Horticulture

Plant growth and yield were modestly reduced but importantly, nitrogen fixation was almost entirely eliminated. Nodule content of ceria approached 11 mg kg−1 in some instances and electron microscopy confirmed the complete absence of symbiotic bacteria. Similarly, Hernandez-Viezcas et al.used synchrotron μXRF and μXANES to observe nanoceria within soybean root nodules and pods, although up to 20% had been transformed from CeIJIV) to Ce. However, the inhibition of bacterial nitrogen fixation did not necessarily result in nitrogen shortage for the plants; soybeans exposed to high doses of nanoceria actually grew better those exposed to low doses of nanoceria in the Priester study,suggesting that the plants successfully used an alternative source of nitrogen for growth. In a related study, Bandyopadhyaya et al.observed that nanoceria at 31–125 mg l−1 significantly inhibited the growth of Sinorhizobium meliloti, the primary symbiotic nitrogen fixing bacteria of alfalfa. The authors reported that the negative impact of nanoceria on nitrogen fixing bacteria resulted from nanoparticle adsorption on the extracellular surface and the subsequent alteration of certain surface protein structures. These changes could potentially affect colonization of symbiotic bacteria on root surfaces and therefore negatively impact plant nitrogen cycling. Notably, this study was conducted in cell culture and more investigation in soil-based systems will be needed.

In a final soil study, Morales et al.noted that nanoceria at concentrations up to 500 mg kg−1 had no impact on cilantro shoot biomass and in some instances,vertical rack increased root growth. However, the authors did report FTIR-detected changes in carbohydrate chemistry, which raises the potentialfor altered nutritional content in edible tissues. A recent study with rice confirmed that exposure of 500 mg nanoceria/kg soil throughout the life cycle of rice substantially altered the nutritional values of rice grains.For examples, the authors reported that nanoceria generally reduced the sulfur and iron content of rice grains and the extent of reduction depended upon the variety of rice types. The authors also reported the alteration of macromolecule contents in rice grains by nanoceria exposure, providing the first direct evidence on the mitigation of nutritional values of agricultural grains by nanoceria.Due to their small sizes, nanoparticles can move through the macro and microporosity of the soil and be detrimental for soil microbial communities.They may have an impact on soil microorganisms via a direct effect , changes in the bioavailability of toxicants or nutrients, indirect effects resulting from their interaction with natural organic compounds and interaction with toxic organic compounds which would amplify or alleviate their toxicity.

In two soils contaminated with nanoceria at 100 mg Ce/kg of dry soil, no significant effect on both microbial biomass C and N were observed after 60 days.However nanoceria decreased microbial C/N ratio and increased the metabolic quotient , probably due to microbial stress and changes in the composition of microbial communities inhabiting soil. They found that nanoceria were associated to small aggregates rich in both labile organic C, microbial biomass and clays, suggesting that nanoparticles can interact with most of microbial communities inhabiting soil.So far, the only two terrestrial organism to have been used to assess nanoceria soil toxicity are the earthworm Eisenia fetidia and the nematode Caenorhabditis elegans. Lahive et al. compared the toxicity of cerium salt and three different nanoceria to E. fetida in exposed in standard Lufa 2.2 soil. While median lethal concentration and effective concentration values of 317.8 and 294.6 mg Ce kg−1 were found for survival and reproduction , respectively, neither of these endpoints were affected by even the highest Cerium particle concentrations of 10 000 mg Ce kg−1 . The three nanoceria used varied in size ranges , with one larger particle and the cerium salt used as controls. However, there was a dose-dependent increase in cerium in the organisms at all exposure concentrations, and for all material types. With earthworms exposed to CeO2 particles interestingly having higher concentrations of total cerium compared to those exposed to ionic cerium, without exhibiting the same toxic effect.

Additionally, histological observations in earthworms exposed to the particulate forms of CeO2 showed cuticle loss from the body wall and some loss of gut epithelium integrity. The data overall suggesting that while nanoceria do not affect survival or reproduction in E. fetida over the relatively short standard test period, then there were histological changes that could indicate possible deleterious effects over longer term exposures. In contrast to E. fetida, then C. elegans is most often exposed in aquatic media rather than soil and so it is also often considered an aquatic toxicity testing organism.Roh et al. assessed the interaction between nanoceria and C. elegans and encountered a marked size-dependent effect on the fertility and survival of C. elegans. Zhang et al. evaluated the in vivo effects of a positively charged coated nanoceria on C. elegans at low concentrations . The results indicated that nanoceria induced ROS accumulation and oxidative damage in C. elegans, and finally lead to a significant decreased lifespan even at the exposure level of 0.172 μg l−1 . Collin et al.2 showed that the toxicity and bio-accumulation of coated nanoceria in C. elegans were dependent on the surface charge of the nanoceria. The positively charged nanoceria were significantly more toxic to C. elegans and bioaccumulated to a greater extent than the neutral and negatively charged nanoceria. They measured a LC50 of 15.5 mg l−1 for L1 stage C. elegans exposed during 24 h to the positively charged coated nanoceria.The presence of NOM has been shown to influence the bioavailability and toxicity of other nanoparticles.The presence of humic acid in the exposure media had been shown to influence Ce bio-accumulation in C. elegans exposed to positively charged coated nanoceria.2 Ce bio-accumulation was influenced by the ratio between HA and nanoceria. For a relevant scenario, i.e. when the concentration of HA was higher than the nanoceria concentration, Ce bio-accumulation decreased. Interestingly, for all tested concentration, the presence of HA in the exposure media significantly decreased the toxicity of nanoceria to C. elegans.

The decrease of toxicity was explained by the profound modifications induced by the adsorption of humic acid such as a change of the ZP or the formation of μ size aggregates, which were too large to be absorbed by C. elegans.First of all, the aggregation state appears to be an important parameter to consider when dealing with exposure of aquatic organisms to nanoceria due to their low solubility. On a large scale, aggregation/sedimentation of nanoceria in aquatic environments will leave a small portion of the total mass of nanoceria available for direct uptake by planktonic organisms , while the majority will be in contact with benthic organisms . In this case, sediments should be regarded as a sink for nanoceria discharged to the aquatic environment. Not only can the exposure pathway be different upon aggregation,vertical farming hydroponic but the mechanisms of internalization can also vary. Like the aggregation, the chemical stability of nanoceria can change in environmental biological pH/Eh conditions. Metals such as Ce exhibit various possible redox states IJCeIJIII), Ce) for which stability is a function of Eh and pH values. Intracellular Eh is controlled by metabolic processes as the oxidative phosphorylation in mitochondria. It is based on a series of redox reactions at near circumneutral pH for which potentials are in a – 0.32 to 0.29 V . Extracellular Eh is generally controlled by thiol/ disulfide redox systems for which Eh vary in a – 0.140/–0.08 V range. In such intra- and extra-cellular Eh conditions, Ce can be redox unstable which lead to electron exchange between nanoparticle surface and surrounding media. This could be the starting point of disequilibrium of the redox balance and then to oxidative stress toward micro- and macro-organisms. Regarding microorganisms, up to now, no undisputable evidence of nanoceria uptake by cells has been obtained. The nanoceria were either found in direct contact with the bacterial wall or trapped in the exopolysaccharides layer surrounding the microorganisms.For instance, studies have shown that Escherichia coli exposed to nanoceria in a simplified exposure media were covered by a thin and regular monolayer of nanoceria surrounding the cell wall. But for Synechocystis, nanoparticles could not form a shell at the cell surface because they were adsorbed onto the protecting layer of EPS bound to cell membranes. These nanoparticlestrapping EPS likely explains the higher level of nanoceria adsorption onto Synechocystis as compared to E. coli. Several studies have been conducted investigating toxicity in microorganisms. The toxicity of nanoceria was found to be strain- and size-dependent for E. coli and B. subtilis, whereas they did not affect S. oneidensis growth and survival.EC was near 5 mg l−1 for E. coli and ranged from 0.27 to 67.5 mg l−1 for Anabaena in pure water.Chronic toxicity to algae P. subcapitata with 10% effect concentrations between 2.6 and 5.4 mg l−1 was observed. Van Hoecke et al.observed that the presence of NOM decreased the toxicity of nanoceria to P. sub-capitata.

They assumed that the adsorption of NOM to the nanoceria surface prevented the particle from directly interacting with algal cells thereby decreasing the bio-availability of the particles. Under exposure to nanoceria, N. europaea cells show larger sedimentation coefficient than the control.The toxicity of nanoceria was either exerted by direct contact with cells,membrane damage,cell disruption,release of free CeIJIII).No oxidative stress response was detected with E. coli or B. subtilis, but nanoceria and CeCl3 alter the electron flow, and the respiration of bacteria.Pelettier et al.also observed the disturbance of genes involved in sulfur metabolism, and an increase of the levels of cytochrome terminal oxidase transcripts known to be induced by iron limitation. Rodger et al.also monitored the growth inhibition of P. sub-capitata and reported EC50 value of 10.3 mg l−1 of a 10- to 20 nm nanoceria. This inhibitory mode of action was mediated by a cell-particle interaction causing membrane damage and likely photochemically induced. Even if free Ce is toxic, release of Ce from the nanoceria did not explain by itself the toxicity observed in these studies . However, the reduction of the Ce into Ce at the surface of the nanoceria correlates with the toxicity. Using XANES at Ce L3-edge, Thill et al.and Auffan et al.98 showed that the cytotoxicity/genotoxicity of nanoceria could be related to the reduction of surface Ce atoms to Ce. But, further research is needed to find out whether the oxidative activity of ceria could be responsible. Regarding inverterbrates, one of the most favorable routes for nanoceria uptake by aquatic organisms is ingestion. For instance, ingestion via food chain was the main route of nanoceria uptake by the microcrustaceans Daphnia pulex. The adsorption of nanoceria on algae during the exposure to sub-lethal doses of nanoceria enhanced by a factor of 3 the dry weight concentration of Ce on the whole D. pulex. Nanoparticles were localized in the gut content, in direct contact with the peritrophic membrane,and on the cuticle.Interestingly, the depuration was not efficient to remove the nanoceria from the organisms. From 40% to 100% of the nanoceria taken up by D. pulex was not release after the depuration process. However, the authors demonstrated that the shedding of the chitinous exoskeleton was the crucial mechanism governing the released of nanoceria regardless of the feeding regime during exposure.Moreover, interspecific toxic effects of nanoceria toward daphnia were explained by morphological differences such as the presence of reliefs on the cuticle and a longer distal spine in D. similis acting as traps for the nanoceria aggregates. Acute ecotoxicity testings showed that D. similis was 350 times more sensitive to nanoceria than D. pulex with 48 h EC50 for D. similis about of 0.3 mg l−1 . 100 In addition, D. similis has a mean swimming velocity twice as fast as D. pulex and thus initially collide with twice more nanoceria aggregates. The effect of the exposure methods, direct and through sorption to phytoplankton was tested on the mussel Mytilus galloprovincialis. 101 Ce uptake was enhanced by the ingestion via the phytoplankton in the first 5 days of exposure but was similar to a direct exposure after 2 weeks. The authors showed that with increasing nanoceria concentration, mussels increased their clearance rates as well as the pseudofeces production in order to prevent the ingestion of nanoceria.

Due to the rotational motion of the water, the levels of turbulence kinetic energy are increased leading to vigorous mixing and hence to uniform exposure to UV radiation. Since the UV lamps are arranged circumferentially around the outside of the quartz cylinder, pathogens that may become imbedded into suspended solids experience increasing probability of receiving UV dose sufficient for their activation since the UV light is radiated from all direction. In a conventional commercial system, where the water flow is linear and parallel to the UV lamps, embedded pathogens are more likely to exit the system before receiving adequate dose. At top of the cylinder, the treated water overflows as though over a radial weir into a collection trough and from there into an outlet tube either to be re-used elsewhere. The passage of the treated water above the weir is associated with significant entrainment of air into the water thereby elevating the percentage of dissolved oxygen while simultaneously lowering the water temperature. Testing of the UV reactor performance was carried out at the National Ornamental Research Site at the Dominican University of California . At this site, quarantine plant pathogens are studied in a mock nursery under field conditions that mimic those found in commercial nurseries . For the experiment,vertical farming tower for sale irrigation water from the research site was collected and stored for 7 days prior to the experiment to have enough volume to run the reactor at a flow of 2.2 l/s and to provide the endogenous population of bacteria, oomycetes and fungi for the test.

Tests were conducted on two separate occasions; in June 2013 and in October 2013. On the day of testing, this water was also spiked with P. ramorum zoospores to ensure its presence in the test water. P. ramorum zoospores were produced as described by Widmer . P. ramorum strain 1,418,886 was grown on CV8-agar at 20 °C for approximately three weeks. Sporangia production was induced by adding 15 ml of soil extract water. Release of zoospores was induced by cold shock, and then zoospores were harvested and counted using a hemacytometer. A total of 5 l of zoospore suspension was added to a water tank of volume 1893 l for a final concentration of 1.3 × 102 spores/ml. The UV reactor was placed in line with the effluent from the collection basin and was tested at a constant flow rate of 2.2 l/s. The tests were conducted under three separate conditions in which 4, 8 and 12 lamps were used. One liter of sample was collected from the influent of the disinfection system and one liter from each lamp condition to test samples for bacterial and fungal counts. The transmittance of the water was 76.4% UVT. Bacterial counts were made from cultures growing on Reasoner’s 2A, Acidified Dextrose Potato Agar , and PARPH-V8. One milliliter of each sample was plated in triplicate on each media type and cultured using standard methods. R2A is the preferred media for culturing bacteria found in treated or potable water sources, water sources with low concentrations of endogenous bacterial populations and long incubation times . ADPA is a media commonly used to culture fungal populations, but will also cultivate some bacterial populations.PARPH-V8 media contains pimaricin, ampicillin, rifamycin, pentachloronitrobenzene , and hymexazol and is a selective media designed to isolate Phytophthora sp. . The UV dose supplied by the system was determined by quantifying its ability to remove the MS2 virus . MS2 is a male-specific RNA virus that infects bacteria.

It has a similar structure to the polio virus and is widely used in water treatment research to assess the efficacy of a particular treatment method for virus removal . In the present application, water inoculated with MS2 to a given concentration was introduced into the UV system where it was exposed to UV light produced from UV lamps that varied in number from 2 to 12. Samples of the treated water were collected and delivered on the same day to Biovir Laboratories Inc. in accordance with the National Water Reuse Institute sampling guidelines . There, collimated beam testing was carried out according to standard methods to generate a dose response curve from which the actual UV dose delivered to the water was deduced. Results were obtained for water samples having UVT of 70 and 95%. The UVT was adjusted using instant coffee, an approved NWRI method . Each trial of the experiment used 1 l of MS2 with a titer of 1014 plaque forming units per milliliter for a final influent concentration of 108 PFU/ml. One liter of sample was collected for the collimated beam testing. The flow rate was kept constant at 2.2 l/s for all the tests.In water-treatment applications, reduction in bacterial counts achieved by a particular treatment method is measured in Blog reduction^ – the number of viable bacterial cells removed expressed on a logarithmic scale. For example, a 4-log reduction is a 10,000-fold decrease in the number of microorganisms present in the sample. A summary of the log reduction of the bacterial counts present in the irrigation water is presented in Table 1. Shown there are the results obtained from each of the bacterial culturing methods, and for three different lamp conditions corresponding to 4, 8 and 12 lamps. The results from the R2A and the PARPH culturing methods indicate that a minimum of 3.7 log reduction is achieved by the UV system. Low bacterial counts were seen on the APDA, because the acidification of the media suppresses bacterial growth.Microbial counts were evaluated using APDA to assess the fungal concentration and PARPH-V8 was used to determine the concentration of P. ramorum.

Whereas true fungi were present in the used nursery water as natural contaminants, P. ramorum was added as described above. One milliliter of each sample was plated in triplicate on each media type and cultured using standard methods. Fungal counts from the APDA media and PARPH media at each UV treatment are listed in Table 2. It can be seen there that the UV system removed about 75%, 91.7%, and 91.7% of fungal counts using 4, 8, and 12 UV lamps, respectively. It was also found that all lamp combinations of the UV system were able to inactivate 100% of the P. ramorum in the effluent.California’s water re-use policy Title 22 requires a demonstration of 5-log removal of MS-2, and the use of at least two reactors in series for redundancy to ensure a minimum level of safety in the system . Since only one reactor was tested,hydroponic vertical farm it must achieve at least 2.5 log removal of MS2 or a UV dose of 50 mJ/ cm2 . A dose of 50 mJ/cm2 is an approximate UV dose for 2.5 log removal. Figure 2a and b show the average log inactivation of MS2 and the average UV dose in the reactor with respect to change in lamp condition for the testing at UC Davis. From these figures, the reactor must operate with at least six lamps at a UVT of 95% and at least eight lamps at a UVT of 70% to achieve aminimum of 2.5 log removal of MS2. If only the 50 mJ/ cm2 UV dose condition is considered, then the UV reactor must operate with at least four lamps at a UVT of 95% and at least six lamps at a UVT of 70%. Figure 2c is a plot that shows the electric energy needed by the reactor to achieve one log of MS 2 inactivation for every 3785 l of irrigation water treated. As the UVT decreases from 95 to 70%, the energy required to inactivate a log of MS2 increases as more lamps would be required to deliver the necessary UV dose. Also, for the 95% UVT case, the energy required for a log removal changes very little when more than six lamps are used. Hence this would be the ideal number of lamps that would be needed for use in these conditions. Using the UVT of the water from NORS-DUC , the results of testing at the Davis were used to determine the UV dose supplied to the irrigation water. Using linear interpolation with the data from Fig. 2b, the UV dose supplied to the NORS-DUC irrigation water was 51, 61, 82, 100 mJ/cm2 for 4, 6, 8, and 12 lamps, respectively. Thus, to achieve a 5-log removal of MS2, two reactors each with four lamps would be needed to deliver the required UV dose. However, from a practical standpoint, since the percent of dissolved organics in the water can cause fluctuations in the UVT, it would be prudent to use six lamps. Achieving a 5-log removal in the irrigation water ensures that plant pathogens are not spread to the natural wildlife and renders the water suitable for reuse within the nursery. Additional low-cost safety measures can also be used in conjunction with UV treatment to help facilitate reuse within the plant nursery, such as filtration and settling. Since UV treatment is a physical process, pathogens embedded in soil particles may be shielded from treatment . In fact, Title 22 requires that the turbidity of the treated water be less than 5 NTU to account for this problem . Minimizing this risk and meeting Title 22 requirements can be achieved by first allowing heavy particles to settle out of the waste stream and then screening out suspended particles with a filter before treatment, typically with a nominal diameter of 1–10 μm.Livestock are an important livelihood source for over 900 million small scale producers in low-and middle-income countries , a source of nutrient-dense animal source foods and income for members of such households .

Livestock production systems in LMICs range from extensive mobile pastoral to semi-intensive urban and peri-urban small-scale production systems. Demand for livestock products is increasing worldwide, particularly in LMIC, fueled by population growth, urbanization, and rising incomes . Per capita consumption of ASF increased four to five-fold from the early 1960s to 2015 , with three fourths of this growth coming from LMIC . By 2050, consumers in LMIC will demand 107 million tons more meat and 5.5 million tons more milk than they did in 2005/2007 . The increase in demand pertains to both quality and quantity, especially as incomes rise from USD 2 to 10 per day, particularly among urban consumers who purchase livestock products from supermarkets .Though the livestock revolution can be an important opportunity for increasing income for small scale producers in LMIC, various constraints limit their ability to take advantage of it. Among many systematic, bio-physical and socio-cultural problems, limited access to quality feed is a major challenge across all production systems in LMIC . Feed affects livestock productivity, profitability, environmental impact, human food and nutrition security, animal welfare and ethics, and animal and human health . Financially, feed costs account for up to 70% of the total variable costs of livestock production and may reach 90% in more intensive systems . The global value of purchased compound feed relative to total animal output is about 30% on average for all production systems, and is 10% for cattle, 40% for pigs and 80% for poultry . Good quality feed improves livestock productivity, resulting in lower age at first calving and shorter inter-calving interval, thus increasing productive life and profitability . Proper feeding improves animal immunity , health, welfare, and reproductive performance; enables higher productivity under a given management regimen and contributes to environmental sustainability by converting energy and nutrients from land that is unusable by humans into highly nutritious food. Much of the feed consumed from livestock is derived from such areas or inedible agro-industrial byproducts. In fact, about 86% of the feed globally consumed by livestock is not edible by humans . Properly implemented forage-based systems including silvopastoral systems can reduce emissions from livestock . Proper feeding can reduce the methane emissions from livestock farming by increasing the ratio of feed used for production to that used for maintenance, also known as the maintenance dilution effect . This is critically important since feed , and enteric fermentation contribute to 45% and 39%, respectively, of the total emissions from livestock production . Lack of availability and access to quality feed continues to be the most important limitation to livestock production in LMIC. For instance, in six African and Asian focus countries of the Feed the Future innovation Lab for Livestock Systems, feeds and feeding-related issues were ranked consistently as the primary constraint to livestock production and consumption of ASF .

Often considered “the elephant in the room,” some of the old prophecies on the existence of biophysical limits to population growth are going to be central in the analysis of the future of food security . It has been argued that there is an urgent need to contain the escalating demand for food commodities by stabilizing the global population . This has revamped the debate on the efficacy of population policies and reproductive health education , as well as other longer-term approaches based on both social and economic development, including empowerment of women and access to education, poverty eradication, and other factors affecting fertility rates .Though food supply may be adequate at the global scale, high levels of undernourishment persist in many parts of the developing world , while habits of over consumption have become commonplace in the United States and Europe . Thus, in addition to production-side solutions that have been proposed for meeting future demand, recent work has pointed toward the need for efforts to draw down per capita demand , particularly in countries with diets with a large fraction from animal products, and to promote better physical and economic access for less integrated markets. Approaches to promote a shift toward healthier and environmentally more sustainable diets can be based on a variety of interventions, including raising awareness, education, “nudge” methods ,plant benches economic incentives, taxation , and law restrictions. The latter three approaches, however, can be difficult to accept in free market economies and liberal societies .

Although a consumption focused approach to food security may be difficult to implement, given the social and cultural associations of diets, new studies have demonstrated linkages between sustainable dietary choices and health and explored sustainable diets. Thus, approaches based on health awareness can also improve environmental sustainability. Other strategies to enhance awareness and education rely on the effect of sustainability labels on food choices or rely on academic institutions to take the lead in evaluating and improving the water, carbon, and nitrogen footprints of the institutions, starting with the food served . Indeed, universities and other nonprofit organizations can be leaders in developing internal food sustainability policies and, in doing so, set the standards for other institutions in a manner similar to that of major divestment initiatives in “unethical” businesses related to apartheid, tobacco, or fossil fuels. Such divestment efforts started from the management of endowments of major university and religious organizations and spread to the broader market . Presently, universities are pioneering efforts aimed at calculating and reducing the nitrogen, carbon, and water footprints within their institutions or promoting low meat diets . There are, however, some major barriers to a dietary shift away from a meat-based diet. The greatest barriers are cultural and are associated with the appreciation of meat by those who are used to having it as the central part of their meals, enjoy its taste, lack knowledge about how to prepare vegetarian meals, or believe that meat has a higher nutritional value than other food types. Moreover, in many societies meat consumption is perceived as a sign of affluence, status, masculinity, authority, and physical strength . These cultural factors shape a society’s consumption patterns and make dietary shifts a difficult task .

Appreciation for meat is typically stronger among men, younger people, families with children, and rural communities in which meat consumption is considered an important part of their tradition, whereas plant-based diets are perceived to have no taste or nutrition value . Knowledge gaps about the environmental and health impacts of meat, and false perceptions about the nutritional properties of vegetables, can constitute important barriers to a shift toward diets that use less meat. There is also a generalized reluctance to the use of meat substitutes because of unfamiliarity with their taste and texture, and many people do not know how to replace meat with proteins from vegetable sources . Although cultural barriers are hard to remove, some of the knowledge gaps listed above could be addressed by educating citizens about the nutritional, health, and environmental implications of their food consumption habits. Attempts at promoting dietary shifts could be more effective if they target specific social groups―namely, the student population, who could start getting used to meat substitutes at a young age; women, who appear to be more inclined to vegetarian diets than men ; or citizens concerned about the health impacts of an excessive use of meat . Other educational initiatives could appeal to concerns about environmental impacts, animal ethics, and welfare . New policies could promote healthier and sustainable diets by setting higher nutritional and environmental standards for school meals . To reduce meat consumption some school districts and workplaces are already adopting meat-free days in their meal plans, while promoting health education to decrease employers’ long-term healthcare costs. In some affluent countries, there are already signs of reduced meat consumption, which indicates that, as societies become wealthier, concerns about health and environment lead to a more moderate consumption of meat, according to a Kuznet-like inverted-U curve . Reductions in meat consumption can be favored by urbanization, education, empowerment of women, or the use of sustainability labels . In the developing world, however, the expected trend is still that of an increase in meat consumption in the next few decades.For instance, less sustainable food types, such as meat or unhealthy processed foods could be taxed, whereas subsidies could be used to reduce the prices of vegetables, meat substitutes, and other more sustainable and healthier food products.

Such policies could ensure that food prices account for environmental costs and use part of the tax revenues for the improvement of taste, texture, and nutritional properties of meat substitutes . The efficacy of policies acting on food prices, however, could be modest in affluent societies where only a relatively small fraction of the income is typically spent on food, a pattern known as Engel’s law.Trends toward greater animal protein in diets mean that livestock production systems need to become far more efficient. In this regard, the ongoing transition toward monogastric production is encouraging. Indeed, recent work has shown that shifting grain-fed beef production entirely to chicken and pork production would feed an additional 367 million people . As with closing crop yield gaps, the industrialization of animal production poses a huge challenge to the sector’s vital and ongoing role in poverty alleviation because livestock, and agriculture in general, are important for various aspects of rural livelihoods . Likewise, the expansion of sustainable forms of aquaculture offers an alternative, and potentially more environmentally sustainable, way to meet some of this future demand for animal products . Even more so than for crop production, the future of the livestock sector is far from clear . The last few decades have seen an increasing reliance on poultry and swine meat, and a decrease in the fraction of ruminant meat consumption, worldwide . This trend allows for a reduction of the land used and carbon footprints of meat per unit calorie .As noted in the previous sections, a possible approach to feed the world with the limited resources of the planet is to reduce the consumption of meat, particularly of the meat types that have the greater environmental footprints. Alexander et al. reviewed a series of alternative meat types,rolling bench including insects, cultured meats , and imitation meats; they found that insects and imitation meats had particularly low land use requirements relative to conventional meat. However, imitation meats had relatively minor reductions in land use requirements compared to poultry and dairy, further emphasizing the importance of dietary change and waste reductions . Other studies have highlighted the environmental, health and economic benefits of eating insects . Most insects have relatively high bio-conversion rates , close to 5 times those of cattle. Moreover, the edible biomass fraction is much higher in insects than in livestock . Therefore, the feed-to-edible meat ratio is much more favorable for insects than for livestock, which explains their smaller land and carbon footprints . Insect meat is also healthier because of its high protein and low fat contents. Further, because insect production requires low technological inputs, it can be practiced by small-scale farmers, thereby improving the food security and nutrition of rural populations, as well as their livelihoods . Insects may be used either for direct human consumption or as feed for the aquaculture and livestock industries. The feed used for insect production can be based on various types of organic waste, including cellulosic materials. Therefore, reliance on insect meat may allow for an effective recycling of waste and favor the establishment of a circular economy , whereby food waste is turned into protein-rich feed and food .

As noted in section 2, about 24% of global food production for human consumption is lost or wasted through the food supply chain . Recent work has demonstrated the environmental benefits of reducing food waste and shown that consumer waste of animal products is particularly costly in terms of land use and crop production . All of these studies provide important insights into how consumers may consider healthier and less environmentally burdensome consumption choices . It has been estimated that food waste accounts for 23% of the arable land, 24% of freshwater resources used for crop production, and an amount of food per capita of roughly 625 kcal per cap per day, including large quantities of nutrients, micro-nutrients, and minerals . These figures speak for themselves. A strategy aimed at improving the use of land and water for food production needs to invest in food waste reduction and reuse. Many studies have investigated how food waste can be reduced by removing inefficiencies in the food supply chain from agricultural production to post harvest storage, processing, distribution, and consumption. Possible actions include crop production planning to avoid surpluses that cannot be placed on the market; improvements in storage, refrigeration, and transportation facilities, particularly in the developing world; changes in the logistics of food retailing and distribution to account for the limited shelf life of perishable products; and consumer education on how to make more effective purchase plans and deal with “expiration” and “sell-by” dates . In developed countries, some of the quality standards for fresh produce overemphasize aesthetic criteria and idealizations about fruit or vegetable size and shape, or product uniformity with the effect of discarding products that are perfectly healthy and edible. Consumer education could encourage the use of substandard or unappealing food products, and products that are unsuitable for human consumption could be repurposed and used as animal feed or for bio-energy production . Food waste can also be contributed to by retailer overbuying, oversized packages, and stores’ compliance with “sell-by” or “use-by” dates. To redress some of these factors, it is possible to act at the retailer and distributor level. Of note is France, where a recent law forbids the destruction of unsold food as it approaches its “best by” date. Rather, supermarkets need to donate these products to food bank charities, though the process of food delivery to humanitarian organizations remains a difficult task as it requires timeliness and coordination . The EU Commission has subsequently established a multi-stake holder platform with the explicit task of developing a strategy to reduce food waste. Another particularly important opportunity in the FEW nexus is to enhance the recovery, treatment, and reuse of wastewater. In terms of water scarcity, wastewater offers a potential alternative source of irrigation in some contexts . Improved access to sanitation is not only a UN Sustainable Development Goal in and of itself, but improved sanitation systems offer massive potential to recover critical plant nutrients, particularly P, to offset agricultural nutrient demands at the global scale and, to some degree, to influence household energy goals through renewable sources such as biogas . Recycling of nutrients from urban waste streams is especially important for the P cycle because of the nonrenewable nature of this resource and the relatively high P content of sewage sludge . However, in addition to the infrastructure needed for urban sanitation, multiple socioeconomic and environmental factors can play a role in the efficacy of nutrient recycling in any given city .Despite all the efforts our societies can make to reduce food waste through more efficient food supply chains, consumer education, and the use of food banks, some of the losses existing in the food system are unavoidable.

Depending on the depth of the deposit, oil sands are extracted using two different methods―surface mining and in situ drilling . In situ technology requires less water than surface mining . Bitumen from mined oil sands is a low-quality product that needs to be upgraded through a water-demanding process into synthetic crude oil before being delivered to refineries . Shale oil and gas extraction is performed through horizontal drilling and hydraulic fracturing, technologies that require a lower amount of water than other fossil fuels. However, shale oil extraction requires a large upfront use of water over a few days, after which oil is produced over several months . Thus, intensive water withdrawals over a short period of time can induce or enhance local water stress. By adopting a hydrologic perspective that considers water availability and demand together, Rosa et al., 2018 presented a global analysis of the impact of shale oil and gas extraction on water resources, 25 liter pot particularly on irrigated crop production. Using a water balance analysis, Rosa et al., 2018 found that 31–44% of the world’s shale deposits are located in areas where water stress would either emerge or be exacerbated as a result of unconventional oil and gas extraction from shale rocks. This analysis is an example of how research can analyze all the three dimensions of the FEW nexus using geospatial data-driven analyses.

Results from these studies can be used by decision makers and local communities to better understand the water and food security implications of energy systems.A small volume of water is required during the drilling and cementing phases. Interestingly, unconventional gas production from shale gas requires the same amount of water as shale oil wells drilled in the same area . However, energy production from shale oil has a lower water footprint than energy from shale gas because of the higher energetic content of oil. Unconventional gas can also be produced from coal bed methane. In this case, deep coal seams undevelopable for mining operations are drilled to extract the natural gas that is absorbed by the organic material in the coal formation. Coal bed methane has a low water footprint and releases substantial volumes of produced water that, if treated, can be recirculated into the water cycle.Coal has not only high GHG emissions per unit of energy produced but also a high water cost . The amount of water used for coal mining varies between underground and surface mines. Water requirements increase as the coal mine operations move deeper underground. An increasing trend in coal mining operations is to wash coal, a process that requires about 3.79–7.58 L/GJ . Coal washing is accomplished by density separation or froth floatation to separate mined coal ore from a mixture of materials . This process aims to improve combustion efficiency to meet environmental standards by reducing sulfur and particulate emission during combustion .

Water can also be used to transport coal as a slurry through pipelines . In an attempt to curb the increasing atmospheric CO2 concentrations, recent energy policies have mandated a certain degree of reliance on renewable energy sources as alternatives to fossil fuels . Thus, gasoline and diesel are now commonly blended with bio-ethanol and bio-diesel. These bio-fuels can be obtained from a variety of crops, including food crops , cellulose-rich crop residues , and algae . To date , the bio-fuels that are commonly used are of the first generation. Bioethanol is mainly made with maize in the United States and sugarcane in Brazil, whereas bio-diesel is produced using vegetable oil . Bioethanol consumption is for most part domestic, and at leastone third of the global bio-diesel is available through international trade, mostly associated with palm oil from Indonesia and Malaysia . The water used for bio-fuels strongly varies with crop type, geographic location, climate, and soil . First-generation bio-fuels have a much higher water footprint than fossil fuels and therefore compete with the food system directly and indirectly . The competition of bio-fuels with food production explains the heated debate on how bio-energy production competes with the food system and the appropriateness of using food crops to fill fuel tanks instead of feeding the poor . Rulli et al. found, however, that to date, only about 4% of the global energy consumption by the transport sector and 0.2% of global energy use in all sectors is utilized for bio-fuels. For the year 2000, bio-fuel production accounted for about 2–3% of the global land and water used for agriculture .

In 2007, bio-fuel production accounted for about 2% of the global production of inorganic phosphorus fertilizer . Second- and third-generation bio-fuels do not compete with food production because they do not rely on biomass that could otherwise be used for food, and they consume relatively small amounts of water .The water footprint of fossil fuels is typically calculated by accounting only for the water used for oil or gas extraction and processing without considering the fact that these hydrocarbons result from the transformation of ancient plant biomass over geological time . Millions of years ago the growth of that biomass was associated with the transpiration of ancient water, similar to the way today’s bio-fuel production entails the consumptive use of the huge amounts of water . For any agricultural commodity , the water consumed in transpiration is the major contributor to the water footprint of fossil fuels. The main difference, in this case, is that the water used for transpiration is ancient water. The omission of ancient water from the calculation of the water footprint of fossil fuels explains the big gap between the water footprint of fossil fuels and bio-fuels . The ancient water component of the water footprint of fossil fuels is difficult to estimate because that water was transpired millions of years ago by plant species and under climate conditions that do not exist anymore and are not known to us. It is possible, however, to estimate the amount of water that it would take today to replace the “burning” of ancient water with present water by shifting from fossil fuels to present biomass . To meet today’s fossil energy need , a consumptive use of water would be close to 7.39 × 1013 m3 year, which is order of magnitude greater than the water used for extraction and processing that is usually accounted for in water footprint calculations of fossil fuels . Thus,to meet its energy needs, humanity is using an amount of ancient water of the same order of magnitude as the annual evapotranspiration from all terrestrial ecosystems . In other words, the energy that is powering industrial societies relies on water from a geological past . Likewise, the use of fossil fuels is relying on past sunlight and land , allowing industrial societies to have access to an unprecedented amount of energy that cannot be replaced with present-day biomass because of constraints imposed by the water cycle and land availability . These findings highlight the need for nonfuel-based sources of renewable energy as future substitutes for fossil fuels.The discussion of ancient water presented in the previous section highlights some limitations in the calculations of the water footprint of fossil fuels. Although the water footprint of bio-fuels and food products accounts for the water used their production, for fossil fuels, the water footprint accounts for the actual water needed in extraction and processing, neglecting the ancient water used millions of years ago . Moreover, previous works have assessed the water footprint of energy production and power generation from the life cycle analysis perspective without considering the impacts on local water resources . In analyses of the hydrologic impacts of fossil fuel production,raspberry cultivation pot an approach that looks at the total water used for extraction and processing may be misleading because these two water needs are typically met with water resources available in two different locations . LCA scientists typically focus on a comprehensive accounting of all water costs associated with production and processing, regardless of where the water comes from. Therefore, there is the need for a more hydrologic-based approach as an alternative to classic LCA calculations of the water footprint .Thermal power generation accounts for 70% of world power generation . Current technologies used for thermoelectric power plants are based on a steam Rankine cycle and heavily rely on water. In these systems, a cooling fluid is needed to cool and condensate the outlet steam of the expanders. In a thermoelectric power plant, water is heated to produce the steam needed to spin the turbines that generate electricity. Thermodynamic limits require cooling the steam into water before it can be reheated to produce steam again. Surface water from a nearby water body typically is used as a refrigerating fluid because of its availability and efficient heat transfer properties.

For this reason, thermoelectric power plants are built close to rivers, lakes, and seas. The volumes of water withdrawn for thermal power generation are staggering. For example, in the United States thermoelectric power plants account for 40% of total freshwater withdrawals and 4% of freshwater consumption . Power plants built along the coast can reduce the use of freshwater and limit the exposure to water stress. However, seawater is more corrosive and requires more resistant materials and higher capital costs . Nuclear power has the highest water consumption among thermoelectric technologies . Water is needed not only to cool the exhaust steam but also to control the temperature of the fission process of uranium. Additionally, uranium mining and processing requires substantial amounts of water . Coal and natural gas-fired power plants, as well as refineries, can be retrofitted with a carbon capture unit . Although carbon capture and storage is a promising technology to limit the climate change impacts of energy production by reducing CO2 emissions from fossil fuels, the actual technology is based on absorption capture units, which rely on large volumes of water to separate CO2 from the flue gas .Energy is used for multiple food system activities, including the operation of farm machinery and the processing, packaging, transporting, refrigerating, and preparing of food . As one example, the U.S. Department of Agriculture estimated that overall food-related energy use in the United States represented 16% of the Nation’s total energy budget . The energy use involved in the food system therefore to some degree links food systems to GHG emissions. Food systems contribute between 19% and 29% of total global anthropogenic GHG emissions, but direct emissions from agricultural production and indirect emissions resulting from land use change contribute much more to total emissions than other food system activities . Even before the food production stage, energy use is required in the production of fertilizers and pesticides; for example, industrial ammonia synthesis using the HaberBosch process for N fertilizer manufacturing uses greater than 1% of energy production worldwide because of its reliance on high temperature and high pressure . Although food is increasingly transported across vast distances, a life cycle assessment of U.S. foods by Weber and Matthews found that transportation represented just 11% of total food-related GHG emissions, meaning that food choice had a higher relative impact on the reduction of overall emissions than the sourcing of local foods to reduce transportation emissions. One of the most obvious ways in which food is linked to energy is the use of food crops as feed stock for bio-fuel production . There is a myriad of cases where the water needs of the energy and food sectors strongly interact with one another through their competition for land and water . As a result, energy prices can also be linked to food prices because of the increased cost of agricultural production and transportation , which was observed particularly with the growing demand for first-generation bio-fuels as a result of higher oil prices in the 2000s . The links between food and first-generation bio-fuels are further discussed in the following section.The production of bio-fuels is one of the more prominent examples of connections between food and energy markets that has raised concerns about diverting resources from one product to the production of another product , which can generate higher returns. These dynamics are further complicated by agricultural subsidies, tariffs, incentives for renewable energy, and opportunities associated with international land investments for agribusiness corporations.

The surface of the bubbles, as shown earlier, is proposed to contain adsorbed hydroxide ions arranged in rhomboid unit cells, and by vector addition it is clear that the least repulsion to oxygen molecule to diffuse through the hydroxide ions would be at the centre of each rhombus, which would be limited to a greatly reduced area for the diffusion to occur through. This restriction would significantly increase the time needed for the gas to diffuse outward, causing the bubble to shrink at a much lower rate. Thus, the electrostatic repulsion would, in theory, be the weakest at the centre of each rhombus, and would presumably permit the number of oxygen molecules that can fit through it, as well as who have the requisite kinetic energy, to diffuse outward. However, the number of the ions adsorbed to the surface causes difference to the limitation of outward diffusion. If, as in the second case, hydroxide ions are assumed to completely saturate the surface, then the diffusion is inhibited by the steric repulsion or steric hindrance of the hydroxide ions on the surface. This in turn will reduce the diffusion to nearly negligible levels, giving the nanobubbles highly increase lifetimes. While both the cases of stationary and moving nanobubbles represent two opposite sides of the spectrum of possible cases, it is clear that the trend of increasing number of adsorbed ions correlates to a decrease in the outward diffusion of gas and thus increased lifetimes of bulk nanobubbles.The repulsion of the ions and the gas molecules is, essentially, a case of repulsion in aqueous solution, however, within the nanobubble, the case of the purely aqueous solution must be replaced with the case that the gas itself is a second medium with an interface. Thus, the solvent within the nanobubble becomes the oxygen gas and the ions are at the interface of the second medium. If the Gouy-Chapman theory of double layers is used, then the Debye length for the oxygen medium will approach infinity,fodder sprouting system and the effect of ionic repulsion extends throughout the nanobubble, allowing the hydroxide ions to repel oxygen molecules away from the interface and keeping them within the nanobubble and enabling them to balance the external pressure.

The strength of the repulsive force would not be of the same level as the repulsion between, for example, two hydroxide ions, since the oxygen molecule is not charged, but the oxygen molecule also has two lone pairs in the valence shells of its constituent atoms, which can be repelled albeit much more weakly than an ion. If this conjecture is true, however, it will remain a valid mechanism for the inhibition of outward diffusion of electronegative gases from their respective nanobubbles. This hypothesis is supported by the work of Meegoda and co-workers, who report increasing size and zeta potential with increasing electronegativity of the gas contained within the nanobubble. They report the largest size and the highest zeta potential for nanobubbles composed of ozone, followed by oxygen, followed by air and lastly of nitrogen. Thus, it is reasonable to suppose that the nature of the bond formed is a stronger version of the standard hydrogen bond between water molecules, due to the dipole moment of the hydroxide ion. At the same time, however, the gas within the nanobubble also is repelled by the oxygen atom, the mechanism of which is by means of ion-lone pair repulsion, which would force the gas molecules to stay within the nanobubble, and hence severely limiting diffusion of the gas into the solvent. However, as recently reported by Ushikubo, nanobubbles of inert gases do possess similar lifetimes and are formed from helium, neon, and argon, and since the only intermolecular forces of note they experience are van der Waal’s forces of attraction, Lifshitz forces and dipole-dipole interactions, it can be assumed that these are also strong enough, and the gases sufficiently inert, for the same mechanism as well as the steric hindrance of the hydroxide ions to apply to the same case.Considering the formation of a 1 μm microbubble which eventually shrinks into a nanobubble, the number of ions available to it for stabilisation from the water it displaces upon formation, at pH 7, is approximately 33 ions, which if all the ions were adsorbed, does not agree with the zeta potentials reported by Takahashi et. al. for microbubbles of comparable size, which by equation is given to be approximately 495 ions.

It follows that the ions which are adsorbed diffuse toward the nanobubble surface from the surrounding bulk fluid, which can explain the apparent generation of free radicals observed by Takahashi et. al., since there is now a minuscule concentration difference present to drive the diffusion. The availability of hydroxide ions also depends on the pH, and at pH 7 it is thus possible for stable nanobubbles to form as is reported by Ushikubo, as well providing a mathematical treatment for their stabilization and the calculation of their surface charge. At lower pH, in the absence of other ions, the concentration of stabilized ions would be lower due to the lower availability of hydroxide ions and the increased time needed for them to diffuse to the surface of the nanobubble, allowing it more time to shrink. The dependence of the size of the bulk nanobubble on external pressure is given by equation . Of the external pressure, the proportion of the atmospheric pressure to the total value of the actual pressure, the rest being the pressure exerted by the fluid. However, the major component to the force contributing to the shrinkage of the nanobubble is the surface tension, which also increase with the size of the nanobubble. Thus, for higher external pressures and given that a limited amount of gas is dissolved in the fluid, the equation gives a trend of increasing nanobubble size with increasing external pressure. However, due to the limited amount of gas available, it is expected that the number of nanobubbles formed, i.e. concentration will decrease, while still giving higher particle size. This is confirmed by Tuziuti and co-workers through their observations of air nanobubbles in water. The temperature term appears only in the term that describes the internal pressure, causing a linear increase with temperature, not taking into account the increase in molecular motion due to heat, as well the increased energy of the surface ions. Thus, it also shows that the internal pressure will increase with the increase in temperature. This will, in turn, cause a reduction in the radius if all other terms are kept the same. Thus, we can say that given a limited amount of gas dissolved in the solvent, an increase in temperature will give smaller nanobubbles, but will also cause an increase in concentration of the nanobubbles in the solvent. It is also possible that zeta potentials may decrease, as thermally agitated hydroxide ions may be more susceptible to de-adsorption and may return to solution more easily. Conversely, as lower temperatures, larger bubbles may form, especially by the method of collapsing microbubbles, and larger numbers of hydroxide ions may be adsorbed on the surface of the nanobubble, giving longer lifetimes. Bulk nanobubbles are, in essence, minuscule voids of gas carried in a fluid medium,microgreen fodder system with the ability to carry objects of the appropriate nature, that is, positively charged for a length of time that is significant, if the nanobubble is left alone, yet is also controllable, since the bubbles can be made to collapse with ultrasonic vibration, or magnetic fields. The applications, then, seem to be limited only by how we can manipulate and design systems that make use of these properties for new technology in several fields. As mentioned before, thus far technology has made use of the uncontrolled collapse and generation of bulk nanobubbles, in the fields of hydroponics, pisciculture, shrimp breeding, and algal growth, while the property of emission of hydroxide ions during collapse has been applied to wastewater treatment. Here and there, there are indications of greater possibilities, as evidenced by research into their ability to remove microbial films from metals, to remove calcium carbonate and ferrous deposits from corroded metal, the use of hydrogen nanobubbles in gasoline to improve fuel efficiency, and the potential application for to serve as nucleation sites for crystals of dissolved salts.

The following sections elaborate on further applications which are possible in the near future. Proton exchange membrane fuel cells, are finding wide application in several fields due to the ease of their deployment, the low start-up times, and the convenience of their size and operating temperatures . However, significant limitations exist for their wider application, which can broadly be classed under the headings of catalysis, ohmic losses, activation losses, and mass transfer losses. The first of these is due to the rate of catalysis of the splitting of hydrogen, which cannot be pushed beyond a certain limit due to the constraints of temperature. But the larger issue is the cost of the catalyst itself, which is a combination of platinum nanoparticles and graphite powder, which provides the electrical conductivity. The inclusion of platinum presents a significant cost disadvantage, and while efforts are ongoing to reduce or replace platinum as a catalyst, these are still experimental and much research is ongoing in this field. The second limitation is due to ohmic losses, which accumulate due the proton exchange membranes, also termed the electrolyte, and can only be reduced by reducing the thickness of the membrane. Current popularly used membranes are usually made of Nafion, a sulphonate-grafted derivative of polytetrafluoroethylene marketed by DuPont, but experimental membranes include the use of graphene, aromatic polymers, and other similar materials which possess a high selective conductivity toward protons [ref]. However, beyond a certain thickness the membranes are unable to mechanically support themselves, and often mechanical failure of the membrane will cause a break in operations. The third limitation is due to the start-up conditions of the fuel cell, and are a matter of the mechanics of operation of the fuel cell itself. The last limitation is due to the transport of hydrogen and oxygen to the triple phase boundaries around the catalyst and the transport of water away from them, and is a significant concern for the operation and efficiency of PEMFCs. However, the current PEMFCs depend on gaseous hydrogen and oxygen, which are released from a compressed source and derived from air respectively. This necessitates a mechanically strong membrane and construction to resist the operating pressures. However, the inclusion of the gas as a nanobubble dissolved in water presents new possibilities, used in combination with microfluidic technology. It becomes possible to also replace both membranes and catalysts with materials that have been hitherto discarded fro being too mechanically weak, such as graphene, and the possibility of using graphene as a combined catalyst and proton exchange membrane, as nanobubbles of hydrogen and air, dissolved in water, to act as the reservoirs for the fuel and oxidant. Such as system would operate on the basis that nanobubbles are negatively charged, and would hence be attracted to the graphene through which current would be passed in order to activate the process. Air and hydrogen nanobubbles would be separated by the graphene membrane, and be adsorbed to opposite sides of it. The graphene membrane would also have a potential difference applied across it in the plane of the graphene layer. This would, in turn, permit the hydrogen to be catalyzed to protons [ref], and hence be conducted across the graphene [ref], allowing it react with the oxygen to form more water, which would be carriedaway with the flow. Microfluidic bipolar plates would enable the construction of such a device, and such fuel cells could become the future source of energy for several applications. The advantages of such a system would be numerous. Firstly, graphene is far cheaper than platinum, and can be used as a catalyst of almost comparable quality, in addition to also being the conductor for the removal of electrons released during catalysis. Secondly, the thickness of a graphene sheet is in the range of nanometers, which would mean that ohmic losses would, quite possibly, be nearly eliminated.

Metal localization and elemental associations in plants were investigated with X-ray and electron microscopies and X-ray microtomography.Particular emphasis was placed on the phenomenon of ‘simultaneous hyper accumulation’and its relationship to metal co-tolerance.Metal localization and elemental associations in A.murale were investigated with SXRF, CMT, SEM-EDS and XAS.Two-dimensional SXRF images of A.murale leaves revealed a distinctive localization pattern for Co relative to Ni and Zn.The Ni distribution in leaves was essentially uniform, although the fluorescence intensity was slightly elevated in the midrib region.A uniform Ni distribution was anticipated for two-dimensional leaf images because Alyssum sequesters Ni within the epidermal layers.The Ni distributions were comparable for both young and old leaves, and Ni localization was not altered in A.murale plants exposed to mixed-metal systems.The Zn distribution in A.murale leaves appeared similar to that of Ni; however, elevated fluorescence intensity in the midrib region was more evident in SXRF images of Zn than Ni because the Zn fluorescence signal was not dominated by the epidermal cell layers.Zinc was not hyper accumulated by A.murale and would not be preferentially compartmentalized in epidermal This issue.The Zn distributions were comparable for both young and old leaves.In contrast to Ni and Zn distributions, Co was preferentially localized at the tips and margins of A.murale leaves.Similar Co localization patterns have been reported for various non accumulator plants investigated using autoradiography.Cobalt localization in A.murale was consistent for young and old leaves, but Co enrichment near the leaf tips was more common on older leaves than on younger leaves.

Spatial associations in leaves were visualized by combining fluorescence data from individual elements into a multi-color image and plotting a line profile.An SXRF image of Co, Ni and Ca localization in an A.murale leaf revealed a color gradient near the leaf tip as a result of the uniform Ni distribution and the irregular Co distribution in the leaf.A line profile generated for a segment from the leaf center towards the leaf tip showed that the substantial increase in the Co signal coincided with a subtle decrease in the Ni signal; Co and Ni were not preferentially co-localized in leaves.Spatial associations of Co with Mn and S coincided with statistically significant correlations in bulk shoot concentrations.Cobalt-treated plants had the highest shoot concentrations of Mn and Sand the highest S TFs.SXRF images of Co and Mn in A.murale leaves revealed co-localization of these elements at leaf trichomes.A Mn-rich zone surrounds the base of A.murale trichomes and could sequester Ni or Co.SXRF images of S and Co in A.murale leaves indicated co-localization of these elements near the leaf tips/margins.S and Co were spatially correlated,hydroponic bucket but preliminary XAS data did not indicate a direct chemical association.F-CMT images of A.murale fine-root segments revealed distinctive metal partitioning patterns as a function of distance from the root tip.Nickel, Co and Fe were localized in the root vasculature0.5 mm from the apex; the root tip appeared most active in the absorption of these metals.In the root segment 6 mm from the apex, these metals were predominantly confined to the epidermis, signifying sorption to surface functional groups, mucilage, bacterial bio-films, or metal oxide plaques on the root surface.Additionally, metal enrichment was discernible in the vasculature but was absent from the pith.Similar localization patterns were reported by Fellows et al., who investigated europiumup take in living roots of Avena sativa and observed maximal fluorescence intensities in the apical This issue at the root tip and within the zone of root maturation , whereas elevated fluorescence from highly differentiated root This issue was observed at the epidermis.Metal localization patterns in the A.murale root segment 3 mm from the apex were intermediate to those observed at 0.5 and 6 mm; elevated Ni and Co levels were observed in the vascular cylinder and at the root epidermis.

Zinc and Mn localization patterns in A.murale roots were distinct from Ni, Co and Fe and from one another.Zinc was predominantly localized in isolated domains within the root at 0.5, 3 and 6 mm from the apex, whereas Mn was sporadically localized at the epidermis.A separate experiment conducted with A.murale plants exposed to a Co-enriched nutrient solution verified that the Co localization phenomenon observed in the metal interaction study was not a result of simultaneous hyper accumulation; thus, Co localization had not been altered in plants exposed to elevated Ni and Zn concentrations.Cobalt accumulated by A.murale was ultimately deposited on leaf surfaces near the tips/margins.Identical Co localization patterns were observed for Co-treated A.corsicum and A.troodii , suggesting similar mechanisms exist in other Ni hyper accumulator species of Alyssum.Cobalt on the surface of leaves was visible by optical microscopy.BSE images showed a coating on the leaf surface near the tips/margins, and X-ray micro-analysis indicated that the coating was a Co-rich phase.The electron microprobe beam penetrated only a few microns into the sample and thus the recorded signals were emitted from the leaf surface or the cuticle layer; a comparison of EDS spectra from the Co-rich and bulk-leaf regions further supports the finding of a Co-rich phase deposited on the exterior of leaves.Leaf images from the optical microscope and SEM corroborate the Co distribution observed with SXRF.Micro-XAS spectra collected from hydrated A.murale leaves revealed that the oxidation state of Co in plants was Co; artificial Co oxidation was not observed in this study, but Co can result from sample alteration in the X-ray beam and by ligand stabilization with multi-dentate amine ligands.Spectra collected at the Co-rich region near the tip showed striking differences from spectra collected at the bulk-leaf region.The Co k3-weighted χ spectrum from the Co-rich region had a beat pattern near 5 Å–1 and a split oscillation between 7 and 8.5 Å–1, whereas the χ spectrum from the bulk-leaf region did not have these characteristic structural features ; spectra with several frequencies are indicative of a long-range ordered binding environment such as that in a mineral structure, whereas spectra dominated by a single frequency are indicative of a short-range ordered environment.

An evaluation of the Co-binding environment for Co-rich spot 1 provided distances and numbers of Co–Co pairs that are characteristic of an edge-sharing layered framework.A strong second-shell feature and a third metal shell at about twice this distance was present in the Fourier transform from the Co-rich region, but was absent in the FT from the bulk-leaf region.Cobalt accumulated by A.murale formed Co-rich mineral precipitate on the leaf surface.Detailed characterization of the Co phase formed on leaves and the ligands involved with Co transport and detoxification in Alyssum are beyond the scope of this study but will be reported in a future publication.DA-CMT images of a hydrated leaf from Co-treated A.murale revealed a lack of metal enrichment in epidermal This issue.Cobalt near leaf tips was localized predominantly on the leaf exterior.In addition to Co enrichment on the leaf surface, DA-CMT slices 20–30 µm below the leaf tip showed Co localized in isolated regions inside the leaf associated with the vascular system.Cobalt was consistently observed on the exterior of leaves, but deposition on leaf surfaces was less prevalent at greater distances from the leaf tip.Images from several hundreds to thousands of microns below the leaf tip showed a predominance of Co distributed around the leaf ground This issue ; the distribution between cells was interpreted as Co in the leaf apoplasm.Cobalt enrichment was more prevalent in the region composed of spongy mesophyll than palisade mesophyll; spongy mesophyll contains fewer chloroplasts and many intercellular spaces linked to the outside via stomata.Regions with elevated Co or Ni were frequently observed in proximity to leaf trichomes; however, Co enrichment was associated with the trichome structures on the leaf surface , whereas Ni was associated with the basal portion of trichomes.DA-CMT images of a hydrated leaf from Nitreated A.murale showed metal enrichment in the epidermis ; this result is consistent with other studies of Ni compartmentalization in Alyssum, which have shown Ni sequestration in epidermal cell vacuoles.In addition to epidermal localization, Ni was observed within leaf ground This issue.In contrast to Co, a fraction of Ni in ground This issue occupied the same spaces as mesophyll cells,stackable planters and this Ni distribution was interpreted as partial metal enrichment of mesophyll This issue.At a fundamental level, mechanisms of metal tolerance and hyper accumulation in Alyssum remain poorly understood.A.murale hyper accumulates Ni and Co, but Zn is not accumulated to abnormal levels.Elevated Co or Zn concentrations do not alter Ni accumulation or localization, and thus A.murale can be used to recover Ni from most metal-enriched soils containing these metal co-contaminants.A.murale is more tolerant to Ni than Co; nickel tolerance is attained via epidermal compartmentalization.A.murale does not sequester Co in epidermal cells; Co in the xylem or leaf apoplasm is excreted from leaves and subsequently sequestered on leaf surfaces as sparingly soluble precipitate.Therefore, the specialized biochemical processes linked to Ni tolerance in A.murale do not confer tolerance to Co.Cobalt is ultimately deposited at the leaf tips/margins, whereas Ni is sequestered in epidermal cells.Vacuolar sequestration is a key strategy for metal tolerance because leaf epidermal cells provide an effective sink for the accumulated metal.Metal concentrations exceeding 0.35 M were measured in epidermal cell vacuoles of a Zn hyperaccumulator.McNear et al.imaged Niin an A.murale leaf and reported, in addition to epidermal enrichment, elevated Ni accumulation on/in the leaf tip; furthermore, it was suggested that leaf tips function as an additional reservoir for Ni when concentrations exceed the finite capacity of cell vacuoles.However, McNear et al.did not consider that their leaf tip was curled such that the epidermal layers near the tip were oriented parallel to the incident X-ray beam regardless of the rotation angle; thus, the beam exclusively ‘sampled’ metal-enriched epidermal This issue in this region, leading to the erroneous impression of elevated Ni on/in the leaf tip.Upper slices from the DA-CMT movie revealed a lack of Ni enrichment near the point of the leaf ; nonetheless, these slices afford a rare glimpse of Ni localization across leaf epidermal This issue.

Clear differences between Ni and Co localization suggest that A.murale uses a different metal sequestration mechanism for Co than for Ni.Whereas Ni is redistributed to leaf epidermal cells and subsequently transported across the tonoplast for long-term sequestration in vacuoles , Co does not have an efficient route of entry into epidermal cells.Considering the serpentine origin of Alyssum, a cellular-level tolerance mechanism for Co may not have been naturally selected because the Ni : Co ratio in these soils is relatively large.Additionally, Co is typically retained more strongly by the soil components than Ni and thereby is less phytoavailable.Therefore, mechanisms other than vacuolar sequestration must be operating to cope with the elevated Co concentrations in plant This issue.A.murale leaves apparently lack the transport system needed to sequester Co in epidermal cells and thus accumulated Co resides in the xylem and the leaf apoplasm.Mass flow and diffusion gradients in the apoplasm will cause Co to redistribute in leaves.Water loss via transpiration will move Co towards the leaf surfaces and margins where transpiration is maximal.Cobalt principally follows the transpiration stream and results in Co enrichment at leaf tips/margins.When transpiration is low , root pressure will cause exudation of xylem sap from the ventilation poreslocated at the points of the leaf margin to where veins extend.Guttation fluids of plants from ultramafic soils have been reported to contain elevated metal concentrations.For example, Minuartia verna grown in metal-contaminated soil accumulated Cu and Zn in leaves and excreted these metals onto leaf surfaces via hydathodes.Cobalt accumulated by A.murale is ultimately deposited on leaf surfaces.Cobalt enrichment on leaf surfaces is evident from X-ray microtomography , SEM-EDS, optical microscopy and visual inspection of Co-treated A.murale leaves.Similar observations were made by Vergnano & Hunter , who noted red-colored leaf tips on plants exposed to Co-enriched nutrient solution.Cobalt deposition at A.murale leaf tips is most prevalent on older leaves.For many plant species, older leaves have the highest concentrations of elements such as Co because evapotranspiration continues as long as the leaf is attached to the plant.Deposition of sparingly soluble Co species near the tips/margins of A.murale leaves is corroborated by in situ microspectroscopic analyses , revealing that weight percent Co is sequestered on leaf surfaces and forms Co-rich mineral precipitate.Metal-tolerant Arabidopsis halleri , grown in a Zn- and Cu-contaminated soil, had mixed-metal precipitate on leaf surfaces.Elevated regions of Ni and Co occur consistently in proximity to the trichomes on A.murale leaves, and the Mn-rich zone surrounding the base of trichomes may be partially responsible for this metal enrichment.Simultaneous hyperaccumulation of Mn and Ni occurred in the basal compartment of the nonglandular trichomes on Alyssum leaves.

OsPRR95 corresponds to Arabidopsis PRR, AtPRR5 or AtPRR9.A report has revealed that triple mutant prr 9–11 prr 7–10 prr 5–10 Arabidopsis exhibit better salt, drought and cold tolerance than wild type, and thus suggested that PRR5, PRR7 and PRR9 are involved in the diurnal cold stress-initiating stress response by mediating the cyclic expression of stress response genes, including DREB1/CBF.Additionally, Mesembryanthemum crystallinumCSP1, which is a class of pseudo-response regulator-like proteins, co-localizes with calcium-dependent protein kinasein the nucleus of NaCl-stressed ice plants, suggesting that it may be regulated by McCDPK1 through reversible phosphorylation.According to the MSU7 database, LOC_Os05g38710, the novel CaM1 target, is annotated as lipin, and the mRNA sequence of LOC_Os05g38710 is annotated as phosphatidate phosphatase.A report has demonstrated that the N- and C-terminal regions of mammalian lipin protein share sequence similarity to yeast PAH1.Phosphatidate phosphatase is the enzyme that converts phosphatidic acid to diacylglycerol and Pi.In Phaseolus vulgaris cotyledons, phosphatidate phosphatase is stimulated by Ca2+ or CaM with Ca2+, and a possible role of Ca2+-second-messenger in membrane-lipid degradation initiation has been suggested.Therefore,hydroponic nft system its identification as a CaM-interacting protein herein suggests that Ca2+/CaM stimulates phosphatidate phosphatase via direct binding.

By protein functional association analysis of each of these CIPs, the GO terms enriched in each set of resulting associated proteins that matched those from OsCam1–1 affected salt-responsive DEGs are presented in Fig.10.Matched GO terms revealed interacting protein candidates that potentially regulate various cellular processes represented by each enriched GO term of the OsCam1–1 affected salt-responsive DEGs.Cadmium , one of the most toxic heavy metals for both plants and humans, accumulates in the human body through the food chain and causes serious health problems.In recent years, the accumulation of Cd in rice grains has become an important agricultural problem in Japan because the Cd content of rice grains sometimes exceeds the limit proposed by the Codex Alimentarius Commission.In addition, Cd intake from rice accounts for about one-half of the intake from food in Japan according to the National Institute of Health Sciences.Therefore, new technologies for reducing the Cd content in rice grains are urgently required.Although the mechanism underlying the uptake and translocation of Cd in plants is not completely understood, some irontransporters, such as OsIRT1 and OsIRT2, are reported to uptake Cd as well as Fe.AtNramp3 and AtNramp4 from Arabidopsis, which belong to the Nramp metal transporter family, function as Fe and Cd transporters.Rice has seven Nramp genes , and OsNramp1 has been reported to function as an Fe transporter.In this study, we investigated the possibility that OsNramp1 also transports Cd.

Full-length OsNramp1 was amplified by RT-PCR using total RNA prepared from hydroponically grown rice shoots.The subcellular localization of OsNramp1 was determined by monitoring the expression of an OsNramp1::GFP fusion protein in onion epidermal cells transformed by DNA particle bombardment.GFP, contained in the vector pH7FWG2, was fused to the 3’-terminus of OsNramp1 using the Gateway system.To test the growth of OsNramp1-expressing yeast, full-length OsNramp1 cDNA was inserted into the expression vector pYH23.The construct was then introduced into yeast strain ycf1 using the lithium acetate method.Ycf1 lacks the YCF1 transporter, which functions in the compartmentation of Cd into vacuoles.Yeast cells transformed with empty pYH23 were used as a control.The transformed yeast cells were grown in synthetic defined medium and spotted onto SD agar containing CdCl2.To measure the metal content of the OsNramp1-overexpressing rice, plants were grown in Cd-contaminated soil for 6 months in a greenhouse.Harvested leaf blades were dried at 70˚C for 1 week.Sample digestion and measurement of the metal content were performed as described previously , except that the digestion time and temperature were changed to 2 h at 230˚C.Manned space exploration missions deploy technologies and products that mitigate crew-safety concerns and that assist with mission accomplishment.These technologies are continuously evaluated for relevance and cost, a term that accounts for launch mass, drawn power, volumetric size, useful product life, astronaut utility, etc.This evaluation is important, because space missions are inherently expensive; every unit mass of payload that is launched into space necessitates the launch of an additional 99 units of mass.Hence, there is an interest in novel technologies that simultaneously decrease cost, reduce risk and increase the probability of mission success.Typically, the cost of these new technologies is reduced through in situ resource utilization which consists of harnessing materials located at a mission’s destination.This paper investigates how current biological techniques and future synthetic biology progress can meet several of the above-mentioned needs.The work reviews existing biological processes to demonstrate that they already constitute a competitive yet non-traditional technology that is capable of processing volatiles and waste resources readily available on two representative space missions in a way that reduces the launch mass of propellant, food and raw material for three-dimensional printing, and also overcomes the decreased product shelf-life of a common therapeutic.

The paper employs these reviewed processes in designs for natural and artificially enhanced biological manufacturing strategies that can be leveraged to saThisfy space input availability and output-desirability constraints.The work then analyses methodological feasibility, technique versatility and the costs and yields of feed stocks and constituents, and compares possible future ‘space synthetic biology’ advances to other new aerospace technologies.Although a novel technique, synthetic biology has already been tapped for its potential to eliminate plastic waste, enrich food, monitor pollution and chemicals and be an ISRU tool.This paper furthers these forays and widens the scope of the technology by indicating its capacity for extensive product applicability in space despite the severe input limitations imposed by the space environment.Of these candidate missions, the fourth warrants an analysis of applicable bio-production techniques for two reasons: a lengthy total time spent on board a spacecraft that is comparable to the long residence time of the asteroid-investigation mission, and an extremely lengthy stay on Mars that makes it vital to explore all technologies that could reduce risk, decrease launch mass and manufacture products with a short shelf-life.Given its possible precursor status for the Martian mission, the lunar mission will also be examined for bio-manufacturing benefits.Representative values of the masses of crew-produced wastes, which serve as potential resources for biology based designs, are listed in table 1 for the previously stated Martian- and lunar-manned space exploration missions.Further inputs for these two missions can be drawn from the Mars atmosphere or the permanently shadowed craters on the south pole of the Moon, respectively,and the Martian soil or lunar regolith, respectively.

The tables suggest that carbon dioxide and nitrogen are somewhat plentiful resources for biology applications over the course of a Mars voyage and stay.However, these resources are significantly scarcer on a Moon mission.Yet, if large enough excavators and bioreactors are deployed , there should be enough of these resources extracted to test the viability of biological techniques prior to a Mars voyage.Hydrogen and oxygen may also be available for bio-manufacturing on both missions as a result of the electrolysis of polar water, but it is expected that water availability will be reduced given its priority to support crew life.If required, hydrogen can be transported to Mars and also stored until use, but this process is considered somewhat difficult and problematic.On the Moon however, hydrogen is already present.Oxygen may also be harvested from the Martian soil or the lunar regolith with post-excavation processing.Hence, bio-production applications for Mars and the Moon need to take as inputs: carbon dioxide, nitrogen , hydrogen and oxygen,nft channel ordered here by their availability.This resource set of elements and simple compounds can conceivably support biological systems because its constituent elements form a subset of the main elements required for life, namely carbon, hydrogen, nitrogen, oxygen, phosphorus and sulfur.The latter two elements are not readily available on both Martian and lunar missions, although sulfur is present on the Moon alone.Soil-based metal resources from Mars and regolith-derived metal resources from the Moon are not considered suitable for biology application in this paper.A cost-based ordering of the items required for a Mars or Moon mission cannot be compiled, because cargo manifests for these future missions are still in flux.Moreover, at this early stage of mission planning, the form of the cost metric itself and the relative weightings it contains is somewhat unclear.This is attributable to changing technology readiness-levels and disparate evaluation criteria.Nevertheless, four targets quantifiably stand out for biological production.Fuel, for instance, is currently projected to be ‘about two-thirds of the mass on an Earth-to-Mars-to Earth mission cost-effective [extraterrestrial]-produced propellant could decrease the mass that must be lifted from Earth by a factor of two to three’.Food is another necessary target, as evidenced by crew meals constituting the bulk of a recent supply mission to the International Space Station.Bio-polymers are a third target, because plastics are included in the list of feed stock materials that can be used for three dimensional printing.The three-dimensional printing of structures to manufacture a spacecraft in space can decrease roughly 30% of the craft’s launch mass by reducing the supporting structural material that is required, and additive manufacturing can also reduce the launch mass cost associated with storing a multitude of spacecraft spare parts.Because the 30% number presumes a launch of necessary printer media, the extraterrestrial production of raw material for three dimensional printing, e.g.bio-polymers, can achieve even greater mass reductions.Further savings can also be realized by deploying additive manufacturing for other purposes, such as the construction of habitats, rocket engine parts, sample containers, spacecraft electronic platforms, etc.Lastly, the accelerated expiration of pharmaceuticals induced by space radiation necessitates the on-demand synthetic manufacture of such pharmaceuticals on long-duration missions.In this paper, a versatile drug to treat infection and pain symptoms, e.g.aspirin, acetaminophen, etc., is targeted for biosynthesis.It is envisioned that this drug will be manufactured when desired by astronauts using bacteria that are activated from a frozen state.The bacteria will not themselves ‘expire’ from space radiation because of storage in a small, lead-lined container while inactive; bacterial spores and rock colonizing eukaryotes can survive with little protection in space for between 1.5 and 6 years.

Quality control of astronaut-activated bacteria can be performed through portable gene sequencers that are in development, and that are already being contemplated for use in space exploration.The choice of four targets outlined in this section is further justified by their inclusion in the list of needs presented in §1 for which NASA seeks promising new technologies.Sections 3–6 confirm the feasibility and benefits of producing each of these desirable endpoints with contemporary biological techniques.Hence, the design problem that is tackled in this paper: design biology processes to go from the inputs listed in the left column of figure 2 to the outputs listed in the right column of figure 2, using the fewest number of intermediates,organisms and steps, with the greatest possible commonality of such intermediates, organisms and steps, and with the goal of substantially reducing launch mass and increasing product shelf-life.The availability of only a few input elements and simple compounds coupled with the predefined desirability of various output products constrain space biology designs.The current state of the technology requires design options to include, as a first step, those organisms that already use the same resources on the Earth.Thereafter, the outputs of these organisms can serve as inputs to other organisms.The yields of such modular designs can be analysed and then improved upon with bioengineering and genetic modification techniques.As synthetic biology matures over the coming decades, it may be possible to build designer organisms from scratch that directly manufacture the desired products efficiently.Because carbon dioxide and nitrogen compounds are the dominant available resources, organisms that harness these resources and the yields and efficiencies at which they do so are of prime importance.Further, the outputs of these organisms will be useful as either the desirable targets of figure 2 or as feed stock intermediates to obtain these targets.Thus, we summarize in electronic supplementary material, table S1 the mechanisms of action and the outputs produced by organisms that take in carbon dioxide, as detailed by.Electronic supplementary material, table S2 provides a similar summary of organisms that use and produce various nitrogen compounds; these organisms also play a role in the microbial nitrogen cycle.A greedy design approach involves employing the lowest-energy carbon dioxide fixation process from electronic supplementary material, table S1, which exists in methanogens and acetogens.Conveniently, the responsible pathway in these organisms, the Wood –Ljungdahl pathway, requires the input of hydrogen and the presence of anoxic conditions.

These toxic elements can be bio-available to terrestrial and aquatic organisms, including crop plants, and could be further bio-accumulated via the food chain causing damage to humans.Since these metals cannot be degraded, current remediation approaches include excavation or capping, with a very high cost and damage to ecosystems.In many cases, these options are not economically feasible, when the contamination is very wide-spread as is the case of many contaminated farmlands and river beds.Compared to ex-situ remediation technologies, in-situ decontamination does not require excavation and transport of contaminated sediment and soil to off-site treatment or disposal facilities, thus it is generally a more practical and economical approach.Conventional insitu soil remediation technologies used for industrial sites contaminated with heavy metals include soil washing/flushing,nft hydroponic chemical immobilization, electro kinetic extraction, and phytoremediation.While these technologies may be appropriate for small scale remediation, they quickly become cost-prohibitive at larger scales.The cost of phytoremediation does not increase much with scale, but the accumulation of metals in the plants presents ecological risks and an eventual disposal cost.

Capping sediments essentially destroys habitat, and the capping may be removed during a large storm event, reexposing the contaminated media.Thus, there is an urgent need to find better methods to sequester heavy metals to reduce human and ecological risk and ensure better food security.Chelating agents, for instance, ethylene diaminetetraacetic acid , are widely used as extractive agents for heavy metals decontamination.Due to its strong metal chelating ability and low cost, EDTA has been used as a metal extraction agent in soil washing.However, soil washing can result in unintended mobilization of metals and other pollutants that can be more easily transported by groundwater, and EDTA itself can pose issues as secondary pollution.Thus, a suitable supporting material for EDTA and other chelating agents would minimize the potential unintended environmental implications.Previously we developed super-paramagnetic EDTA-functionalized nanoparticle adsorbents for water treatment, which were shown to remove a wide range of metal ions with high sorption capacity.To date, most nanoscale adsorbents have been applied to the decontamination of aquatic systems, while very few studies have investigated sediment and soil remediation.We have also demonstrated that nanoparticles can readily transport vertically into deeper soil, driven by gravity.Thus, we set out to develop a new type of high density nanoscale adsorbent, which can remove heavy metal ions during its downward transport, significantly reducing their bioavailability.For this study, we selected tungsten oxidenanoparticlesas the dense core, which is a relatively low-cost material with high density and low ecotoxicity, to develop the dense nanocomposites that can transport vertically through the porous medium.

We first report on the synthesis of EDTA-based Ligand DNPs.We then demonstrate the sorption capacity of Ligand DNPs for Cd2+ and Pb2+.Next, we evaluate the removal efficiency of Ligand DNPs for Cd2+ and Pb2+ in two different natural porous matrices.Finally, we report on the insitu remediation performance of Ligand DNPs for Cd2+ and Pb2+ during gravity-driven vertical transport in these media.The results demonstrate that Ligand DNPs can be applied for effective in-situ metal decontamination from soils and sediments.Pyridine and toluene were purchased from Alfa Aesar.triethoxysilanewas purchased from Sigma-Aldrich.Cadmium chloride anhydrous, lead chloride, ethylenediaminetetraacetic acid , and tris aminomethane were purchased from Fisher Scientific.Diethyl ether and sodium dihydrogen phosphate were purchased from Acros Organics.Standard Suwannee River natural organic matter was obtained from the International Humic Substances Society.A NOM stock solution was prepared by mixing a known amount of NOM with DIwater for 24 h.The pH of the stock solutions was adjusted to 8 with 0.1 M and 0.01 M NaOH and HCl.All chemicals were used as received, without further purification.All solutions were prepared with deionized water from a Barnstead NANOpure Diamond water purification system.Similar to our previous synthesis strategies, the core-shell Ligand DNPs were prepared in two steps.The WO3 nanoparticles were coated with APTES to form a silane polymer layer via hydrolysis reaction.Then, the surface was modified with EDTA by forming the amide bonds between the EDTA’s carboxylic acid groups and APTES coating’s amino groups.

WO3 nanoparticles were dispersed into 40 mL toluene in a flask.After mixing well, 0.4 mL APTES was added to attach an amino group to the WO3 particles.Then the flask was connected to a reflux system , which was then rotated at 30 rpmin a water bath at 90˚C, and refluxed for 2 h.After the solution cooled to room temperature , 2 mM EDTA and 60 mL pyridine were added.The mixture was again rotated at 30 rpm in a water bath at 90˚C in the reflux system for 2 h.After the solution cooled down to room temperature, 100 mL sodium bicarbonate was added to adjust pH to 8.0.Deionized water was used to rinse the particles twice and then decanted.The same rinsing procedure was performed twice with ethanol and then diethyl ether.The particles were dried at room temperature for 24 h, and stored in a capped bottle prior to use.Transmission electron microscopy images were obtained using a JEOL 1230 Transmission Electron Microscope operated at 80 kV.Scanning electron microscopy studies were performed on a FEI XL40 Sirion FEG Digital Scanning Microscope.The surface area and pore volume of Ligand DNPs were determined using a Micromeritics 3Flex Porosimeter.The functional groups of the Ligand DNPs were detected using a Fourier transform infrared spectrometer on a Nicolet iS 10 FT-IR Spectrometer.Two representative soils were used in this study, as examples of the application of Ligand DNPs to treat contaminated porous media.A grassland soil was collected from a flat, well drained grassy area at the Sedgwick Reserve in Santa Ynez, CA , and farmland soil was collected from a fallow field at an organic farm in Carpinteria, CA.The permit for collecting soil samples was authorized by Brenda Juarez.Soil properties can be found in the Supporting Information , in S1 Table in S1 File.Soils were air dried, sieved through a 2 mm mesh, and stored at 4˚C until use.The physicochemical properties of the sieved soil samples, including pH, texture, saturation percent, soluble salts, cation exchange capacity , conductivity, organic content, bulk density, and exchangeable NH4, NO3, K, and PO4, were characterized in our previous study, and available in the SI, shown as S1 Table in S1 File.Total W, Cd, and Pb concentrations of each soil were measured by digesting ~0.3 g soil samples in 10 mL 1:3 HNO3: HCl at 200˚C for 1.5 h in a microwave digestion system , followed by analysis via inductively coupled plasma mass spectroscopy.In order to simulate Cd or Pb contamination, 20 g of each type of soil were placed in 50 mL conical test tubes, mixed with 40 mL of 10 mg/L Cd2+ or Pb2+ solution on an end-over-end shaker with a speed of 70 rpm at room temperature for 7 days to ensure sufficient equilibration time.Then, the tubes were centrifuged at 10,000 rpm for 20 min to separate soil and the residual Cd2+ or Pb2+ solution, and the supernatant was collected for residual Cd2+ or Pb2+ concentration determination by ICP-MS.Soil saturated with Cd2+ or Pb2+ was preserved at 4˚C for the sorption studies.Air dried soil saturated with Cd2+ or Pb2+ was digested with 1:3 HNO3:HCl at 200˚C for 1.5 h in a microwave digestion system,nft system then analyzed via ICP-MS to determine the total Cd or Pb content.For batch sorption experiments, 20.0 mg of Ligand DNPs were first dispersed in 5 mL DI water, then mixed with 10 g of Cd2+ or Pb2+ contaminated soil , in 50 mL conical tubes at pH = 7.Then, these tubes were mixed on the end-over-end system with a speed of 70 rpm at room temperature for 7 days, to ensure sufficient equilibration time.Adsorption kinetics studies were carried out at the previously stated conditions but for a set amount of time, varying from 6-h, to 12-h, 24-h, 2-day, 3-day, and 7-day.The dosage of Ligand DNPs ranged from 3, to 5, 10, 15 and 20 mg to study the adsorption isotherms at pH 7.To evaluate the potential effect of NOM on the remediation performance of Ligand DNPs, the adsorption isotherms were conducted by first dispersing 3, 5, 10, 15 or 20 mg of Ligand DNPs in 5 mL NOM solution , then mixing with 10 g of each type of contaminated saturated soil for 7 days.

After mixing the Ligand DNPs with contaminated saturated soil for the specified time, the supernatant and soil were separated by centrifugation.Due to the high density, the immobilized heavy metals adsorbed by Ligand DNPs would be spun down.The treated soil was collected from the top layer to avoid the possible heavy metal binding Ligand DNPs, and then dried in an oven at 60˚C for 72 h, then digested for total Cd or Pb content analysis via ICP-MS.All experiments were conducted at ambient temperature.To investigate the decontamination capability of Ligand DNPs during gravity-driven transport through soil saturated with Cd2+ or Pb2+, first the contaminated soil was packed into 15 mL conical tubes.An opening with a diameter of 2 cm was made at the bottom of the tubes as the outlet of the system.Suspensions of 20, 40, 60, 80 and 100 mg of Ligand DNPs were dispersed in 5 mL DI water, respectively, and then evenly applied onto the top of each conical tube.After applying the Ligand DNP suspension and allowing the suspension to drip out, the soil columns were placed in horizontal position and air dried overnight then oven dried at 60˚C for 72 h.The dried soil was carefully removed from the conical tube in 3 cm segments, labeled top, middle, and bottom section.Sub-samples were weighed, then digested for Cd or Pb content analysis.The Ligand DNPs were mixed with the two Cd or Pb contaminated soils at pH 7 for 7 days to evaluate their isothermal sorption performance.As shown in Fig 2, the removal efficiency gradually increased as the dosage of Ligand DNPs increased, since this increases the number of active sites.The Ligand DNPs exhibited higher Cd or Pb removal efficiency when applied to farmland soil compared to grassland soil.As shown in S1 Table in S1 File, grassland and farmland soil exhibited significantly different physicochemical characteristics, particularly the organic and ionic concentrations.The CECof grassland soilis considerably higher than the CEC of farmland soil , which results in higher retention of cations, including Cd2+ and Pb2+, leading to much lower desorption from the contaminated soil to the soil-water interface.In addition, as shown in S1 Table in S1 File, the electrical conductivity was 142.1 μm/cm for farmland soil and 18.9 μm/cm for grassland soil, indicating a higher concentration of ionsin the leachate of farmland soil compared to grassland soil.Thus, there can be a higher soil-water interface concentration of Cd2+ or Pb2+ in farmland soil compared to grassland soil, which increases the accessibility and interaction between the active sites of Ligand DNPs and Cd2+ or Pb2+.In both Cd2+ and Pb2+ contaminated soil remediation scenarios, Ligand DNPs achieved higher removal efficiencies on contaminated farmland soil than grassland soil.Ligand DNPs exhibited higher removal efficiencies of Pb2+ from both farmland and grassland soils compared to Cd2+, which agrees with the sequence of their EDTA complex formation constants : 18.04 for Pb2+ and 16.46 for Cd2+.It suggests that the complexation between Pb2+ or Cd2+ and the EDTA-functionalized surface is the dominant removal mechanism.The time-dependent removal of Pb2+ or Cd2+ by Ligand DNPs in contaminated soil was evaluated in batch studies, as shown in Fig 3.Ligand DNPs showed quick removal of Pb2+ in contaminated farmland soils, with over 75% of maximum removal efficiency achieved in the first 6 hours, and a minor increase from 1 to 7 days, when Pb2+ in contaminated grassland soils were treated with Ligand DNPs.Thus, the sorption equilibrium of bio-available Pb2+ with Ligand DNPs can be rapidly reached within 1–2 days, with mixing, in both farmland and grassland soils.Similar removal performance was observed when applying Ligand DNPs for Cd2+ soil remediation, as over 70% of the maximum removal efficiency could be achieved in the first 6 hours for both soils.However, it took up to 3 days of mixing to achieve Cd2+ sorption equilibrium , suggesting Ligand DNPs exhibit a faster removal rate for Pb2+ than Cd2+, which is due to the stronger binding constant with EDTA.NOM concentration in the soil typically ranges from 0.5% to 5%.In the current study, the original grassland soil had a higher organic content than the farmland soil , showing a relatively wide range of organic content.In addition, soluble NOM can interfere with, or compete for, metal cation sorption.In order to evaluate the effect of soluble NOM on the removal of Pb2+ or Cd2+ using Ligand DNPs, an extra 1% NOM was spiked into the Pb2+ or Cd2+ contaminated soils.

Taken together, these data indicate that MsLEC1 and MsLEC2 genes, as well as the orthologous gene MaLEC, are expressed in the youngest cells of indeterminate nodules of both alfalfa and white sweetclover. Moreover, the genes that encode these soluble lectins are also expressed in root tips. MsENOD40 gene expression. We hypothesized that expressing the lectin transgenes would not only have an effect on overall nodule phenotype, but also on downstream nodulin gene expression. We analyzed the expression of MsENOD40, which is expressed within a few hours after rhizobial inoculation . Similar to MsLEC1, MsENOD40 is expressed in the youngest cells of indeterminate nodules and also in root meristems . Accumulation of MsENOD40 RNA was significantly higher in RNA isolated from LEC1AS and LEC2AS nodules than in RNA derived from vector controls, in spite of high variability . The highest mean MsENOD40 RNA accumulation was found for LEC1AS plants, which exhibited the severest symbiotic abnormalities.Lectin concentration is very low in the roots of Phaesolus vulgaris in the absence of Rhizobium phaseoli, but infection causes an increase in the quantity of lectin in the roots . However, lectin levels remain relatively constant in pea roots upon inoculation with R. leguminosarum bv. viciae . Thus,flood and drain table the significance of changes or lack thereof in the regulation of lectin expression levels during nodulation of legume roots is unclear.

Earlier, we found that MsLEC2 mRNA accumulated in uninoculated alfalfa roots , and here, we report that its accumulation increased in the roots of nodulated plants as well, possibly due to the initiation of nodule primordia or, in the case of sweet clover, more lateral roots . Nevertheless, it is difficult to assess the importance of MsLEC2 function in the alfalfa-S. meliloti symbiosis. Most LEC2AS plants did not differ from vector controls in the symbiotic parameters examined, nor did they show any obvious alterations in nodule development or morphology. However, symbiotic parameters measured in some of the LEC2AS plants were at a level intermediate between that of the vector controls and LEC1AS plants. These data collectively suggest that MsLEC2 may have a subtle role in the alfalfa-S. meliloti symbiosis. In contrast, all of the symbiotic parameters that were examined in LEC1AS plants were clearly abnormal. In addition, MsLEC2 mRNA accumulation was up-regulated in many of the LEC1AS nodules and roots of nodulated plants. Moreover, the most abnormal LEC1AS nodule used for Northern blot analysis had the highest level of MsLEC2 mRNA accumulation. These results suggest that the MsLEC1 gene product may repress MsLEC2 expression during symbiosis and that there may be a relationship between the level of MsLEC2 expression and normal symbiotic development. The Mtlec2 gene, which is 93% homologous to MsLEC2, may be inessential for nodulation in M. truncatula because it apparently is a pseudogene.

Although the Mtlec2 promoter was active in mature Medicago varia nodules, it was not active in uninoculated or nodulated M. varia roots . In alfalfa, MsLEC3 is a pseudogene whereas MsLEC2 is not, a finding that is consistent with our results showing MsLEC2 mRNA accumulation in nodules, as well as in uninoculated and nodulated alfalfa roots. . This lectin has been proposed to function as a storage protein. We did not detect MsLEC1 mRNA accumulation in nodules using Northern blots containing total RNA, but we were able to detect it in both root and nodule meristems using in situ hybridization methods. These data correlate with those whereby a promoter-GUS fusion of the Mtlec1 gene was found to be active in mature nodules of transgenic M. varia plants but not with its localization in the nodule peripheral tissue instead of the nodule meristem . However, blue color indicating GUS expression was observed in developing nodule primordia of the M. varia Mtlec1–gusA transgenic plants . Our in situ data also demonstrated MsLEC1 mRNA accumulation in root tips, but the Mtlec1–gusA fusion was not expressed in the root meristems of transgenic M. varia. To help resolve these differences, we examined the expression of MaLEC, which codes for a putative soluble lectin, in white sweet clover. We detected MaLEC mRNA in white sweet clover nodule and root meristematic regions, suggesting that these are the main sites of expression for soluble lectin genes in these organs. The symbiotic abnormalities of LEC1AS plants are consistent with the in situ hybridization data, demonstrating that MsLEC1 is expressed in alfalfa nodules. It appears that MsLEC1 expression at the correct level at nodule initiation and in cells in zones I and II of the nodule may be important for regulation of nodule number , as well as for the regulation of nodule size and persistence . Interestingly, the antisense-MsLEC1 mRNA also accumulated in nodules at almost undetectable levels , in spite of its transcription being driven by the strong cauliflower mosaic virus 35S promoter.

These results suggest that the accumulation of the antisense-MsLEC1 mRNA is regulated in some unknown manner. The abnormally large number and size of nodules seen on LEC1AS plants were unexpected. Based on studies in which lectins promoted nodulation and nodulation-related responses , we predicted that smaller, uninfected nodules would have developed. Indeed, the majority of nodules produced by LEC1AS plants were small, undeveloped, and senesced prematurely . However, although infection thread formation appeared normal, at least based on the organized arrangement of rhizobia in the curled root hairs , it is not known whether or not the premature senescence exhibited by the LEC1AS nodules is due to a lack of MsLEC1 transcript accumulation in nodule meristematic tissues or to a defect in persistence of the infection threads. The latter seems less likely because the rhizobia were not affected by their course through the nodules; large numbers of bacteria were recovered from both LEC1AS and LEC1ST nodules . Based on our previous studies, when the MsLEC1 gene is disturbed, a disorganized proliferation of embryonic and vegetative tissues results . In this report, we have shown that following inoculation with Rm1021, the LEC1AS transgenic root nodules that result are also highly aberrant. In contrast, no abnormal vegetative or reproductive development was detected in the LEC1ST plants , although some abnormal nodulation was observed. This result is compatible with the finding that sensesuppression-induced symbiotic abnormalities are usually milder than those from antisense suppression and further suggests that symbiotic processes may be more sensitive to alterations in MsLEC1 expression. In addition to finding that MsLEC2 gene expression was upregulated in the LEC1AS transgene-containing tissues, we also found that MsENOD40, an early nodulin gene, was expressed at relatively high levels in LEC1AS nodules. The MsENOD40 gene has been shown to be up-regulated in response to cytokinin application, and it has been proposed that nodule development may be influenced by changes in the endogenous cytokinin to auxin ratio . LEC1AS plants exhibit excessive nodule formation, a result that is consistent with an increase in the level or responsiveness to cytokinin. Moreover, LEC1AS plantlets frequently formed severe teratomas with minimal root development , and mature LEC1AS roots were poorly developed , further suggesting an excessive cytokinin response. A mechanism whereby lectin could mediate phytohormone levels and interactions is hypothetical at this time, but hydrophobic ligands, including auxins and cytokinins,rolling bench are known to bind to some soluble legume lectins, albeit to sites independent of the sugar-binding site . Taken together, our findings indicate that the expression of the MsLEC1 and MsLEC2 genes, especially the MsLEC1 gene, is important in the compatible symbiotic interaction between alfalfa and S. meliloti. This hypothesis is consistent with both lectin gain-of-function and loss-of-function experiments. How lectins promote compatible symbiotic interactions is unclear, particularly because lectins with similarity to legume lectins have been found in plant families in addition to the Fabaceae . In Arabidopsis moreover, numerous genes encoding receptor kinases with legume lectin domains have been uncovered , and similar proteins have now been identified in Medicago truncatula . The finding that other plant families have genes that encode proteins with legume lectin domains implies that legume lectins are derived from a lectin gene that was already present in an ancestral flowering plant . Indeed, many proteins that appear to be specific to the legumeRhizobium interaction seem to be recruited from proteins that are common to both legumes and nonlegumes, e.g., NORKand HAR1/ NARK . NORK extracellular sequencelike genes are found not only in nonlegumes, including several grasses and Arabidopsis, but also in a gymnosperm . Similarly, HAR1/NARK genes are very similar to CLAVATA1, a serinethreonine kinase that is important for restricting the floral meristem in Arabidopsis.

It is clear that duplication of NSL, CLAVATA1, and other genes found in nonlegumes has taken place, along with a specialization of their respective proteins for the Rhizobium-legume symbiosis. Gene duplication events often result in the development of new functions for the new proteins. Thus, what makes the rhizobialegume interaction specific may rely more on the details of the interactions between various legume proteins including lectinsand their ligands. Finding a lectinless mutant in an indeterminate nodule-forming legume such as white sweetclover, which appears to have only one gene coding for a soluble lectin, might be one strategy for testing this hypothesis. Alternatively, introducing a gene for a legume lectin, e.g., SBL or PSL into a nonlegume such as Arabidopsis, may also help elucidate whether or not legume lectins can promote colonization of a nonlegume root by rhizobia. Construction of transgenic plants was described previously . Briefly, the MsLEC1 transgene contained 420 bp of DNA encompassing the 3 portion of the open reading frame plus 76 bp of predicted 3 untranslated region. The MsLEC2 transgene contained 400 bp from the 5 portion of the open reading frame beginning 74 bp downstream of the predicted initiator codon . Sense or antisense orientations of transgenes were confirmed using DNA sequence analysis. The CaMV 35S promoter drove transcription of all the transgenes. One plant line of alfalfa cv. Regen SY was used for transformation and regeneration of dozens of independent transformant lines of LEC1AS, LEC1ST, LEC2AS and LEC2ST plants, some of which were grown to maturity for use in nodulation assays. Further control lines containing only the vector used for lectintransgene plants but lacking inserted genes following the promoter were also constructed and were used in nodulation assays. Stable transgene integration and activity, as well as transgene-specific phenotypic effects, have been clearly demonstrated . For nodulation tests, stem cuttings of the transgenic alfalfa plants were placed in sterile 11-liter pans that contained 6 liters of a 1:1 mix of perlite/vermiculite saturated with 2.5 liters of complete Hoagland’s 1 /4-strength nutrient solution and were allowed to root. Stem cuttings were from independent, primary transformant lines because they demonstrated developmental abnormalities that were very similar to progeny resulting from selfing . However, because alfalfa is an outcrossing tetraploid and shows inbreeding depression, it was difficult to obtain progeny plants that survived to maturity. The cuttings were transferred to sterilized Magenta jars containing a similar mix of perlite and vermiculite watered with Hoagland’s 1 /4-strength nutrient solution minus nitrogen. A 5-ml suspension of Sinorhizobium meliloti wild-type strain 1021 cells at an optical density of 600 nm equal to approximately 0.1 to 0.2, labeled either with GUS or with GFP , was added to the Magenta jars after the bacteria were rinsed and diluted in sterile water. One and two weeks after inoculation, the roots were carefully removed from the Magenta jars, were rinsed, and were prepared either for GUS-staining or for viewing under a Zeiss Axiophot fluorescent microscope. Stem cuttings of the transgenic alfalfa plants were made as described above and allowed to root. Cuttings were placed in pots with approximately 400 cm3 of potting soil in a greenhouse. One week before inoculation, nitrogen nutrition was withdrawn from the plants, but other macronutrients were supplied. The potting soil was leached with large quantities of tap water four and one days before inoculation. Rm1021 cells were grown in RDM medium , containing 100 mg of streptomycin per liter to an OD600 of 0.11 or 0.13, depending on the experiment. Rhizobia were pelleted in a clinical centrifuge and were suspended in sterile milli-Q water to an OD600 of 0.1 . Rm1021 suspension was placed on the surface of the potting soil of each plant.

Microfluidic platforms have also been successfully employed to study the interactions between the root, microbiome and nematodes in real time . In the systems, additional vertical side channels are connected perpendicularly to the main micro-channel to enable introduction of microorganisms and solutes to the roots in a spatially and temporally defined manner . A recent microfluidic design incorporated a nano-porous interface which confines the root in place while enabling metabolite sampling from different parts of the root . These studies demonstrated the potential of microfluidics in achieving spatiotemporal insights into the complex interaction networks in the rhizosphere. Despite several advantages of microfluidics in rhizosphere research as described above, some challenges remain. All the microfluidic applications grow plants in hydroponic systems where clear media is necessary for the imaging applications and packing solid substrates in the micro-channels is not trivial.Thus,vertical grow rack interrogating the micro-scale interactions in bigger, more developed plants is not possible with current micro-fluidic channel configurations.

In addition, technical challenges such as operating the micro-valves and micro-fabrication present a barrier to device design and construction for non-specialists. Fabricated ecosystems aim to capture critical aspects of ecosystem dynamics within highly controlled laboratory environments . They hold promise in accelerating the translation of lab-based studies to field applications and advance science from correlative and observational insights to mechanistic understanding. Pilot scale enclosed ecosystem chambers such as EcoPODs, EcoTrons and EcoCELLs have been developed for such a purpose . These state-of-the-art systems offer the ability to manipulate many parameters such as temperature, humidity, gas composition, etc., to mimic field conditions and are equipped with multiple analytical instruments to link below ground rhizosphere processes to above ground observations and vice versa . Currently, however, accessibility to such systems is low as there are only several places in the world which can host such multifaceted facilities due to the requirement of significant financial investments. Switching back to lab-scale systems, a recent perspective paper calls for the need to standardize devices, microbiomes and laboratory techniques to create model ecosystems to enable elucidation of molecular mechanisms mediating observed plant-microbe interactions e.g., exudate driven bacterial recruitment . Toward this goal, open source 3D printable chambers, termed Ecosystem Fabrication devices, have been released with detailed protocols to provide controlled laboratory habitats aimed at promoting mechanistic studies of plant-microbe interactions . Similar to a rhizotron setup, these flowth rough systems are designed to provide clear visual access to the rhizosphere with flexibility of use with either soil or liquid substrates.

Certainly, there are many limitations to these devices in that they are limited to relatively small plants and limit the 3D architecture of the root system. Still, an advantage with the EcoFAB is that its 3D printable nature allows for adaptations and modifications to be made and shared on public data platforms such as Github for ease of standardization across different labs and experiments . In fact, a recent multi-lab effort showed high reproducibility of root physiological and morphological traits in EcoFAB-grown Brachypodium distachyon plants . The development of comparable datasets through the use of standardized systems is crucial to advancing our understanding of complex rhizosphere interactions. Open science programs such as the EcoFAB foster a transparent and collaborative network in an increasingly multidisciplinary scientific community. Specialized plant chamber systems are necessary for nondestructive visualization of rhizosphere processes and interactions as all destructive sampling approaches tend to overestimate the rhizosphere extent by 3–5 times compared to those based on visualization techniques . Nonetheless, plants in such chambers are still grown in defined boundaries and suffer from inherent container impacts. For instance, studies have pointed out that container design significantly  influences root growth during early developmental stages and leaves lasting impacts on plant health and phenotype . The majority of the lab-based chambers are also centimeter scale and are unlikely to replicate exact field conditions in terms of soil structure, water distribution, redox potential or root zone temperatures . While comparisons between chamber-grown and pot-grown plants show similar outputs , studies comparing plants grown in confined spaces to those directly grown in the field are missing. A recent review mapped the gradient boundaries for different rhizosphere aspects and found that despite the dynamic nature of each trait, the rhizosphere size and shape exist in a quasi-stationary state due to the opposing directions of their formation processes . The generalized rhizosphere boundaries were deducted to be within 0.5–4 mm for most rhizosphere processes except for gases which exceeds > 4 mm and interestingly, they are independent of plant type, root type, age or soil . Bearing this in mind, our assessment of the different growth chambers revealed possible overestimation of rhizosphere ranges in some chamber set ups.

For instance, the use of root-free soil pouches representing rhizosphere soil despite being cm-distance away from the rhizoplane. This prompts the need for careful evaluation of new growth chamber designs to ensure accurate simulation of natural rhizosphere conditions. To date, many rhizosphere microbiome studies and growth chambers systems focus on the impact of plant developmental stage, genotype and soil type on microbial composition and function . On the other hand, predation as a driver in the rhizosphere microbiome remains understudied. For instance, protists are abundant in the soil and are active consumers of bacteria and fungi and play a role in nutrient cycling yet remain an overlooked part of the rhizosphere . Viruses are also pivotal in modulating host communities thereby affecting bio-geochemical cycles but their  influence in the rhizosphere is poorly studied . These predatorprey interactions in the rhizosphere deserve in-depth studies which can be facilitated by these specialized growth chambers. Another area worth investigating in the rhizosphere is in anaerobic microbial ecology. At microbially relevant scales, soils primarily exist as aggregates . Aggregation creates conditions different from bulk soil, particularly in terms of oxygen diffusion and water flow resulting in anoxic spaces within aggregates and  influences the microbial community.The rhizosphere is also rich in a wide range of compounds which can serve as alternative electron acceptors such as nitrate, iron, sulfate and humic substances in the absence of oxygen . However, most anaerobic studies in the rhizosphere focus only on aqueous environments such as water-logged paddy soils despite biochemical and metatranscriptomic evidence pointing to the possibility of anaerobic respiration in the rhizosphere . To fully understand biogeochemical cycles in the rhizosphere, it is imperative to investigate rhizosphere processes in the microscale and to include localized redox conditions as one of the influencing parameters. Microfluidic platforms with its fast prototyping capabilities can be helpful in creating growth chambers designed to stimulate these redox changes. In the study of the rhizosphere microbiome, genetic manipulation strategies are foundational in deep characterization of microbial mechanisms and current manipulation techniques require axenic isolates. However, the uncultivability of a significant portion of soil microorganisms continues to hamper efforts in gaining mechanistic knowledge. Even for culturable isolates,vertical grow table the process of isolation introduces selective pressure and disturbance to the community with inevitable loss of information on spatial interactions. A recent innovation in gene editing technologies using CRISPR-cas systems demonstrated in situ editing of genetically tractable bacteria within a complex community . Coupled with the use of transparent soil-like substrates , the application of such a technique for the editing of in situ rhizosphere microbiome while preserving spatial and temporal associations would indeed bring invaluable insights. Specialized growth chambers using 3D fabrication and microfluidic technologies are primed to facilitate such innovations. Finally, this review revealed that while similarities exist among the different growth chamber systems, many of these systems are bespoke. This makes it difficult to replicate experiments and determine reproducibility which are important cornerstones of scientific advancement. The complexity of rhizosphere interactions also warrant that computational models are essential to gain a better understanding of system level processes . However, predictive modeling requires data from standardized approaches to be comparable between experiments. Thus, future growth chamber systems and designs are encouraged to follow the open science framework to enable standardization to an extent, such as in the case of EcoFABs .

In the 1960s, the state of Punjab led in the adoption of new high-yielding varieties of wheat and rice. Production of these new varieties required innovations in the use of fertilizer and water, which occurred in a complementary manner to the innovation in seed choices. Mechanization of several aspects of farming also became a supporting innovation. Agricultural extension services based in Punjab’s public universities guided farmers in their transition to the new modes of production. Furthermore, an infrastructure of local roads and market towns had been developed by the state government: these, along with central government procurement guarantees, gave farmers access and security in earning income from their produce. In the private sector, new providers of seeds and fertilizer, as well as farm equipment and equipment maintenance services also arose. All of these conditions together created what has been known as the Green Revolution economy. With the Green Revolution, Punjab quickly became the state with the highest per capita income. This ranking persisted into the 1990s, but underlying conditions became less favorable well before then. Gains in agricultural yields and productivity slowed, due to diminishing returns. While India began to grow faster after trade and industrial policy liberalization of 1991 and subsequent creeping reforms in other sectors, agriculture remained locked into the old policies, and Punjab mostly into the old equilibrium. The relative failure of Punjab to transition from agriculture to industry or to modern services means that the state still faces a major challenge in effecting this classical structural transformation needed for growth. This failure has been a major reason in the state’s decline toward the middle of the per capita income rankings of India’s major states. However, agriculture also desperately needs attention, even if it cannot be the only sector that must change to address Punjab’s economic problems. The reasons for not neglecting agriculture are several. First, there is the immediate problem of economic distress in the sector, concentrated among small farmers and agricultural laborers. Second, the current pattern of cropping and water use is leading to a rapid decline in the groundwater table, threatening complete ecological collapse of much of the state’s agriculture. Third, the Green Revolution economy has little or no room for further innovation that would enhance productivity and rural incomes. Any one of these reasons is significant, but put together, they imply a compelling case for considering how innovation in Punjab agriculture can be spurred. This paper considers five challenges to effecting meaningful innovation in Punjab’s agricultural economy. It does not present solutions, but it is hoped that an analysis of obstacles to change can provide fundamental inputs into the process of seeking positive change. The first challenge to innovation is that, in contrast to the 1960s Green Revolution, a post-innovation agricultural economy will be much more complex, with a wider range of crops, requiring more sophisticated production technologies, as well as greater complexity in the entire supply chain. The second challenge is that this more complex agriculture will need more sophisticated infrastructure, since fruits and vegetables are much more perishable than grains such as wheat and rice . Other complementary inputs, such as water, fertilizer, farm equipment and management, will also need to be provided in innovative ways. A third challenge flows from the first two characteristics of complexity and complementarity: the costs of switching to new products and modes of production will entail significant one-time switching costs, as well as new and ongoing risks. Future risks, even if partly covered by insurance, represent a kind of switching cost, albeit less direct than explicit expenditures on shifting farm operations from one set of routines and activities to another. The fourth challenge considered here is more subtle, in that it concerns questions of appropriate balance, rather than movement to a well-defined post-innovation future. Indeed, the challenge is to assess what kinds of innovations can best be implemented in which contexts or situations: in some cases, incremental innovations or adaptation of existing frontier techniques from elsewhere may work, while in other cases, frontier innovations spurred by fundamental research may be required.