Characterization of childhood exposure to environmental contaminants using stool in a semi-urban middle-class cohort from eastern Canada.

Children are exposed to various environmental organic and inorganic contaminants with effects on health outcomes still largely unknown. Many matrices (e.g., blood, urine, nail, hair) have been used to characterize exposure to organic and inorganic contaminants. The sampling of feces presents several advantages; it is non-invasive and provides a direct evaluation of the gut microbiome exposure to contaminants. The gut microbiome is a key factor in neurological development through the brain-gut axis. Its composition and disturbances can affect the neurodevelopment of children. Characterization of children exposure to contaminants is often performed on vulnerable populations (e.g., from developing countries, low-income neighborhoods, and large urban centers). Data on the exposure of children from middle-class, semi-urban, and mid-size populations to contaminants is scarce despite representing a significant fraction of the population in North America. In this study, 73 organics compounds from different chemical classes and 22 elements were analyzed in 6 years old (n = 84) and 10 years old (n = 119) children's feces from a middle-class, semi-urban, mid-size population cohort from Eastern Canada. Results show that 67 out of 73 targeted organics compounds and all elements were at least detected in one child's feces. Only caffeine (97% & 80%) and acetaminophen (28% & 48%) were detected in more than 25% of the children's feces, whereas all elements besides titanium were detected in more than 50% of the children.

In Vivo Temperature Dependency of Molybdenum and Vanadium Nitrogenase Activity in the Heterocystous Cyanobacteria Anabaena variabilis.

The reduction of atmospheric dinitrogen by nitrogenase is a key component of terrestrial nitrogen cycling. Nitrogenases exist in several isoforms named after the metal present within their active center: the molybdenum (Mo), the vanadium (V), and the iron (Fe)-only nitrogenase. While earlier in vitro studies hint that the relative contribution of V nitrogenase to total BNF could be temperature-dependent, the effect of temperature on in vivo activity remains to be investigated. In this study, we characterize the in vivo effect of temperature (3-42 °C) on the activities of Mo nitrogenase and V nitrogenase in the heterocystous cyanobacteria Anabaena variabilis ATTC 29413 using the acetylene reduction assay by cavity ring-down absorption spectroscopy. We demonstrate that V nitrogenase becomes as efficient as Mo nitrogenase at temperatures below 10-15 °C. At temperatures above 22 °C, BNF seems to be limited by O2 availability to respiration in both enzymes. Furthermore, Anabaena variabilis cultures grown in Mo or V media achieved similar growth rates at temperatures below 20 °C. Considering the average temperature on earth is 15 °C, our findings further support the role of V nitrogenase as a viable backup enzymatic system for BNF in natural ecosystems.

Molybdenum threshold for ecosystem scale alternative vanadium nitrogenase activity in boreal forests.

Biological nitrogen fixation (BNF) by microorganisms associated with cryptogamic covers, such as cyanolichens and bryophytes, is a primary source of fixed nitrogen in pristine, high-latitude ecosystems. On land, low molybdenum (Mo) availability has been shown to limit BNF by the most common form of nitrogenase (Nase), which requires Mo in its active site. Vanadium (V) and iron-only Nases have been suggested as viable alternatives to countering Mo limitation of BNF; however, field data supporting this long-standing hypothesis have been lacking. Here, we elucidate the contribution of vanadium nitrogenase (V-Nase) to BNF by cyanolichens across a 600-km latitudinal transect in eastern boreal forests of North America. Widespread V-Nase activity was detected (∼15-50% of total BNF rates), with most of the activity found in the northern part of the transect. We observed a 3-fold increase of V-Nase contribution during the 20-wk growing season. By including the contribution of V-Nase to BNF, estimates of new N input by cyanolichens increase by up to 30%. We find that variability in V-based BNF is strongly related to Mo availability, and we identify a Mo threshold of ∼250 ng·glichen-1 for the onset of V-based BNF. Our results provide compelling ecosystem-scale evidence for the use of the V-Nase as a surrogate enzyme that contributes to BNF when Mo is limiting. Given widespread findings of terrestrial Mo limitation, including the carbon-rich circumboreal belt where global change is most rapid, additional consideration of V-based BNF is required in experimental and modeling studies of terrestrial biogeochemistry.

Assessing pesticides exposure effects on the reproductive performance of a declining aerial insectivore.

In the context of increasing global environmental changes, it has become progressively important to understand the effects of human activity on wildlife populations. Declines in several avian populations have been observed since the 1970s, especially with respect to many farmland and grassland birds, which also include some aerial insectivores. Changes in farming practices referred to as agricultural intensification coincide with these major avian declines. Among those practices, increased pesticide use is hypothesized to be a key driver of avian population declines as it can lead to both toxicological and trophic effects. While numerous laboratory studies report that birds experience acute and chronic effects upon consuming pesticide treated food, little is known about the effects of the exposure to multiple pesticides on wildlife in natural settings. We monitored the breeding activities of Tree Swallows (Tachycineta bicolor) on 40 farms distributed over a gradient of agricultural intensification in southern Québec, Canada, to evaluate the presence of pesticides in their diet and quantify the exposure effects of those compounds on their reproductive performance between 2013 and 2018. We first assessed the presence of 54 active agents (or derivatives) found in pesticides in 2,081 food boluses (insects) delivered to nestlings by parents and documented their spatial distribution within our study area. Second, we assessed the effect of pesticide exposure through food (number of active agents detected and number of contaminated boluses on a given farm for a given year, while controlling for sampling effort) on clutch size as well as hatching and fledging successes and nestling's mass upon fledging. Pesticides were ubiquitous in our study system and nearly half (46%) of food boluses were contaminated by at least one active agent. Yet we found no relationship between our proxies of food contamination by pesticides and Tree Swallow reproductive performance. More studies are needed to better understand the putative role of pesticides in the decline of farmland birds and aerial insectivores as potential sublethal effects of pesticides can carry over to later life stages and impact fitness.

Efficiencies of selected biotreatments for the remediation of PAH in diluted bitumen contaminated soil microcosms.

Unconventional oils such as diluted bitumen from oil sands differs from most of conventional oils in terms of physiochemical properties and PAHs composition. This raises concerns regarding the effectiveness of current remediation strategies and protocols originally developed for conventional oil. Here we evaluated the efficiency of different biotreatment approaches, such as fungi inoculation (bioaugmentation), sludge addition (bioaugmentation/biostimulation), perennial grasses plantation (phytoremediation) and their combinations as well as natural attenuation (as control condition), for the remediation of soil contaminated by synthetic crude oil (a product of diluted bitumen) in laboratory microcosms. We specifically monitored the PAHs loss percentage (alkylated PAHs and unsubstituted 16 EPA Priority PAHs), the residue of PAHs and evaluated the ecotoxicity of soil after treatment. All treatments were highly efficient with more than ~ 80% of ∑PAHs loss after 60 days. Distinctive loss efficiencies between light PAHs (≤ 3 rings, ~ 96% average loss) and heavy PAHs (4-6 rings, ~ 29% average loss) were observed. The lowest average PAHs residue (0.10 ± 0.02 mg·kg-1, for an initial concentration of 0.29 ± 0.12 mg·kg-1) was achieved with the "sludge-plants (grasses)" combination. Sludge addition was the only treatment that achieved significantly lower ecotoxicity (3% ± 4% of growth inhibition of L. sativa) than the control (natural attenuation, 13% ± 4% of inhibition). Sludge addition, grasses plantation and "sludge-fungi combination" treatments could result in lower PAH exposure (than other treatments) in post-treated soil when using the Canadian Soil Quality Guidelines for the protection of environmental and human health for potentially carcinogenic and other PAHs.

Bacillus subtilis Modulates Its Usage of Biofilm-Bound Iron in Response to Environmental Iron Availability.

Iron (Fe) is one of the most important micronutrients for most life forms on earth. While abundant in soil, Fe bioavailability in oxic soil is very low. Under environmental conditions, bacteria need to acquire sufficient Fe to sustain growth while limiting the energy cost of siderophore synthesis. Biofilm formation might mitigate this Fe stress, since it was shown to accumulate Fe in certain Gram-negative bacteria and that this Fe could be mobilized for uptake. However, it is still unclear if, and to what extent, the amount of Fe accumulated in the biofilm can sustain growth and if the mobilization of this local Fe pool is modulated by the availability of environmental Fe (i.e., Fe outside the biofilm matrix). Here, we use a nondomesticated strain of the ubiquitous biofilm-forming soil bacterium Bacillus subtilis and stable Fe isotopes to precisely evaluate the origin of Fe during growth in the presence of tannic acid and hydroxides, used as proxies for different environmental conditions. We report that this B. subtilis strain can accumulate a large quantity of Fe in the biofilm, largely exceeding Fe associated with cells. We also report that only a fraction of biofilm-bound Fe is available for uptake in the absence of other sources of Fe in the vicinity of the biofilm. We observed that the availability of environmental Fe modulates the usage of this pool of biofilm-bound Fe. Finally, our data suggest that consumption of biofilm-bound Fe relates to the efficacy of B. subtilis to transport Fe from the environment to the biofilm, possibly through siderophores.IMPORTANCE Recent pieces of evidence suggest that Fe bound to the biofilm could assume at least two important functions, a local source of Fe for uptake and a support to extracellular metabolism, such as extracellular electron transfer. Our results show that B. subtilis can use biofilm-bound Fe for uptake only if it does not compromise Fe homeostasis of the biofilm, i.e., maintains a minimum Fe concentration in the biofilm for extracellular purposes. We propose a theoretical framework based on our results and recent literature to explain how B. subtilis manages biofilm-bound Fe and Fe uptake in response to environmental Fe availability. These results provide important insights into the management of biofilm-bound and environmental Fe by B. subtilis in response to Fe stress.

Differential gene expression associated with fungal trophic shifts along the senescence gradient of the moss Dicranum scoparium.

Bryophytes harbour microbiomes, including diverse communities of fungi. The molecular mechanisms by which perennial mosses interact with these fungal partners along their senescence gradients are unknown, yet this is an ideal system to study variation in gene expression associated with trophic state transitions. We investigated differentially expressed genes of fungal communities and their host Dicranum scoparium across its naturally occurring senescence gradient using a metatranscriptomic approach. Higher activity of fungal nutrient-related (carbon, nitrogen, phosphorus and sulfur) transporters and Carbohydrate-Active enZyme (CAZy) genes was detected toward the bottom, partially decomposed, layer of the moss. The most prominent variation in the expression levels of fungal nutrient transporters was from inorganic nitrogen-related transporters, whereas the breakdown of organonitrogens was detected as the most enriched gene ontology term for the host D. scoparium, for those transcripts having higher expression in the partially decomposed layer. The abundance of bacterial rRNA transcripts suggested that more living members of Cyanobacteria are associated with the photosynthetic layer of D. scoparium, while members of Rhizobiales are detected throughout the gametophytes. Plant genes for specific fungal-plant communication, including defense responses, were differentially expressed, suggesting that different genetic pathways are involved in plant-microbe crosstalk in photosynthetic tissues compared to partially decomposed tissues.

Iron Homeostasis in Bacillus subtilis Requires Siderophore Production and Biofilm Formation.

Iron (Fe) is the most important metal in biology. Despite its abundance, Fe is mostly present as a ferric form in soils, strongly limiting its bioavailability. To overcome the challenge of Fe acquisition, many microorganisms produce siderophores to retrieve Fe from natural sources. Another ubiquitous feature of bacteria in natural environments is biofilm formation. Previous studies showed that external Fe strongly influenced biofilm formation in several bacteria, suggesting that this microenvironment plays a mechanistic role in micronutrient acquisition for bacteria. Here, we applied a complementary set of analytical methods and deletion mutants to evaluate the role of biofilm formation, siderophore production, and their interaction in Fe homeostasis in Bacillus subtilis We observed that Fe homeostasis, i.e., active growth at a constant intracellular Fe concentration, requires both siderophore production and biofilm formation. Also, we report that in B. subtilis, both biofilm formation and siderophore production are required to achieve active Fe acquisition from the medium and to sustain normal growth. Furthermore, we provide evidence that the formation of biofilm slightly enhances the kinetics of Fe complexation by catechol siderophores and markedly improves siderophore use efficiency. These results provide new perspectives on the mechanism underlying siderophore-based acquisition of Fe in biofilm-forming bacteria.IMPORTANCE Iron acquisition is of fundamental importance for microorganisms, since this metal is generally poorly bioavailable under natural conditions. In the environment, most bacteria are found tightly packed within multicellular communities named biofilms. Here, using the soil Gram-positive bacterium Bacillus subtilis, we show that biofilm formation and the production of siderophores, i.e., small molecules specifically binding metals, are both essential to ensure Fe uptake from the medium and maintain cellular Fe homeostasis. The biofilm matrix appears to play an important role favoring the efficient usage of siderophores. Taken together, our results demonstrate a close link between biofilm formation and iron acquisition in B. subtilis, allowing a better comprehension of how bacteria can cope with metal limitation under environmental conditions.

Monitoring of prenatal exposure to organic and inorganic contaminants using meconium from an Eastern Canada cohort.

Evaluating in utero exposure to inorganic and multiclass organic contaminants is critical to better evaluate potential harmful effects on prenatal and postnatal development. The analysis of meconium, the first bowel discharge of the newborn, has been proposed as a non-invasive way to assess cumulative prenatal exposure. The aim of this study was to implement an analytical method for quantifying 72 targeted organic compounds, including pesticides, pharmaceutical compounds and daily life xenobiotics, in meconium in addition to selected elements (17 elements). We report initial monitoring results based on the analysis of 396 meconium samples from an Eastern Canada cohort (Quebec, Canada). Element contents in meconium were analysed by mass spectrometry after digestion in nitric acid and peroxide. Targeted organic compounds were extracted and purified from meconium samples by a solid-liquid extraction followed by a dispersive-SPE purification before tandem mass spectrometry analysis. Concentrations of targeted elements were within the range of concentration reported in European and US studies but were lower than concentrations found in a developing country cohort (i.e., Pb, Cd). Out of the 72 targeted organic compounds, 31 were detected at least once and 30 were quantified. Compounds with the highest frequency of detection were caffeine, detected in all samples (from 2.80 to 6186 ng g-1), followed by acetaminophen detected in 53% of the samples (up to ~402 µg g-1) and methyl paraben detected in 20% of the samples (up to ~10 µg g-1). Pesticides were detected in low frequencies (< 2%) and low concentration (< 35 ng g-1). Results show that meconium can be used to monitor prenatal exposure of foetus to a wide array of inorganic and organic contaminants.

Molybdenum and phosphorus limitation of moss-associated nitrogen fixation in boreal ecosystems.

Biological nitrogen fixation (BNF) performed by moss-associated cyanobacteria is one of the main sources of new nitrogen (N) input in pristine, high-latitude ecosystems. Yet, the nutrients that limit BNF remain elusive. Here, we tested whether this important ecosystem function is limited by the availability of molybdenum (Mo), phosphorus (P), or both. BNF in dominant mosses was measured with the acetylene reduction assay (ARA) at different time intervals following Mo and P additions, in both laboratory microcosms with mosses from a boreal spruce forest and field plots in subarctic tundra. We further used a 15 N2 tracer technique to assess the ARA to N2 fixation conversion ratios at our subarctic site. BNF was up to four-fold higher shortly after the addition of Mo, in both the laboratory and field experiments. A similar positive response to Mo was found in moss colonizing cyanobacterial biomass. As the growing season progressed, nitrogenase activity became progressively more P limited. The ARA : 15 N2 ratios increased with increasing Mo additions. These findings show that N2 fixation activity as well as cyanobacterial biomass in dominant feather mosses from boreal forests and subarctic tundra are limited by Mo availability.

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.

Intracellular Enzymes Contribution to the Biocatalytic Removal of Pharmaceuticals by Trametes hirsuta.

The use of white rot fungi (WRF) for bioremediation of recalcitrant trace organic contaminants (TrOCs) is becoming greatly popular. Biosorption and lignin modifying enzymes (LMEs) are the most often reported mechanisms of action. Intracellular enzymes, such as cytochrome P450 (CYP450), have also been suggested to contribute. However, direct evidence of TrOCs uptake and intracellular transformation is lacking. The aim of this study was to evaluate the relative contribution of biosorption, extracellular LMEs activity, TrOCs uptake, and intracellular CYP450 on the removal of six nonsteroidal anti-inflammatories (NSAIs) by Trametes hirsuta. Results show that for most tested NSAIs, LMEs activity and biosorption failed to explain the observed removal. Most tested TrOCs are quickly taken up and intracellularly transformed. Fine characterization of intracellular transformation using ketoprofen showed that CYP450 is not the sole intracellular enzyme responsible for intracellular transformation. The contribution of CYP450 in further transformation of ketoprofen byproducts is also reported. These results illustrate that TrOCs transformation by WRF is a more complex process than previously reported. Rapid uptake of TrOCs and intracellular transformation through diverse enzymatic systems appears to be important components of WRF efficiency toward TrOCs.

Is vanadium a biometal for boreal cyanolichens?

Molybdenum (Mo) nitrogenase has long been considered the predominant isoenzyme responsible for dinitrogen fixation worldwide. Recent findings have challenged the paradigm of Mo hegemony, and highlighted the role of alternative nitrogenases, such as the vanadium-nitrogenase. Here, we first characterized homeostasis of vanadium (V) along with other metals in situ in the dinitrogen fixing cyanolichen Peltigera aphthosa. These lichens were sampled in natural sites exposed to various levels of atmospheric metal deposition. These results were compared with laboratory experiments where Anabaena variabilis, which is also hosting the V-nitrogenase, and a relatively close relative of the lichen cyanobiont Nostoc, was subjected to various levels of V. We report here that V is preferentially allocated to cephalodia, specialized structures where dinitrogen fixation occurs in tri-membered lichens. This specific allocation is biologically controlled and tightly regulated. Vanadium homeostasis in lichen cephalodia exposed to various V concentrations is comparable to the one observed in Anabaena variabilis and other dinitrogen fixing organisms using V-nitrogenase. Overall, our findings support current hypotheses that V could be a more important factor in mediating nitrogen input in high latitude ecosystems than previously recognized. They invite the reassessment of current theoretical models linking metal dynamics and dinitrogen fixation in boreal and subarctic ecosystems.

Metallophore mapping in complex matrices by metal isotope coded profiling of organic ligands.

Metal isotope coded profiling (MICP) introduces a universal discovery platform for metal chelating natural products that act as metallophores, ion buffers or sequestering agents. The detection of cation and oxoanion complexing ligands is facilitated by the identification of unique isotopic signatures created by the application of isotopically pure metals.

Indoor environment, physiological factors, and diet as predictors of halogenated flame retardant levels in stool and plasma of children from a Canadian cohort.

Exposure to halogenated flame retardants (HFRs) has been associated with various adverse effects on human health. Human exposure to HFRs mainly occurs through diet, ingesting contaminated dust, and inhaling contaminated air. Understanding and characterizing the variables linked to these exposure pathways is essential for developing effective risk assessment and mitigation strategies. We investigated indoor environment quality, physiological factors, and diet as potential predictors of HFRs concentration in children's plasma and stool. A selected number of HFRs, including polybrominated diphenyl ethers (PBDEs), Dechlorane-like compounds, and emerging halogenated flame retardants, were measured in children from eastern Quebec (Canada). Information on indoor environment quality, physiological factors, and diet was obtained through self-report questionnaires. Our results show that lower brominated compounds, which are more volatile, were primarily correlated to indoor environment quality. Notably, the use of air purifiers was associated with lower BDE47 and BDE100 levels in blood and newer residential buildings were associated with higher concentrations of BDE47. A significant seasonal variation was found in stool samples, with higher levels of lower brominated PBDEs (BDE47 and BDE100) in samples collected during summer. No association between household income or maternal education degree and HFRs was found. Among emerging compounds, Dec602 and Dec603 were associated with the most variables, including the use of air dehumidifiers, air conditioning, and air purifiers, and the child's age and body fat percentage.

Effects of tungsten and titanium oxide nanoparticles on the diazotrophic growth and metals acquisition by Azotobacter vinelandii under molybdenum limiting condition.

The acquisition of essential metals, such as the metal cofactors (molybdenum (Mo) and iron (Fe)) of the nitrogenase, the enzyme responsible for the reduction of dinitrogen (N(2)) to ammonium, is critical to N(2) fixing bacteria in soil. The release of metal nanoparticles (MNPs) to the environment could be detrimental to N(2) fixing bacteria by introducing a new source of toxic metals and by interfering with the acquisition of essential metals such as Mo. Since Mo has been reported to limit nonsymbiotic N(2) fixation in many ecosystems from tropical to cold temperate, this question is particularly acute in the context of Mo limitation. Using a combination of microbiology and analytical chemistry techniques, we have evaluated the effect of titanium (Ti) and tungsten (W) oxide nanoparticles on the diazotrophic growth and metals acquisition in pure culture of the ubiquitous N(2) fixing bacterium Azotobacter vinelandii under Mo replete and Mo limiting conditions. We report that under our conditions (≤10 mg·L(-1)) TiO(2) NPs have no effects on the diazotrophic growth of A. vinelandii while WO(3) NPs are highly detrimental to the growth especially under Mo limiting conditions. Our results show that the toxicity of WO(3) NPs to A. vinelandii is due to an interference with the catechol-metalophores assisted uptake of Mo.

Complementarity of plasma and stool for the characterization of children's exposure to halogenated flame retardants: Update on analytical methods and application to a Canadian cohort.

Sixteen halogenated flame retardants including Polybrominated diphenyl ethers (PBDEs), Dechlorane-like compounds, and emerging halogenated flame retardants were measured in stool and plasma samples from children aged 8.9-13.8 years old. Samples were obtained from a Canadian cohort investigating the effect of contaminants on children's neurodevelopment in the Estrie region, Québec, Canada. The method for stool analysis developed for this study showed good recovery for all targeted compounds (73%-93%) with associated relative standard deviation (RSD) in the range of 16.0%-30.7% for most compounds except for the thermosensitive BDE209, OBTMBI, and BTBPE, which showed slightly higher RSD, i.e., 49.3%, 37.2%, and 34.9% respectively. Complementarity investigation of stool and blood samples allowed us to better characterize human exposure to these halogenated flame retardants. Exposure patterns differed significantly between stool and blood, notably in the relative abundance of BDE47, BDE100, BDE99, and BDE153 and the detection frequencies of BDE209, syn-DP, anti-DP, and DBDPE. There was no correlation between the two matrices' PBDEs concentration levels except for BDE153 (rho = 0.44, p < 0.01). Our results indicate that future epidemiological studies may benefit from the use of stool as a complementary matrix to blood, especially investigations into chemical impacts on the gut microbiome. Results also revealed that children from the GESTE cohort, an Eastern Canadian semi-rural cohort, are exposed to both historical and emergent flame retardants.

Pulcherriminic acid modulates iron availability and protects against oxidative stress during microbial interactions.

Siderophores are soluble or membrane-embedded molecules that bind the oxidized form of iron, Fe(III), and play roles in iron acquisition by microorganisms. Fe(III)-bound siderophores bind to specific receptors that allow microbes to acquire iron. However, certain soil microbes release a compound (pulcherriminic acid, PA) that, upon binding to Fe(III), forms a precipitate (pulcherrimin) that apparently functions by reducing iron availability rather than contributing to iron acquisition. Here, we use Bacillus subtilis (PA producer) and Pseudomonas protegens as a competition model to show that PA is involved in a peculiar iron-managing system. The presence of the competitor induces PA production, leading to precipitation of Fe(III) as pulcherrimin, which prevents oxidative stress in B. subtilis by restricting the Fenton reaction and deleterious ROS formation. In addition, B. subtilis uses its known siderophore bacillibactin to retrieve Fe(III) from pulcherrimin. Our findings indicate that PA plays multiple roles by modulating iron availability and conferring protection against oxidative stress during inter-species competition.

Short-term N2 fixation kinetics in a moss-associated cyanobacteria.

N(2) fixation by moss-associated cyanobacteria plays an important role in the nitrogen cycling of terrestrial ecosystems. Recent studies have mainly focused on boreal ecosystems; little is known about such association in other ecosystems. Moss-associated cyanobacteria are subject to rapid changes (hourly or less) in environmental conditions that may affect N(2) fixation kinetics. Using a recently developed method (Acetylene Reduction Assays by Cavity ring-down laser Absorption Spectroscopy, ARACAS) with higher sensitivity and sampling frequency than the conventional method, we characterize short-term kinetics of N(2) fixation by cyanobacteria on moss carpets from warm and cold temperate forests. We report the identification of a heretofore unknown multispecies true-moss-cyanobacteria diazotrophic association. We demonstrate that short-term change in abiotic variables greatly influences N(2) fixation. We also show that difference in relative proportion of two epiphytic diazotrophs is consistent with divergent influences of temperature on their N(2) fixation kinetics. Further research is needed to determine whether this difference is consistent with a latitudinal trend.

N2 fixation estimates in real-time by cavity ring-down laser absorption spectroscopy.

The most common currency for estimating N(2) fixation is acetylene reduction to ethylene. Real-time estimates of nitrogen fixation are needed to close the global nitrogen budget and these remain a critical gap in both laboratory and field experiments. We present a new method for continuous real-time measurements of ethylene production: Acetylene Reduction Assays by Cavity ring-down laser Absorption Spectroscopy (ARACAS). In ARACAS, air in the headspace of an incubation chamber is circulated with a diaphragm pump through a cavity ring-down ethylene spectrometer and back to the incubation chamber. This paper describes the new approach and its benefits compared to the conventional detection of ethylene by flame ionization detector gas chromatography. First, the detection of acetylene reduction to ethylene is non-intrusive and chemically non-destructive, allowing for real-time measurements of nitrogenase activity. Second, the measurements are made instantaneously and continuously at ppb levels, allowing for observation of real-time kinetics on time intervals as short as a few seconds. Third, the instrument can be automated for long time periods of measurement. Finally, the technique will be widely accessible by the research community as it can be readily adapted to most existing acetylene reduction protocols and is based on a modestly priced, commercially available instrument. We illustrate its use for measuring N(2) fixation using two species, the diazotrophic bacterium Azotobacter vinelandii and the lichen Peltigera praetextata. We also discuss potential limitations of the approach, primarily the implications of leaks in the analyzer, as well as future improvements.