Size Fractionated Chlorophyll a Response from an Open. Ocean Ecosystem Iron Addition Experiment

Transcription

1 Size Fractionated Chlorophyll a Response from an Open Ocean Ecosystem Iron Addition Experiment by Zackary I. Johnson Submitted to the Department of Civil and Environmental Engineering in partial fulfillment of the requirements for the degree of Bachelor of Science at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY 13 May 1994 Massachusetts Institute of Technology, All Rights Reserved. Author... Department of Civil and Environmental Engineering 13 May 1994 Certified by... Professor Sallie W. Chisholm Department of Civil and Environmental Engineering Thesis Supervisor

2 Abstract Size Fractionated Chlorophyll a Response from an Open Ocean Ecosystem Iron Addition Experiment by Zackary I. Johnson Submitted to the Department of Civil and Environmental Engineering on 13 May 1994, in partial fulfillment of the requirements for the degree of Bachelor of Science Considerable evidence suggests that major portions of the world s oceans are limited by Fe, which supports the notion that new production and subsequent CO 2 drawdown are less than predicted based on available macronutrients (NO 3, PO 4 ). To test this hypothesis, a purposeful addition of Fe was performed on the ecosystem level in a high nutrient, low chlorophyll region near the Galapagos Islands to determine the effect of Fe in limiting phytoplankton production. A second experiment was to observe phytoplankton communities in upstream and downstream locations within the naturally fertilized Galapagos Island plume. In the first experiment, a response to Fe addition was observed in all size classes, except for 10-18µm, after one day as an increase in chlorophyll a concentrations. Concentrations continued to increase on day two, but subsequently began to decline. The 10-18µm size class began to increase after two days and continued to increase four days after Fe addition. All size classes are limited by Fe, though other factors such as grazer regulation for the smaller size classes, sinking for the larger size classes, and alteration of cellular machinery can affect overall phytoplankton community structure. In the second experiment, increases the large size classes were observed in the downstream locations compared with the upstream locations. No increases were observed in the <1µm size fraction, which is thought to be limited by grazer regulation. Sinking terms also limit phytoplankton production in deep, nutrient replete areas. Thesis Supervisor: Sallie W. Chisholm Title: Professor of Civil and Environmental Engineering

3 Acknowledgments A little less than four years ago I came to MIT with all the wonder, excitement, and nervousness that every freshman exhibits. But I now get ready to leave MIT with new skills, confidence and pride, hopeful about the challenges that are to face me in the future. Certainly this growth was not without help and I owe a great deal of both my academic and personal knowledge to my advisor, Penny Chisholm. From initially luring me into the field of oceanography with a UROP that offered the prospect of a cruise to the Sargasso Sea (the cruise turned out to be in 30-40ft seas and everyone got sick), to facilitating a trip to San Diego to attend an ASLO conference, she has given me far more opportunities than I had ever hoped for. Thank you for everything. I also have to thank Jeff Dusenberry, my best UROP mentor, for not only teaching me many new skills, but also for putting up with all of my dumb questions. In addition, the others in the lab including Sheila, Brian, Lisa, Karina and Amy have helped me with everything from where to buy organic beets to how to plot data without introducing biases. In all, the Chisholm Lab has been a wonderful place to work, learn and grow. Thanks. Finally, I owe a great deal to my friends and brothers at Phi Delta Theta for helping have fun while engaging in all of the ordeals that MIT is famous for. My family has also been extremely supportive, especially during the tough times when everything is going wrong, as has Debby, who helps me realize that sometimes there are more important things than getting an A on that problem set. In short, my career at MIT has not been an individual pursuit, but a journey aided by many.

4 Table of Contents 1 Introduction Methods Filter Fractionation Replication/Retentate Bottle Experiments Results Leg One: Fe addition Leg Two: Galapagos Islands Plume Fertilization Bottle Experiments Discussion Conclusions...38 Bibliography...41 Appendix...43

5 List of Figures Figure 1: Map of Sampling Stations (note station 9 is mis-plotted) (Martin et al. 1994)12 Figure 2: Results from Replication and Filtrate/Retentate Experiments. a. Filtrate replication, b. Retentate replication, c. Calculated size classes based on filtrate and retentate values Figure 3: Chlorophyll a concentrations two days before the addition of Fe. Note the majority of phytoplankton reside in the <5µm size classes Figure 4: a. Time course plot of a. in-patch and b. out-of-patch chlorophyll a concentration of size classes versus time after Fe addition. Note the peak values in all but one size class on day 2, and the increasing concentrations of the 10-18µm size class on day 3, in the inpatch data, while the out-of-patch concentrations remain relatively constant over the course of ten days...18 Figure 5: a.chlorophyll a concentrations and b. percent of total chlorophyll a of in-patch and out-of-patch stations one day after Fe addition at 10m...20 Figure 6: a.chlorophyll a concentrations and b. percent of total chlorophyll a of in-patch and out-of-patch stations two days after Fe addition at 10m...21 Figure 7: a.chlorophyll a concentrations and b. percent of total chlorophyll a of in-patch and out-of-patch stations three days after Fe addition at 10m...22 Figure 8: a.chlorophyll a concentrations and b. percent of total chlorophyll a of in-patch and out-of-patch stations four days after Fe addition at 10m...23 Figure 9: Comparison of chlorophyll a concentrations at 10m for the controls in the patch/ plume experiment. Note the fractions concentrations are relatively constant as are the percentage of each fraction, though both do show slight variations...25 Figure 10: Comparison of mean upstream/downstream chlorophyll a concentrations at 10m for the plume experiment. Note that the while the <1µm size fraction remains constant in the up and downstream stations, all other size fractions increase markedly in the downstream stations...27 Figure 11: Results from the bottle experiment from the first leg. Note the time lag to initiate pseudo-exponential increase in chlorophyll a concentration...29 Figure 12: Results from the size fractionated bottle experiments from the second leg with added germanium. Note that the large size classes do not increase substantially...30 Figure 13: In Patch vs. Downstream Stations at 10m. Note that increases in chlorophyll a concentration are not due to similar size classes Figure 14: Fractionated Chlorophyll a data vs. Total Chlorophyll a concentration. Note that as chlorophyll a concentration increases the smaller size classes reach a threshold beyond which only increases in the larger size classes (>10µm) will cause increases in total chlorophyll a concentrations...40

6 List of Appendices Appendix 1: Tabulated results from Fe addition (patch) study...44 Appendix 2: Tabulated results from the Galapagos Plume Study...45

7 Introduction In some regions of the world s oceans, e.g. Southern Ocean, Equatorial Pacific, Antarctic Waters, conditions exist where major plant nutrients (NO 3, PO 4, SiO 3 ) are not depleted despite adequate light. These high nutrient (e.g. NO 3 > 7µm, PO 4 > 1µm), low chlorophyll (Chl < 0.5ug/l) (HNLC) areas (Chisholm and Morel, 1991), seem to contain all of the necessary components of a high new production system (i.e. high carbon flux to the deep ocean), yet achieve only a fraction of their potential new production (Chavez and Barber, 1987). Several hypotheses have been proposed to explain the high nutrient low phytoplankton paradox in these regions including: trace metal inhibition (Barber and Ryther, 1969); increased grazing pressure (Walsh 1976, Frost 1992); NO 3 regulation of phytoplankton growth rates (Wilkerson and Dugdale 1992); limited source of bloom forming diatoms (Chavez 1989), and iron limitation (Martin 1990b). Of these hypotheses, iron limitation and grazing have received the most attention. Perhaps the most controversial explanation for the HNLC regions, that of iron limitation suggested by Martin and co-workers (Martin and Fitzwater, 1988; Martin 1990b, Martin et al. 1990a, Martin et al. 1991), is based on the physiological Fe requirements of phytoplankton compared with low available concentrations. Although available Fe concentrations are difficult to determine (Wells 1991), Fe concentrations in HNLC areas are generally thought to be quite low (Martin et al. 1989), typically being <1 nmol Fe, because they are isolated from continental sources of Fe and receive only periodic atmospheric aerosol inputs (Duce and Tindale 1991, Wells 1991). In addition, it is hypothesized that phytoplankton in HNLC areas require higher Fe concentrations because of the excess of the nutrients available (Martin et al. 1991). However because phytoplankton evolved from an anoxic environment rich in Fe (10-3 M) (Brand 1991), and because of Fe s wide range of redox potential depending on which complex surrounds it (Morel et al. 1991b), Fe is 7

8 used extensively by phytoplankton in a variety of cellular systems including synthesis of DNA, RNA, chlorophyll, electron transport, oxygen metabolism, nitrogen reductase, and nitrogen fixation (Weinberg, 1989). Despite this universal need, all phytoplankton do not require similar ambient concentrations of Fe, because of different size (see below) and nutrition (Price et al. 1991) requirements. For example, marine algae grown on NH 4, NO 3, and N 2 typically require 10, 15, and >200 µmolfe/molc, respectively (Morel et al. 1991b) indicating that Fe requirements vary species to species depending on nitrogen sources. However, Fe concentration requirements are not absolute and can change under Fe stress or limitation. Because phytoplankton have no Fe storage mechanism (Weinberg 1989) and in response to low available Fe concentrations, phytoplankton have several adaptations to reduce Fe requirements. Reduction of internal Fe concentration by replacement with other metals that can act as substitutes in some Fe pathways has the two fold effect of decreasing a cell s Fe requirement and increasing Fe kinetics in favor of the cell. Another technique employed by cells is increasing uptake kinetics by increasing surface-bound ligands that specifically bind Fe (Hudson and Morel 1990). However, cell size, i.e. surface area to volume ratio, ultimately becomes the growth rate limiting factor because Fe uptake becomes efficient such that uptake nears the physical limits of diffusion (Hudson and Morel 1990). Despite the reduction of Fe intake and increasing uptake kinetics, many cells can still be Fe limited because some cellular enzymes such as nitrogen fixation and nitrogen reductase can only use Fe (Hudson and Morel, 1990). Thus, at one end of the spectrum are small cells that grow on NH 4 which require smaller Fe concentrations and at the other end of the spectrum are large cells growing on NO 3 which require a higher Fe concentration. 8

9 Kinetic theory and laboratory observations have been supported by field data that shows that HNLC areas such as the equatorial Pacific are dominated by small (< 5 µm) phytoplankton (Peña et al. 1990, Chavez 1989, Murray 1992). Trace metal limitation such as Fe could be an important selective force on the phytoplankton, favoring certain species in areas with limited Fe concentrations (Brand 1983). However, small size fractions have negligible sinking rates (Takahashi 1983) and may be dominant because of sinking rate evolutionary forces structuring phytoplankton communities (Chavez 1989). The Fe hypothesis has further been tested using ship-board bottle experiments. These tests have shown that nmol amounts of Fe addition result in several fold increases in phytoplankton standing stocks relative to controls (ex. Helbling et al. 1991, Martin et al. 1990a, Martin and Fitzwater 1988). While the interpretation of these tests is controversial (Banse 1991), mainly because of their relevance to the unbottled ocean systems which contain higher trophic levels, Fe does seem to increase the total chlorophyll in these waters. In addition, it appears that Fe has a dramatic impact on phytoplankton species composition in HNLC regions (Chavez et al. 1991). Further controversy fueling Fe limitation hypotheses has resulted from the hypothesis that fertilizing HNLC areas such as the Southern Ocean with Fe could cause a huge draw down of CO 2 which could possibly have global climate implications (Martin 1990b). While most scientists agree Fe plays a role in phytoplankton population dynamics, increasing CO 2 draw down through Fe fertilization, and other effects such as altered trophic interactions are uncertain and remain difficult to prove (Broecker 1990, Brandini 1993). A competing or perhaps complimentary hypothesis (Price et al. in press, Morel et al. 1991b) to Fe limitation is that of grazer regulation having a role in limiting production in HNLC regions. Several researchers have noted that despite increases in total standing 9

10 stocks of phytoplankton in Fe enrichment bottle experiments, specific growth rates do not increase following Fe addition (Banse 1991, others) -- specific growth rates in the equatorial Pacific have been found to be relatively high (0.7 day -1 ) before Fe addition (Price et al. 1991). This points to grazing as a potential limiting factor, allowing specific growth rates to remain high, while keeping standing stock populations low. Grazing theories have also been combined with Fe limitation such that grazing could be limiting phytoplankton in the smaller categories that are present in the HNLC regions, while other factors including inhibitory NH 4 levels or Fe limitation could be limiting the larger size classes (Price et al. in press, Morel et al. 1991). Despite extensive energies devoted to these hypotheses, it is difficult to draw direct conclusions because of the inherent limitations of bottle experiments. Consequently, a purposeful mesoscale Fe addition experiment was performed to determine Fe s role as a limiting factor in HNLC regions. Here we document size fractionation of Fe addition and control samples to determine the response of different classes of phytoplankton to a pulse injection of Fe. 10

11 Methods To determine the effects of Fe addition in situ, two ecosystem level experiments were carried out near the Galapagos Islands. In the first experiment 7800 moles of 0.5M Fe II solution was dispersed over an eight by eight kilometer patch. An inert tracer gas, SF 6, was simultaneously released with the Fe and a Global Positioning System (GPS) equipped buoy was used to provide a LaGrangian reference point, both to track the patch. (For further Fe addition discussion see Martin et al. 1994) In the second experiment, the Galapagos Island plume extending 500 kilometers to the north was used as a natural fertilization experiment. Upstream stations, out of the plume and beyond eolian dust inputs from the island were compared to downstream stations located in varying degrees within the naturally fertilized plume. Furthermore, stations were taken from shelf locations, in a channel between the Galapagos s two islands (Figure 1). Samples for both experiments were collected ultra-clean trace metal sampling techniques (30-liter Go-Flo bottles on Kevlar Line (Fitzwater et al. 1982)). Filter Fractionation Filter fractionations using chlorophyll a as an indicator of biomass (Takahashi et al. 1988) were performed with varying size fractionations, typically <0.8, <1, <3, <5, <10, <18µm, and Total for the first leg, and <1, <5, <10µm, and Total for the second leg. Chlorophyll a extractions were performed on the filtrate using the chlorophyll a extraction method outlined in Parson et al Samples ranged in volume from ~ ml, and were performed using Poretics polycarbonate membrane filters with gravity filtration. Absorbance readings were taken using a Turner Designs model R Fluorometer calibrated with commercial chlorophyll a. Samples were also acidified (Parsons et al. 1984) and phaeo-pigment analyses were performed. 11

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13 Replication/Retentate A replication experiment was performed using all size fractions to determine the reproducibility of individual samples. While no replicates were performed for individual samples during the cruise except for station 16 on the second leg, standard deviation from the mean value on this day is only 3% for the worst size fraction (Total Chl a), indicating that individual measurements are reliable. The mean standard deviation from mean chlorophyll a values is 2.1% (Figure 2a). A comparison of filtrate/retentate values for individual samples was also performed to determine the bias of calculating discrete fractions utilizing each method. Results from this experiment suggest that individual values are reproducible using both methods (Figure 2a,b), but that inter-method comparison does not yield similar absolute chlorophyll a values for discrete size fractions (Figure 2c) Differences in chlorophyll a concentrations could be due to bias based on calculations in assigning specific size classes, especially in the end categories such as <0.8µm or >10µm. Another possibility is that pressure differences caused by the presence of the Whatman GF/F under the Poretics filter could have effects on a organisms affinity to the filter, altering whether it would go through the filter. Notable differences occur in the <0.8µm size fraction with the retentate method yielding results that are ~3X that of the filtrate values. Conversely, the 1-5µm and >10µm size classes are larger in the filtrate samples by the same absolute chlorophyll a amount. All data including in-patch, out-of-patch, upstream, and downstream stations were collected using the filtrate method. Chlorophyll a measurements using the filtrate method from the controls, namely upstream and out-ofpatch, show levels in accordance with previously published values. 13

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15 Bottle Experiments Bottle Experiments were performed on both the first and second leg of the cruise. Aliquots of the water sampled were placed in 2 liter acid washed polycarbonate bottles (Fitzwater et al. 1982). Fe amended samples had 5nM un-chelated Fe added at time zero. Both experiment and control bottles were maintained at surface sea water temperature with running seawater. Total chlorophyll a concentrations were measured using the above technique. A second set of bottle experiments were performed during the second leg of the cruise, using similar techniques to that of leg one. Three identical sets of bottles plus one set of bottles with added germanium of 2nM Mn (to retard diatom growth) were treated with Fe in a similar manner as the previous bottle experiment. An identical set of four bottles (three plus one Germanium addition control/no Fe) with out Fe addition were utilized as controls. Individual bottles were sacrificed daily and filter fractionated, according to the above technique. 15

16 Results Leg One: Fe addition The size distribution of chlorophyll a concentrations at 10m shows that the majority of phytoplankton standing stock biomass in this region before Fe addition resides in the <5µm size fraction (Figure 3). These values are in accordance with previously reported values from the subtropical Hawaiian waters having a size structure of 80% in the <3µm fraction (Takahashi et al. 1983) and Equatorial Pacific waters having the <1µm fraction containing >40% of the biomass (Peña et al. 1990, DiTullio et al. 1993). Total chlorophyll a values of ± µgchla/liter at 10m also are in accordance with previous observations (Peña et al. 1990) A time course plot over the eight days of tracking the fertilized patch shows that chlorophyll a values from out of the patch did not change considerably (Figure 4a). Chlorophyll a had a standard deviation over ten days of 16, 22, 29, 42 and 42% of the mean total chlorophyll a in the <1µm, 1-5µm, 5-10µm, 10-18µm and >18µm size classes, respectively. No daily size class measurement exceeded two standard deviations from the mean value. Thus, despite the patch moving, surrounding waters remained similar in phytoplankton community structure and can be compared to patch data as controls. Fe concentrations before addition were below the detection level of 1nM dissolved Fe (DFe), of which most was Fe(III) (Millero and Sotolongo 1988). Post fertilization values were raised to a peak of 6.2nM DFe in the center of the patch on the day of fertilization, yet quickly diffused to become more homogeneous. One day after fertilization the highest value of Fe in the center of the patch was 3.6nM DFe with subsequent days values decreasing ~15% reaching a level of <0.5nM on the fourth day. The SF 6 used to trace the patch behaved similarly to the Fe addition with concentrations remaining between 16

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19 40-50fM over the entire range of tracking the patch. Ambient SF 6 concentrations were approximately 0.05fM (Martin et al. 1994). After Fe addition, all size classes but the 10-18µm size fraction showed an unequivocal increase in chlorophyll a concentrations. The first day after Fe addition, total chlorophyll a at 10m had increased by 65%. Size fraction response to the Fe addition at 10m was fairly uniform in the increasing size fractions with the 10-18µm size fraction remaining within experimental error values of pre-addition levels (Figure 5). Size structure was not altered considerably because of the uniform increases in most size classes. Similar results were obtained for the second day after fertilization. Chlorophyll a concentrations were markedly larger over control samples with the total chlorophyll a concentrations at 10m 143% larger than the control (Figure 6). A time course plot of in-patch stations versus time reveals that total chlorophyll a concentrations at 10m increased to a maximum level of 0.638µgChla/liter on the second day after fertilization (Figure 4b). Percentage increases over controls increased notably in the 1-5µm and >18µm size classes while the other size classes remained or increased only slightly over day one levels. By the third day, total chlorophyll a concentrations at 10m had decreased slightly as a result of decreases in the <1µm, 1-5µm, 5-10µm, and >18µm size fractions, though at this time point the 10-18µm size class shows increases over previous levels for the first time since addition (Figure 7). Despite this increase, chlorophyll a concentrations dropped to 0.567µgChla/liter at 10m on day three and remained at that level (0.571 µgchla/liter) four days after Fe addition (FIgure 8), in spite of continuing increases in the 10-18mm size class and decreases in the other classes. Although all but one size class in fertilized waters initially increased in chlorophyll a concentrations over out-of-patch stations, continual increases in chlorophyll a in 10-18µm size fraction over time are apparent. Percentage of total chlorophyll a also increases for 19

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24 this size fraction. However, percent of total increases in this size class are a partial result of the slight decrease in the smaller size classes after their initial increase, compared with the continued increasing of the 10-18µm size fraction. Patch integrity was well maintained through the initial four days of the experiment after addition of Fe, expanding to a 8x12km rectangle. Unfortunately, by the fifth day the patch of Fe fertilized water became subducted to a depth of ~35m, by a front of low salinity water. This makes comparison with previous days in-patch and out-of-patch data difficult. Even so, trends from the center of the subducted region, i.e. approximately 35m, suggest that the 10-18µm size class continues to increase in spite of being subducted, implying that had the patch remained on the surface, the general trend of increasing 10-18µm size class exhibited during the first four days at 10m would have continued. This assumption should be taken with caution, however, as it is difficult to extrapolate chlorophyll a data to a different depth. Leg Two: Galapagos Islands Plume Fertilization The size distributions of chlorophyll a for samples that were outside of the plume are similar, though not identical, to the out-of-patch samples from the Fe fertilization (Figure 9). While the mean chlorophyll a concentrations of ± 0.008, ± 0.000, ± 0.003, ± 0.002, ± 0.007µgChla/liter for the respective size classes of <1µm, 1-5µm, 5-10µm, >10µm, and Total are ~20% smaller than the out-of-patch concentrations, the size structure is consistent with the out-of-patch data (Appendix 2). Stations within the Galapagos plume, similarly to the in-patch stations of the fertilization experiment, exhibited large increases in chlorophyll a concentrations. All size fractions increased chlorophyll a concentrations several fold except for the <1µm fraction which remained constant compared to upstream values (Figure 10). Size fraction response 24

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26 to the plume at 10m resulted in 4, 299, 288, 738, and 186% increases in the <1µm, 1-5µm, 5-10µm, >10µm, and Total size classes, respectively Station location had a profound influence on the chlorophyll a concentrations at 10m and size structure of the phytoplankton community. Station sixteen, located on a shelf which is 500 meters deep, behaved differently from other stations that were located offshore in deep locations. Near-shore, shelf stations had the highest total chlorophyll a concentrations. In addition, these stations had a two fold increase in the 1-5µm size class compared with other downstream stations. An extreme example of station location effect was noted with a station that was located in-between the two major islands of the Galapagos Islands. Station twenty (Figure 1), located on a shallow shelf, in warm waters and in a protected area had chlorophyll a concentrations that were as high as 8.134µgChla/liter for total chlorophyll a with only 0.180µgChla/liter residing in the <1µm size fraction, indicating an upper limit of the chlorophyll a concentrations for this region. Thus, stations exhibited a wide variability in chlorophyll a concentrations based on location, with generally higher readings occurring as proximity to land neared, though stations with similar proximity had similar chlorophyll a concentrations. Bottle Experiments Initial bottle experiments tracking total chlorophyll a over time show several fold increases over initial values by the seventh day after Fe addition (end of experiment). Control and Fe addition bottles show increases from initial chlorophyll readings of to ± µgchla/liter for Fe addition and ± µgchla/liter for the control after the first day, representing a 41% and 50% increase, respectively. Gradual increases in both control and Fe addition bottles continued until four days after Fe addition, reaching a level of ± and ± µgchla/liter for the Fe addition and control bot 26

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28 tles three days after addition, respectively. After four days, chlorophyll a concentrations increased in exponential fashion, and peaked 6 and 7 days after addition, at maximum values of ± 1.67 and 6.73 ± 1.79 µgchla/liter for Fe addition and control bottles, respectively (Figure 11). Size fractionation bottle experiments performed on the second leg of the cruise yielded similar total chlorophyll a concentration results, with the exponential increase occurring at day 3. Size fractionation from this experiment shows a large increase in chlorophyll a concentrations in the >10µm size fraction over control, however, the rest of the data is patchy and no conclusions were drawn from it. Increases in the larger size classes corresponds to previous bottle experiments showing large increases in the diatom size range over the controls after an initial waiting period of approximately four days (Helbling et al. 1991). Size fractionation bottle experiments with added germanium (diatom inhibitor) show increases in both control and Fe addition bottles. Unlike other bottle experiments, bottles with added germanium did not show large increases in the >10µm size fraction (Figure 12). Instead, the majority of the increase in the total chlorophyll a concentration came from several fold increases in the 1-5µm size fraction in both the germanium control and Fe addition bottles. However, the Fe addition bottles were 35% higher than the germanium controls. The 5-10µm size class also had increases in chlorophyll a concentrations in both bottles with Fe addition bottles 157% larger than the germanium controls. 28

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31 Discussion The results presented in the first experiment indicate that all but one size class in the area of study initially increased upon the addition of Fe. This change was evident 24hr following the addition of Fe. While change was apparent in most size classes, the most dramatic initial change occurred in the 5 10µm fraction, with an 129% increase. The larger size fractions also generally increased, though not as greatly. However, when size fractionation data is considered in small intervals, it is shown that the 10-18µm size class achieved no increase in chlorophyll a. This size class remains constant over the first two days following addition, but by the third day the 10-18µm class shows increases. Immediate increases in the chlorophyll a concentration for all but one size class is indicative of Fe limitation in these size classes. Fe uptake rates have been shown to be high in Fe-limited phytoplankton (Hudson and Morel 1990), even on the order of 40 times that of coastal species (Price et al. 1991). Thus, despite long term Fe processing rates eventually subsiding well below initial rates (Price et al. in press), cells that are truly Fe limited should be able to process any sporadically available Fe immediately. This was seen as an increase in most of the size classes. However, the 10-18µm size class shows no such increase until three days after Fe addition. While smaller size classes appear to be limited by Fe, the time lag experienced by the 10-18µm size class indicates that in addition to possible Fe limitation, other factors could be influencing growth. Bottle experiments performed on this cruise and by others have shown that chlorophyll a increases following Fe addition follow a time lag similar to the 10-18µm class (Price et al. 1991, Martin 1990a, Helbling et al. 1991). Fe uptake limitation caused by diffusional limitation is an unlikely explanation to this situation as the larger size class of >18µm shows increases in chlorophyll a concentration immediately following addition, (and continues to increase on day 2). Impact from grazers is also unlikely in 31

32 this size class. While the protozoans that graze on smaller phytoplankton can respond quickly to any increases in phytoplankton abundance in the smaller size categories (Miller 1991, Banse 1982), larger zooplankton are not able to respond as quickly. Furthermore, the sudden increase on day three is not indicative of grazer control. A third possibility initially affecting this size class is that of physiological acclimation. Because of their history of Fe limitation, the physiology of the phytoplankton may be configured to use NH 4 instead of NO 3. Upon the receipt of Fe, phytoplankton in this size class may alter their cellular processes to initiate the use of NO 3, partly because of decreased ammonium concentrations after the sudden growth burst from more efficient ammonium users namely smaller phytoplankton and partly due to the abundance of Fe which is necessary for the production of nitrogen reductase (Weinberg, 1989). This alteration may introduce a lag time of a couple of days. This is supported by bottle experiment data showing that, commensurate with the couple day lag time until pseudo-exponential increases in chlorophyll a concentrations, there is no net increase in the NH 4 utilization, but rather an increase in NO 3 utilization (Morel et al. 1991a). Fast repetition rate fluorometry has demonstrated that the photosynthetic energy conversion efficiency of all size classes increases one day after Fe addition (Kolber et al. in press). This suggests that Fe is limiting all size classes, including the 10-18µm size class which shows no apparent increase in chlorophyll a concentrations until three days after addition. While ammonium concentrations decrease 0.1 ± 0.07µm (approximately in half) indicating a physiological stimulation (Martin et al. 1994), NO 3 concentrations do not initially drop in the detectable range (0.5µm). In addition, it is difficult to attribute the ammonium reduction to all size classes or only certain size classes. Thus, it is distinctly possible that certain size classes are responsible for the majority of the ammonium draw down 32

33 whereas other size classes are readjusting their inner cellular mechanisms to utilize other sources of nitrogen as substrate. Despite experiencing an initial time lag, the 10-18µm size class continued to increase along with the other classes, while the <1µm size class remains static two days after Fe addition. Increasing chlorophyll a patterns also begin to degrade for the 1-5µm size class, despite showing increases on the first and second day, with levels beginning to return to ambient concentrations on day three. The initial increase in the <1µm and 1-5µm size class demonstrates that some limiting factor was quenched by the introduction of Fe. Because these small phytoplankton enjoy rapid uptake kinetics, inherent from their small size (Hudson and Morel 1990, Takahashi et al. 1983), sudden increases in chlorophyll a following Fe addition is the result of Fe limitation. However, another factor quickly becomes limiting to the small phytoplankton size classes, owing to Fe concentrations still being elevated beyond ambient levels at three and four days after the addition, yet chlorophyll a values not increasing. Grazer response to increased chlorophyll a concentrations is a likely limiting factor, that could explain the observed decrease in these size classes. Not only will small unicellular protozoans respond quickly to increasing phytoplankton stocks by increasing numbers thus increasing grazing pressure (not experienced by larger phytoplankton), but there could also be an increase in diel grazers, that vertically migrate to surface waters at night (Johnson, personal communication). Diel grazers that find a phytoplankton rich source will stay in a region through the day time to take advantage of higher food stocks. This grazer response from lower depths, i.e. migratory grazers, is supported by an increase in chlorophyll a concentrations in lower waters, i.e. 55m, under the patch. Despite these deep areas not receiving any Fe addition, there is a general increase in the total chlorophyll a 33

34 concentration over time with notable increases occurring in the larger size fraction, indicative of grazer regulation. Fast repetition rate fluorometry has shown that while increases in the photosynthetic conversion energy efficiency occur, maximum levels are achieved by two days after the addition of Fe, and subsequently decrease over time (Kolber et al. in press). This corresponds to the total chlorophyll a peaks, along with peaks in the <1, 1-5, 5-10, and >18µm size classes and subsequent decrease in these size classes, though the 10-18µm size class shows increases on the second day and continues to increases through the fourth day. Grazing pressure is not likely to account for the decrease in photosynthetic conversion energy efficiency, but limitation by a micro or macro nutrient could. Perhaps lower levels of ammonium act as a feedback to cue phytoplankton to slow down their growth rate. The bulk of the phytoplankton that were initially growing on NH 4 could be switching their inner cellular machinery just as the 10-18µm size class initially did to preferentially take up the more abundant NO 3 nitrogen source. Fe could also become a limiting again as Fe concentrations rapidly decreased after the third day. The second leg of the cruise dealing with the plume to the north off the coast of the Galapagos Islands deals with natural fertilization from the island sources. While it is presumed that Fe is the limiting agent, there are certainly other trace metals and nutrients that are contributing to the plume. In addition, the patch experiment location and Galapagos island experiment location can not be considered identical bodies of water, making interexperiment comparisons strained. Thus, it is difficult to directly compare the patch fertilization experiment with the Galapagos plume study, but certain analogies can be made. Notable increases in total chlorophyll a are present when downstream data is compared with upstream data. Increases in total chlorophyll a are on the order of those experienced in the patch fertilization experiment (approximately 3X). This suggests if continual 34

35 fertilization were carried out, there would not be huge increases in chlorophyll a concentration over what was observed during the first couple of days following the fertilization. However, increases in chlorophyll a concentration in the patch and downstream are not attributable to the same size classes increasing (Figure 13), suggesting that different factors are controlling each situation. While in-patch data suggests that all size classes are limited by Fe, downstream data shows only increases in the larger size classes, with no apparent increase in the <1µm size class over controls. It is unlikely that these populations are Fe limited in light of the Fe addition experienced from the plume. Grazer control would seem the most probable explanation, resulting in low observed chlorophyll a concentrations despite adequate Fe and other major nutrients. Because fertilization from the plume is a continuous process, smaller grazers would have ample time to grow and keep these populations in check. In addition, the initial response of grazers to increased phytoplankton levels was seen in the smaller size classes after the addition of Fe. For the downstream stations, a similar situation exists, but at this time point after addition (time = infinity) grazer population has achieved a steady state level, keeping the small phytoplankton population in check. As Frost has pointed out in chemostat models (Frost 1992), increased grazing pressure yet low standing stocks, predicts that phytoplankton in the downstream location have higher specific growth rates than their upstream counterparts. However, division rates in other off-shore areas with persistent high nutrients but low chlorophyll have shown elevated rates of division and do not suggest Fe deficiency (Price et al. 1991, Banse 1991). In addition, several bottles experiments have been performed to determine the effect of Fe on specific growth rates. These experiments also suggest that Fe does not effect the rate at which phytoplankton divide (Price et al. 1991). It seems unlikely that the region downstream of the Galapagos is limited by any micronutrient and nutrient analysis indicates 35

36

37 that macronutrients are abundant. Clearly then, grazing is an important factor in these waters because it is necessary to achieve a very high specific growth rate and the low standing stock observed in the <1µm size class. The large increases in chlorophyll a concentrations due to increases in the larger size classes are most likely due to the physiological stimulation of these size classes by Fe. Why then are there higher chlorophyll a concentrations in these size classes, while in the smaller size class grazers are able to keep phytoplankton in check? Grazers that feed on large phytoplankton are simply not able to maintain the large numbers needed to keep the rate of production supported by the high levels of nutrients in check. In this system (an open ocean area high in nutrients but also fertilized by Fe) chlorophyll a concentrations can remain high, much like a coastal system. Thus, as large phytoplankton species dominate coastal systems (Chavez 1989), large phytoplankton also dominate in this situation. While concentrations of the larger phytoplankton size fractions dominate the downstream stations, it appears that there is a limiting factor even to these fractions when ambient nutrients are considered. While most downstream stations had chlorophyll a concentrations in the range of to 0.903µgChla/liter, station twenty that was on a shelf location had a total chlorophyll a concentration of 8.134µgChla/liter! While its proximity to land was much greater than the other stations measured, the defining factor that made this station achieve such a high concentration of chlorophyll a was its depth. Phytoplankton population dynamics are profoundly influenced by sinking rates; while cells that are <3µm have relatively negligible sinking rates (Takahashi et al. 1983), larger cells such as diatoms, have sinking rates that have been a major evolutionary force in structuring phytoplankton communities (Hutchinson et al, 1967). Station twenty is sufficiently shallow in depth such that larger phytoplankton that would be removed in open ocean systems due to sinking are continually replaced, thus the sinking is not important at this loca- 37

38 tion, and large phytoplankton can increase the total chlorophyll a concentration. Thus, while open ocean phytoplankton in the larger size classes may not be limited by factors such as nutrient limitation or grazing, populations are kept low due to sinking terms. Conclusions It becomes obvious that no one factor alone determines the number and species composition of organisms in an ecosystem (Hansson and Carpenter 1993). Macronutrients such as NO 3 and NH 4, micronutrients such as Fe, grazing, and even sinking can all have profound influences on phytoplankton concentrations and speciation. In addition, there is often a dynamic nature to these limiting factors such that one factor will quickly replace another factor if conditions are even slightly altered. In the first experiment Fe was added in nm amounts, resulting in chlorophyll a concentration increases in all but one size class, indicating an immediate response, thus Fe limitation. The 10-18µm size class did not increase until three days after addition, curiously corresponding to the decreases in the other size classes. This lag-time is also observed in bottle experiments, indicating some consistent process that is limiting this size class from immediately increasing. Alteration of cellular physiology, to account for changing physical surroundings, provides a possible explanation for the observed lag-time. But it is unclear if this size class is directly Fe limited, though fast repetition rate fluorometry indicates that physiological stimulation occurs upon the addition of Fe. Regardless, the pulse injection of Fe creates a system that is constantly in flux, first rising to higher chlorophyll a concentrations, then quickly decreasing chlorophyll a concentrations. The natural fertilization experiment offered by the plume from the Galapagos Islands, represents a situation that provides a constant source of Fe (along with other micronutrients), despite being in a different body of water. Constant fertilization seems to indicate that in the long run, all the larger size classes increase upon Fe addition, except 38

39 for the <1µm fraction. Fast growing micro-heterotrophs offer an explanation for relatively static levels of the <1µm size fraction. Thus, it appears that all size fractions are to at least some degree affected by the addition of Fe, and can be considered Fe-limited. However, sinking also limits the standing stock of phytoplankton, most notably in the larger size fractions. Fe addition in the patch experiment caused increases in the phytoplankton concentrations, but if Fe were continually added as in the plume experiment, phytoplankton standing stocks would reach some critical level where the sinking terms would dominate and additional Fe addition would not promote continued phytoplankton production. When fractionated chlorophyll a concentration is plotted against total chlorophyll a concentration, it is apparent that to increase total chlorophyll a, increases in larger size classes are necessary (Chisholm, 1992). While the data set analyzed here does not span a continuous range of total chlorophyll a concentrations, it does suggest that the larger size classes become the limiting populations as total chlorophyll a concentrations increase (Figure 14), implying that sinking becomes important as other limiting factors such as Fe are quenched. Thus, while a great a deal was learned from the pulse injection of Fe in ecosystem experiment, further experiments of this nature are needed to help distinguish between long-term and short-term Fe effects on phytoplankton communities. Continual Fe addition experiments and further determination of nutrient usage by individual size classes of phytoplankton could be particularly informative in describing phytoplankton community structures in HNLC regions. 39

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41 References [1] Banse, K. Cell volumes, maximal growth rates of unicellular algae and ciliates, and the role of ciliates in the marine pelagial. Limnology and Oceanography (1982). [2] Banse, K. Rates of phytoplankton cell division in the f... iron enrichment experiments. Limnology and Oceanography 36(8) (1991). [3] Barber, R., Ryther, J. Organic chelators: factors affecting primary production in the Cromwell Current upwelling. Journal of Experimental Biology and Ecology (1969). [4] Brand, L. Minimum iron requirements of marine phytoplankton and the implications for the biogeochemical control of new production. Limnology and Oceanography 36(8) (1991). [5] Brand, L., Sundra, W., Guillard, G. Limitation of marine phytoplankton reproductive rates by zinc, manganese, and iron. Limnology and Oceanography 28(6) (1983). [6] Brandini, F. Phytoplankton biomass in an Antarctic coastal environment during stable water conditions - implications for the iron limitation theory. Marine Ecology Progress Series (1993). [7] Broecker, W. Comment on Iron Deficiency limits phytoplankton growth in Antarctic waters by John H. Martin et al. Global Biogeochemical Cycles 4(1) 3-4 (1990). [8] Chavez, F. Size distribution of phytoplankton in the central and eastern tropical pacific. Global Biogeochemical Cycles 3(1) (1989). [9] Chavez, F. and Barber, R. An estimate of new production in the Equatorial Pacific. Deep Sea Research (1987). [10] Chavez, F., Buck, K., Barber, R. Phytoplankton taxa in relation to primary production in the equatorial Pacific. Deep Sea Research in press. [11] Chavez, F., Buck, K., Coale, K., Martin, J., DiTullio, G., Welschmeyer, N., Jacobson, A., Barber, R. Growth rates, grazing, sinking and iron limitation of equatorial Pacific phytoplankton. Limnology and Oceanography 36(8) (1991). [12] Chisholm, S. Phytoplankton Size. in Primary Productivity and Biogeochemical Cycles in the Sea ed. P. Falkowski and A. Woodhead. Plenum Press, N.Y. [13] Chisholm, S. and Morel, F. Preface: What controls phytoplankton in nutrient-rich areas of the open sea? ASLO symposium in Limnology and Oceanography (1991). [14] DiTullio, D., Hutchins, D., Bruland, K. Interaction of iron and major nutrients controls phytoplankton growth and species composition in the tropical North Pacific Ocean. Limnology and Oceanography 38(3) (1993). [15] Duce, R. and Tindale, N. Atmospheric transport of iron and its deposition in the ocean. Limnology and Oceanography 36(8) (1991). [16] Dugdale, R., Wilkerson, F., Barber, R. Chavez, F. Estimating New Production in the Equatorial Pacific Ocean at 150 o W. Journal of Geophysical Research (1992). 41

42 [17] Fitzwater, S., Knauer, G., Martin, J. Metal contamination and its effect on primary production measurement. Limnology and Oceanography 27, (1982). [18] Frost, B. and Frazen, N. Grazing and iron limitation in the control of phytoplankton stock and nutrient concentration: a chemostat analogue of the Pacific equatorial upwelling zone. Marine Ecology Progress Series (1992). [19] Hansson, L.-A. and Carpenter, S. Relative importance of nutrient availability and food chain for size and community composition in phytoplankton. Oikos (1993). [20] Helbling, E., Villafañe, V., Holm-Hansen, O. Effect of iron on productivity and size distribution of Antarctic phytoplankton. Limnology and Oceanography 36(8) (1991). [21] Hutchinson, G. A Treatise on Limnology Wiley, New York (1967). [22] Hudson, R. and Morel, F. Iron transport in marine phytoplankton: Kinetics of cellular and medium coordination reactions. Limnology and Oceanography 35(5) (1990). [23] Jochem, F., Zeitzchel, B. Productivity regime and phytoplankton size structure in the tropical and subtropical North Atlantic in spring Deep Sea Research II 40(1/2) (1993). [24] Johnson, K. Personal Communication. 27 March [25] Kolber, Z., Barber, R., Chisholm, S., Coale, K., Greene, R., Johnson, K., Lindley, S., Falkowski, P. Iron limits photosynthetic conversion efficiency in the equatorial pacific ocean. In Press (1994). [26] Martin, J. Glacial-interglacial CO 2 change: The iron hypothesis. Paleoceanography 5(1) 1-13 (1990b). [27] Martin, J. and Fitzwater, S. Iron deficiency limits phytoplankton growth in the northeast Pacific subarctic. Nature (1988). [28] Martin, J., Fitzwater, S., Gordon, R. Iron Deficiency limits phytoplankton growth in antarctic waters. Global Biogeochemical Cycles 4 (1) 5-12 (1990a). [29] Martin, J. Gordon, R., Fitzwater, S. The case for iron. Limnology and Oceanography 36(8) (1991). [30] Martin, J., Gordon, R., Fitzwater, S., Broenhow, W. VERTEX: Phytoplankton/iron studies in the Gulf of Alaska. Deep Sea Research (1989). [31] Martin, J. et al. The Iron Hypothesis: Ecosystem Tests in Equatorial Pacific Waters. In Press (1994). [32] Miller, C., Frost, B., Booth, B., Wheeler, P., Landry, M., Welschmeyer, N. Ecological Processes in the Subarctic Pacific: Iron Limitation cannot be the whole story. Oceanography (1991). [33] Millero, F. and Sotolongo, S. The oxidation of Fe(II) with H 2 O 2 in seawater. Geochim. Cosmochim. Acta 53, (1989). [34] Morel, F., Hudson, R., Price, N. Limitation of productivity by trace metals in the sea. Limnology and Oceanography 36(8) (1991). 42