea is used in the traditional sense to monitor the enrichment of the particulates while l4C-urea activity is used to estimate the 1SN urea remaining in solution. The fractional change in l4C activity in solution combined with changes in measured urea concentrations deter­mine the isotope dilution of the substrate.

Bronk and Glibert (1991) recently described a method for the isolation of dissolved organic nitrogen (DON) for tracer studies of DON release from phytoplankton. Procedures have also been described for the isolation and isotope analysis of oxidized N-forms (nitrate, nitrite) (e.g., Schell, 1978; K a to re /a / . , 1992). but these will not be considered because oxidation rates have generally been thought to be much lower in the euphotic zone than production rates of the reduced N-forms (ammonium and urea) and lower than phytoplankton N- demand (Olson, 1981). Some recent studies, however, have shown significant nitrification above the nitracline (Ward et a l . , 1989; Eppley and Renger, 1986; Eppley et

a l . , 1990) and suggest that the importance of mixed layer production of oxidized nitrogen forms be re-evaluated.

Distribution of nutrient regeneration

An important assumption in Dugdale and Goering’s (1967) conceptual model of nitrogen cycling is that the supply rate of regenerated nitrogen to the ocean eupho­tic zone is in balance with consumption. Indirect evi­dence supporting this view came from observations that the limited range of concentrations locally and patterns (or lack of) in the distribution of ammonium, in contrast to those of nitrate, implied that ammonium supply and consumption were in approximate balance. In vitro

isotope tracer measurements have made it possible to test this generalization based on concurrent measure­ments of ammonium utilization and regeneration. A compilation of available measurements shows that util­ization and regeneration strongly co-vary over a wide range of fluxes representing regions from productive coastal to oligotrophic open ocean waters (Fig. 2). A closer inspection of the data shows that U/R ratios can vary considerably at individual locations, depths, and times, but fluxes may balance with appropriate averag­ing (e.g. Harrison et a l . , 1983). A similar pattern is seen for phosphate fluxes. Hansell and Goering (1989) and Fuhrman (1987) have also noted a close correspondence

152 W. G. Harrison I C E S m ar . Sei. S y m p . , 197 ( 1993)

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Figure 2. Relationship between in vitro measurements of am­monium (top panel) and phosphate (lower panel) utilization (U) and regeneration (R) based on a survey of the literature. The data span five orders of magnitude, covering productive coastal to oligotrophic open ocean waters.

between urea and amino acid utilization and regener­ation. One implication of these findings is that nutrient regeneration (and regenerated production) does not simply represent a regionally invariant “basal” metab­olism, but is linked to the general productivity level of the community. Eppley et al. (1979) were first to draw attention to this relationship from their seasonal study of nitrogen cycling in coastal waters. They explained this as a response of nutrient regeneration to increased new primary production which may be more directly linked to the system productivity level (Dugdale and Goering, 1967). A more extensive analysis combining coastal and oceanic data confirms this pattern (Fig. 3) and supports the general view that regenerated production, although increasing with productivity level, represents pro­portionally less of the community production as the productivity increases (Eppley and Peterson, 1979).

Regionally, nutrient regeneration has been shown to vary with time of day, depth , and season. Although daily supply and removal processes may be in balance, diel variations in ammonium utilization and regeneration are commonly observed with utilization dominating during daylight hours and regeneration dominating at night (Caperon et al., 1979; Glibert, 1982; Wheeler et a i , 1989). Depth variations are also common with both utilization and regeneration decreasing with depth (Har­rison, 1978; Harrison etal., 1983; Probyn, 1987; Hanson and Robertson, 1988). Regeneration rates often de­crease more slowly than utilization, resulting in pro­portionally lower U/R ratios with depth. Under strati­fied conditions, ammonium regeneration usually peaks in the upper mixed layer, above the subsurface chloro­phyll maximum (Harrison et a i , 1983). A m o re exten­sive analysis has revealed this pattern to be a general

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Figure 3. Relationship between regenerated production (ammonium utilization rate) and total ( 14C photosynthetic rate, left panel) and new (nitrate utilization rate, right panel) production. Data arc from temperate coastal and oceanic studies carried out by the author and colleagues in his laboratory. Dashed line in least squares (Model II) regression fit to the data; both regressions are statistically significant (p < 0.05). Solid line in right panel is 1:1 relationship.

IC E S m ar . Sei. S y m p . . 197 (1993) Nutrient recycling in production experiments 153

one; the relationships between the primary productivity and subsurface chlorophyll maximum layers and the vertical distributions of new and regenerated production suggest that the shallower productivity maximum is fueled largely by nutrient regeneration, whereas the biomass maximum is tied more closely to new nutrients (Fig. 4; see also Longhurst and Harrison, 1989; Harri­son, 1990). Seasonal studies of nutrient regeneration are few and limited to coastal waters but show a relatively consistent pattern of low fluxes during winter and elev­ated rates during spring and summer (Taft et al., 1975; Glibert, 1982; LaRoche, 1983; Harrison, 1983a). Gli­bert (1982), for example, showed that ammonium utiliz­ation and regeneration rates were in balance during summer but that regeneration often exceeded utilization in spring and fall in Vineyard Sound. LaRoche (1983) found that ammonium regeneration rarely balanced utilization (the latter always in excess) in her seasonal study of a coastal embayment, but that regeneration exceeded utilization on a few occasions, in spring and late summer, similar to Glibert’s findings. Harrison (1983a), working in the same system as LaRoche, also found that phosphorus utilization usually exceeded re­generation but did observe maximum regeneration rates in the spring coincident with the decline of the algal bloom and in the fall, consistent with results of the previously mentioned investigators.

Causative organisms

Elucidating which components of the plankton commu­nity are important in nutrient regeneration has been one of the major goals of contemporary research on nutrient cycles (Harrison, 1992). The earliest views were that nutrient regeneration was mediated principally by bac­teria below the euphotic zone (Harrison, 1980). Later, interest focused on the larger heterotrophs, mesozoo- plankton, resident in the surface ocean. Contemporary views are that the community of protozoans which prey on bacteria and phytoplankton and are food for the larger grazers may be the most important nutrient regen­erators (Reid et al. , 1990; Capriulo, 1991). Much of the evidence for the dominance of the ’‘micro-grazer” proto­zoans in nutrient regeneration has been circumstantial: theoretical considerations (allometric principles) and laboratory physiological studies. A number of re­searchers, however, have combined tracer measure­ments (isotope-dilution method) with size-fractionation techniques to attempt a direct assessment of the contri­bution of the various size categories to nutrient regener­ation in natural plankton communities. Because of the normal screening procedures (see above), mesozoo- plankton are not generally included in these estimates. A compilation of representative results from these field

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Figure 4. Relationship between fractional regenerated pro­duction (regenerated production/total production) in the pri­mary productivity and chlorophyll maximum layers of coastal and oceanic waters (see Harrison, 1990). Line represents 1:1 relationship. Note that the primary productivity and chloro­phyll maximum layers were at the same depth at 34% of the stations.

studies reveals that all major size categories (picoplank- ton, nanoplankton, microplankton) contribute to nutri­ent regeneration but that the nano (2-20 urn) and picoplankton (<2 //m) appear to dominate (Fig. 5). Although these results are largely consistent with cur­rent notions of the partitioning of nutrient regeneration among the components of the plankton community, i.e . , dominance of the nanoplankton (protozoans) and their picoplankton prey (bacteria), there are some inconsis­tencies with evidence using other approaches. Specifi­cally, the relatively large contribution of the picoplank­ton is difficult to reconcile based on both theory and experimental evidence (Goldman et al., 1987; King, 1987). One explanation points to a potential methodolo­gical problem, i.e., the necessity in isotope dilution experiments to pre-screen samples before incubation. This procedure eliminates many natural community interactions (large predator-small prey) that may affect the nutrient regeneration process (Caron et al., 1988). Glibert et al. (1992) suggest that this problem may be the basis for the often observed high nutrient regeneration rates in the smallest size categories, often in excess of rates in the total (unfractionated) samples. This may result, for example, from the release of “grazing press­ure” , allowing the smaller prey to grow and metabolize at rates much higher than when in the presence of their predators. Glibert and co-workers go on to suggest that the significance of mesozooplankton in nutrient regener­ation may have been underestimated historically for similar reasons, the argument being that mesozoo­plankton may contribute more to the nutrient regener-

154 W. G. Harrison IC E S m ar . Sei. S y m p . , 197 (1993)

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Figure 5. Particle size-partitioning of ammonium regeneration from in vitro size-fractionation experiments taken from the literature (see Harrison, 1992). The data were grouped into three general size categories, <2 fim (picoplankton), < 2 0 ^m (pico + nanoplankton) and < 2 0 0 /im (pico + nano + micro­plankton). A fourth category, >200 ,um, representing organ­isms normally excluded from in vitro incubations (see text), is based on literature estimates of ammonium regeneration by mesozooplankton (Harrison, 1980; Bidigare, 1983); these values were added to the in vitro values. Results are given as a percentage (mean and standard deviation) of the total (unfrac­tionated) in vitro rates.

ation of the community through selective grazing control of the smaller forms than through their direct metabolic contribution, i.e., “top-down” control (Roman et a i ,

1988; Glibert et a l., 1991). As a result, isotope tracer methods currently employed may inadvertently over­represent some components (picoplankton), while under-representing others (mesozooplankton).

Consequences to primary productivity

Although the disturbance of natural nutrient regener­ation processes is only one of many possible pertur­bations in conventional in vitro productivity experi­ments ( Sheldon et a i , 1973 ; Venrick et a l . , 1977 ; Gieskes é ta l . , 1979; Carpenter and Lively, 1980; F itzw atere ïa/., 1982), there has been considerable work in recent years on the short-term response of primary productivity to nutrient supply in an attempt to understand the underly­ing biochemical basis of nutrient deficiency and its direct effects on photosynthesis (Goldman and Dennett, 1983; Elrifi and Turpin, 1987; Turpin, 1991). A detailed sum­mary of that work is beyond the scope of this paper; however, it is important to evaluate based on existing data the extent to which in vitro incubation procedures alter the natural nutrient supply processes and how this might affect productivity measurements.

Goldman et al. (1981) were among the first to draw attention to the possible consequences of nutrient exhaustion on productivity during prolonged in vitro

incubations. Photosynthetic rates were not significantly affected by nutrient exhaustion in their field experi­ments; however, productivity based on nitrogen utiliz­ation (the limiting nutrient) clearly was, resulting in significant underestimates of true fluxes. In general, manifestations of nutrient deficiency on in vitro photo­synthetic rates have been difficult to demonstrate in the field (Glibert et a l ., 1985). Indeed, a general survey of the literature reveals that nutrients more often than not are present in measurable quantities, even over long incubations and when initial concentrations are ex­tremely low (e.g., McCarthy and Eppley, 1972; Price et

a i , 1985; Harrison and Harris, 1986). This is largely a consequence of the fact that nutrient regeneration is significant in incubation bottles. In fact, results summar­ized in Figure 1 suggest that nutrient depletion in vitro

may be the exception rather than the rule.Although the effects of nutrient exhaustion on pro­

ductivity estimates based on utilization of the depleted nutrient are evident (Goldman et a i , 1981; see also Fisher et a i , 1981), apparent non-linearities in utiliz­ation are common even when depletion does not occur (Goldman et a i , 1981; their Fig. 7). This can often be traced to nutrient regeneration and the effects of isotope dilution of the substrate on computations of utilization rates. Conventional equations are based on the assump­tion that the substrate isotope enrichment factor (specific activity in the case of radiotracers) is constant over the incubation period (Dugdale and Goering, 1967). Glibert e ta l. (1982) were first to show that isotope dilution resulted in an altered substrate isotope enrich­ment factor, leading to errors (underestimates) in am­monium uptake rates by as much as a factor of two or more in coastal and oceanic waters. Subsequent studies have confirmed their findings (Selmer, 1988) and ex­tended the studies to include phosphorus (Harrison, 1983a) and urea (Hansell and Goering. 1989; Slawyk et

a l., 1990). Furthermore, Harrison and Harris (1986) showed from detailed time-series measurements that isotope dilution effects are strongly time-dependent, as would be expected (Garside and Glibert, 1984), and are strongly influenced by the dynamics (growth/mortality) of the resident plankton communities. Kanda et al.

(1987) have attempted to generalize the isotope dilution errors for the much more extensive historical data sets of ammonium utilization. Their analysis largely confirmed the existing direct experimental evidence, suggesting that the errors are greatest in coastal waters and de­crease offshore (Fig. 6). Errors in computing utilization rates will obviously bias estimates of regeneration when mass balance approaches are used, as discussed above (see also Dugdale and Wilkerson, 1986). Isotope dilu­

I C E S m ar . Sei. S y m p . , 197 ( 1993) Nutrient recycling in production experiments 155

2 . 5 -

U / R = 0 . 5

/U

.5 - ,

C oasta l O ceanic

----------------------------- P r o d u c t i v i t y

Figure 6 . Predicted errors in estimates of ammonium utiliz­ation resulting from substrate “ isotope dilution" under circum­stances where regeneration rates (R) are equivalent to (U/R = 1.0) or twice (U/R = 0.5) utilization rates; U = utilization rate corrected for isotope dilution, u = conventional, uncorrected rate. Data are those summarized by Kanda et al. (1987) from coastal and oceanic waters, ranked within region according to productivity level.

tion errors not only impact nutrient utilization estimates p e r se (Glibert el a l . , 1982), but may also bias estimates of the “f-ratio” (Eppley and Peterson, 1979), i.e., new production/[new + regenerated production] (Harrison et a l . , 1987; Ward et a l . , 1989) or complicate attempts to derive kinetics parameters using conventional concen­tration addition or substrate disappearance techniques (Garside, 1984; Morrissey and Fisher, 1988).

The availability of data on concentrations and tracer content of substrate as well as particulate matter in isotope dilution experiments has brought to light another potential problem with conventional nutrient tracer techniques, namely isotope inventory. Isotope dilution models initially used were based on the assump­tion that all of the substrate utilized was recovered in the particulate matter (Caperon et a l , 1979; Glibert el a l. ,

1982). Glibert et al. (1982), however, noted that utiliz­ation rates computed by tracer disappearance from the substrate almost always exceeded those determined by tracer enrichment of the particulates, i.e., ~40% of the isotope leaving the ammonium pool was not recovered in the particulates, suggesting either analytical problems or the involvement of an additional unknown dissolved N-pool not routinely measured (Harrison, 1983b; Laws, 1984). This isotope discrepancy has been found in other studies of ammonium (Ward et a l., 1989; Wheeler and Kokkinakis, 1990; but see Glibert et a l . , 1991) and urea cycling (Hansell and Goering, 1989; Slawyk e ta l . , 1990) and may have a direct bearing on the often observed discrepancy between utilization rates determined by substrate disappearance and rates determined by iso­

tope incorporation (Dugdale and Wilkerson, 1986). Analytical problems have generally been discounted (but see Altabet, 1990), which has prompted researchers to consider the involvement of additional N-pools and to employ more sophisticated models (Laws, 1985; La­Roche and Harrison, 1987; Price and Harrison, 1988). Researchers studying the dynamics of the phosphorus cycle seem to have recognized the need for more com­plex models well before nitrogen researchers (Watt and Hayes, 1963; Taft e ta l . , 1975; Smith e t a l . , 1985). These new analyses point to the importance of an additional dissolved nitrogen pool(s) (e.g. dissolved organic nitro­gen) with which nitrogen exchange may be significant, i.e., comparable in magnitude to ammonium utilization and regeneration. Examples in the literature are regret­tably few, because conventional data sets are generally inadequate for this type of analysis. Moreover, because of the nature and quantity of data required, it is unlikely that this approach will become routine. On the other hand, new techniques have recently been described by which tracer exchange with dissolved organic-N pools can be directly measured (Bronk and Glibert, 1991, 1993). Clearly, however, a more comprehensive com- partmental analysis approach will be necessary to im­prove our understanding of the cycling of nutrients in productivity experiments and in nature.

Conclusions

The disturbance of natural nutrient regeneration pro­cesses has been one of the principal concerns in conven­tional in vitro productivity experiments. Little evidence, however, is available supporting arguments that nutri­ent depletion is important in short-term incubations or when it does occur, that photosynthesis is significantly affected. Effects are clear and substantial, on the other hand, when utilization of the limiting nutrient is used as the index of productivity. Nutrient depletion in incu­bation experiments is relatively uncommon; nutrient regeneration in bottles is significant and is generally equivalent to or greater than utilization in a wide range of environments. One consequence of the latter, how­ever, is that productivity estimates based on conven­tional nutrient utilization models may significantly underestimate true fluxes due to substrate “isotope dilution” . Questions persist about the reliability of cur­rent experimental methods and models used to deter­mine nutrient regeneration: (1) do size-fractionation procedures inherently bias the results, (2) how import­ant are the unmeasured dissolved organic pools in nutri­ent regeneration? Although contemporary measure­ments have substantially improved our understanding of nutrient cycling in productivity experiments as well as in nature, we still have a long way to go.

156 W. G. Harrison IC E S m ar . Sei. S y m p . . 197 (1993)

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