Systematic evaluation of methods for iron-impregnation of biochar and effects on arsenic in flooded soils.

There is a need for innovative strategies to decrease the mobility of metal(loids) including arsenic (As) and cadmium (Cd) in agricultural soils, including rice paddies, so as to minimize dietary exposure to these toxic elements. Iron (Fe)-modified biochars (FBCs) are used to immobilize As and Cd in soil-water systems, but there is a lack of clarity on optimal methods for preparing FBCs because there are only limited studies that directly compare BCs impregnated with Fe under different conditions. There is also a lack of information on the long-term performance of FBCs in flooded soil environments, where reductive dissolution of Fe (oxy)hydroxide phases loaded onto biochar surfaces may decrease the effectiveness of FBCs. This study uses material characterization methods including FTIR, SEM-EDX, BET, and adsorption isotherm experiments to investigate the effects of Fe-impregnation methods (pH, pyrolysis sequence, and sonication) on the morphology and mineralogy of Fe loaded onto the biochar surface, and to FBC adsorbent properties for arsenate (As(V)), arsenite (As(III)), and Cd. Acidic impregnation conditions favored the adsorption of As(III) onto amorphous Fe phases that were evenly distributed on the biochar surface, including within the biochar pore structure. The combination of sonication with acidic Fe-impregnation conditions led to the best adsorption capacities for As(V) and As(III) (4830 and 11,166 µg As g-1 biochar, respectively). Alkaline Fe-impregnation conditions led to the highest Cd adsorption capacity of 3054 µg Cd g-1 biochar, but had poor effectiveness as an As adsorbent. Amending soil with 5% (w/w) of an acid-impregnated and sonicated FBC was more effective than an alkaline-impregnated FBC or ferrihydrite in decreasing porewater As concentrations. The acid-impregnated FBC also had greater longevity, decreasing As by 54% and 56% in two flooded phases, probably due to the greater stability of Fe(III) within the biochar pore structure that may have a direct chemical bond to the biochar surface. This study demonstrates that FBCs can be designed with selectivity towards different As species or Cd and that they can maintain their effectiveness under anaerobic soil conditions. This is the first study to systematically test how impregnation conditions affect the stability of FBCs in soils under multiple drying-rewetting cycles.

Multiple Rounds of In Vivo Random Mutagenesis and Selection in Vibrio natriegens Result in Substantial Increases in REE Binding Capacity.

Rare earth elements (REE) are essential ingredients in many modern technologies, yet their purification remains either environmentally harmful or economically unviable. Adsorption, or biosorption, of REE onto bacterial cell membranes offers a sustainable alternative to traditional solvent extraction methods. But in order for biosorption-based REE purification to compete economically, the capacity and specificity of biosorption sites must be enhanced. Although there have been some recent advances in characterizing the genetics of REE-biosorption, the variety and complexity of bacterial membrane surface sites make targeted genetic engineering difficult. Here, we propose using multiple rounds of in vivo random mutagenesis induced by the MP6 plasmid combined with plate-throughput REE-biosorption screening to improve a microbe's capacity and selectivity for biosorbing REE. We engineered a strain of Vibrio natriegens capable of biosorbing 210% more dysprosium compared to the wild-type and produced selectivity improvements of up to 50% between the lightest (lanthanum) and heaviest (lutetium) REE. We believe that mutations we observed in ABC transporters as well as a nonessential protein in the BAM outer membrane ß-barrel protein insertion complex likely contribute to some─but almost certainly not all─of the biosorption changes we observed. Given the ease of finding significant biosorption mutants, these results highlight just how many genes likely contribute to biosorption as well as the power of random mutagenesis in identifying genes of interest and optimizing a biological system for a task.

Genomic characterization of rare earth binding by Shewanella oneidensis.

Rare earth elements (REE) are essential ingredients of sustainable energy technologies, but separation of individual REE is one of the hardest problems in chemistry today. Biosorption, where molecules adsorb to the surface of biological materials, offers a sustainable alternative to environmentally harmful solvent extractions currently used for separation of rare earth elements (REE). The REE-biosorption capability of some microorganisms allows for REE separations that, under specialized conditions, are already competitive with solvent extractions, suggesting that genetic engineering could allow it to leapfrog existing technologies. To identify targets for genomic improvement we screened 3,373 mutants from the whole genome knockout collection of the known REE-biosorbing microorganism Shewanella oneidensis MR-1. We found 130 genes that increased biosorption of the middle REE europium, and 112 that reduced it. We verified biosorption changes from the screen for a mixed solution of three REE (La, Eu, Yb) using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in solution conditions with a range of ionic strengths and REE concentrations. We identified 18 gene ontologies and 13 gene operons that make up key systems that affect biosorption. We found, among other things, that disruptions of a key regulatory component of the arc system (hptA), which regulates cellular response to anoxic environments and polysaccharide biosynthesis related genes (wbpQ, wbnJ, SO_3183) consistently increase biosorption across all our solution conditions. Our largest total biosorption change comes from our SO_4685, a capsular polysaccharide (CPS) synthesis gene, disruption of which results in an up to 79% increase in biosorption; and nusA, a transcriptional termination/anti-termination protein, disruption of which results in an up to 35% decrease in biosorption. Knockouts of glnA, pyrD, and SO_3183 produce small but significant increases (≈ 1%) in relative biosorption affinity for ytterbium over lanthanum in multiple solution conditions tested, while many other genes we explored have more complex binding affinity changes. Modeling suggests that while these changes to lanthanide biosorption selectivity are small, they could already reduce the length of repeated enrichment process by up to 27%. This broad exploratory study begins to elucidate how genetics affect REE-biosorption by S. oneidensis, suggests new areas of investigation for better mechanistic understanding of the membrane chemistry involved in REE binding, and offer potential targets for improving biosorption and separation of REE by genetic engineering.

Toward Integrating EBPR and the Short-Cut Nitrogen Removal Process in a One-Stage System for Treating High-Strength Wastewater.

Enhanced biological phosphorus removal (EBPR) is an economical and sustainable process for phosphorus removal from wastewater. Despite the widespread application of EBPR for low-strength domestic wastewater treatment, limited investigations have been conducted to apply EBPR to the high-strength wastewaters, particularly, the integration of EBPR and the short-cut nitrogen removal process in the one-stage system remains challenging. Herein, we reported a novel proof-of-concept demonstration of integrating EBPR and nitritation (oxidation of ammonium to nitrite) in a one-stage sequencing batch reactor to achieve simultaneous high-strength phosphorus and short-cut nitrogen removal. Excellent EBPR performance of effluent 0.8 ± 1.0 mg P/L and >99% removal efficiency was achieved fed with synthetic high-strength phosphorus wastewater. Long-term sludge acclimation proved that the dominant polyphosphate accumulating organisms (PAOs), Candidatus Accumulibacter, could evolve to a specific subtype that can tolerate the nitrite inhibition as revealed by operational taxonomic unit (OTU)-based oligotyping analysis. The EBPR kinetic and stoichiometric evaluations combined with the amplicon sequencing proved that the Candidatus Competibacter, as the dominant glycogen accumulating organisms (GAOs), could well coexist with PAOs (15.3-24.9% and 14.2-33.1%, respectively) and did not deteriorate the EBPR performance. The nitrification activity assessment, amplicon sequencing, and functional-based gene marker quantification verified that the unexpected nitrite accumulation (10.7-21.0 mg N/L) in the high-strength EBPR system was likely caused by the nitritation process, in which the nitrite-oxidizing bacteria (NOB) were successfully out-selected (<0.1% relative abundance). We hypothesized that the introduction of the anaerobic phase with high VFA concentrations could be the potential selection force for achieving nitritation based on the literature review and our preliminary batch tests. This study sheds light on developing a new feasible technical route for integrating EBPR with short-cut nitrogen removal for efficient high-strength wastewater treatment.

Effects of alternate wetting and drying on oxyanion-forming and cationic trace elements in rice paddy soils: impacts on arsenic, cadmium, and micronutrients in rice.

Rice is a global dietary staple and its traditional cultivation under flooded soil conditions leads to accumulation of arsenic (As) in rice grains. Alternate wetting and drying (AWD) is a widely advocated water management practice to achieve lower As concentrations in rice, water savings, and decreased methane emissions. It is not yet clear whether AWD leads to tradeoffs between concentrations of As and micronutrient elements (e.g., zinc, manganese, molybdenum) in rice grain. We analyzed pore water chemistry and rice grain composition data from a field experiment conducted in Arkansas, USA, in 2017 and 2018 to test the hypothesis that AWD will have diverging effects on oxyanion-forming (arsenic, molybdenum) vs. cationic (cadmium, zinc, manganese, copper) trace elements. This was hypothesized to occur via decreases in soil pH and/or precipitation of iron oxide minerals during oxidizing conditions under AWD. Solubility of all trace elements, except zinc, increased in more reducing conditions. Consistent with our hypothesis, AWD tended to increase grain concentrations of cationic elements while decreasing grain concentrations of oxyanionic elements. Decreases in total As in rice grains under AWD were mainly driven by changes in dimethylarsinic concentrations, with negligible changes in inorganic As. Linear mixed-effects modeling showed that effects of AWD on grain composition were more significant in 2017 compared to 2018. These differences may be related to the timing of dry-downs in the developmental stage of rice plants, with dry-downs during the heading stage of rice development leading to larger impacts on grain composition of certain elements. We also observed significant interannual variability in grain elemental composition from continuously-flooded fields and postulate the warmer temperatures in 2018 may have played a role in these differences.

Climate change effects on denitrification performance of woodchip bioreactors treating agricultural tile drainage.

Denitrifying woodchip bioreactors (WBRs) are a nature-based technology that are increasingly used to control nonpoint source nitrate (NO3-) pollution in agricultural catchments. The treatment effectiveness of WBRs depends on temperature and hydraulic retention time (HRT), both of which are affected by climate change. Warmer temperatures will increase microbial denitrification rates, but the extent to which the resulting benefits to treatment performance may be offset by intensified precipitation and shorter HRTs is not clear. Here, we use three years of monitoring data from a WBR in Central New York State to train an integrated hydrologic-biokinetic model describing links among temperature, precipitation, bioreactor discharge, denitrification kinetics, and NO3- removal efficiencies. Effects of climate warming are assessed by first training a stochastic weather generator with eleven years of weather data from our field site, and then adjusting the distribution of precipitation intensities according to the Clausius-Clapeyron relationship between water vapor and temperature. Modeling results indicate, in our system, faster denitrification rates will outweigh the influence of intensified precipitation and discharge under warming, leading to net improvements in NO3- load reductions. Median cumulative NO3- load reductions at our study site from May - October are projected to increase from 21.7% (interquartile range 17.4%-26.1%) under baseline hydro-climate to 41.0% (interquartile range 32.6-47.1%) with a + 4 °C change in mean air temperature. This improved performance under climate warming is driven by strong nonlinear dependence of NO3- removal rates on temperature. Temperature sensitivity may increase with woodchip age and lead to stronger temperature-response in systems like this one with a highly aged woodchip matrix. While the impacts of hydro-climatic change on WBR performance will depend on site-specific properties, this hydrologic-biokinetic modeling approach provides a framework for assessing climate impacts on the effectiveness of WBRs and other denitrifying nature-based systems.

Oxic-anoxic cycling promotes coupling between complex carbon metabolism and denitrification in woodchip bioreactors.

Denitrifying woodchip bioreactors (WBRs) are increasingly used to manage the release of non-point source nitrogen (N) by stimulating microbial denitrification. Woodchips serve as a renewable organic carbon (C) source, yet the recalcitrance of organic C in lignocellulosic biomass causes many WBRs to be C-limited. Prior studies have observed that oxic-anoxic cycling increased the mobilization of organic C, increased nitrate (NO3 - ) removal rates, and attenuated production of nitrous oxide (N2 O). Here, we use multi-omics approaches and amplicon sequencing of fungal 5.8S-ITS2 and prokaryotic 16S rRNA genes to elucidate the microbial drivers for enhanced NO3 - removal and attenuated N2 O production under redox-dynamic conditions. Transient oxic periods stimulated the expression of fungal ligninolytic enzymes, increasing the bioavailability of woodchip-derived C and stimulating the expression of denitrification genes. Nitrous oxide reductase (nosZ) genes were primarily clade II, and the ratio of clade II/clade I nosZ transcripts during the oxic-anoxic transition was strongly correlated with the N2 O yield. Analysis of metagenome-assembled genomes revealed that many of the denitrifying microorganisms also have a genotypic ability to degrade complex polysaccharides like cellulose and hemicellulose, highlighting the adaptation of the WBR microbiome to the ecophysiological niche of the woodchip matrix.

Time-Dependent Biosensor Fluorescence as a Measure of Bacterial Arsenic Uptake Kinetics and Its Inhibition by Dissolved Organic Matter.

Microbe-mediated transformations of arsenic (As) often require As to be taken up into cells prior to enzymatic reaction. Despite the importance of these microbial reactions for As speciation and toxicity, understanding of how As bioavailability and uptake are regulated by aspects of extracellular water chemistry, notably dissolved organic matter (DOM), remains limited. Whole-cell biosensors utilizing fluorescent proteins are increasingly used for high-throughput quantification of the bioavailable fraction of As in water. Here, we present a mathematical framework for interpreting the time series of biosensor fluorescence as a measure of As uptake kinetics, which we used to evaluate the effects of different forms of DOM on uptake of trivalent arsenite. We found that thiol-containing organic compounds significantly inhibited uptake of arsenite into cells, possibly through the formation of aqueous complexes between arsenite and thiol ligands. While there was no evidence for competitive interactions between arsenite and low-molecular-weight neutral molecules (urea, glycine, and glyceraldehyde) for uptake through the aquaglyceroporin channel GlpF, which mediates transport of arsenite across cell membranes, there was evidence that labile DOM fractions may inhibit arsenite uptake through a catabolite repression-like mechanism. The observation of significant inhibition of arsenite uptake at DOM/As ratios commonly encountered in wetland pore waters suggests that DOM may be an important control on the microbial uptake of arsenite in the environment, with aspects of DOM quality playing an important role in the extent of inhibition. IMPORTANCE The speciation and toxicity of arsenic in environments like rice paddy soils and groundwater aquifers are controlled by microbe-mediated reactions. These reactions often require As to be taken up into cells prior to enzymatic reaction, but there is limited understanding of how microbial arsenic uptake is affected by variations in water chemistry. In this study, we explored the effect of dissolved organic matter (DOM) quantity and quality on microbial As uptake, with a focus on the role of thiol functional groups that are well known to form aqueous complexes with arsenic. We developed a quantitative framework for interpreting fluorescence time series from whole-cell biosensors and used this technique to evaluate effects of DOM on the rates of microbial arsenic uptake. We show that thiol-containing compounds significantly decrease rates of As uptake into microbial cells at environmentally relevant DOM/As ratios, revealing the importance of DOM quality in regulating arsenic uptake, and subsequent biotransformation, in the environment.

Effects of organic sulfur and arsenite/dissolved organic matter ratios on arsenite complexation with dissolved organic matter.

The speciation and fate of arsenic (As) in soil-water systems is a topic of great interest, in part due to growing awareness of As uptake into rice as an important human exposure pathway to As. Rice paddy and other wetland soils are rich in dissolved organic matter (DOM), leading to As/DOM ratios that are typically lower than those in groundwater aquifers or that have been used in many laboratory studies of As-DOM interactions. In this contribution, we evaluate arsenite (As(III)) binding to seven different DOM samples at As/DOM ratios relevant for wetland pore waters, and explore the chemical properties of the DOM samples associated with high levels of As(III)-DOM complexation. We integrate data from wet chemical analysis of DOM chemical properties, dialysis equilibrium experiments, and two-site ligand binding models to show that in some DOM samples, 15-60% of As(III) can be bound to DOM at environmentally-relevant As/DOM ratios of 0.0032-0.016 µmol As/mmol C. Binding decreases as the As(III)/DOM ratio increases. The organic sulfur (Sorg) content of the DOM samples was strongly correlated with levels of As(III)-DOM complexation and "strong" binding site densities, consistent with theories that thiols are strong binding ligands for As(III) in natural organic matter. Finally, a whole-cell E. coli biosensor assay was used to show that DOM samples most effective at complexing As(III) also led to decreased microbial As(III) uptake at low As/DOC ratios. This work demonstrates that naturally-occurring variations in the Sorg content of DOM has a significant impact on As(III) binding to DOM, and has implications for As(III) availability to microorganisms.

Meta-omics-aided isolation of an elusive anaerobic arsenic-methylating soil bacterium.

Soil microbiomes harbour unparalleled functional and phylogenetic diversity. However, extracting isolates with a targeted function from complex microbiomes is not straightforward, particularly if the associated phenotype does not lend itself to high-throughput screening. Here, we tackle the methylation of arsenic (As) in anoxic soils. As methylation was proposed to be catalysed by sulfate-reducing bacteria. However, to date, there are no available anaerobic isolates capable of As methylation, whether sulfate-reducing or otherwise. The isolation of such a microorganism has been thwarted by the fact that the anaerobic bacteria harbouring a functional arsenite S-adenosylmethionine methyltransferase (ArsM) tested to date did not methylate As in pure culture. Additionally, fortuitous As methylation can result from the release of non-specific methyltransferases upon lysis. Thus, we combined metagenomics, metatranscriptomics, and metaproteomics to identify the microorganisms actively methylating As in anoxic soil-derived microbial cultures. Based on the metagenome-assembled genomes of microorganisms expressing ArsM, we isolated Paraclostridium sp. strain EML, which was confirmed to actively methylate As anaerobically. This work is an example of the application of meta-omics to the isolation of elusive microorganisms.

Inhibition of methanogenesis leads to accumulation of methylated arsenic species and enhances arsenic volatilization from rice paddy soil.

Flooded soils are important environments for the biomethylation and subsequent volatilization of arsenic (As), a contaminant of global concern. Conversion of inorganic to methylated oxyarsenic species is thought to be the rate-limiting step in the production and emission of volatile (methyl)arsines. While methanogens and sulfate-reducing bacteria (SRB) have been identified as important regulators of methylated oxyarsenic concentrations in anaerobic soils, the effects of these microbial groups on biovolatilization remain unclear. Here, microcosm and batch incubation experiments with an Arkansas, USA, rice paddy soil were performed in conjunction with metabolic inhibition to test the effects of methanogenic activity on As speciation and biovolatilization. Inhibition of methanogenesis with 2-bromoethanesulfonate (BES) led to the accumulation of methylated oxyarsenic species, primarily dimethylarsinic acid (DMAs(V)), and a four-fold increase in As biovolatilization compared to a control soil. Our results support a conceptual model that methanogenic activity suppresses biovolatilization by enhancing As demethylation rates. This work refines understanding of biogeochemical processes regulating As biovolatilization in anaerobic soil environments, and extends recent insights into links between methanogenesis and As metabolism to soils from the mid-South United States rice production region.

Generation of a Gluconobacter oxydans knockout collection for improved extraction of rare earth elements.

Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency.

Associations between inorganic arsenic in rice and groundwater arsenic in the Mekong Delta.

There is growing concern regarding human dietary exposure to arsenic (As) via consumption of rice. The concentration and speciation of As in rice are highly variable, and models describing rice As speciation as a function of environmental covariates remain elusive. We conducted a survey of paddy rice and soil in the Mekong Delta with the objective of linking patterns in rice As content to soil chemical variables or hydrogeological parameters. The sum of As species (ΣAs) in husked rice averaged 243 µg/kg and the average inorganic As (iAs) content was 84%. There was no relationship found between rice As concentration or speciation and As levels in soil. However, mean As concentrations in groundwater near rice sampling locations were strongly correlated with grain ΣAs and iAs over a large part of the study region, despite the fact that groundwater is not commonly used for rice paddy irrigation in this region. We hypothesize that surficial sediments with high concentrations of soluble and plant-available As also serve as sources of arsenic to downgradient shallow aquifers, explaining the observed associations between rice and groundwater As. This study suggests that shallow groundwater As concentrations may serve as a useful indicator for locations at risk of elevated iAs concentrations in rice.

Kinetics of nitrous oxide mass transfer from porewater into root aerenchyma of wetland plants.

The creation and/or restoration of wetlands is an important strategy for controlling the release of reactive nitrogen (N) via denitrification, but there can be tradeoffs between enhanced denitrification and the production of nitrous oxide (N2 O), a potent greenhouse gas. A knowledge gap in current understanding of belowground wetland N dynamics is the role of gas transfer through the root aerenchyma system of wetland plants as a shortcut emission pathway for N2 O in denitrifying wetland soils. This investigation evaluates the significance of mass transfer into gas-filled root aerenchyma for the N2 O budget in wetland mesocosms planted with Sagittaria latifolia Willd. and Schoenoplectus acutus (Muhl. ex Bigelow) Á. Löve & D. Löve. Dissolved gas tracer push-pull tests with N2 O and the nonreactive gas tracers helium, sulfur hexafluoride, and ethane were used to estimate first-order rate constants for gas transfer into roots and microbial N2 O reduction and thereby disentangle the effects of root-mediated gas transport from microbial metabolism on N2 O balances in saturated soils. Root-mediated gas transport was estimated to account for up to 37% of overall N2 O removal from the wetland soils. Rates of microbial N2 O reduction varied widely based on the organic matter content of the soil media and served as a key control on the fraction of N2 O that transferred into roots. This research identifies transport through root aerenchyma as a potential shortcut pathway for N2 O emission from wetland soils and sediments and indicates that this process should be considered in both measurements and mechanistic modeling of belowground wetland N dynamics.

Nitrous Oxide and Methane Dynamics in Woodchip Bioreactors: Effects of Water Level Fluctuations on Partitioning into Trapped Gas Phases.

Woodchip bioreactors (WBRs) are low-cost, passive systems for nonpoint source nitrogen removal at terrestrial-aquatic interfaces. The greenhouse gases nitrous oxide (N2O) and methane (CH4) can be produced within WBRs, and efforts to reduce N2O and CH4 emissions from WBR systems require improved understanding of the biogeochemical and physical-chemical mechanisms regulating their production, transport, and release. This study evaluates the impact of trapped gas-filled void volumes as sinks of dissolved gases from water and as sources of episodic fluxes when water levels fall. Dissolved gas tracer experiments in a laboratory bioreactor were used to parameterize nonequilibrium advection-dispersion-gas transfer models and quantify trapping of gas-filled voids as a function of antecedent hydrological conditions. Experiments following a water-level rise revealed that up to 24% of the WBR pore volume was occupied by trapped gas phases, which were primarily located in pore spaces inside woodchips. This finding was confirmed with X-ray-computed microtomography. N2O (3.3-10%) and CH4 (4.3-14%) injected into the reactor following a water table rise partitioned into gas-filled voids and were released when water tables fell. In the case of N2O, partitioning into trapped gas phases makes N2O unavailable for enzymatic reduction, potentially enhancing N2O fluxes under fluctuating water levels.

Simultaneous measurements of dissolved CH4 and H2 in wetland soils.

Biogeochemical processes in wetland soils are complex and are driven by a microbiological community that competes for resources and affects the soil chemistry. Depending on the availability of various electron acceptors, the high carbon input to wetland soils can make them important sources of methane production and emissions. There are two significant pathways for methanogenesis: acetoclastic and hydrogenotrophic methanogenesis. The hydrogenotrophic pathway is dependent on the availability of dissolved hydrogen gas (H2), and there is significant competition for available H2. This study presents simultaneous measurements of dissolved methane and H2 over a 2-year period at three tidal marshes in the New Jersey Meadowlands. Methane reservoirs show a significant correlation with dissolved organic carbon, temperature, and methane emissions, whereas the H2 concentrations measured with dialysis samplers do not show significant relationships with these field variables. Data presented in this study show that increased dissolved H2 reservoirs in wetland soils correlate with decreased methane reservoirs, which is consistent with studies that have shown that elevated levels of H2 inhibit methane production by inhibiting propionate fermentation, resulting in less acetate production and hence decreasing the contribution of acetoclastic methanogenesis to the overall production of methane.

Arsenic Methylation Dynamics in a Rice Paddy Soil Anaerobic Enrichment Culture.

Methylated arsenic (As) species represent a significant fraction of the As accumulating in rice grains, and there are geographic patterns in the abundance of methylated arsenic in rice that are not understood. The microorganisms driving As biomethylation in paddy environments, and thus the soil conditions conducive to the accumulation of methylated arsenic, are unknown. We tested the hypothesis that sulfate-reducing bacteria (SRB) are key drivers of arsenic methylation in metabolically versatile mixed anaerobic enrichments from a Mekong Delta paddy soil. We used molybdate and monofluorophosphate as inhibitors of sulfate reduction to evaluate the contribution of SRB to arsenic biomethylation, and developed degenerate primers for the amplification of arsM genes to identify methylating organisms. Enrichment cultures converted 63% of arsenite into methylated products, with dimethylarsinic acid as the major product. While molybdate inhibited As biomethylation, this effect was unrelated to its inhibition of sulfate reduction and instead inhibited the methylation pathway. Based on arsM sequences and the physiological response of cultures to media conditions, we propose that amino acid fermenting organisms are potential drivers of As methylation in the enrichments. The lack of a demethylating capacity may have contributed to the robust methylation efficiencies in this mixed culture.

Direct measurements of methane emissions from abandoned oil and gas wells in Pennsylvania.

Abandoned oil and gas wells provide a potential pathway for subsurface migration and emissions of methane and other fluids to the atmosphere. Little is known about methane fluxes from the millions of abandoned wells that exist in the United States. Here, we report direct measurements of methane fluxes from abandoned oil and gas wells in Pennsylvania, using static flux chambers. A total of 42 and 52 direct measurements were made at wells and at locations near the wells ("controls") in forested, wetland, grassland, and river areas in July, August, October 2013 and January 2014, respectively. The mean methane flow rates at these well locations were 0.27 kg/d/well, and the mean methane flow rate at the control locations was 4.5 × 10(-6) kg/d/location. Three out of the 19 measured wells were high emitters that had methane flow rates that were three orders of magnitude larger than the median flow rate of 1.3 × 10(-3) kg/d/well. Assuming the mean flow rate found here is representative of all abandoned wells in Pennsylvania, we scaled the methane emissions to be 4-7% of estimated total anthropogenic methane emissions in Pennsylvania. The presence of ethane, propane, and n-butane, along with the methane isotopic composition, indicate that the emitted methane is predominantly of thermogenic origin. These measurements show that methane emissions from abandoned oil and gas wells can be significant. The research required to quantify these emissions nationally should be undertaken so they can be accurately described and included in greenhouse gas emissions inventories.

Global methane emissions from pit latrines.

Pit latrines are an important form of decentralized wastewater management, providing hygienic and low-cost sanitation for approximately one-quarter of the global population. Latrines are also major sources of the greenhouse gas methane (CH4) from the anaerobic decomposition of organic matter in pits. In this study, we develop a spatially explicit approach to account for local hydrological control over the anaerobic condition of latrines and use this analysis to derive a set of country-specific emissions factors and to estimate global pit latrine CH4 emissions. Between 2000 and 2015 we project global emissions to fall from 5.2 to 3.8 Tg y(-1), or from ∼ 2% to ∼ 1% of global anthropogenic CH4 emissions, due largely to urbanization in China. Two and a half billion people still lack improved sanitation services, however, and progress toward universal access to improved sanitation will likely drive future growth in pit latrine emissions. We discuss modeling results in the context of sustainable water, sanitation, and hygiene development and consider appropriate technologies to ensure hygienic sanitation while limiting CH4 emissions. We show that low-CH4 on-site alternatives like composting toilets may be price competitive with other CH4 mitigation measures in organic waste sectors, with marginal abatement costs ranging from 57 to 944 $/ton carbon dioxide equivalents (CO2e) in Africa and 46 to 97 $/ton CO2e in Asia.

A push-pull test to measure root uptake of volatile chemicals from wetland soils.

This paper introduces a novel modification of the single-well "push-pull" test that uses nonvolatile and multiple volatile tracers to investigate the transport and root uptake kinetics of volatile chemicals in saturated soils. This technique provides an estimate of potential volatilization fluxes without relying on enclosure-based measurements. The new push-pull methodology was validated with mesocosm experiments, and bench-scale hydroponic measurements were performed to develop an empirical relationship for scaling root uptake rates between chemicals. A new modeling approach to interpret data using sulfur hexafluoride and helium as dual volatile tracers was developed and shown to decrease errors relative to existing analytical techniques that utilize bromide as a conservative tracer. Root uptake of the volatile tracers was diffusion-limited, and uptake rate constants (kv) in vegetated experimental mesocosms ranged from 0.021 ± 9.0 × 10(-4) h(-1) for CFC-12 to 2.41 ± 0.98 h(-1) for helium. Hydroponic and mesocosm experiments demonstrate that the molecular diameter is a robust empirical predictor of kv.