Such pollution is pervasive worldwide because increasing populations and associated economic growth are diminishing available freshwater, thus leading to increased irrigation of farmlands with waste waters.In the initial soil, copper occurs in two morphological forms . One form decorates coarse organic particles that have some recognizable structures from reticular tissue , and the other occurs in the fine clayey matrix in areas that show organic particulate shapes only at high µ-XRF resolution. In the two phytoremediated soils, similar Cu-organic particulate associations, but also, hot spots of Cu grains 5-20 µm in size were observed in the thin-section maps . In the rhizosphere of P. australis, the Cu hot spots exist outside and in roots and specifically in cortical parenchyma, but not in central vascular cylinders from the stele that contain vascular bundles through which micro-nutrients are transported to reproductive and photosynthetic tissues. In contrast, the main roots and rhizome of I. pseudoacorus do not contain detectable Cu grains, but in the surrounding soil Cu grains are aligned, suggesting that they are associated with biological structures. Under an optical microscope filamentous and ramified organic structures,vertical hydroponic garden similar to root hairs or hyphae from endomycorrhizal fungi, are visible in places where the Cu spots were observed by µ-XRF . Fungal forms are more likely because mycorrhizal hyphae typically are anastomosing, whereas root hairs are not. Fungi may also be implicated in the formation of Cu grains in the cortex of P. australis since roots of this plant are known to be colonized by arbuscular endomycorrhizae in contaminated environments .

These hypotheses are consistent with the capacity of mycorrhizae to accumulate metals and with the storage of Cu in secondary feeder roots of the water hyacinth Eichhornia crassipes . The Cu grains have about the same size as the electron-dense Cu granules in cells of E. splendens placed in a CuSO4 solution for 30 days .Organic Cu.While biological activity clearly modified the original distribution of Cu in the rhizospheres, the Cu species could not be identified from the µ-XRF maps but instead were elucidated using EXAFS spectroscopy. All eight µ-EXAFS spectra from areas in the original soil containing the particle morphologies and chemical compositions observed with µ-XRF can be superimposed on the soil’s bulk EXAFS spectrum , indicating that the initial Cu speciation occurred uniformly. If the initial soil contained various assemblages of Cu species that were distributed unevenly, then we might expect that the proportions of species also would have varied among analyzed areas and been detectable by µ-EXAFS spectroscopy ; however, this was not observed. These spectra match those for Cu2+ binding to carboxyl ligands in natural organic matter, as commonly observed . Elemental Cu. In contrast, only the reference spectrum of elemental copper matches the µ-EXAFS spectra of the 12 hot-spot Cu grains, which are statistically invariant . Photo reduction of Cu2+ in the X-ray beam cannot explain the formation of Cu0 because no elemental Cu was detected in the initial soil by powder and µ-EXAFS, and Cu0 was detected in the two phyto remediated soils at 10 K by bulk EXAFS, and all individual spectral scans from the same sample could be superimposed. At 10 K, radiation damage is delayed , and if Cu had been reduced in the beam, the proportion of Cu0 would have increased from scan to scan which was not observed. Spectra for rhizosphere Cu grains and reference metallic copper have the same phase and overall line shape, but they have significant differences in fine structure and amplitude, which provide details about the nature of the Cu grains.

In the soil Cu grains, shoulders at 5.8 and 7.3 Å-1 are weak and the spectral amplitude is reduced by about 35% and attenuated, relative to metallic copper. The decreased amplitude of the EXAFS signal for the Cu grains relative to well-crystallized metallic copper cannot arise from over absorption because the spectra of the grains were recorded in transmission mode and because the amplitude reduction from over absorption would be uniform in R-space, as demonstrated for ZnS and MnO2 particles , which does not occur in this case. Derived radial structure–functions share the four-peak character of Cu metal . However, long-distance pair correlations are progressively diminished in the soil spectra, indicating multiple interatomic distances , reduced coordination numbers from small particles , and/or abundant micro-structural defects, such as grain and twin boundary dislocations or atomic-scale vacancies. Simulation of the data using multiple-scattering ab initio FEFF calculations showed no evidence for structural disorder .Although the rhizosphere Cu grains are not structurally disordered, their CNs are only 65% , 45% , 38% , and 32% of those in the Cu metal spectrum, indicating that structural order is limited in extent. The lower CN values are consistent with small particles having incompletely coordinated surface atoms. If closed-shell packed and monodispersed, these particles would have a minimum size of 10–15 Å assuming a spherical cuboctahedron shape and 15–20 Å assuming a hemispherical shape, as reported for nanoscale platinum particles . If Cu atoms were missing , as reported for Fe metal powders with a first-shell CN reduced by up to 50% of that in metallic Fe, the effective particle size could be as high as ca. 100 Å in diameter . A defective nanoparticle model is appealing because the surface area-to-volume ratio increases with only a lattice-vacancy parameter.

Constitutive nanoparticles in the micrometer-sized Cu grains are joined at particle or so-called grain boundaries, which might contain stabilizing organic molecules.Complexation to oxygen ligands is possible because EXAFS has relatively low sensitivity to low-Z atoms. However, linear combinations of experimental spectra for organically bound Cu2+ and nanometallic Cu show that the fraction of potential organic Cu in the Cu grains is less than 15%, if present at all . Amounts of Organic and Elemental Cu in the Rhizospheres. Composite bulk EXAFS spectra of the two remediated soils and spectra from the two species identified by µ-EXAFS intersect at the same k values, confirming that only two main Cu species exist in both rhizospheres. Fractional amounts of organically bound Cu from the original soil and metallic Cu formed during phytoremediation were estimated by reconstructing the bulk spectra with linear combinations of the two single species spectra. The best fit for the rhizosphere of P. australis was obtained with 75 . X-ray Diffraction. Featureless two-dimensional µ-XRD patterns from eight Cu hot spots confirm that the Cu grains are aggregates of nanoparticles. However,vertical home farming three patterns display a faint continuous diffraction ring at the Bragg angle for the brightest 111 reflection of Cu metal, indicating larger individual particles with a domain size of 130–150 Å . About 25×104 larger particles would be needed to produce these XRD patterns, but they would comprise only about 0.01% of the analyzed volume . Thus, the diffracting Cu hot spots may have sufficient big particles to yield a powder ring, but they are not enough for their 2NN, 3NN, and higher Cu-Cu shells to contribute significantly to the EXAFS signal. Also, the big particles are undetected by µ-EXAFS because EXAFS signal intensity is linearly proportional to the number of atoms whereas XRD intensity is proportional to the number of atoms squared.The rhizospheres were oxidizing as indicated by the presence of iron oxyhydroxide , absence of sulfide minerals, and the fact that P. australis and I. pseudoacorus are typical wetlands plants with aerenchyma that facilitate oxygen flow from leaves to roots . Thermodynamic calculations using compositions of soil solutions collected below the rhizosphere indicate that Cu+ and Cu2+ species should have been dominant . These points along with the occurrences of nanocrystalline Cu0 in plant cortical cells and as stringer morphologies outside the roots together suggest that copper was reduced biotically. Ecosystem ecology of the rhizosphere indicates synergistic or multiple reactions by three types of organisms: plants, endomycorrhizal fungi, and bacteria. Normally, organisms maintain copper homeostasis through cation binding to bio-active molecules such as proteins and peptides.

When bound, the Cu2+/Cu1+ redox couple has elevated half-cell potentials that facilitate reactions in the electron-transport chain. Even though average healthy cell environments are sufficiently reducing , there are enough binding sites to maintain copper in its two oxidized states. Copper is also important in controlling cell-damaging free radicals produced at the end of the electron-transport chain, for example in the superoxide dismutase enzyme Cu-Zn-SOD, which accelerates the disproportionation of superoxide to O2 and hydrogen peroxide. However, unbound copper ions can catalyze the decomposition of hydrogen peroxide to water and more free radical species. To combat toxic copper and free radicals, many organisms overproduce enzymes such as catalase, chelates such as glutathione, and antioxidants . Mineralizationcould also be a defense against toxic copper, but reports of Cu+ and Cu2+ bio-minerals are rare; only copper sulfide in yeast and copper oxalate in lichens and fungi are known. Atacamite 3Cl in worms does not appear to result from a biochemical defense. Bio-mineralization of copper metal may have occurred by a mechanism analogous to processes for metallic nanoparticle synthesis that exploit ligand properties of organic molecules. In these processes, organic molecules are used as templates to control the shape and size of metallic nanoparticles formed by adding strong reductants to bound cations. For copper nanoparticles and nanowires, a milder reductants as corbic acidshas been used. Ascorbic acid, a well-known antioxidant, reduces Cu cations to Cu0 only when the cations are bound to organic substrates such as DNA in the presence of oxygen in the dark or via autocatalysis on Cu metal seeds in the absence of stabilizing organic ligands . As an example of synthetic control, pH dependent conformation of histidine-rich peptides has led to larger nanocrystals of Cu0 at pH 7–10 than at pH 4–6 . Plants produce ascorbic acid for many functions and rhizospheres often contain the breakdown products of ascorbic acid, which facilitates electron transfer during mineral weathering . Plants produce more ascorbic acid when grown in soils contaminated with heavy metals including copper . Fungi, which proliferate over plants and bacteria in metal-contaminated soils, can stabilize excess copper by extracellular cation binding or oxalate precipitation , but mechanisms probably also require enzymes, thiol-rich proteins and peptides, and antioxidants . The formation of electron-dense Cu granules within hyphae of arbuscular mycorrhizal fungi isolated from Cu- and As-contaminated soil suggests that fungi also can produce nanoparticulate copper. Some copper reduction possibly occurred in response to the European heat wave of the summer of 2003 . Elevated expression of heat shock protein HSP90 and metallothionein genes has been observed in hyphae of an arbuscular mycorrhizal fungus in the presence of 2 × 10–5 M CuSO4 in the laboratory . This suggests that a single driving force can trigger a biological defense mechanism that has multiple purposes. Thus, reduction of toxic cations to native elements may increase as rhizosphere biota fight metal stress and stresses imposed by elevated temperatures expected from global warming, bacteria , and algae can transform other more easily reducible metals, including Au, Ag, Se , Hg, and Te, to their elemental states both intra and extracellularly. When mechanisms have been proposed, they typically have involved enzymes; however, ascorbic acid was implicated when Hg2+ was transformed to Hg0 in barley leaves . Theoretically all of these metal cations could be transformed by a reducing agent weaker than ascorbic acid . However, binding appears to stabilize cationic forms in the absence of a sufficiently strong reductant such as ascorbic acid. Processes used in materials synthesis that were developed with biochemical knowledge might yield clues to other possible, but presently unknown, biologically mediated reactions in different organisms.The discovery of nanoparticulate copper metal in phytoremediated soil may shed light on the occurrence of copper in peats.However, swamps by definition are more oxidizing with neutral to alkaline pHs, and they may be ideal sites for biotic formation of metallic Cu nanoparticles. For example, in swamp peats near Sackville, New Brunswick, Canada, copper species unidentifiable by XRD were dissolved only with corrosive perchloric acid , suggesting they may have been nanoparticulate metal formed by active root systems of swamp plants. If swamp peats evolve to bog peats the Cu reduction mechanism could convert to autocatalysis on the initial nanocrystals . The addition of peats that either act as templating substrates or contain nanoparticulate copper could enhance the effectiveness of using wetlands plants for phytoremediation.

The glycerin “waste” from processing can be utilized to produce soaps, fertilizers and in some cases a component in animal feeds . One possible problem with bio-diesel is long-term storage in warm humid environments, but this can be overcome either by closely matching production to use or with biocides added to reduce microbial degradation of the fuel if long-term storage is necessary. Bio-diesel also offers wide application as a fuel for transport, farm equipment, manufacturing machinery and electric generation.While ethanol and methane can be utilized for electric generation, diesel-electric generation is the worldwide standard for power generation at all levels below large-scale developed world power-grids. From power generation for a small to medium sized developing world community to backup power for large building in the developed world, diesel-electric generators are the most well developed and common method for electric power generation . Species and crops utilized in a FFP system can and will vary widely depending on the location of the operation,vertical aeroponpic tower garden available resources, and the needs of the organization operating the facility and associated community. The FFP approach allows each operation to be tailored to local circumstances and in most cases carefully fitting the facility design and the animal and plant species under culture to local environmental and social circumstances will be necessary to realize the full potential of the FFP approach. In general a FFP system will incorporate fish ponds rearing a finfish or shellfish product. The nutrient-rich water from the fish ponds then flows into an agricultural product.

Possible options include a food algae crop, a water intensive crop such as rice, or a hydroponic system which can incorporate a wide variety of traditional terrestrial agricultural crops. The waste water from all crops then flows into algae ponds designed specifically for algal culture for bio-fuel production. Algae are then harvested from these ponds and in the case of bio-diesel production algae oil is extracted from the crop and converted to fuel. The remaining algal material is then utilized in animal feeds, fertilizers, or for the production of other bio-fuels. Waste water leaving a FFP system should be quite nutrient-poor and have a low pathogen load “clean” .Based on the most current research results the High Rate Pond design seems to be the best performing large-scale algae culture system for the species of algae tested as candidates for bio-fuel production to date . It has been used effectively for both microalgae and macroalgae production. Typical design is a large oval raceway with a center divider. Water flow is maintained in the HRP via a paddle wheel and CO2 is injected in the case of high-density culture operations. HRPs are typically shallow in the case of microalgae culture and deeper in the case of macroalgae culture .The major, and I would argue reasonable, assumption this estimate hinges upon is easily achieving a 1% photosynthetic available radiation efficiency from algae under cultivation. PAR efficiencies of 2% and above have been reported for Miscanthus. Large scale algal culture in early pond systems have achieved 10% PAR efficiencies and relatively long-term trials under laboratory conditions have reported greater than 20% PAR efficiencies with microalgae . Given a moderate level of investment in refining current algal culture technology directed at bio-fuel production, PAR efficiencies of 2% should easily be achievable at a commercial production level and in the near future 10% PAR efficiencies may be commercially achievable.From 1978 to 1996 the US Department of Energy’s National Renewable Energy Laboratory ran the Aquatic Species Program . The main focus of this program was the production of bio-diesel from high lipid-content algae grown in ponds, utilizing waste CO2 from coal fired power plants . In the early years of the ASP program a collection of over 3,000 oil producing strains of organisms was amassed from samples taken from sites in the west, northwest and southwest continental US, and Hawaii.

After screening and characterization efforts the collection was reduced to around 300 promising species, mostly green algae and diatoms. The collection is now housed at the University of Hawaii and is available to researchers . At the height of the ASP program much of the work was focused on the physiology and biochemistry of algae as it related to improving oil production in algal organisms—particularly nutrient deficiency as a trigger for increased oil production . While the ASP program found contradicting results using nutrient deficiency culture techniques, their work clearly provided the foundation for more recent work which has demonstrated the utility of nutrient deficiency as a mechanism to increase oil content in algal cells The latter years of the ASP program were mostly focused on molecular biology and genetic engineering techniques for improved oil production in microalgae, and the development of large-scale algae production systems. This work was a major factor in refining the design of the High Rate Pond system currently used by a number of commercial ventures for the production of Spirulina and other commercial algae species, and the algal culture system suggested for food and fuel poly culture in this paper.The majority of public research conducted during the period the Aquatic Species Program was active was either directly part of the program or contract work funded through the ASP. The program’s basic conclusion was microalgae production of bio-diesel was technically feasible but economically unfeasible. The program concluded the factors effecting cost the most were biological, and not engineering related. Even with favorable assumptions of biological productivity, their projected costs for bio-diesel were two times higher than petroleum diesel fuel costs at the time . At the time this conclusion was made oil was at approximately $25 per barrel. The program was closed by the US Department of Energy in 1996, but a number of the researcher involved continued to work in the area of energy production through the culture of microorganisms.

After the closure of the Aquatic Species Program the majority of public research work on microalgae for bio-diesel production shifted to academic institutions—mostly in the US, Japan, Israel, and more recently China. The vast majority of this work has been high-tech in nature and only really appropriate for further development and application in industrialized nations. The focus of this recent work is split to opposite ends of the spectrum. The majority of the work is narrowly focused on lab-scale research on the characteristics of individual microalgae species – such as lipid profiles– or oil extraction and processing techniques . A much smaller collection of work has focused on rough calculations of the economics and engineering of utilizing microalgae for bio-fuel production as a method to replace large portions of the world’s energy needs—such as replacing all petroleum used by the US for transportation . Through an extensive literature search I was unable to find any public research work moving towards application of algae culture for bio-fuel production to small or medium scale ideas/projects. Though it does appear that some research at this level is being conducted in the private sector, but results –for obvious reasons– have not been published.A primary hurdle encounter culturing microalgae outside of a lab environment –particularly large-scale production in open ponds– are problems with species dominance and predation. Indigenous species of algae frequently will out-compete domesticated species under culture replacing a crop of high-lipid algae with an unusable product. Similarly zooplankton predators can invade a pond system and consume a significant portion of the target species under culture. This has been a significant issue for all research work on large-scale production of microalgae for bio-fuels . Huntley and Redalje discuss the use a two part culture system that seems to address the problem of species dominance and predation effectively. In their system a permanent colony of the algae under culture are maintained in a closed photo bioreactor at high densities. The colony in the photo bioreactor is used to inoculate grow-out ponds with an algae harvest cycle of 3-4 days. This method advances target algae species growth in the grow-out pond which allows harvest to occur before problems of species dominance and predation can take hold. This appears to be a major advance in large scale microalgae production. Other difficulties with large-scale culture of microalgae for bio-fuel production include problems maintaining suspension in the water column of the grow-out pond, difficulties with harvesting and extraction of lipids, DO supersaturation, and photo inhibition where algal cells collect moresolar energy than can be utilized in photosynthesis causing damage to the cell . A further hurdle to application to the FFP system is the generally technical nature of current large-scale microalgae culture systems for bio-fuels. Example, most designs utilize CO2 injection to achieve maximum production per given area and to stabilize pH. Some, like the two part culture system described above,vertical gardening in greenhouse incorporate large lab like components in the facility. I would argue that microalgae culture for bio-fuel production holds great promise for the industrialized world, but in the short-term it is likely too technically and engineering intensive for the food and fuel poly culture concept. Long-term, as the culture technology develops and matures, microalgae for bio-fuels could be retooled for use in small to medium scale projects in the developing world.Given the current state of algae culture technology, I would argue that large-scale macroalgae culture for the production of bio-fuels offers a number of significant advantages over microalgae culture and is likely the best candidate for the food and fuel poly culture concept. The poly culture system remains basically the same.

With the only major addition being attachment structures in the High Rate Pond system such as float or rope attachments similar to what is currently already in wide commercial use . Macroalgae culture technology is well established at the commercial level and grown commercially in many parts of the world. Japan has produced around a half million tons annually of Porphyra, Undaria and Laminaria sp. for decades . Culture difficulties such as maintaining target species dominance within the grow-out pond and predation by unwanted organisms are greatly reduced in macroalgae culture when compared to microalgae. Macroalgae harvesting can be accomplished through simple hand or mechanical means, and extraction of lipids would likely be very similar to the techniques used with terrestrial grain crops such as soybeans. In the case of application to a FFP system, the macroalgae component could be designed with the goal of efficient moderate intensity production as apposed to maximum productivity. This would replace the need for CO2 injection with a simpler aeration system, and should eliminate the problems of DO supersaturation and photo inhibition. While a few reviews of the potential for macroalgae to bio-fuels have been published , actual research work on macroalgae culture for bio-fuel production appears to be completely lacking. This leaves two major questions unanswered regarding utilization of macroalgae culture in a FFP system. First, are achievable PAR efficiencies under culture at least close to that of microalgae? At first glance, I would argue macroalgae PAR efficiencies for some species are high enough to fit in the FFP system. Example, Gao and Mckinley projected the production of Laminaria japonica on an annualized basis to be 6.5 times the maximum projected yield for sugarcane on an areal basis. Second, what are the oil and sugar profiles of species with acceptable PAR efficiencies? At this time I am unable to find any published work detailing oil or sugar profiles of macroalgae.The food and fuel polyculture concept offers two distinct opportunities as a conservation tool. First, human generated nutrient-rich waste streams pose a significant threat to aquatic ecosystems around the world. Remediation of nutrient-rich waste streams is inherent to the FFP system—the concept values agriculture and aquaculture “waste” as a commodity to be captured and converted into fuel. The conservation action of reducing discharge of nutrient-rich waste should occur with little or no political/economic input. Second, production of world capture based fisheries has peaked and continued over exploitation likely will lead to even lower production in the future. Aquaculture will be key to meeting future world demand for fisheries products and reducing fishing pressure on the world’s aquatic ecosystems. Reduction of fishing pressure on local ecosystems is an opportunity presented within the FFP concept, but NOT inherent to the concept.

Recent global increases in food demand have been largely driven by demographic growth and improvements in income . Since World War II, population has more than tripled from 2.4 billion to 7.3 billion people ; South and East Asia experienced the most substantial increases . Rising incomes have allowed households to afford richer diets with higher calorie and protein intake per capita but often with stronger burdens on natural resources and the environment . A typical trend observed in countries undergoing economic development is that, as the average income increases, there is a growth in the consumption of non-starchy food such as vegetables, dairy,meat, and consumable oil , a pattern that is also known as “Bennett’s law” . Indeed, per capita consumption of animal food has been increasing in the last few decades . Dairy and meat production are expected to increase by 65% and 76%, respectively, by 2050 . Such an increase in the consumption of animal products can impede humanity’s ability to meet greenhouse gas emission targets . The general improvement in household economic status has meant that 123 million people in developing countries were able to escape undernourishment between 1990 and 2015 alone . Yet substantial nutrition deficiencies persist with roughly one in seven people receiving inadequate protein and calories and still more lacking access to important micro-nutrients .Despite massive increases in crop production over the past 50 years,aeroponic tower garden system a growing share of this output is not being used for direct human consumption. The growth in demand for animal products , combined with a shift toward a more crop-dependent livestock sector, has substantially increased competition for crop use between direct human consumption and feed to support livestock.

Indeed, the excess in crop production afforded by the technological advances of the green revolution has allowed for the use of crops as feed, thereby dramatically increasing the rates of livestock production, a phenomenon known as the “livestock revolution” . This new system of livestock production has increasingly relied on concentrated animal feed operations as an alternative to range land production . Owing in large part to the usage of energy-rich oil cakes as feed, 51% of the world’s crop calories are currently devoted to animal production . This trend has meant that countries with emerging economies and a rising middle class have had to depend more heavily on feed imports, mainly from the United States, Brazil, and Argentina, in order to support domestic animal production . Likewise, the global demand for seafood has increased and has been met by increased fish and seafood production in aquaculture operations, while increasing the pressure on wild fisheries . In addition to demographic and dietary drivers, there has been a rapid increase in demand for crop-based bio-fuels since the start of the 21st century , driven in part by clean energy mandates in the United States and the EU Parliament . This has led to the growing diversion of crop supply, mainly maize in the United States, sugarcane in Brazil, rapeseed in Europe, and oil palm in Indonesia and Malaysia, toward the production of bioethanol and bio-diesel . Although in 2000 only about 3% of crop supply was used for bio-fuel production, diversion of human-edible calories to crop-based bio-fuels increased dramatically during 2000–2010 . Rulli et al. estimate that the crops diverted to bio-fuel use could feed nearly 300 million people if they were used as food. In addition, the rise in bio-fuel demand has had an important influence on food commodity markets; several studies provide evidence that bio-fuels have contributed substantially to higher food prices, as well as increased market volatility .

Thus, it is clear that these first-generation bio-fuels have served to further increase competition for crop use and the resources to support food production. Another key component in the fate of global crop production is that of food waste. Roughly one-quarter of food production is lost or wasted at various steps along the food supply chain, from losses during production to uneaten food on a person’s plate, with distinct regional patterns . In Asia and sub-Saharan Africa, the vast majority of food losses occur during the early stages of the supply chain as a result of large production losses from dry spells, flooding, and tropical disease, as well as inadequate storage. In contrast, for Europe and North America, approximately one third of food waste occurs at either the retailer level or the consumer level .The wide diffusion of fertilizers and high-yielding crop varieties has led to much of the tripling in food supply, which to some degree has likely avoided even greater expansion of croplands. However, this intensification of agriculture to prevent the widespread conversion of natural systems has come with important trade-offs , promoting cultivation practices with extensive environmental consequences that were often inadvertently supported by policies and subsidies . For instance, over application of fertilizers, pesticides, and herbicides is a major contributor to non-point source pollution, eutrophication of water bodies, loss of soil biodiversity, GHG emissions, and acid rain . As a result, the global food system has become one of the most extensive ways by which humanity has modified the environment .More than one half of the accessible runoff is withdrawn for human use , and nearly all of the anthropogenic consumptive water use is for agriculture . The mechanization of agricultural production has allowed for intensified soil tillage, thereby increasing the rates of soil loss, which by far exceed those of soil formation .

Fertilizer production has more than doubled the amount of reactive nitrogen in the environment , and GHG emissions from food production and land use change contribute 19–30% of humanity’s GHG emissions . GHG emissions from agricultural activities increased annually by 1.1% from year 2000 to 2010 . The livestock sector contributes disproportionately to the environmental burden of food production . Although animal production makes up 25% of the world’s food supply by weight, 18% of dietary calories, and 39% of protein , it accounts for approximately 75% of agricultural land area , 29–43% of the total agricultural water footprint , 46–74% of agricultural GHG emissions , and 34–58% of total nitrogen use . The overall greater footprint of livestock production is in large part attributable to the inefficiencies by which plant biomass can be incorporated into animal tissue, particularly for cattle . Owing in large part to the efficient feed conversion ratios of monogastric digestion, as well as the inherent variability in range land biomass production , the world’s livestock systems have been transitioning from an extensive, beef-dominated system toward a focus on concentrated, feed-reliant pig and chicken production . This trend has led to important environmental trade-offs that have occurred within the livestock sector, where improvements in land use efficiency and GHG emissions per unit of animal production have been offset by the increasing water and nitrogen requirements of feed production . Animal agriculture is a major source of GHG emissions, land use,dutch bucket for sale and water consumption. Interestingly, pets such as dogs and cats are also major contributors to the demand for animal products. A recent study for the United States has shown that dogs and cats account for roughly 25–30% of the land, water, and phosphate footprint of animal production . A decrease in the reliance on animal-derived products can reduce environmental impacts and increase food security. A recent study, which modeled the U.S. agricultural system without farmed animals, found that, without animal food production, the total food production of the United States would increase by 23% and total agricultural GHG emissions would decrease by 28% . However, in this system modeled without farmed animals, population diet of the United States resulted in the absence of essential nutrients that are present only in animal products .Global food production is facing mounting constraints to its continued growth. These limitations fall into two broad categories related to changing climate and bounds imposed by plant physiology and production decisions. Regarding the first, there is evidence of reductions in food production resulting from climate change in recent decades, though overall production gains have been able to overcome these reductions so far . Early work on this topic showed that between 1981 and 2002 the combined production of three major crops—barley, maize, and wheat—was reduced by 40 Mt/year, compared to a case with no climate effect . From 1980 to 2008, global wheat and maize production fell 4% and 6%, respectively, below what would be expected without climate trends; these effects varied widely across crops and countries . It has been estimated that, without accounting for the effect of CO2 fertilization, each degree Celsius of mean global temperature increase is expected to induce a 6.0% drop in the global yield of wheat, 3.2% of rice, 7.4% of maize, and 3.1% of soybean .

Other work has shown that as much of one third of global crop yield variability can be explained by inter annual fluctuations in temperature or precipitation, with climate variability explaining as much as 60% of yield variability in certain breadbasket areas . Moreover, extreme droughts and heat waves, which are expected to intensify under climate change, can strongly reduce crop production . Recent modeling efforts created an ensemble of models that consider a different configuration of carbon dioxide under most recent climate projections . Regarding livestock, there has been substantial investigation of the effects of heat stress on animal production , but to date, no studies have examined the relation between animal productivity and inter annual climate variability. Even though historical effects of climate trends on food systems have been modest and masked by overall gains in production from specific regions, it is expected that climate change impacts on food production will become more pronounced in the coming decades, depending on the GHG emissions trajectory considered . The second set of constraints to production, which are related to crop physiology and production decisions, play an important role as well. Many places around the world—23–37% of maize, rice, soybean, and wheat areas—are experiencing a plateau or collapse of major crop yields from a combination of biophysical and socioeconomic factors . In areas that continue to realize overall yield gains, there are emerging indications that these improvements are being disproportionately contributed by a small fraction of highly productive cropland, whereas yields in other cultivated areas have increased more slowly. Pointing to this, a recent study focused on maize in the U.S. Midwest showed that the greatest yield improvements are being provided by a narrowing area of cropland . Along with these features of yield trends, the efficient use of fertilizers for cereal production has also plateaued, as the highest returns on nutrient inputs occur when yields are low . The nutritional quality of global cereal production has declined steadily with time, as nutrient-rich cereals have been supplanted by high-yielding rice, wheat, and maize varieties . This increase in high-yielding crop production has been in part driven by the increasing prevalence of large farms, which generally produce a less nutritionally diverse set of crops , and has resulted in dwindling amounts of key nutrients, such as protein, iron, and zinc per tons of cereal crop . Enhancements of atmospheric CO2 concentrations are expected to exacerbate these declines by adversely affecting crop nutrient content in plant tissue, especially in C3 crops . Though food supply remains largely nutritionally adequate at the global scale, the persisting challenges of food access, widespread malnourishment, and nutrient deficiencies amplify these trends of declining nutritional quality. Though not explored in depth here, other important factors also serve to curtail food production. For instance, desertification and soil salinization have rendered large amounts of arable land and grazing areas unusable . Urbanization has removed a fraction of fertile cropland from active production . Excess surface ozone has further led to relative yield decreases of between 3% and 16% for maize, rice, soybeans, and wheat .Human societies rely on freshwater resources for a variety of activities, including drinking, household usage, and industrial and agricultural production . Agricultural uses, however, by far exceed any other form of human appropriation of freshwater resources .

The organic solvent in the sample may also interfere with the icELISA. Sample dilution is a frequently used method to reduce the interference effects on ELISA analysis. Although the high sensitivity of the mAb can afford for up to 200,000-, 400,000-, and 10,000-fold dilutions for the DHA, ATS, and ATM drug samples, respectively, matrix effects on the assay accuracy were evaluated using the spike studies before analysis of drug samples. An amount of 2 mg/mL extracted ART-based drug samples, of which the active ingredient contents were quantified by icELISA, was spiked with corresponding standard substance at 2 and 4 mg, respectively. The extracted ART-based drug samples with no corresponding standard substance added were used as the blank control. The drug samples were added and disposed according to the icELISA procedure as described in the previous section. Three separate samples were taken for each drug sample, and each sample was analyzed in triplicate.Poor quality medicines, both substandard and counterfeit, constitute a major burden on the public health in resource poor countries. The use of such drugs not only severely jeopardizes the health of patients but also thwarts control efforts. Extensive investigations documented such epidemics of counterfeit ART drugs in Southeast Asia,and there is clear evidence showing that such threats have also emerged in other continents.In resource-poor countries, other neglected tropical diseases suffer similar fate,hydroponic fodder system and a recent report of poor-quality generic drug for the treatment of visceral leishmaniasis in the national elimination program of Bangladesh is another vivid example.

Although these examples stress the requirement for strict quality assurance by the government regulatory authorities, the development of simple and rapid methods to assess drug quality convenient methods for quality control at the field sites are desperately needed. Based on our success of generating specific antibodies for ART and its derivatives, we developed an icELISA for accurate measuring of ART drug contents. Here, we further validated the icELISA method using both standard and 22 commercial ART drugs sampled from various hospitals and pharmacies. The contents of ARTs in these drugs determined by icELISA and the gold standard HPLC method showed a borderline significant difference . In particular, the variation of the icELISA results was significantly higher than that of the HPLC method , suggesting that performance of the icELISA needs to be improved. In addition, we want to acknowledge that the convenience samples represented a disparate collection of pills, and some were from known sources of good-quality drugs. Therefore, testing of the method using samples of counterfeit and substandard drugs may be needed for further validation purpose.Commercial drugs contain matrix materials that might interfere with the assay. We showed that the icELISA method was highly sensitive for ARTs, which allows the samples to be highly diluted. This could eliminate the potential interference from the matrices of the commercial drugs. With all drug formulations tested, we did not detect significant interference of the matrices with either method. Furthermore, the use of chromatographically pure acetonitrile for the sample extraction may enhance assay tolerance against matrix interference. In addition, sample extraction may be repeated to increase ART recovery rates. A potential use of the icELISA method is for quantification of ARTs in commercial ACT drug formulations, which contain other partner antimalarial drugs. In our tested samples, the partner drugs did not interfere with the assay, suggesting the icELISA method is specific to detect ARTs in the antimalarial drugs. Although the cross-reactivity of mAb 3H2 with ATS, DHA, and ATM prevents differential detection ofART and its derivatives in the same samples, it does not constitute a major problem for our purpose of using the icELISA for quality assurance of ART drugs because all ART drugs contain a single target analyte of ART or its derivatives.

Further applications of the icELISA under a variety of field settings are needed to validate its value for quality control of ART drugs. At this point, there is no intent for commercialization of the icELISA, and collaborations with colleagues on further testing of the icELISA are encouraged.In order to compare the stability of PSII under Mn deficiency with the response observed when plants were exposed to other relevant nutritional disorders, a multi-elemental deficiency study was conducted, using the Mn-inefficient genotype Antonia. The induction of the different nutrient deficiencies was followed by visual inspection of leaf symptom development and by measuring Chl a fluorescence on the youngest fully expanded leaves, two to three times per week. Plants were sampled for analysis when the first leaf symptoms appeared or if no distinct symptoms developed, when Fv/Fm was reduced to 0.55 . Very distinct leaf symptoms were noticed on Mg, S, Cu and Fe deficient plants before any significant decline in Fv/Fm was observed, whereas no symptoms developed for Mn even at a Fv/Fm value at 0.55. Cu and S deficiency resulted in a marked reduction in shoot growth and Cu deficiency resulted in the development of necrotic tips commonly referred to as “yellow tip” whereas S deficiency resulted in anthocyanosis of lower stems . The chlorophyll content was reduced significantly in plants exposed to Mg-, S- and Fe-deficiency, but only marginally in Cu deficient plants despite a very low Cu concentration . Mn deficiency was the only nutritional disorder, which did not result in a decreased content of Chl nor any visible leaf symptoms and yet the Fv/Fm was reduced to 0.55 when plants were exposed to Mn deficiency . The successful induction of the individual nutrient deficiencies were confirmed by an ICP-MS based multi-elemental leaf tissue analysis. The Mg, S, Mn, Cu and Fe treatments were found to be well below the reported critical threshold limits for the development of deficiencies, being 1300, 1500, 17, 5 and 35 µg g-1 dry weight , respectively . The impact of Mg, S, Fe, Cu and Mn deficiency on selected key-proteins in PSII and PSI was examined and compared with the control treatment . The amount of the central core protein of PSII, PsbA, was significantly reduced for Mn and Fe deficient plants compared to the control treatment, resulting in a 63% and 37% reduction, respectively. However, Mg, S and Cu deficiency did not affect the content of PsbA significantly. To further confirm damage to the central core of PSII, an antibody directed against PsbP, which is a subunit of the OEC, was used.

The relative decrease in PsbP followed that of PsbA under Mn deficiency, however, it should be noted that the reduction in PsbP was insignificant under Fe-deficiency . The amount of Lhcb1, a subunit of the major light harvesting complex LHCII was insignificantly changed in Mg, S, Mn and Cu deficient plants. However, Fe-deficient plants appeared to have considerably less Lhcb1 compared to the control treatment and there was a tendency towards an increase in the amount of Lhcb1 for the S and Cu deficient plants. Likewise,fodder system the amount of the PSI core subunit PsaF was significantly increased in the S and Cu deficient plants by 69% and 87% respectively. We describe a theoretical framework for predicting bulk nanobubble size of any given combination of a gas and water, based upon the force balance at the gas/liquid interface. We show how this balance can develop between the internal pressure, external pressure and surface tension, and the electrostatic repulsion of hydroxide ions adhered to the surface of the nanobubble that gives rise to their relatively high negative zeta potential. We also analyse the adsorption of hydroxide ions at the surface of the nanobubble and the dependence of nanobubble formation on pH and the required initial size of a bubble that leads to the formation of a stable nanobubble. Further analysis is carried out on the velocity of the bulk nanobubble due to Brownian motion, and its effects on the rates of diffusion of the gas into the water, as well analysis on the interaction between hydroxide ions and oxygen molecules to infer the inhibition of their diffusion. Future applications and methodologies for applications, based on the equations proposed are also discussed. The dictionary definition of the prefix ‘nano’ indicates that the object or dimension it describes is in the range of 10-9 of the dimensions it is described by, and it is as such that nanobubbles have become popular, under a misnomer. Many prefer the name ultrafine bubbles, since the size range of nanobubbles begins at one micron, and usually goes down much further to only as few as 10 nanometers in diameter. Since their discovery as the remains of collapsing microbubbles and of their persistence after formation, attempts have been consistently to understand the mechanics of their dissolution and stability, to enable the design of systems that use them to our advantage. The first field to experience benefits due to micro- and nanobubbles was agriculture, and the use of nanobubble water was well-documented by several studies since the year 2000, showing increased growth and quality of root vegetables grown in hydroponic systems, as well as the cultivation of tomatoes in soil. Further benefits were also demonstrated with pisciculture, showing increased sizes of the fish cultured in nanobubble water, due to an increase in the dissolved oxygen content. Similar benefits were also demonstrated in the case of shrimp breeding, due to the same phenomenon.

However, all of these systems were simply a case of using the equipment for generating microbubbles without much control, and to permit them to dissolve into the water without regard for optimization. Indeed, without any parameters to measure the rate of dissolution and the generation and stability of the generated bubbles, it is not possible to optimize such a system. Thus, ongoing research has focused on the generation, stability and control of these bubbles for diverse application in the fields of drug delivery, water treatment, energy storage, and various others. 1 The second technological application of microbubbles was for the treatment of water based on the release of hydroxide ions from collapsing microbubbles, which shed light on one particular area, which was a promising candidate for explaining the stability of the nanobubble. The focus of stability was discovered by measurement of the zeta-potential of the first microbubbles of about 2 microns in diameter, which was found to be about -35 mV, and is still thought to be the cloud of ions that exists around a nanobubble. This suggested a role of the surrounding cloud of ions in their stability, in particular their ability to inhibit diffusion of the gas into the fluid, which has given rise to several theories regarding the mechanism of the ions’ stabilizing influence. Several approaches have also been made for specific cases such as surface nanobubbles and electrochemically generated bubbles, which involve several scenarios of diffusion and shrinkage. The work that is outlined here will summarize and look for theoretical evidence and alternatives to the presented theories, as well as present a new argument for the mechanism of stability of bulk nanobubbles, which seeks to incorporate and explain as many of the observed behaviors of nanobubble systems as possible. A further analysis of possible future applications is also presented. Where pint is the internal pressure, pext is the external pressure, γ is the surface tension, and r is the radius of the nanobubble. The scientific consensus is that the internal pressure of the bubble is not high enough to balance both the external pressure, which is a combination of the pressure exerted by the water column above the bubble as well as atmospheric pressure, and the pressure exerted by the surface tension, which seeks to reduce the surface area of the nanobubble. Both of these contribute to the pressure differential across the nanobubble surface that essentially forces the bubble to collapse, with the gas leaving the bubble by diffusing into the bulk solvent. Given the small amounts of gas within the bubbles, this should, in theory, take a very small amount of time to diffuse and disperse, causing the nanobubble to shrink to nothing almost instantly. However, the lifetime of nanobubbles has bene measured in the hours, if not days, which shows that the process of diffusion should be inhibited in some way, and that the pressure is, in effect, balanced or very nearly so.

To produce 18 m3 of three-dimensional printed habitat structure on Mars encompassing a volume of 120 m3 for six crew, a current estimate of shipped mass cost is 24 T, of which 11.4 T is dry salts to mix with the MgO in the Martian soil for printer feed stock.Substituting this mixture with PHB, which has a density of 1250 kg m23 , the required 18 m3 of habitat has a mass of 22 500 kg.Consider the following unoptimized bio-manufacture scenario: two 2000 l bioreactors, each at a working volume of 1500 l, resulting in the production of 111.6 kg d21 of PHB.In this scenario, 202 days are needed to synthesize 22 500 kg of PHB, an identical duration as that required for methane bio-manufacture.The total empty mass of the bioreactors is 1.6 T , an 86% savings over the 11.4 T of shipped dry salts.Drawn power can be calculated to be 7.221 W using, and the volumetric size of the bioreactors works out to be 9.896 m3.It should be possible to improve the PHB accumulation percentage of A.platensis with synthetic biology approaches, because such techniques have allowed the cyanobacterium Synechococcus sp.PCC7002 to stably accumulate PHA at up to 52% of the cell dry mass when using the complementation of a cyanobacterial recA null mutation with the Escherichia coli recA gene on a plasmid expression system.Synthetic biology efforts to combine the benefits of C.necator with the cyanobacterium SynechocysThis sp.PCC 6803 of §3.3 have also been documented.Such efforts are important because they lend credence to the idea of one organism that is capable of producing multiple commodities: fuel, biopolymers and as we shall see,dutch bucket hydroponic possibly pharmaceuticals as well.At present, workarounds for accelerated pharmaceutical expiry involve the launch of one or more small unmanned spacecraft to restock crew supplies as needed.

However, the practicality of these missions hinges on the location of a supply depot, conducive launch opportunities, travel time, etc., with corresponding implications on astronaut health in the event of a medical emergency.Thus, the cost of pharmaceutical shelf-life includes the cost of crew safety in addition to the cost of a resupply mission.The former is not easy to quantify but is regarded as paramount to reduce.The advantageous role of biotechnology in pharmaceutical manufacture is known, and examples include antibiotics, antibiotic alternatives such as bacteriophages , and even cancer chemotherapy.Biotechnology can also facilitate the synthesis of antibiotics such as doxycycline and tetracycline from an acetate substrate, albeit with involved steps.To tackle the typically complicated substrate sequence and pathway identification problem to desirable chemical targets, a computerized suite of tools has been recently developed.This platform also suggests the amount of synthetic biological engineering that is necessary to accomplish the manufacture of the target.For instance, Anderson et al.explain how acetaminophen can be produced by modifying the chorismate pathway , wherein p-aminobenzoic acid that is made from chorismate is transformed into 4-aminophenol using the 4ABH gene taken from a mushroom, and this 4-aminophenol can then be used as a substrate to produce acetaminophen with the E.coli nhoA gene.Unfortunately, this strain is not directly suitable for space application, on account of the reduced availability of the resources that the strain uses.However, it should be possible to implement the above path to acetaminophen by harnessing a similar chorismate pathway in an autotroph of electronic supplementary material, table S1 that uses readily available inputs on space missions.The cyanobacterium SynechocysThis sp.PCC 6803, for example, has the genes for chorismate synthase and the trpG and trpE genes that are homologues of the respective E.coli pabA and pabB genes, allowing it to generate an analogue of p-aminobenzoic acid called anthranilate.Moreover, anthranilate can also be acted upon by the 4ABH gene and 4-aminobenzoate hydroxylase.It is therefore plausible that acetaminophen can be synthesized by SynechocysThis sp.PCC 6803 after the insertion of the 4ABH and nhoA genes , and testing of this hypothesis in the near-future is anticipated.

As Ungerer et al.indicate, unnatural gene insertions into SynechocysThis sp.PCC 6803 and subsequent organism optimization can allow for a substantial rate of production of a desired target.It is possible to estimate the volumetric yield of acetaminophen bio-manufacture in SynechocysThis sp.PCC 6803 with the data in: an equivalent carbon output for the reported productivity rate of 171 mg l21 d21of ethylene occurs when 1.5 mmol l21 d21 of acetaminophen is produced, i.e.230 mg l21 d21.This yield is sufficient, because pharmaceuticals require low concentrations of an active ingredient, for example, a single tablet dose of acetaminophen is 325 mg.Hence, even accounting for losses, only a few days of acetaminophen manufacture starting from protected, inactive bacteria will be required to replenish stocks of the pharmaceutical that have expired early because of space radiation.This renders crews independent from resupply spacecraft that could take at least 210 days to arrive after Earth launch.Moreover, the anticipated productivity rate should render the pharmaceutical production apparatus eminently portable and suitable for use on long voyages, because the required bioreactor working volumes need only be around 2 l or less.According to Stoker, this volume in a 3 l bioreactor would draw about 43.7 mW.A calcareous soil with low organic matter content was collected, air-dried and sieved through a 5-mm screen.Sewage sludge was collected, air dried and grounded to about 5 mm granules, and applied as such to each soil according to the treatments.The soil and SS were analyzed for general characteristics, nutrients and heavy metals according to the standard procedures.The major soil and SS characteristics are showed in Table 1.Greenhouse pot experiments with the following treatments were conducted in a randomized complete block design with four replications: 1) zero SS application ; 2) 20 ton SS ha-1; 3) 40 ton SS ha-1; 4) 80 ton SS ha-1; 5) 160 ton SS ha-1; 6) 80 ton Cd-enriched SS ha-1; 7) 160 ton Cd-enriched SS ha-1; and 8) 80 kg DAPha-1 that represented the recommended fertilizer rate.The SS was enriched with Cd by addition of CdCl2 to a Cd concentration of 50 mg kg-1.Each pot was filled with 7 kg air dry soil, air dry soil-SS mixture or air dry soil fertilized with DAP per treatments.Three radish seedlings per pot were planted in seven-liter pots.Plants were watered on alternate days to maintain water content at approximately field capacity.At the end of the growing period , the whole plants were harvested from each pot.

The fresh weights of both shoots and tubers were recorded.Plant parts were oven-dried at 700C for 48 hours, then dry weights recorded.Plant parts were ground to a fine powder using a laboratory mill with 0.5 mm sieve.The milled plant samples were analyzed for nutrients and heavy metals by standard procedures.The soil at the end of the experiment was analyzed for the same parameters mentioned above.Addition of original and Cd-enriched SS increased shoot N content compared to both control and DAP fertilizer treatments.All rates from 40 to 160 ton SS ha-1 of both original and Cd-enriched SS gave the highest shoot and tuber N contents with no significant differences among each treatment.The tuber N content was the lowest for the DAP fertilizer treatment and was even lower than that of the non fertilized control treatment; possibly this decline could be attributed to the dilution effect caused by the higher growth with DAP fertilizer application.Shoot P content increased with increasing rates of SS and was the lowest for the control,dutch buckets system and then the DAP fertilizer treatments.Tuber P content was higher with SS application being the highest with higher rates of SS.The lowest tuber P content was obtained with the control treatment and the DAP fertilizer treatment gave a value higher than that obtained by the control and the SS rate of 20 ton ha-1.Both shoot and tuber K contents were not affected by SS application except by the highest rate of the Cd-enriched SS which might attributed to the increased concentration caused by the stunted growth.Increased plant concentrations of N, P and K with SS application is mainly attributed to the increased levels of the available forms of these nutrients in the soil following SS application.Moreover, Oudeh et al.reported that sludge contains large amounts of N and P, and found an increase in their levels in SS-treated soil when compared to a control or even to the chemical fertilizer treatments.It should be mentioned however, that excessive application of N and P by high rates of sludge application can be detrimental to crop production and the environment.On the other hand, other researchers reported that little differences were found between Thissue concentrations of N, P, and K in corn grown on SS amended soil versus the control.However, it has been reported by Rubin et al.that applying SS at a rate sufficient to meet the plant’s N requirement would provide an excess of P but insufficient K.Therefore, they stated that balancing the amount of nitrogen applied in the SS with the plant’s requirement is an important management factor to avoid nutrient imbalance.Shoot Fe concentration increased with application of SS at rates equal to or higher than 40 ton ha-1.The highest two application rates resulted in lower shoot Fe when the SS was enriched with Cd.Similar trends, but with lower values, was obtained for tuber Fe concentration.Concentrations of Mn in the shoot increased with SS addition, with the highest values obtained by the highest two rates.The treatments effect on tuber Mn concentration was not significant.Application of SS at rates equal to or higher than 40 ton ha-1, similarly or equivalently increased both shoot and tuber Zn concentrations over other treatments.Addition of 80 and 160 ton ha-1 of SS, with or without Cd enrichment, resulted in the highest Cu concentrations in both the shoot and the tuber of radish.The highest shoot Cd concentration was obtained with the application of the highest rate of the Cd-enriched SS followed by the next highest rate.Application of original SS increased shoot Cd only at the highest rate of 160 ton SS ha-1.A similar trend was observed with tuber Cd concentration.SS may facilitate higher mobility for heavy metals through the effect of dissolved organic compounds from the applied SS that form soluble complexes with Cd and Zn and to a lesser extent with Cu and Pb.It is well documented that most of the heavy metal accumulation occurs in the roots of vegetable crops.Differences between root and leaf content was larger for Cd than for Pb, indicating that the translocation of Pb from roots to leaves is more limited than for Cd.

Frost and Ketchum found higher Cd in leaves with SS application.On the other hand, Oudeh et al.found that Zn and Cu but not Cd concentrations were higher in roots than in shoots.In this study, such a differentiation in Pb and Cd distribution were not observed.DTPA-extractable Fe, Mn, Zn and Cu increased with increasing SS application rates.Addition of 160 ton of both SS and Cd-enriched SS ha-1 resulted in the highest values for DTPA-extractable micro-nutrients.On the other hand, DTPA-extractable Pb increased similarly by all rates of SS application rates.Both the control and the DAP fertilizer treatments resulted in the lowest values of the DTPA-extractable Pb.Addition of the Cden riched SS resulted in the highest DTPA-extractable Cd being higher at the higher application rate.Comparatively, much lower DTPA-extractable Cd levels were obtained for the application of the original SS.However, the highest two rates of the original SS yielded higher values of DTPA-extractable Cd compared to the lower rates, but the highest levels of Cd were found with the addition of the Cd-enriched SS.The relationships between the DTPA-extractable Cd and the Cd concentration in radish shoot and tuber were significant and followed quadratic equations.The Cd concentration tended to slowly increase with increasing DTPA-extractable Cd within the range from 0.05 to 0.15 mg kg-1, after that a steep increase was observed.It can be summarized that application of SS to calcareous soil improved soil organic matter, available P, and micro-nutrients as well as the plant growth.However, application of higher rates was unnecessary for plant growth and yet has the potential to cause nutrient imbalance in the soil.Cd-enriched SS resulted in stunted growth at the highest rate and tend to accumulate Cd in the soil and plant parts.Therefore, it can be concluded that SS application to calcareous soils improves plant growth and soil fertility but at high rates of SS and SS rich in Cd should be avoided.

Differences in root impedance have been demonstrated to have a major impact on penetration resistance.Fifteen blocks measuring 33 x 33m were marked out in an even grid with five blocks in each of three north-south columns representing the three treatment replicates.Blocks were separated from each other by 3m wide strips which were sown with grass seed after the first trial year was sown.Within each block, trial plots measured 1.55m wide by 2.1m long and were sown at 250 seed m-2 in early November 2007 or at 360 seed m-2 in early April 2008 for winter and spring varieties, respectively.Fifty-six lines of winter barley and 64 lines of spring barley, identified as a population useful for association genetics , were sown to each cultivation treatment.Prior to the year of sampling, the trial was sown in three successive seasons.The data presented in this paper were derived from samples taken from only the plough treatment, which represents the typical agricultural practice for the region,strawberry gutter system and minimum tillage, which is gaining popularity because of fuel costs and perceived benefits to soil conservation.Flag leaf samples were taken at growth stage 21 from both barley plants grown in pots and each genotype grown in the field and samples were kept on ice until they could be frozen.Samples were freeze dried before milling to a flour.One hundred mg of shoot material was digested in 5 mL of 18 M H2SO4 at 360 °C for 20 mins, after which an excess of 30% hydrogen peroxide was added until digests cleared.

Concentrations of P in diluted digests were determined by reaction with malachite green.All data are presented as the mean of three replicates and bars represent standard errors of the mean.Significant differences were established using ANOVA, and treatment means compared by LSD.Relationships between both shoot biomass and P concentration in the glasshouse experiment were related to the P added using regression.From these regression it was possible to determine the critical P concentration for barley to reach 75% growth in this soil.Mean data from the different cultivation treatments within each population of genotypes were compared using regression and r2 and 95% confidence intervals derived.The critical P concentration to achieve 75% growth was also plotted.All data were tested for normality prior to analysis.The shoot dry weight of spring barley cultivar Optic increased significantly to an asymptote with increasing P addition to the soil.This growth response was described by an exponential rise to a maximum of the form y = a + b.Using this equation it was possible to calculate the amount of P that needs to be added to this soil to achieve a critical proportion of maximal growth.For the purposes of this experiment 75% of maximal growth was used to define the critical growth of the barley plants, which was achieved by an addition of 175 mg P kg-1 soil.The shoot P-concentration of these plants also increased significantly with the addition of P to soil,however this did not reach an asymptote with the relationship being described by a linear equation.This allowed the calculation of the critical P concentration in the shoots to achieve 75% growth which, on this soil, was 2.56 µg P g-1 DW.These results demonstrate that soils typical of the arable north east of Scotland are responsive to the addition of P when barley is grown and that reasonably large additions of P are required to achieve growth approaching the physiological maximum for the barley.When grown in the field, winter barley genotypes had significantly greater P concentration in flag leaves than spring barley genotypes.This may be an indication of the greater root growth associated with winter germplasm which would allow greater access to P resources.

Within the spring and winter genotypes there was significant variation in shoot P concentration ranging from 1.7 to 3.0 and 1.5 to 2.5 µg P g-1 DM, respectively.Of particular interest was that some of the winter barley genotypes were able to achieve the critical shoot P concentration necessary to achieve 75% maximal growth in this soil under conventional cultivation.In contrast none of the spring genotypes were able to achieve this.Moreover, none of either winter or spring varieties passed the critical level in the minimum till cultivation treatment.There was also a significant impact of the cultivation treatment on the concentration of P in shoots, however this effect was dependent on which set of genotypes were considered.In the winter germplasm the minimum till cultivation treatment caused a decline in the shoot P concentration while in the spring germplasm there was a slight increase in the average shoot P concentration.The impact of the cultivation treatment on the ability of the various genotypes to acquire P is demonstrated even more strongly when the shoot P concentration of the various genotypes is correlated between the different cultivation treatments.The lack of correlation demonstrates that there is no significant relationship between the genotypes, either winter or spring germplasm, when grown in the different cultivation treatments.In fact, only 32% of the winter genotypes fall within the 95% confidence intervals of the relationship, while even fewer, only 19%, of the spring genotypes fall within the corresponding confidence interval.This suggests that the vast majority of barley genotypes have a differential response in P-nutrition to cultivation treatments, with approximately equal numbers being more suited to minimum tillage and vice versa.Interestingly, of the five winter genotypes which showed P concentrations greater than the critical for 75% maximal growth in conventional cultivation treatments, only one demonstrated a suitability for growth in minimum till cultivation.The availability and cycling of P for the conventionally cultivated and minimum till treatments are likely to be quite different due to differences in the biological and physiocochemical conditions of these treatments.For example microbial community structure and size, rooting depth, and water relations are all likely to be different.These differences will go some way to explaining why there is no relationship between genotypes of the association mapping populations between the two treatments.It is likely that the traits which allow some genotypes to successfully acquire P in the conventional plough cultivation treatment will be quite different to those of which benefit P-nutrition in minimum tillage treatment, therefore compromising the use of the genetic variation to predict useful genotypes for the different treatments.Results here are analogous with results on studies on genotypic variation in P-use efficiency in wheat which demonstrate that the ability of the wheat lines to acquire P was greatly dependent on soil type and only a small proportion of the variability in shoot growth and P content was attributable to genotypic differences.

It is therefore imperative that screening for P-use efficiency in cereals be performed on soils rather than in hydroponics or agarand ideally on a range of different soil types and under different agronomic treatments as the genetic control of this trait on one soil is likely to be different under a range of conditions.The development of SNP based high throughput genome wide assays which use hundreds of SNP’s to identify single markers from association mapping populations are a powerful tool to allow genotypic variation identified in studies like this to identify markers for P-use efficiency in barley.However, while there may be significant genotypic variation in these association mapping populations, the genetic component of this variation is not always robust between treatments.In conclusion, we have shown that genotypic variation in P-use efficiency is present in association mapping populations grown in the field.However, such variation is not related between soil cultivation treatments, where differences in root abiotic stresses will have a large impact on root growth and nutrient acquisition.It is therefore important that when screening for markers for multi-mechanistic traits, such as P-use efficiency, that screening occurs in a number of environments so that the robustness of the genotypic variation can be tested.In recent decades,grow strawberry in containers ubiquitous use of groundwater in California and other parts of the world have led to chronic groundwater overdraft and water quality issues.Worldwide recognition of groundwater depletion and its adverse effects on human and environmental well-being has increasingly led to actions, policy, and legislative change to manage water resources jointly and sustainably.For example, the Sustainable Groundwater Management Act formed in 2014 in California requires groundwater users to achieve long term groundwater sustainability by managing groundwater extraction and intentionally replenishing water in groundwater aquifers.One possible technique for groundwater replenishment is agricultural managed aquifer recharge in which farmland is flooded using excess surface water in order to recharge the underlying aquifer.As most agricultural fields have lower infiltration capacities compared with dedicated recharge basins, Ag-MAR is designed to capture high-volume excess surface water by flooding large areas of farmland at relatively low recharge rates of less than one meter per Ag-MAR event.Ideally, flooding for Ag-MAR is preferably done on fallow fields or during crop dormancy periods, when agricultural fields have the potential to serve as percolation basins for groundwater recharge.O’Geen et al.recommended potential areas in California for Ag-MAR using an index that combines five soil characteristics: deep percolation, root zone residence time, chemical properties, topography, and surface conditions.An ideal Ag-MAR site will comprise an effective deep percolation , adequate crop tolerance for flooding, low soil salinity, leveled soil surface, and lack of compaction and erosion.O’Geen et al.identified an area of 22,500 km2 of agricultural land , mostly in the Central Valley, as having excellent to moderately good potential for Ag-MAR.Root zone residence time is defined as the duration of saturated conditions in the soil root zone after water is applied.It is a key factor in Ag-MAR, as prolonged saturated conditions in the rhizosphere can damage perennial crops due to O2 deficiency— hypoxia—a well-known situation in agricultural soils under intensive irrigation.

Root functioning, nutrient and water uptake, vegetative growth, and crop yield are all affected by low O2 concentration.Moreover, in flooded fields, complete depletion of O2—anoxia— may occur, which affects crops severely and can lead to plant mortality.Therefore, quantifying the soil aeration status is of great importance for implementation of Ag-MAR on fields with perennial cropsD.A.Webb], grapes [ViThis vinifera L.], alfalfa [Medicago sativa L.]) with potentially no yield lost.Soil aeration is essential to support aerobic soil respiration which includes O2 consumption by plant roots and microbial population.The O2 level in unsaturated soils depends highly on the gas phase since the O2 concentration in atmospheric air is ∼250 mg L−1 while water in equilibrium with the atmosphere contains dissolved O2of only ∼8 mg L−1.The two transport mechanisms for meeting soil oxygen demand are diffusion of O2 in both the liquid and gas phases and convection of O2 in flowing water or air.However, low diffusion rates in water and the low solubility of O2 in water make the liquid phase contribution for O2 replenishment negligible.Therefore, gas transport is considered the main mechanism for supplying soil O2, and gas diffusion, driven by the O2 concentration gradient between the atmosphere and the soil, is considered the dominant transport process.The composition and magnitude of the gas phase in soils determines the soil aeration status, which controls O2 availability to soil respiration.Soil aeration status can be evaluated by the following quantifiers: volumetric air content , O2 concentration in the gas or liquid phase , O2 diffusion rate and soil redox potential.Under flooded conditions, as expected during Ag-MAR, O2 supply by gas transport is suppressed by soil saturation, as water occupies most of the air-filled soil pores.If ponding occurs, the ponded water layer at the soil surface will act as a barrier that effectively blocks soil gas exchange with the atmosphere, because the diffusivity of O2 in water is 10,000 times lower than in air.Under these conditions, hypoxia is expected to develop rapidly as the result of root respiration, microbial activity, displacement of air by water and impeded soil gas exchange.The depletion rate of O2 from the soil solution and entrapped air pockets will depend on temperature and respiration activity, so depletion will be slow at low temperatures and low organic-matter content.Upon water logging, the rate of decline in soil O2 from ∼21% to 0% can vary, ranging from one to several days.To minimize crop damage during Ag-MAR due to poor soil aeration, an adequate supply of O2 to the root zone must be provided.This can be achieved by natural- or forced-aeration of the root zone.

Nitrogen was applied as NH4N03 by continuous injection with the irrigation water at constant concentrations of 25, 50, and 75 mg Nil. The two irrigation treatments allowed the soil water suction at a 25 em depth to drop to approximately 10 or 30 cb at time of irrigation. Pragmatically the two irrigation treatments resulted in irrigation of approximately every day compared to every other day applications. There was no effect of the irrigation treatment on the yields. During the period of peak production, the lowest yields occurred under the lowest N application treatments. However, this yield deficit appears to have been partially offset by greater production of these low N treatments during the earlier part of the season. The total yields of the lowest N treatment were 8~ to 90 per cent of the maximum and yields were not consistently improved by applying N at higher rates. As the rate of N application increased, an increasingly greater proportion of the N taken up by the crop was partitioned into the vines and foliage. In contrast,flood tray an increase in N application rate from 120 to 585 kg/ha increased the amount of N removed in the fruit by only 40kg/ha. Thus, the total amount of N actually removed from the field depended very little on the rate of N fertilizer application. It is apparent from the fruit yield and N uptake data that although total N removal continued to increase with increasing N application, the additional N assimilated in the two higher treatments did little to improve fruit yield but served primarily to elaborate vegetative material and increase the storage of N in the vines.

Therefore, N fertilizer efficiency expressed in terms of the per cent recovery of applied N, the amount of N used by the plant to increase yield, or the economic return for unit of N applied, decreased substantially at the higher N rates. It should be noted, however, that since almost all of the excess N taken up was partitioned into the vines, a large amount of N could be returned to the soil and should serve as an important N source for subsequent crops. The results of the tomato experiment as well as some of the sprinkler-celery experiments illustrate that relatively high N concentration during the initial stages of growth is important. Even though the plant removal is small in total quantity, the plant has a very small root system and must, therefore, be provided with nitrogen in relatively high concentration to prevent nitrogen deficiency at the early stages of growth. On the other hand, as the plant grows larger and the root system becomes more extensive, supplying nitrogen in the amount necessary for uptake appears to be adequate. Nitrogen applied in excess of this amount may potentially be lost through denitrification or leaching depending upon water-soil interactions.Furrow-irrigated sweet corn was grown on the UCR Experiment Station to test the effects of a nitrification inhibitor and three rates of irrigation water application . On a sandy soil, the nitrification inhibitor significantly increased the average weight of the corn stalks. On a sandy loam soil, the nitrification inhibitor increased average N concentrations per stalk at low rates of water application but had little effect at higher application rates. Generally soil N levels were maintained higher with the nitrification inhibitor, and the effect was more pronounced at higher rates of water application .As an example , researchers at Berkeley Lab use 3D printing to create molds for casting the biocompatible polymer polydimethylsiloxane into the upper portion of a fluidics chamber.

This is subsequently attached to a microscope slide, completing the chamber. This is then placed into a sterile container, providing a gnotobiotic system for studying microbial interactions. This chamber design includes a port for growing the model grass Brachypodium distachyon, tubing that allows sterile introduction and sampling of microbes and metabolites, and a lighting system. EcoFABs have been tested with different growth media , different microbes , and plants . Data collected in duplicated systems in different labs had excellent reproducibility .An EcoPOD is a larger-scale fabricated ecosystem that allows direct and intensive monitoring and manipulation of replicated plant-soil-microbe-atmosphere interactions over the complete plant life cycle. The EcoPODs will be equipped with environmental controls to carefully manipulate and control temperature, humidity, and other important climatic parameters both above and below ground. The EcoPODs will also be outfitted with sensors capable of monitoring soil moisture, oxygen, and specific nutrients, and the output from these sensors will be integrated using computer models to gain a coherent understanding of the environment inside the EcoPOD. Multiple EcoPODs will allow scientists to examine the impact of differences in types of soils, microbes and plants on ecosystem interactions. The first prototype EcoPOD will be installed at Berkeley Lab in November 2020, based on a model developed at the German Centre for Integrative Biodiversity Research and manufactured by the German company Umwelt Geräte Technik . This prototype will be used as a base-case for the development of further sensors and imaging capabilities. A vision for 16 EcoPODs, to be placed in the BioEPIC building at Berkeley Lab, is being developed; with a full complement of EcoPODS up to 4 binary variables can be investigated at one time was composed of a brief introduction, followed by breakout into three groups . By holding it at the Genomic Sciences PI meeting between sessions, we aimed to capture broad perspectives from across the Biological and Environmental Science Biological Systems Science /Bioenergy Research Center portfolio. Each group discussed three different topics, guided by facilitators, and recorded by note-takers: Science that can be enabled by fabricated mesocosms; Defining the key technological needs for these systems How to use fabricated mesocosms to bridge the scale gap between lab and field.

We encouraged broad discussion including criticism. The charge questions which were provided to all participants are included in Appendix 3.Discussions at the workshop included identifying which scientific questions would benefit from these technologies, challenges in implementing the experiments, benefits of de-risking experiments at smaller scales prior to testing in more complex experimental systems, missing capabilities, as well as the experimental limitations of fabricated ecosystems. Additionally, there was extensive discussion about data standards and sharing. Finally, we discussed approaches for steering and community access.EcoPODs present a larger challenge to understand reproducibility, due to the complexity of the system. EcoPODs are lower-throughput/lower-replication and it would help future users design and plan their experiments if data on reproducibility was available. In addition, some concerns about the reproducibility or relevance of past Ecotron-like experiments were raised. Therefore, it was suggested that a set of EcoPOD experiments are performed which show the extent of reproducibility of data collected. This would include experiments which test reproducibility within one unit between lysimeters . These would test single variables and would be paired with field work. One such project is currently underway funded by Berkeley Lab internal research funding , but further investment in this type of project was considered highly beneficial. These experiments could also provide valuable bench marking datasets which can be used as references for field work. For example, iron-deficient experiments in the EcoPOD would generate sets of signature plant and microbial transcripts and metabolites that can be used to survey for iron deficiency in the field. Because of issues of bio-availability, soil micro-nutrient concentrations are poor indicators of nutrient sufficiency,ebb and flow tray but these signatures would provide insights into what each organism is experiencing. Finally, these experiments would act as a pilot for developing data standards and collaborating with system modelers and field researchers, to ensure that data sets that are collected both in fabricated ecosystems and at field sites are One of the key drivers behind developing the fabricated ecosystem infrastructure at Berkeley Lab was to provide a platform for computational modelers to work with experimental biologists to improve our understanding of these complex ecologies. There is a strong scientific need to try to reconcile models driven by lab data and those driven by field data. By identifying which variables are driving the discrepancies between the field and lab data, it will help to prioritize research to understand why we are not able to accurately describe these processes. As fabricated ecosystems move to more complex microbial communities, the combinatorial possibilities will preclude investigating all possible communities within EcoFABs. Therefore it is critical to make effective use of simulations to predict the most informative community structures. KBase capabilities in flux balance modeling will be central to construction of community models to predict microbial interactions and activities. In particular, it was clear that fabricated ecosystems would be valuable for improving our understanding of the role that heterogeneity has on models. Effects seen in the lab are often stronger than those observed in the field e.g., nutrient responses due to masking from heterogeneity. For example, how “strong” does an effect in the lab have to be to be important in the field. Can we predict this? In addition, the flow of lab to EcoFAB to EcoPOD and field give some unique opportunities to consider the challenges of modeling across scales using data from the different systems. Discussions also emphasized that modelers with experience working with experimental data should be included in experimental teams from the beginning, so that the modeling work is fully integrated with the proposed work. In addition, teams should also include those with experience of collecting and working with field data.

This will enable the scientists to learn to speak the same language from the start, as well as to design experiments that inform different aspects of plantmicrobial-soil science. The Berkeley Lab team are encouraged to help facilitate these interactions, and funding to support these interdisciplinary teams is considered a priority.Experiments using the EcoPODs will control a number of environmental parameters, including light intensity, relative humidity, above and below ground temperature, soil moisture and, potentially, gas concentrations. Sensors used to control these parameters, as well as measures of plant and microbial response to these conditions have the potential to generate terabytes of information for each replication of each experiment. Many of the comments centered on what to do with this flood of data and how to ensure that it was stored in a way to make it fully accessible and understandable to future researchers. There was significant discussion on the best way to proceed for standardization of data. There are already a number of ecology community data and metadata standardization groups out there that are working in this area. Two of the leaders in this area are the National Microbiome Data Collaborative and micro BEnet for metadata standards. It is important to communicate between different groups, such as experimentalists and modelers, so that the collected data will be useful to the larger scientific community. Additionally, some coordination between different experimental disciplines will be necessary.Some data-intensive applications have the potential to create computational bottlenecks. Examples given included multi-spectral imaging of root architecture and next-gen sequencing outputs. Can we leverage efforts by other groups that are working in this area, such as ARPA-E, to manage data on this scale? We will also need to develop more efficient ways of sharing these datasets with other members of the scientific community.Below ground sensing in the field is a major challenge due to access. The EcoFABs have the advantage of allowing visual access to the root system including use of optodes for monitoring pH, CO2, O2, and etc throughout the experimental time course. This allows the user to gather both spatial and temporal information of both root,microbe, and chemistry, as well as any introduced perturbation such as nutrient source. However, the spatial scale is necessarily limited. This is particularly challenging when looking at interactions that take weeks to develop, such as between roots and mycorrhizal fungi, or using larger plant species, such as sorghum. Additionally, there are concerns about the effect of light on the below-group ecosystem which is being addressed by using plastics for the root chamber that don’t transmit light in the range sensed by roots. Future challenges include developing miniaturized sensors, e.g., for soil matrix moisture, that are compatible with the EcoFAB. The EcoPODs have the advantage of being much larger, but with necessarily less visual access to the below ground processes. The capture of the growing root architecture in 3D continues to be a challenge.

Electrical Resistivity Tomography and ElectroMagnetic Induction approaches have been used to image and monitor soil resistivity changes associated with the Root Water Uptake.Recent studies explored the use of multi-frequency Electrical Impedance Tomography to take advantage of the root polarizable nature.Despite these advantages, geophysical methods to date share common limitations regarding root characterization. Geophysical methods developed to investigate geological media: in the case of roots they measure the root response as part of the soil response, see Fig. 1a for the ERT acquisition. Because of the natural soil heterogeneity and variability the resolution and signal characteristics of geophysical methods strongly depend on soil type and conditions. As such, interpretation of the root soil system response is non-unique, hindering the differentiation between roots of close plants and the extraction of specific information about root physiology from the electrical signals. Unlike geophysical methods, the BIA for root investigation developed to specifically target the impedance of plant tissues, limiting the influence of the growing medium. A practical consequence is that BIA involves the application of sensors into the plant to enhance the method sensitivity.The measured BIA responses have been used to estimate root characteristics, such as root absorbing area and root mass . Estimation of these root traits is based on assumptions on the electrical properties of roots . A key assumption is that current travels and distributes throughout the root system before exiting to the soil,mobile grow rack with no leakage of current into the soil in the proximal root position . It is only in the former case, that the BIA signal would be sensitive to root physiology.

Despite the physiological relevance of the BIA assumptions and the number of BIA studies, a suitable solution for the characterization of the current pathways in roots is missing. Thus far, only indirect information obtained from invasive and time-consuming experiments have been available to address this issue . Mary et al. , and Mary et al. 2020 tested the combined use of ERT and Mise A La Masse methods for imaging grapevine and citrus roots in the field. An approach hereafter called inversion of Current Source Density was used to invert the acquired data. The objective of this inversion approach is to image the density and position of current passing from the plant to the soil. The current source introduced via the stem distributes into “excited” roots that act as a distributed network of current sources . Consequently, a spatial numerical inversion of these distributed electric sources provides direct information about the root current pathways and the position of the roots involved in the uptake of water and solutes. The numerical approach used to invert for the current source density is a key component required for such an approach. Mary et al. used a nonlinear minimization algorithm for the inversion of the current source density. The algorithm consisted of gradient-based sequential quadratic programming iterative minimization of the objective functions described in Mary et al. .Because no information about the investigated roots was available, the authors based these inversion assumptions and the interpretation of their results on the available literature data on grapevine root architecture. Consequently, Mary et al. highlighted the need for further iCSD advances and more controlled studies on the actual relationships between current flow and root architecture. In this study, we present the methodological formulation and evaluation of the iCSD method, and discussits applications for in-situ characterization of current pathways in roots. We perform our studies using laboratory rhizotron experiments on crop roots. The main goals of this study were: 1) develop and test an iCSD inversion code that does not rely on prior assumptions on root architecture and function; 2) design and conduct rhizotron experiments that enable an optimal combination of root visualization and iCSD investigation of the current pathways in roots to provide direct insight on the root electrical behavior and validate the iCSD approach; and 3) perform experiments to evaluate the application of the iCSD method on different plant species and growing medium that are common to BIA and other plant studies.

The relationship between hydraulic and electrical pathways has been the object of scientific debate because of its physiological relevance and methodological implications for BIA methods . A key and open question concerns the distribution of the current leakage . The distribution of the current leakage is controlled by 1) the electrical radial and longitudinal conductivities , and 2) by the resistivity contrast between root and soil. With regard to σcr and σcl, when σcl is significantly higher than σcr, the current will predominantly travel through the xylems to the distal “active” roots, which are mostly root hairs. Based on the link between hydraulic and electrical pathways, this is consistent with a root water uptake process where root hairs play a dominant role while the more insulated and suberized roots primarily function as conduits for both water and electric current . On the contrary, if the σcr is similar to σcl, the electrical current does not tend to travel through the entire root system but rather starts leaking into the surrounding medium from root proximal portions. The coexistence of proximal and distal current leakage is in line with studies that suggest the presence of a more diffused zone of RWU, and a more complex and partial insulation effect of the suberization, possibly resulting from the contribution of the cell-to-cell pathways . Soil resistivity can affect the distribution of the current leakage by influencing the minimum resistance pathways, i.e., whether roots or soil provide the minimum resistance to the current flow. In addition, soil resistivity strongly relates to the soil water content, which, as discussed, affects the root physiology. Therefore, information on the soil resistivity, such as the ERT resistivity imaging, has the potential for supporting the interpretation of both BIA and iCSD results. Dalton proposed a model for the interpretation of the plant root capacitance results in which the current equally distributes over the root system. Because of the elongated root geometry this model is coherent with the hypothesis of a low resistance xylem pathway . Numerous studies have applied Dalton’s model documenting the predicted correlation between root capacitance and mass . In fact, recent studies with wheat, soy, and maize roots continue to support the capacitance method . Despite accumulating studies supporting the capacitance method, hydroponic laboratory results of Dietrich et al. and other studies have begun to uncover potential inconsistencies with Dalton’s assumptions. In their work, Dietrich et al. explored the effect of trimming submerged roots on the BIA response and found negligible variation of the root capacitance. Cao et al. reached similar conclusions regarding the measured electrical root resistance .

Urban et al. discussed the BIA hypotheses and found that the current left the roots in their proximal portion in several of their experiments. Conclusions from the latter study are consistent with the assumption that distal roots have a negligible contribution on root capacitance and resistance. Because of the complexity of the hydraulic and electrical pathways, their link has long been the object of scientific research and debate. For recent reviews see Aroca and Mancuso ; for previous detailed discussions on pathways in plant cells and tissues see Fensom , Knipfer and Fricke , and Findlay and Hope ; see Johnson and Maherali et al. in regard to xylem pathways. See Jackson et al. and Hacke and Sperry for water pathways in roots. Thus, above discrepancies in the link between electrical and hydraulic root properties can be, at least to some degree,ebb and flow table attributed to differences among plant species investigated and growing conditions. Among herbaceous plants, maize has been commonly used to investigate root electrical properties . For instance, Ginsburg investigated the longitudinal and radial current conductivities of excited root segments and concluded that the maize roots behave as leaking conductors. Similarly, Anderson and Higinbotham found that σcr of maize cortical sleeves was comparable to the stele σcl. Recently, Rao et al. found that maize root conductivity decreases as the root cross-sectional area increases, and that primary roots were more conductive than brace roots. By contrast, BIA studies on woody plants have supported the hypothesis of a radial isolation effect of bark and/or suberized tissues . Plant growing conditions have been shown to affect both water uptake and solute absorption due to induced differences in root maturation and suberization . Redjala et al. observed that the cadmium uptake of maize roots grown in hydroponic conditions was higher than in those grown aeroponically. Tavakkoli et al. demonstrated that the salt tolerance of barley grown in hydroponic conditions differed from that of soil-grown barley. Zimmermann and Steudle documented how the development of Casparian bands significantly reduced the water flow in maize roots grown in mist conditions compared to those grown hydroponically. During their investigation on the effect of hypoxia on maize, Enstone and Peterson reported differences in oxygen flow between plants grown hydroponically and plants grown in vermiculite. The results reported above and in other investigations are conducive to the hypothesis that root current pathways are affected by the growing conditions, as suggested in Urban et al. . For example, the observations by Zimmermann and Steudle and Enstone and Peterson may explain the negligible contributions to the BIA signals from distal roots under hydroponic conditions . At the same time, the more extensive suberization in natural soil and weather conditions could explain the good agreement between the rooting depth reported by Mary et al. based on the iCSD and the available literature data for grapevines in the field. To minimize these ambiguities and to develop a more robust approach for non-invasive in-situ root imaging, we aim to develop iCSD inversion code that does not rely on prior assumptions on root architecture and function and use rhizotron experiments to validate the iCSD approach.The phrase “inversion of Current Source Density” was introduced by Łęski et al. to describe the 2D imaging of current sources associated with the brain neural activation. Similar inversion methodologies have been developed for the interpretation of the self-potential data, where the distribution of naturally occurring currents is investigated.

With regard to active methodologies, Binley et al. developed an analogous approach for detecting pollutant leakage from environmental confinement barriers. Although there are physical and numerical intrinsic differences between application of the iCSD to detect brain neuronal activity and current pathways in roots, we decided to adopt the term iCSD as the general physical imaging of current source density remains valid. With iCSD, we indicate the coupling of ERT and MALM through the proposed numerical inversion procedure for the imaging of the current source density, and its correlation with root architecture. We introduce the necessary aspects regarding the ERT and MALM methods in this section. However, we direct the interested readers to more in-depth discussion about the ERT method , and to Schlumberger and Parasnis with regard to the MALM method. In the following discussion we use ρmed to represent the 2D or 3D distribution of the electrical resistivity in the growing medium . CSD represents the 2D, or 3D, distribution of the Current Source Density within the same medium. In the case of roots, the CSD is controlled by the current conduction behavior of the roots, specifically by the leakage pattern of the root system . Both ERT and MALM are active methods. In these methods the current is forced through the medium by applying a potential difference between two current electrodes. In ERT, both current electrodes are positioned in the investigated medium, while for MALM the positive current pole is installed in the plant stem,similar to BIA . The potential field resulting from the current injection depends on CSD, resistivity of the medium , and boundary conditions. The boundary conditions are known a priori and their impact on the potential field can be properly modeled. In ERT, the current sources correspond to the electrodes used to inject current, allowing us to invert for ρmed. Then, the iCSD accounts for the obtained ρmed and explicitly inverts the MALM data to obtain current source distribution.The rhizotrons used in this study were designed to enable the concurrent direct visualization of the roots and electrical measurements. Rhizotron dimensions were 52 cm × 53 cm × 2 cm , see Fig. 2. Figure 2a shows the rhizotron setup with 64 silver/silver chloride electrodes located on the back viewing surface.

For culture on agar plates, seeds of different genotypes were sterilized with 75% ethanol for 10 min, washed in sterilized water three times, and sown on the Murashige and Skoog medium containing 2% sucrose and 0.8% phytoblend . The plates were incubated at 4 ◦C in darkness for two days and then were positioned vertically in the growth chamber at 22 ◦C under 12 h light/12 h dark photoperiod. After germination, five-day-old seedlings were transferred onto agar-solidified MS media supplemented with Na+ at the indicated concentrations and were grown at 22 ◦C under 12 h light/12 h dark photoperiod. For hydroponic culture, after germination and being grown on the MS plate for ten days, the seedlings were transferred to 1/6-strength MS liquid solutions and were grown under the 12 h light/12 h dark photoperiod in the plant growth chamber. Fresh liquid solutions were replaced once a week. After two-week culture, the plants were treated with 1/6 MS solutions supplemented with a range of NaCl concentrations and were grown under 12 h light/12 h dark photoperiod. We show a representative A. mangium root under hydroponic condition in Figure 1a. A cap-like structure, which was apparently consisted of the plant tissues, covered the entire root apex up to the 5 mm region behind the tip. After the structure was readily sloughed off , the root was covered again with a new one of similar size in a week after the detachment . To evaluate roles of the structure on root growth,stacking pots supplier the whole structure and resulting tissues on root surface were carefully removed with forceps, and then the roots were exposed to 0.5 mM calcium solution containing 0, 100, or 500 μM Al for 48 h.

Al induced a root bending in both roots immediately after the exposure, and the proportion in the occurrence of root bending was higher in Al-treated roots without the cap-like structure . The addition of Al slightly reduced the elongation of roots both with and without a cap-like structure, and the degree in the reduction was larger in structure-less roots . In the absence of Al, the removal of a cap-like structure hardly decreased the root elongation, although it slightly increased the occurrence of root bending . In roots without cap-like structures, this facilitation of root bending may result in a sight decrease in the Al-resistant root elongation. The present study revealed that the presence of a cap-like structure around root apices in A. mangium ameliorates an Al-facilitated bending of root growth direction. Root caps play major roles in root gravitropism through the localization of amyloplast granules and/or the basipetal flow of auxin . Our further analysis of the cap-like structure identified that a major attached position of a structure to the root was confined to columella root cap region, and the rigidity between this connection was enhanced during prolonged Al exposure periods . The undetached state of a cap-like structure may have a role in protecting inner root cap cell function from instant Al-caused damages. An apparent root bending induced by Al seems to require high concentrations, since no similar root bending in Al-sensitive plants has been reported. It is likely that a much lower concentration of Al may be sufficient for the complete arrest of root elongation until root cap cell dysfunction become recognizable as a root bending.

The present study did not find any detrimental role of a floating, cap-like structure around the root in Al-resistant root elongation, similar to previous findings on root caps . In the present study, however, we did not evaluate potential roles of undetached tissues on Al tolerance mechanisms. A cycle of detachment and formation of caplike structures imply that non-detached original tissues in the root surface area of root apex may play roles in the Al-resistant root elongation in A. mangium. Another study of A. mangium revealed that more than half of fibrillary tissue initials separated from the root surface were present beyond the entire root elongation region . In summary, we have found a novel cap-like structure of tissues for the protection of root cap cells at the root apex of A. mangium. Further characterization of the detachment pattern of the tissues from the root surface should provide better understanding of their roles in high Al tolerance mechanisms. Potassium and calcium are key elements for plant physiology, as evidenced by being the highest concentration cations in plants and, together with N and P, among the most crucial elements for plant growth and productivity. K is centrally involved in a wide range of crucial biophysical and biochemical processes in plants, including cell osmotic pressure regulation, growth, regulation of photosynthesis, ion homeostasis, control of cell membrane polarization, enzyme conformation/activation, water conservation and salt resilience. Ca has its own crucial roles including as a significant component in cellular walls, the maintenance of membrane integrity against the passive transfer of H+ , K+ , Na+ , and other monovalent cations, and biochemical signaling. Due to these various essential roles, much research has focused on the mechanisms of K and Ca uptake by and transport within plants. For Ca and Mg, another key nutrient element, isotopic fractionation by plants has been investigated both experimentally in the lab to elucidate processes of acquisition and transport, but also in the field ) as monitors of the local and global biogeochemical cycling of these elements.

The isotopic fractionation by plants of micro-nutrients such as Fe, Cu, Zn, and Mo have also been measured. In the case of K, it has been suggested that terrestrial plants represent a significant reservoir for K, with as much as 40– 70% or more of the dissolved K in the world’s rivers coming from the decay of plant matter, resulting from its crucial role as a plant nutrient. Therefore, potassium isotopic systematics potentially provides a new tool for tracking and quantifying nutrient cycling in ecological systems ; a proxy for global geochemical cycling; and a research avenue for understanding the optimal fertilization for efficient agricultural food production. To fulfill that potential, the degree of K isotopic fractionation by plants needs to be established. Two published studies do suggest that plants may fractionate K isotopes. Deviations in the 41K/39K ratio of ∼1‰ from an in-house reference material favoring the light isotope have been measured by Li et al. in several plant materials , but with no measurement of the K sources. Likewise, in their broad survey of natural geologic and organic materials Morgan et al. found that a banana and a potato purchased in a Scottish grocery had 41K/39K ∼ 0.4‰ lighter and ∼0.1 ‰ heavier respectively than seawater K, but again with no measurements of the K sources. Here we use hydroponically grown plants, with isotopically characterized K and Ca nutrient sources, to quantify and compare the isotopic fractionation of K and Ca by three species of vascular plants: soybean , rice , and wheat . These three plants were chosen to cover a selection of important food crops; and each are C3 plants. We choose to compare K and Ca isotopic fractionation for several reasons. First, the atomic weights of K and Ca are relatively close so that mass fractionations can be easily compared. Second, K and Ca have contrasting roles in plant physiology as described above, and have contrasting valencies. Third, there is a body of experimental data on Ca isotopic fractionation by plants that we can compare our Ca isotopic results to . Methods Soybean, rice, and wheat were grown from seeds and cultivated in a large hydroponic system at the U.C. Berkeley Oxford Facility greenhouse. The hydroponic solution, a modified Hoagland’s solution replete with Ca nitrate and K nitrate was replenished periodically to provide an isotopically constant source of Ca and K during plant growth.Harvested plants were divided broadly for soybeans into root, stem,grow lights supplier and leaf samples, and for rice and wheat plants into samples of roots and leaves. The plant samples were rinsed, dried, and weighed, and then ashed before complete dissolution in high-purity nitric acid. Aliquots were taken of the sample solutions for K and Ca isotopic analyses. K was separated from the sample aliquots using AG50×8 cation resin and 1 M HNO3. The K isotopic analyses were conducted on an IsoProbe MC-ICPMS at Lawrence Berkeley National Lab using a sample-standard bracketing technique where Ar-based mass interferences were removed by the introduction of Ne + H2 gas to the hexapole collision cell. Results are reported as per mil deviations of the 41K/39K ratio relative to an inhouse K standard using a spectroscopic concentration standard . We estimate that our in-house standard on the Bulk Silicate Earth scale of Wang and Jacobsen has an approximate δ41KBSE of +0.5‰. For Ca isotopic analysis, the sample aliquots were spiked with a 42Ca–48Ca double spike before chemical separation using Eichrom DGA resin eluted with 3 N HNO3 and separated Ca collected with DI H2O. The spiked Ca separates were then analyzed for isotopic composition using thermal ionization mass spectrometry with a Triton multicollector instrument using a multi-dynamic Faraday cup routine. The Ca isotopic results are reported as per mil deviations of the 44Ca/40Ca ratio from the Bulk Silicate Earth 44Ca/40Ca of 0.0212035. On this scale our long-term average δ44Ca of SRM915A is −1.0 ± 0.1 2s. Further details of the K and Ca separation and isotopic analyses are presented in the Supporting Information.

Another possible effect to consider with transport across cellular membranes is that ions would need to be desolvated before their passage. Hoffman et al. suggest through molecular dynamics simulations, that water exchange rates for solvated ions has a rate dependence on the mass of the solvated ion such that the lighter isotope is favored in desolvation, resulting in a predicted ∼2‰ fractionation in 41K/39K between a precipitating solid and the solution. However, a further feature of K+ specific channels lies in the process of desolvation/solvation of K+ during transmembrane transport. Solvated K + enters the channel where the K+ is handed off to eight protein bonded oxygen atoms replacing the role of the solvating H2O molecules, minimizing the energy required for desolvation, with resolvation occurring with the exit of K+ from the channel.Therefore, we suggest that isotopic fractionation due to dehydration of K+ accompanying K+channel transport may be minimal. Instead we hypothesize that their size selectivity as argued above is the main source of fractionation of K isotopes. We propose that the HATS, operative under low external K+ , would fractionate K isotopes less that the LATS, which in large part depends on highly selective K+ ion channels. This suggestion is supported by experiments involving cells of a marine diatom where under low external Zn concentration , the observed isotopic fractionation of Zn is four times less than under high Zn external concentration . A difference between HA vs LA transport has also been called upon by Deng et al. to explain differences in Zn isotopic fractionation between Zn nonaccumulating and Zn hyperacumulating plants. For isotopic fractionation of K in plants, a test of this would be examining K isotopic fractionation as a function of external solution K+ concentration. Another possible test would be experiments involving genetically modified plants that are capable of only either HATS or LATS K+ transport to look for contrasts in K isotopic fractionation. Roots are not only vital for anchorage and for acquisition of water and nutrients from the soil, but are also engaged in complex physical and chemical interactions with the soil. Plant roots release approximately 11–40% of their photosynthetically fixed carbon, commonly known as root exudates, into the soil . Root exudates and mucilage act as nutrient sources and as signaling molecules for soil microorganisms, thus shaping the microbial community in the immediate vicinity of the root system . In turn, microbial processes promote plant growth by aiding in nutrient acquisition, plant growth hormone production and bio-control of plant pathogens . The physicochemical characteristics of the surrounding soil are also affected by interactions between roots and the microbial community. This interplay between the different rhizosphere components is affected by spatio-temporal processes, which culminates in dynamic feedback loops that maintain the complex rhizosphere environment with physical, chemical and biological gradients that are distinct from the bulk soil .