Controlling In Planta Gold Nanoparticle Synthesis and Size for Catalysis.

Gold nanoparticles (Au-NPs) are used as catalysts for a diverse range of industrial applications. Currently, Au-NPs are synthesized chemically, but studies have shown that plants fed Au deposit, this element naturally as NPs within their tissues. The resulting plant material can be used to make biomass-derived catalysts. In vitro studies have shown that the addition of specific, short (∼10 amino acid) peptide/s to solutions can be used to control the NP size and shape, factors that can be used to optimize catalysts for different processes. Introducing these peptides into the model plant species, Arabidopsis thaliana (Arabidopsis), allows us to regulate the diameter of nanoparticles within the plant itself, consequently influencing the catalytic performance in the resulting pyrolyzed biomass. Furthermore, we show that overexpressing the copper and gold COPPER TRANSPORTER 2 (COPT2) in Arabidopsis increases the uptake of these metals. Adding value to the Au-rich biomass offers the potential to make plant-based remediation and stabilization of mine wastes financially feasible. Thus, this study represents a significant step toward engineering plants for the sustainable recovery of finite and valuable elements from our environment.

Inventing hyperaccumulator plants: improving practice in phytoextraction research and terminology.

Toxic metals and metalloids, especially from anthropogenic sources, now pollute substantial areas of our planet. Phytoextraction is a proven technology with the potential to reduce metal/metalloid pollution, and where financially viable, recover valuable metals ('phytomining'). Toward these aims, there has been a surge of publications over the last two decades. While important progress is being made, ongoing propagation of poor practice, and the resultant drain from funding sources, is hindering this promising research area. This includes mis-ascribing hyperaccumulator species, hydroponics with extremely high dose levels, misuse of Bioconcentration Factors, use of food or biomass crops with low accumulation for phytoextraction, the phenomenon of 'template papers' in which a known hyperaccumulator for element X is dosed with element Y, or a common weed species dosed with any variety of elements to make it 'hyperaccumulate'. Here we highlight these misconceptions with the hope that this will help to: (i) disseminate accurate definitions for in planta metal accumulation; (ii) quash the propagation of poor practice by limiting the inflation of unnecessary publications via the practice of 'template paper' writing; (iii) be used by journal editors and reviewers to validate their reasoning to authors; and (iv) contribute to faster progress in delivering this technology to in-the-field practitioners.

In this note, we highlight some common misconceptions with the hope that this will help to disseminate accurate definitions for hyperaccumulation, promote the appropriate use of hydroponics, and limit template paper writing.

Plasma-Membrane-Localized Transporter NREET1 is Responsible for Rare Earth Element Uptake in Hyperaccumulator Dicranopteris linearis.

Rare earth elements (REEs) are critical for numerous modern technologies, and demand is increasing globally; however, production steps are resource-intensive and environmentally damaging. Some plant species are able to hyperaccumulate REEs, and understanding the biology behind this phenomenon could play a pivotal role in developing more environmentally friendly REE recovery technologies. Here, we identified a REE transporter NRAMP REE Transporter 1 (NREET1) from the REE hyperaccumulator fern Dicranopteris linearis. Although NREET1 belongs to the natural resistance-associated macrophage protein (NRAMP) family, it shares a low similarity with other NRAMP members. When expressed in yeast, NREET1 exhibited REE transport capacity, but it could not transport divalent metals, such as zinc, nickel, manganese, or iron. NREET1 is mainly expressed in D. linearis roots and predominantly localized in the plasma membrane. Expression studies in Arabidopsis thaliana revealed that NREET1 functions as a transporter mediating REE uptake and transfer from root cell walls into the cytoplasm. Moreover, NREET1 has a higher affinity for transporting light REEs compared to heavy REEs, which is consistent to the preferential enrichment of light REEs in field-grown D. linearis. We therefore conclude that NREET1 may play an important role in the uptake and consequently hyperaccumulation of REEs in D. linearis. These findings lay the foundation for the use of synthetic biology techniques to design and produce sustainable, plant-based REE recovery systems.

Genetic modification of western wheatgrass (Pascopyrum smithii) for the phytoremediation of RDX and TNT.

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.

A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.

OBJECTIVES: To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants. RESULTS: To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His6-tagged recombinant proteins enabled in vitro determination of Michaelis-Menten kinetic parameters with each dinitrotoluene substrate. CONCLUSIONS: Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.

Right on target: using plants and microbes to remediate explosives.

While the immediate effect of explosives in armed conflicts is frequently in the public eye, until recently, the insidious, longer-term corollaries of these toxic compounds in the environment have gone largely unnoticed. Now, increased public awareness and concern are factors behind calls for more effective remediation solutions to these global pollutants. Scientists have been working on bioremediation projects in this area for several decades, characterizing genes, biochemical detoxification pathways, and field-applicable plant species. This review covers the progress made in understanding the fundamental biochemistry behind the detoxification of explosives, including new shock-insensitive explosive compounds; how field-relevant plant species have been characterized and genetically engineered; and the major roles that endophytic and rhizospheric microorganisms play in the detoxification of organic pollutants such as explosives.

Industry-Informed Workshops to Develop Graduate Skill Sets in the Circular Economy Using Systems Thinking.

Increasing demand for chemicals worldwide, depleting resources, consumer pressure, stricter legislation, and the rising cost of waste disposal are placing increasing pressure on chemical and related industries. For any organization to survive in the current arena of growing climate change laws and regulations, and increasing public influence, the issue of sustainability must be fundamental to the way it operates. A sustainable manufacturing approach will enable economic growth to be combined with environmental and social sustainability and will be realized via collaboration between a multidisciplinary community including chemists, biologists, engineers, environmental scientists, economists, experts in management, and policy makers. Hence, employees with new skills, knowledge, and experience are essential. To realize this approach, the design and development of a series of workshops encompassing systems thinking are presented here. After close consultation with industry, an annual program of interactive workshops has been designed for graduate students to go beyond examining the "greening" of chemical reactions, processes, and products, and instead embed a systems thinking approach to learning. The workshops provide a valuable insight into the issues surrounding sustainable manufacturing covering change management, commercialization, environmental impact, circular economy, legislation, and bioresources incorporating the conversion of waste into valuable products. The multidisciplinary course content incorporates industrial case studies, providing access to real business issues, and is delivered by experts from academic departments across campus and industry.

Phytodetoxification of TNT by transplastomic tobacco (Nicotiana tabacum) expressing a bacterial nitroreductase.

KEY MESSAGE: Expression of the bacterial nitroreductase gene, nfsI, in tobacco plastids conferred the ability to detoxify TNT. The toxic pollutant 2,4,6-trinitrotoluene (TNT) is recalcitrant to degradation in the environment. Phytoremediation is a potentially low cost remediation technique that could be applied to soil contaminated with TNT; however, progress is hindered by the phytotoxicity of this compound. Previous studies have demonstrated that plants transformed with the bacterial nitroreductase gene, nfsI have increased ability to tolerate and detoxify TNT. It has been proposed that plants engineered to express nfsI could be used to remediate TNT on military ranges, but this could require steps to mitigate transgene flow to wild populations. To address this, we have developed nfsI transplastomic tobacco (Nicotiana tabacum L.) to reduce pollen-borne transgene flow. Here we have shown that when grown on solid or liquid media, the transplastomic tobacco expressing nfsI were significantly more tolerant to TNT, produced increased biomass and removed more TNT from the media than untransformed plants. Additionally, transplastomic plants expressing nfsI regenerated with high efficiency when grown on medium containing TNT, suggesting that nfsI and TNT could together be used to provide a selectable screen for plastid transformation.

Expression in grasses of multiple transgenes for degradation of munitions compounds on live-fire training ranges.

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.

Expression of a Drosophila glutathione transferase in Arabidopsis confers the ability to detoxify the environmental pollutant, and explosive, 2,4,6-trinitrotoluene.

The explosive 2,4,6-trinitrotoluene (TNT) is a significant, global environmental pollutant that is both toxic and recalcitrant to degradation. Given the sheer scale and inaccessible nature of contaminated areas, phytoremediation may be a viable clean-up approach. Here, we have characterized a Drosophila melanogaster glutathione transferase (DmGSTE6) which has activity towards TNT. Recombinantly expressed, purified DmGSTE6 produces predominantly 2-glutathionyl-4,6-dinitrotoluene, and has a 2.5-fold higher Maximal Velocity (Vmax ), and five-fold lower Michaelis Constant (Km ) than previously characterized TNT-active Arabidopsis thaliana (Arabidopsis) GSTs. Expression of DmGSTE6 in Arabidopsis conferred enhanced resistance to TNT, and increased the ability to remove TNT from contaminated soil relative to wild-type plants. Arabidopsis lines overexpressing TNT-active GSTs AtGST-U24 and AtGST-U25 were compromised in biomass production when grown in the absence of TNT. This yield drag was not observed in the DmGSTE6-expressing Arabidopsis lines. We hypothesize that increased levels of endogenous TNT-active GSTs catalyse excessive glutathionylation of endogenous substrates, depleting glutathione pools, an activity that DmGST may lack. In conclusion, DmGSTE6 has activity towards TNT, producing a compound with potential for further biodegradation. Selecting or manipulating plants to confer DmGSTE6-like activity could contribute towards development of phytoremediation strategies to clean up TNT from polluted military sites.

Toward Financially Viable Phytoextraction and Production of Plant-Based Palladium Catalysts.

Although a promising technique, phytoextraction has yet to see significant commercialization. Major limitations include metal uptake rates and subsequent processing costs. However, it has been shown that liquid-culture-grown Arabidopsis can take up and store palladium as nanoparticles. The processed plant biomass has catalytic activity comparable to that of commercially available catalysts, creating a product of higher value than extracted bulk metal. We demonstrate that the minimum level of palladium in Arabidopsis dried tissues for catalytic activity comparable to commercially available 3% palladium-on-carbon catalysts was achieved from dried plant biomass containing between 12 and 18 g·kg-1 Pd. To advance this technology, species suitable for in-the-field application: mustard, miscanthus, and 16 willow species and cultivars, were tested. These species were able to grow, and take up, palladium from both synthetic and mine-sourced tailings. Although levels of palladium accumulation in field-suitable species are below that required for commercially available 3% palladium-on-carbon catalysts, this study both sets the target, and is a step toward, the development of field-suitable species that concentrate catalytically active levels of palladium. Life cycle assessment on the phytomining approaches described here indicates that the use of plants to accumulate palladium for industrial applications has the potential to decrease the overall environmental impacts associated with extracting palladium using present-day mining processes.

Arabidopsis Glutathione Transferases U24 and U25 Exhibit a Range of Detoxification Activities with the Environmental Pollutant and Explosive, 2,4,6-Trinitrotoluene.

The explosive 2,4,6-trinitrotoluene (TNT) is a major worldwide military pollutant. The presence of this toxic and highly persistent pollutant, particularly at military sites and former manufacturing facilities, presents various health and environmental concerns. Due to the chemically resistant structure of TNT, it has proven to be highly recalcitrant to biodegradation in the environment. Here, we demonstrate the importance of two glutathione transferases (GSTs), GST-U24 and GST-U25, from Arabidopsis (Arabidopsis thaliana) that are specifically up-regulated in response to TNT exposure. To assess the role of GST-U24 and GST-U25, we purified and characterized recombinant forms of both enzymes and demonstrated the formation of three TNT glutathionyl products. Importantly, GST-U25 catalyzed the denitration of TNT to form 2-glutathionyl-4,6-dinitrotoluene, a product that is likely to be more amenable to subsequent biodegradation in the environment. Despite the presence of this biochemical detoxification pathway in plants, physiological concentrations of GST-U24 and GST-U25 result in only a limited innate ability to cope with the levels of TNT found at contaminated sites. We demonstrate that Arabidopsis plants overexpressing GST-U24 and GST-U25 exhibit significantly enhanced ability to withstand and detoxify TNT, properties that could be applied for in planta detoxification of TNT in the field. The overexpressing lines removed significantly more TNT from soil and exhibited a corresponding reduction in glutathione levels when compared with wild-type plants. However, in the absence of TNT, overexpression of these GSTs reduces root and shoot biomass, and although glutathione levels are not affected, this effect has implications for xenobiotic detoxification.

Harnessing microbial gene pools to remediate persistent organic pollutants using genetically modified plants--a viable technology?

It has been 14 years since the international community came together to legislate the Stockholm Convention on Persistent Organic Pollutants (POPs), restricting the production and use of specific chemicals that were found to be environmentally stable, often bioaccumulating, with long-term toxic effects. Efforts are continuing to remove these pollutants from the environment. While incineration and chemical treatment can be successful, these methods require the removal of tonnes of soil, at high cost, and are damaging to soil structure and microbial communities. The engineering of plants for in situ POP remediation has had highly promising results, and could be a more environmentally-friendly alternative. This review discusses the characterization of POP-degrading bacterial pathways, and how the genes responsible have been harnessed using genetic modification (GM) to introduce these same abilities into plants. Recent advances in multi-gene cloning, genome editing technologies and expression in monocot species are accelerating progress with remediation-applicable species. Examples include plants developed to degrade 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), trichloroethylene (TCE), and polychlorinated biphenyls (PCBs). However, the costs and timescales needed to gain regulatory approval, along with continued public opposition, are considerable. The benefits and challenges in this rapidly developing and promising field are discussed.

Analysis of the xplAB-containing gene cluster involved in the bacterial degradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine.

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 explosive-degrading cytochrome P450 XplA: biochemistry, structural features and prospects for bioremediation.

XplA is a cytochrome P450 that mediates the microbial metabolism of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It has an unusual structural organisation comprising a heme domain that is fused to its flavodoxin redox partner. XplA along with its partnering reductase XplB are plasmid encoded and the gene xplA has now been found in divergent genera across the globe with near sequence identity. Importantly, it has only been detected at explosives contaminated sites suggesting rapid dissemination of this novel catabolic activity, possibly within the 50-year period since the introduction of RDX into the environment. The X-ray structure of XplA-heme has been solved, providing fundamental information on the heme binding site. Interestingly, oxygen is not required for the degradation of RDX, but its presence determines the final degradation products, demonstrating that the degradation chemistry is flexible with both anaerobic and aerobic pathways resulting in the release of nitrite from the substrate. Transgenic plants expressing xplA are able to remove saturating levels of RDX from soil leachate and may provide a low cost sustainable remediation strategy for contaminated military sites.

A gene-fusion approach to enabling plant cytochromes p450 for biocatalysis.

Cytochromes P450 from plants have the potential to be valuable catalysts for industrial hydroxylation reactions, but their application is hindered by poor solubility, the lack of suitable expression systems and the requirement of P450s for auxiliary redox-transport proteins for the delivery of reducing equivalents from NAD(P)H. In the interests of enabling useful P450 activity from plants, we have developed a suite of vectors for the expression of plant P450s as non-natural genetic fusions with reductase proteins. First, we have fused the P450 isoflavone synthase (IFS) from Glycine max with the bacterial P450 reductase domain (Rhf-RED) from Rhodococcus sp., by using our LICRED vector developed previously (F. Sabbadin, R. Hyde, A. Robin, E.-M. Hilgarth, M. Delenne, S. Flitsch, N. Turner, G. Grogan, N. C. Bruce, ChemBioChem 2010, 11, 987-994) creating the first active bacterial-plant fusion P450 enzyme. We have then created a complementary vector, ACRyLIC for the fusion of selected plant P450 enzymes to the P450 reductase ATR2 from Arabidopsis thaliana. The applicability of this vector to the creation of active P450 fusion enzymes was demonstrated using both IFS1 and the cinnamate-4-hydroxylase (C4H) from A. thaliana. Overall the fusion vector systems will allow the rapid creation of libraries of plant P450s with the aim of identifying enzyme activities with possible applications in industrial biocatalysis.

Plants to mine metals and remediate land.

Engineered plants can clean up pollution and recover technology-critical metals.

Engineering plants for the phytoremediation of RDX in the presence of the co-contaminating explosive TNT.

The explosive compounds hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) are widespread environmental contaminants commonly found as co-pollutants on military training ranges. TNT is a toxic carcinogen which remains tightly bound to the soil, whereas RDX is highly mobile leaching into groundwater and threatening drinking water supplies. We have engineered Arabidopsis plants that are able to degrade RDX, whilst withstanding the phytotoxicity of TNT. Arabidopsis thaliana (Arabidopsis) was transformed with the bacterial RDX-degrading xplA, and associated reductase xplB, from Rhodococcus rhodochrous strain 11Y, in combination with the TNT-detoxifying nitroreductase (NR), nfsI, from Enterobacter cloacae. Plants expressing XplA, XplB and NR remove RDX from soil leachate and grow on soil contaminated with RDX and TNT at concentrations inhibitory to XplA-only expressing plants. This is the first study to demonstrate the use of transgenic plants to tackle two chemically diverse organic compounds at levels comparable with those found on contaminated training ranges, indicating that this technology is capable of remediating concentrations of RDX found in situ. In addition, plants expressing XplA and XplB have substantially less RDX available in aerial tissues for herbivory and potential bioaccumulation.

Field trial demonstrating phytoremediation of the military explosive RDX by XplA/XplB-expressing switchgrass.

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.

How synthetic biology can help bioremediation.

The World Health Organization reported that "an estimated 12.6 million people died as a result of living or working in an unhealthy environment in 2012, nearly 1 in 4 of total global deaths". Air, water and soil pollution were the significant risk factors, and there is an urgent need for effective remediation strategies. But tackling this problem is not easy; there are many different types of pollutants, often widely dispersed, difficult to locate and identify, and in many cases cost-effective clean-up techniques are lacking. Biology offers enormous potential as a tool to develop microbial and plant-based solutions to remediate and restore our environment. Advances in synthetic biology are unlocking this potential enabling the design of tailor-made organisms for bioremediation. In this article, we showcase examples of xenobiotic clean-up to illustrate current achievements and discuss the limitations to advancing this promising technology to make real-world improvements in the remediation of global pollution.