Iron acquisition from various ferric chelates and colloids was studied using iron‐limited cells of Anabaena flos‐aquae (Lyng.) Brèb UTEX 1444, a cyanobacterial strain that produces high levels of siderophores under iron limitation. Various chelators of greatly varying affinity for Fe3+ (HEDTA, EDDHA, desferrioxamine mesylate, HBED, 8‐hydroxyquinoline) were assayed for the degree of iron acquisition by iron‐limited cyanobacterial cells. Iron uptake rates (measured by graphite furnace atomic absorption spectrometry) varied approximately inversely with calculated [Fe3+] (calculated as pFe) and decreased with increasing chelator‐to‐iron ratio. No iron uptake was observed when Fe3+ was chelated with HBED, the strongest of the tested chelators. Iron‐limited Anabaena cells were able to take up iron from 8‐hydroxyquinoline (oxine or 8HQ), a compound sometimes used to quantify aquatic iron bioavailability. Iron bound to purified humic acid was poorly available but did support some growth at high humic acid concentrations. These results suggest that for cyanobacteria, even tightly bound iron is biologically available, including to a limited extent iron bound to humic acids. However, iron bound to some extremely strong chelators (e.g. HBED) is likely to be biologically unavailable.

N,N′-di(2-hydroxybenzoyl)-ethylenediamine-N,N′-diacetic acid (HBED) is a very strong Fe3+ chelator. Strategy I vascular plants, which use a reductive system for iron acquisition, similar to many green algae, are able to access iron from HBED (R.L. Chaney. 1988. J. Plant Nutr. 11: 1033–1050). However, iron-limited cells of the Strategy I green alga Chlamydomonas reinhardtii Dangeard were unable to access iron present as Fe3+–HBED. In contrast, Fe3+ chelated with hydroxyethylethylenediaminetriacetic acid (HEDTA; a weaker chelator) was rapidly taken up by iron-limited Chlamydomonas cells. Chlamydomonas ferric reduction rates with Fe3+–HBED were approximately 15% of the rate observed with Fe3+–HEDTA, suggesting that low reduction rates with Fe3+–HBED might be one factor in the low rate of iron acquisition. By contrast, iron-limited cells of the Strategy I green alga Chlorella kessleri Fott et Nováková were able to rapidly assimilate Fe3+ chelated by HBED, although ferric reduction rates with Fe3+–HBED were approximately 38% the rate of activity with Fe3+–HEDTA. Similar differential iron uptake rates for the two algal species were obtained using the strong Fe3+ chelator (and siderophore analogue) desferrioxamine B mesylate and the cyanobacterial siderophore schizokinen. These results suggest that there are differences among Strategy I green algae in their abilities to acquire Fe3+ from various ferric chelates: not all Strategy I algae can equally access tightly complexed Fe3+. Chlamydomonas appears to be the first documented Strategy I organism that is unable to acquire iron from Fe3+–HBED. These results also suggest that green algal iron acquisition from siderophores is species dependent. Finally, we suggest that iron acquisition from Fe3+–HBED might serve as an assay for an organisms’ ability to access tightly complexed iron.

Iron acquisition by iron-limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron-chelating molecules released by iron-limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron-limited cells of the cyanobacterium Anabaena flos-aquae (L.) Bory was investigated in cells resuspended in siderophore-free medium. Removal of siderophores decreased iron-uptake rates by 60% compared to siderophore-replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N-(2-hydroxyethyl) ethylenediamine-N,N¢,N¢-triacetic acid (HEDTA); log K cond = 28.64 for Fe(III)HEDTA(OH) ) ] than from a very strong chelator [N,N¢-bis(2-hydroxybenzyl)-ethylenediamine-N,N¢-diacetic acid (HBED); log K cond = 31.40 for Fe(III)HBED ) ], and increasing chelator:Fe(III) ratios decreased the Fe(III)-uptake rate; these results were evident in both short-term (4 h; absence of siderophores) and long-term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore-free conditions. The results of the short-term experiments are consistent with an Fe(III)-binding ⁄ uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature-shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)-binding mechanism. Overall, these results point to an important, siderophore-independent iron-acquisition mechanism by iron-limited cyanobacterial cells.

Cells of the green alga Chlorella kessleri Fott et Nováková use a reductive mechanism for iron acquisition. Iron-limited cells acquired iron more rapidly from a chelator with a lower stability constant for Fe3+ (hydroxyethylethylenediaminetriacetic acid (HEDTA)) than from a chelator with a higher stability constant (N,N′-di[2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED)). Furthermore, iron uptake rates decreased with increasing chelator concentrations at constant iron concentration. The negative effects of elevated HBED levels on iron uptake could be partly alleviated by the addition of Ga3+, which suggests that iron-free chelator has a negative effect on iron acquisition by competing for Fe2+ with the ferrous transport system. Furthermore, ferric reductase activity progressively decreased with increasing concentrations of both chelators (in the iron-free form). This effect was not alleviated by Ga3+ addition and was apparently caused by the direct inhibition of the reductase. Overall, we conclude that chelators with high stability constants for Fe3+ decrease iron acquisition rates by Strategy I organisms via three separate mechanisms.