McRose, D. L., A. Lee, S. H. Kopf, O. Baars, A.M.L. Kraepiel, D. M. Sigman, F. M. M. Morel, and X. Zhang. “Effect of Iron Limitation on the Isotopic Composition of Cellular and Released Fixed Nitrogen in Azotobacter vinelandii.Geochimica et Cosmochimica Acta 244 (2019): 12-23. DOI: 10.1016/j.gca.2018.09.023Abstract
Most biological nitrogen transformations have characteristic kinetic isotope effects used to track these processes in modern and past environments. The isotopic fractionation associated with nitrogen fixation, the only biological source of fixed nitrogen (N), provides a particularly important constraint for studies of nitrogen cycling. Nitrogen fixation using the ‘canonical’ Mo-nitrogenase produces biomass with a δ15N value of ca. −1‰ (vs. atmospheric N2). If the ‘alternative’ V- and Fe-only nitrogenases are used, biomass δ15N can be between −6‰ and −7‰. These biomass values are assumed to be relatively invariant and to reflect the cellular level expressed isotope effect of nitrogen fixation. However, field and laboratory studies report wide ranges of diazotrophic biomass δ15N (from −3.6‰ to +0.5‰ for Mo-based nitrogen fixation). This variation could be partly explained by the release of dissolved organic N (DON) that is isotopically distinct from biomass. The model nitrogen fixer Azotobacter vinelandii secretes siderophores, small molecules that aid in Fe uptake and can comprise >30% of fixed nitrogen. To test whether siderophores (and other released N) can decouple biomass δ15N from the isotope effect of nitrogen fixation we measured the isotopic composition of biomass and released N in Fe-limited A. vinelandii cultures fixing nitrogen with Mo- and V-nitrogenases. We report that biomass δ15N was elevated under Fe limitation with a maximum value of +1.2‰ for Mo-based nitrogen fixation. Regardless of the nitrogenase isozyme used, released nitrogen δ15N was also 2–3‰ lower than biomass δ15N. Siderophore nitrogen was found to have a slightly higher δ15N than the rest of the DON pool but was still produced in large enough concentrations to account for increases in biomass δ15N. The low δ15N of siderophores (relative to biomass) is consistent with what is known from compound specific isotope studies of the amino acids used in siderophore biosynthesis, and indicates that other amino-acid derived siderophores should also have a low δ15N. The implications for studies of nitrogen fixation are discussed.
Zhang, X., O. Baars, and F. M. M. Morel. “Genetic, structural, and functional diversity of low- and high-affinity siderophores in strains of nitrogen fixing Azotobacter chroococcum. doi:10.1039/c8mt00236c.” Metallomics (2018). DOI: 10.1039/C8MT00236CAbstract
To increase iron (Fe) bioavailability in surface soils, microbes secrete siderophores, chelators with widely
varying Fe affinities. Strains of the soil bacterium Azotobacter chroococcum (AC), plant-growth
promoting rhizobacteria used as agricultural inoculants, require high Fe concentrations for aerobic
respiration and nitrogen fixation. Recently, A. chroococcum str. NCIMB 8003 was shown to synthesize
three siderophore classes: (1) vibrioferrin, a low-affinity a-hydroxy carboxylate (pFe = 18.4),
(2) amphibactins, high-affinity tris-hydroxamates, and (3) crochelin A, a high-affinity siderophore with
mixed Fe-chelating groups (pFe = 23.9). The relevance and specific functions of these siderophores
in AC strains remain unclear. We analyzed the genome and siderophores of a second AC strain,
A. chroococcum str. B3, and found that it also produces vibrioferrin and amphibactins, but not crochelin
A. Genome comparisons indicate that vibrioferrin production is a vertically inherited, conserved strategy
for Fe uptake in A. chroococcum and other species of Azotobacter. Amphibactin and crochelin
biosynthesis reflects a more complex evolutionary history, shaped by vertical gene transfer, gene gain
and loss through recombination at a genomic hotspot. We found conserved patterns of low vs. highaffinity
siderophore production across strains: the low-affinity vibrioferrin was produced by mildly Fe
limited cultures. As cells became more severely Fe starved, vibrioferrin production decreased in favor of
high-affinity amphibactins (str. B3, NCIMB 8003) and crochelin A (str. NCIMB 8003). Our results show
the evolution of low and high-affinity siderophore families and conserved patterns for their production
in response to Fe bioavailability in a common soil diazotroph.
Baars, Oliver, François M. M. Morel, and Xinning Zhang. “The purple non-sulfur bacterium Rhodopseudomonas palustris produces novel petrobactin related siderophores under aerobic and anaerobic conditions.” Environmental Microbiology 20, no. 5 (2018): 1667-1676. DOI:10.1111/1462-2920.14078Abstract
Many bacteria produce siderophores to bind and take up Fe(III), an essential trace metal with extremely low solubility in oxygenated environments at circumneutral pH. The purple non‐sulfur bacterium Rhodopseudomonas palustris str. CGA009 is a metabolically versatile model organism with high iron requirements that is able to grow under aerobic and anaerobic conditions. Siderophore biosynthesis has been predicted by genomic analysis, however, siderophore structures were not identified. Here, we elucidate the structure of two novel siderophores from R. palustris: rhodopetrobactin A and B. Rhodopetrobactins are structural analogues of the known siderophore petrobactin in which the Fe chelating moieties are conserved, including two 3,4‐dihydroxybenzoate and a citrate substructure. In the place of two spermidine linker groups in petrobactin, rhodopetrobactins contain two 4,4′‐diaminodibutylamine groups of which one or both are acetylated at the central amine. We analyse siderophore production under different growth modes and show that rhodopetrobactins are produced in response to Fe limitation under aerobic as well as under anaerobic conditions. Evaluation of the chemical characteristics of rhodopetrobactins indicates that they are well suited to support Fe acquisition under variable oxygen and light conditions.
Baars, Oliver, Xinning Zhang, Marcus I. Gibson, Alan T. Stone, François M. M. Morel, and Mohammad R. Seyedsayamdost. “Crochelins, siderophores with a novel iron-chelating moiety from the nitrogen-fixing bacterium Azotobacter chroococcum.” Angewandte Chemie 57, no. 2 (2017): 536-541. DOI: 10.1001/anie.201709720/epdfAbstract
Microbes utilize siderophores to access essential iron resources. Over 500 siderophores are known, but they utilize a small set of common moieties to bind iron. Azotobacter chroococcum expresses iron-rich nitrogenases with which it reduces N2. Though an important agricultural inoculant, the structures of its iron-binding molecules remain unknown. Herein, we examine the 'chelome' of A. chroococcum using a small molecule discovery and bioinformatics approach. We find that it produces vibrioferrin and amphibactins as well as a novel family of siderophores, the crochelins. Detailed characterization shows that the most abundant member, crochelin A, binds iron in a hexadentate fashion using a new iron-chelating γ-amino acid. Insights into the biosynthesis of crochelins and the mechanism by which iron may be removed upon import of the holo-siderophore are presented. Our work expands the repertoire of iron-chelating moieties in microbial siderophores.
McRose, Darcy L., Xinning Zhang, A.M.L. Kraepiel, and F. M. M. Morel. “Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments.” Frontiers in Microbiology 8:, no. 267 (2017). DOI:10.3389/fmicb.2017.00267Abstract

The nitrogenase enzyme, which catalyzes the reduction of N2 gas to NH4+, occurs as three separate isozyme that use Mo, Fe-only, or V. The majority of global nitrogen fixation is attributed to the more efficient 'canonical' Mo-nitrogenase, whereas Fe-only and V-('alternative') nitrogenases are often considered 'backup' enzymes, used when Mo is limiting. Yet, the environmental distribution and diversity of alternative nitrogenases remains largely unknown. We searched for alternative nitrogenase genes in sequenced genomes and used PacBio sequencing to explore the diversity of canonical (nifD) and alternative (anfD and vnfD) nitrogenase amplicons in two coastal environments: the Florida Everglades and Sippewissett Marsh (MA). Genome-based searches identified an additional 25 species and 10 genera not previously known to encode alternative nitrogenases. Alternative nitrogenase amplicons were found in both Sippewissett Marsh and the Florida Everglades and their activity was further confirmed using newly developed isotopic techniques. Conserved amino acid sequences corresponding to cofactor ligands were also analyzed in anfD and vnfD amplicons, offering insight into environmental variants of these motifs. This study increases the number of available anfD and vnfD sequences ∼20-fold and allows for the first comparisons of environmental Mo-, Fe-only, and V-nitrogenase diversity. Our results suggest that alternative nitrogenases are maintained across a range of organisms and environments and that they can make important contributions to nitrogenase diversity and nitrogen fixation.

Darnajoux, Romain, Xinning Zhang, Darcy L. McRose, Jolanta Miadlikowska, Francois Lutzoni, AnneM.L. Kraepiel, and Jean-Philippe Bellenger. “Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current BNF estimates.” New Phytologist 213, no. 2 (2017): 680-689. DOI: 10.1111/nph.14166Abstract
  • Cryptogamic species and their associated cyanobacteria have attracted the attention of biogeochemists because of their critical roles in the nitrogen cycle through symbiotic and asymbiotic biological fixation of nitrogen (BNF). BNF is mediated by the nitrogenase enzyme, which, in its most common form, requires molybdenum at its active site. Molybdenum has been reported as a limiting nutrient for BNF in many ecosystems, including tropical and temperate forests. Recent studies have suggested that alternative nitrogenases, which use vanadium or iron in place of molybdenum at their active site, might play a more prominent role in natural ecosystems than previously recognized.
  • Here, we studied the occurrence of vanadium, the role of molybdenum availability on vanadium acquisition and the contribution of alternative nitrogenases to BNF in the ubiquitous cyanolichen Peltigera aphthosa s.l.
  • We confirmed the use of the alternative vanadium‐based nitrogenase in the Nostoc cyanobiont of these lichens and its substantial contribution to BNF in this organism. We also showed that the acquisition of vanadium is strongly regulated by the abundance of molybdenum.
  • These findings show that alternative nitrogenase can no longer be neglected in natural ecosystems, particularly in molybdenum‐limited habitats.
Zhang, Xinning, Darcy L. McRose, Romain Darnajoux, J-P Bellenger, François M. M. Morel, and AnneM.L. Kraepiel. “Alternative nitrogenase activity in the environment and nitrogen cycle implications.” Biogeochemistry 127 (2016): 189-198. DOI: 10.1007/s10533-016-0188-6Abstract
Biological nitrogen fixation, the main natural input of fixed nitrogen into the biosphere, is catalyzed by Mo-, V-, or Fe-only nitrogenase metalloenzymes. Although “alternative” V- and Fe-only nitrogenase genes are found in many environments, the contribution of these isoenzymes to N2 fixation is unknown. Here we present a new method (ISARA, isotopic acetylene reduction assay) that distinguishes canonical Mo and alternative nitrogenase activities based on in vivo 13C fractionation of acetylene reduction to ethylene (13εMo = 13.1–14.7 ‰, 13εV = 7.5–8.8 ‰, 13εFe = 5.8–6.5 ‰). ISARA analyses indicate significant contributions of alternative nitrogen fixation in boreal cyanolichens and salt marshes (~10–40 % acetylene reduction, ~20–55 % N2 fixed). These results affect the quantitative interpretation of natural abundance 15N data or traditional acetylene reduction assays. They also invite a reexamination of the conditions under which the different nitrogenase isozymes are active and suggest significant interactions between the cycles of nitrogen and trace metals.
Baars, Oliver, X. Zhang, François M. M. Morel, and M. Seyedsayamdost. “The siderophore metabolome ofAzotobacter vinelandii.” Applied and Environmental Microbiology 82, no. 1 (2015): 27-39. DOI: 10.1128/AEM.03160-15Abstract
In this study, we performed a detailed characterization of the siderophore metabolome, or “chelome,” of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.
Zhang, Xinning, Daniel M. Sigman, François M. M. Morel, and A.M.L. Kraepiel. “Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia.” Proceedings of the National Academy of Sciences 111, no. 13 (2014): 4782-4787. DOI:10.1073/pnas.1402976111Abstract
Biological nitrogen fixation constitutes the main input of fixed nitrogen to Earth’s ecosystems, and its isotope effect is a key parameter in isotope-based interpretations of the N cycle. The nitrogen isotopic composition (δ15N) of newly fixed N is currently believed to be ∼–1‰, based on measurements of organic matter from diazotrophs using molybdenum (Mo)-nitrogenases. We show that the vanadium (V)- and iron (Fe)-only “alternative” nitrogenases produce fixed N with significantly lower δ15N (–6 to –7‰). An important contribution of alternative nitrogenases to N2 fixation provides a simple explanation for the anomalously low δ15N (<–2‰) in sediments from the Cretaceous Oceanic Anoxic Events and the Archean Eon. A significant role for the alternative nitrogenases over Mo-nitrogenase is also consistent with evidence of Mo scarcity during these geologic periods, suggesting an additional dimension to the coupling between the global cycles of trace elements and nitrogen.
Matson, E.G., A.Z. Rosenthal, X. Zhang, and J.R. Leadbetter. “Genome-wide effects of selenium and translational uncoupling on transcription in the termite gut symbiontTreponema primitia.mBio 4, no. 6 (2013): 1-9. DOI: 10.1128/mBio.00869-13Abstract
When prokaryotic cells acquire mutations, encounter translation-inhibiting substances, or experience adverse environmental conditions that limit their ability to synthesize proteins, transcription can become uncoupled from translation. Such uncoupling is known to suppress transcription of protein-encoding genes in bacteria. Here we show that the trace element selenium controls transcription of the gene for the selenocysteine-utilizing enzyme formate dehydrogenase (fdhFSec) through a translation-coupled mechanism in the termite gut symbiont Treponema primitia, a member of the bacterial phylum Spirochaetes. We also evaluated changes in genome-wide transcriptional patterns caused by selenium limitation and by generally uncoupling translation from transcription via antibiotic-mediated inhibition of protein synthesis. We observed that inhibiting protein synthesis in T. primitia influences transcriptional patterns in unexpected ways. In addition to suppressing transcription of certain genes, the expected consequence of inhibiting protein synthesis, we found numerous examples in which transcription of genes and operons is truncated far downstream from putative promoters, is unchanged, or is even stimulated overall. These results indicate that gene regulation in bacteria allows for specific post-initiation transcriptional responses during periods of limited protein synthesis, which may depend both on translational coupling and on unclassified intrinsic elements of protein-encoding genes.
Rosenthal, A.Z., X. Zhang, K.S. Lucey, E.A. Ottesen, V. Trivedi, H.M.T. Choi, N.A. Pierce, and J.R. Leadbetter. “Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy.” Proceedings of the National Academy of Sciences 110, no. 40 (2013): 16163-16168. DOI: 10.1073/pnas.1307876110Abstract



Zhang, X., and J.R. Leadbetter. “Evidence for cascades of perturbation and adaptation in the metabolic genes of higher termite gut symbionts.” mBIO 3, no. 4 (2012): e00223-12. DOI: 10.1128/mBio.00223-12Abstract
Termites and their gut microbes engage in fascinating dietary mutualisms. Less is known about how these complex symbioses have evolved after first emerging in an insect ancestor over 120 million years ago. Here we examined a bacterial gene, formate dehydrogenase (fdhF), that is key to the mutualism in 8 species of “higher” termite (members of the Termitidae, the youngest and most biomass-abundant and species-rich termite family). Patterns of fdhF diversity in the gut communities of higher termites contrasted strongly with patterns in less-derived (more-primitive) insect relatives (wood-feeding “lower” termites and roaches). We observed phylogenetic evidence for (i) the sweeping loss of several clades of fdhF that may reflect extinctions of symbiotic protozoa and, importantly, bacteria dependent on them in the last common ancestor of all higher termites and (ii) a radiation of genes from the (possibly) single allele that survived. Sweeping gene loss also resulted in (iii) the elimination of an entire clade of genes encoding selenium (Se)-independent enzymes from higher termite gut communities, perhaps reflecting behavioral or morphological innovations in higher termites that relaxed preexisting environmental limitations of Se, a dietary trace element. Curiously, several higher termite gut communities may have subsequently reencountered Se limitation, reinventing genes for Se-independent proteins via convergent evolution. Lastly, the presence of a novel fdhF lineage within litter-feeding and subterranean higher (but not other) termites may indicate recent gene “invasion” events. These results imply that cascades of perturbation and adaptation by distinct evolutionary mechanisms have impacted the evolution of complex microbial communities in a highly successful lineage of insects.
Zhang, X., E.G. Matson, and J.R. Leadbetter. “Genes for selenium dependent and independent formate dehydrogenase in the gut microbial communities of 3 lower, wood-feeding termites and a wood-feeding roach.” Environmental Microbiology 13, no. 2 (2011): 307-23. DOI: 10.1128/mBio.00223-12Abstract

The bacterial Wood–Ljungdahl pathway for CO2-reductive acetogenesis is important for the nutritional mutualism occurring between wood-feeding insects and their hindgut microbiota. A key step in this pathway is the reduction of CO2 to formate, catalysed by the enzyme formate dehydrogenase (FDH). Putative selenocysteine- (Sec) and cysteine- (Cys) containing paralogues of hydrogenase-linked FDH (FDHH) have been identified in the termite gut acetogenic spirochete, Treponema primitia, but knowledge of their relevance in the termite gut environment remains limited. In this study, we designed degenerate PCR primers for FDHH genes (fdhF) and assessed fdhF diversity in insect gut bacterial isolates and the gut microbial communities of termites and cockroaches. The insects examined herein represent three wood-feeding termite families, Termopsidae, Kalotermitidae and Rhinotermitidae (phylogenetically ‘lower’ termite taxa); the wood-feeding roach family Cryptocercidae (the sister taxon to termites); and the omnivorous roach family Blattidae. Sec and Cys FDHH variants were identified in every wood-feeding insect but not the omnivorous roach. Of 68 novel alleles obtained from inventories, 66 affiliated phylogenetically with enzymes from T. primitia. These formed two subclades (37 and 29 phylotypes) almost completely comprised of Sec-containing and Cys-containing enzymes respectively. A gut cDNA inventory showed transcription of both variants in the termite Zootermopsis nevadensis (family Termopsidae). The gene patterns suggest that FDHH enzymes are important for the CO2-reductive metabolism of uncultured acetogenic treponemes and imply that the availability of selenium, a trace element, shaped microbial gene content in the last common ancestor of dictyopteran, wood-feeding insects, and continues to shape it to this day.

Zhang, X., Matson, E.G., and J.R. Leadbetter. “Selenium controls transcription of paralogous formate dehydrogenase genes in the termite gut acetogen, Treponema primitia.” Environmental Microbiology 12 (2010): 2245-2258. DOI: 10.1111/j.1462-2920.2010.02330.xAbstract

The termite gut spirochete, Treponema primitia, is a CO(2)-reductive acetogen that is phylogenetically distinct from other distantly related and more extensively studied acetogens such as Moorella thermoacetica. Research on T. primitia has revealed details about the role of spirochetes in CO(2)-reductive acetogenesis, a process important to the mutualism occurring between termites and their gut microbial communities. Here, a locus of the T. primitia genome containing Wood-Ljungdahl pathway genes for CO(2)-reductive acetogenesis was sequenced. This locus contained methyl-branch genes of the pathway (i.e. for the reduction of CO(2) to the level of methyl-tetrahydrofolate) including paralogous genes for cysteine and selenocysteine (Sec) variants of formate dehydrogenase (FDH) and genes for Sec incorporation. The FDH variants affiliated phylogenetically with hydrogenase-linked FDH enzymes, suggesting that T. primitia FDH enzymes utilize electrons derived directly from molecular H(2). Sub-nanomolar concentrations of selenium decreased transcript levels of the cysteine variant FDH gene. Selenium concentration did not markedly influence the level of mRNA upstream of the Sec-codon in the Sec variant FDH; however, the level of transcript extending downstream of the Sec-codon increased incrementally with increasing selenium concentrations. The features and regulation of these FDH genes are an indication that T. primitia may experience dynamic selenium availability in its H(2)-rich gut environment.

Zhang, X., A. L. Gillespie, and A.L. Sessions. “Large D/H variations in bacterial lipids reflect central metabolic pathways.” Proceedings of the National Academy of Sciences USA 106 (2009): 12580-12586. DOI: 10.1073/pnas.0903030106Abstract

Large hydrogen-isotopic (D/H) fractionations between lipids and growth water have been observed in most organisms studied to date. These fractionations are generally attributed to isotope effects in the biosynthesis of lipids, and are frequently assumed to be approximately constant for the purpose of reconstructing climatic variables. Here, we report D/H fractionations between lipids and water in 4 cultured members of the phylum Proteobacteria, and show that they can vary by up to 500‰ in a single organism. The variation cannot be attributed to lipid biosynthesis as there is no significant change in these pathways between cultures, nor can it be attributed to changing substrate D/H ratios. More importantly, lipid/water D/H fractionations vary systematically with metabolism: chemoautotrophic growth (approximately −200 to −400‰), photoautotrophic growth (−150 to −250‰), heterotrophic growth on sugars (0 to −150‰), and heterotrophic growth on TCA-cycle precursors and intermediates (−50 to +200‰) all yield different fractionations. We hypothesize that the D/H ratios of lipids are controlled largely by those of NADPH used for biosynthesis, rather than by isotope effects within the lipid biosynthetic pathway itself. Our results suggest that different central metabolic pathways yield NADPH—and indirectly lipids—with characteristic isotopic compositions. If so, lipid δD values could become an important biogeochemical tool for linking lipids to energy metabolism, and would yield information that is highly complementary to that provided by 13C about pathways of carbon fixation.

Warnecke, F., P. Luginbühl, .., Zhang, X., and et al. “Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.” Nature 450 (2007): 560-565. DOI: 10.1038/nature06269Abstract

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms1 and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis2. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding ‘higher’ Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-μl environment can be.

Vadas, T.M., X. Zhang, A.M. Curran, and B.A. Ahner. “Fate of DTPA, EDTA and EDDS in hydroponic media and effects on plant mineral nutrition.” Journal of Plant Nutrition 30 (2007): 1229- 1246. DOI: 10.1080/01904160701555119Abstract

Synthetic chelators are commonly used in hydroponic media to solubilize iron (Fe); however, the fate of these chelators is unknown. This study examined the persistence of three synthetic chelators, ethylenediaminetetraacetate (EDTA), diethylenetriaminepentaacetate (DTPA), and ethylenediaminedisuccinate (EDDS) in a bench-scale lettuce production system. The EDDS concentration decreased rapidly within 7d, most likely due to biodegradation. The EDTA and DTPA concentrations stayed steady throughout the experiments despite additions to maintain a constant volume and loss of chelator may have been due to either plant uptake or photodegradation of the chelator. Despite large differences in solution chemistry, the final shoot concentrations of iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) were similar among chelator treatments, whereas root concentrations of these same elements were highly variable. The concentration of DTPA in a commercial lettuce production system was measured and highly variable concentrations were found.