Anne M. L. Kraepiel

Geochemistry of trace metals in the Gironde estuary

Abstract Using clean techniques, we measured the dissolved, particulate, and (by cross-flow filtration) colloidal fractions of Cd, Ni, Zn, Cu, Pb, Mn, and Fe in the Gironde, an estuary in southwestern France. The fractions of the particulate riverine metals that are apparently mobilized in the estuary vary from >90% for Cd to less than 2% for Pb. Observed mid-salinity maxima for Cd, Ni, and Zn are well reproduced by a simple steady-state conservation model which accounts for the inorganic complexation of the metals by seawater anions. The concentration profiles of other metals, except Fe, can also be modeled by choosing an appropriate desorbable fraction and maintaining equilibrium between particles and solution. While colloidal iron decreases rapidly at low salinities, the colloidal concentrations of the other metals are quasi-conservative in the estuary. It appears that the colloidal fraction contains both iron oxide particles that separate from the rest by coagulation and organic macromolecules which bind most of the other metals and remain in solution.

Acquisition of inorganic carbon by the marine diatom Thalassiosira weissflogii

Recent data on the physiology of inorganic carbon acquisition by the model marine diatom Thalassiosira weissflogii (Grunow) demonstrate the importance of the catalytic equilibration of HCO3-and CO2by carbonic anhydrases located in the periplasm and in the cytoplasm. These enzymes can use Zn, Co or Cd as their metal centre, and their activity increases at low ambient CO2. The silica frustule provides buffering for extracellular CA activity, The transmembrane transport of CO2 may occur by passive diffusion. Under CO2 limitation, the cytoplasmic HCO3-is used to form malate and oxaloacetic acid via phosphoenolpyruvate carboxylase. It appears that subsequent decarboxylation of these compounds in the chloroplast regenerates CO2 near the site of Rubisco, and thus provides the organism with an effective unicellular C4 photosynthetic pathway. These results, together with other published data, bring up two major questions regarding inorganic carbon acquisition in diatoms: What is the major species of inorganic carbon (CO2 or HCO3-) transported across the membrane under natural conditions? And what is the form of carbon (inorganic or organic) accumulated by the cells?

Multiple roles of siderophores in free-living nitrogen-fixing bacteria

Free-living nitrogen-fixing bacteria in soils need to tightly regulate their uptake of metals in order to acquire essential metals (such as the nitrogenase metal cofactors Fe, Mo and V) while excluding toxic ones (such as W). They need to do this in a soil environment where metal speciation, and thus metal bioavailability, is dependent on a variety of factors such as organic matter content, mineralogical composition, and pH. Azotobacter vinelandii, a ubiquitous gram-negative soil diazotroph, excretes in its external medium catechol compounds, previously identified as siderophores, that bind a variety of metals in addition to iron. At low concentrations, complexes of essential metals (Fe, Mo, V) with siderophores are taken up by the bacteria through specialized transport systems. The specificity and regulation of these transport systems are such that siderophore binding of excess Mo, V or W effectively detoxifies these metals at high concentrations. In the topsoil (leaf litter layer), where metals are primarily bound to plant-derived organic matter, siderophores extract essential metals from natural ligands and deliver them to the bacteria. This process appears to be a key component of a mutualistic relationship between trees and soil diazotrophs, where tree-produced leaf litter provides a living environment rich in organic matter and micronutrients for nitrogen-fixing bacteria, which in turn supply new nitrogen to the ecosystem.

Molybdenum and phosphorus interact to constrain asymbiotic nitrogen fixation in tropical forests.

Biological di-nitrogen fixation (N2) is the dominant natural source of new nitrogen to land ecosystems. Phosphorus (P) is thought to limit N2 fixation in many tropical soils, yet both molybdenum (Mo) and P are crucial for the nitrogenase reaction (which catalyzes N2 conversion to ammonia) and cell growth. We have limited understanding of how and when fixation is constrained by these nutrients in nature. Here we show in tropical forests of lowland Panama that the limiting element on asymbiotic N2 fixation shifts along a broad landscape gradient in soil P, where Mo limits fixation in P-rich soils while Mo and P co-limit in P-poor soils. In no circumstance did P alone limit fixation. We provide and experimentally test a mechanism that explains how Mo and P can interact to constrain asymbiotic N2 fixation. Fixation is uniformly favored in surface organic soil horizons - a niche characterized by exceedingly low levels of available Mo relative to P. We show that soil organic matter acts to reduce molybdate over phosphate bioavailability, which, in turn, promotes Mo limitation in sites where P is sufficient. Our findings show that asymbiotic N2 fixation is constrained by the relative availability and dynamics of Mo and P in soils. This conceptual framework can explain shifts in limitation status across broad landscape gradients in soil fertility and implies that fixation depends on Mo and P in ways that are more complex than previously thought.

Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia.

Significance Biological nitrogen fixation is the main route by which nitrogen enters the biosphere. This reaction is catalyzed by nitrogenase, a metalloenzyme that exists in forms containing molybdenum, vanadium, or iron only. The contribution of the “alternative” vanadium and iron-only nitrogenases to nitrogen fixation in the present and the past is unknown. Here we show that the nitrogen isotopic composition (15N to 14N ratio) of biomass generated from nitrogen fixation by alternative nitrogenases is significantly and characteristically lower than biomass produced by molybdenum nitrogenases. In light of these results, nitrogen isotope measurements in ancient sediments imply an important role for iron-only nitrogenases in nitrogen fixation within certain anoxic, molybdenum-limited ancient environments. 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.

Essential metals for nitrogen fixation in a free-living N2-fixing bacterium: chelation, homeostasis and high use efficiency

Biological nitrogen fixation, the main source of new nitrogen to the Earths ecosystems, is catalysed by the enzyme nitrogenase. There are three nitrogenase isoenzymes: the Mo-nitrogenase, the V-nitrogenase and the Fe-only nitrogenase. All three types require iron, and two of them also require Mo or V. Metal bioavailability has been shown to limit nitrogen fixation in natural and managed ecosystems. Here, we report the results of a study on the metal (Mo, V, Fe) requirements of Azotobacter vinelandii, a common model soil diazotroph. In the growth medium of A. vinelandii, metals are bound to strong complexing agents (metallophores) excreted by the bacterium. The uptake rates of the metallophore complexes are regulated to meet the bacterial metal requirement for diazotrophy. Under metal-replete conditions Mo, but not V or Fe, is stored intracellularly. Under conditions of metal limitation, intracellular metals are used with remarkable efficiency, with essentially all the cellular Mo and V allocated to the nitrogenase enzymes. While the Mo-nitrogenase, which is the most efficient, is used preferentially, all three nitrogenases contribute to N₂ fixation in the same culture under metal limitation. We conclude that A. vinelandii is well adapted to fix nitrogen in metal-limited soil environments.

Vanadium requirements and uptake kinetics in the dinitrogen-fixing bacterium Azotobacter vinelandii.

ABSTRACT Vanadium is a cofactor in the alternative V-nitrogenase that is expressed by some N2-fixing bacteria when Mo is not available. We investigated the V requirements, the kinetics of V uptake, and the production of catechol compounds across a range of concentrations of vanadium in diazotrophic cultures of the soil bacterium Azotobacter vinelandii. In strain CA11.70, a mutant that expresses only the V-nitrogenase, V concentrations in the medium between 10−8 and 10−6 M sustain maximum growth rates; they are limiting below this range and toxic above. A. vinelandii excretes in its growth medium micromolar concentrations of the catechol siderophores azotochelin and protochelin, which bind the vanadate oxoanion. The production of catechols increases when V concentrations become toxic. Short-term uptake experiments with the radioactive isotope 49V show that bacteria take up the V-catechol complexes through a regulated transport system(s), which shuts down at high V concentrations. The modulation of the excretion of catechols and of the uptake of the V-catechol complexes allows A. vinelandii to precisely manage its V homeostasis over a range of V concentrations, from limiting to toxic.

Natural and anthropogenic processes contributing to metal enrichment in surface soils of central Pennsylvania

Metals in soils may positively or negatively affect plants as well as soil micro-organisms and mesofauna, depending on their abundance and bioavailability. Atmospheric deposition and biological uplift commonly result in metal enrichment in surface soils, but the relative importance of these processes is not always resolved. Here, we used an integrated approach to study the cycling of phosphorus and a suite of metals from the soil to the canopy (and back) in a temperate watershed. The behavior of elements in these surface soils fell into three categories. First, Al, Fe, V, Co, and Cr showed little to no enrichment in the top soil layers, and their concentrations were determined primarily by soil production fluxes with little influence of either atmospheric inputs or biological activity. Second, P, Cu, Zn and Cd were moderately enriched in surface soils due to a combination of atmospheric deposition and biological uplift. Among the metals we studied, Cu, Zn and Cd concentrations in surface soils were the most sensitive to changes in atmospheric deposition fluxes. Finally, Mo and Mn showed strong enrichment in the top soil layer that could not be explained strictly by either current atmospheric deposition or biological recycling processes, but may reflect both their unique chemistry and remnants of past anthropogenic fluxes. Mn has a long residence time in the soil partly due to intense biological uplift that retains Mn in the top soil layer. Mo, in spite of the high solubility of molybdate, remains in the soil because of strong binding to natural organic matter. This study demonstrates the need to consider simultaneously the vegetation and the soils to understand elemental distribution within soil profiles as well as cycling within watersheds.

Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current biological nitrogen fixation estimates

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.

Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments

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.