Divergence of Biocrust Active Bacterial Communities in the Negev Desert During a Hydration-Desiccation Cycle.

Rain events in arid environments are highly unpredictable and intersperse extended periods of drought. Therefore, tracking changes in desert soil bacterial communities during rain events, in the field, was seldom attempted. Here, we assessed rain-mediated dynamics of active bacterial communities in the Negev Desert biological soil crust (biocrust). Biocrust samples were collected during, and after a medium rainfall and dry soil was used as a control; we evaluated the changes in active bacterial composition, potential function, potential photosynthetic activity, and extracellular polysaccharide (EPS) production. We hypothesized that rain would activate the biocrust phototrophs (mainly Cyanobacteria), while desiccation would inhibit their activity. In contrast, the biocrust Actinobacteria would decline during rewetting and revive with desiccation. Our results showed that hydration increased chlorophyll content and EPS production. As expected, biocrust rewetting activated Cyanobacteria, which replaced the former dominant Actinobacteria, boosting potential autotrophic functions. However, desiccation of the biocrust did not immediately change the bacterial composition or potential function and was followed by a delayed decrease in chlorophyll and EPS levels. This dramatic shift in the community upon rewetting led to modifications in ecosystem services. We propose that following a rain event, the response of the active bacterial community lagged behind the biocrust water content due to the production of EPS which delayed desiccation and temporarily sustained the biocrust community activity.

Transformation of 2, 4, 6-trinitrotoluene by Stenotrophomonas strain SG1 under aerobic and anaerobic conditions.

TNT, or 2,4,6-trinitrotoluene, is a common explosive that can contaminate soil and groundwater in production sites, military training areas, and disposal locations. The compound is highly toxic; therefore, there is an urgent need to rehabilitate the impacted environments. Harnessing the microbial ability to biodegrade TNT into environmentally harmless compound(s) is one approach to remediating contaminated sites. In our study, we report on the genomic and metabolic ability of Stenotrophomonas strain SG1 to degrade TNT under aerobic and anaerobic conditions. The bacterial strain SG1 was first isolated as a contaminant from a culture of Diaphorobacter sp. strain DS2 over minimal media supplemented with TNT. The draft genome assembly of strain SG1 is ∼4.7 Mb and is distributed among 358 contigs. The homology search against the custom database of enzymes responsible for TNT biodegradation revealed the presence of three N-ethylmaleimide reductases (NemA) with a defined KEGG ortholog and KEGG pathway of TNT degradation. The presence of respiratory nitrate reductases has also been mapped, which supports denitrification under anaerobic conditions. Experimentally, the TNT transformation rate accelerated when carbon sources, such as sodium acetate, sodium citrate, sodium succinate, sucrose, and glucose (final concentration of 5 mM), were added. Citrate promoted the highest growth and TNT transformation ratio (88.35%) in 120 h. With the addition of 5 mM ammonium chloride, TNT completely disappeared in the citrate and sucrose-containing treatments in 120 h. However, higher biomass was obtained in the sucrose and glucose-containing treatments in 120 h. During incubation, the formation of amino dinitrotoluene isomers, dinitrotoluene isomers, trinitrobenzene, azoxy isomers, diaryl hydroxylamines, and corresponding secondary amines was confirmed by GC/MS and UPLC/MS. 2-Amino-4-nitrotoluene, 4-amino-2-nitrotoluene, and 2-amino-6-nitrotoluene were also identified in the culture supernatant by GC/MS. Under anaerobic conditions, TNT completely disappeared in the citrate and citrate plus nitrate treatments. Since the strain shows the ability to remove TNT, this research should be useful in basic research and practical applications for removing TNT from wastewater.

Inhibition effect of 2,4,6-trinitrotoluene (TNT) on RDX degradation by rhodococcus strains isolated from contaminated soil and water.

2,4,6-trinitrotoluene (TNT) is a highly toxic explosive that contaminates soil and water and may interfere with the degradation of co-occurring compounds, such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We proposed that TNT may influence RDX-degrading bacteria via either general toxicity or a specific effect on the |RDX degradation mechanisms. Thus, we examined the impact of TNT on RDX degradation by Rhodococcus strains YH1, T7, and YY1, which were isolated from an explosives-polluted environment. Although partly degraded, TNT did not support the growth of any of the strains when used as either sole carbon or sole nitrogen sources, or as carbon and nitrogen sources. The incubation of a mixture of TNT (25 mg/l) and RDX (20 mg/l) completely inhibited RDX degradation. The effect of TNT on the cytochrome P450, catalyzing RDX degradation, was tested in a resting cell experiment, proving that TNT inhibits XplA protein activity. A dose-response experiment showed that the IC50/trans values for YH1, T7, and YY1 were 7.272, 5.098, and 9.140 (mg/l of TNT), respectively, illustrating variable sensitivity to TNT among the strains. The expression of xplA was also strongly suppressed by TNT. Cells that were pre-grown with RDX (allowing xplA expression) and incubated with ammonium chloride, glucose, and TNT, completely transformed into their amino dinitrotoluene isomers and formed azoxy toluene isomers. The presence of oxygen-insensitive nitroreductase that enable reduction of the nitro group in the presence of O2 in the genomes of these strains suggests that they are responsible for TNT transformation in the cultures. The experimental results concluded that TNT has an adverse effect on RDX degradation by the examined strains. It inhibits RDX degradation due to the direct impact on cytochrome P450, xplA, or its expression. The tested strains can transform TNT independently of RDX. Thus, degradation of both compounds is possible if TNT concentrations are below their IC50 values.

Continuous monitoring of dissolved inorganic nitrogen (DIN) transformations along the waste-vadose zone - groundwater path of an uncontrolled landfill, using multiple N-species isotopic analysis.

Landfill leachates contain a heavy load of dissolved inorganic nitrogen (DIN), posing a threat to water resources. Therefore, it is highly important to understand the processes that control its evolution (speciation, accumulation, or attenuation) during the percolation of leachates through the unsaturated zone, finally affecting the groundwater. However, tracking DIN transformations in this complex and inaccessible environment is challenging, and knowledge concerning this important topic under field conditions is scarce. The presented study used a unique monitoring system that allows sampling of repetitive samples from within the waste and the unsaturated zone. An array of 8 wells penetrating the underlying aquifer completed the spatial observation. Multiple N-species isotopic approach was applied to discern the dominating N-involving processes over the continuum - from the waste mound through the unsaturated zone and the underlying aquifer. Despite the considerable heterogeneity observed throughout the profile, the results provided a cohesive and valuable reflection of the evolution of the inorganic nitrogen pool in this highly contaminated environment. Leachates inside the waste had reducing characteristics with high accumulation of ammonium (up to 360 mg/l NH4+-N), and a distinct δ15N-NH4+ range (-3‰ to +10‰). The upper layers of the unsaturated zone underneath the landfill margins found to be aerated, promoting N oxidation which resulted in the accumulation of nitrate in the leachates (up to 490 mg/l NO3-N). Exceptionally high concentrations of nitrite (up to 126 mg/l NO2-N) were found as oxygen levels decreased in deeper sections of the vadose zone. Enrichment of δ15N-NO2- compared to δ15N-NO3- indicated the significance of autotropic nitrite reduction, controlling the DIN composition, correlated with NO2- accumulation and net DIN attenuation. The δ15N: δ18O ratio implied co-occurrence of denitrification in the leachates, even in the more oxidized sections, further contributing to N-attenuation in the unsaturated zone. In the aquifer, δ15N-NH4+ values and δ15N: δ18O ratio linked N contamination to the leachates source. The encounter with the oxidized groundwater promoted intensive nitrification. δ15N-NO2- values in the groundwater were lighter than both δ15N-NH4+ and δ15N-NO3- by 22‰ to 62‰, implying the co-occurrence of nitrification-denitrification processes. The effect of denitrification grew with decreasing dissolved oxygen (DO) levels below 0.5 mg/l towards the center of the plume, contributing to net DIN attenuation in the plume. The findings are significant for any consideration of the risk posed by DIN, as well as remediation measures, in a landfill environment and other sites with a heavy load of degrading organic matter.

Optimization of compound-specific chlorine stable isotope analysis of chloroform using the Taguchi design of experiments.

RATIONALE: Chloroform, a probable human carcinogen, is commonly detected in various concentration levels in many surface water and groundwater sources. Compound-specific chlorine stable isotope analysis (Cl-CSIA) is significant in investigating the fate of chlorinated contaminants in the environment. Analytical conditions should, however, be thoroughly examined for any isotopic fractionation. In this study, we simultaneously optimize three analytical parameters for a robust online Cl-CSIA of chloroform using the Taguchi design of experiments. METHODS: For Cl-CSIA, a purge-and-trap autosampler coupled to a gas chromatograph in tandem with a quadrupole mass spectrometer, with electron ionization in selected ion monitoring (SIM) mode, was used. Using the Taguchi method, the dominant parameter affecting the results of Cl-CSIA for chloroform was identified through concurrent investigation of the signal-to-noise ratios (S/N) of three parameters, each at three levels: purging time (5, 10, 15 min), transfer time (80, 120, 160 s), and dwell time (20, 60, 100 ms). Moreover, the optimum combination of the levels was identified. RESULTS: The purging time, with a maximum S/N, resulted in the highest influence on the isotope ratios determined. It was further refined through additional experiments to sufficiently extract chloroform from the aqueous phase. Accordingly, 8 min of purging time, 120 s transfer time and 100 ms dwell time were the optimum conditions for Cl-CSIA of chloroform. Post-optimization, a precision of ±0.28 ‰ was achieved for 8.4 nmol of chloroform (equivalent to 0.89 µg or approx. 25 nmol Cl-mass on column). CONCLUSIONS: A simple online method for Cl-CSIA of chloroform was optimized with the Taguchi design of experiments. The Taguchi method was very useful for the optimization of the analytical conditions. However, the purging conditions should be fine-tuned and selected so that sufficient extraction of a target compound is confirmed to acquire a stable and higher precision of the method.

Holistic approach for evaluation of landfill leachate pollution potential - From the waste to the aquifer.

Leachate contamination from uncontrolled landfills is a long-lasting environmental hazard that threatens groundwater resources worldwide. We developed a holistic site-characterization approach that incorporates leachate data from the waste body, unsaturated zone, and groundwater with information on landfill geomorphology and climatic conditions. An advanced vadose-zone monitoring system was used to characterize the percolation patterns and chemical properties of the leachates in the waste body and underlying unsaturated zone; a set of observation wells was used to characterize the contaminants' distribution in the groundwater. Multivariate analysis of the chemical composition overcame multiparameter complexity, and pinpointed the dominant factors controlling contaminant migration dynamics. The landfill's mound morphology, constructed with steep slopes, led to runoff generation, limited water infiltration through the waste, and enhanced infiltration of contaminated water at the landfill margins. Aerated conditions in the unsaturated zone under the margins induced leachate degradation and oxidation processes. The chemical composition of leachates under the center of the landfill remained typically anaerobic (high DOC, NH4+, Fe2+) despite the low water penetration. The limited water-percolation rates through the waste body and substantial mixing of the leachates with the oxidizing aquifer water led to almost complete degradation of the organic matter and significant nitrogen attenuation in the groundwater. Bromide release from decomposing waste served as an effective tracer for leachate distribution in the subsurface. The holistic approach implemented in this study provides robust and valuable insights into the factors that control landfill contamination potential and can be implemented in other sites with different climatic and geomorphological features.

Multi-elemental C-Br-Cl isotope analysis for characterizing biotic and abiotic transformations of 1-bromo-2-chloroethane (BCE).

Multi-elemental C-Br-Cl compound-specific isotope analysis was applied for characterizing abiotic and biotic degradation of the environmental pollutant 1-bromo-2-chloroethane (BCE). Isotope effects were determined in the model processes following hydrolytic dehalogenation and dihaloelimination pathways as well as in a microcosm experiment by the microbial culture from the contaminated site. Hydrolytic dehalogenation of BCE under alkaline conditions and by DhaA enzyme resulted in similar dual isotope slopes (ɅC/Br 21.9 ± 4.7 and 19.4 ± 1.8, respectively, and ɅC/Cl ~ ∞). BCE transformation by cyanocobalamin (B12) and by Sulfurospirillum multivorans followed dihaloelimination and was accompanied by identical, within the uncertainty range, dual isotope slopes (ɅC/Br 8.4 ± 1.7 and 7.9 ± 4.2, respectively, and ɅC/Cl 2.4 ± 0.3 and 1.5 ± 0.6, respectively). Changes over time in the isotope composition of BCE from the contaminated groundwater showed only a slight variation in δ13C values and were not sufficient for the elucidation of the BCE degradation pathway in situ. However, an anaerobic microcosm experiment with the enrichment cultures from the contaminated groundwater presented dual isotope slopes similar to the hydrolytic pathway, suggesting that the potential for BCE degradation in situ by the hydrolytic dehalogenation pathway exists in the contaminated site.

Isotope analysis method for the herbicide bromoxynil and its application to study photo-degradation processes.

Bromoxynil is an increasingly applied nitrile herbicide used for post-emergent control of annual broadleaved weeds. Compound-specific isotope analysis (CSIA) of the compound is of interest for studying its environmental fate, yet is challenging following its polar nature. We present a CSIA method for bromoxynil that includes offline thin-layer chromatography purification followed by an elemental analyzer isotope ratio mass spectrometer (EA-IRMS). This method was shown to be accurate and precise for δ13C and δ15N analysis of the compound (standard deviation of replicate standards <0.5‰). The method was applied to photodegraded samples, either radiated under laboratory condition with a UV lamp, or exposed to sunlight under environmental conditions. Dominating degradation products were similar in both cases. Nevertheless, isotope effects differed, presenting a strong inverse carbon isotope effect (εC = 4.74 ±â€¯0.82‰) and a weak inverse nitrogen isotope effect (εN = 0.76 ±â€¯0.12‰) for the laboratory experiment, and an insignificant carbon isotope effect (εC = 0.34 ±â€¯0.44‰) and a normal nitrogen isotope effect (εN = -3.70 ±â€¯0.30‰) for the natural conditions experiment. The differences in δ13C vs. δ15N enrichment trends suggest different mechanism for the two processes. Finally, the obtained dual isotope trend for natural conditions provide the basis for studying the dominance of photodegradation as a degradation route in the environment.

Potential for co-metabolic oxidation of TCE and evidence for its occurrence in a large-scale aquifer survey.

Trichloroethylene (TCE) is a groundwater pollutant that is prevalent worldwide. In contaminated groundwater, TCE can be biodegraded following either reductive dechlorination or aerobic co-metabolic oxidation. However, since the co-metabolic process is not accompanied by indicative and easily detectable transformation products, little is known about its prominence in the environment. To estimate the environmental importance of the oxidative process, a regional groundwater survey was conducted. In this survey, polluted water from 100 wells along the Israeli Coastal Aquifer was sampled. Geochemical data indicated oxic conditions prevailing in most sites. The sampled groundwater was used for microcosm experiments, functional gene analysis, and TCE compound-specific isotope analysis (δ13C and δ37Cl). Enrichments of methane and toluene oxidizers in microcosms indicated the high potential of the indigenous microbial community to co-metabolically oxidize TCE. This was further reinforced by the high abundance of mmoX and PHE functional genes quantified in some of the sites (yet lower abundance of TOD functional gene was found). Finally, compound-specific isotope analysis was used to assess the magnitude of TCE oxidation in practice. Applying the isotopic tool for scattered points on a regional scale demanded the consideration of a wide δ13C range of source TCE, hampering the ability to detect small shifts of a single permil. Thus, despite the high potential for the oxidation process, no evidence was attained for the natural occurrence of the process, and significant isotopic shifts were restricted to actively treated sites only. This limitation should be considered in future regional scale studies, in which no single source is defined.

Isotope Fractionation (δ13C, δ15N) in the Microbial Degradation of Bromoxynil by Aerobic and Anaerobic Soil Enrichment Cultures.

Bromoxynil is an increasingly applied nitrile herbicide. Under aerobic conditions, hydration, nitrilation, or hydroxylation of the nitrile group commonly occurs, whereas under anaerobic conditions reductive dehalogenation is common. This work studied the isotope effects associated with these processes by soil cultures. The aerobic soil enrichment culture presented a significant increase in Stenotrophomonas, Pseudomonas, Chryseobacterium, Achromobacter, Azospirillum, and Arcticibacter, and degradation products indicated that nitrile hydratase was the dominant degradation route. The anaerobic culture was dominated by Proteobacteria and Firmicutes phyla with a significant increase in Dethiosulfatibacter, and degradation products indicated reductive debromination as a major degradation route. Distinct dual-isotope trends (δ13C, δ15N) were determined for the two routes: a strong inverse nitrogen isotope effect (εN = 10.56 ± 0.36‰) and an insignificant carbon isotope effect (εC = 0.37 ± 0.36‰) for the aerobic process versus a negligible effect for both elements in the anaerobic process. These trends differ from formerly reported trends for the photodegradation of bromoxynil and enable one to distinguish between the processes in the field.

Application of vadose-zone monitoring system for real-time characterization of leachate percolation in and under a municipal landfill.

Leachates from solid-waste landfills are considered a severe threat to groundwater quality. The fate of pollutants in the waste and underlying unsaturated zone is crucial for evaluating environmental risks and selecting a restoration strategy. In this study, a vadose-zone monitoring system (VMS) installed in a municipal landfill was used, for the first time, to continuously track leachates percolation dynamics and assess their chemical transformation across the entire thickness of the waste body (15m) and underlying unsaturated zone (16m) to the water table. Winter rains were found to quickly infiltrate through the waste and underlying vadose zone despite a clay cover that was implemented as part of a restoration and leachate-prevention strategy. Within the waste body, the flow pattern was controlled by preferential flow paths, which changed frequently. It is hypothesized that ongoing decomposition of the waste creates dynamic variations in the waste's physical structure and flow pattern. Water samples collected from the waste layer indicated the formation of highly polluted leachates. The chemical composition in the waste body showed extreme variability between sampling points with respect to DOC (407-31,464mg/L), BOD/COD ratios (0.07-0.55), Fe2+ (6.8-1154mg/L), ammonium (68-2924mg/L) and heavy metal concentrations. Environmental hot spots creating concentrated, aggressive, "acid-phase" leachates still exist in the waste more than 13years after closing the landfill. However, continuous changes in the flow pattern and moisture distribution affected the creation and decay of such environments. In the underlying sandy vadose zone, some sections repeatedly exhibited stronger and faster flow characteristics than others. These local fluxes of concentrated leachates rapidly transported heavy contaminant loads toward the groundwater. However results showed evidence of continual attenuation processes in the deep vadose zone, with the anaerobic digestion of organic matter, as well as the significant retention of heavy metals.

Using the natural biodegradation potential of shallow soils for in-situ remediation of deep vadose zone and groundwater.

In this study, we examined the ability of top soil to degrade perchlorate from infiltrating polluted groundwater under unsaturated conditions. Column experiments designed to simulate typical remediation operation of daily wetting and draining cycles of contaminated water amended with an electron donor. Covering the infiltration area with bentonite ensured anaerobic conditions. The soil remained unsaturated, and redox potential dropped to less than -200mV. Perchlorate was reduced continuously from ∼1150mg/L at the inlet to ∼300mg/L at the outlet in daily cycles. Removal efficiency was between 60 and 84%. No signs of bioclogging were observed during three operation months although occasional iron reduction observed due to excess electron donor. Changes in perchlorate reducing bacteria numbers were inferred from an increased in pcrA gene abundances from ∼105 to 107 copied per gram at the end of the experiment indicating the growth of perchlorate-reducing bacteria. We proposed that the topsoil may serve as a bioreactor to treat high concentrations of perchlorate from the contaminated groundwater. The treated water that infiltrates from the topsoil through the vadose zone could be used to flush perchlorate from the deep vadose zone into the groundwater where it is retrieved again for treatment in the topsoil.

Mercury isotope signatures as tracers for Hg cycling at the New Idria Hg mine.

Mass-dependent fractionation (MDF) and mass-independent fractionation (MIF) of Hg isotopes provides a new tool for tracing Hg in contaminated environments such as mining sites, which represent major point sources of Hg pollution into surrounding ecosystems. Here, we present Hg isotope ratios of unroasted ore waste, calcine (roasted ore), and poplar leaves collected at a closed Hg mine (New Idria, CA, U.S.A.). Unroasted ore waste was isotopically uniform with δ(202)Hg values from -0.09 to 0.16‰ (± 0.10‰, 2 SD), close to the estimated initial composition of the HgS ore (-0.26‰). In contrast, calcine samples exhibited variable δ(202)Hg values ranging from -1.91‰ to +2.10‰. Small MIF signatures in the calcine were consistent with nuclear volume fractionation of Hg isotopes during or after the roasting process. The poplar leaves exhibited negative MDF (-3.18 to -1.22‰) and small positive MIF values (Δ(199)Hg of 0.02 to 0.21‰). Sequential extractions combined with Hg isotope analysis revealed higher δ(202)Hg values for the more soluble Hg pools in calcines compared with residual HgS phases. Our data provide novel insights into possible in situ transformations of Hg phases and suggest that isotopically heavy secondary Hg phases were formed in the calcine, which will influence the isotope composition of Hg leached from the site.

Mercury localization and speciation in plants grown hydroponically or in a natural environment.

Better understanding of mercury (Hg) accumulation, distribution, and speciation in plants is required to evaluate potential risks for the environment and to optimize phytostabilization strategies for Hg-contaminated soils. The behavior of Hg in alfalfa (Medicago sativa) plants grown under controlled conditions in a hydroponic system (30 µM HgCl2) was compared with that of naturally occurring Horehound (Marrubium vulgare) plants collected from a mining soil polluted with Hg (Almadenejos, Spain) to characterize common mechanisms of tolerance. Synchrotron X-ray Fluorescence microprobe (µ-SXRF) showed that Hg accumulated at the root apex of alfalfa and was distributed through the vascular system to the leaves. Transmission electron microscopy (TEM) implied association of Hg with cell walls, accompanied by their structural changes, in alfalfa roots. Extended X-ray absorption fine structure (EXAFS) determined that Hg was principally bound to biothiols and/or proteins in M. sativa roots, stems, and leaves. However, the major fraction of Hg detected in M. vulgare plants consisted of mineral species, possibly associated with soil components. Interestingly, the fraction of Hg bound to biothiols/proteins (i.e., metabolically processed Hg) in leaves of both plants (alfalfa and M. vulgare) was similar, in spite of the big difference in Hg accumulation in roots, suggesting that some tolerance mechanisms might be shared.