Agricultural soils are important sources of two potent greenhouse gases, nitrous oxide (N2O) and methane (CH4), the atmospheric levels of which are steadily increasing. Increases in N2O also threaten the earth's protective ozone layer. Only two reactions are known to degrade N2O and CH4 without requiring high temperatures and high energy inputs: those catalyzed by bacterial N2O reductase (N2OR) and methane monooxygenase (MMO). Plants genetically engineered to constitutively express N2OR and MMO could potentially remove both gases from soil and atmosphere. Although the cytosolic expression of these genes in plants has proven unsuccessful, new biotechnologies allow introduction of protein complexes into eukaryotic mitochondria, a platform that shares many similarities to the membranes and intermembrane space of bacteria. Based on these theoretical considerations, we set out to model the potential impact of N2OR- and MMO-transformed plants on emissions and uptake of N2O and CH4. Our calculations suggest that such plants could prevent 50% of soil emissions of N2O and CH4 and take up substantial N2O from the atmosphere. If planted globally, N2OR- and MMO-engineered crop plants could potentially stop increases in N2O and CH4 in the atmosphere and slow increases in global warming, providing a strong incentive for research into this biotechnology.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a major component of munitions, is used extensively on military training ranges. As a result, widespread RDX pollution in groundwater and aquifers in the United States is now well documented. RDX is toxic, but its removal from training ranges is logistically challenging, lacking cost-effective and sustainable solutions. Previously, we have shown that thale cress (Arabidopsis thaliana) engineered to express two genes, xplA and xplB, encoding RDX-degrading enzymes from the soil bacterium Rhodococcus rhodochrous 11Y can break down this xenobiotic in laboratory studies. Here, we report the results of a 3-year field trial of XplA/XplB-expressing switchgrass (Panicum virgatum) conducted on three locations in a military site. Our data suggest that XplA/XplB switchgrass has in situ efficacy, with potential utility for detoxifying RDX on live-fire training ranges, munitions dumps and minefields.

Main conclusion Transgenic western wheatgrass degrades the explosive RDX and detoxifies TNT. Contamination, from the explosives, hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), and 2, 4, 6-trinitrotoluene (TNT), especially on live-fire training ranges, threatens environmental and human health. Phytoremediation is an approach that could be used to clean-up explosive pollution, but it is hindered by inherently low in planta RDX degradation rates, and the high phytotoxicity of TNT. The bacterial genes, xplA and xplB , confer the ability to degrade RDX in plants, and a bacterial nitroreductase gene nfsI enhances the capacity of plants to withstand and detoxify TNT. While the previous studies have used model plant species to demonstrate the efficacy of this technology, trials using plant species able to thrive in the challenging environments found on military training ranges are now urgently needed. Perennial western wheatgrass ( Pascopyrum smithii ) is a United States native species that is broadly distributed across North America, well-suited for phytoremediation, and used by the US military to re-vegetate military ranges. Here, we present the first report of the genetic transformation of western wheatgrass. Plant lines transformed with xplA , xplB, and nfsI removed significantly more RDX from hydroponic solutions and retained much lower, or undetectable, levels of RDX in their leaf tissues when compared to wild-type plants. Furthermore, these plants were also more resistant to TNT toxicity, and detoxified more TNT than wild-type plants. This is the first study to engineer a field-applicable grass species capable of both RDX degradation and TNT detoxification. Together, these findings present a promising biotechnological approach to sustainably contain, remove RDX and TNT from training range soil and prevent groundwater contamination.

SummaryThe nitrogen (N) fertilizer required to supply a bioenergy industry with sufficient feedstocks is associated with adverse environmental impacts, including loss of oxidized reactive nitrogen through leaching and the production of the greenhouse gas nitrous oxide (N2O). We examined effects on crop yield, N fate and the response of ammonia‐oxidizing bacteria (AOB) and ammonia‐oxidizing archaea (AOA) to conventional fertilizer application or intercropping with N‐fixing alfalfa, for N delivery to switchgrass (Panicum virgatum), a potential bioenergy crop. Replicated field plots in Prosser, WA, were sampled over two seasons for reactive nitrogen, N2O gas emissions, and bacterial and archaeal ammonia monooxygenase gene (amoA) counts. Intercropping with alfalfa (70:30, switchgrass:alfalfa) resulted in reduced dry matter yields compared to fertilized plots, but three times lower N2O fluxes (≤ 4 g N2O‐N ha−1 d−1) than fertilized plots (12.5 g N2O‐N ha−1 d−1). In the fertilized switchgrass plots, AOA abundance was greater than AOB abundance, but only AOB abundance was positively correlated with N2O emissions, implicating AOB as the major producer of N2O emissions. A life cycle analysis of N2O emissions suggested the greenhouse gas emissions from cellulosic ethanol produced from switchgrass intercropped with alfalfa cultivation would be 94% lower than emissions from equivalent gasoline usage.

The indoor air in urban homes of developed countries is usually contaminated with significant levels of volatile organic carcinogens (VOCs), such as formaldehyde, benzene, and chloroform. There is a need for a practical, sustainable technology for the removal of VOCs in homes. Here we show that a detoxifying transgene, mammalian cytochrome P450 2e1 can be expressed in a houseplant, Epipremnum aureum, pothos ivy, and that the resulting genetically modified plant has sufficient detoxifying activity against benzene and chloroform to suggest that biofilters using transgenic plants could remove VOCs from home air at useful rates.

Most agricultural N2 O emissions are a consequence of microbial transformations of nitrogen (N) fertilizer, and mitigating increases in N2 O emission will depend on identifying microbial sources and variables influencing their activities. Here, using controlled microcosm and field studies, we found that synthetic N addition in any tested amount stimulated the production of N2 O from ammonia-oxidizing bacteria (AOB), but not archaea (AOA), from a bioenergy crop soil. The activities of these two populations were differentiated by N treatments, with abundance and activity of AOB increasing as nitrate and N2 O production increased. Moreover, as N2 O production increased, the isotopic composition of N2 O was consistent with an AOB source. Relative N2 O contributions by both populations were quantified using selective inhibitors and varying N availability. Complementary field analyses confirmed a positive correlation between N2 O flux and AOB abundance with N application. Collectively, our data indicate that AOB are the major N2 O producers, even with low N addition, and that better-metered N application, complemented by selective inhibitors, could reduce projected N2 O emissions from agricultural soils.

Expression of the bacterial nitroreductase gene, nfsI, in tobacco plastids conferred the ability to detoxify TNT.

A controlled field study was performed to evaluate the effectiveness of transgenic poplars for phytoremediation. Three hydraulically contained test beds were planted with 12 transgenic poplars, 12 wild type (WT) poplars, or left unplanted, and dosed with equivalent concentrations of trichloroethylene (TCE). Removal of TCE was enhanced in the transgenic tree bed, but not to the extent of the enhanced removal observed in laboratory studies. Total chlorinated ethene removal was 87% in the CYP2E1 bed, 85% in the WT bed, and 34% in the unplanted bed in 2012. Evapotranspiration of TCE from transgenic leaves was reduced by 80% and diffusion of TCE from transgenic stems was reduced by 90% compared to WT. Cis-dichloroethene and vinyl chloride levels were reduced in the transgenic tree bed. Chloride ion accumulated in the planted beds corresponding to the TCE loss, suggesting that contaminant dehalogenation was the primary loss fate.

The discharge of the synthetic estrogen, 17α-ethinylestradiol (EE2), in wastewater treatment plant (WWTP) effluents is an environmental concern as this compound can alter the reproductive system of aquatic wildlife at low ng L-1 concentrations. The impact of EE2 at such low concentrations indicates the need to identify activated sludge (AS) process designs that minimize WWTP effluent EE2 concentrations. An EE2 fate and transformation model was developed based on the following mechanism: (1) EE2 production from deconjugation of EE2-3-sulfate, a conjugated form excreted from humans, (2) EE2 removal from biodegradation by heterotrophic biomass growing on other substrates, and (3) EE2 removal from sorption to activated sludge. These mechanisms were incorporated into the International Water Association (IWA) Activated Sludge Model No. 2d (ASM2d) to model the fate of EE2 across aerobic and biological nutrient removal (BNR) AS systems. The model was calibrated and evaluated for mixed liquor solids, nutrients, and EE2 using lab-scale aerobic and BNR AS reactors fed primary effluent and amended with ng.L-1 estrogen concentrations. A sensitivity analysis predicted effluent EE2 concentrations were most sensitive to the biodegradation rate coefficient (kbio,H), the influent biodegradable chemical oxygen demand to EE2 ratio, and the aerobic solids retention time and were least sensitive to the deconjugation rate coefficient (kcle,H) and the solidliquid partitioning coefficient. Predicted effluent EE2 concentrations were more sensitive to the kcle,H/kbio,H ratio for AS systems with low kbio,H values.

The deposition of toxic munitions compounds, such as hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), on soils around targets in live-fire training ranges is an important source of groundwater contamination. Plants take up RDX but do not significantly degrade it. Reported here is the transformation of two perennial grass species, switchgrass (Panicum virgatum) and creeping bentgrass (Agrostis stolonifera), with the genes for degradation of RDX. These species possess a number of agronomic traits making them well equipped for the uptake and removal of RDX from root zone leachates. Transformation vectors were constructed with xplA and xplB, which confer the ability to degrade RDX, and nfsI, which encodes a nitroreductase for the detoxification of the co-contaminating explosive 2, 4, 6-trinitrotoluene (TNT). The vectors were transformed into the grass species using Agrobacterium tumefaciens infection. All transformed grass lines showing high transgene expression levels removed significantly more RDX from hydroponic solutions and retained significantly less RDX in their leaf tissues than wild-type plants. Soil columns planted with the best-performing switchgrass line were able to prevent leaching of RDX through a 0.5-m root zone. These plants represent a promising plant biotechnology to sustainably remove RDX from training range soil, thus preventing contamination of groundwater.

Removal rates of benzene and formaldehyde gas by houseplants reported by several laboratories varied by several orders of magnitude. We hypothesized that these variations were caused by differential responses of soil microbial populations to the high levels of pollutant used in the studies, and tested responses to benzene by plants and soils separately. Five houseplant species and tobacco were exposed to benzene under hydroponic conditions and the uptake rates compared. Among the test plants, Syngonium podophyllum and Chlorophytum comosum and Epipremnum aureum had the highest benzene removal rates. The effects of benzene addition on populations of soil bacteria were determined using reverse transcription quantitative PCR (RT-qPCR) assays targeting microbial genes involved in benzene degradation. The total bacterial population increased as shown by increases in the levels of eubacteria 16S rRNA, which was significantly higher in the high benzene incubations than in the low benzene incubations. Transcripts (mRNA) of genes encoding phenol monooxygenases, catechol-2,3-dioxygenase and the housekeeping gene rpoB increased in all soils incubated with high benzene concentrations. Therefore the enrichment of soils with benzene gas levels typical of experiments with houseplants in the literature artificially increased the levels of total soil bacterial populations, and especially the levels and activities of benzene-degrading bacteria.

Application of nitrogen fertilizer has altered terrestrial ecosystems. Ammonia is nitrified by ammonia and nitrite-oxidizing microorganisms, converting ammonia to highly mobile nitrate, contributing to the loss of nitrogen, soil nutrients and production of detrimental nitrogen oxides. Mitigating these costs is of critical importance to a growing bioenergy industry. To resolve the impact of management on nitrifying populations, amplicon sequencing of markers associated with ammonia and nitrite-oxidizing taxa (ammonia monooxygenase-amoA, nitrite oxidoreductase-nxrB, respectively) was conducted from long-term managed and nearby native soils in Eastern Washington, USA. Native nitrifier population structure was altered profoundly by management. The native ammonia-oxidizing archaeal community (comprised primarily by Nitrososphaera sister subclusters 1.1 and 2) was displaced by populations of Nitrosopumilus, Nitrosotalea and different assemblages of Nitrososphaera (subcluster 1.1, and unassociated lineages of Nitrososphaera). A displacement of ammonia-oxidizing bacterial taxa was associated with management, with native groups of Nitrosospira (cluster 2 related, cluster 3A.2) displaced by Nitrosospira clusters 8B and 3A.1. A shift in nitrite-oxidizing bacteria (NOB) was correlated with management, but distribution patterns could not be linked exclusively to management. Dominant nxrB sequences displayed only distant relationships to other NOB isolates and environmental clones.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) fill key roles in the nitrogen cycle. Thus, well-vetted methods for characterizing their distribution are essential for framing studies of their significance in natural and managed systems. Quantification of the gene coding for one subunit of the ammonia monooxygenase (amoA) by polymerase chain reaction is frequently employed to enumerate the two groups. However, variable amplification of sequence variants comprising this conserved genetic marker for ammonia oxidizers potentially compromises within- and between-system comparisons. We compared the performance of newly designed non-degenerate quantitative polymerase chain reaction primer sets to existing primer sets commonly used to quantify the amoA of AOA and AOB using a collection of plasmids and soil DNA samples. The new AOA primer set provided improved quantification of model mixtures of different amoA sequence variants and increased detection of amoA in DNA recovered from soils. Although both primer sets for the AOB provided similar results for many comparisons, the new primers demonstrated increased detection in environmental application. Thus, the new primer sets should provide a useful complement to primers now commonly used to characterize the environmental distribution of AOA and AOB.

Ammonia-oxidizing thaumarcheota (AOA) and ammonia-oxidizing bacteria (AOB) differentially influence soil and atmospheric chemistry, but soil properties that control their distributions are poorly understood. In this study, the ammonia monooxygenase gene (amoA) was used to identify and quantify presumptive AOA and AOB and relate their distributions to soil properties in two experimental fields planted with different varieties of switchgrass (Panicum virgatum), a potential bioenergy feedstock. Differences in ammonia oxidizer diversity were associated primarily with soil properties of the two field sites, with pH displaying significant correlations with both AOA and AOB population structure. Percent nitrogen (%N), carbon to nitrogen ratios (C:N), and pH were also correlated with shifts nitrifier population structure. Nitrosotalea-like and Nitrosospira cluster II populations were more highly represented in acidic soils, whereas populations affiliated with Nitrososphaera and Nitrosospira cluster 3A.1 were relatively more abundant in alkaline soils. AOA were the dominant functional group in all plots based on quantitative polymerase chain reaction and high-throughput sequencing analyses. These data suggest that AOA contribute significantly to nitrification rates in carbon and nitrogen rich soils influenced by perennial grasses.

Biodegradation is the primary removal mechanism of the potent endocrine-disrupting estrogen 17-ethinylestradiol (EE2) during activated sludge (AS) wastewater treatment. Analysis of AS treatment process designs to optimize EE2 removal requires knowledge of EE2 biodegradation kinetics. However, there is little information on EE2 biodegradation kinetics for different types of systems and under long-term studies. EE2 biodegradation kinetics were investigated at 20 degrees C using laboratory-scale sequencing batch reactors simulating aerobic, anaerobic/aerobic enhanced biological phosphorus removal, and anoxic/aerobic biological nitrogen removal processes fed synthetic wastewater. Three sets of reactor experiments were conducted using different municipal AS plant seed sources and with solid retention times (SRTs) ranging from 8 to 13 days. EE2 biodegradation was described by a pseudo first-order biodegradation rate model with a rate coefficient (k(b)) normalized to the reactor volatile suspended solids (VSS). EE2 k(b) values were determined from batch degradation tests and from calibration of the model to the reactor process. Significant EE2 biodegradation occurred only under aerobic conditions. Observed EE2 k(b) values for aerobic, anaerobic/aerobic, and anoxic/aerobic operations ranged from 4 to 22 L/g VSS-day, 4 to 19 L/g VSS-day, and 3 to 20 L/g VSS-day, respectively. Model simulations showed that the EE2 removal efficiency could range from 72% to over 99% for these coefficients at a 10-day SRT. Predicted EE2 removal efficiency at a given k(b) value can be improved by operating at a higher SRT upto a limit, by having a greater number of aerobic reactor stages, and by having a higher influent biodegradable chemical oxygen demand concentration.

ABSTRACT Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera ( Rhodococcus , Microbacterium , Gordonia , and Williamsia ) isolated from geographically distinct locations and established that the xplA and xplB ( xplAB ) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB , the Rhodococcus species studied here share a 14-kb region flanking xplAB ; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB -flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX.

The removal of the potent endocrine-disrupting estrogen hormone, 17α-ethinylestradiol (EE2), in municipal wastewater treatment plant (WWTP) activated sludge (AS) processes can occur through biodegradation by heterotrophic bacteria growing on other organic wastewater substrates. Different kinetic and metabolic substrate utilization conditions created with AS bioselector processes can affect the heterotrophic population composition in AS. The primary goal of this research was to determine if these changes also affect specific EE2 biodegradation kinetics. A series of experiments were conducted with parallel bench-scale AS reactors treating municipal wastewater with estrogens at 100-300 ng/L concentrations to evaluate the effect of bioselector designs on pseudo first-order EE2 biodegradation kinetics normalized to mixed liquor volatile suspended solids (VSS). Kinetic rate coefficient (kb) values for EE2 biodegradation ranged from 5.0 to 18.9 L/g VSS/d at temperatures of 18 °C to 24 °C. EE2 kb values for aerobic biomass growth at low initial food to mass ratio feeding conditions (F/Mf) were 1.4 to 2.2 times greater than that from growth at high initial F/Mf. Anoxic/aerobic and anaerobic/aerobic metabolic bioselector reactors achieving biological nutrient removal had similar EE2 kb values, which were lower than that in aerobic AS reactors with biomass growth at low initial F/Mf. These results provide evidence that population selection with growth at low organic substrate concentrations can lead to improved EE2 biodegradation kinetics in AS treatment.

Abstract : Our overall goals were to identify RDX-degrading bacteria in plant rhizospheres, discover the factors that control their abundance and diversity, and develop probes that can be used in the field to detect them. We suggest that important controlling variables in determining RDX persistence in soil are carbon and nitrogen availability. This implies that soil bacteria do not effectively degrade energetic materials in situ unless they are associated with a carbon-rich environment that selects for populations active in either direct or cometabolic degradation of RDX. Further, we expected that activity of RDX-degrading bacteria would be favored by specific carbon sources in the root exudate spectrum, allowing the rhizosphere to be manipulated to enhance populations of RDX-degrading bacteria.

: We have transformed a plant, Arabidopsis thaliana, with the bacterial genes for the three subunit proteins of the soluble methane monooxygenase and with two associated genes, the mmoG and the mmoC. The presence of the genes and the transcripts in selected lines of the transformed plants was confirmed at the genomic, transcript and protein levels. The transformed plants removed more methane compared to the wild-type, but only slightly, about 5% in 7 days . The production of 1-naphthol from naphthalene was 2-3 times greater in transformed plant lines compared to untransformed wild types. These results suggest that the sMMO was properly assembled and partially functional, however, additional controls are needed to confirm. We discuss the results and suggest follow-on experiments.