Metabolic Performance and Fate of Electrons during Nitrate-Reducing Fe(II) Oxidation by the Autotrophic Enrichment Culture KS Grown at Different Initial Fe/N Ratios.

Autotrophic nitrate-reducing Fe(II)-oxidizing (NRFeOx) microorganisms fix CO2 and oxidize Fe(II) coupled to denitrification, influencing carbon, iron, and nitrogen cycles in pH-neutral, anoxic environments. However, the distribution of electrons from Fe(II) oxidation to either biomass production (CO2 fixation) or energy generation (nitrate reduction) in autotrophic NRFeOx microorganisms has not been quantified. We therefore cultivated the autotrophic NRFeOx culture KS at different initial Fe/N ratios, followed geochemical parameters, identified minerals, analyzed N isotopes, and applied numerical modeling. We found that at all initial Fe/N ratios, the ratios of Fe(II)oxidized to nitratereduced were slightly higher (5.11 to 5.94 at Fe/N ratios of 10:1 and 10:0.5) or lower (4.27 to 4.59 at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1) than the theoretical ratio for 100% Fe(II) oxidation being coupled to nitrate reduction (5:1). The main N denitrification product was N2O (71.88 to 96.29% at Fe/15N ratios of 10:4 and 5:1; 43.13 to 66.26% at an Fe/15N ratio of 10:1), implying that denitrification during NRFeOx was incomplete in culture KS. Based on the reaction model, on average 12% of electrons from Fe(II) oxidation were used for CO2 fixation while 88% of electrons were used for reduction of NO3- to N2O at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1. With 10 mM Fe(II) (and 4, 2, 1, or 0.5 mM nitrate), most cells were closely associated with and partially encrusted by the Fe(III) (oxyhydr)oxide minerals, whereas at 5 mM Fe(II), most cells were free of cell surface mineral precipitates. The genus Gallionella (>80%) dominated culture KS regardless of the initial Fe/N ratios. Our results showed that Fe/N ratios play a key role in regulating N2O emissions, for distributing electrons between nitrate reduction and CO2 fixation, and for the degree of cell-mineral interactions in the autotrophic NRFeOx culture KS. IMPORTANCE Autotrophic NRFeOx microorganisms that oxidize Fe(II), reduce nitrate, and produce biomass play a key role in carbon, iron, and nitrogen cycles in pH-neutral, anoxic environments. Electrons from Fe(II) oxidation are used for the reduction of both carbon dioxide and nitrate. However, the question is how many electrons go into biomass production versus energy generation during autotrophic growth. Here, we demonstrated that in the autotrophic NRFeOx culture KS cultivated at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1, ca. 12% of electrons went into biomass formation, while 88% of electrons were used for reduction of NO3- to N2O. Isotope analysis also showed that denitrification during NRFeOx was incomplete in culture KS and the main N denitrification product was N2O. Therefore, most electrons stemming from Fe(II) oxidation seemed to be used for N2O formation in culture KS. This is environmentally important for the greenhouse gas budget.

Capturing In Situ Glyphosate (De)sorption Kinetics in Floodplain Aquifer Sediment Columns: Geophysical Measurements and Reactive Transport Modeling.

Glyphosate, an ionizable organic herbicide, is frequently detected in soils and groundwater globally despite its strong retention via sorption. Understanding its apparent mobility hinges on our ability to quantify its system-specific sorption behavior, hindered by its affinity to adsorb onto sediments, yielding very low aqueous concentrations. Here, we present findings from a saturated flow-through column experiment in which we monitored glyphosate sorption onto a natural calcareous aquifer sediment, using the noninvasive geophysical method spectral induced polarization (SIP). Our kinetic sorption reactive transport model predicted the strong nonlinear reversible retention of glyphosate and reproduced the spatial profile of retained glyphosate in the sediment, with a measured maximum of 0.06 mg g-1. The strong contribution of sorption to pore fluid conduction masked the expected variations in imaginary conductivity, σ″. However, time constants derived from a Cole-Cole model matched the timing and spatial distribution of model-predicted sorbed concentration changes, increasing from 0.8 × 10-3 to 1.7 × 10-3 s with an increase in sorbed glyphosate of 0.1 mg g-1. Thus, glyphosate sorption modified the surface charging properties of the sediment proportional to the solid-bound concentrations. Our findings link SIP signal variations to sorption dynamics and provide a framework for improved monitoring of charged organic contaminants in natural sediments.

Toward Improved Bioremediation Strategies: Response of BAM-Degradation Activity to Concentration and Flow Changes in an Inoculated Bench-Scale Sediment Tank.

Compound-specific isotope analysis (CSIA) can reveal mass-transfer limitations during biodegradation of organic pollutants by enabling the detection of masked isotope fractionation. Here, we applied CSIA to monitor the adaptive response of bacterial degradation in inoculated sediment to low contaminant concentrations over time. We characterized Aminobacter sp. MSH1 activity in a flow-through sediment tank in response to a transient supply of elevated 2,6-dichlorobenzamide (BAM) concentrations as a priming strategy and took advantage of an inadvertent intermittence to investigate the effect of short-term flow fluctuations. Priming and flow fluctuations yielded improved biodegradation performance and increased biodegradation capacity, as evaluated from bacterial activity and residual concentration time series. However, changes in isotope ratios in space and over time evidenced that mass transfer became increasingly limiting for degradation of BAM at low concentrations under such stimulated conditions, and that activity decreased further due to bacterial adaptation at low BAM (µg/L) levels. Isotope ratios, in conjunction with residual substrate concentrations, therefore helped identifying underlying limitations of biodegradation in such a stimulated system, offering important insight for future optimization of remediation schemes.

Anaerobic Neutrophilic Pyrite Oxidation by a Chemolithoautotrophic Nitrate-Reducing Iron(II)-Oxidizing Culture Enriched from a Fractured Aquifer.

Neutrophilic microbial pyrite (FeS2) oxidation coupled to denitrification is thought to be an important natural nitrate attenuation pathway in nitrate-contaminated aquifers. However, the poor solubility of pyrite raises questions about its bioavailability and the mechanisms underlying its oxidation. Here, we investigated direct microbial pyrite oxidation by a neutrophilic chemolithoautotrophic nitrate-reducing Fe(II)-oxidizing culture enriched from a pyrite-rich aquifer. We used pyrite with natural abundance (NA) of Fe isotopes (NAFe-pyrite) and 57Fe-labeled siderite to evaluate whether the oxidation of the more soluble Fe(II)-carbonate (FeCO3) can indirectly drive abiotic pyrite oxidation. Our results showed that in setups where only pyrite was incubated with bacteria, direct microbial pyrite oxidation contributed ca. 26% to overall nitrate reduction. The rest was attributed to the oxidation of elemental sulfur (S0), present as a residue from pyrite synthesis. Pyrite oxidation was evidenced in the NAFe-pyrite/57Fe-siderite setups by maps of 56FeO and 32S obtained using a combination of SEM with nanoscale secondary ion MS (NanoSIMS), which showed the presence of 56Fe(III) (oxyhydr)oxides that could solely originate from 56FeS2. Based on the fit of a reaction model to the geochemical data and the Fe-isotope distributions from NanoSIMS, we conclude that anaerobic oxidation of pyrite by our neutrophilic enrichment culture was mainly driven by direct enzymatic activity of the cells. The contribution of abiotic pyrite oxidation by Fe3+ appeared to be negligible in our experimental setup.

Mass-Transfer-Limited Biodegradation at Low Concentrations-Evidence from Reactive Transport Modeling of Isotope Profiles in a Bench-Scale Aquifer.

Organic contaminant degradation by suspended bacteria in chemostats has shown that isotope fractionation decreases dramatically when pollutant concentrations fall below the (half-saturation) Monod constant. This masked isotope fractionation implies that membrane transfer is slow relative to the enzyme turnover at µg L-1 substrate levels. Analogous evidence of mass transfer as a bottleneck for biodegradation in aquifer settings, where microbes are attached to the sediment, is lacking. A quasi-two-dimensional flow-through sediment microcosm/tank system enabled us to study the aerobic degradation of 2,6-dichlorobenzamide (BAM), while collecting sufficient samples at the outlet for compound-specific isotope analysis. By feeding an anoxic BAM solution through the center inlet port and dissolved oxygen (DO) above and below, strong transverse concentration cross-gradients of BAM and DO yielded zones of low (µg L-1) steady-state concentrations. We were able to simulate the profiles of concentrations and isotope ratios of the contaminant plume using a reactive transport model that accounted for a mass-transfer limitation into bacterial cells, where apparent isotope enrichment factors *ε decreased strongly below concentrations around 600 µg/L BAM. For the biodegradation of organic micropollutants, mass transfer into the cell emerges as a bottleneck, specifically at low (µg L-1) concentrations. Neglecting this effect when interpreting isotope ratios at field sites may lead to a significant underestimation of biodegradation.

AQDS and Redox-Active NOM Enables Microbial Fe(III)-Mineral Reduction at cm-Scales.

Redox-active organic molecules such as anthraquinone-2,6-disulfonate (AQDS) and natural organic matter (NOM) can act as electron shuttles thus facilitating electron transfer from Fe(III)-reducing bacteria (FeRB) to terminal electron acceptors such as Fe(III) minerals. In this research, we examined the length scale over which this electron shuttling can occur. We present results from agar-solidified experimental incubations, containing either AQDS or NOM, where FeRB were physically separated from ferrihydrite or goethite by 2 cm. Iron speciation and concentration measurements coupled to a diffusion-reaction model highlighted clearly Fe(III) reduction in the presence of electron shuttles, independent of the type of FeRB. Based on our fitted model, the rate of ferrihydrite reduction increased from 0.07 to 0.19 µmol d-1 with a 10-fold increase in the AQDS concentration, highlighting a dependence of the reduction rate on the electron-shuttle concentration. To capture the kinetics of Fe(II) production, the effective AQDS diffusion coefficient had to be increased by a factor of 9.4. Thus, we postulate that the 2 cm electron transfer was enabled by a combination of AQDS molecular diffusion and an electron hopping contribution from reduced to oxidized AQDS molecules. Our results demonstrate that AQDS and NOM can drive microbial Fe(III) reduction across 2 cm distances and shed light on the electron transfer process in natural anoxic environments.

Linking Spectral Induced Polarization (SIP) and Subsurface Microbial Processes: Results from Sand Column Incubation Experiments.

Geophysical techniques, such as spectral induced polarization (SIP), offer potentially powerful approaches for in situ monitoring of subsurface biogeochemistry. The successful implementation of these techniques as monitoring tools for reactive transport phenomena, however, requires the deconvolution of multiple contributions to measured signals. Here, we present SIP spectra and complementary biogeochemical data obtained in saturated columns packed with alternating layers of ferrihydrite-coated and pure quartz sand, and inoculated with Shewanella oneidensis supplemented with lactate and nitrate. A biomass-explicit diffusion-reaction model is fitted to the experimental biogeochemical data. Overall, the results highlight that (1) the temporal response of the measured imaginary conductivity peaks parallels the microbial growth and decay dynamics in the columns, and (2) SIP is sensitive to changes in microbial abundance and cell surface charging properties, even at relatively low cell densities (<108 cells mL-1). Relaxation times (τ) derived using the Cole-Cole model vary with the dominant electron accepting process, nitrate or ferric iron reduction. The observed range of τ values, 0.012-0.107 s, yields effective polarization diameters in the range 1-3 µm, that is, 2 orders of magnitude smaller than the smallest quartz grains in the columns, suggesting that polarization of the bacterial cells controls the observed chargeability and relaxation dynamics in the experiments.

Sensing Coated Iron-Oxide Nanoparticles with Spectral Induced Polarization (SIP): Experiments in Natural Sand Packed Flow-Through Columns.

The development of nanoparticle-based soil remediation techniques is hindered by the lack of accurate in situ nanoparticle (NP) monitoring and characterization methods. Spectral induced polarization (SIP), a noninvasive geophysical technique, offers a promising approach to detect and quantify NPs in porous media. However, its successful implementation as a monitoring tool requires an understanding of the polarization mechanisms, the governing NP-associated SIP responses and their dependence on the stabilizing coatings that are typically used for NPs deployed in environmental applications. Herein, we present SIP responses (0.1-10 000 Hz) measured during injection of a poloxamer-coated superparamagnetic iron-oxide nanoparticle (SPION) suspension in flow-through columns packed with natural sand from the Borden aquifer. An advective-dispersive transport model is fitted to outflow SPION concentration measurements to compute average concentrations over the SIP spatial response domain (within the columns). The average SPION concentrations are compared with the real and imaginary components of the complex conductivity. Excellent correspondence is found between the average SPION concentrations in the columns and the imaginary conductivity values, suggesting that NP-mediated polarization (that is, charge storage) increases proportionally with increasing SPION concentration. Our results support the possibility of SIP monitoring of spatial and temporal NP distributions, which can be immediately deployed in bench-scale studies with the prospect of future real-world field applications.

Dynamics of suspended and attached aerobic toluene degraders in small-scale flow-through sediment systems under growth and starvation conditions.

The microbially mediated reactions, that are responsible for field-scale natural attenuation of organic pollutants, are governed by the concurrent presence of a degrading microbial community, suitable energy and carbon sources, electron acceptors, as well as nutrients. The temporal lack of one of these essential components for microbial activity, arising from transient environmental conditions, might potentially impair in situ biodegradation. This study presents results of small scale flow-through experiments aimed at ascertaining the effects of substrate-starvation periods on the aerobic degradation of toluene by Pseudomonas putida F1. During the course of the experiments, concentrations of attached and mobile bacteria, as well as toluene and oxygen were monitored. Results from a fitted reactive-transport model, along with the observed profiles, show the ability of attached cells to survive substrate-starvation periods of up to four months and suggest a highly dynamic exchange between attached and mobile cells under growth conditions and negligible cell detachment under substrate-starvation conditions. Upon reinstatement of toluene, it was readily degraded without a significant lag period, even after a starvation period of 130 days. Our experimental and modeling results strongly suggest that aerobic biodegradation of BTEX-hydrocarbons at contaminated field sites is not hampered by intermittent starvation periods of up to four months.

Does It Pay Off to Explicitly Link Functional Gene Expression to Denitrification Rates in Reaction Models?

Environmental omics and molecular-biological data have been proposed to yield improved quantitative predictions of biogeochemical processes. The abundances of functional genes and transcripts relate to the number of cells and activity of microorganisms. However, whether molecular-biological data can be quantitatively linked to reaction rates remains an open question. We present an enzyme-based denitrification model that simulates concentrations of transcription factors, functional-gene transcripts, enzymes, and solutes. We calibrated the model using experimental data from a well-controlled batch experiment with the denitrifier Paracoccous denitrificans. The model accurately predicts denitrification rates and measured transcript dynamics. The relationship between simulated transcript concentrations and reaction rates exhibits strong non-linearity and hysteresis related to the faster dynamics of gene transcription and substrate consumption, relative to enzyme production and decay. Hence, assuming a unique relationship between transcript-to-gene ratios and reaction rates, as frequently suggested, may be an erroneous simplification. Comparing model results of our enzyme-based model to those of a classical Monod-type model reveals that both formulations perform equally well with respect to nitrogen species, indicating only a low benefit of integrating molecular-biological data for estimating denitrification rates. Nonetheless, the enzyme-based model is a valuable tool to improve our mechanistic understanding of the relationship between biomolecular quantities and reaction rates. Furthermore, our results highlight that both enzyme kinetics (i.e., substrate limitation and inhibition) and gene expression or enzyme dynamics are important controls on denitrification rates.

Chromium (VI) removal kinetics by magnetite-coated sand: Small-scale flow-through column experiments.

Magnetite nanoparticles are promising materials for treating toxic Cr(VI), but safe handling is challenging due to their small size. We prepared flow-through columns containing 10% or 100% (v/v) magnetite-coated sand. Cr(VI) removal efficiency was determined for different Cr(VI) concentrations (0.1 or 1.0 mM), neutral or alkaline pH, and oxic/anoxic conditions. We formulated a reactive-transport model that accurately predicted total Cr removal, accounting for reversible and irreversible (chemi)sorption reactions. Our results show that the material removes and irreversibly sequesters Cr(VI). For the concentration range used 10% and 100% (v/v) -packed columns removed > 99% and 72% of influent Cr(VI), respectively. Two distinct parameter sets were necessary to fit the identical model formulation to the 10 or 100% (v/v) columns (e.g., maximum sorption capacities (qmax) of 1.37 µmol Cr/g sand and 2.48 µmol Cr/g, respectively), which we attributed to abrasion-driven magnetite micro-particle detachment during packing yielding an increase in reactive surface area. Furthermore, experiments under oxic conditions showed that, even when handled in the presence of O2, the magnetite-coated sand maintained a high removal capacity (47%). Our coupled experimental and modelling analyses indicates that magnetite-coated sand is a promising and suitable medium for treating Cr(VI)-contaminated water in fixed-bed reactors or permeable reactive barriers.

Spatiotemporal geo-electrical sensing of a Pluronic-coated cobalt ferrite nanoparticle slug in natural sand flow-through columns.

Rising industrial interest in the application of nanomaterials for the remediation of contaminated sites has led to concern over the environmental fate of the nanoremediation agents used. A critical requirement in evaluating and understanding nanoparticle (NP) behaviour in porous media is the development of analytical methods capable of in situ monitoring of complex NP transport dynamics. Spectral induced polarization (SIP), a non-invasive geo-electrical technique, offers a promising tool for detecting and quantifying NPs in soil and aquifer media. However, its application for monitoring the spatial migration and attachment behaviour of NPs remains uninvestigated. Here, we present results from flow-through experiments where we monitored the transport of cobalt ferrite nanoparticles (CoFe-NPs) coated with Pluronic, an amphiphilic polymer, in natural aquifer sand columns. We coupled concentration breakthrough curve analysis with SIP monitoring and reactive transport modeling to relate spatiotemporal NP concentration distributions to geo-electrical signals. Changes in the real (σ') conductivity at three different locations along the columns closely correlated with model-computed total (solid plus aqueous phase) NP concentrations during the propagation of a NP slug. The imaginary conductivity (σ″) correlated closely with the arrival of the NP-slug. However, during the receding front, bimodal σ″-signal peak behaviour was observed propagating through the columns, indicating the existence of complex in situ NP transport dynamics, potentially revealing the rupture of nanoclusters upon straining and their effect on bulk charge storage that may not be obvious from breakthrough curve data alone. Fitting of a double Cole-Cole relaxation model yielded distinct shifts in relaxation time (τ) associated with the polarization of smaller length-scale particles. Post-NP pulse τ and σ″ did not return to pre-injection values; these lingering signals were caused by retained NP concentrations as low as 8.8 mg kg-1. Our results support the applicability of SIP for spatial and temporal monitoring of NP distributions, with implications for the investigation of NP transport and nanoremediation strategies.

Immobilizing magnetite onto quartz sand for chromium remediation.

Magnetite nanoparticles are often promoted as remediation agents for heavy metals such as chromium due to their reactivity and high surface area. However, their small size also makes them highly mobile increasing the risk that reacted pollutants will be transported to different locations rather than being safely controlled. Released to aquatic environments, aggregation leads to a loss of their nano-specific properties and contaminant-removal capacity. We immobilized magnetite onto sand to overcome these issues whilst maintaining reactivity. We compare biogenic magnetite and abiogenic magnetite coated sand against magnetite nanoparticles. Magnetite coatings mostly exhibited a Fe(II)/Fe(III) ratio close to stoichiometry (0.5). We tested the efficacy of the magnetite-coated sand to adsorb chromium, with respect to biogenic/abiogenic nanoparticles. Langmuir-type sorption of Cr(VI) onto magnetite (4.32 mM total Fe) was observed over the tested concentration range (10-1000 µM). Biogenic nanoparticles showed the highest potential for Cr(VI) removal with maximum adsorption capacity (Qmax) of 1250 µmol Cr/g Fe followed by abiogenic nanoparticles with 693 µmol Cr/g Fe. All magnetite coated sands exhibited similar sorption behavior with average Qmax ranging between 257-471 µmol Cr/g Fe. These results indicate coating magnetite onto sand may be more suitable than free nanoparticles for treating environmental pollutants such as chromium.