Room-to-Room Variability of Airborne Polychlorinated Biphenyls in Schools and the Application of Air Sampling for Targeted Source Evaluation.

Airborne polychlorinated biphenyl (PCB) concentrations are higher indoors than outdoors due to their historical use in building materials and their presence in modern paints and surface treatments. For some populations, including school children, PCB levels indoors result in inhalation exposures that may be greater than or equivalent to exposure through diet. In a school, PCB exposure may come from multiple sources. We hypothesized that there are both Aroclor and non-Aroclor sources within a single school and that PCB concentration and congener profiles differ among rooms within a single building. To evaluate this hypothesis and to identify potential localized sources, we measured airborne PCBs in nine rooms in a school. We found that schoolroom concentrations exceed outdoor air concentrations. Schoolroom concentrations and congener profiles also varied from one room to another. The concentrations were highest in the math room (35.75 ng m-3 ± 8.08) and lowest in the practice gym (1.54 ng m-3 ± 0.35). Rooms in the oldest wing of the building, originally constructed between 1920 and 1970, had the highest concentrations. The congener distribution patterns indicate historic use of Aroclor 1254 as well as modern sources of non-Aroclor congeners associated with paint pigments and surface coatings. Our findings suggest this noninvasive source identification method presents an opportunity for targeted source testing for more cost-effective prioritization of materials remediation in schools.

Metabolism and Photolysis of 2,4-Dinitroanisole in Arabidopsis.

New insensitive munitions explosives, including 2,4-dinitroanisole (DNAN), are replacing traditional explosive compounds to protect soldiers and simplify transport logistics. Despite the occupational safety benefits of these new explosives, feasible strategies for cleaning up DNAN from soil and water have not been developed. Here, we evaluate the metabolism of DNAN by the model plant Arabidopsis to determine whether phytoremediation can be used to clean up contaminated sites. Furthermore, we evaluate the role of photodegradation of DNAN and its plant metabolites within Arabidopsis leaves to determine the potential impact of photolysis on the phytoremediation of contaminants. When exposed to DNAN for three days, Arabidopsis took up and metabolized 67% of the DNAN in hydroponic solution. We used high resolution and tandem mass spectrometry in combination with stable-isotope labeled DNAN to confirm ten phase II DNAN metabolites in Arabidopsis. The plants separately reduced both the para- and ortho-nitro groups and produced glycosylated products that accumulated within plant tissues. Both DNAN and a glycosylated metabolite were subsequently photolyzed within leaf tissue under simulated sunlight, and [15N2]DNAN yielded 15NO2- in leaves. Therefore, photolysis inside leaves may be an important, yet under-explored, phytoremediation mechanism.

Biotransformation of 2,4-dinitroanisole by a fungal Penicillium sp.

Insensitive munitions explosives are new formulations that are less prone to unintended detonation compared to traditional explosives. While these formulations have safety benefits, the individual constituents, such as 2,4-dinitroanisole (DNAN), have an unknown ecosystem fate with potentially toxic impacts to flora and fauna exposed to DNAN and/or its metabolites. Fungi may be useful in remediation and have been shown to degrade traditional nitroaromatic explosives, such as 2,4,6-trinitrotoluene and 2,4-dinitrotoluene, that are structurally similar to DNAN. In this study, a fungal Penicillium sp., isolated from willow trees and designated strain KH1, was shown to degrade DNAN in solution within 14 days. Stable-isotope labeled DNAN and an untargeted metabolomics approach were used to discover 13 novel transformation products. Penicillium sp. KH1 produced DNAN metabolites resulting from ortho- and para-nitroreduction, demethylation, acetylation, hydroxylation, malonylation, and sulfation. Incubations with intermediate metabolites such as 2-amino-4-nitroanisole and 4-amino-2-nitroanisole as the primary substrates confirmed putative metabolite isomerism and pathways. No ring-cleavage products were observed, consistent with other reports that mineralization of DNAN is an uncommon metabolic outcome. The production of metabolites with unknown persistence and toxicity suggests further study will be needed to implement remediation with Penicillium sp. KH1. To our knowledge, this is the first report on the biotransformation of DNAN by a fungus.

Antecedent moisture controls on stream nitrate flux in an agricultural watershed.

Evaluating nitrate-N fluxes from agricultural landscapes is inherently complex due to the wide range of intrinsic and dynamic controlling variables. In this study, we investigate the influence of contrasting antecedent moisture conditions on nitrate-N flux magnitude and dynamics in a single agricultural watershed on intra-annual and rainfall-event temporal scales. High temporal resolution discharge and nitrate concentration data were collected to evaluate nitrate-N flux magnitude associated with wet (2009) and dry (2012) conditions. Analysis of individual rainfall events revealed a marked and consistent difference in nitrate-N flux response attributed to wet/dry cycles. Large-magnitude dilutions (up to 10 mg N L) persisted during the wet antecedent conditions (2009), consistent with a dominant baseflow contribution and excess groundwater release in relation to precipitation volume (discharge > > precipitation). Smaller-magnitude concentrations (<7 mg N L) were observed during the drought conditions of 2012, consistent with a quickflow-dominated response to rain events and infiltration/storage of precipitation resulting in discharge < precipitation. Nitrate-N loads and yields from the watershed were much higher (up to an order of magnitude) in the wet year vs. the dry year. Our results suggest that the response of nitrate-N loading to rain events is highly dependent on intra-annual antecedent moisture conditions and subsurface hydrologic connectivity, which together dictate the dominant hydrologic pathways for stream recharge. Additionally, the results of our study indicate that continued pronounced wet/dry cycles may become more dominant as the short-term driver of future nitrate-N exports.

Nitrogen removal from wastewater by an aerated subsurface-flow constructed wetland in cold climates.

The objective of this study was to assess the role of cyclic aeration, vegetation, and temperature on nitrogen removal by subsurface-flow engineered wetlands. Aeration was shown to enhance total nitrogen and ammonia removal and to enhance removal of carbonaceous biochemical oxygen demand, chemical oxygen demand, and phosphorus. Effluent ammonia and total nitrogen concentrations were significantly lower in aerated wetland cells when compared with unaerated cells. There was no significant difference in nitrogen removal between planted and unplanted cells. Effluent total nitrogen concentrations ranged from 9 to 12 mg N/L in the aerated cells and from 23 to 24 mg N/L in unaerated cells. Effluent ammonia concentrations ranged from 3 to 7 mg N/L in aerated wetland cells and from 22 to 23 mg N/L in unaerated cells. For the conditions tested, temperature had only a minimal effect on effluent ammonia or total nitrogen concentrations. The tanks-in-series and the PkC models predicted the general trends in effluent ammonia and total nitrogen concentrations, but did not do well predicting short-term variability. Rate coefficients for aerated systems were 2 to 10 times greater than those for unaerated systems.

Feature Engineering and Supervised Machine Learning to Forecast Biogas Production during Municipal Anaerobic Co-Digestion.

Municipalities with excess anaerobic digestion capacity accept offsite wastes for co-digestion to meet sustainability goals and create more biogas. Despite the benefits inherent to co-digestion, the temporal and compositional heterogeneity of external waste streams creates operational challenges that lead to upsets or conservative co-digestion. Given the complex microbial bioprocesses occurring during anaerobic digestion, prediction and modeling of the outcomes can be challenging, and machine learning has the potential to improve understanding and control of co-digestion processes. Biogas flows are a surrogate for process health, and here, we predicted biogas production from historical data collected by a water resource recovery facility (WRRF) during normal operation. We tested a daily lab and operational data set (n = 1089 after cleaning) and a minute-by-minute supervisory control and data acquisition (SCADA) operational data set (n = 491,761 after cleaning) to determine if forecasting biogas flow for a 24 h time horizon is feasible without collecting additional data. We found that a multilayer perceptron (MLP) neural network model outperformed tree-based and multiple linear regression models. Using a high-resolution SCADA data set for the first time, we showed that MLP neural networks could predict biogas production with an adjusted coefficient of determination (R2) of 0.78 and a mean absolute percentage error of 13.4% on a holdout test set. Adding daily laboratory analyses to the model did not appreciably improve the prediction of biogas flows. Feature engineering was essential to an accurate prediction, and 11 of the 15 most important features in the SCADA model were calculated from raw SCADA outputs. In summary, this paper demonstrates that minute-scale SCADA information collected at a municipal co-digestion facility can forecast biogas production, as a first step toward a digital twin model, without additional data collection.

Congener-Specific Emissions from Floors and Walls Characterize Indoor Airborne Polychlorinated Biphenyls.

To reconcile the federal regulation of material polychlorinated biphenyl (PCB) concentrations with recently implemented state regulations of airborne PCBs, there is a need to characterize the relationship between PCB emissions from surfaces and air concentrations. We hypothesized that the magnitude and congener distribution of emissions from floors and walls fully account for the airborne PCBs measured in rooms constructed during the height of PCB production and sales. We measured emissions of PCB congeners from various wall and floor materials using polyurethane foam passive emission samplers before and after hexane wiping. Our results revealed that PCB emissions from flooring adequately predicted the magnitude and congener distribution of PCBs observed in the room air. Emissions varied by material within a single building (5 × 103 ng m-2 day-1 from wood panel walls to 3 × 104 ng m-2 day-1 from vinyl tile) and within the same room. Yet congener distributions between material emission PCB profiles and room air PCB profiles were statistically similar. Hexane wiping significantly reduced PCB emissions (>60%), indicating the importance of surface films as an ongoing source of airborne PCBs. The magnitude and congener distribution of material bulk concentrations did not explain that of material emissions or air concentrations. Passive measurements of polychlorinated biphenyl emissions from floors in a university building predict the concentrations of PCBs in room air.

Photolysis of 3-Nitro-1,2,4-triazol-5-one: Mechanisms and Products.

Insensitive munitions formulations that include 3-nitro-1,2,4-triazol-5-one (NTO) are replacing traditional explosive compounds. While these new formulations have superior safety characteristics, the compounds have greater environmental mobility, raising concern over potential contamination and cleanup of training and manufacturing facilities. Here, we examine the mechanisms and products of NTO photolysis in simulated sunlight to further inform NTO degradation in sunlit surface waters. We demonstrate that NTO produces singlet oxygen and that dissolved oxygen increases the NTO photolysis rate in deionized water. The rate of NTO photolysis is independent of concentration and decreases slightly in the presence of Suwannee River Natural Organic Matter. The apparent quantum yield of NTO generally decreases as pH increases, ranging from 2.0 × 10-5 at pH 12 to 1.3 × 10-3 at pH 2. Bimolecular reaction rate constants for NTO with singlet oxygen and hydroxyl radical were measured to be (1.95 ± 0.15) × 106 and (3.28 ± 0.23) × 1010 M-1 s-1, respectively. Major photolysis reaction products were ammonium, nitrite, and nitrate, with nitrite produced in nearly stoichiometric yield upon the reaction of NTO with singlet oxygen. Environmental half-lives are predicted to span from 1.1 to 5.7 days. Taken together, these data enhance our understanding of NTO photolysis under environmentally relevant conditions.

Reconnaissance of Oxygenic Denitrifiers in Agriculturally Impacted Soils.

Row crop production in the agricultural Midwest pollutes waterways with nitrate, and exacerbates climate change through increased emissions of nitrous oxide and methane. Oxygenic denitrification processes in agricultural soils mitigate nitrate and nitrous oxide pollution by short-circuiting the canonical pathway to avoid nitrous oxide formation. Furthermore, many oxygenic denitrifiers employ a nitric oxide dismutase (nod) to create molecular oxygen that is used by methane monooxygenase to oxidize methane in otherwise anoxic soils. The direct investigation of nod genes that could facilitate oxygenic denitrification processes in agricultural sites is limited, with no prior studies investigating nod genes at tile drainage sites. Thus, we performed a reconnaissance of nod genes at variably saturated surface sites, and within a variably to fully saturated soil core in Iowa to expand the known distribution of oxygenic denitrifiers. We identified new nod gene sequences from agricultural soil and freshwater sediments in addition to identifying nitric oxide reductase (qNor) related sequences. Surface and variably saturated core samples displayed a nod to 16S rRNA gene relative abundance of 0.004% to 0.1% and fully saturated core samples had relative nod gene abundance of 1.2%. The relative abundance of the phylum Methylomirabilota increased from 0.6% and 1% in the variably saturated core samples to 3.8% and 5.3% in the fully saturated core samples. The more than 10-fold increase in relative nod abundance and almost 9-fold increase in relative Methylomirabilota abundance in fully saturated soils suggests that potential oxygenic denitrifiers play a greater nitrogen cycling role under these conditions. IMPORTANCE The direct investigation of nod genes in agricultural sites is limited, with no prior studies investigating nod genes at tile drains. An improved understanding of nod gene diversity and distribution is significant to the field of bioremediation and ecosystem services. The expansion of the nod gene database will advance oxygenic denitrification as a potential strategy for sustainable nitrate and nitrous oxide mitigation, specifically for agricultural sites.

Uses and biases of volunteer water quality data.

State water quality monitoring has been augmented by volunteer monitoring programs throughout the United States. Although a significant effort has been put forth by volunteers, questions remain as to whether volunteer data are accurate and can be used by regulators. In this study, typical volunteer water quality measurements from laboratory and environmental samples in Iowa were analyzed for error and bias. Volunteer measurements of nitrate+nitrite were significantly lower (about 2-fold) than concentrations determined via standard methods in both laboratory-prepared and environmental samples. Total reactive phosphorus concentrations analyzed by volunteers were similar to measurements determined via standard methods in laboratory-prepared samples and environmental samples, but were statistically lower than the actual concentration in four of the five laboratory-prepared samples. Volunteer water quality measurements were successful in identifying and classifying most of the waters which violate United States Environmental Protection Agency recommended water quality criteria for total nitrogen (66%) and for total phosphorus (52%) with the accuracy improving when accounting for error and biases in the volunteer data. An understanding of the error and bias in volunteer water quality measurements can allow regulators to incorporate volunteer water quality data into total maximum daily load planning or state water quality reporting.

Metagenomic analysis of nitrogen-cycling genes in upper Mississippi river sediment with mussel assemblages.

We investigated the impact of native freshwater mussel assemblages (order Unionoida) on the abundance and composition of nitrogen-cycling genes in sediment of an upper Mississippi river habitat. We hypothesized that the genomic potential for ammonia and nitrite oxidation would be greater in the sediment with mussel assemblages, presumably due to mussel biodeposition products, namely ammonia and organic carbon. Regardless of the presence of mussels, upper Mississippi river sediment microbial communities had the largest genomic potential for nitrogen fixation followed by urea catabolism, nitrate metabolism, and nitrate assimilation, as evidenced by analysis of nitrogen cycling pathway abundances. However, genes encoding nitrate and nitrite redox reactions, narGHI and nxrAB, were the most abundant functional genes of the nitrogen cycling gene families. Using linear discriminant analysis (LDA), we found nitrification genes were the most important biomarkers for nitrogen cycling genomic potential when mussels were present, and this presented an opposing effect on the abundance of genes encoding nitric oxide reduction. The genes involved in nitrification that increased the most were amoA associated with comammox Nitrospira and nxr homologs associated with Nitrospira. On the other hand, the most distinctive biomarkers of microbial communities without mussels were norB and nrfA, as part of denitrification and dissimilatory nitrate reduction to ammonium pathways, respectively. Ultimately, this research demonstrates the impact of native mollusks on microbial nitrogen cycling in an aquatic agroecosystem.

The Genomic Potentials of NOB and Comammox Nitrospira in River Sediment Are Impacted by Native Freshwater Mussels.

Freshwater mussel assemblages of the Upper Mississippi River (UMR) sequester tons of ammonia- and urea-based biodeposits each day and aerate sediment through burrowing activities, thus creating a unique niche for nitrogen (N) cycling microorganisms. This study explored how mussels impact the abundance of N-cycling species with an emphasis on Candidatus Nitrospira inopinata, the first microorganism known to completely oxidize ammonia (comammox) to nitrate. This study used metagenomic shotgun sequencing of genomic DNA to compare nitrogen cycling species in sediment under a well-established mussel assemblage and in nearby sediment without mussels. Metagenomic reads were aligned to the prokaryotic RefSeq non-redundant protein database using BLASTx, taxonomic binning was performed using the weighted lowest common ancestor algorithm, and protein-coding genes were categorized by metabolic function using the SEED subsystem. Linear discriminant analysis (LDA) effect sizes were used to determine which metagenomes and metabolic features explained the most differences between the mussel habitat sediment and sediment without mussels. Of the N-cycling species deemed differentially abundant, Nitrospira moscoviensis and "Candidatus Nitrospira inopinata" were responsible for creating a distinctive N-cycling microbiome in the mussel habitat sediment. Further investigation revealed that comammox Nitrospira had a large metabolic potential to degrade mussel biodeposits, as evidenced the top ten percent of protein-coding genes including the cytochrome c-type biogenesis protein required for hydroxylamine oxidation, ammonia monooxygenase, and urea decomposition SEED subsystems. Genetic marker analysis of these two Nitrospira taxons suggested that N. moscoviensis was most impacted by diverse carbon metabolic processes while "Candidatus Nitrospira inopinata" was most distinguished by multidrug efflux proteins (AcrB), NiFe hydrogenase (HypF) used in hydrogen oxidation and sulfur reduction coupled reactions, and a heme chaperone (CcmE). Furthermore, our research suggests that comammox and NOB Nitrospira likely coexisted by utilizing mixotrophic metabolisms. For example, "Candidatus Nitrospira inopinata" had the largest potentials for ammonia oxidation, nitrite reduction with NirK, and hydrogen oxidation, while NOB Nitrospira had the greatest potential for nitrite oxidation, and nitrate reduction possibly coupled with formate oxidation. Overall, our results suggest that this mussel habitat sediment harbors a niche for NOB and comammox Nitrospira, and ultimately impacts N-cycling in backwaters of the UMR.

Effect of freshwater mussels on the vertical distribution of anaerobic ammonia oxidizers and other nitrogen-transforming microorganisms in upper Mississippi river sediment.

Targeted qPCR and non-targeted amplicon sequencing of 16S rRNA genes within sediment layers identified the anaerobic ammonium oxidation (anammox) niche and characterized microbial community changes attributable to freshwater mussels. Anammox bacteria were normally distributed (Shapiro-Wilk normality test, W-statistic =0.954, p = 0.773) between 1 and 15 cm depth and were increased by a factor of 2.2 (p < 0.001) at 3 cm below the water-sediment interface when mussels were present. Amplicon sequencing of sediment at depths relevant to mussel burrowing (3 and 5 cm) showed that mussel presence reduced observed species richness (p = 0.005), Chao1 diversity (p = 0.005), and Shannon diversity (p < 0.001), with more pronounced decreases at 5 cm depth. A non-metric, multidimensional scaling model showed that intersample microbial species diversity varied as a function of mussel presence, indicating that sediment below mussels harbored distinct microbial communities. Mussel presence corresponded with a 4-fold decrease in a majority of operational taxonomic units (OTUs) classified in the phyla Gemmatimonadetes, Actinobacteria, Acidobacteria, Plantomycetes, Chloroflexi, Firmicutes, Crenarcheota, and Verrucomicrobia. 38 OTUs in the phylum Nitrospirae were differentially abundant (p < 0.001) with mussels, resulting in an overall increase from 25% to 35%. Nitrogen (N)-cycle OTUs significantly impacted by mussels belonged to anammmox genus Candidatus Brocadia, ammonium oxidizing bacteria family Nitrosomonadaceae, ammonium oxidizing archaea genus Candidatus Nitrososphaera, nitrite oxidizing bacteria in genus Nitrospira, and nitrate- and nitrite-dependent anaerobic methane oxidizing organisms in the archaeal family "ANME-2d" and bacterial phylum "NC10", respectively. Nitrosomonadaceae (0.9-fold (p < 0.001)) increased with mussels, while NC10 (2.1-fold (p < 0.001)), ANME-2d (1.8-fold (p < 0.001)), and Candidatus Nitrososphaera (1.5-fold (p < 0.001)) decreased with mussels. Co-occurrence of 2-fold increases in Candidatus Brocadia and Nitrospira in shallow sediments suggests that mussels may enhance microbial niches at the interface of oxic-anoxic conditions, presumably through biodeposition and burrowing. Furthermore, it is likely that the niches of Candidatus Nitrososphaera and nitrite- and nitrate-dependent anaerobic methane oxidizers were suppressed by mussel biodeposition and sediment aeration, as these phylotypes require low ammonium concentrations and anoxic conditions, respectively. As far as we know, this is the first study to characterize freshwater mussel impacts on microbial diversity and the vertical distribution of N-cycle microorganisms in upper Mississippi river sediment. These findings advance our understanding of ecosystem services provided by mussels and their impact on aquatic biogeochemical N-cycling.

Simulated mussel mortality thresholds as a function of mussel biomass and nutrient loading.

A freshwater "mussel mortality threshold" was explored as a function of porewater ammonium (NH4+) concentration, mussel biomass, and total nitrogen (N) utilizing a numerical model calibrated with data from mesocosms with and without mussels. A mortality threshold of 2 mg-N L-1 porewater NH4+ was selected based on a study that estimated 100% mortality of juvenile Lampsilis mussels exposed to 1.9 mg-N L-1 NH4+ in equilibrium with 0.18 mg-N L-1 NH3. At the highest simulated mussel biomass (560 g m-2) and the lowest simulated influent water "food" concentration (0.1 mg-N L-1), the porewater NH4+ concentration after a 2,160 h timespan without mussels was 0.5 mg-N L-1 compared to 2.25 mg-N L-1 with mussels. Continuing these simulations while varying mussel biomass and N content yielded a mortality threshold contour that was essentially linear which contradicted the non-linear and non-monotonic relationship suggested by Strayer (2014). Our model suggests that mussels spatially focus nutrients from the overlying water to the sediments as evidenced by elevated porewater NH4+ in mesocosms with mussels. However, our previous work and the model utilized here show elevated concentrations of nitrite and nitrate in overlying waters as an indirect consequence of mussel activity. Even when the simulated overlying water food availability was quite low, the mortality threshold was reached at a mussel biomass of about 480 g m-2. At a food concentration of 10 mg-N L-1, the mortality threshold was reached at a biomass of about 250 g m-2. Our model suggests the mortality threshold for juvenile Lampsilis species could be exceeded at low mussel biomass if exposed for even a short time to the highly elevated total N loadings endemic to the agricultural Midwest.

Partial nitritation ANAMMOX in submerged attached growth bioreactors with smart aeration at 20 °C.

Submerged attached growth bioreactors (SAGBs) were operated at 20 °C for 30 weeks in smart-aerated, partial nitritation ANAMMOX mode and in a timer-controlled, cyclic aeration mode. The smart-aerated SAGBs removed 48-53% of total nitrogen (TN) compared to 45% for SAGBs with timed aeration. Low dissolved oxygen concentrations and cyclic pH patterns in the smart-aerated SAGBs suggested conditions favorable to partial nitritation ANAMMOX and stoichiometrically-derived and numerically modeled estimations attributed 63-68% and 14-44% of TN removal to partial nitritation ANAMMOX in these bioreactors, respectively. Ammonia removals of 36-67% in the smart-aerated SAGBs, with measured oxygen and organic carbon limitations, further suggest partial nitritation ANAMMOX. The smart-aerated SAGBs required substantially less aeration to achieve TN removals similar to SAGBs with timer-controlled aeration. Genomic DNA testing confirmed that the dominant ANAMMOX seed bacteria, received from a treatment plant utilizing the DEMON® sidestream deammonification process, was a Candidatus Brocadia sp. (of the Planctomycetales order). The DNA from these bacteria was also present in the SAGBs at the conclusion of the study providing evidence for attached growth and limited biomass washout.

The effects of individual PCB congeners on the soil bacterial community structure and the abundance of biphenyl dioxygenase genes.

Polychlorinated biphenyls (PCBs) are toxic environmental contaminants that represent a class of 209 congeners characterized by different degrees of chlorination and substitution patterns. Most of experimental studies about microbial degradation of PCBs have been conducted on PCB mixtures, even though evidence accumulated in bacteria and other organisms shows that exposure to different congeners may have different biological effects. Microcosm experiments were conducted using aerobic agitated soil slurries individually exposed to PCB congeners with different degrees of chlorination: PCB-3, 15, 28, and 77, and the commercial mixture Aroclor 1242. After four weeks of incubation, PCBs were analyzed by gas chromatography/mass spectrometry (GC/MS) showing different transformation extents: With the exception of PCB-15 that was not significantly transformed (7%), biodegradation rates decreased with the degree of chlorination, from 75% for PCB-3 to 22% for PCB-77 and Aroclor 1242. The bacterial abundance, as measured by colony counting and 16S rDNA quantification by real-time PCR, was lower (of about 40%) in soil microcosms exposed to the higher-chlorinated congeners, PCB-28, PCB-77, and Aroclor 1242, as compared to non-exposed soils and soils exposed to the lower-chlorinated congeners, PCB-3 and PCB-15. The relative abundance of different taxonomic groups, as determined by real-time PCR, revealed an increase of ß-Proteobacteria and Actinobacteria in all microcosms exposed to PCBs, as compared with non-exposed soil. In addition, exposure to PCB-77 and Aroclor 1242 resulted in a higher abundance of α-Proteobacteria and Acidobacteria. Globally, these results suggest that exposure to PCBs (and especially to higher-chlorinated congeners and Aroclor 1242) selected bacterial groups involving most known PCB degraders, i.e., ß-Proteobacteria and Acidobacteria. The quantification of biphenyl dioxygenase (BPH) genes--involved in the aerobic degradation of PCBs--using real-time PCR showed that exposure to all PCB congeners and Aroclor 1242 resulted in a marked increase of two out of the four BPH genes tested, similarly suggesting the selection of PCB-degrading bacteria. This paper showed that exposure to different PCB congeners leads to different structures of the soil bacterial community and BPH genes expression patterns.

Gene expression and microscopic analysis of Arabidopsis exposed to chloroacetanilide herbicides and explosive compounds. A phytoremediation approach.

Understanding the function of detoxifying enzymes in plants toward xenobiotics is of major importance for phytoremediation applications. In this work, Arabidopsis (Arabidopsis thaliana; ecotype Columbia) seedlings were exposed to 0.6 mm acetochlor (AOC), 2 mm metolachlor (MOC), 0.6 mm 2,4,6-trinitrotoluene (TNT), and 0.3 mm hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In vivo glutathione (GSH) conjugation reactions of AOC, MOC, RDX, and TNT were studied in root cells using a multiphoton microscope. In situ labeling with monochlorobimane, used as a competitive compound for conjugation reactions with GSH, confirmed that AOC and MOC are conjugated in Arabidopsis cells. Reverse transcription-PCR established the expression profile of glutathione S-transferases (GSTs) and nitroreductases enzymes. Genes selected for this study were AtGSTF2, AtGSTU1, AtGSTU24, and two isoforms of 12-oxophytodienoate reductase (OPR1 and OPR2). The five transcripts tested were induced by all treatments, but RDX resulted in low induction. The mRNA level of AtGSTU24 showed substantial increase for all chemicals (23-fold induction for AOC, 18-fold for MOC, 5-fold for RDX, and 40-fold for TNT). It appears that GSTs are also involved in the conjugation reactions with metabolites of TNT, and to a lesser extent with RDX. Results indicate that OPR2 is involved in plant metabolism of TNT (11-fold induction), and in oxidative stress when exposed to AOC (7-fold), MOC (9-fold), and RDX (2-fold). This study comprises gene expression analysis of Arabidopsis exposed to RDX and AOC, which are considered significant environmental contaminants, and demonstrates the importance of microscopy methods for phytoremediation investigations.

Phytophotolysis of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in leaves of reed canary grass.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was degraded in reed canary grass leaves exposed to simulated sunlight to primary products nitrous oxide and 4-nitro-2,4-diazabutanal. This is the first time that 4-nitro-2,4-diazabutanal, a potentially toxic degradate, has been measured in plant tissues following phytotransformation of RDX. These compounds, along with nitrite and formaldehyde, were also detected in aqueous RDX systems exposed to the same simulated sunlight. Results showed that the initial products of RDX photodegradation in translucent plant tissues were similar to products formed from aqueous photolysis of RDX. Combustion analysis of leaves following 14C-RDX uptake and subsequent light exposure revealed the presence of tissue-bound material that could not be extracted with acetonitrile. No detectable formaldehyde was emitted from the leaves. The detection of similar RDX degradation products in both aqueous and plant-based systems suggests that RDX may be initially transformed by similar mechanisms in both systems. Direct photolysis of RDX via ultraviolet irradiation passing into the leaves is hypothesized to be responsible for the observed transformations. In addition, membrane-bound "trap chlorophyll" in the chloroplasts may shuttle electrons to RDX as an indirect photolysis transformation mechanism. Results from this study indicate that reed canary grass facilitates photochemical degradation of RDX, and this mechanism should be considered along with more established phytoremediation processes when assessing the fate of contaminants in plant tissues. Plant-mediated phototransformation of xenobiotic compounds is a process that may be termed "phytophotolysis".

Metabolism and mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine inside poplar tissues (Populus deltoides x nigra DN-34).

Poplar tissue cultures and leaf crude extracts (Populus deltoides x nigra DN-34) were exposed to [U-14C]hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and incubated under light and in the dark. Poplar tissue cultures were able to partially reduce RDX to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), regardless of the presence or absence of light. However, further transformation of RDX, MNX, and DNX required exposure to light and resulted in the formation of formaldehyde (CH2O), methanol (CH3OH), and carbon dioxide (CO2). Similarly, transformation of RDX by poplar leaf crude extracts required exposure to light. Neither reduction of RDX to MNX and DNX nor mineralization into CO2 were recorded in crude extracts, even when exposed to light, suggesting that both processes were light-independent and required intact plant cells. Control experiments without plant material showed that RDX was partially transformed abiotically, by the sole action of light, but to a lesser extent than in the presence of plant crude extracts, suggesting the intervention of plant subcellular structures through a light-mediated mechanism. Poplar tissue cultures were also shown to mineralize 14CH2O and 14CH3OH, regardless of the presence or absence of light. These results suggest that transformation of [U-14C]RDX by plant tissue cultures may occur through a three-step process, involving (i) a light-independent reduction of RDX to MNX and DNX by intact plant cells; (ii) a plant/light-mediated breakdown of the heterocyclic ring of RDX, MNX, or DNX into C1-labeled metabolites (CH2O and CH3OH); and (iii) a further light-independent mineralization of C1-labeled metabolites by intact plant cells. This is the first time that a significant mineralization of RDX into CO2 by light-exposed plant tissue cultures is reported.