iron chemistry in seawater and its relationship to ...kbruland/manuscripts/wells/...iron input...

Marine Chemistry 48 (1995) 157-182

Iron chemistry in seawater and its relationship to phytoplankton: a workshop report

Mark L. Wells”, Neil M. Priceb, Kenneth W. Bruland”

ahWute of Marine Sciences, University of California, Santa Cruz, Santa Cruz, CA 95064, UsA bBiology Department, McGill University, Montreal, Canada

Received 26 July 1994; revision accepted 6 October 1994

1. Introduction

The ongoing debate about iron limitation of phytoplankton in the open ocean has highlighted how little we know about the marine chemistry of iron and its relationship to iron uptake by microorganisms. The idea that iron may be limiting phytoplankton was suggested and investigated by Gran (1931) but the topic suffered from analytical problems for almost sixty years (de Baar, 1994). The concept is now treated more seriously since the advent of trace metal clean procedures which permit the accurate measurement of dissolved and particulate iron (Bruland et al., 1979; Gordon et al., 1982; Landing and Bruland, 1987). These measurements demonstrate that in some ocean habitats dissolved iron concentrations in surface waters are as low as 20-30 pM (Martin et al., 1991), concentrations that are unlikely to support high phytoplankton biomass. The controversy over the role of iron in ocean productivity has stimu- lated the development of new methodologies for rapidly analyzing low-level iron concentrations in seawater (Elrod et al., 1991; O’Sullivan et al., 1991; Yokoi and van den Berg, 1992; Obata et al., 1993). Unfortunately, most of these novel methods provide little information about the ambient speciation of iron and hence its accessibility to phytoplankton. Critical assessment of the inter-

active relationship between iron and marine phytoplankton assemblages is hindered greatly by our current analytical resources.

Most of the iron debate centers on the remote HNLC regions of the subarctic Pacific, the equatorial Pacific and the Southern Ocean. These regions are characterized by a persistence of excess major nutrients (N, P) and low biomass relative to coastal systems having similar major nutrient concentrations. Because the root cause(s) for this situation may have important ramifications to the ocean-atmosphere exchange of CO2 and global climate cycles (Martin, 1990), a special symposium was held (February 1991) to address what controls phytoplankton production in nutrient-rich areas of the open sea (see Limnology and Oceanography, Vol. 36). John Martin and his colleagues argued that an inadequate iron supply was the major factor (Martin et al., 1991). Results from bottle enrichment studies of theirs and others support this hypothesis (Martin and Fitzwater, 1988; de Baar et al., 1990; Martin et al., 1990a, 1994; Hel- bling et al., 1991; Greene et al., 1994; Kolber et al., 1994; Price et al., 1994), though alternate inter- pretations have suggested that other factors also play an important role (Dugdale and Wilkerson, 1990; Banse, 1991; Buma et al., 1991; Mitchell et al., 1991; Nelson and Smith, 1991) [see the reviews of Cullen (1991) and de Baar (1994)]. The recent

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158 M.L. Wells et aLlMarine Chemistry 48 (1995) 157-182

mesoscale iron fertilization experiment south of the Galapagos Islands demonstrated that enhanced iron input indeed increases phytoplankton growth rates and biomass (Kolber et al., 1994; Martin et al., 1994). Even so, the ecosystem response to the transient iron addition was far less dramatic than seen in bottle experiments, which illustrates our incomplete understanding of iron:plankton interactions in HNLC systems.

But iron also may play an important role in the other oceanic domains, namely the subtropical

gyres, continental shelves, coastal upwelling zones, and the transitional waters separating these regions. For example, colonies of the oceanic nitrogen-fixing cyanobacterium Trichodesmium observed in sub-tropical gyres may “capture” and extract iron from freshly deposited mineral aerosols, thus contributing “new” iron and nitrogen to the ecosystem (Rueter, 1988, abstract). In contrast, shelf and coastal regions have much higher iron concentrations and supply rates but phytoplankton iron requirements also are higher and differ greatly among species (Brand et al., 1983; Murphy et al., 1984). Iron may thus exert selective control over species dominance within algal assemblages, thereby regulating the food web structure of these ecosystems (e.g. Hansen et al., 1994). Clearly, the relationship between iron and phytoplankton may have significance beyond its role in HNLC regions.

2. Workshop summary

Improving our understanding of the relationship between iron and phytoplankton in the oceans has been hampered by the complexity of the biological and chemical aspects of the problem. There was a strong need to identify what currently is known with certainty about iron speciation in seawater, the metabolic requirements of phytoplankton for iron, the various strategies phytoplankton employ to obtain iron, and how they adapt to low iron conditions. This critical assessment could then serve as a springboard for focusing efforts on the research issues of greatest importance.

A major challenge facing biological and chemical oceanographers is to develop techniques to quantify those fractions of iron that are

accessible to phytoplankton and how this accessibility influences marine ecosystems. Traditionally, however, there has been little direct interaction among theoretical marine chemists, analytical marine chemists and algal physiologists and eco- logists working on this problem. To overcome this obstacle, we (Mark Wells and Ken Bruland) were funded by NSF, in cooperation with ONR, NASA, NOAA, and DOE, to organize a workshop (May I-5, 1994) composed of such individuals (see Table 1) to address these central issues. This report is a summation of the workshop findings.

The workshop did not address the role of iron in limiting regional or global scale primary production. The intent instead was to facilitate cross- disciplinary interaction to identify productive research avenues for developing the tools needed to better examine these and other questions about the marine biogeochemistry of iron.

To help establish a conceptual framework of iron biogeochemistry, each participant prepared an extended abstract of their newest research and viewpoints. The titles of these abstracts are listed in Appendix 1; full abstracts are available by contact- ing the Institute of Marine Sciences, University of California, Santa Cruz. After two days of individual presentations, the participants split into working groups to identify important unresolved problems which could produce significant advances in understanding the marine biogeochemistry of iron. Afterwards, the working group findings were discussed in a series of plenary sessions. This report is derived largely from the working notes of these group discussions and thus is a summed reflection of the ideas and insights of many individuals. In addition to this summary and the available abstracts, a series of articles based on presentations at the workshop will appear shortly in a special issue of Marine Chemistry.

2.1. Dejining iron availability

A key aim of the workshop was to better define biological availability of iron in seawater to help focus the development of analytical tools to quantify this fraction. However, it became apparent during the course of discussions that

M.L. Wells et al.lkfarine Chemistry 48 (1995) 157-182 159

Table 1 List of participants

Anderson, Donald, M. - Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA Barber, Richard - Duke University, Marine Laboratory, Beaufort, NC 28516, USA Brand, Larry, E. - Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami,

FL 33149, USA Bruland, Kenneth, W. - Institute of Marine Sciences, University of California, Santa Cruz, Santa Cruz, CA 95064, USA Butler, Alison - Department of Chemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA Coale, Kenneth, H. - Moss Landing Marine Laboratories, P.O. Box 450, Landing, CA 95039-0450, USA Crumbliss, Alvin, L. - Chemistry Department, P.M. Gross Chemistry Laboratory, Duke University, Durham, Box 90346, NC 27708-

0346, USA de Baar, Hein, J.W. - Netherlands Institute for Sea Research (NIOZ), P.O. Box 59, 1790 AB Den Burg, Texel, The Netherlands Ditullio, Giacome, R. - Ecology Department, University of Tennessee, 108 Hoslins, Knoxville, TN 37996-l 191, USA Geider, Richard, J. - Cannon Laboratory, University of Delaware, Lewes, DE 19958-1298, USA Gledhill, Martha - Department of Oceanography, University of Liverpool, Liverpool, Great Britain L69 3BX Goldberg, Edward, D. - Marine Research Division, 0220, Scripps Institution of Oceanography, La Jolla, CA 92093-0220, USA Greene, Richard, M. - Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA Haygood, Margo, G. - Marine Biology Division, 0202, Scripps Institution of Oceanography, La Jolla, CA 92093-0202, USA Hudson, Robert, J. M - Institute of Marine Sciences, University of California, Santa Cruz, Santa Crux, CA 95064, USA Hutchins, David, A. - Marine Sciences Research Center, State University of New York, Stony Brook, NY 11794, USA Johnson, Kenneth, S. - Moss Landing Marine Laboratories, P.O. Box 450, Moss Landing, CA 95039-0450, USA Kester, Dana, R. - University of Rhode Island, Graduate School of Oceanography, Narragansett, RI 02882, USA King D., Whitney - Colby College, Department of Chemistry, Waterville, ME 04901, USA Landing, William, M. - Department of Oceanography, Florida State University, Tallahassee, FL 32306, USA Luther, George, W. - College of Marine Science, University of Delaware, Lewes, DE 19958, USA Matsunaga, Katsuhiko - Department of Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate 041, Japan Measures, Chris, I. - Department of Oceanography, University of Hawaii, 1000 Pope Rd., Honolulu, HI 96822, USA Millero, Frank, J. - University of Miami, Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami,

FL 28516, USA Morel, Fragois, F.M.M. - Ralph M. Parsons Laboratory, Massachusetts Institute of Technology, Building 48-423, Cambridge, MA

02139, USA Price, Neil, M. - Biology Department, McGill University, Montreal, Canada Rue, Eden, L. - Department of Chemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA Rueter, John - Department of Biology, Portland State University, P.O. Box 751, Portland, OR 97207, USA Sulzberger, Barbara - Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), Dtibendorf, Switzerland Sunda, William, G. - National Marine Fisheries Service, Southeast Fisheries Center, Beaufort Laboratory, Beaufort, NC 28516, USA Takeda, Shigenobu - Central Research Institute of Electric Power Industry, Biology Department, Abiko Research Laboratory, 1646,

Abiko, Abiko-city, Chiba, 270- 11, Japan Trick, Charles, G. - University of Western Ontario, Department of Plant Sciences, London, Ont., Canada van den Berg, Constant, M.G. - Department of Oceanography, University of Liverpool, Liverpool, Great Britain L69 3BX Voelker, Bettina, M. - Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), CH-8600, Diibendorf,

Switzerland Waite, T., David - Department of Water Engineering, University of New South Wales, Sydney, NSW 2052, Australia Wells, Mark, L. - Institute of Marine Sciences, University of California, Santa Cruz, Santa Cruz, CA 95064, USA Zhuang, Guoshun - Environmental Sciences Program, Univ. of Massachusetts at Boston, 100 Morrissey Blvd., Boston, MA 02125,

USA

this task presently is largely beyond our capability. The perspective that developed during the workshop is that iron availability will be a function of: (1) the various chemical forms of iron in seawater, (2) the preference of the uptake mechanism of each organism for one or another of these forms, and (3) the balance between the reaction kinetics of iron

exchange among chemical species, the iron uptake kinetics of a given organism, and the iron demand of each member population within the phytoplankton assemblage. Clearly, the complexity of the natural seawater system frustrates attempts to define a general “biological availability” of iron based on a singular analytical measurement of


Biological Component

l Concentrations of Fe species

l Mechanisms and kinetics of exchange among species

l Thermodynamic and kinetic modelllng

l Ecosystem structure l Fe and C exports l External and internal Fe

inputs

Chemical Component EcologicaVBiogeochemical Component

Fig. 1. Conceptual diagram of the interaction among the biological, chemical and ecological/biogeochemical components of the marine biogeochemistry of iron.

“reactive” metal. Moreover, our present ignorance of many aspects of iron chemistry in seawater makes it unlikely that we can accomplish this task within the near future.

2.2. Conceptual diagram

A measure of our limited understanding of the inter-relationship between iron chemistry and the biota was the difficulty participants experienced in formulating a simplified conceptual framework within which to identify the most pressing questions. Eventually, a conceptual diagram comprising three main components was chosen: (1) a biological component encompassing the biochemical and physiological aspects of single-celled organisms, (2) a chemistry component comprising various forms and chemical species of iron in seawater along with the abiotic processes regulating the interchange among them, and (3) an ecological/biogeochemical component reflecting both ecosystem structure and transfer of matter into and out of the upper ocean (Fig. 1).

The considerable overlap among these com-

ponents is illustrated by the processes interlinking them. It is expected that biology will influence iron speciation by releasing organic molecules that chelate or complex iron, by mediating redox transformations at the cell surface, and by selectively taking up various iron species. The resultant “availability” of iron can influence the size structure and species composition of the algal assemblage and hence the community food web. The ecology of the system, in turn, will control both the form and magnitude of regenerated iron in surface waters as well as influence the cycling and removal of other elements.

A vital but external component to this conceptual framework is the input of energy as light. Variations in the intensity and spectral qualities of light not only influence photosynthetic rates, and hence the overall biological demand for iron, but also will modify the solution, colloidal, and particulate chemistry of iron via various photochemical transformations.

Of the many questions surrounding this simplified conceptual model, five emerged as the most pressing during the workshop discussions:

M.L. Wells et al./Marine Chemistry 48 (1995) 157-182 161

(1) What are the sources and sinks of new and regenerated soluble iron in the upper ocean?

(2) What is the chemical speciation of iron among the soluble, colloidal and particulate fractions, including the rates and mechanisms of transformations among these forms?

(3) What are the inter-relationships between the chemical speciation of iron and the microbial community (e.g. how the chemical speciation influences iron uptake by the biotic community and how the community influences iron speciation)?

(4) How do microbes adapt biochemically and physiologically to variations in iron availability in the ocean?

(5) What ecological and biogeochemical implications arise from temporally and spatially changing iron supply and availability in the oceans?

Participants sought to provide guidance for answering these broad questions by dissecting each into a set of more specific questions, some of which already have answers and others useful for posing testable hypotheses. Wherever possible, recommendations were made regarding specific methods or new analytical approaches that would be promising tools for attacking these problems.

(1) What are the sources and sinks of new and regenerated soluble iron in the upper ocean?

Iron is a particle reactive trace metal existing at extremely low concentrations in the oceans. Its nutrient-type characteristics are inferred from vertical profiles [low surface values, higher deepwater values (e.g. Martin and Gordon, 1988)] and from rapid biological recycling (Hutchins et al., 1993, abstract). Iron also exhibits particle-sorption (scavenging) characteristics (decreasing concentrations with increasing age of deepwaters) with relatively short residence times both in surface waters (weeks, months, to a year depending upon the regime) and in the oceans as a whole (less than a century). Much of the iron remineralized in the intermediate and deep waters is scavenged and removed to the sediments and is not mixed back

into the surface waters, leading to a continuous depletion of iron relative to the major nutrients nitrate and phosphate. Thus, it is particularly relevant to examine new external sources of iron.

A. What are the sources of iron?

Sources of dissolved iron to the photic zone include both “new” sources from external inputs as well as “regenerated” sources recycled in-situ from various particulate phases. These sources include:

. wet and dry deposition of atmospheric aerosols,

- vertical mixing and upwelling, . inputs from rivers and bottom sediments, and . biogenic recycling of cellular iron in surface

waters

In remote oceanic regions, wet and dry deposition of aerosols is an important external source of new iron. The aerosol iron deposited is a mixed assemblage of terrestrially weathered particles, solid forms reworked by photochemical/ reprecipitation processes, and dissolved metal species. The delivery of wet and dry deposition to the surface ocean varies markedly both spatially and temporally (Moore et al., 1984; Duce, 1986). A consensus of the workshop was that we need a better understanding of the precise chemical nature of deposited iron and the spatial and temporal variability of the atmospheric source. In addition, the various processes that act to solubilize iron from the mineral aerosols, both in the atmosphere and within the surface ocean, need to be quantified (see belowj.

Another important source of iron to surface waters is vertical mixing and upwelling, which also varies markedly on spatial and temporal scales.

Atmospheric deposition of iron generally has been considered to be the dominant source of iron to surface waters of the open ocean. For example, the subarctic Pacific lies in the path of an extended aerosol plume originating in China (Young and group, 1991). Martin et al. (1989) estimated that atmospheric deposition accounted for 84-93% of the external iron input to these


surface waters. A similar predominance of atmospheric iron input occurs in the major subtropical gyres, where a high degree of stratification strongly limits vertical mixing of iron from below the thermocline. However, in the HNLC regions of the equatorial Pacific and Southern Ocean, iron input from upwelling and vertical mixing over- whelms the meager aerosol deposition in these remote areas (Bruland, this meeting; de Baar, abstract).

In coastal and shelf waters, substantial external inputs of iron come from riverine sources and bottom sediments, leading to markedly higher dissolved and particulate iron concentrations. Much of the particulate iron in nearshore waters is inorganic and processes that solubilize this reservoir, making it accessible to phytoplankton, are especially relevant (see below). The greater iron inputs to coastal and shelf regions compared to the open ocean are accompanied by high iron requirements of neritic phytoplankton species (Brand et al., 1983; Sunda et al., 1991). These systems therefore might be strongly influenced as the comparatively rich iron resource is diminished by phytoplankton blooms.

The single largest reservoir of iron in the surface waters of HNLC regions may be the biota itself. New evidence indicates that this biological pool of iron is recycled on the time scale of days, much like N and P (Hutchins et al., 1993). This “input” of regenerated iron to surface waters is estimated to be more than an order of magnitude greater than the external supply rate of iron (Bruland, this meeting; Morel, this meeting) and may largely satisfy the iron-demand of phytoplankton in these systems. This view is supported by recent results of Price et al. (1994) showing that iron uptake rates of plankton in the equatorial Pacific are sufficient to entirely turn over the dissolved iron pool within half a day or less. Presently, there is no indication of the chemical forms of this regenerated iron or whether these forms are directly reassimilated by phytoplankton.

B. What are the sinks of iron?

The removal of iron from surface waters is fairly

well constrained within a geochemical (i.e. mean residence time) perspective, however, the mechanisms and dynamics of this removal is not well understood. Mechanisms for removing iron from surface waters include:

* sorption and precipitation, 0 biological assimilation, - aggregation of inorganic or organic colloids,

and . sinking of mineral and biogenic particles.

While much of the particulate iron introduced via rivers, sediment resuspension, or as mineral aerosols will be removed by settling, ascertaining the underlying basis for the removal of “dissolved” iron forms is much more difficult. In regions with a high sinking flux of inorganic mineral particles (e.g. in some coastal and well mixed shelf waters), dissolved iron may be removed abiotically by sorption to surfaces of these particles. Similarly, sinking organic particles also can scavenge soluble iron from surface waters (Morel and Hudson, 1985). Dissolved iron also is “removed” via direct assimilation by phytoplankton. The subsequent sinking of live cells or fecal matter will transport a portion of this biogenic iron from surface waters.

In addition to direct assimilation and sorption onto sinking (mineral and biogenic) particles, iron may sorb to colloidal organic matter which is abundant in surface waters (Wells and Goldberg, 1992, 1994). The stability of this colloidal phase is a topic of much dispute (Honeyman and Santschi, 1989; Bauer et al., 1992; Moran and Buesseler, 1992; Wells and Goldberg, 1993), but the extremely large colloidal surface area combined with the particle reactive nature of iron suggests that aggregation of organic colloids could be important for removing iron.

Significant unresolved issues regarding iron removal include (1) identifying the specific mechanisms of iron sorption to abiotic and biotic sinking particles, which will shed light on how changes in iron speciation may affect this removal pathway, (2) changes in the iron “export efficiency” of the assimilation pathway with shifts in primary productivity or species assemblage, and (3) the abundance and reactivity of iron in the colloidal reservoir. The relative importance of abiotic, bio-

M.L. Wells et al.iMarine Chemistry 48 (199.5) 157-182 163

tic and colloid aggregation removal processes will vary from regime to regime and with season.

(2) What are the chemical speciation and forms of iron among the soluble, colloidal and particulate fractions, including the rates and mechanisms of transformations among these forms?

There are some large gaps in our knowledge of iron chemistry in seawater. The development of trace metal clean techniques for seawater col- lection and analysis over the past decade (Bruland et al., 1979; Gordon et al., 1982; Landing and Bruland, 1987) has given us reliable profiles for iron in the traditional categories of particulate (> 0.4 pm) and “dissolved” (< 0.4 pm) fractions; however, the chemical forms of iron within these fractions has been largely speculative. For example, there now is evidence that iron exists, at least partially, in the small colloidal phase (Wells and Goldberg, 1991; Wu and Luther, 1994; Powell and Landing, abstract) which is included in operationally defined “dissolved” fractions. Deter- mining how iron is partitioned among these phases, and among various chemical forms within each phase, is central to understanding iron speciation in seawater.

A. Soluble iron

What is the solubility and speciation of Fe(III) in seawater?

The major issues that remain unresolved include:

- the disputed existence of the soluble Fe(OH)i species,

. the degree of organic complexation of Fe(III), and

. the source, supply, and chemical nature of Fe(III)-binding organic ligands.

It is an embarrassing observation that despite decades of studies on the marine chemistry of iron, and despite increasing evidence that iron may play a singularly significant role in ocean ecosystems, there is no clear consensus among marine chemists on the thermodynamic solubility of inorganic Fe(II1) in oxic seawater. Solubility estimates

for Fe(II1)’ (the sum of dissolved inorganic species) vary from N 10e8 M to N lo-” M depending on the disputed presence of the dissolved Fe(OH)i species (Byrne and Kester, 1976; Morel, 1983). Experimental evidence from filtration studies suggest that solubility lies near the upper end of this range, but these findings are equivocal because small colloidal forms of iron may pass through even small pore-size filters. It is crucial that we ascertain the upper limit of iron solubility in seawater because natural “dissolved” concentrations of iron span the disputed solubility range, i.e. colloidal iron precipitates are expected or not depending on the solubility estimate chosen. Better quantification of the thermodynamic formation constants of the Fe(OH)i3-‘) species as a function of ionic strength, pH, temperature and pressure would bring us closer to this goal.

But the relationship between iron chemistry and its uptake by phytoplankton is more than just a question of solubility; the coordination exchange kinetics of the various species exert a primary control over the rate of iron uptake (Morel et al., 199 1, Crumbliss, abstract; Hudson and Bruland, abstract). For example, while iron uptake generally correlates with “free metal ion” activity in well defined, chelate buffered media of laboratory studies, this correlation results from the rapid inter- equilibration of Fe3+ with the more abundant and kinetically labile hydrolysis species and the constant proportionality between concentrations of these species, Fe(II1)’ and Fe3+, in constant pH seawater (Hudson et al., 1992). In natural waters, the concentration of Fe(II1)’ is the relevant factor to consider with respect to the uptake of inorganic iron.

One of the most intriguing new aspects of iron chemistry presented at the workshop were very recent data indicating that up to 99.9% of the dissolved iron in surface waters is bound within organic complexes in both nearshore and open ocean environments (Gledhill and van den Berg, 1994; Rue and Bruland, submitted; van den Berg, submitted; Wu and Luther, submitted), resulting in extremely low (subpicomolar) concentrations of dissolved Fe(II1)‘. A similar degree of complexation has been shown for both Cu and Zn in surface waters, though complexation of Cu and Zn

164 M.L. Wells et al./Marine Chemistry 48 (199.5) 157-182

decreases significantly in deep waters (Coale and Bruland, 1988; Bruland, 1989) while preliminary results indicate iron complexation remains high (Rue and Bruland, submitted; van den Berg, submitted). Both the high stability of this ligand class for iron, and its significant concentration in seawater appear to be remarkably consistent in the North Pacific, North Sea and western Mediterra- nean. The source of these ligands is unknown, though preliminary results suggest that they may originate from phytoplankton (van den Berg, submitted). Strong iron-binding proteins also have been isolated and identified from the marine bivalve Mytilis edulis (Taylor et al., 1994), though the extent that these compounds are released to coastal waters is unknown.

It is crucial that preliminary measurements of strong Fe(II1) complexing organic ligands in seawater be verified. If these ligands indeed are in ubiquitous excess over dissolved iron concentrations in the oceans, it will profoundly alter our perception of iron speciation in seawater and raise more questions than provide answers. What is the composition and source of this ligand class? Do they exist within the soluble or colloidal organic phase? Do these organic ligands serve to “buffer” dissolved iron concentrations and thereby limit its rapid removal from seawater (Rue and Bruland, submitted)? Most importantly, are they an assemblage of siderophores released to facilitate iron uptake (see below) or do they comprise general cell exudation and degradation products? If a major fraction of these organic ligands are siderophores, then iron speciation in seawater would reflect a vigorous com- petition among various autotrophic and heterotrophic microorganisms for control of a scarce resource.

Insight into the potential role of these newly found organic ligands will come from better characterization of their sources, sinks (algal uptake, microbial degradation, photolysis, colloid aggregation), residence times, and their complexing characteristics with iron and other metals. Competitive ligand exchange voltammetry techniques combined with cell culture studies offer one the more promising directions for future research.

What factors injkence the redox state of iron in

seawater?

Though Fe(II1) is the thermodynamically stable oxidation state of iron in oxic seawater, a substantial body of evidence now suggests that a significant fraction of dissolved iron in surface waters exists as Fe(I1). Inorganic Fe(I1) oxidation pathways are well described for inorganic seawater and are rapid (Miller0 and Sotolongo, 1989) but considerably less is known about rates of Fe(II1) reduction. Some reductive processes responsible for maintaining measurable Fe(I1) concentrations in oxic surface waters may include:

- photochemical reduction, - thermal reduction (i.e. by organics), . enzymatic reduction, - reduction within microenvironments, and - retardation of Fe(I1) oxidation rates by for-

mation of Fe(I1) organic complexes.

Photoreduction of Fe(III) The deposition of photochemically altered aero-

sols may be a significant source of Fe(I1) to surface waters. Both direct and indirect photoprocesses can potentially transform iron redox states during aerosol transport. For example, soluble Fe(I1) has been measured in fog and cloud aerosols (Behra and Sigg, 1990; Erel et al., 1993; Zhu et al., 1993) and in aerosols from remote ocean regions (Zhuang et al., 1992; Zhu et al., 1993). In addition, the photoreduction of soluble and synthetic colloidal Fe(II1) has been measured in seawater under laboratory and field conditions (Waite and Morel, 1984a; Rich and Morel, 1990; Wells et al., 1991a; Waite and Szymczak, 1993; Miller and Kester, 1994; Zhang et al., submitted; Kester, abstract; King, abstract; Gledhill and van den Berg, abstract) and diurnal variations in surface water concentrations of Fe(I1) and reactive Fe(II1) species have been observed (Hong and Kester, 1986; O’Sullivan et al., 1991; Johnson et al., 1994), though at somewhat higher iron concentrations than expected for open ocean environments.

While photoreduction of Fe(II1) inorganic ligand complexes occurs readily at short UV wave- lengths (King et al., 1993, abstract; Miller et al.,

M.L. Wells et al.lMarine Chemistry 48 (1995) 157-182 165

submitted), natural organic chromophores can greatly enhance iron reduction under earth-surface light regimes (Erel et al., 1993; Waite and Morel, 1984b; Wells et al., 1991a). Reduction of Fe(III)-organic ligand complexes may occur by direct ligand-to-metal charge transfer or from reaction with secondary photolysis products. Work- shop participants heard the first results from laboratory studies showing that the photo- produced superoxide radical (02) rapidly reduces soluble Fe(III)-inorganic ligand complexes (Voelker and Sedlak, submitted) but apparently does not react significantly with colloidal iron oxyhydroxides (Voelker, this meeting). In HNLC waters, where iron concentrations are below solubility estimates, reduction by superoxide was argued to maintain up to 30-75% of dissolved iron as Fe(I1). However, complexation of Fe(II1) by natural organic ligands may sharply curtail, though not eliminate, iron reduction by 0,.

There is a pressing need to identify and characterize: (1) the nature and source of natural Fe(II1) reducing chromophores, (2) the relative importance of direct and indirect photoprocesses to iron chemistry in surface waters, and (3) the spectral dependence and kinetics of these reactions at ambient concentrations of iron and chromophores.

Thermal reduction of Fe(III) Several organic ligands can reduce Fe(II1) upon

complexation (e.g. tannic acid) and it is plausible that similar redox reactions might occur in seawater. Though thermal reduction of iron by organic ligands in surface seawater is thought to be of much less consequence than direct or indirect photolysis, the significance of this reduction pathway below the upper photic zone is presently unknown.

Enzymatic reduction of Fe(III) Fe(II1) reduction also may be enzymatically-

mediated either by plasmalemma (membrane bound) redox proteins or by intracellular enzymes secreted as a result of cell damage. Both mechanisms may enhance iron acquisition by phytoplankton and yet have distinct implications for biological effects on iron speciation. On one hand, release of intracellular enzymes should be linked to grazing

activity and thus correlated positively with phytoplankton growth rates [i.e. extracellular Fe(II1) reduction rates would increase as cell growth rates and grazing rates increase]. Con- versely, if reduction at the cell surface by electron pumping is important, Fe(II1) reduction rates should be an inverse function of growth rates. Cells continue to produce reducing power (as NADPH) by photosynthesis even when nutrient limitation inhibits cell division, and this non- utilized reducing capacity may be eliminated from the cell by pumping electrons across the membrane (i.e. via plasmalemma redox proteins). Rates of extracellular Fe(II1) reduction increase as iron- limited growth rates decrease in Thalassiosira oceanica, suggesting that the reductant pumping mechanism may be related to iron-deficiency (Maldonado and Price, unpubl. data).

Fe (III) reduction in microenvironments Reducing microenvironments are more common

in oxic waters than is generally recognized (All- dredge and Cohen, 1987). Detrital aggregates (“marine snow”), diatom mats, the feeding struc- tures of larvaceans, fecal pellets and Trichodes- mium colonies all can harbor sub-oxic internal environments which could facilitate Fe(I1) production. One acidic, sub-oxic environment often not considered is the digestive systems of planktonic grazers. Protozoan feeding vacuoles may drop to pH N 3 and copepod guts can reach pH N 5 during the initial stages of digestion. Because cell turnover rates in open ocean and nearshore waters are on the order of days or less, grazers could have a major effect on iron speciation.

An additional reducing microenvironment that needs to be explored is that within organic colloidal matrices (Goldberg, abstract). There is preliminary evidence that marine colloidal organic matter can harbor thermodynamically unstable species such as iodide and arsenite, suggesting that higher oxidation states of elements are reduced within the electron rich colloids (Gold- berg, abstract).

Fe(II) oxidation in seawater The oxidation of inorganic Fe(I1) species in sur-

face seawater is rapid and well quantified, with a

166 M.L. Wells et al./kfarine Chemistry 48 (1995) 157-182

reaction half-time of a few minutes. The dominant inorganic oxidation pathway for Fe(I1)’ in surface seawater is by reaction with photo-produced H202 (Moffet and Zika, 1987) which is N 4-40 times faster than oxidation by O2 (Miller0 and Sotolongo, 1989). In deep waters, where H202 concentrations are much lower, oxidation of Fe(I1)’ is dominated by reactions with Oz. Because H202 forms in surface waters from disproportionation of photochemically-generated superoxide radical (02) it is expected that sunlight enhances rates of iron reduction and oxidation leading to rapid photoredox cycling in surface seawater. Such photoredox cycling should enhance ambient concentrations of kinetically labile inorganic species of Fe(I1)’ and Fe(II1)’ (Morel et al., 1991; Wells et al., 1991a; Sunda, 1989; Kester, abstract) and thereby increase cellular iron uptake by membrane transport ligands (see below).

Though the inorganic speciation of Fe(I1)’ in seawater is better characterized than that for Fe(III)‘, recent measurements of significant concentrations of Fe(I1) in surface seawater implies that it may be considerably more stable than predicted. It is possible that Fe(I1) species are stabi- lized by complexation with organic ligands that impede oxidation (Kester, abstract; Gledhill and van den Berg, submitted). The apparent stability of Fe(I1) in surface waters is of particular importance because Fe(I1)’ species are substantially more soluble in seawater than Fe(II1)’ and are kinetically more labile. Thus, a well buffered Fe(I1)’ system might increase iron availability markedly. One meth- odological avenue which shows much promise for elucidating Fe(I1) speciation in seawater is competitive equilibrium/adsorptive cathodic stripping voltammetry using Fe(I1) selective added ligands.

B. Colloidal iron

The marine colloidal phase may serve as a reservoir of iron in seawater, though its significance within the different oceanographic domains has yet to be established. The central issues needing attention are:

- the abundance and distribution of colloidal iron in ocean waters,

. the forms of iron occurring in the colloidal phase,

- rates of exchange between colloidal iron and soluble species,

- photochemical reactivity of colloidal iron, and - the dynamics of colloid behavior.

Without precise knowledge about the inorganic solubility of Fe(III)‘, the degree of organic complexation of soluble Fe(III), and the fraction of total “dissolved” iron existing as Fe(I1) species, it is unclear where and when iron is likely to precipi- tate in seawater. While colloidal oxyhydroxides occur in nearshore surface waters (Wells and Gold- berg, 1992) and in hydrothermal plumes (Feely et al., 1990; Cowen, 1992) most colloidal matter in seawater has a largely organic matrix (Koike et al., 1989; Longhurst et al., 1992; Wells and Goldberg, 1992; Wells and Goldberg, 1994). Pure forms of colloidal iron oxyhydroxides, such as those typically used in experimental studies, are rare in surface seawaters (Wells and Goldberg, 1994). Even so, preliminary results indicate that 20-40% of the filterable iron in surface waters of the North Atlantic and Greenland Sea occurs in the colloidal phase (Wu and Luther, 1994; Powell and Landing, abstract), presumably associated with colloidal organic matter.

There is a need to better characterize the oceanographic distributions of marine colloids and the concentrations of iron within this phase. Ultra- filtration studies with systems specially modified to minimize contamination would be a good step towards this goal. Verification of preliminary findings showing iron associated with marine colloidal matter must be followed by determination of the chemical nature of this association. Are inorganic precipitates of iron agglomerated into the organic matrix, or do soluble Fe(II1)’ hydrolysis species sorb or become complexed at the organic colloid surface? Do soluble organic-Fe(II1) complexes become associated with colloidal surfaces, or partition into the colloid matrix? Exploring the mechanisms for iron-colloid association will require development of new clever methods, however, these results will be crucial for predicting the subsequent reactivity of natural “colloidal” iron with respect

M.L. Wells et aLlMarine Chemistry 48 (1995) 157-182 161

to solution phase chemical processes.

C. Particulate iron

thermochemical and photo-

Particulate iron is generally ignored when con- templating iron “availability”. Though some phytoplankton are known to eat bacteria (Bird and Kalff, 1986) there is presently no evidence that phytoplankton directly engulf inorganic particles. Nevertheless, as noted above particulate substances may become accessible by facilitated dissolution in specialized microenvironments (e.g. within Trichodesmium colonies). But even outside these microenvironments, particles may rapidly equilibrate thermochemically with solution species to provide a ready source of iron to phytoplankton (Wells et al., 1983; Rich and Morel, 1990). For example, measurements of labile iron in coastal and shelf seawaters by reaction with a chelating agent showed the particulate fraction to contain the majority of easily exchangeable iron in these waters (Wells and Mayer, 1991b). The source, distribution, and input rates of particulate iron may therefore have important consequences for the supply of iron to phytoplankton.

Unfortunately, while reliable data now exist for particulate iron distributions in seawater, very few data exist on the specific composition and chemical reactivity of this size fraction. For example, though Fe/Al ratios in atmospheric dusts often are similar to average crustal values calculated from silicate mineral abundance, more iron occurs as oxyhydroxides in these mineral aerosols than within the parent (crustal) silicates due to weathering (Murad and Fischer, 1988; Goldberg, this meeting). Moreover, spatially distinct terrestrial weathering patterns, combined with apparently extensive photochemical and acidic reprocessing of mineral aerosol particles (Behra and Sigg, 1990; Zhuang et al., 1992; Erel et al., 1993; Zhu et al., 1993) makes it likely that the chemistry of these solid phases varies as a function of source point and atmospheric residence time. Better characterization of the chemistry of particulate inputs is essential to understanding the interaction between particulate and soluble iron forms in seawater.

(3) What are the inter-relationships between the chemical speciation of iron and the microbial community?

As discussed previously, preliminary evidence suggests that a major fraction of soluble iron is chelated with organic ligands. The microbial community may therefore play a key role in controlling the speciation and redox state of iron in seawater. But in addition to indirect effects arising from the inadvertent release of cell metabolites and cell surface reactions, microorganisms may actively attempt to regulate iron speciation to facilitate its acquisition. The form such manipulation might take will depend on the various iron uptake strategies employed by marine microorganisms.

Strategies for iron acquisition; how do these AifSer among organisms and across environments?

Phytoplankton may utilize a variety of strategies for extracting iron from the surrounding environments, including:

. uptake of Fe’ by membrane bound porter sites, - uptake of Fe-siderophore chelates, - extracellular reduction, * excess (or luxury) uptake and storage, and . solid-phase Fe acquisition.

Oceanic microorganisms have evolved at least two different mechanisms for acquiring iron from seawater at extremely low concentrations. Diatoms and other eukaryotic phototrophs are thought to primarily utilize dissolved inorganic Fe(II1)’ and Fe(I1)’ (Morel et al., 1991) by a transport ligand process involving cell surface complexation and internalization. This conceptual model is founded on a very limited study of 4 species and thus may not be applicable to all organisms. In this model, dissolved labile inorganic Fe(I1) and Fe(II1) species diffuse to the cell surface where they undergo ligand exchange reactions with membrane porter sites whose nature is unknown (Hudson and Morel, 1990, 1993). The iron is then transported into the cell. If high levels of iron chelation (Gledhill and van den Berg, 1994; Rue and Bruland, submitted; van den Berg, submitted; Wu and Luther, submitted) are characteristic of all ocean surface waters, iron uptake strategies based


solely on Fe(II1)’ acquisition may be inefficient because Fe(II1)’ concentrations (- 0.1 PM) would be too low for diffusion to support the typical growth rates of many oceanic phytoplankton (Sunda and Huntsman, submitted).

But strong organic complexation of iron in seawater and Fe’-based uptake strategies are not necessarily exclusive. Cell surface-mediated reduction of these organic-iron complexes by reduced biochemicals such as thiols or plasmalemma redox proteins (Price and Maldonado, abstract) may convert them to transportable labile Fe(I1)’ and Fe(II1)’ species. In addition, there now is ample evidence for a dynamic iron redox cycling in seawater (Waite and Morel, 1984b; O’Sullivan et al., 1991; Wells et al., 1991a; King et al., 1993; Waite and Szymczak, 1993; Miller and Kester, 1994; Miller et al., submitted; King et al., abstract). Organisms may promote this process by releasing extracellular organic compounds which thermally or photochemically reduce Fe(II1) (Waite et al., abstract). Photochemical redox cycling enhances steady state concentrations of both Fe(I1)’ and Fe(II1)’ and thus would facilitate iron uptake via membrane-bound, Fe’- based transport systems (Sunda, 1989; Morel et al., 1991; Wells and Mayer, 1991a).

The extent of photochemical enhancement of Fe’ concentrations is currently unknown. However, a mere 5 % steady-state photoconversion of organically bound Fe(II1) species to Fe’ at a filterable iron concentration of 50 pM would increase Fe’ concentrations by 20-30 fold to a value of 2.5 pM (Sunda, unpubl. data). An Fe(II1)’ concentration of 2.5 pM would be sufficient to support specific growth rates of 0.5-0.7 day-’ which are typical for oceanic phytoplankton (Sunda and Huntsman, in prep.). Thus, photoredox cycling may well buffer Fe’ at sufficiently high levels to support observed algal growth rates, even if high levels of Fe(II1) chelation greatly diminishes Fe’ in the dark under equilibrium conditions.

Redox cycling notwithstanding, preliminary evidence suggests that a substantial portion of tilter- able (i.e. “dissolved”) iron is inaccessible to phytoplankton in equatorial Pacific waters. Using a novel approach, Fe’ in this surface seawater was manipulated by titration with a terrestrial fungal

siderophore (Wells et al., 1994). The rapidly formed siderophore-Fe complex was unavailable to the marine microorganisms and thus could be used to test the availability of ambient iron species. Less than 40% of the N 25 pM filterable iron concentration appeared to be available to increase picoplankton biomass, suggesting that Fe’ species were not the dominant form of iron in these waters, and that the remaining forms were resistant against conversion to biologically accessible species.

Production of siderophores represents a second strategy for microbial iron acquisition and is common in soil and enteric habitats subject to iron deficiency (Neilands, 1974; Winkelmann et al., 1987; Matzanke et al., 1989; Crumbliss, 1991). In this mechanism, microbes produce strong Fe(II1) chelating ligands, primarily with multiple catechol and hydroxamate functional groups, which effectively out compete other ligands for Fe(II1). These Fe-siderophore chelates are then taken up into the cell by membrane transport proteins and the iron is released internally for metabolism. Iron release involves either degradation of the siderophore, reduction of Fe(II1) to Fe(I1) or both. Following iron release, the siderophore typically is excreted to complex more iron. Permeations on this siderophore mechanism include release of the siderophore bound iron at the cell surface (often by reduction; Crumbliss, abstract; Trick, abstract) and transport of a new internal Fe-chelate.

Such ligands may play similar roles in seawater (Butler, abstract; Crumbliss, abstract; Trick, abstract). Indeed, microorganisms isolated from coastal and oceanic habitats produce siderophores in iron deficient culture media (Trick et al., 1983; Trick, 1989; Reid and Butler, 1991; Haygood et al., 1993) and aqueous extracts of coastal blue green algal mats demonstrate the presence of hydroxamate siderophores (Estep et al., 1975). In no cases, however, have soluble siderophore complexes been identified in field water samples, although little effort has been directed toward their detection. The preliminary findings of strong iron-binding ligands in the North Atlantic (Gledhill and van den Berg, 1994), North Pacific (Rue and Bruland, submitted), and Mediterranean (van den Berg, submitted) should rekindle interest in marine siderophore research.


In soils, siderophores facilitate iron uptake by the siderophore producer but limit access to iron by other organisms. However, many terrestrial microorganisms possess the capability of utilizing Fe-siderophore complexes produced by other organisms, gaining the benefits without incurring the considerable metabolic costs involved in siderophore synthesis. Interspecific utilization of Fe- siderophore complexes may entail “copying” siderophore transport sites or involve surface reductive dissociation of Fe(lII)-siderophore complexes by catechols at the cell membrane followed by uptake of the released iron (Trick, abstract). The preva- lence of such Fe-siderophore “pirates” in seawater is entirely unknown, but verification of their presence would greatly complicate attempts to quantify the availability of iron to specific members of the phytoplankton assemblage. As a further complication, terrestrial bacteria often have multiple uptake systems for iron that can operate simultaneously; marine microorganisms likely also have this capability.

Because iron input can be sporadic, the ability of microorganisms to store iron may be an important adaptive strategy. Sunda and Huntsman (in prep.) found that 5 out of 6 algal species tested had at least some capability to store excess iron. The capacity for “luxury uptake” differed among species, in accordance with the higher, more variable iron levels in neritic environments. Luxury uptake was highest for coastal diatoms (Thalussiosira pseudo- nana and T. weissjlogii), which were able to take up a 20-40 fold excess of cellular iron over levels needed for maximum growth (i.e. enough iron to fuel 5 cell divisions). The ability to store large levels of excess iron may be an important strategy for organisms which bloom episodically.

Several lines of circumstantial evidence suggest that the nitrogen-fixing cyanobacterium Trichodes- mium can help meet its high iron requirement by the adsorption and leaching of atmospheric dust particles. In the North Tropical Atlantic, Saharan dust may support the population of Trichodesmium (Rueter et al., 1990) and a Trichodesmium bloom was observed at the Hawaii ocean times series station in the North Pacific following a storm (i.e. deposition) event (Hutchins, Ditullio, Hudson, Wells, unpubl. data). A mechanism of direct con-

tact between the iron source (dust particle) and the buoyant Trichodesmium colonies would avoid the problems associated with dilute iron chemistry and loss of particulate iron from the photic zone. Based on dust flux measurements, dissolution rates and Trichodesmium productivity’s, Rueter and his colleagues (Rueter et al., 1992) estimated that half of the total iron used biologically in the North Tropi- cal Atlantic could be introduced via this route.

Characteristics of the environment may influence the strategies employed

It is likely that the above strategies by which phytoplankton may acquire iron shift in importance among different oceanographic environments, not only because of changes in the phytoplankton assemblage but also because of different external inputs and chemical speciation of iron. Ultimately, iron uptake strategies will have a pivotal role in phytoplankton/iron interactions in seawater, but this level of understanding is presently far from our grasp. A blend of laboratory and field experimentation focused on isolating and quantifying the different pathways for iron uptake by phytoplankton should provide the first steps towards this goal.

(4) How do microbes adapt biochemically and physiologically to Fe availability in the ocean?

Understanding how cells adapt to low iron conditions in seawater depends on identifying the biochemical roles for iron and which cellular processes are most strongly dependent on iron. This information then can be used to develop specific tools for evaluating the iron-related physiological state of organisms in different oceanographic regimes and over various times scales (hours to months).

Cellular processes most strongly regulated by iron Very few studies of the biochemical roles of iron

have examined marine phytoplankton (e.g. Glover, 1977; Sandmann, 1985; Rueter and Ades, 1987; Doucette and Harrison, 1991; Greene et al., 1991; Geider et al., 1993) and even fewer have involved marine heterotrophic bacteria (Haygood, abstract). Nonetheless, several candidate meta-

170 M.L. Wells et al./Marine Chemistry 48 (1995) 157-182

bolic processes are expected to be particularly catalysts be replaced by other molecules of sensitive to the iron supply to phytoplankton, similar function, and how are iron require- including: ments regulated?

. nitrogen assimilation,

. N2 fixation, - photosynthetic and respiratory electron trans-

port, - porphyrin biosynthesis, and . removal of reactive oxygen species.

Nutrient limitation affects microbial growth

(4

(4

How does the biochemistry of oceanic plankton with low Fe:C ratios differ from that of other organisms? What are the kinetics of physiological and biochemical responses to changes in iron availability?

rates by impairing essential biochemical functions. Major nutrients, such as C, N, and P, have essential roles as structural components (carbo- hydrates and proteins), energy currency (ATP), and information storage (nucleic acids). Like other micronutrients, iron plays a largely catalytic role as a cofactor of enzymes and redox proteins including those required for the assimilation of the major nutrients. Iron-containing proteins are essential for photosynthetic and respiratory electron transport, and are directly involved in nitrate and nitrite reduction, sulfate reduction, N2 fixation, chlorophyll synthesis, and a number of other biosynthetic and degradative reactions, including those involved in detoxification of O2 radicals (Geider and La Roche, 1994, abstract). Because of the multiplicity of functions of iron, many cellular processes are likely to be affected simultaneously by iron-limitation. Specific metabolic pathways, however, are known to require large amounts of iron, including N2 fixation and NO, reduction (Raven, 1988). Laboratory and field evidence suggests that these cellular processes may be restricted in iron-poor environments (Rueter, 1988; Price et al., 1991, 1994; Timmer- mans et al., 1994).

“Use efficiency” is a term employed by plant physiologists to describe the rate of photosynthesis (or biomass accumulation) per unit of limiting resource (Raven, 1988). In autotrophs, the rate of carbon dioxide fixation per unit cellular iron, or the iron use efficiency (IUE), is the product of the carbon to iron ratio (C:Fe, with units of mol organic carbon per mol cellular iron) and the specific growth rate (p, with units of dd’): IUE = (C:Fe) p. It thus incorporates stoichiometric information on elemental composition with kinetic information on growth rates into a single term. Like the assimilation number, which is the rate of CO2 fixation per unit chlorophyll per unit time, the IUE is affected by intra- and interspecific factors.

While obtaining a better understanding of the pathways for metabolic use of iron is a central goal for marine algal physiologists, there are several areas that are particularly lacking in information:

Raven (1988, 1990) calculated theoretical, maximum iron-use efficiencies for phytoplankton by considering the iron content and specific reaction rate of iron-containing catalysts in the photosynthetic and respiratory pathways and in N assimi- latory pathways. Though the calculations derived from data on Fe-sufficient organisms which were not selected for genetic acclimation to low iron conditions, they demonstrate the high cost of N2 fixation and the moderate cost of NO; assimilation relative to growth on NH:. Nitrate assimilation and reduction increases the iron requirement for growth (relative to NH,$ assimilation) by about 60% and N2 fixation increases this requirement by lOO-fold. Light also is expected to alter the iron requirements of phototrophs, increasing it by as much as 50 times at low photon flux densities of 1 uE rnp2 s-l. Experimental tests using Thalassio- sira weissflogii support the predicted increase of Fe quota under low light (Strzepek and Price, in

prep.).

(a>

(b)

How flexible is “iron use efficiency” (i.e. C:Fe/time) in the heterotrophic and autotrophic biota and how does it respond to iron availability? What are the minimum iron requirements for growth of organisms, to what extent can iron

Photoautotrophs may decrease their Fe requirements and still grow quickly (i.e. increase their


IUE) by replacing Fe catalysts with those which contain no metal and by altering metabolic pathways. For example, if ATP for biosynthesis is generated directly by photophosphorylation (e.g. as opposed to CO2 fixation into carbohydrate followed by ATP synthesis during mitochondrial oxidation of carbohydrate) IUE could be increased by about 25% (Raven, 1988). The maxima could be further increased if flavodoxin and plastocyanin substitute for ferredoxin and cytochrome c in the photosynthetic electron transfer chain. Raven (1988) argued for a modest (- 10%) decrease in Fe requirement with this substitution, however, Anderson and Erder (this workshop) estimate a much greater savings (> 80%) using lower Fe:C ratios. Even given this intraspecific and pheno- typic plasticity, the discrepancies between observed and theoretical iron-use efficiencies of the phototrophs that have been studied cannot be resolved (Sunda et al., 1991). Future research must address the fundamental issues of iron biochemistry and physiology in a wider range of oceanic and coastal phytoplankton isolates.

Although the information on C:Fe and IUE of relevant oceanic phytoplankton clones is limited (Geider and La Roche, 1994), there is currently no equivalent information for other members of the microbial loop. It is anticipated that for a given growth rate, the iron requirements of heterotrophs will be lower (i.e. C:Fe higher) than those of autotrophs because they lack iron-rich photosynthetic membranes (Raven, 1988, 1990). However, bacteria and ciliates have the potential to achieve specific growth rates that exceed those of the phytoplankton. High cellular iron contents (i.e. low C:Fe) may thus be required to sustain these high growth rates, even though heterotrophs have a higher intrinsic IUE.

The metabolic iron requirement of different phytoplankton species varies widely. The calculated, minimum iron requirements for algal growth (Raven, 1988, 1990) are greater than measured values by many times, but the organisms from which these values were calculated were not iron stressed. Carbon to iron ratios vary from < 2000 C:Fe in the freshwater Nz-fixing cyanobacterium Anabaena sp. (Hutchins et al., 1991) to N 500,000 C:Fe in the oceanic diatom Thalassiosira

oceanica (Sunda et al., 1991). With these iron quotas, the organisms achieve specific growth rates of N 0.03 d-’ and N 1 .O d-' , respectively, demonstrating the great variability in IUE. Exclud- ing the N2 fixers, which have a very high iron- requirement (Raven, 1988), the range of C:Fe ratios is still 50-fold. The red tide dinoflagellate, GJlmnodinium sarzguineum, has a C:Fe ratio of < 10,000 (Doucette and Harrison, 1991) and in some coastal cyanobacterial strains (Brand, 1991a), and in diatoms from coastal upwelling regimes (Bruland et al., 1991) the C:Fe ratio is N 20,000. The high iron requirements of coastal isolates, and their lower IUE compared to oceanic species, suggests the potential for iron-limitation in coastal seawaters even considering the substantially higher quantities of total iron in these waters.

While cellular iron requirements provide guidance towards identifying species which have economized their iron use, iron use efficiencies have greater ecological relevance because they quantify potential growth rates per unit iron quota. But physical constraints which regulate iron transport into the cell also must be considered. Decreasing cell size is accompanied by larger surface area to volume ratios and better diffusional characteristics, so that small cells having low IUE can compete with larger cells display- ing higher IUE. For example, three different oceanic species in culture ranging in size from 1.5 to 6 pm all had similar iron-limited growth rates at the lowest Fe’ concentration despite having very different cellular iron requirements (Sunda and Hunstman, in prep.). This result was achieved by an inverse relationship between iron transport kinetics (per unit carbon) and iron use efficiency. Larger cells, such as T. oceanica, must then evolve higher use efficiency to overcome the unfavorable transport kinetics. Thus, cellular iron quotas and use efficiencies alone are not enough to anticipate the relative success of different members of the algal assemblage.

Explanations for the tremendous variation in C:Fe ratios of phytoplankton are presently largely speculative as we yet have little information on the biochemical differences among algae. This speculation forms the basis for seeking indicators of iron


stress in phytoplankton which is discussed below. Similarly, we have little information on the kinetics of physiological and biochemical responses to changes in iron availability. For example, we cannot define clearly between physiological and biochemical responses that represent short term acclimation to iron limitation and more long range genetic “acclimation” patterns to a low iron environment. These issues will be important for predicting population responses to fluctuations in iron supply originating from changes in external iron inputs or short term variations in iron speciation.

Biochemical, molecular and physiological indicators of Fe status of plankton communities

The metabolic consequences of iron deprivation to marine algae and heterotrophic bacteria are poorly understood. In higher plants, the activity of iron-containing enzymes has been examined under conditions of iron deficiency. Catalase (Leidi et al., 1986) and lipoxygenase (Boyer and Ploeg, 1986) activities, for example, decrease under low iron and provide a measure of iron stress in these organisms. In marine phytoplankton, the ratio of cytochrome f: chlorophyll a declines in iron-limited cultures of Phaeodactylum tricornutum and Zsochrysis galbana (Glover, 1977). Variations in ferredoxin content also have been used to infer iron-limitation for zooxanthellae on Davies Reef, Australia (Entsch et al., 1983) and Trichodesmium colonies from the Caribbean (Rueter et al., 1992). In the approaches outlined above, a decline in enzyme activity or change in biochemical content is observed in response to iron-limitation. Iron limitation also appears to alter the cellular abundance of photosystem I and II, increase the number of non-functional reaction centers, impair energy transfer efficiency in photosystem II, and reduce intersystem electron flow (Greene et al., 1992; Geider et al., 1993; Geider and La Roche, 1994; Green et al., abstract). However, all of the above indicators may be susceptible to interferences by factors other than iron-limitation, such as toxicity or limitation by other elements.

Promising candidate indicators of iron stress and iron limitation in marine algae include:

. cytochrome c/plastocyanin ratios,

. ferredoxin/flavodoxin ratios,

. fluorescence characteristics, - iron transport rates and proteins, and - immuno-probes, mRNA.

Experimental tests to establish the importance of iron limitation in the sea have relied largely on enrichment assays in bottles (Martin et al., 1989, 1991; de Baar et al., 1990; Buma et al., 1991; Coale, 1991; Helbling et al., 1991; Takeda et al., submitted) and most recently on enrichment of a 60 km* patch of surface ocean (Martin et al., 1994). In these experiments, iron concentrations are arti- ficially increased and the response of the plankton community is assayed for a variety of physiological indicators, most of which measure the bulk community response to the iron additions, rather than the response of individual species or classes of organisms. More recently, size-fractionation of the induced response as well as pigment analyses have shown that iron additions differentially affect separate components of the planktonic assemblage (Buma et al., 1991; DiTullio et al., 1993; Price et al., 1994; Barber et al., abstract). Improved methods which instantaneously detect and quantify the nature and extent of iron limitation of the separate components of the diverse planktonic assemblage are needed (i.e. an autecological rather than a systems approach).

A number of biochemicals may potentially serve as diagnostic indicators of iron-limitation. One candidate pathway worthy of investigation is the cytochrome c/plastocyanin system, whereby iron limitation results in the substitution of copper- containing plastocyanin for cytochrome c, which contains iron (Sandmann and Boger, 1980). Another promising indicator for heterotrophic bacteria as well as eukaryotic and prokaryotic phytoplankton is based on the substitution of the non-iron containing protein flavodoxin for the catalytic protein ferredoxin (the terminal electron donor to NADP in the photosynthetic electron transport chain) under conditions of iron limitation (e.g. Zumft and Spiller, 1972; Entsch et al., 1983; Laudenbach et al., 1988; La Roche et al., 1993).

The power and utility of the “diagnostic indicator” approach is already apparent in ongoing


studies of ferredoxin and flavodoxin by several research groups (Geider and La Roche, 1994; Anderson and Erdner, abstract; La Roche and Geider, abstract). Although early studies of this indicator suffered from the need to collect large quantities of algae, tools now are under development to detect these two target proteins in micro- gram quantities using HPLC, at nanogram levels using Western blots of protein extracts, and potentially at picogram levels in individual cells using immunofluorescence. These methods potentially can separate the pelagic community response at several different ecological levels. Whereas HPLC can only provide data for the bulk community or for size fractions of that community, antibody detection on Western blots can resolve class- or species-specific responses. Immunocytochemical techniques have the potential to reveal the physiological status of individual cells; flow cytometric analyses would then help establish the overall health of that species population. Attention also is being directed to molecular characterization of the two proteins, which can lead to probes for their genes, or to the mRNA transcripts of those genes, which could offer similar levels of resolution and specificity.

Direct application of HPLC and antibody-based techniques to natural waters has been constrained by the large amount of water that must be filtered to provide the biomass for HPLC measurements, and by the lack of broad cross-reactivity in the antibodies that have thus far been obtained. Nevertheless, the concept is sound and soon should be applicable to natural communities. Results from laboratory studies already demonstrate the information that can be uniquely provided by the use of diagnostic indicators. For example, one surprising observation from studies of laboratory cultures is that expression of the flavodoxin gene occurs in a wide range of marine phytoplankton species, including isolates from coastal regions where iron has been presumed to be in high abundance (Anderson and Erdner, abstract; La Roche and Geider, abstract). The implication is that iron limitation may be a problem common to many oceanic regimes, not just those where ambient iron concentrations are very low (see below).

One new approach yielding exciting insight into the effects of iron on natural phytoplankton populations is fast repetition rate (FRR) fluorometry (Kolber and Falkowski, 1992). The FRR fluorometer measures the change in the quantum yield of in vivo chlorophyll fluorescence resulting from exciting photosystem II (PSII) with a train of subsaturating flashes. The cumulative stimulation of PSI1 by these flashes induces a saturation profile, the amplitude of which is quantitatively proportional to the quantum efficiency of PSII. The rate of saturation is quantitatively proportional to the functional photon absorption cross section of the PSI1 reaction center. In other words, FRR fluorometry provides an instantaneous measurement of the community-scale efficiency of algal light harvesting systems which infers a metabolic status of the phytoplankton assemblage. Low photosynthetic efficiency can result from a poor supply of nutrients, toxic metal (e.g. Cu) effects, and photo damage of the light harvesting system. For example, iron limitation leads to a marked decrease in quantum efficiency and an increase in absorption cross section in laboratory cultures (Greene et al., 1992). In the HNLC waters of the equatorial Pacific, there was a high degree of inactivation of PSI1 reaction centers and low photosynthetic energy conversion efficiency in all phytoplankton size classes, but after mesoscale iron enrichment, in-situ photosynthetic efficiency increased by 70% and the absorption cross section decreased by a factor of two, consistent with an alleviation of iron stress (Kolber et al., 1994). These changes were observed in all size classes including picoplankton, implying that small size may offer no refuge from iron limitation in this low iron environment. Similar effects of iron enrichment to these waters also have been found in on-deck culture experiments (Greene et al., 1994). This rapid, non-intrusive methodology is certain to be an important tool for probing the physiological state of natural phytoplankton populations. Its further refinement in conjunction with studies of iron speciation may offer the best approach in the future for linking algal physiology and the ambient iron chemistry in seawater.

174 M.L. Wells et aLlMarine Chemistry 48 (199.5) 1.57-182

The applicability of laboratory studies to natural plankton communities

Two central issues must be considered when extrapolating laboratory test results to natural phytoplankton communities.

(1) How general are the responses measured with individual organisms and what is the extent of intraspecific and interspecific variability?

(2) How representative of the indigenous micro- biota are the organisms that we presently study in culture?

Field studies (Martin and Gordon, 1988; de Baar et al., 1990; Martin et al., 1990b, 1991; Buma et al., 1991; Helbling et al., 1991; Takeda et al., submitted) have documented how iron can influence total phytoplankton biomass and productivity, yet we know that the phytoplankton community com- prises a diversity of species with different adaptations and iron requirements. Although we still cannot culture many phytoplankton, particularly oceanic species, we do have in culture a number of clones, both neritic and oceanic, that are dominant in their communities and appear to be reason- ably representative. By contrast, analysis of ribosomal RNA isolated from bulk water samples has revealed a previously unrecognized diversity of bacteria and Arches, of which no representatives are in culture (Giovannoni et al., 1990; Fuhrman et al., 1992). Because many of these microbes had diverged from the primary lineage prior to the development of an oxidizing atmosphere, when iron became much less available, novel adaptations to deal with subsequent low iron conditions may have arisen in the different groups. Thus, extrapolating from one group to another may be unwise.

Broad differences among phytoplankton in their adaptations to iron availability have been identified. Laboratory studies indicate that cyanobacteria have a higher cellular iron requirement than most eukaryotic phytoplankton (Brand, 1991a) which is due in part to a higher PSI/PSI1 ratio (Raven, 1990). Yet to be examined are the iron growth requirements of the widespread and abundant prochlorophytes. Related to this question of iron requirements of representative groups of phytoplankton is the question of whether or not we have even identified all the major groups of

phytoplankton in the ocean. The fact that the two most numerically abundant groups of phytoplankton, the cyanobacteria and prochlorophytes, have only recently been discovered does not give us much confidence. The laboratory observation of higher iron requirements in cyanobacteria than in most eukaryotic phytoplankton agrees well with the global distribution of these two groups with respect to the relative inputs of iron and major nutrients to the photic zone (Brand, 1991a). How- ever, this observation has not successfully predicted the outcome of bottle and mesoscale experimental enrichments. In bottle enrichments, cyanobacteria populations often decline with iron additions (DiTullio, abstract) while all phylo- genetic groups, including both cyanobacteria and eukaryotes, increased in the mesoscale iron enrichment experiment in the equatorial Pacific (Barber et al., 1994). Current efforts to explain the discrepancies between single species laboratory studies, bottle enrichments in the field, and mesos- tale enrichments have focused on the complex interactions between phytoplankton, their grazers and the microbial loop.

In addition to the importance of studying representative species of the phytoplankton community, it is important to study representative genotypes. Genetic variability within populations and genetic differentiation between populations are well known and probably occur in all phytoplankton species (Brand, 1991b). It is therefore important to use local isolates in any site specific laboratory studies. An excellent example of this with respect to trace metals is the study of Jensen et al. (1974) in which a clone of Skeletonema costatum from a metal polluted fjord was much more tolerant of zinc toxicity than a clone of the same species from a relatively pristine fjord.

While genetic variation can be a complicating factor, it can also be used to our advantage as an indicator of past selective pressure. Brand et al. (1983) used the differences in iron requirements between neritic and oceanic phytoplankton species to argue that iron limitation of growth rates must have been the selective force leading to such differences.

It is important that iron manipulation experiments with natural population or laboratory

M.L. Wells et al./Marine Chemistry 48 (1995) 157-182 115

cultures be conducted as near to ambient concen- Morel (1993) argued that transport limitation at trations as possible. Experiments using metal con- low concentrations of iron largely explain why centrations lo-100 fold above natural levels may HNLC ecosystems are dominated by picoplank- affect the speciation and reactivity of not only iron ton. They apparently flourish because favorable but also that of other bioactive metals, particularly surface area to volume ratios and diffusion charac- if iron solubility is exceeded. For oceanic clones, teristics optimize iron acquisition per unit of bio- where little work has been done, the new evidence mass, though elevated iron transport rates (Price et of strong iron-binding ligands may complicate cul- al., 1994) and FRR fluorometry (Greene et al., turing studies which employ nutrient-enriched sea- 1994; Kolber et al., 1994) suggest that picoplank- water media and synthetic ligands to buffer metal ton in the equatorial Pacific still are experiencing activity. Development of standardized protocols iron stress. Larger cells do not compete effectively for media preparation and growing open ocean for the scarce iron resource and are curtailed to low clones should help reduce these uncertainties. growth rates.

(5) What are the ecological and biogeochemical implications of changing iron availability in the oceans?

Of all of the aspects discussed during the workshop, the possible implications of changing iron availability on the ecology and biogeochemistry of ocean systems were perhaps the most speculative. Iron will become an important factor only in those places or times when the supply rates approach or are below the level of iron demand by the plankton community. Temporal or spatial variations in iron supply could influence:

- phytoplankton biomass and production rates, . size structure of the phytoplankton assem-

blage, - species composition within size fractions, and - trophic dynamics and thus export production.

The present focus of iron research in the sea concentrates on HNLC regions where relatively low and constant phytoplankton biomass coexists with high concentrations of the major nutrients. These HNLC regions, including the subarctic Paci- fic, the equatorial Pacific and the Southern Ocean, are characterized by a high input of major nutrients via upwelling or vertical mixing but a low external supply of iron. Characteristics of the algal assemblages occurring in these regions are consistent with iron (and other trace metals) being an important factor. By virtue of their small size, picoplankton (< 2.0 pm) are extremely well adapted to maximize iron uptake. In fact, Hudson and

It also is expected that iron can strongly influence the resultant food web structure of the ecosystem. Picoplankton are fed upon by small herbivores which can grow faster than their prey (Banse, 1992) and thus can quickly respond to any increase in picoplankton growth rates. This quick response is argued to maintain biomass at a low and nearly constant level in HNLC regions (Banse, 1991; Frost, 1991; Miller et al., 1991; Banse and English, 1994), though the effects of light and macronutrients also may play an important role in the Southern Ocean (Dugdale and Wilkerson, 1990, 1991; Mitchell et al., 1991; Nelson and Smith, 1991). In other words, phytoplankton populations might be grazer-controlled in an iron-limited ecosystem (the so-called ecumenical hypothesis). However, picoplankton biomass levels still can increase when perturbations in nutrient inputs are sufficiently large, such as in the mesoscale iron enrichment experiment (Martin et al., 1994), suggesting that iron may be more important than grazing in controlling algal biomass in these waters.

In HNLC systems, temporary increased inputs of iron (by a series of major storms, volcanic erup- tions, etc.) may cause a shift towards an ultra- plankton (- 2-5 pm) dominated system (Martin and Fitzwater, 1988; Martin et al., 1990a,b; de Baar et al., submitted) or simply increase the production of all size classes of phytoplankton (Kolber et al., 1994; Martin et al., 1994). The net effect in both cases should be to increase export production until the iron resource is returned to pre disturb- ance levels and the ecosystem moves back to one sustained primarily by regenerated iron (unless


another resource first becomes limiting). Such periodic inputs of iron would serve to maintain an assemblage of phytoplankton species whose iron requirements range from extremely low to moderate, as appears to be the case in remote HNLC regions.

In subtropical waters, increases in iron input could have a similar effect but for entirely different reasons. Because phytoplankton in these systems appear to be N-limited, most species should experience no direct effect from an increased supply of iron. However, Trichodesmium sp. are not limited by low concentrations of NO; but are likely stressed or limited by iron given their extremely high iron requirements for N2 fixation (see above). Increased iron inputs thus might increase their production significantly and indirectly stimulate overall productivity and export production.

Even though iron concentrations in coastal and shelf regions are substantially higher than in oceanic surface waters, changing iron availability could limit certain populations and lead to a shift in the dominant species within the phytoplankton assemblage. For example, Doucette and Harrison (1990) obtained results demonstrating the importance of iron for dinoflagellates and Wells et al. (1991b) have argued that low iron conditions may limit the distribution of dinoflagellates in the Gulf of Maine. The observation that flavodoxin is expressed in coastal diatom species (Anderson and Erdner, abstract: La Roche and Geider, abstract) strongly suggests, from what we know about the substitution of flavodoxin for ferredoxin, that these cells are biochemically prepared to deal with iron limitation. It seems unlikely that coastal phytoplankton would have this adaptive ability without the need to express it occasionally.

Unlike the stable HNLC regions, iron depletion in coastal and shelf waters may result from organism growth and the seasonal accumulation of high biomass levels (e.g. the coastal spring bloom). This represents a different type of iron limitation than seen in open ocean systems. Such bloom related depletion has two important implications, first that cells which can store excess levels of iron prior to the bloom (e.g. diatoms) may have a competitive advantage, and second, that use of

iron sparing mechanisms (e.g. flavodoxin for ferredoxin) may be needed to help continue growth after iron depletion. This pattern of iron depletion following growth presumably also is prevalent in other environments where the chlorophyll and standing stocks vary, such as the edges of river plumes and along ice margins (de Baar et al., submitted). Similar considerations also should apply in non-HNLC open ocean environments which characteristically experience spring time blooms, such as in the North Atlantic.

3. Summary

The perceptions of ocean chemists and algal physiologists have shifted enormously over the past two decades, from attempts to correlate metal uptake by phytoplankton with bulk measurements of total “dissolved” metals, to recognizing that metal speciation is the critical factor. This evolution in thinking began with well-defined, metal buffered laboratory culture experiments and the principles learned there have appeared to work well for predicting the biological effects of various metals (e.g. Cu, Zn, and Mn) in natural systems; until iron. It became apparent during the workshop discussions that two main factors con- tribute to the vexing difficulty in characterizing the relationship between iron chemistry and phytoplankton dynamics.

First, the marine chemistry of iron is more complex and less explored than many other bioactive metals. The central issues that elicited surprise and chagrin at the workshop were that: (1) there is no clear consensus about the inorganic speciation of Fe(II1) in seawater and as a result we still do not know the inorganic solubility of iron in seawater with respect to any of the solid iron phases present, (2) inorganic iron species may in fact be a minor component of soluble iron chemistry with organic complexation instead being the dominant form, and (3) as yet we cannot explain the apparent persistence or speciation of Fe(I1) in surface waters.

The second complicating factor is the diversity of iron uptake strategies likely employed by phytoplankton. Up until now, we have modeled iron acquisition by phytoplankton in much the same

M.L. Wells et al./Marine Chemistry 48 (199.5) 157-182 111

way as for other metals, namely the reaction of inorganic metal species with cell surface ligand sites followed by transport into the cell. This “classical” uptake mechanism has been verified for iron and well characterized in laboratory culture and modeling studies. We now realize that while iron uptake typically is correlated with free hydrated Fe 3+ in constant pH seawater containing different synthetic chelators, it is the more abundant, kinetically labile hydrolysis species comprising Fe(II1)’ and Fe(I1)’ that actually control uptake rates. However, very recent findings suggesting that the bulk of soluble iron may be organically bound in natural systems means that labile inorganic species might fall to extremely low levels, perhaps requiring photochemical or other redox mechanisms to provide a constant supply of metal.

In addition to direct uptake of Fe(III)/, we now have glimpsed several other tricks marine algae might use to acquire iron when this resource becomes scarce. Cells might employ the release and uptake of siderophores, become siderophore “pirates” by utilizing the siderophore-Fe complexes of other species, or reductively dissociate “generic” organic-iron complexes at the cell surface. There presently is no direct evidence as to the significance of these alternate iron uptake strategies in marine waters.

Though a central goal of the workshop was to better define iron availability to help develop analytical tools for quantifying this bioavailable fraction in seawater, this task is not possible given the foundation of our present knowledge. Quantification of iron “availability” in natural waters awaits better characterization of the marine chemistry of iron in conjunction with exploration of the uptake strategies used by different members of the algal community.

An issue raised repeatedly during the discussions was that while interest in the role of iron has focused on HNLC regions, there is increasing evidence that iron also may be important in other oceanic and even neritic domains. At least some neritic phytoplankton species have the ability to switch from iron-rich to non-iron containing electron transfer proteins under iron deplete conditions; an adaptive strategy which implies that these nearshore organisms become limited or

stressed by iron availability at certain times. By influencing algal species composition, iron chemistry and supply may interact with other biological factors in regulating the food web structure of many marine ecosystems.

What emerged then from this workshop was a shift in the paradigm of iron biogeochemistry in seawater, from one in which iron/phytoplankton interactions behave similarly as for other bioactive metals to one in which iron stands as truly unique. It is certain that unraveling the rich complexity of interaction between iron chemistry and the biota will need the frank cross-disciplinary discussion and collaboration that characterized this workshop.

Acknowledgements

We wish to thank the staff at the Bermuda Bio- logical Station for Research for their gracious and friendly help before, during and after the workshop. We thank the participants for their unbridled enthusiasm and openness which repeatedly led to exciting speculation and new conceptual fantasies; an interaction unusual in the present difficult funding situation. We express our appreciation to Eden Rue for her additional assistance before and during the workshop. We thank the working group leaders - Kenneth Coale, Whitney King, George Luther, Frank Millero, and Charlie Trick - who helped guide the working group discussions towards formulating a cohesive list of issues. We also thank Bill Sunda, Dorothy Swift, Richard Greene and Hein de Baar for their detailed comments and suggestions on earlier versions of this manuscript as well as other participants who provided their views. We give particular thanks to Neil Andersen for spearhead- ing the search for funding to make this workshop possible. This workshop was sponsored by NSF in cooperation with ONR, NASA, NOAA, and DOE.

Appendix 1. List of abstract titles

Alison Butler - Mechanisms of iron acquisition by marine micro-

organisms: bacterial siderophores

Donald M. Anderson and Deana L. Erdner - Molecular probes

for iron nutritional status in marine phytoplankton

178 M.L. Wells et al.lMarine Chemistry 48 (1995) 157-182

Richard Barber and the Iron Ex group - The in situ phytoplankton response to natural and experimental iron enrichment

Larry E. Brand - Simultaneous nutrient limitation by iron, zinc and manganese in marine phytoplankton

Kenneth Coale and the Iron Ex group - Mesoscale iron enrichment and Galapagos plume studies in the equatorial Pacific

Alvin L. Crumbliss - Chemical models related to siderophore mediated iron bioavailability

Hein J.W. de Baar, Maria A. van Leeuwe, Renate Scharek, Jeroen T.M. de Jong, Klaas R. Timmermans, Bettina M. Liischer and Rob F. Nolting - Iron and plankton in the Antarctic Circumpolar Current

Giacome R. DiTullio - Importance of Fe-availability on size- fractionated 14C :59Fe uptake ratios and phytoplankton ecology in the tropical Pacific ocean

Martha Gledhill and Constant M.G. van den Berg - The speciation of iron in the North Sea measured by cathodic stripping voltammetry

Richard M. Greene, Zbigniew Kolber and Paul Falkowski - Photosynthetic energy conversion efficiency and iron limitation in the equatorial Pacific

David A. Hutchins - Bioavailability of biological iron in high and low iron environments

Margo G. Haygood - Iron physiology of marine bacteria Robert Hudson and Kenneth W. Bruland - Iron transport

mechanisms in marine microorganisms: chemical and physical constraints

Kenneth S. Johnson and the MLML group - Iron photochemistry and analytical chemistry

Dana R. Kester - Marine redox cycle of iron Julie La Roche and Richard J. Geider - Evaluating the use of

flavodoxin as a molecular marker of phytoplankton iron status

D. Whitney King, Robb A. Aldrich and Heather A. Lounsbury - Investigation of the redox dynamics of iron in aquatic systems

Katsuhiko Matsunaga, Kenshi Kuma, Yoshihiro Suzuki and I. Kudo - Bioavailable iron in submicron colloids

Chris I. Measures, J. Juan and J.A. Resing - Determination of iron in seawater by flow injection analysis using in-line preconcentration and spectophotometric detection

Francois M.M. Morel - Cycling and availability of iron in the equatorial Pacific

Rodney T. Powell and William M. Landing - Colloidal iron: oceanic and estuarine size distributions and reactivity

Neil M. Price and M.T. Maldonado - Iron acquisition by marine diatoms consumming different nitrogen substrates

Eden L. Rue and Kenneth W. Bruland - Complexation of iron(II1) by natural organic ligands in the central North Pacific as determined by competitive equilibration/ adsorptive cathodic stripping votammetry

John Rueter - Iron nutrition for two strains of oceanic cyanobacteria: the role of aeolian dust

Barbara Sulzberger - Kinetics of photoreductive dissolution of colloidal iron(II1): its dependence on the type of colloidal iron(II1)

William G. Sunda and Susan A. Huntsman - Iron uptake and growth limitation in oceanic and coastal phytoplankton

Shigenobu Takeda - Iron enrichment experiments in the equatorial Pacific: availability of added iron to phytoplankton

Steven W. Taylor, George W. Luther III, J. Herbert Waite and Brent Lewis - Polarographic and spectrophotometric investigation of iron(II1) complexation to 3,4-dihydroxyphenylalanine- containing peptides and proteins from Mytilus edulis, and I-nitroso-2-naphthol

Charles G. Trick - Physiological changes in the marine cyanobacterium Synechococcus sp. PCC7002 exposed to low ferric ion levels

Constant van den Berg - Determination of organic complexation of iron in the western Mediterranean

Bettina M. Voelker and D.L. Sedlak - Iron reduction by photo- produced superoxide in seawater

T. David Waite, R. Szymczak and Q. Espey - Recent observations of photochemically mediated iron transformations in seawater

Jingfeng Wu and George W. Luther III - Size- fractionated iron concentrations in the water column of the Northwest Atlantic ocean

Guoshun Zhuang, Zhen Yi and Gordon T. Wallace - An improved HPLC method for the determination of Fe(H) in aerosols, rainwater and seawater

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