Marine bacteria and biogeochemical cycling of iron in the oceans

FEMS Microbiology Ecology 29 (1999) 1^11

MiniReview

Marine bacteria and biogeochemical cycling of iron in the oceans Philippe D. Tortell a; *, Maria T. Maldonado b , Julie Granger b , Neil M. Price a

b

Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544, USA b Department of Biology, McGill University, Montreal, Que. H3A 1B1, Canada Received 18 June 1998; received in revised form 2 December 1998 ; accepted 8 December 1998

Abstract Prokaryotic microbes play a critical role in oceanic Fe cycling. They contain most of the biogenic Fe in offshore waters and are responsible for a large portion of the Fe uptake by the plankton community. In the subarctic North Pacific, surface populations of heterotrophic species assimilate more than 50% of the dissolved Fe and thus compete directly with phytoplankton for this limiting resource. In oligotrophic tropical and subtropical waters, photosynthetic bacteria become more important in Fe cycling as the number of unicellular cyanobacteria increases and the nitrogen-fixing Trichodesmium, which contains most of the biogenic Fe in the mixed layer, becomes abundant. Like their terrestrial counterparts, heterotrophic and phototrophic marine bacteria produce Fe-binding siderophores that are involved in Fe acquisition. Evidence exists that bacteria may modify Fe chemistry in the sea through the production of these ligands and thereby play a significant role in regulating production of eukaryotic phytoplankton. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Iron chemistry; Marine bacterium ; Iron cycling

1. Introduction Over the past decade and a half, it has become apparent that trace metals can a¡ect oceanic primary productivity as limiting nutrients and toxic inhibitors [1]. As a result, the biogeochemical cycling of trace metals in the oceans has become a subject of great interest and research. Of all trace metals, Fe has thus far received most attention. Shipboard bioassay experiments [2^4] as well as two mesoscale in situ Fe fertilizations [5,6] have demonstrated that low Fe availability constrains phytoplankton growth in several large open ocean regions where primary production is low despite high concentrations of major nu* Corresponding author.

trients (nitrate, phosphate, silicate). Iron limitation in these regions decreases the e¤ciency of the `biological carbon pump' through which CO2 is consumed in surface waters and transported as sinking particulate organic carbon to the deep sea. Given the importance of this process in controlling atmospheric CO2 [7], Fe biogeochemistry in the oceans has important implications for global carbon cycling and climate studies. At present, our understanding of Fe biogeochemistry is incomplete. Many of the processes that are thought to control dissolved and particulate Fe concentrations in the oceans are known from laboratory studies that mimic natural conditions with varying degrees of success. One fact that is clear, however, is that biological processes are of great importance.

0168-6496 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 1 1 3 - 5

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Most investigations of Fe-biota interactions to date have focused on eukaryotic phytoplankton, which account for the bulk of CO2 export. Relatively little attention has been given to the role of phototrophic and heterotrophic bacteria in Fe cycling. This is in marked contrast to studies of ocean carbon and nitrogen dynamics where these organisms have been shown to play a central role [8,9]. In this review, we discuss the biogeochemistry of Fe in the oceans with speci¢c emphasis on the role of prokaryotes. In particular, we highlight several mechanisms through which photosynthetic cyanobacteria and heterotrophic bacteria may a¡ect Fe bioavailability and cycling. Despite the recently discovered abundance of Archaea in the oceans [10,11] we shall not explicitly consider these organisms for lack of relevant physiological data. Given the tremendous advances in aquatic microbiology and rapid development of new molecular techniques, it may soon be possible to examine bacterial-Fe interactions across the broad taxonomic and biochemical diversity of marine prokaryotes. Our discussion points to several important questions that will need to be addressed by such future research.

2. Biogeochemistry of dissolved iron in the sea Although Fe is the fourth most abundant element in the earth's crust, its concentration in most ocean waters is vanishingly low. Until recently, ubiquitous contamination of samples during collection and analysis prevented accurate determination of true dissolved Fe levels. The advent of ultra-trace metal clean techniques [12], however, facilitated accurate and systematic measurements of Fe in the world's oceans [13] and provided new insight into the role of marine biota in Fe cycling. Below, we brie£y summarize the most salient features of dissolved Fe distributions and chemistry, and discuss recent evidence for bacterial control of Fe speciation and solubility. Like nearly all trace metals, dissolved Fe concentrations show strong horizontal and vertical gradients, decreasing by as much as several hundredfold from coastal to o¡shore waters and increasing signi¢cantly with depth in the upper 500 meters [13]. Coastal waters receive large inputs of Fe from rivers and anoxic sediments [13] whereas o¡shore regions

Fig. 1. Vertical distribution of dissolved iron and of two classes of iron-binding ligands, L1 and L2, in the central North Paci¢c. The data are replotted from [22].

rely mainly on atmospheric dust deposition and/or upwelling of deep waters as sources of Fe [14,15]. We shall focus our discussion on oceanic (o¡shore) regions that account for s 90% of marine primary productivity and are characterized by persistently low Fe availability. Such low Fe levels demonstrably limit primary productivity in high-nutrient oceanic regions [6] and may impose a signi¢cant stress on phytoplankton in oligotrophic subtropical gyres. Recent work has demonstrated that coastal upwelling regimes can also be Fe-de¢cient [16]. Dissolved Fe concentrations in o¡shore waters of the Paci¢c, Atlantic, and Southern Oceans average 0.07 þ 0.04 nmol kg31 at the surface ( 6 200 m) and 0.76 þ 0.25 nmol kg31 at depth ( s 500 m) [13]. Such surface depletion is typical of `bioactive' micro- and major nutrients and results from biological uptake in the upper water column followed by regeneration during oxidation of organic detrital matter as it sinks to the sea £oor. Heterotrophic bacteria in surface and subsurface layers of the oceans mediate this latter process [9].

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Despite its nutrient-type pro¢le, the oceanic distribution of Fe di¡ers signi¢cantly from that of other essential trace metals such as Zn [17]. Most of these metals are highly soluble in seawater, whereas Fe is present to a large extent in particulate phases (silicates, aluminosilicates and oxyhydroxides) (see [18]). The `dissolved' Fe fraction (operationally de¢ned as 6 0.4 Wm) consists largely of colloidal hydrolysis species such as Fe(OH)3 which are rapidly scavenged out of the water column by coagulation and adsorption onto sinking particulate material. As a result, the dissolved Fe pool in deep waters turns over rapidly with an ocean residence time on the order of 100 years [19]. Unlike other particle reactive metals (e.g. Al, Mn, Pb) whose dissolved concentrations decrease with depth due to removal by particulate adsorption, deep water ( s 500 m) dissolved Fe concentrations appear to be relatively invariant (Fig. 1). This anomalous behavior is at least partially attributable to the presence of high a¤nity organic ligands which speci¢cally complex Fe(III) and increase its apparent solubility by enhancing colloid dissolution and reducing scavenging rates [13]. Indeed, UV oxidation of the organic ligands in open ocean water samples, has been shown to decrease the relative proportion of truly dissolved ( 6 0.025 Wm), i.e. non-colloidal, Fe [20]. Several groups have examined organic Fe complexation using highly sensitive electrochemical techniques [21^23]. Their results suggest that greater than 99.9% of dissolved Fe is bound by ligands which appear to fall into two classes. The stronger ligand class (L1) is present in surface waters at concentrations of 0.4^1.0 nM and has an inorganic Fe conditional stability constant1 of approximately 1013 M31 while the L2 class is found throughout the water column at approximately 1.5 nM with an inorganic Fe conditional stability constant of about 3U1011 M31 (Fig. 1). As a result of this organic complexation, inorganic Fe concentrations are less than 0.1 pM (i.e. 0.0001 nM) in surface waters and 1 The conditional stability constant (KP) is de¢ned as: [FeL]/ ([FeP]W[LP]); where [FeL] is the concentration of organically complexed Fe, and [FeP] and [LP] are the concentrations of unbound Fe and ligand respectively. KP expresses the relative thermodynamic stability of organic Fe complexes at speci¢ed values of pH and ionic strength (8.1 and 0.7 M in seawater).

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show more pronounced vertical gradients than total dissolved Fe [22]. As discussed below, this has important implications for biological Fe acquisition. The sources and chemical structures of the Fe complexing agents are presently unknown although it is clear that these organic compounds must be of biogenic origin. A prominent hypothesis is that they are bacterial siderophores ^ highly speci¢c Fe-binding compounds utilized for Fe acquisition [24]. Several lines of indirect evidence are consistent with this hypothesis. Laboratory cultures of oceanic heterotrophic and phototrophic bacteria produce siderophores when grown under low Fe levels typical of oceanic environments [25,26]. In the ¢eld, evidence of in situ production of hydroxamate containing siderophores has been obtained from coastal cyanobacterial mats [27]. Furthermore, the conditional stability constant of the strong organic ligand class (L1) in oceanic waters is similar to that of the well studied siderophore desferrioxamine B (1016:5 M31 ) [22]. Although the siderophores of a number of cultured marine strains have been isolated and studied (e.g. [28,29]), the low concentration of Fe-binding ligands in seawater precludes the precise structural characterization necessary to trace their biotic origins. Identifying the biological sources and chemical structures of organic Fe ligands in seawater will be one of the great challenges facing oceanographers over the next decade.

3. Biological Fe acquisition If, indeed, the organic Fe chelators in seawater are predominantly of bacterial origin, prokaryotes may be largely controlling the availability of dissolved Fe to eukaryotic phytoplankton through complexation and assimilation. Laboratory studies indicate that certain strains of heterotrophic marine bacteria utilize Fe bound to siderophores, including those that do not produce them [30]. At least some marine bacteria may thus rely on Fe-siderophore complexes as the sole Fe source in situ. Measurements of the chemical speciation of Fe during the second Equatorial Paci¢c Fe enrichment experiment [6] showed a rapid ( 6 1 day), four-fold increase in L1 concentration that resulted in the complexation of nearly all the added Fe [31]. As the complexation preceded the

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increase in phytoplankton biomass it may have in£uenced the community response to Fe enrichment by a¡ecting Fe bioavailability. The chemical speciation of Fe has been shown to determine the extent to which it can be taken up by phytoplankton [32]. While early work suggested that phytoplankton access only inorganic forms of Fe, we have recently learned that diatoms are able to utilize Fe bound to a number of organic chelators including siderophores via a cell surface reductase mechanism [33]. Evidence of Fe-siderophore use by natural assemblages of plankton in the subarctic Paci¢c has also been obtained [34]. Both autotrophic and heterotrophic plankton are apparently able to take up Fe bound to desferrioxamine B and E, and large phytoplankton reduce the Fe in the chelates extracellularly. The ability of indigenous plankton to utilize Fe complexed to siderophores provides indirect evidence that these bacterial compounds may be important in Fe cycling in situ. Very recent work [35] indicates that marine photosynthetic £agellates may acquire Fe by ingesting bacteria which, as discussed below, constitute a large pool of particulate Fe in open ocean waters.

4. Biogenic iron in oceanic surface waters The majority of Fe in seawater resides in particulate phases whose biogeochemical cycling is as poorly understood as that of the dissolved species. Total particulate Fe ( s 0.4 Wm) shows high spatial variability and is only weakly correlated with dissolved Fe levels [13]. Understanding the dynamics of the particulate Fe reservoir requires information on its partitioning between lithogenic (aluminosilicate), detrital (non-living organic), and biogenic (living) pools. Recent data indicate that bacteria comprise a large part of the biogenic Fe in open ocean waters and play a critical role in Fe cycling. Quantifying the size of biogenic Fe pools requires estimates of biomass (mol C) and Fe requirements (commonly referred to as Fe quota, expressed as mol Fe cell31 or Wmol Fe mol C31 ) of all producers and consumers in the ecosystem. While biomass data are available from several long-term oceanographic stations, relatively few measurements of Fe:C ratios have been reported for representative open ocean

plankton groups. Nonetheless, it appears that marine prokaryotes (cyanobacteria and heterotrophic bacteria) have a signi¢cantly higher Fe content than eukaryotic phytoplankton. Under Fe-de¢cient culture conditions, oceanic diatoms (eukaryotic algae) have an average Fe quota of 3.0 þ 1.5 Wmol Fe mol C31 [36] while open ocean heterotrophic and photosynthetic bacteria have Fe:C ratios of approximately 7.5 þ 1.7 (¢ve isolates) and 19 (one isolate), respectively [37,38]. Some of these laboratory data have been substantiated by ¢eld studies at Station Papa in the subarctic North Paci¢c Ocean, an o¡shore, Fe-limited region [2]. At this station, Fe:C ratios of eukaryotic phytoplankton and heterotrophic bacteria are 3.7 þ 2.3 and 6.1 þ 2.5 Wmol Fe mol C31 , respectively [34,37], values similar to those measured in Felimited cultures. To our knowledge, no ¢eld measurements of Fe:C ratios of coccoid cyanobacteria have yet been reported. The high Fe quotas of marine bacteria, although possibly surprising to oceanographers, are not unexpected based on the biochemistry and physiology of these organisms. Both phototrophic and heterotrophic prokaryotes require large quantities of Fe as a redox catalyst in their respective photosynthetic and respiratory electron transport chains. Indeed, simple computation of the Fe content of a heterotrophic bacterium, derived from the concentration of Fe-requiring catalysts, indicates that the majority of the cellular Fe is compartmentalized in the respiratory electron chain (Table 1). This conclusion is supported by empirical data from Corynebacterium diphtheriae [39]. More data are needed to determine whether the Fe quotas of photosynthetic marine bacteria are truly higher than those of their heterotrophic counterparts. High bacterial Fe:C ratios change our conceptual models of biological Fe cycling in the open ocean. Tortell et al. [37] constructed a preliminary biogenic Fe budget for Station Papa by summing the Fe content (biomass multiplied by Fe quota) of eukaryotic phytoplankton, cyanobacteria and heterotrophic bacteria. Their results suggested that the prokaryotes constitute a striking 80% of the biogenic Fe in this system with heterotrophic bacteria alone accounting for half of this Fe pool. We have recalculated the Station Papa biogenic Fe budget (Table 2) using 3year average plankton Fe:C ratios recently deter-

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Table 1 Theoretical Fe requirement of Escherichia coli growing aerobically in Fe-replete medium containing yeast extract as a C substrate Enzyme complex

[Complex]a (Wmol g protein31 )

Fe contentb (atom/complex)

NADH-Q reductase

0.206

Succinate-Q dehydrogenase

0.335

Cytochrome b1 Cytochrome oxidase Aconitase Superoxide dismutase Catalase Total Total measurede

0.335 0.124 0.018 0.530 0.011 ^

2U2Fe-2S 6U4Fe-4S 1U2Fe-2S 1U3Fe-4S 1U4Fe-4S 1 2 1U4Fe-4S 1 1 ^

[Fe]c (mol Fe/cellU10320 )

Fe quotad (Wmol Fe mol C31 )

89.4

46.7 5.19 3.84 1.15 8.22 0.17 155 216

43 61

Note that 94% of the cellular Fe is found in the respiratory chain, associated with NADH-Q reductase, succinate-Q reductase, cytochrome b1 , and cytochrome oxidase complexes. a Concentrations Fe containing catalysts were obtained from published values and from measurements of catalytic activity and turnover. NADH-Q reductase was determined from the di¡erence between total £avin (0.541 nmol mg protein31 [63]) minus £avin associated with succinate dehydrogenase, where one £avin equivalent exists per NADH-Q or succinate dehydrogenase equivalent. Succinate dehydrogenase was assumed to be equimolar with cytochrome b1 . Cytochrome b1 and cytochrome oxidase were obtained from [63]. Aconitase concentration was determined from enzyme activity of E. coli (63.5 nmol cis-aconitate min31 mg protein31 [64]), turnover of puri¢ed enzyme in yeast (50 Wmol cis-aconitate min31 ), and molecular weight (68 500 g mol31 [65]). Fe-SOD concentration was determined from enzyme activity for E. coli (5.6 U mg protein31 [66]), turnover of puri¢ed bovine Cu/Zn-SOD (3300 U mg protein31 [67]), and molecular mass of Fe-SOD (40 000 g mol31 ) and Cu/Zn-SOD (32 000 g mol31 ) [68]. Concentration of catalase was obtained from Corynebacterium diphtheridae [39]. b Includes heme and non-heme (Fe-S clusters) Fe. c Iron content normalized per cell assuming 1.55U10313 g protein cell31 . d Iron content normalized per cellular C assuming 0.17 pg C Wm33 [69] and a cell volume of 2.5 Wm3 [70]. e [70].

mined for this oceanic region [34]. The result is very similar to that obtained previously [37]. The total biogenic Fe that we calculated (V17 pM) falls well below the 560 pM particulate Fe previously reported for the subarctic Paci¢c Ocean [40] suggesting that lithogenic and/or detrital Fe pools are large and/or the biogenic Fe pool is signi¢cantly underestimated.

Fig. 2 illustrates our current understanding of Fe cycling among biogenic pools in these waters. Recently, Price and Morel [18] expanded the Station Papa biological Fe budget to include metazoan and protozoan grazers using Fe:C ratios [41] and biomass estimates [42,43]. Addition of the Fe contained in these organisms increases the calculated

Table 2 Iron content and steady-state Fe assimilation rates of autotrophic and heterotrophic plankton at Station Papa in the subarctic Paci¢c Ocean (after [37]) Plankton group

Biomass (Wmol C l31 )

Fe quotaa (Wmol Fe mol C31 )

Biogenic Fe (pmol l31 )

Turnover rate (day31 )

Fe uptake rate (fmol Fe l31 h31 )

Eukaryotic phytoplankton Cyanobacteria Heterotrophic bacteria Total

1.41 þ 0.49 0.24 þ 0.16 1.18 þ 0.37 2.83

3.7 þ 2.3 19 6.05 þ 2.5

5.22 4.56 7.13 16.9

0.25 0.2 0.06

54 38 18 110

a Eukaryotic phytoplankton and heterotrophic bacteria Fe quotas measured at Station Papa in the subarctic Paci¢c Ocean during three consecutive years (average þ S.D. [34]). Cyanobacteria Fe quotas are laboratory measurements [38].

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Fig. 2. Biological Fe cycle in the upper and deep ocean in the open subarctic Paci¢c Ocean. Circles represent the relative biogenic iron pools in the upper ocean derived from ¢eld measurements of Fe:C ratios of plankton and annual averages of their C standing stocks, as reported in Table 1. Arrows indicate the £ow among Fe pools, including iron uptake by heterotrophic bacteria, phototrophic cyanobacteria and eukaryotic phytoplankton. Iron inputs to the surface ocean are restricted to aeolian deposition [14] and deep water upwelling [15]. Loss from the surface is mediated by sinking particles. Note that protozoa and metazoa, which contribute to primary production and graze on smaller plankton, are not included in this model. They likely represent a signi¢cant biogenic iron pool and contribute to the sinking particle £ux and surface remineralization of Fe [18].

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Table 3 Biogenic Fe budget for surface waters of the northern Sargasso Sea ( 6 200 m) Plankton group

Biomassa (Wmol C l31 )

Fe quotab (Wmol Fe mol C31 )

Biogenic Fe (pmol l31 )

Eukaryotic phytoplankton Cyanobacteria Prochlorococcus Heterotrophic bacteria Heterotrophic protozoa Total Trichodesmium Total

0.47 þ 0.09 0.16 þ 0.48 0.39 0.57 þ 0.12 0.31 þ 0.01 1.90 0.12 þ 0.1 colonies l31

3.0 7.5^19 7.5^19 7.5 12

1.41 1.2^3.04 2.92^7.41 4.27 3.72 13.52^19.85 27.6 41.12^47.45

0.23 nmol colony31

a

Data are averages of spring and fall data as reported [46,47]. Biomass of Trichodesmium is reported as abundance in colonies l31 [51]. Data for eukaryotic phytoplankton and heterotrophic bacteria are from laboratory measurements as in Tortell et al. [37]. Iron quotas for protozoa are taken from Chase and Price [41]. For the photosynthetic prokaryotes, we have assumed upper and lower limits for Fe:C ratios corresponding to laboratory measurements of cyanobacteria and heterotrophic bacteria. Iron content of Trichodesmium is reported per colony [53]. b

biogenic Fe to V38 pM with bacteria accounting for 40% of the total. The biogenic Fe budget of the subarctic North Paci¢c may not be representative of tropical and subtropical oceanic ecosystems where photosynthetic prokaryotes dominate phytoplankton biomass [44] and primary productivity [45]. In these waters, bacteria may be even more important in Fe cycling. To test this idea, we have constructed a preliminary biological Fe budget for the northern Sargasso Sea (Table 3) using published biomass data [46,47]. and laboratory Fe quota measurements (which include a number of Sargasso Sea isolates [37]). A salient feature of this open ocean region is the abundance of Prochlorococcus [48,49] that are not found at Station Papa. Because no Fe quotas have been reported for these organisms and the cyanobacterial estimates are based on a single Paci¢c Ocean strain we have chosen upper and lower limits of 19 and 7.5 Wmol Fe mol C31 for photosynthetic prokaryotes. (These values represent Fe:C ratios of cyanobacteria and heterotrophic bacteria measured in the laboratory.) If we include in the budget only unicellular organisms that assimilate Fe directly from solution, prokaryotes account for 86^91% of the biogenic Fe depending on our choice of Fe:C ratios (Table 3). Considering protozoan grazers in the model reduces the contribution of prokaryotes to 62^74% and brings the total biological Fe to ca. 20 pM. This value is similar to the biogenic Fe calculated for the subarctic Paci¢c yet substantially less than the 750 pM particulate Fe measured in the northwest Atlantic [50]. Despite the

higher abundance of photosynthetic prokaryotes in the Sargasso Sea, heterotrophic bacteria appear to contribute substantially to the biological Fe pool constituting between ca. 30^50% of the Fe stocks in producers (Table 3). The above calculation does not include photosynthetic Trichodesmium colonies that are abundant in tropical waters [51,52] and have an elevated Fe demand associated with N2 ¢xation [53]. Such high Fe requirements may possibly limit the abundance of these organisms and the extent of N2 ¢xation in the sea [53]. As shown in Table 3, it appears that Trichodesmium could potentially constitute the majority of biological Fe in subtropical gyres containing as much Fe as all other organisms combined. The means by which they acquire this Fe is uncertain although some novel mechanisms such as utilization of colloidal Fe have been proposed [53]. In addition to contributing substantially to standing stocks of biogenic Fe, prokaryotes appear to assimilate a large fraction of the dissolved Fe. Steady-state Fe uptake rates calculated from the total Fe in each biological pool and published estimates of mean turnover times suggest that heterotrophic bacteria and cyanobacteria account for approximately 20 and 30% of total community uptake respectively at Station Papa [37] (Table 2, Fig. 2). These calculations are supported by direct measurements of size-fractionated 55 Fe uptake rates which show that 55 þ 20% of Fe assimilation at this station is due to prokaryotes (0.2^1.0 Wm) [34]. Normalized to carbon biomass, bacterial Fe uptake rates

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are signi¢cantly faster, by 1.6 times, than those of eukaryotic phytoplankton, presumably re£ecting their high Fe requirements for growth [37]. Spatial and temporal variability in biomass, growth rates, and Fe quotas of various plankton groups will undoubtedly in£uence their contributions to biogenic Fe pools and assimilation rates. Maldonado and Price [34] found that the relative contribution of bacteria and phytoplankton to community Fe uptake in the subarctic Paci¢c was signi¢cantly (P 6 0.001) correlated with their biomass ratios, but independent of their production rates. In a multiyear survey of ¢ve stations, they showed that bacteria accounted for an average of 58 þ 23% of community Fe uptake in this ocean region. Over larger spatial scales in bodies of water di¡ering greatly in trophic status, Fe assimilation by bacteria appears to vary widely and correlate poorly to bacterial production or biomass [54]. Much more data will be needed to quantify and understand the patterns of biological Fe uptake.

5. Iron limitation of bacteria in the sea While a number of studies have examined the effects of Fe de¢ciency on phytoplankton growth in coastal and oceanic waters, very little is known about the potential for Fe limitation of bacterial production. The high Fe demand of prokaryotes and the observation that Fe:C ratios of Fe-limited cultures are very similar to those in the ¢eld suggest that bacteria may su¡er Fe de¢ciency in situ [37]. Indeed, Behrenfeld et al. [55]. have shown that the indigenous populations of photoautotrophs composed primarily of Synechococcus and Prochlorococcus are Fe-stressed in the equatorial Paci¢c Ocean, even though the biomass of these organisms does not appear to increase greatly in response to Fe enrichments [6,56]. E¤cient grazing by rapidly growing protozoans seems to be at least partly responsible for this apparent paradox [57]. Despite high grazing pressure, the abundance of heterotrophic bacteria has been shown to increase in Fe-amended Equatorial Paci¢c samples [57]. This e¡ect, however, was attributed to elevated dissolved organic matter (DOM) levels associated with the stimulation of large phytoplankton growth rather than Fe limita-

tion per se. By comparison, Fe enrichment of pre¢ltered Southern Ocean water (free of phytoplankton and grazers) signi¢cantly stimulated the growth of heterotrophic bacterial populations suggesting that they are truly Fe-de¢cient [58]. It is important to note that photosynthetic and heterotrophic prokaryotes have unique requirements for light and DOM respectively whose availability can partially determine the extent to which they may be Fe-limited (e.g. [59]). Future ¢eld studies will need to consider these interactions. Tortell et al. [37]. examined the physiological effects of Fe limitation on open ocean heterotrophic bacteria and the relationship between Fe and C metabolism in these organisms. Under Fe-de¢cient culture conditions, respiratory electron transport activity was reduced resulting in a signi¢cant decrease in carbon growth e¤ciencies and an e¡ective co-limitation by Fe and C. Recent ¢eld studies examining the responses of heterotrophic bacteria to Fe and C additions have provided some preliminary support of this co-limitation hypothesis ([54]). It appears, therefore, that Fe availability may a¡ect the pathways of carbon metabolism in heterotrophic bacteria and in£uence the relative importance of the microbial loop as a `carbon sink' or `carbon link' (sensu Azam et al. [8])

6. Conclusions and future prospects It is now becoming apparent that marine bacteria play a critical role in the biogeochemical cycling of Fe in the oceans. By virtue of their high, Fe-rich biomass in o¡shore waters these organisms take up and sequester large quantities of Fe thereby competing directly with phytoplankton for this potentially limiting resource. This competition may be mediated in part through the production of siderophores by which bacteria may be largely controlling Fe speciation and solubility in seawater. Our current understanding of Fe-bacterial interactions is based on a very small amount of data. We need much more information on the Fe quotas of a variety of bacteria (and phytoplankton) in the laboratory and in the ¢eld as well as characterization of the Fe-binding ligands. This should provide insight into how bacteria satisfy their nutritional Fe require-

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ments and elucidate the competitive interactions among di¡erent plankton groups. Future studies need to examine the extent to which bacteria are Fe-limited in situ. The use of physiological rate measurements [37] and molecular probes [60] will be critical in addressing this question. While we have focused our attention on Fe in this review, we believe that bacteria are likely to be important in the oceanic cycling of other trace metals as well. Most bioactive metals thus far examined are largely complexed by strong organic ligands of unknown origin [1]. Cyanobacteria have already been implicated as producers of Cu-speci¢c ligands [61]. Recent work suggests that limitation by trace metals other than Fe may possibly constrain phytoplankton growth in the sea [62]. The extent to which this may be true of bacteria remains to be discovered.

Acknowledgments Franc°ois Morel, Phoebe Lam, Klaus Keller and Anne Krapiel provided helpful comments. Funding for this work was provided by grants from the Natural Sciences and Engineering Research Council of Canada.

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