Natural solution for the remediation of multi-metal contamination: application of natural amino acids, Pseudomonas fluorescens and Micrococcus yunnanensis to increase the phytoremediation efficiency.

Natural amino acids (NAA) have been rarely investigated as chelators, despite their ability to chelate heavy metals (HMs). In the present research, the effects of extracted natural amino acids, as a natural and environmentally friendly chelate agent and the inoculation of Pseudomonas fluorescens (PF) and Micrococcus yunnanensis (MY) bacteria were investigated on some responses of quinoa in a soil polluted with Pb, Ni, Cd, and Zn. Inoculation of PGPR bacteria enhanced plant growth and phytoremediation efficiency. Pb and Cd were higher in quinoa roots, while Ni and Zn were higher in the shoots. The highest efficiencies were observed with NAA treatment and simultaneous inoculation of PF and MY bacteria for Ni, Cd, Pb, and Zn. The highest values of phytoremediation efficiency and uptake efficiency of Ni, Cd, Pb, and Zn were 21.28, 19.11, 14.96 and 18.99 µg g-1, and 31.52, 60.78, 51.89, and 25.33 µg g-1, respectively. Results of present study well demonstrated NAA extracted from blood powder acted as strong chelate agent due to their diversity in size, solubilizing ability, abundant functional groups, and potential in the formation of stable complexes with Ni, Cd, Pb, and Zn, increasing metal availability in soil and improving phytoremediation efficiency in quinoa.

This study focused on an underexplored topic, the potential of natural amino acids (NAA) and plant growth-promoting rhizobacteria (PGPRs) to enhance phytoremediation efficiency of quinoa in a multi-metal contaminated soil with the waste recycling approach. Despite their chelating abilities, NAA have been rarely studied in this context. In the present study, the effects of extracted NAA, acting as environmentally friendly chelating agents, and the inoculation of Pseudomonas fluorescens (PF) and Micrococcus yunnanensis (MY) bacteria were examined on the responses of quinoa in a soil contaminated with Pb, Ni, Cd, and Zn.

Transformation of Iminodi(methylene phosphonate) on Manganese Dioxides - Passivation of the Mineral Surface by (Formed) Mn2.

Aminopolyphosphonates (APPs) are strong chelating agents with growing use in industrial and household applications. In this study, we investigated the oxidation of the bisphosphonate iminodi(methylene phosphonate) (IDMP) - a major transformation product (TP) of numerous commercially used APPs and a potential precursor for aminomethylphosphonate (AMPA) - on manganese dioxide (MnO2). Transformation batch experiments at pH 6 revealed AMPA and phosphate as main TPs, with a phosphorus mass balance of 80 to 92% throughout all experiments. Our results suggest initial cleavage of the C-P bond and formation of the stable intermediate N-formyl-AMPA. Next, C-N bond cleavage leads to the formation of AMPA, which exhibits lower reactivity than IDMP. Reaction rates together with IDMP and Mn2+ sorption data indicate formation of IDMP-Mn2+ surface bridging complexes with progressing MnO2 reduction, leading to the passivation of the mineral surface regarding IDMP oxidation. Compound-specific stable carbon isotope analysis of IDMP in both sorbed and aqueous fractions further supported this hypothesis. Depending on the extent of Mn2+ surface concentration, the isotope data indicated either sorption of IDMP to the mineral surface or electron transfer from IDMP to MnIV to be the rate-limiting step of the overall reaction. Our study sheds further light on the complex surface processes during MnO2 redox reactions and reveals abiotic oxidative transformation of APPs by MnO2 as a potential process contributing to widespread elevated AMPA concentrations in the environment.

Expanding the calibration range of compound-specific chlorine isotope analysis by the preparation of a 37 Cl-enriched tetrachloroethylene.

RATIONALE: The recent development of reliable GC/qMS methods for δ37 Cl compound-specific stable isotope analysis (CSIA) paves the way for dual carbon-chlorine isotope analysis of chlorinated ethenes and thus allows deeper insights into underlying transformation processes/mechanisms. A two-point calibration is indispensable for the precise and correct conversion of raw data to the international δ37 ClSMOC scale. The currently available calibration standards for tetrachloroethylene (PCE) span only a very narrow range from -2.52‰ (EIL2) to +0.29‰ (EIL1), which is considerably smaller than observed δ37 Cl isotope enrichment in (bio-)transformation studies (up to 12‰). METHODS: We describe the preparation and evaluation of a new 37 Cl-enriched PCE standard to avoid bias in δ37 Cl CSIA arising from extrapolation beyond the calibration range. The preparation comprised: (i) partial PCE reduction by zero-valent zinc in a system of PCE, ethanol (initial volume ratio 3/5) and trace amounts of water followed by (ii) liquid-liquid extraction and (iii) a subsequent fractional distillation to purify the 37 Cl-enriched PCE. RESULTS: The obtained PCE (PCEenriched ) showed a purity of 98.8% (mole fraction) and a δ37 ClSMOC value of +10.8 ± 0.5‰. The evaluation of an experimental dataset with and without extrapolation showed no significant variation. CONCLUSIONS: The new PCE standard (PCEenriched ) expands the calibration range to 13.3‰ (previously 2.8‰) and thus prevents potential bias introduced by extrapolation beyond the calibration range.

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.

Two Pathways Compete in the Mn(II)-Catalyzed Oxidation of Aminotrismethylene Phosphonate (ATMP).

Mn(II)-catalyzed oxidation by molecular oxygen is considered a relevant process for the environmental fate of aminopolyphosphonate chelating agents such as aminotrismethylene phosphonate (ATMP). However, the potential roles of Mn(III)ATMP-species in the underlying transformation mechanisms are not fully understood. We combined kinetic studies, compound-specific stable carbon isotope analysis, and equilibrium speciation modeling to shed light on the significance of such Mn-ATMP species for the overall ATMP oxidation by molecular oxygen. The fraction of ATMP complexed with Mn(II) inversely correlated with both (i) the Mn(II)-normalized transformation rate constants of ATMP and (ii) the observed carbon isotope enrichment factors (εc-values). These findings provide evidence for two parallel ATMP transformation pathways exhibiting distinctly different reaction kinetics and carbon isotope fractionation: (i) oxidation of ATMP present in Mn(III)ATMP complexes (εc ≈ -10 ‰) and (ii) oxidation of free ATMP by such Mn(III)ATMP species (εc ≈ -1 ‰) in a catalytic cycle. The higher reaction rate of the latter pathway implies that aminopolyphosphonates can be trapped in catalytic Mn-complexes before being transformed and suggests that Mn(III)ATMP might be a potent oxidant also for other reducible solutes in aqueous environments.

Denitrifier Method for Nitrite Removal in Electrochemical Analysis of the Electron Accepting Capacity of Humic Substances.

Humic substances (HSs) are important electron acceptors and donors in soils and aquifers. The coupling of anoxic nitrogen (N) cycling to the function of HSs as a redox battery, however, remains poorly understood. Mediated electrochemical analysis is an emerging tool to determine the redox properties (i.e., electron donating capacity (EDC), electron accepting capacity (EAC), and redox state) of HS. However, the presence of nitrite (NO2-), a central intermediate of the N-cycle, interferes with the electrochemical determination of the EAC. To eliminate this interference, we developed a bioassay to remove nitrite in HS samples using the denitrifying bacterium Pseudomonas nitroreducens. Cell suspensions of P. nitroreducens completely removed NO2- at various concentrations (1, 2, and 5 mM) from humic acid samples (1 g HA/L) of different redox states. As P. nitroreducens is not able to exchange electrons with dissolved humic acids, the procedure allows an accurate and reliable determination of the EAC of humic acid samples. The proposed method thus opens new perspectives in biogeochemistry to study interactions between HSs and N cycling.

Deciphering the Variability of Stable Isotope (C, Cl) Fractionation of Tetrachloroethene Biotransformation by Desulfitobacterium strains Carrying Different Reductive Dehalogenases Enzymes.

Kinetic isotope effects have been used successfully to prove and characterize organic contaminant transformation on various scales including field and laboratory studies. For tetrachloroethene (PCE) biotransformation, however, causes for the substantial variability of reported isotope enrichment factors (ε) are still not deciphered (εC = -0.4 to -19.0‰). Factors such as different reaction mechanisms and masking of isotope fractionation by either limited intracellular mass transfer or rate-limitations within the enzymatic multistep reaction are under discussion. This study evaluated the contribution of these factors to the magnitude of carbon and chlorine isotope fractionation of Desulfitobacterium strains harboring three different PCE-transforming enzymes (PCE-RdhA). Despite variable single element isotope fractionation (εC = -5.0 to -19.7‰; εCl = -1.9 to -6.3‰), similar slopes of dual element isotope plots (ΛC/Cl values of 2.4 ± 0.1 to 3.6 ± 0.1) suggest a common reaction mechanism for different PCE-RdhAs. Cell envelope properties of the Desulfitobacterium strains allowed to exclude masking effects due to PCE mass transfer limitation. Our results thus revealed that different rate-limiting steps (e.g., substrate channel diffusion) in the enzymatic multistep reactions of individual PCE-RdhAs rather than different reaction mechanisms determine the extent of PCE isotope fractionation in the Desulfitobacterium genus.

Electron Hopping Enables Rapid Electron Transfer between Quinone-/Hydroquinone-Containing Organic Molecules in Microbial Iron(III) Mineral Reduction.

The mechanism of long-distance electron transfer via redox-active particulate natural organic matter (NOM) is still unclear, especially considering its aggregated nature and the resulting low diffusivity of its quinone- and hydroquinone-containing molecules. Here we conducted microbial iron(III) mineral reduction experiments in which anthraquinone-2,6-disulfonate (AQDS, a widely used analogue for quinone- and hydroquinone-containing molecules in NOM) was immobilized in agar to achieve a spatial separation between the iron-reducing bacteria and ferrihydrite mineral. Immobilizing AQDS in agar also limited its diffusion, which resembled electron-transfer behavior of quinone- and hydroquinone-containing molecules in particulate NOM. We found that, although the diffusion coefficient of the immobilized AQDS/AH2QDS was 10 times lower in agar than in water, the iron(III) mineral reduction rate (1.60 ± 0.28 mmol L-1 Fe(II) d-1) was still comparable in both media, indicating the existence of another mechanism that accelerated the electron transfer under low diffusive conditions. We found the correlation between the heterogeneous electron-transfer rate constant (10-3 cm s-1) and the diffusion coefficient (10-7 cm2 s-1) fitting well with the "diffusion-electron hopping" model, suggesting that electron transfer via the immobilized AQDS/AH2QDS couple was accomplished through a combination of diffusion and electron hopping. Electron hopping increased the diffusion concentration gradient up to 106-fold, which largely promoted the overall electron-transfer rate during microbial iron(III) mineral reduction. Our results are helpful to explain the electron-transfer mechanisms in particulate NOM.

Preferential Sorption of Tannins at Aluminum Oxide Affects the Electron Exchange Capacities of Dissolved and Sorbed Humic Acid Fractions.

Natural organic matter and humic substances (HS) in soils and sediments participate in numerous biogeochemical processes. Sorption to redox-inert aluminum oxide (Al2O3) was recently found to affect the redox properties of HS both in sorbed and dissolved state. With this study, we aim to decipher the molecular basis for these observations by applying Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS) and mediated electrochemical analysis to Elliott soil, Pahokee peat, and Suwannee river humic acid (HA) samples before and after sorption to polar Al2O3 and a nonpolar sorbent (DAX-8 resin). The FT-ICRMS data provided evidence of preferential sorption of specific HA fractions, primarily tannin-like compounds, to Al2O3. These oxygen-rich compounds bear a high density of redox-active functional groups, and their adsorption leads to a depletion of electron exchange capacity in dissolved HAs and enrichment of HAs adsorbed at Al2O3. Sorption of HAs to DAX-8 was less selective and caused only slight changes in electron exchange capacities of dissolved and sorbed HA fractions. By combining FT-ICRMS and electrochemical approaches, our findings suggest that a selective sorption of oxygen-rich compounds in HA fractions to mineral oxides is a decisive factor for the different redox properties of dissolved and sorbed HA fractions.

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.

Stable carbon isotope analysis of polyphosphonate complexing agents by anion chromatography coupled to isotope ratio mass spectrometry: method development and application.

Compound-specific carbon isotope analysis (carbon CSIA) by liquid chromatography/isotope ratio mass spectrometry (LC-IRMS) is a novel and promising tool to elucidate the environmental fate of polar organic compounds such as polyphosphonates, strong complexing agents for di- and trivalent cations with growing commercial importance over the last decades. Here, we present a LC-IRMS method for the three widely used polyphosphonates 1-hydroxyethane 1,1-diphosphonate (HEDP), amino tris(methylenephosphonate) (ATMP), and ethylenediamine tetra(methylenephosphonate) (EDTMP). Separation of the analytes, as well as ATMP and its degradation products, was carried out on an anion exchange column under acidic conditions. Quantitative wet chemical oxidation inside the LC-IRMS interface to CO2 was achieved for all three investigated polyphosphonates at a comparatively low sodium persulfate concentration despite the described resilience of HEDP towards oxidative breakdown. The developed method has proven to be suitable for the determination of carbon isotope fractionation of ATMP transformation due to manganese-catalyzed reaction with molecular oxygen, as well as for equilibrium sorption of ATMP to goethite. A kinetic isotope effect was associated with the investigated reaction pathway, whereas no detectable isotope fractionation could be observed during sorption. Thus, CSIA is an appropriate technique to distinguish between sorption and degradation processes that contribute to a concentration decrease of ATMP in laboratory batch experiments. Our study highlights the potential of carbon CSIA by LC-IRMS to gain a process-based understanding of the fate of polyphosphonate complexing agents in environmental as well as technical systems.

Capillary electrophoresis-mass spectrometry for the direct analysis of glyphosate: method development and application to beer beverages and environmental studies.

In this study, we developed and validated a CE-TOF-MS method for the quantification of glyphosate (N-(phosphonomethyl)glycine) and its major degradation product aminomethylphosphonic acid (AMPA) in different samples including beer, media from toxicological analysis with Daphnia magna, and sorption experiments. Using a background electrolyte (BGE) of very low pH, where glyphosate is still negatively charged but many matrix components become neutral or protonated, a very high separation selectivity was reached. The presence of inorganic salts in the sample was advantageous with regard to preconcentration via transient isotachophoresis. The advantages of our new method are the following: no derivatization is needed, high separation selectivity and thus matrix tolerance, speed of analysis, limits of detection suitable for many applications in food and environmental science, negligible disturbance by metal chelation. LODs for glyphosate were < 5 µg/L for both aqueous and beer samples, the linear range in aqueous samples was 5-3000 µg/L, for beer samples 10-3000 µg/L. For AMPA, LODs were 3.3 and 30.6 µg/L, and the linear range 10-3000 µg/L and 50-3000 µg/L, for aqueous and beer samples, respectively. Recoveries in beer samples for glyphosate were 94.3-110.7% and for AMPA 80.2-100.4%. We analyzed 12 German and 2 Danish beer samples. Quantification of glyphosate and AMPA was possible using isotopically labeled standards without enrichment, purification, or dilution, only degassing and filtration were required for sample preparation. Finally, we demonstrate the applicability of the method for other strong acids, relevant in food and environmental sciences such as N-acetyl glyphosate, N-acetyl AMPA (present in some glyphosate resistant crop), trifluoroacetic acid, 2-methyl-4-chlorophenoxyacetic acid, glufosinate and its degradation product 3-(methylphosphinico)propionic acid, oxamic acid, and others.

Effects of Sorption on Redox Properties of Natural Organic Matter.

Natural organic matter (NOM) is an important redox-active component of natural porous media and predominantly occurs in the sorbed state. Nevertheless, the effects of NOM sorption at minerals on its redox properties are unknown and thus are the major objective of this study. We report how adsorption of three different humic acids (HAs) to redox-inert sorbents (polar Al2O3 and nonpolar DAX-8 resin) affects their electron-exchange capacities (EEC) and redox states. The electron-donating capacity of HAs sorbed at Al2O3 increased by up to 200%, whereas the EEC of the remaining dissolved HA fractions decreased compared with their initial properties. Sorption at DAX-8, however, did not affect significantly the EEC of HAs. We rationalize these results by (i) preferential sorption of NOM components rich in redox-active groups (e.g., quinone, polyphenols) and (ii) surface-catalyzed polymerization of polyphenolic compounds. Our results demonstrate that even in the absence of electron exchange with the sorbent, adsorption to polar mineral surfaces considerably affects the redox properties of NOM. Quantification of the redox state and EEC of adsorbed NOM is thus crucial for assessing electron-transfer processes as well as organic carbon stabilization and sequestration in soils and sediments.

Calibration bias of experimentally determined chlorine isotope enrichment factors: the need for a two-point calibration in compound-specific chlorine isotope analysis.

RATIONALE: The recent development of compound-specific online chlorine isotope analysis (37 Cl-CSIA) methods has fostered dual chlorine-carbon isotope studies to gain better insights into sources and environmental transformation reactions of chlorinated ethenes. One-point and two-point calibration schemes are currently used to convert raw data to the international δ37 ClSMOC scale, but a critical evaluation of best practices to arrive at reliable δ37 ClSMOC signatures and enrichment factors was missing and is presented here. METHODS: Aqueous solutions of neat perchloroethylene and trichloroethylene (TCE) and aqueous samples from a TCE biodegradation experiment with pure cultures of Desulfitobacterium hafniense Y51 were analysed for their chlorine isotope ratios using GC/qMS and GC/IRMS. The δ37 ClSMOC values were obtained using one-point and two-point calibration schemes. Chlorine isotope enrichment factors, εCl , were calculated using both approaches and the corresponding bias of δ37 ClSMOC values introduced by the different types of calibration was determined. RESULTS: Different calibration methods resulted in significant differences (up to 30%) in both δ37 Cl signatures and εCl values. CONCLUSIONS: Our results demonstrate that a two-point calibration together with comprehensive information on reference materials is indispensable and should become standard practice for reliable 37 Cl-CSIA of organic compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Experimental Determination of Isotope Enrichment Factors - Bias from Mass Removal by Repetitive Sampling.

Application of compound-specific stable isotope approaches often involves comparisons of isotope enrichment factors (ε). Experimental determination of ε-values is based on the Rayleigh equation, which relates the change in measured isotope ratios to the decreasing substrate fractions and is valid for closed systems. Even in well-controlled batch experiments, however, this requirement is not necessarily fulfilled, since repetitive sampling can remove a significant fraction of the analyte. For volatile compounds the need for appropriate corrections is most evident, and various methods have been proposed to account for mass removal and for volatilization into the headspace. In this study we use both synthetic and experimental data to demonstrate that the determination of ε-values according to current correction methods is prone to considerable systematic errors even in well-designed experimental setups. Application of inappropriate methods may lead to incorrect and inconsistent ε-values entailing misinterpretations regarding the processes underlying isotope fractionation. In fact, our results suggest that artifacts arising from inappropriate data evaluation might contribute to the variability of published ε-values. In response, we present novel, adequate methods to eliminate systematic errors in data evaluation. A model-based sensitivity analysis serves to reveal the most crucial experimental parameters and can be used for future experimental design to obtain correct ε-values allowing mechanistic interpretations.

Optimization of a large-volume injection method for compound-specific isotope analysis of polycyclic aromatic compounds at trace concentrations.

RATIONALE: Compound-specific isotope analysis (CSIA) of persistent organic contaminants can be used for source apportioning in the environment if appropriate sensitivity can be achieved. This paper describes the optimization and validation of a sensitive analytical approach for the determination of the carbon isotope composition of semi-volatile organic compounds, such as polycyclic aromatic hydrocarbons (PAHs). METHODS: Analyses are based on the introduction of up to 150 µL of organic extracts by means of programmed temperature vaporization-large-volume injection combined with gas chromatography coupled to isotope ratio mass spectrometry (PTV-LVI-GC/IRMS). To allow for the analysis of more volatile, low-molecular-weight PAHs, the PTV injector was equipped with a sub-ambient/cryogenic cooling. Accuracy, precision, linearity and determination limits for application in isotope analysis were evaluated for a set of individual PAHs ranging from two- to five-ring molecular structures. The method was exemplified by determining the δ(13) C values of individual PAHs in soil samples in a source apportionment study at a contaminated site. RESULTS: The choice of PTV injection parameters is crucial to prevent isotope fractionation during injection and largely depends on the analytes to be determined. The observed isotope fractionation effect on semi-volatiles depends on the applied solvent and injection temperature and demonstrates the importance of performing appropriate tests with given PTV parameters for each of the compounds of interest. The proposed PTV-LVI-GC/IRMS method allows the carbon isotope ratio (δ(13) C value) of individual PAHs to be determined accurately and precisely at concentrations of 0.04-0.1 ng µL(-1) even for volatile PAHs such as naphthalene or acenaphthene. CONCLUSIONS: LVI with PTV injector cooling allows for the isotopic analysis of volatile and semi-volatile PAHs at trace concentrations, thus considerably expanding the applicability of CSIA in environmental studies.

Resiliency of Stable Isotope Fractionation (δ(13)C and δ(37)Cl) of Trichloroethene to Bacterial Growth Physiology and Expression of Key Enzymes.

Quantification of in situ (bio)degradation using compound-specific isotope analysis requires a known and constant isotope enrichment factor (ε). Because reported isotope enrichment factors for microbial dehalogenation of chlorinated ethenes vary considerably we studied the potential effects of metabolic adaptation to TCE respiration on isotope fractionation (δ(13)C and δ(37)Cl) using a model organism (Desulfitobacterium hafniesne Y51), which only has one reductive dehalogenase (PceA). Cells grown on TCE for the first time showed exponential growth until 10(9) cells/mL. During exponential growth, the cell-normalized amount of PceA enzyme increased steadily in the presence of TCE (up to 21 pceA transcripts per cell) but not with alternative substrates (<1 pceA transcript per cell). Cultures initially transferred or subcultivated on TCE showed very similar isotope fractionation, both for carbon (εcarbon: -8.6‰ ± 0.3‰ or -8.8‰ ± 0.2‰) and chlorine (εchlorine: -2.7‰ ± 0.3‰) with little variation (0.7‰) for the different experimental conditions. Thus, TCE isotope fractionation by D. hafniense strain Y51 was affected by neither growth phase, pceA transcription, or translation, nor by PceA content per cell, suggesting that transport limitations did not affect isotope fractionation. Previously reported variable ε values for other organohalide-respiring bacteria might thus be attributed to different expression levels of their multiple reductive dehalogenases.

Diffusive fractionation of BTEX and chlorinated ethenes in aqueous solution: quantification of spatial isotope gradients.

Laboratory experiments were performed to investigate and quantify the extent of diffusive isotope fractionation of organic contaminants in aqueous solution. We selected petroleum hydrocarbons (toluene and ethylbenzene, in 1:2 mixtures of labeled (perdeuterated) and nonlabeled isotopologues) and chlorinated solvents (trichloroethene, TCE, and cis-dichloroethene, cis-DCE, at their natural isotopic abundance) as model compounds. The experimental approach using gel diffusion tubes allowed us to resolve concentration and isotopic gradients induced by isotopologue-specific diffusion and to determine aqueous diffusion coefficients in agreement with the values calculated using published empirical correlations. The experimental results were quantitatively evaluated with numerical simulations to determine the aqueous diffusion coefficients, D, and the exponent of the inverse power-law relation between D and the molecular mass of the isotopologues. The results show remarkable diffusive isotope fractionation for all the investigated organic compounds; however, the extent of fractionation was found to be smaller for the chlorinated ethenes and remarkably deviating from an inverse square root relationship between the isotopologues diffusion coefficients and their molecular mass. The outcomes of this study are relevant for the interpretation of isotopic signatures of organic contaminants in environmental systems and for the quantitative application of compound specific isotope analysis (CSIA) that needs to take into account the fractionation effects of both physical and transformation processes.

Dispersive liquid-liquid microextraction as a novel enrichment approach for compound-specific carbon isotope analysis of chlorinated phenols.

Compound-specific isotope analysis (CSIA) via gas chromatography-isotope ratio mass spectrometry (GC-IRMS) is a potent tool to elucidate the fate of (semi-)volatile organic contaminants in technical and environmental systems. Yet, due to the comparatively low sensitivity of IRMS, an enrichment step prior to analysis often is inevitable. A promising approach for fast as well as economic analyte extraction and preconcentration prior to CSIA is dispersive liquid-liquid microextraction (DLLME) - a well-established technique in concentration analysis of contaminants from aqueous samples. Here, we present and evaluate the first DLLME method for GC-IRMS exemplified by the analysis of chlorinated phenols (4-chlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol) as model compounds. The analytes were simultaneously acetylated with acetic anhydride and extracted from the aqueous phase using a binary solvent mixture of acetone and tetrachloroethylene. With this method, reproducible δ13C values were achieved with errors ≤ 0.6‰ (n = 3) for aqueous concentrations down to 100 µg L-1. With preconcentration factors between 130 and 220, the method outperformed conventional liquid-liquid extraction in terms of sample preparation time and resource consumption with comparable reproducibility. Furthermore, we have demonstrated the suitability of the method (i) for the extraction of the analytes from a spiked river water sample and (ii) to quantify kinetic carbon isotope effect for 2,4,6-trichlorophenol during reduction with zero-valent zinc in a laboratory batch experiment. The presented work shows for the first time the potential of DLLME for analyte enrichment prior to CSIA and paves the way for further developments, such as the extraction of other compounds or scaling up to larger sample volumes.

Efficient Immobilization of heavy metals using newly synthesized magnetic nanoparticles and some bacteria in a multi-metal contaminated soil.

Simultaneous application of modified Fe3O4 with biological treatments in remediating multi-metal polluted soils, has rarely been investigated. Thus, a pioneering approach towards sustainable environmental remediation strategies is crucial. In this study, we aimed to improve the efficiency of Fe3O4 as adsorbents for heavy metals (HMs) by applying protective coatings. We synthesized core-shell magnetite nanoparticles coated with modified nanocellulose, nanohydrochar, and nanobiochar, and investigated their effectiveness in conjunction with bacteria (Pseudomonas putida and Bacillus megaterium) for remediating a multi-metal contamination soil. The results showed that the coatings significantly enhanced the immobilization of heavy metals in the soil, even at low doses (0.5%). The coating of nanocellulose had the highest efficiency in stabilizing metals due to the greater variety of surface functional groups and higher specific surface area (63.86 m2 g-1) than the other two coatings. Interestingly, uncoated Fe3O4 had lower performance (113.6 m2 g-1) due to their susceptibility to deformation and oxidation. The use of bacteria as a biological treatment led to an increase in the stabilization of metals in soil. In fact, Pseudomonas putida and Bacillus megaterium increased immobilization of HMs in soil successfully because of extracellular polymeric substances and intensive negative charges. Analysis of metal concentrations in plants revealed that Ni and Zn accumulated in the roots, while Pb and Cd were transferred from the roots to the shoots. Treatment Fe3O4 coated with modified nanocellulose at rates of 0.5 and 1% along with Pseudomonas putida showed the highest effect in stabilizing metals. Application of coated Fe3O4 for in-situ immobilization of HMs in contamination soils is recommendable due to their high metal stabilization efficiency and suitability to apply in large quantities.