Horticulture

The process of Velaglucerase alpha was design to modify the glycosylation profile of the protein toward oligomannose N-glycans to improve mannose-receptor mediated uptake of the drug into macrophages, the target cells. Here, we determined the optimal concentration of kifunensine and demonstrate that kifunensine addition at a concentration of 0.375 µM in the Agrobacterium infiltration solution of N. benthamiana plants during the vacuum infiltration process allows the production of exclusively high-mannose recombinant proteins. The anti-CD20 monoclonal antibody rituximab, approved for the treatment of non-Hodgkin’s lymphoma, was selected to evaluate the effectiveness of kifunensine for the production of an anti-cancer antibody with enhanced antibody-dependent cell mediated cytotoxicity . ADCC efficacy of rituximab is inversely correlated with the content of core fucose, suggesting that a rituximab variant with altered glycosylation would lower dosing requirements. More importantly, we demonstrate that afucosylated high-mannose decorated antibody, derived from the treated plants, exhibits increased ADCC effector function, as compared with rituximab derived from non-treated plants. The increased ADCC activity was verified using effector cells carrying both FcγRIIIa-V158 and FcγRIIIa-F158 allotypes. Several strategies could be implemented to modulate the plant-specific glycans: Protein containment in the Endoplasmic Reticulum using specific signal sequences, knockdown of fucosyltransferase and xylosyltransferase enzymesin N. benthamiana with RNA interference technology,hydroponic growing system knockout of fucosyltransferase and xylosyltransferase enzymes in N. benthamiana using gene editing, and replacement of plant glycans with human glycans through glyco-remodeling.

Gene editing using sequence-specific transcription-activator-like effector nucleases was only partially effective, while the use of transgenic knockdown lines at manufacturing require more exigent containment and cleaning procedures. Our approach eliminates the need for modification of the primary sequence or the use of transgenic, regulated material for manufacturing. Combined with the scalability and low manufacturing cost associated with the N. benthamiana transient expression system, this method represents an excellent alternative to the use of either glycoengineered or kifunensine-treated mammalian cell lines for the production of afucosylated anti-cancer antibody.We have hypothesized that a treatment with kifunensine would inhibit trimming of mannose residues in the endoplasmic reticulum , subsequently preventing the addition of α1,3-fucose and β1,2-xylose residues on the polysaccharide core . To do so, 60 plants per conditions were vacuum infiltrated in a solution of Agrobacteria with or without kifunensine, ranging from 0 to 5 µM . Visual observation of infiltrated plants from three to seven days post infiltration revealed no noticeable phenotypic or morphological differences between treated and untreated control plants . All leave and stems were collected from each infiltration and pooled for protein extraction and purification. Rituximab expression levels were quantified at 7 dpi and revealed a low to moderate increase in antibody expression between untreated and treated plants, with the average rituximab expression level ranging from 288 mg/kg to 385 mg/kg whole plant fresh weight in treated plants, compared to 287 mg/kg whole plant FW in untreated plants . These observations demonstrated that the kifunensine treatments were not detrimental to plant growth or protein expression. To evaluate the effect of kifunensine treatments on the integrity and assembly of rituximab, SDS-PAGE analysis was carried out under reduced and non-reduced conditions.

As illustrated in Figure 1D, rituximab derived from kifunensine treated and untreated conditions appeared intact and fully assembled. Non-reduced rituximab migrated at the expected molecular weight of ~145 kDa, while the reduced heavy and light chains migrated at the expected MW of ~50 kDa and ~25 kDa, respectively . Infiltration experiments were run in duplicates.The N-glycosylation profiles of purified rituximab expressed in N. benthamiana were evaluated by LC-MS/MS analysis . The major glycoforms were compared based on the relative intensity of the Asn297 glycopeptide masses identified by LC-MS/MS.As previously described in the literature, this plant-derived rituximab control exhibited primarily complex-type N-glycans, with the most abundant N-glycan structure being GlcNAc2Man3GlcNAc2 . On the other hand, there were significant differences in rituximab N-glycan profiles between untreated and treated samples. Complete conversion of plant complex glycans to oligomannose-type glycans was observed when N. benthamiana plants were infiltrated with ‘higher range’ and ‘medium range’ concentrations of kifunensine . The GlcNAc2Man9 and GlcNAc2Man8 were the major glycoforms observed with Man9 being the most abundant. In fact, the same oligomannose-type glycoform distribution was observed whether 0.375 µM or higher kifunensine concentration was used, indicating that 0.375 µM is sufficient to provide homogeneous rituximab with oligomannose-type glycans . When lower concentrations of kifunensine were used, a mixture of oligomannose, hybrid, and complex glycans was detected . For instance, the glycosylation profifile of rituximab from plants treated with 0.25 µM contained more than ~48% hybrid/complex glycan modififications and ~52% oligomannose glycosylation .

Importantly, no α1,3-fucose or β1,2-xylose residues were detected in rituximab derived from plants treated with ≥0.375 µM kifunensine.In this study, we have investigated the use of kifunensine in a plant expression platform that is established for large-scale manufacturing for recombinant protein. More specifically, we evaluated the concentration of kifunensine sufficient for the production of an afucosylated rituximab with enhanced biological activity. The vacuum infiltration of plants in a solution containing Agrobacterium culture supplemented with kifunensine at a concentration varying from 0.0625 µM to 5 µM did not affect the seven-day post-infiltration growth of the plants or the expression of rituximab. In fact, the expression of rituximab was slightly higher from kifunensine-treated plants. Similar tolerance to kifunensine treatment has also been described for the expression of antibodies in Chinese Hamster Ovary cells. However, it has been reported in one instance that kifunensine treatment of N. benthamiana plants via the growth medium led to a decrease in expression of a recombinant protein]. When applied during plant vacuum infiltration in the Agroinfiltration solution, kifunensine enters the interstitial spaces of the leaf tissue in contact with the host cells where recombinant protein expression occurs, rather than through uptake via the root system. The positive impact of infiltrated kifunensine on host cell tolerance and protein production may be due to its suppressing effects on the ER-associated degradation pathway where proteins with trimmed oligomannose glycans may be degraded if the polysaccharide chain is not further processed or proteins transported to the Golgi apparatus.Kifunensine treatment during the agroinfiltration ultimately results in protein afucosylation as it stops mannose trimming in the endoplasmic reticulum, yielding Man5-Man9 N-glycan structures. When delivered in this fashion, a minimum concentration of 0.375 µM kifunensine was sufficient to generate rituximab harboring only oligomannose glycan structures lacking fucose residues. In agreement with reports using mammalian cell cultures, the minimum required kifunensine concentration to generate antibody devoid of fucose residues in N. benthamiana falls somewhere between 0.25 and 0.375 µM . Kifunensine has the practical advantage of being active at 2-to-4-fold-lower concentrations than other inhibitors of the glycosylation pathway making it more cost-effective. Moreover, as described here and in mammalian cell cultures, treatments with kifunensine leads to a highly homogeneous product, with ultimately no formation of core-fucosylated hybrid structures.Proteins produced with glycan-engineering technologies not only lack potentially immunogenic plant-specific glycoforms,hydroponic growing but also provide enhanced effector function. The in vitro bioassay described in this study demonstrated enhanced ADCC activity from rituximab containing high-mannose glycoforms. It is expected that the reason for this increased ADCC activity lies in the absence of fucose residues on the glycosylation core rather than the high content of mannose residues, as many studies have reported the effect of afucosylation on the ADCC activity of anti-cancer antibodies including rituximab. In fact, similar ADCC results were obtained with antibodies derived from CHO cell cultures treated with kifunensine. It is important to note that ADCC activity was linearly proportional to the relative abundance of oligomannose glycoforms. This was particularly evident with ‘low-range’ concentrations of kifunensine applied, which generated a mixture of complex, hybrid, and oligomannose structures. With rituximab derived from a treatment of 0.25 µM kifunensine, generating a relatively small increase of oligomannose glycoforms, there was a significant but lower increase in ADCC activity . Importantly, plant-derived high-mannose rituximab glycoforms exhibited the same affinity for CD20 as Rituxan®, the commercial standard. Thus, the kifunensine treatment does not affect the paratope conformation of the plant-derived antibody.

As the glycosylation profile of an anti-cancer antibody is correlated to its biological activity, it is therefore considered as a critical quality attribute that needs to be maintained during the manufacturing process. To that end, the glycosylation profile of kifunensine-treated antibodies is homogeneous, consistent, and easy to control at scale, which represent a significant advantage for this technology. There are reports that antibodies carrying high mannose glycans have a shorter serum half-life, as compared with other glycoforms. However, other pharmacokinetic studies with afucosylated high mannose antibodies indicated no impact on clearance. Thus, the pharmacokinetics property of any antibody will have to be evaluated based on its biological activity, the target indication, and dosage regimen. In conclusion, the application of kifunensine during transient agroinfiltration of the N. benthamiana host leverages the scalability and cost-effectiveness of the plant expression platform for the production of biobetter anti-cancer antibodies. First, the scale-up of rituximab expression was also demonstrated at the iBio CDMO facility using manufacturing procedures without affecting the expression or product quality. Second, using the process model described by Holtz et al. 2015, and the findings from this study , the cost of cGMP -grade kifunensine to produce high-mannose, afucosylated antibodies at manufacturing scale was estimated to be less than $0.80/g of antibody produced. Thus, kifunensine can be incorporated into already established manufacturing protocols without affecting production cost significantly. Further studies will focus on determining how long the inhibition effect of kifunensine lasts during and after the plant infiltration process. This attempt to increase anti-cancer efficacy of recombinant antibodies through in-process glycan engineering represents a promising alternative to meet unmet medical needs.The ADCC reporter assay was performed using Wil2-S cells as targets along with Jurkat-CD16 reporter cell lines. Two reporter cell lines stably expressing the FcγRIIIa receptor, V158 or F158 variants were used. Wil2-S cells were plated in a 96-well white bottom assay plate at 5000 cells per well. Serial dilutions of test antibodies were added to the plates containing the target cells and incubated at 4 ◦C for 15 min to allow opsonization. Jurkat-CD16 reporter cells were then added to assay plates already containing Wil2-S cells and antibodies. The fifinal concentration of antibodies ranged from 2 to 0.0003 µg/mL following several 3-fold dilutions. The effector:target cell ratio was 10:1. After a 6 h. incubation at 37 ◦C, One-Glo™ Luciferase Assay Reagent was added and luminescence was determined using a Gen5 microplate reader. Samples and controls were tested in triplicate, and the mean reporter signals of sample dilutions in Relative Luminescence Units were plotted against the antibody concentration. Antibody independent cellular cytotoxicity was measured in wells containing target and effector cells without antibodies. GraphPad prism software was used to plot normalized RLU versus Log10. The half maximal effective concentration values of plant-made rituximab and Fc variants were derived as dose responses obtained from non-linear regression curves. Fold of induction was calculated by taking the ratio of background subtracted induced RLU and background subtracted untreated control. Nitrogen is a crucial plant nutrient; to encourage large yields, farmers tend to apply excess nitrogen fertilizers to their crops. However, crop plants are generally inefficient at nitrogen uptake from the soil, with as much as 50 to 75% of applied N being unused by the plants . Crop plants compete for soil N against soil microbes involved with denitrification and nitrification, volatization to the atmosphere, as well as loss of N by leaching into waterways . It is important to breed and/or design nitrogen use efficient crop plants that can produce the same, or higher yields with less applied N fertilizer. Growth of NUE crop plants, coupled with implementation of best fertilizer management practices, would allow for a reduction of applied N fertilizer per hectare. This would both greatly reduce the N fertilizer expense for the farmer and greatly reduce the environmental pollution from excess N fertilizers. We have recently developed genetically engineered rice by introducing a barley alanine aminotransferase cDNA driven by a rice tissue specific promoter, OsAnt1 . This modification significantly increased biomass and grain yield in the transgenic plants compared to control plants when the plants were grown at a fixed, high amount of ammonium as the N source. As well, we analysed the transcriptomic profile of these transgenic plants grown at the fixed N concentration using Affymetrix Rice GeneChip microarrays to provide further insights into the nature of increased NUE of these transgenics . In this study, we compared various physiological and genetic data from alanine aminotransferase over-expressing transgenic plants to control plants grown at three different nitrogen levels and demonstrated significant changes between them.

These products pass through an elaborate biochemical cycle that eventually forms one molecule of a 6-carbon sugar and regenerates RuBP. The reaction of RuBP with oxygen oxidizes the RuBP, splits it into one molecule of a 3-carbon compound and one molecule of a 2-carbon compound , and subsequently releases carbon dioxide, hence the names C2 pathway or, more commonly, photo respiration. In total, photo respiration consumes biochemical energy, but does not result in any net production of sugar . Thus, photo respiration has been viewed as a wasteful process, a vestige of the high carbon dioxide atmospheres under which plants evolved . The balance between C3 carbon fixation and photo respiration depends on the relative amounts of carbon dioxide and oxygen entering the active site of rubisco and the affinity of the enzyme for each gas . At current atmospheric levels of carbon dioxide and oxygen , photo respiration in most crops dissipates over a quarter of the organic carbon produced during carbon dioxide assimilation . In contrast, C4 crops , which have a metabolic carbon dioxide pump that increases the concentration of this compound at the catalytic site of rubisco, minimize photo respiration at the expense of the additional energy required for pumping. Elevated levels of atmospheric carbon dioxide inhibit photo respiration in C3 plants,vertical greenhouse making photosynthesis more efficient. Initially, this accelerates both their photosynthetic carbon dioxide assimilation and their growth by about a third.

After a few days or weeks, however, carbon dioxide assimilation and growth both slow down until they are accelerated in the long term by only about 12% and 8%, respectively . Moreover, leaf nitrogen and protein concentrations ultimately decrease more than 12% under carbon dioxide enrichment . Such a loss of nitrogen and protein significantly diminishes the value of this plant material as food for animals and humans. Fig. 6. Differences in leaf carbon fixation capacity versus total nitrogen concentration between C3 plants grown at elevated and ambient carbon dioxide concentrations. Each symbol designates the mean ratio for a species. Shown are the regression line and 1:1 line . This data suggests that changes in photosynthesis from carbon dioxide enrichment derive from changes in plant nitrogen levels under carbon dioxide enrichment . Together these trends are known as carbon dioxide acclimation.According to this hypothesis, plants under carbon dioxide enrichment initially assimilate more carbon dioxide into carbohydrates than they can incorporate into their growing tissues. In response, they diminish carbon dioxide assimilation by decreasing their rubisco levels . This change in rubisco levels, however, is not necessarily selective; the decrease may instead just be part of the overall decline in protein and nitrogen concentrations .Another hypothesis for carbon dioxide acclimation is that shoots accumulate carbohydrates faster than roots can absorb nitrogen from soils, making leaf nitrogen concentrations decrease .

As these leaves senesce and drop to the ground, plant litter quality declines, microbial immobilization of soil nitrogen increases because of the high carbon-to-nitrogen ratios in the litter, soil nitrogen availability to plants further diminishes because more soil nitrogen is tied up in microorganisms, plants become even more nitrogen limited, plant protein levels decline and plant processes including photosynthesis slow down . This hypothesis, however, has difficulty in explaining the variation in carbon dioxide acclimation among sites and among methods of carbon dioxide enrichment .We have discovered another explanation for carbon dioxide acclimation: in C3 plants, shoot assimilation of nitrate into organic nitrogen compounds depends on photo respiration, so any condition that inhibits photo respiration also inhibits shoot nitrate assimilation . Thus, at elevated carbon dioxide concentrations, C3 plants that rely on nitrate as a nitrogen source suffer severe deprivation of organic nitrogen compounds such as proteins. The resulting decline in organic nitrogen compounds reduces the plants’ yield and biomass production. While high applications of nitrogen fertilizer may partially compensate for this, the plants’ nitrogen and protein concentrations still diminish . Ammonium and nitrate are the two main sources of nitrogen that are accessible to plants from the soil. Plants show a wide range of responses to carbon dioxide enrichment because the balance between nitrate and ammonium availability varies over seasons, years, locations and plant species. In an annual California grassland where nitrate was the predominant nitrogen source, net primary productivity diminished under carbon dioxide enrichment . This was presumably because elevated carbon dioxide inhibited plant nitrate assimilation , and the grasses became deprived of organic nitrogen.

In contrast, ammonium is the major form of nitrogen available to plants in marshes because wet, anaerobic soils promote denitrification and nitrate leaching . For example, the dominant C3 plant in the Chesapeake Bay marsh showed little carbon dioxide acclimation ; even after a decade of treatment, photosynthesis and growth remained about 35% greater under carbon dioxide enrichment , with little change in nitrogen concentrations . In wheat, another C3 plant, elevated carbon dioxide atmospheres stimulated less growth under nitrate than under ammonium nutrition .Several physiological mechanisms appear to be responsible for the dependency of nitrate assimilation on photo respiration. First, the initial biochemical step of nitrate assimilation is the conversion of nitrate to nitrite in leaves. This step is powered by the high-energy compound NADH , and photo respiration increases the availability of this compound . In contrast, C4 plants generate ample amounts of NADH in leaves via a different biochemical pathway. This explains why shoot nitrate assimilation is relatively independent of carbon dioxide concentrations in C4 plants . Second, the subsequent biochemical step of nitrate assimilation is the conversion of nitrite to ammonium in the chloroplasts of leaf cells, which requires the transport of nitrite into the chloroplast. Elevated carbon dioxide inhibits this transport . Third, this subsequent step also requires chemical energy from the oxidation of a different high-energy compound called ferredoxin. Several other processes — in particular, carbon dioxide assimilation — depend on the same energy source and seem to have priority in using it. Ferredoxin becomes involved in nitrate assimilation only when carbon dioxide availability limits C3 photosynthesis .Many crops in California depend on nitrate as their primary nitrogen source. As atmospheric carbon dioxide concentrations rise and nitrate assimilation diminishes, these crops will be depleted of organic nitrogen, including protein, and food quality will suffer . Wheat, rice and potato provide 21%, 14% and 2%, respectively, of protein in the human diet . At elevated carbon dioxide and standard fertilizer levels, wheat had 10% less grain protein . Similarly, grain protein in rice and tuber nitrogen in potato declined by about 10% at elevated carbon dioxide concentrations. Several approaches could mitigate these declines in food quality under carbon dioxide enrichment. Increased yields may compensate to some degree for total protein harvested . Several-fold increases in nitrogen fertilization could eliminate declines in food quality , but such fertilization rates would not be economically or environmentally feasible given the anticipated higher fertilizer prices and stricter regulations on nitrate leaching and nitrous oxide emissions. Greater reliance on ammonium fertilizers and inhibitors of nitrification might counteract food quality decreases. Nevertheless, the widespread adoption of such practices would require sophisticated management to avoid ammonium toxicity,vertical grow towers which occurs when plants absorb more of this compound than they can assimilate into amino acids and free ammonium then accumulates in their tissues . Several of these issues might be simultaneously addressed by fertigation, or frequent additions of small amounts of ammonium-based fertilizers in water delivered through micro-irrigation. These findings have broad implications for the future of plant distributions and food production. Enriched carbon dioxide atmospheres will not enhance the performance of C3 plants to the extent originally envisioned. A 10% decline in food protein content will further burden regions of the world already affected by hunger. With a better understanding of ammonium and nitrate use by crops and careful nitrogen management, we can turn these phenomena to our advantage.With its Mediterranean climate of moist, mild winters and dry moderate summers, a broad range of fruit and vegetable crops can be grown year round on the central coast of California. Monterey and Santa Cruz counties combined produced $912 million gross value of strawberries and over $2.7 billion worth of vegetables in 2011.

As the interest in organic farming and the demand for organic produce has increased during the last decade, organic farming on the central coast has also greatly increased. There were over 9,300 certified organic hectares in Monterey and Santa Cruz Counties in 2011, five times the number recorded in 1998 . The total farm gate revenue from organic farming in these counties was over $197 million in 2011, representing a dramatic 12-fold increase in 13 years . This trend is also true for organic strawberry production. In 2000, 77 ha of organic strawberries were grown in central coastal California, but by 2012 this had increased to 509 ha, representing 8.3% of the total strawberry production in the area . Continued growth of organic strawberry production in this area, however, faces the challenge of managing soil-borne diseases without the use of synthetic fumigants and fungicides. Verticillium wilt is a soil-borne disease caused by Verticillium dahliae that can damage a wide range of important crops in California. Host crops include lettuce, tomatoes, potatoes, apples, cotton, artichokes, and strawberries . Due to its resilient overwintering structure , this pathogen can survive many years in soil even without host plants . In the premethyl bromide era, Verticillium wilt was a major limiting factor to strawberry production in California . Today, Verticillium wilt is one of several key soil-borne diseases facing California strawberry production and poses a long-term threat for organic strawberry production in the state.To avoid Verticillium wilt and other soil-borne diseases, as well as meet the requirements of the USDA National Organic Program , organic strawberry growers must implement crop rotation. Due to its high sensitivity to the disease, several years between strawberry plantings are necessary . For specialized strawberry growers in California, establishing a crop rotation system implies a major change in the design and management of the farming system. Due to the high costs of production and the high leasing fees of crop lands , specialized organic strawberry growers need to minimize the break time between strawberry crops as much as possible to stay in business. The following biological and cultural approaches to soil-borne disease management in strawberries have been tested: host resistance ; small cell transplants ; organic amendments such as compost ; high nitrogen organic fertilizers ; broccoli residues ; mustard residues , Sudan grass , and other cover crops ; microbial amendments including vesicular arbuscular mycorrhizal fungi ; plant growth promoting rhizobacteria ; crop rotations with broccoli, lettuce or Brussels sprouts; mustard seed meal ; soil-less trough production ; and anaerobic soil disinfestation . Further, a minimum of a three-year rotation is recommended for strawberries that do not use chemical fumigants in Europe , the Northeast and Midwest United States, and in eastern Canada . However, no research has yet integrated multiple biological and cultural practices for different rotation periods of organic strawberries in California. The objective of this project was to demonstrate the effects of strawberry planting frequency in organic strawberry/vegetable rotations and combined biological and cultural practices on fruit yield and disease level. We hypothesized that the use of non-host rotation crops for Verticillium wilt plus bio-fumigation with broccoli, mustard cover crop residues, relatively resistant strawberry cultivars, and compost application would suppress disease sufficiently to grow strawberries in rotation every two or three years. To test the above hypothesis, in 2001, we initiated a five year organic strawberry/vegetable rotation experiment in a commercial California field.The loss of soil productivity when crops are grown repeatedly on the same land resulting in poor plant growth and reduced yields is called “yield decline” , “soil sickness” , or “replant problem” . Such losses, called here “yield decline,” have been reported in many crops worldwide including strawberries . Biotic and abiotic factors can cause yield decline. Further, although one factor may possibly be responsible for yield decline, it is more likely that a combination of factors interact to cause the effect . This study also demonstrated the challenges researchers face when using a participatory process where farmer involvement is a key part of the experiment.

The sensitivity of XRD profiles to the amount, coordination, and position of high-Z interlayer scatterers, and to the number of vacant layer sites is illustrated next.Except for one sample obtained by metal sorption on poorly crystalline Mn oxides , the new Zn-rich phyllomanganate contains higher amounts of vacant layer sites and transition elements than any other natural and synthetic variety described so far . The constant Zn:Mn ratio of Mn-Zn precipitate suggests that Zn co-precipitated with Mn by a yet unknown mechanism to form a chemically well-defined phase as natural solids formed by metal sorption on preexisting mineral surfaces are chemically heterogeneous . Birnessite and vernadite minerals were given different names because the basal reflections of birnessite at 7.2-7.0 Å and 3.6-3.5 Å were not observed originally in the diffraction pattern of vernadite. However, recent studies have shown that natural vernadite and its biogenic and chemical analogs most often display a 001 reflection when their XRD pattern is recorded on modern diffractometers , thus confirming the view of Arrhenius et al. and Giovanoli that this mineral is a c-disordered variety of birnessite. Villalobos et al. showed that basal reflections are present when the diffracting crystallites have only 2-3 layers, on average. Here, this number is as low as 1.2 layers,vertical grow rack system meaning that Mn-Zn precipitate is essentially an assemblage of isolated layers.

Measurements of the Mn edge jumps on different Mn-Zn precipitates provide an inkling of how the constitutive nanoparticles are joined at particle or so-called grain boundaries. The Mn edge jump was typically between 0.2 and 0.3 for aggregates ~15-25 µm in diameter, which indicates that the phyllomanganate represents only a small fraction of the black precipitates, thus revealing a high micro-porosity. This porosity is possibly filled, at least partly, by organics that may help disrupt the parallelism of the layers, but also to tie them together.Since biological oxidation of Mn is generally faster than abiotic oxidation, most natural Mn oxides are considered to be biogenic. Manganese oxidation and the subsequent precipitation of Mn bioxides by microscopic fungi is also well documented . Here, we showed that Mn can be biomineralized also in higher living organisms, such as plants. Except for its atypical high Zn content and the structural consequences thereof, this new manganese biomineral is no exception to the intrinsic nanocrystalline nature of biogenic phyllomanganates. Although the mechanism of Mn to Mn oxidation is presently unknown, the constant Zn:Mn ratio of the new Mn biooxide suggests the existence of a well-defined bioactive process, likely in response to metal toxicity. The occurrence of Zn-Mn precipitate only in the root epidermis and the absence in the roots of any Zn-rich species from the soil matrix suggest that Mn oxidation did not occur in the rhizosphere, and thus does not result from bacterial activity or abiotic reaction.

Divalent manganese may have been complexed and transported to the roots by phytosiderophores , and then oxidized by the plant itself or by endomycorrhizal fungi, as shown for wheat and soybean .Knowing how to stimulate the formation of this new phase in biological systems, or how to synthesize it abiotically, would be a significant progress towards Zn immobilization in contaminated environments and their remediation. Formation of this new phase could in particular facilitate the growth of plants in highly contaminated environments in lowering the concentration of bio-available Zn in the rhizosphere. Rice is the primary staple food source for over half the world’s population. The crop is cultivated in at least 114 countries and is the primary source of income and employment for more than 100 million households in Asia and Africa. In recent years, especially in developing countries, rice production has not matched the food demands of an increasing population. To meet this growing demand, rice production has to be raised by at least 70% over the next three decades. The land area devoted to rice cultivation is limited and production cannot be increased by more acreage, so additional, applied research is needed to find other ways of increasing productivity. With limited land resources and increased demand for enhanced production attention is turning towards intensification through higher fertilizer inputs, which is predicted to result in higher yields. Despite a sound logic base supporting increased fertilizer inputs in some rice cropping systems, possible indirect adverse effects of increased fertilizer inputs were highlighted by Heong. In the last decade, plant hopper outbreaks in rice fields have intensified across Asia resulting in heavy yield losses. Over the past decade yield losses substantially increased due to a widespread outbreak of the brown plant hopper.

For example, the Central Plains of Thailand suffered from persistent Plant hopper outbreaks for 10 consecutive growing seasons from 2008 to 2012 and caused losses worth $52 million or equivalent to about 173,000 tons in 2010. The same pest was responsible for losses of around 1 million tons in Vietnam in 2007, and resulted in a government freeze on rice exports. Relationships among fertilizer applications and insect pest outbreaks are widely described in the scientific literature, especially in response to nitrogen fertilization. Specifically regarding rice-based cropping systems, there is a considerable body of research highlighting the indirect effects of fertilizer applications on crop susceptibility to pests. As an example, BPH prefer to feed and oviposit on rice plants supplied with nitrogen. BPH reared on plants with high nitrogen content had high feeding rates and honeydew excretion, less probing behavior, higher survival rates and population build-ups, higher fecundity and oöcyte production, and higher risk of economically important BPH outbreaks. Nitrogen fertilization has also significantly increased the populations of white-backed plant hoppers , green leaf hoppers, and small brown plant hoppers. Finally, Pandey reported higher incidence of rice leaf rollers damage at higher levels of nitrogen fertilization. Phosphorus fertilization has been reported to markedly increase population growth of BPH. Phosphorus alone or combinations with nitrogen and nitrogen-phosphorus-potassium treatments are reported to support moderate leaf hopper populations. Treatments with phosphorus alone and phosphorus in combination with nitrogen also increased populations of ear head bugs and associated grain damage. It has been suggested that phosphorus tends to increase abundance of yellow stem borers in rice, but to a lesser degree than nitrogen. High fertilization levels of both nitrogen and phosphorus caused higher levels of damage by blue beetles in rice crops. Regarding potassium fertilization, there are reports of negative associations between application rates and prevalence of insect pests in rice. As an example, Kulagold et al. reported that higher potassium fertilization of rice plants led to reduced densities of green leaf hoppers, yellow stem borers, blue beetles, rice leaf rollers and ear head bugs. The rate of rice stem borer infestation was greatest when there was no supply of potassium, but decreased in response to increased potassium concentration in rice plants. Silicon content in rice is reported several folds higher than N, P, and K, also promoting a beneficial effect in rice. Recently Guntzer et al. reported that Si increases resistance against insect pests,vertical farming companies pathogens and abiotic stresses including salinity, drought and storms. In this research, N*P*K interactions in a factorial experiments upon Si content of rice is one of the measured attributes. Ample evidence supports a general hypothesis that excessive crop fertilization regimes affects both risk of infestation and severity of economically insect-induced crop losses. However, there are few studies where the combined effects of nitrogen, phosphorous, and potassium fertilizers are studied in detail regarding BPH infestations and its life history characteristics on rice, and also as collectively altered by varying Si content of rice plant tissues. Our previous study showed that biochemical constituents of BPH varied with nutrient levels at different growth stages, and changes in relative water content of rice plants. Moreover, concentrations of N and P were found much higher in the BPH body than in its host rice plants, and this elemental mismatch is an inherent constraint on meeting nutritional requirements of BPH. In this study, rice plants were grown in pots under factorial combinations of fertilizer regimes and subsequently assessed as host plants for BPH. The following specific objectives were to explore direct effects of NPK fertilizer regimes on physiological characteristics of rice plants, and indirect effects of these regimes on fitness traits of BPH.The principal component analysis of fertilizer regimes, nutritional elements of rice plants, and BPH fitness trait responses showed that 78% of the total variance could be explained by the two principal axes, PCA1 and PCA2 . To improve visualization, four variables were not included in Fig. 1, but they were located within the space denoted “cluster 1”.

Due to the high level of variance explained in the two-dimensional space defined by the principal axes, we could make fairly strong inferences about the relative associations of the explanatory variables to show that: 1) there were very close associations between fertilizer regimes and the corresponding content of the same macro elements in rice plants; 2) all agronomic rice plant traits were positively associated with nitrogen fertilization ; 3) nitrogen fertilization was strongly associated with PCA1 explaining most of the variance in the data set; 4) the PCA2 axes was clearly associated with a negative relationship between free soluble sugar and potassium in rice plants.Nitrogen fertilization significantly increased BPH survival from egg to nymph or to adult and all other fitness traits were positively correlated, which indicated these traits improved significantly with the increase of nitrogen inputs . Fitness responses by BPH to phosphorus showed the same trend as nitrogen, but the level of association was more modest. There was a negative association between potassium and soluble sugar , and these two plant nutrition traits were orthogonal to the BPH fitness traits located along the principal axis, PCA1. This suggested that potassium and soluble sugar had only a minor influence on BPH fitness traits. Very interestingly, BPH nymphal development time was positively associated with silicon plant content and negatively correlated with all other principal BPH fitness traits.BPH is one of the most important insect pests of rice in Asia, and it has been an extremely severe pest of rice for decades. However, BPH was a relatively minor rice pest prior to the advent of high-input rice farming which became major after the development and widespread adoption of new high yielding varieties that required increased inputs of fertilizers and pesticides. These practices altered the crop microclimate and accentuated the pest problem such as shifting of the BPH from a minor to a major insect pest. Our data demonstrate that variation of fertilizer inputs to rice plants significantly affected fitness traits of BPH. Firstly, higher nitrogen input increased the survival rate from egg to nymph or adult stages. Secondly, nitrogen excess increased fresh body weight and enhanced the development of BPH. Thus, higher nitrogen input increased the all fitness traits of BPH. Phosphorus and potassium fertilizers had no significant effect BPH. But if phosphorus is supplied with higher nitrogen input , phosphorus significantly influenced several fitness traits including fecundity , fresh body weight and total number of adult BPH developed from one pair of BPH . Potassium has no significant effect on any fitness traits of BPH either alone or combined with higher nitrogen input , but may as shown by MANOVA and stylized 3D plots , exhibit subtle effects that merit further experiments. These results corroborate the first aim of this study by making it clear that altered fertilizer inputs to rice plants can trigger a bottom-up effect on the fitness traits of BPH. Thus, three questions remain to be answered. What are the physiological changes in the host plants that could explain the effects on the fitness traits of BPH? Do our findings support or refute the “Plant vigor hypothesis” which suggests that herbivorous insects prefer and perform better on rapidly-growing plants? Do our findings support or refute the “Plant stress hypothesis”and what can learn from this study to better manage rice production by fertilizer by pest relationships? Different fertilizer treatment combinations influenced rice plant physiology and in turn affected the fitness traits of BPH. When higher doses of nitrogen fertilizer were applied, plants accumulated higher amounts of nitrogen and soluble protein content in their plant tissue, which ultimately influenced herbivore growth and development . Egg hatch ability also increased with nitrogen content, resulting in more BPH produced with higher dry body weights.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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