Tag: vertical farming

High-Frequency Nitrate required separate injections of monoammonium phosphate and Ca2 in order to avoid precipitation of Ca32. All fertigations lasted 24 h, starting and ending at 7:00 AM, and injection of the non-MAP fertilizers took place between 11:00 PM and 3:00 AM. The soil at the site is classified as a Milham sandy loam derived from granitic and sedimentary alluvium, deep and well-drained. In an existing experiment , five blocks are laid out as a randomized complete blocks design, with eight strip-plot treatments of 15 trees. Treatments assessed had K applications of 224.17 kg ha 1 and P applications of 74.46 kg ha 1. Four blocks were studied for N2O surface emissions, and three of these included soil gas and solute measurements.Static gas chambers were used, of 20.3 cm diameter PVC piping 11 cm high placed over PVC collars installed 5 cm deep directly after fertigation, with aluminum insulation, rubber bottom seals, and manual internal fans. Two drippers were sampled in each plot per event, and different drippers were studied at each fertigation event; those studied were situated about 2 m from the nearest tree trunk, to represent average temperature, moisture and root presence. Chambers were positioned at 4 distances from drippers, along a transect into the center of the alley, at 0, 20, 50 and 90 cm. The maximum extent of the wetting zone at surface rarely exceeded 60 cm from drippers. Emissions from each collar were used to describe emissions from rings around the dripper,hydroponic growing systems with borders midway between distances. Three gas samples were taken over 10-min intervals. To calculate fluxes the ideal gas law was used as by Parkin and Venterea including ambient temperatures from a CIMIS weather station 2.5 km from the orchard, and an elevation of 120 m, corresponding to 0.985 atm of pressure.

Nonlinearity was not apparent with these short flux times.Surface emissions of N2O were expected to derive almost entirely from N fertigations and the first subsequent irrigations in the Standard treatment , with strong seasonal dependence that informed the sampling schedule . Output rates of drippers were checked. Gas samples were taken around peak emissions on the first day after fertigation, and thenceforth from 8:00–14:00 to represent daily averages. Emissions for statistical comparisons were the sums of observed measurements per plot, considered as describing a whole day. However, in order to be comparable to Standard, emissions from the HF applications required interpolations of unmeasured events, which were averages of each measured event’s emissions with the previous or the next. Previous experience of the patterns of emission demanded a different approach in order to estimate total growing season emissions per treatment. First day emissions were calculated assuming that N2O emissions increased linearly from baseline at 7 A.M. until peak ambient temperature, which was close to the time of measurement, then a Q10 adjustment was made using hourly ambient temperatures for the remainder of the day and night . Both of these manipulations sought to avoid overestimation. Q10 values were derived from seasonal diurnal measurements. To estimate the emissions over two additional days after sampling, an exponential curve of decline was fit to measured points averaged by treatment, as emissions = y0 + a, which had the best fit to data. Baseline rates were calculated by treatment to describe time periods when the upper soil was dry, starting 5–7 days after each fertigation, where the curve of decline ended. These were extended until the first day of fertigation for emissions calculations.Soil gas, moisture, temperature and N-distribution profiles were assessed in 3 experimental blocks. 1/800 OD brass tubing was used to take gas samples from depths of 5,10, 15, 20, 30, 45, 60 and 80 cm in the soil, at 20 cm from drippers.

The tubing was reinforced with steel wire, for insertion, after which the wire was withdrawn and replaced with a second, close-fitting internal brass tube of 3/ 3200 OD to minimize internal air volume. Tubes were topped with septa consisting of Nalgene tubing of 1/800 inner diameter injected with a dual plug of silicone and butyl rubber; butyl rubber blocks N2O diffusion while silicone maintains a good seal through syringe use. A volume of air corresponding to the tube volume was extracted before sampling. 5-mL gas samples were taken from 5 and 10 cm depth, and 10-mL samples from 15 cm and greater depths. For samples at 5 and 10 cm, tubes with closed ends and lateral cuts near their tip were inserted at 45 to the soil surface, allowing the surface soil to be tamped down at the entrance point without compacting the soil to be sampled. On this sandy loam, samples could be taken 5 h after irrigation without apparent resistance due to soil water. Gas samples were stored in glass vials with silicone sealant applied over the butyl septum before evacuation to 45 mTorr. All samples were analyzed for N2O using a 63Ni electron capture detector , and larger vials were tested for CH4 and CO2 using a flame ionizing detector , both on the same gas chromatograph . Simultaneously, a custom-built thermo couple probe of diameter 3/1600 took soil temperature at 5, 10, 15, 20, 30, 45 and 60 cm . A neutron probe provided measurements of volumetric water content at 20, 30, 45, 60 and 90 cm, using aluminum access tubes, and a soil moisture count curve fitted to measurements from this soil . A Theta probe was used to measure uvol in the upper 10 cm. Texture and bulk density were tested at each site at 0–20, 20– 30, 30–45, 45–60 and 60–90 cm. To compare the vertical distribution of dissolved NH4 + and NO3
in the soil after fertigations, high-flow ceramic soil solution samplers were installed at 15, 30 and 60 cm depth, 20 cm from the dripper laterally. Evacuation down to 600 mm Hg of vacuum was accomplished with an automotive brake pump, and samples were collected 6 h later, but on the third days after fertigation, samples adequate for analysis were only extracted from 38% of samplers, with fewer in summer, and very few in general from 15 cm depth.

Beginning in June, exchangeable NH4 + and NO3
were also assessed using soil samples from 0 to 5, 12.5–17.5 and 27.5–32.5 cm on each day. Approx. 20 g fresh weight soil was extracted in 80 mL of 2 M KCl, with one hour of shaking. These and the samples of soil solution were run for colorimetric analysis of NH4 + and of NO3.N2O concentrations by depth were combined with moisture readings, bulk density and texture to estimate the soil diffusion coefficient for nitrous oxide using the Unified Diffusivity Model— Buckingham Burdine Campbell . The Campbell parameter “b” was estimated using the clay fraction . Net production or consumption of N2O was estimated for each measured point in the soil except the deepest, using the one-dimensional mathematical model of Yoh et al. and field measurements described above.The soils were collected in late August after a month of irrigations without fertilizer. One replicate per plot was used, and results analyzed as an RCB consistent with the field experiment. pH was tested with 1 g:2 mL slurry in deionized distilled water, using samples from 0 to 20 cm depth at 20 cm from drippers. Treatment effects on nitrification rate were tested with the same samples using an ammonium oxidation assay citing Berg and Rosswall. Treatment effects on denitrification were tested with the same samples using a modified denitrification enzyme activity procedure with N2 as a flushing gas, with glucose, and without chloramphenicol. 5% head space acetylene, generated from calcium carbide,hydroponic farming was added to a round of duplicate samples; in the first test on the soils, 10% head space acetylene had appeared to impede the entire denitrification process. Head space gas samples were nonlinear by 60 min, so data from 20 and 40 min were used. The microcosms were also tested at 24 h to describe Denitrification Potential ; by 48 h, N2O declined in some microcosms. Soils from UAN treatments were tested for N2O production over a 36-h incubation at 3% O2 and 50% WFPS according to the method of Zhu et al. with some modifications. Samples of 15 g dry weight underwent a 24-h pre-wetting at 25% WHC, then were put into 180 mL Erlenmeyer flasks with butyl rubber stoppers and twice evacuated to 1000 mTorr and flushed for a minute with N2. Flasks were then injected with O2 for a 3% O2 head space, then fertigated to 50% WHC with NH4 + or NO3, with and without acetylene, and pure water . 5 mL head space samples assessed N2O production rates through measurements at 5 and 15 h; by 25 h they had declined in NH4 + microcosms, although N2O production continued.Estimated growing season N2O emissions were greatest from HF UAN fertigation, followed by Standard UAN, and lowest from the HF NO3 treatment, but comparisons between the two UAN and the two HF treatments only showed a significant difference between the HF systems . The failure to find differences in the Standard UAN—HF UAN comparison may have been due to high variability within and between blocks for the Standard UAN plots; in the UAN comparison the significance of blocking was p < 0.69 vs. p < 0.12 in the HF comparison. CVs over the measured period were 52% in Standard, 24% in HF UAN and 27% in HF NO3. Emissions from the irrigations following Standard UAN fertigations totaled 26% of the treatment’s growing-season emissions. In previous work, little effect had been seen in subsequent irrigations, even in loam soils . In the HF treatments such residual effects were indistinguishable from effects of the next fertigation.

There were important seasonal effects; higher temperature caused earlier and higher peaks of N2O emission, as well as higher event totals .The average precipitation at the site is 18 cm. The winter of 2013–2014 which followed our work was the driest on record in California; the experimental site received only 2 cm of precipitation and so was not monitored for emissions. However, previous work in the orchard , monitoring UAN-derived N2O emissions from static sprinklers at similar application rates, included a winter with 12 cm precipitation. Of the 730 g N2O-N yearly average emission, those data allowed an estimate that 18.5 g N2O-N ha
1 were emitted in winter. The latter quantity, if it had been seen in all the treatments studied here, would increase estimated N2O emissions by 3.7–8.5%. At the same time, Mediterranean perennial crop systems usually see more winter rainfall, and are capable of supporting cover crops in the winter. In such cases, post-season emissions can be expected to constitute a larger portion of the total and cover crop management, can be an important control, as studied in vineyards .To analyze the transport and fate of applied fertilizer, NH4 + and NO3 in soil solution were sampled by suction lysimeters and in soil by KCl extraction . Urea from Standard UAN was hydrolyzed to NH4 + and further oxidized to mobile NO3 in the soil profile, which by the third day led to 14x higher total NO3
concentrations at 60 cm depth under Standard UAN than under HF NO3 . Low dissolved NO3 in the HF NO3 treatment may be ascribed to higher diffusion to outer parts of the wetting zone , greater root uptake of nitrate through mass flow soon after fertigation, and the observed high retention of nitrate in the upper part of the soil . NH4 + in solution near surface agreed with differences in application strength , but such differences were not apparent in KCl-extractable NH4 + , which described much greater quantities.In general, a better understanding of the spatial origin and fate of N2O under different conditions should lead to improved fertilization and fertigation practices. In this investigation, net N2O production by depth was calculated from soil gas concentrations, soil diffusion parameters, temperatures and water contents  along a one-dimensional vertical profile at 20 cm from the dripper. A 1-D model cannot give a mass balance for N applied with water from a point source; unaccounted lateral diffusion will probably amount to a net loss and lead to underestimates of production. However this problem was judged to be ameliorated by conditions observed before the experiment.

Plants were harvested 45 days after germination when the differences in root characters could be efficiently measured among different recombinant lines. The data for different shoot characters were recorded and the tubes containing roots were stored at 4°C until processing. The roots were washed and recovered without damage using a Xotation technique . The shoot characters measured were longest leaf length , maximum width of the longest leaf , leaf area , plant height , number of tillers , and dry shoot biomass . The root characters measured were number of roots greater than 30 cm , longest root length , total length of roots greater than 30 cm , shallow root weight , deep root weight , dry root biomass , and root biomass to shoot biomass ratio .The shoot and root data were subjected to the analysis of variance for each year .The overall rooting ability of each genotype was calculated by ranking each genotype for individual root traits in each replication in each of the 3 years. Genotypes with the highest values were ranked 5 and those with the lowest values were ranked 1. Subsequently, all the ranks of root characters for each genotype were summed providing a measure of the rooting ability index for each genotype at different replications . The genotypic rank sums averaged across the years were subjected to the non-parametric Quade analysis developed for randomized complete block designs to differentiate genotypes for overall rooting ability.Phenotyping of recombinants was necessary to test the general applicability of the consensus 1RS-1BS map in locating a 1RS region showing better rooting ability. Various shoot and root traits were studied using two parents and three recombinants covering the whole 1RS-1BS map. There were significant differences among years for all shoot characters measured, except for the maximum width of the longest leaf .

Significant differences were found among the genotypes for shoot characters, except for maximum width of the longest leaf and leaf area. Genotype £ year interaction was significant only for the number of tillers per plant . This interaction was due to changes in the magnitude of the genotypic means across different years rather than changes in ranking of the means. Therefore, shoot characters in Table 2 are represented by means averaged across years. Pavon 1RS.1BL was taller,growing tomatoes hydroponically had longer leaf length and a greater root-to-shoot ratio than Pavon 76. Since Pavon 1RS.1BL and Pavon 76 had similar shoot biomass , greater root-to-shoot biomass ratio in the former genotype indicated greater root biomass in 1RS.1BL than Pavon 76 . Leaf area in 1B + 2 was the highest followed by Pavon 1RS.1BL . Despite significant differences observed among the genotypes for shoot characters, the differences were relatively small, except for the shoot biomass in 1B + 2 in the third year which was due to greater number of tillers per plant and plant height . Otherwise, the rest of the genotypes did not show large differences for combined as well as for individual years for most of the shoot traits. There were significant differences among years for all root characters measured . Significant differences were found among the genotypes for all the root characters measured, except for longest root length. The genotype £ year interaction was significant only for the number of roots greater than 30 cm. Therefore, means for root characters were averaged across 3 years . The number of roots greater than 30 cm and root biomass in Pavon 1RS.1BL was greater than those in Pavon 76, which confirmed the results reported earlier . The number of roots greater than 30 cm in Pavon 1RS.1BL, 1B + 2 and 1B + 38 was similar, but greater than those in Pavon 76 and T-14 . A similar trend was observed for the total length of roots greater than 30 cm. Shallow root weight was highest in Pavon 1RS.1BL followed by 1B + 2 , and 1B + 38 . The lowest shallow root weight belonged to T-14 . Deep root weight in Pavon 1RS.1BL, 1B + 2, and 1B + 38 were similar, but greater than those in Pavon 76 and T-14.

The greater dry root biomass observed in Pavon 1RS.1BL as compared to Pavon 76 was because of a combination of greater shallow and deep root weight in the former than in the latter genotype .Genetic mapping in wide hybrids has been performed for many plant species, particularly in diploids including barley , chickpea , lentils , onion , Nicotiana species , and tomato . In the early days of genetic mapping with molecular markers, wide hybrids were the approach of choice, for it guaranteed much higher levels of polymorphism than in intra-specific hybrids. Despite notable instances of non-Mendelian segregation and skewed distribution of recombination, wide hybrids produced useful genetic maps with higher marker saturation at considerably less cost and eVort . The use of wide hybrids in allopolyploids is more complicated than in diploids as allopolyploids tend to have some kind of chromosome pairing control system in place that limits crossing over to homologues. Hence, homoeologous pairing may be low or even non-existent. In this study, recombinants produced by crossing over were used. These recombinants were selected from a population of 103 such recombinants developed by Lukaszewski . The entire recombinant population was selected from a population of ca. 17,000 progeny with the Ph1 system disabled. If the assumption is made that crossing over in the Ph1+ and Ph1¡ wheats is the same, and they appear to be, except for the absence of multiple crossovers per arm , then the sample analyzed here would be equivalent to a population of 136 back cross progeny, a sensible number giving the maximum resolution level of 0.7 cM. The physical distribution of 68 recombinant breakpoints used in the present study is shown in Fig. 3. With a total of 20 physical and molecular markers, we constructed the combined genetic map of 1RS-1BS recombinant breakpoints in Pavon 76 background. The genetic map produced here has an average density of 2.5 cM. The maps shown in Figs. 2 and 4 represent only the physical 40% of the distal ends of the 1S arms as no recombination took place in the proximal 60% of the arm. Any loci in this region would show complete linkage with the centromere.

The advantage of using these recombinant breakpoints in developing a genetic map is in their physical differences from one another. Here, we used two methods to map. Firstly, we used the recombinant breakpoints to develop a genetic map of the arm, and secondly, we used the map to classify the breakpoints. Each interval or genetic distance between two markers is represented by one or more recombinant breakpoints that most often include both reciprocal configurations of the chromosomes. This physical separation of the breakpoints further refines the genetic map to 0.7 cM level. The reciprocal nature of chromosomes with breakpoints in any given interval will permit allocation of identified genetic loci to very narrow physical segments of rye chromatin. This may be a useful tool in dissecting the genetic components of a particular gene of interest or an important agronomic trait present on 1RS. Somers et al. detected an average of one single nucleotide polymorphism per 540 bp ESTs in wheat and demonstrated the reliability of designing PCR primers for locus-specific amplicon production. In this study, we targeted a single chromosome arm, 1BS,hydroponic growing supplies and expected that most of the 91 EST loci allocated to this arm could be converted to 1BS locus-specific amplicon markers. Given that the recombination was intergeneric, we expected that most of these markers would be polymorphic between wheat and rye, and so would produce a highly saturated map. However, only four PCR-based markers showed reliable polymorphism, though we cannot rule out sequence-based polymorphisms that we could not detect. The sequences of the 91 EST loci on wheat 1S arms were used to search rice orthologs using TIGR rice version 4.0 gene models, and 54 of these sequences had rice blastx hits of e-20 or better, comprising 52 rice gene models. Of the 52 models, were clustered on chromosome 5 of rice, which is known to besyntenic to chromosomes 1S of Triticeae . In the present study, two gene models with genebank accessions viz; BE497177 and BF483588 were found to be represented as markers, Xucr_4 and Xucr_8, respectively . The rice annotation of Xucr_8 is described as a drought induced 19 protein which can be helpful in studying drought. However, a high frequency of insertions and deletions in rice and maize make these genomes more Xuid at the DNA sequence level than indicated by Southern analyses . Testing a total of 16 SSR and eSSR markers yielded four polymorphic amplified products, which were comparable to the previous studies . Röder et al. found different band sizes for cultivar and synthetic wheat, though the differences were comparable. Only 1 out of 9 eSSR markers could be mapped on 1BS-1RS. This low success rate may be due to low polymorphism between wheat and rye 1S arms. Whether this indicates high conservation of genic regions across species we cannot tell at this point. Peng and Lapitan showed amplification of 15 wheat eSSR markers in rye as well as barley. They detected polymorphism between wheat and barley but did not mention polymorphism between wheat and rye. A comparison of the current 1RS- 1BS map with the SSR and eSSR maps of the 1B chromosome shows a good agreement in marker order, location, and relative positions in its distal part, regardless of the projection mode used. A similar approach was used to generate a genetic map of ph1b-induced 2R-2B intergeneric recombinants, with a similar success , validating the wide-hybrid approach to mapping. The integration of physical and molecular markers in the present 1RS-1BS map also provided the alignment of recombinant breakpoints over each map interval.

This information offers great potential to study agronomic traits affected by the introduction of alien 1RS chromatin into wheat. Our working hypothesis was if a 1B + line shows some specific trait then this trait should be absent in its complementary T-line, and vice versa. To check the applicability of this concept, we looked at different root characters in wheat. Studies during the past few years showed an increased root biomass in wheat with the alien 1RS chromosome arm over standard spring wheat Pavon 76 and found a positive correlation of increased root biomass with grain yield . Recent studies in rice and maize have also correlated the QTLs for root traits with QTLs for yield under Weld conditions . A major limitation to study roots is the difficulty in making observations as the process is very laborious and time consuming. In the present study, we tested only three recombinant chromosomes with breakpoints selected to divide the recombining portion of the arm into three segments of roughly similar lengths. The experiments were conducted in 3 years at the same months of the year, in replicated trials, to determine the magnitude of the genotype £ year interaction and the repeatability of results from the sand-tube technique. Significant variation was observed from year to year , with considerably higher means in the third year. This might have been due to relatively lower temperatures in the third year providing better conditions for vegetative growth. The significant genotype £ year interaction observed for number of roots greater than 30 cm was due to only one genotype, T-14, producing more roots only in year 2. Otherwise, other genotypes showed similar trends for this character across the 3 years. The lack of signiWcant genotype £ year interaction for most of the root characters examined indicated high repeatability for these root traits. Quade analysis used on rank sums separated the five genotypes in two groups viz., Pavon 1RS.1BL, 1B + 2 and 1B + 38 containing distal rye chromatin with higher rooting ability and Pavon 76 and T-14 lacking the distal rye segment with lower rooting ability . Overall, the wave genotypes examined showed relatively small variation for shoot characters but they differed in root characteristics including root biomass.

Conversely, while nCuO did not cause changes in mitochondrial membrane potential , CuSO4 significantly decreased MMP even at 1 mg/L. Interestingly, nCuO and CuSO4 acted as a chemosensitizers and increased the developmental toxicity of vinblastine, a cell division inhibitor . In an evaluation of 24 different metal oxides, nCuO was found to rank among the top most toxic within this group of NPs . Human bronchial epithelial and murine myeloid cell lines were used to assess oxidative stress and acute pulmonary inflammation. Exposure to nCuO resulted in a marked decline in cell viability even at 0.4 μg/L and a dose-dependent decline in the cellular energy levels . Additionally, nCuO also affected ROS production, intracellular calcium flux, mitochondrial membrane potential, and surface membrane permeability in BEAS-2B and RAW 264.7 cells . To corroborate the in vitro assays, in vivo toxicity assays of acute pulmonary inflammation were conducted with C57 BL/6 mice. nCuO induced statistically significant increases in neutrophil cell counts, monocyte chemo attractant protein-1 , and interleukin-6 levels, compared to less toxic NPs, indicating a strong immune system defense response.Pseudofeces can be ingested by other organisms in the water column or sediments, potentially resulting in Cu bio-availability and bio magnification. Given the likelihood that a fraction of Cu NPs will pass through WWTPs , the microbial community in a decentralized wastewater treatment system was subjected to 3 individual experiments lasting 3 weeks to a low uniform dose of different Cu particles: nCu, nCu2-b, and μCu . The three particles disrupted septic tank function,hydroponic grow system which was unable to meet treatment objectives in terms of 5- day biochemical oxygen demand and pH . There were also noticeable fluctuations in microbial community structure and phenotypes. However, 3 weeks after the end of exposure, the treatment system returned to baseline conditions .

This indicates that while a regular low dose of Cu from the NPs can result in sporadic system disruption, overall the treatment system maintains proper function. The Cu NPs were transformed to copper phosphate and sulfurized compounds, reducing the toxicity of the effluent by 95% or more . To more broadly assess the nanotoxicity of Cu NPs, dozens of studies with different organisms, including many in the USEPA ECOTOX database, were evaluated . Only acute toxicity in freshwater was considered, given the paucity in marine in general and freshwater chronic toxicity data.This information was used to construct species sensitivity distributions for freshwater organisms . The toxicity threshold was much higher for nCuO relative to nCu or Cu+ 2 ions . The SSDs for nCu and Cu+ 2 almost overlapped, indicating similar toxicity thresholds . Of the species used to develop the nCuO and nCu SSDs, the LC50 values tend to be lowest for various daphnia species , indicating high sensitivity, while crustaceans and fish tolerated higher concentrations of nCuO and nCu . The lower toxicity of nCuO in freshwater may reflect its slower dissolution at low ionic strengths and Cu+ 2 complexation in the presence of organic matter . Other studies indicate that the toxic effect observed from nCuO strongly correlates with the fraction of NP dissolved in aquatic media . In freshwater and marine systems, nCu and nCuO were found to cause some toxicity across a range of toxicological endpoints at concentration < 1 mg/L , while others only found toxicity at greater exposure concentrations .While lettuces did not exhibit any reduction in catalase activity, indicating no increased formation of ROS, alfalfa tissues exhibited a considerable change at 10 mg/L nCuO, and in alfalfa roots at 5 mg/L nCu2-a and μCuO . Surprisingly, ascorbate peroxidase activity in lettuce roots increased in all treatments except μCuO, and it also increased in alfalfa roots in all treatments except the two nCu2 nanopesticides. These early growth stage results indicate some noticeable effects from all Cu NPs, although no clear pattern was exhibited with regards to toxicity ranking based on physiological or biochemical indicators. Lettuce grown from seed to maturity in soil, and exposed through foliar spray to nCu2-b during the last 4 weeks before harvesting at 1050 mg/L to 2100 mg/L, exhibited no visible damage . In fact, leaf biomass, which is the harvestable product, increased significantly . However, metabolomics analysis revealed that nCu2-b altered metabolite levels in the lettuce leaves .

The tricarboxylic cycle and several amino acid-related biological pathways were disturbed. Some antioxidant levels were significantly decreased compared to the control, indicating that oxidative stress and a defense response occurred. Nicotianamine, a chelator, increased by 12 to 27 times compared to the control, which may represent a detoxification mechanism. The up-regulation of polyamines and increased potassium may mitigate oxidative stress and enhance tolerance . These changes in antioxidants within the leaves may alter their nutritional value.There were no visible signs of toxicity in the leaves. In contrast, for nCu applied to C. sativus, significant translocation of Cu was observed. When plants were exposed to 10 mg/L to 20 mg/L nCu in hydroponic media, Cu uptake in the roots was greater than in stems and leaves , but in all cases there was significant increase relative to unexposed controls . When plants were exposed to 200–800 mg nCu/kg in soil, Cu was also highest in the roots, but there was significant translocation to stems, leaves and even cucumber fruits . However, the translocation factor was lower in plants exposed to nCu and those only exposed to bio-available Cu in the soil , suggesting that not all the Cu taken up in the roots was available for translocation when it was supplied as nCu . Although the plants showed no visible damage, exposure to nCu reduced photosynthetic rate , and increased transpiration rate . The concentrations of Na, P, S, K, Mo, Fe and Zn decreased in all cucumber tissues in exposed plants . Formation of Cu-phosphate complexes at the root surface decreased P uptake. In addition, meta bolomics demonstrated that C. sativa engages an active defense mechanism in the root zone against nCu stress: up-regulation of amino acids to sequester/exclude Cu/nCu; down-regulation of citric acid to reduce the mobilization of Cu ions; ascorbic acid up-regulation to combat reactive oxygen species; and upregulation of phenolic compounds to increase antioxidant activity . nCu spiked in soil also up- or down-regulated 15 metabolic pathways in cucumber fruits: carbohydrate metabolism , amino acid synthesis and metabolism , and pathways related to lipid metabolism, biosynthesis of other secondary metabolites, and energy metabolism . Despite these metabolic changes, root, stem, leaf and fruit bio-masses were not impacted by exposure to nCu, so the effect may be only on fruit quality and needs further exploration . While crop plants are the intended recipients of nanopesticides and are more likely to be exposed to Cu NPs in biosolids, impacts to surrounding wild plants were also considered.

A study compared the effects of nCu2-b on a wild herbaceous annual plant , radish , and wheat under the same conditions. The uptake and toxicity of nCu2-b grown in potting, grassland, or agricultural soils were found to be dependent on plant species, soil type, soil nutrient levels, and illumination intensity . Exposure to soil nCu2-b concentrations as low as 10 mg/L caused stunted growth in C. unguiculata and resulted in a complete cessation of photosynthesis during the period of peak growth in individuals under high stress growth conditions . These plants also had elevated fractions of oxidized photosystem II reaction centers, which is consistent with known mechanisms of photosynthetic disruption caused by ionic Cu . Uptake and translocation patterns also suggest that nCu2-b underwent partial or total dissolution in the potting soil used , and were being taken up as ionic Cu rather than as particulate Cu2 . However, C. unguiculata, radish, and wheat grown in grassland or agricultural soils had leaf Cu concentrations 1 to 2 orders of magnitude lower than plants grown in potting soil, with the highest Cu concentrations in roots. Few physiological impacts due to nCu2- b exposure were seen in these plants, possibly as a result of decreased translocation to leaves. One exception was seen in wheat grown in grassland soil under low illumination levels during the 8th week of exposure to 25 mg nCu2-b/L·week, which had decreased PsiI quantum yield efficiencies, photochemical quenching, electron transport rates,hydroponic growing systems and ratios of oxidized PsiI reaction centers, compared to control plants. Additionally, radish plants grown under high light conditions and exposed to nCu2-b had significantly larger hypocotyls than control plants, but there was significantly lower grain yield in wheat plants grown under the same conditions. In summary, although effects were observed in seedlings and young plants when exposed to the various Cu particles and dissolved Cu2+, mature plants generally exhibited fewer apparent symptoms, and in several cases produced more biomass, particularly in tissues with commercial value . The terrestrial plants tolerated the exposure to Cu NPs better than aquatic organisms, with higher levels needed to produce measurable effects. This may be due to lower dissolution of Cu in terrestrial environments due to interactions with soil and natural organic matter , and the need for translocation through the root systems. In general, exposure to Cu compounds resulted in alteration of metabolite profiles, inducing anti-oxidant response, potential defense mechanisms , reduced photosynthetic rates and increased transpiration rates in some species. There was no clear trend in toxicity ranking, in some cases nCu being the most toxic, but in many others nCuO was as toxic or more. Only a few studies have been conducted that directly test the toxicity of terrestrial organisms to Cu NPs in soil media. Generally, growth inhibition does not occur until relatively high exposure concentrations, indicating that other toxic endpoints would not occur at likely environmental concentrations for either nCu or nCuO. Rousk et al. found an acute toxic response to soil bacteria from nCuO that did not occur upon exposure to bulk CuO. The growth inhibition EC50 was found to be around 2254 mg/kg for soil bacteria .

Dimkpa et al. investigated the effects of nCuO on sand grown wheat and beans and found both reduced root and shoot growth at 500 mg/kg, with again a greater effect seen from the nano scale rather than the bulk particles . These effects were not observed until relatively high exposure concentrations were reached . Unrine et al. found no adverse effect to E. fedita to nCu at exposure concentrations as high as 65 mg/kg across a range of ecologically relevant endpoints . A number studies have been conducted on the sensitivity and toxicity of microbes and plants to nCuO, however the majority of these were tested in growth media and not in soil. For example, Baek and An found the 24-hr microbial growth inhibition EC50 to fall between 28.6 and 65.9 mg/L . Future studies should consider the effects of Cu NP speciation and transformation, once they are released from the product into different environmental matrices, on toxicity. For example, the toxicity of most Cu NPs was decreased by > 95% after passing through the WWTP, and the speciation was very different from the input NPs. Studies are needed that go beyond the original, pristine NPs. Further work is needed to understand Cu NP toxicity in soils and in particular microbial communities.Aquaporins , also known as MIPs , are integral membrane proteins that increase the permeability of membranes to water, as well as small uncharged molecules. Of all kingdoms, the plant kingdom contains the largest known AQP family consisting over 30 members. There are AQPs in Arabidopsis , in maize and in tomato , tonoplast intrinsic proteins , NOD26-like intrinsic proteins , small basic intrinsic proteins and X intrinsic proteins. Plant PIPs can be divided into two major groups, PIP1 and PIP2, on the basis of their sequences and water-channel activity. PIP2 proteins exhibit high levels of water-channel activity in Xenopus oocytes and yeast vesicles; whereas PIP1 proteins often have relatively low permeability to water. Evidence for the role of PIP1 aquaporin in planta has come from mutant analyses and the manipulation of PIP1 expression in plants. Analysis of Arabidopsis mutants has shown that AtPIP1,2 can account for a significant portion of aquaporin-mediated leaf water transport. The antisense expression of AtPIP1,2 in Arabidopsis has been associated with reductions in the membrane hydraulic conductivity of isolated protoplasts and decreased total root hydraulic conductivity.

Our experimental results demonstrate that Cipro may interfere with the photosynthetic bio-energetic pathway, in addition to causing morphological deformities in higher plants. The range of EC50 values estimated from concentration-response models of the increase in inactive excited PSII Chl units and the decrease in root length are, respectively, 10.3-111.7 and 0.532-0.776 µM, both of which are higher than the respective LOEC values . Although these concentrations are higher than those typically found in surface waters , they are comparable with those found near industrial effluents . Therefore, the observed adverse in vivo response demonstrated here may be relevant to sensitive plants located near high-FQ-containing waters. Finally, the present study not only stresses the need, as pointed out previously , to consider sublethal molecular targets as potential meaningful phytotoxicity end points, but also takes a step toward the evaluation of FQ cumulative effects, a current challenge in the environmental risk assessment of pharmaceuticals , by providing correlations between FQ structures and specific molecular mechanisms of toxic action.Increasing population and consumption have raised concerns about the capability of agriculture in the provision of future food security.Recent estimates suggested that global agricultural production should increase by 70% to meet the food demands of a world populated with ca. 9.1 billion people in 2050. Food security is particularly concerning in developing countries, as production should double to provide sufficient food for their rapidly growing populations. Whether there are enough land and water resources to realize the production growth needed in the future has been the subject of several global-scale assessments. Te increase in crop production can be achieved through extensification and/or intensification. At the global scale, almost 90% of the gain in production is expected to be derived from improvement in the yield, but in developing countries,vertical plant rack land expansion would remain a significant contributor to the production growth.

Land suitability evaluations, yield gap analysis, and dynamic crop models have suggested that the sustainable intensification alone or in conjugation with land expansion could fulfil the society’s growing food needs in the future. Although the world as a whole is posited to produce enough food for the projected future population, this envisioned food security holds little promise for individual countries as there exist immense disparities between regions and countries in the availability of land and water resources, and the socio-economic development. Global Agro-Ecological Zone analysis suggests that there are vast acreages of suitable but unused land in the world that can potentially be exploited for crop production; however, these lands are distributed very unevenly across the globe with some regions, such as the Middle East and North Africa , deemed to have very little or no land for expansion. Likewise, globally available fresh water resources exceed current agricultural needs but due to their patchy distribution, an increasing number of countries, particularly in the MENA region, are experiencing severe water scarcity. Owing to these regional differences, location-specific analyses are necessary to examine if the available land and water resources in each country will surface the future food requirements of its nation, particularly if the country is still experiencing significant population growth.As a preeminent agricultural country in the MENA region, Iran has long been pursuing an ambitious plan to achieve food self- sufficiency. Iran’s self- sufficiency program for wheat started in 1990, but the low rate of production increase has never sustainably alleviated the need for grain imports. Currently, Iran’s agriculture supplies about 90% of the domestic food demands but at the cost of consuming 92% of the available freshwater. In rough terms, the net value of agricultural import is equal to 14% of Iran’s current oil export gross revenue. Located in a dry climatic zone, Iran is currently experiencing unprecedented water shortage problems which adversely, and in some cases irreversibly, affect the country’s economy, ecosystem functions, and lives of many people. Te mean annual precipitation is below 250 mm in about 70% of the country and only 3% of Iran, i.e. 4.7 million ha, receives above 500 mm yr−1 precipitation.

The geographical distribution of Iran’s croplands shows that the majority of Iran’s cropping activities take place in the west, northwest, and northern parts of the country where annual precipitation exceeds 250 mm . However, irrigated cropping is practiced in regions with precipitations as low as 200 mm year−1 , or even below 100 mm year−1 . To support agriculture, irrigated farming has been implemented unbridled, which has devastated the water scarcity problem. The increase in agricultural production has never been able to keep pace with raising demands propelled by a drastic population growth over the past few decades, leading to a negative net international trade of Iran in the agriculture sector with a declining trend in the near past . Although justified on geopolitical merits, Iran’s self-sufficiency agenda has remained an issue of controversy for both agro-ecological and economic reasons. Natural potentials and constraints for crop production need to be assessed to ensure both suitability and productivity of agricultural systems. However, the extents to which the land and water resources of Iran can meet the nation’s future food demand and simultaneously maintain environmental integrity is not well understood. With recent advancement in GIS technology and availability of geospatial soil and climate data, land suitability analysis now can be conducted to gain insight into the capability of land for agricultural activities at both regional and global scales. Land evaluation in Iran has been conducted only at local, small scales and based on the specific requirements of a few number of crops such wheat, rice and faba bean. However, there is no large scale, country-wide analysis quantifying the suitability of Iran’s land for agricultural use. Herein, we systematically evaluated the capacity of Iran’s land for agriculture based on the soil properties, topography, and climate conditions that are widely known for their relevance with agricultural suitability. Our main objectives were to: quantify and map the suitability of Iran’s land resources for cropping, and examine if further increase in production can be achieved through agriculture expansion and/or the redistribution of croplands without expansion. The analyses were carried out using a large number of geospatial datasets at very high spatial resolutions of 850m and 28m .

Our results will be useful for estimating Iran’s future food production capacity and hence have profound implications for the country’s food self-sufficiency program and international agricultural trade. Although the focus of this study is Iran, our approach is transferrable to other countries,what is a vertical farm especially to those in the MENA region that are facing similar challenges: providing domestic food to a rapidly growing population on a thirsty land.We classified Iran’s land into six suitability categories based on the soil, topography, and climate variables known to be important for practicing a profitable and sustainable agriculture. These suitability classes were unsuitable, very poor, poor, medium, good, and very good . This classification provides a relative measure for comparing potential crop yields across different lands. Four major land uses that were excluded from the suitability analysis comprised 19.3 million ha of Iran’s land , leaving 142.8 million ha available for agricultural capability evaluation .When land suitability was evaluated solely based on the soil and topographic constraints , 120 million ha of land was found to have a poor or lower suitability ranks . Lands with a medium suitability cover 17.2 million ha whilst high-quality lands do not exceed 5.8 million ha . Te spatial distribution of suitability classes shows that the vast majority of lands in the center, east and, southeast of Iran have a low potential for agriculture irrespective of water availability and other climate variables . As shown in Fig. 2, the potential agricultural productivity in these regions is mainly constrained by the low amount of organic carbon and high levels of sodium contents . Based on soil data, Iran’s soil is poor in organic matters with 67% of the land area estimated to have less than 1% OC. Saline soils, defined by FAO as soils with electrical conductivity >4 dS/m and pH<8.2, are common in 41 million ha of Iran. Although many plants are adversely affected by the saline soils , there are tolerant crops such as barley and sugar beet that can grow almost satisfactorily in soils with ECs as high as 20 dS/m, which was used as the upper limit of EC in this analysis .Although sodic soils are less abundant in Iran , soils that only have high ESP covers ~30 million ha . We used an ESP of 45% as the upper limit for cropping since tolerant crops such as sugar beet and olive can produce acceptable yield at such high ESP levels.As shown in Fig. 2, EC is not listed among the limiting factors, while we know soil salinity is a major issue for agriculture in Iran. This discrepancy can be explained by the higher prevalence of soils with ESP>45% compared to those with EC>20 dS/m, which can spatially mask saline soils. Tat is, the total area of soils with EC>20 dS/m was estimated to be about 6.4 million ha , while soils exceeding the ESP threshold of 45 constituted ~12 million ha i.e. almost double the size of saline soils. Iran’s high-quality lands for cropping are confined to a narrow strip along the Caspian Sea and few other provinces in the west and northwest .

In the latter provinces, the main agricultural limitations are caused by high altitude and steep slopes as these regions intersect with the two major mountain ranges in the north and west .Thus far, the land suitability analysis was based on soil and terrain conditions only, reflecting the potential agricultural productivity of Iran’s without including additional limitations imposed by the water availability and climatic variables. However, Iran is located in one of the driest areas of the world where water scarcity is recognized as the main constraint for agricultural production. Based on aridity index, our analysis showed that 98% of Iran could be classified as hyper-arid, arid, or semi-arid . August and January are the driest and wettest months in Iran, respectively, as shown in Fig. 3. Over half of the country experiences hyper-arid climate conditions for five successive months starting from June . This temporal pattern of aridity index has dire consequences for summer grown crops as the amount of available water and/or the rate of water uptake by the crop may not meet the atmospheric evaporative demand during these months, giving rise to very low yields or total crop failure. It must be noted that the high ratio of precipitation to potential evapotranspiration in humid regions could also result from low temperature rather than high precipitation. There is a high degree of overlap between regions that experience wetter conditions for most of the year and those identified as suitable for agriculture based on their soil and terrain conditions . This spatial overlap suggests that some of the land features relevant to cropping might be correlated with the climate parameters. In fact, soil organic carbon has been found to be positively correlated with precipitation in several studies. To incorporate climate variables into our land suitability analysis, we used monthly precipitation and PET as measures of both overall availability and temporal variability of water. We derived, from monthly precipitation and PET data, the length of the growing period across Iran . Growing period was defined as the number of consecutive months wherein precipitation exceeds half the PET. As shown in Fig. 3, areas where moisture conditions allow a prolonged growing period are predominately situated in the northern, northwestern, and western Iran with Gilan province exhibiting the longest growing period of 9 months. For over 50% of the lands in Iran, the length of the moist growing period is too short to support any cropping unless additional water is provided through irrigation . However, these areas, located in the central, eastern, and southeastern Iran, suffer from the shortage of surface and groundwater resources to support irrigated farming in a sustainable manner. Taking into account daily climate data and all types of locally available water resources can improve the accuracy of the length of growing period estimation.

Synergistic effects on plant growth under several conditions when PGPR and AMF are coinoculated are reported . In our study, however, the effects of combined inocculation with two PGPRs or with PGPRs and AMF never exceeded the sum of the effects of single inocculation with PGPRs and AMF. This indicates additive effects of PGPRs and AMF rather than clear synergistic effects. Roots of tomato after application of both bacteria strains were not only healthier but also showed a significantly higher colonization by AMF Glomus intraradices, indicating that AMF infection in the soils was suppressed directly by pathogens or indirectly as consequence of poor root development. Azcón and Linderman reported that unidentified PGPR have a strong stimulatory effect on the growth of AMF and increased mycelia growth of G. mosseae spores. P. fluorescens 92rk, alone or co-inoculated with P. fluorescent P190r, increased mycorrhizal colonization of tomato roots by G. mosseae BEG12 . Similarly to the results obtained by Marulanda-Aguirre et al. , where Bacillus megaterium inoculated with G. intraradices showed the highest percentage of mycorrhizal root length of Lactuca sativa plants compared to the single inoculation of G. intraradices. These results suggest that PGPR and AMF might be co-inoculated, at least in soils with a low AMF status,vertical hydroponic to optimize the formation and function of the mycorrhizal symbiosis. Both PGPR and AMF inoculation treatments directly and indirectly improved the nutrients acquisition and allocation to the shoots of tomato plants. The concentrations of P, Mn and Zn in tomato shoots were higher after inoculation with P. sp. ”Proradix®” and B. amyloliquefaciens FZB42 when compared to the untreated control. The ability of P. fluorescens and AMF to promote plant growth by improved nutrient acquisition and suppression of soil borne pathogens is well documented.

Both functions may promote plant growth but by different mechanisms. AMF facilitated mineral and water uptake, and increased the defense against soil borne pathogens . PGPRs induced the release of plant growth regulators . Siddique reported that Pseudomonas spp. can synthesize certain enzymes that can modulate plant hormone levels, might limit the available iron via siderophore production and can also kill pathogens by production of certain antibiotics. Our study confirmed that, B. amyloliquefaciens FZB42 can act as a PGPR, as described by De Freitas et al. , Kokalis-Burelle et al. , Kishore et al. and Marulanda-Aguirre et al. . Phae et al. reported that B. subtilis NB22 significantly reduce the occurrence of crown and root rot disease of tomato. Another aspect in the present study was to test if the mixtures of different bacteria species improve the control against FORL compared to one bacterium species alone. Our results did not confirm Pierson and Weller and Schisler et al. who proposed a strategy to increase the efficacy and the consistency of disease control by mixed application of antagonistic microorganisms with different modes of actions. Cordier et al. stated that dual or multiple inoculations of beneficial microorganisms can be neutral, positive or negative depending on the inoculants used. However, our study showed that combined application of two PGPRs improved tomato growth and suppressed FORL to the same extent as single application or further increases P shoot concentrations. Our results are in agreement with studies by Raupach et al. , Pierson and Weller and Duffy et al. , all of which demonstrated that certain mixtures of PGPR were significantly suppressive to cucumber pathogens and take-all disease. Different mechanisms of action for different PGPR strains may explain why combinations of P. sp. ”Proradix®” and Bacillus amyloliquefaciens FZB42 suppress disease similar to inoculation with single strains. Sung and Chung demonstrated that chitinase-producing Streptomyces spp. and B. cereus isolates used in combination with antibiotic-producing P. fluorescens and Burkholderia cepacia isolates had a synergistic effect on the suppression of rice sheath blight and Szczech and Dyśko who reported that among tested bacterial inoculations, only mixture of the bacteria B125 and PT42 tended to affect positively the growth of the plants and to reduce the density of Fusarium spp. in the rhizosphere of tomato plants.

These results indicate that the consistency of biocontrol agents in suppression of soil borne pathogens influenced by many factors, i.e. bacterial strains, soil borne pathogen species, species of plant, etc. Protein expression in plant systems has the potential to provide a safe, cost-effective, and scalable method to meet the increasing need for therapeutic protein production. Plant-based expression offers several advantages to the bio-pharmaceutical industry, including decreased cost of production, scalability, lack of susceptibility to mammalian pathogens, elimination of animal- or human-sourced raw materials, and the production of complex proteins with post-translational modifications such as N-glycosylation. For many therapeutic proteins, N-glycosylation is essential for protein folding, oligomerization, quality control, enzyme activity, ligand interactions, localization, and trafficking. Despite its potential, a possible barrier to the commercialization of plant-made glyco protein drugs is the difference between the N-glycan structures of human and plants. Of particular concern are plant-specific structures contained in complex type N-glycans, namely, α1,3 core fucose, β1,2 bisecting xylose, and the Lewis a epitope. Even though there is no definitive proof of adverse effects from plant-specific glycan structures, the presence of nonhuman glycans could potentially cause unwanted immunogenicity in humans, and the lack of sialic acid termination may lead to reduced blood circulatory half-life. A change in glycan structure could also potentially alter the protein’s structure or accessibility of its epitopes and, consequently, its function. Therefore, to ensure the efficacy of a plant-made bio-similar therapeutic, it is important that the N-glycans are compatible with both the protein’s function and the human immune system. Several strategies exist to modify a glyco protein’s N-glycan structures in planta, such as glycoengineering of the host cells using CRISPR/Cas9 genome editing to knock outβ-xylosyltransferase genes and α-fucosyl transferase genes and RNA interference technology to down regulate XylT and FucT genes, targeting of the protein to specific organelles, addition of compounds to alter the function of glycan-modifying enzymes, and in vitro glycan remodeling using chemoenzymatic reactions. In this work, we utilize kifunensine, a potent and highly specific inhibitor of α-mannosidase I in both plant and animal cells resulting in production of glycoproteins containing predominantly Man8GlcNAc2 and Man9GlcNAc2 structures, to a rice cell suspension culture grown in a bioreactor to inhibit α-mannosidase I activity. More than a few studies of kifunensine treatment in whole Nicotiana benthamiana plants successfully produced predominant Man9 structure glycoproteins; however, the study of kifunensine treatment in plant cell suspension cultures, including transgenic rice cell suspensions, is very limited.

In transgenic rice cell suspensions treated with 5 µM kifunensine cultivated in shake flasks, the productivity of a target glycoprotein, acid α-glucosidase , was significantly lower than the control, but the relative abundance of high-mannose structure GAA increased by 65% compared to the control. Here, we report the effects of kifunensine treatment on production and N-glycosylation of a glycoprotein conducted in a bioreactor. The culture media, method of cultivation, degree of glycosylation, and multi-merization of the product were different from the study by Choi et al.. Rice is generally recognized as safe by the FDA, and the rice alpha amylase 3D promoter, what is vertical farming a metabolically-regulated strong promoter, used in this study was well studied. In addition, semicontinuous bioreactor operations of transgenic rice cell suspensions proved the stability and robustness of transgenic rice cells under the RAmy3D promoter system for long-term recombinant protein production. In this study, we use a transgenic rice cell suspension culture to produce recombinant human butyrylcholinesterase , a bioscavenger hydrolase enzyme that can be used as a therapeutic and prophylactic treatment to counter organophosphorus nerve agents, as a model glycoprotein. Human BChE is a tetrameric glycoprotein with four identical 69-kDa monomers containing nine N-glycosylation sites per each monomer, with its activity, stability, and blood circulatory half-life highly dependent on the presence and structure of these glycans. The production of recombinant human BChE in transgenic rice cell suspension cultures is controlled by the RAmy3D promoter that is highly activated in the absence of sugar. Like other glycoproteins, N-glycosylation of a nascent rrBChE starts in the ER by co- or post-translational transfer of Glc3Man9GlcNAc2 from a dolichol lipid carrier onto Asn-X-Ser/Thr residues, where X is any amino acid except Pro. Since our rrBChE gene construct contains the RAmy3D signal peptide, a glycosylated rrBChE follows the secretory pathway for secretion, involving removal of glucose and mannose residues and addition of new sugar residues and N-acetylglucosamine , leading to complex-type N-glycans of rrBChE, as previously reported.Our goal in this study is to investigate the effects of kifunensine on N-glycosylation modification and the production of rrBChE in a transgenic rice cell culture bioreactor. By adding kifunensine to the medium during bioreactor cultivation at the end of the growth phase and throughout the induction phase, we demonstrate the production and N-glycosylation pattern of rrBChE in culture medium and within the cell aggregates .As shown in Figure 2a, the overall production level of active rrBChE increased by up to 80 µg/g FW, at least 1.5-fold higher than recently reported, using the same two-stage cultures. Table 2 also shows a significant improvement in the volumetric productivity and specific productivity . The increase in total active rrBChE, volumetric productivity, and specific productivity may be the result of increasing sugar-free medium concentration by 1.25 times and decreasing the bioreactor working volume during the media exchange by 1.25 times.Our hypothesis is that the rate of mannose trimming by ER and/or Golgi α-mannosidase I is faster than the rate of diffusion of kifunensine from cell aggregates to individual cells and from individual cells to Golgi and ER compartments.Although the concentration of rrBChE of 7.5 mg/L in the transgenic rice cell suspension culture in this study was significantly lower than the concentration of recombinant BChE of 1–5 g/L from the milk of transgenic goats, it is important to consider the long development time required between gene transfer and lactation for transgenic goats. There is limited quantitative information on production costs for rBChE from the milk of transgenic goats. However, since mammalian production systems can harbor and propagate human pathogens, regulatory requirements for the characterization of the transgenic founder, as well as feeding, housing, health monitoring, genetic stability assessment, and regulated disposal of ex-producer animals, could increase the production costs of human therapeutics in transgenic animals compared with plant-based systems. The expression vector design, cloning, transformation and selection of the callus, and media components were previously described. In brief, rice calli derived from Oryza sativa cv. Taipei 309 embryo/scutellum were co-cultured with Agrobacterium tumefaciens containing the binary vector with the RAmy3D promoter, codon-optimized human BChE gene to express in rice, the RAmy3D signal peptide, and the RAmy3D terminator. After eight rounds of screening starting with more than 300 transformation events, a stable transgenic rice cell line “9-2” was established, which was previously used in other studies as well as this study. The inoculum cultures were grown in 250 mL sugar-rich media in 1 L-shake flasks for 6 days in an Innova 4000 incubator/shaker at 140 rpm and 27 ◦C in the dark.Combined shake flasks described in Section 3.1 were inoculated at about 20% v/vin a 5 L stirred-tank bioreactor equipped with a pitched blade impeller and containing 2.5 L of sterile NB + S added through the head plate inoculation port inside a biosafety cabinet. Bioreactor conditions were controlled at 27 ◦C, 75 rpmagitation speed, 40% dissolved oxygen of air saturation , and 0.2 vvm of the overall mixed gas flow rate. The oxygen uptake rate was measured by the change in DO level in the culture when aeration is stopped but with continued agitation. The culture pH was monitored but not controlled. The cultures grown in the glass bioreactor were exposed to ambient light. Freshly prepared kifunensine solution was added to the bioreactor at 5 µM final concentration in the bioreactor medium at day 6 of cultivation, 24 h before the media exchange. At day 7 of cultivation, media exchange was performed to replace spent sugar-rich medium with sugar-free medium using the same method as previously described. Kifunensine solution was added at day 0 , day 2, and day 4 following induction, assuming 0 µM of kifunensine in the bulk medium prior to each kifunensine addition.

We identify key the data gaps that need to be filled in order to proceed with meaningful ecological risk assessments, whether they are more global/ regional in nature, or for site specific assessments. Finally, we attempt to draw conclusions from the literature about the relative sensitivity of different model organisms, as well as the importance of particle properties on fate, transport and effects.Nanoceria is used in electronic and optical devices, polishing agents for glass and of silicon wafers, exterior paints, metallurgy, and diesel fuel additives.Additionally, nanoceria is used in automotive catalytic converters.It is also used in catalysts in petroleum refining, in the fluid catalytic cracking process . Based on the amount of total global CeO2 annual production and global nanoceria production rates,roughly 15–25% of total CeO2 production is nano . Cerium oxide is used in these applications in both nano and non-nano form and quantitative estimates of cerium oxide use within specific applications do not distinguish between nanoceria and its bulk counterpart.There are few studies that quantify the release of engineered nanomaterials during use, and even less nanoceria specific studies. One of the few studies by Park et al., indicates that 6–100% of CeO2 will be released during the use phase of diesel fuel additives.This has not yet been validated by other researchers. In laboratory conditions, particles filters from diesel cars removed 99.9% of Ce present in fuel additives.However the manuscript does not specify whether the Ce additive was in the nanoscale. Considering the applications and the likelihood that the nanomaterials are released to the environment, the following assumptions were made. For example,vertical growers nanoceria in batteries is enclosed within a protective casing, which is likely to minimize release during use.

If the batteries are disposed of improperly, the most likely environmental compartment would be soil, with negligible release to air, water or wastewater treatment plants . Similar assumptions were made for metallurgical products, catalysts in FCC, polishing powders used in industry , and other applications. Experimental studies have been conducted to measure the release of various manufactured nanoparticles from surface paints on exterior facades. Kaegi et al. measured concentrations as high as 600 μg L−1 of nano-TiO2 in runoff from newly-painted building facades,and estimated that as much as 30% of nano-Ag is released from surface paints within a year of paint application.However, no data exist on nanoceria released from paints. Based on similar information, estimated nanoceria concentrations in treated WWTP effluent discharged to waterbodies are expected to be in the range of 0.003–1 μg L−1 . In biosolids, nanoceria concentrations are expected to be around 0.53–9.10 mg kg−1 .These estimated concentrations are expected to increase as nanoceria is used more widely, and there will likely be accumulation of CeO2 in soils and sediments, further increasing exposure concentrations in these media.The detection and characterization of nanoceria under conditions relevant to environmental, toxicological and biological systems remains a challenging, and frequently impossible, task. However, there is little or nothing that is ceria-specific, but applies to all nanomaterials. However, aspects of characterization are included here since it is fundamental to understanding of all nanomaterials, including nanoceria. Essential general aspects are listed below: i) In environmental systems, the specific and accurate detection and characterization of manufactured nanoceria remains essentially impossible,due to the gap between metrology and analysis and the complexity of the system . Total Ce detection is useful as it acts an upper limit of nano-ceria concentrations for risk assessment, but is not synonymous with manufactured nanoceria.

The discussion below applies primarily to spiked materials, mainly in the laboratory or mesocosm. ii) As with other nanomaterials, nanoceria should be fully characterized using suitable preparation methods and a multi-method metrological approach. In a multi-method approach, independent techniques operating on independent measuring principles provide cross-validation of measured properties. The source of the nanomaterial also needs to be fully reported, given the likely effects on properties. Fuller discussion is given elsewhere.iii) A number of properties require characterization which can be grouped as size, shape, morphology, aggregation/ agglomeration, surface charge and dissolution . These groups, or classes, contain several individual properties. For instance, for size, an average size should be reported, along with some measure of spread .iv) Given the changes that are well known to occur upon storage or changing media,it is essential to perform appropriate measurement over temporal and spatial scales which adequately capture the dynamics of the nanomaterial system. Although, none of the points above are ceria specific, nanoceria is capable of oxygen storage, which is size and shape dependent.Nanoceria is generally thought to have low solubility in water,although this is size and oxidation state dependent. Where dissolution and solubility are low, study is rendered simpler because dissolved ions should have little impact on toxicity. However, recent work has shown potential effects of even low level dissolution.Nano-ceria has two stable oxidation states IJCeIJIII) and Ce) under environmental conditions and cerium has the ability to transition readily between these two states.This redox activity gives nanoceria some of its key properties.However, oxidation state and morphology are usually poorly controlled or defined and spatially variable within an individual particle,giving rise to poorly reproducible data and uncertainties in understanding toxicity or exposure. These uncertainties, along with dynamic changes that occur in complex media, could explain the variable environmental and toxicological results that are seen in the literature for nanoceria.Table 1 shows a non-definitive selection of studies of nanoceria in a variety of different environmental, toxicological and standard complex media. These studies are examples of some of the most complete characterization in the literature, although there is still little consistency between studies and it is often not clear which nanomaterial properties require analysis because it is not well understood how each affects biological or environmental processes.

Lastly, because of logistical or other constraints, characterization is often not performed as fully as necessary to interpret such processes. Some of the most powerful techniques for the visualization of nanoparticles are transmission electron microscopy , atomic force microscopy and scanning electron microscopy . These techniques not only provide direct visual images but can be used to quantify other properties such aggregation, dispersion, sorption, size, structure and shape of the nanoparticles,although the sample preparation may alter considerably the sample. These techniques have been extensively applied to nanoceria, occasionally in complex media. Van Hoecke et al.and Rodea-Palomares et al. used TEM to visualize the interaction between the nanoceria and algal cells in order to test whether the nanoparticles are taken up or adsorbed by the algal cell wall. Zhang et al.used TEM to further investigate the needle like clusters on the epidermis and in the intercellular spaces of cucumber roots after treatment with nanoceria over 21 days. In some cases, TEM has been coupled with spectroscopy, for instance TEM coupled with EDS was used to determine the elemental composition of ceria clusters on both the root epidermis and in the intercellular regions of the cucumber plant.Merrifield et al.used AFM to image and quantify the size of PVP-coated nanoceria while compared them using TEM and DLS in toxicology exposure media. TEM confirmed that the larger particles are aggregates composed of smaller individual particles , but that nanoceria properties did not measurably change in the exposure media tested. In the same study, EELS was used to quantify the oxidation states showing that the smallest and the largest samples were composed of entirely CeIJIII),vertical grow with only small amounts of Ce present in the largest sample. Such spectroscopy is essential to microscopy imaging in complex media and is required to unambiguously identify the nanoparticles of interest in the presence of materials with similar sizes, shapes and electron densities/tip interactions. Microscopy, although a powerful single particle method, remains challenging when attempting to provide statistically meaningful measurements. Much data reported in the literature is pictorial and non-quantitative; careful design and timeconsuming analysis are required to be able to determine representative parameters with confidence. Nanoparticle tracking analysis is another widely used characterization technique which utilises microscopy to determine size distributions and number concentration of nanoparticles in liquid samples. NTA has been infrequently used for nanoceria, for instance to determine the mean size of nanoceria in green alga and crustaceans and to better understand the effect of natural organic matter on the particle-size distribution of nanoceria settling in model fresh water as a function of time.However, the methodology has some limitations in complex and realistic media.X-ray photoelectron spectroscopy has been used in only one relevant study, to our knowledge, in this case to understand the antioxidant capacity of nanoceria to DNA. The calculation of CeIJIII) : CeIJIV) ratios was performed,in an analogous manner to EELS, within a multi-method approach. Similarly, synchrotron-based X-ray spectroscopy has been used in several studies to assess Ce speciation. Studies using micro X-ray fluorescence coupled with X-ray absorption near edge structure in natural matrices have been conducted concluding that nanoceria can undergo biotransformations within a matrix, so the modifications, the mechanism and extent of these transformations should be fully addressed.Scanning transmission X-ray microscopy is an analytical microscopy which, with extended X-ray absorption fine structure spectroscopy, provided 2D quantitative maps of chemical species at concentrations which are environmentally relevant.

X-ray microscopy can in principle provide a spatial resolution down to ~30 nm while imaging the specimen in the aqueous state without the need for sample preparation.Synchrotron-based techniques provide direct structural information regarding the nanoparticles and their interaction with the environment.It is clear that X-ray spectroscopy, XPS and EELS are complementary methods for oxidation state analysis and combination may prove fruitful. Field flow fractionation has also been used on nanoceria to measure the size distribution of nanoceria in synthesized samples as well as to understand the aggregation behavior of other nanoparticles in the presence and absence of humic substances.ICP-MS can be used as a detector for FFF, but has not been for environmental or toxicological studies of nano-ceria, to our knowledge. Preliminary studies have shown the feasibility of ICP-MS for nanoceria analysis in single particle mode, although its further application in real systems has yet to be demonstrated. Infrared spectroscopy has also been used to study biotransformations in plants by comparing the molecular environment of the sample before and after exposure hence concluding that cerium speciation changes after incubation of nanoceria in different exposure media over 21 days. Ultraviolet-visible spectroscopy has been used to monitor the dynamic aggregation process of nanoceria in various waters with time along with DLS and TEM.Nanoparticles properties are altered by the water chemistry such as pH, ionic strength, nature of electrolytes or presence of NOM. One of the most important changes may be aggregation of nanoparticles: between the same nanoparticle, homoaggregation, or between nanoparticles and an environmental particle, heteroaggregation. The increase in size of the aggregates affects their transport, behavior, reactivity, uptake by organisms, and toxicity. In pure water, the stability of non-coated nanoparticles in solution depends on their surface charge. Nanoparticles brought into close contact via Brownian diffusion processes will repel each other if the charge is strong enough to overcome attractive forces. Nanoceria surface charge is dependent of the pH; nanoceria are positively charged at low pH, negatively charged at high pH and have an isoelectric point at approximately pH 8.The methods of synthesis and the cleanup of nanoceria have been shown to play a role in affecting the experimental point of zero net charge for nanoceria suspensions, which range from 6.5 to 8.1.Differences in the reported PZC may also come from differences in the method applied to determine the PZC, the order of titration process, and sorption of anions used in the titration.These authors measured the aggregation kinetic of nanoceria and compared to the theoretical prediction of Derjaguin–Landau–Verwey– Overbeek . At pH 11, the experimental critical coagulation concentration was higher for the monovalent , than the divalent salts, 80 mM and 16 mM respectively. They showed that DLVO theory could predict quite well the stability of nanoceria at this pH. However, this model fails to explain aggregation behavior as solution conditions become more environmentally relevant and non- DLVO forces may also play important roles between particles.In a water-saturated column packed with sand, water composition has also been shown to control the transport and deposition of nanoceria.

Reducing the “need” to fish by increasing available aquaculture derived food and materials does not necessarily equal reduced fishing pressure. Political/economic input will likely be needed to achieve the conservation goal of reduced fishing pressure as an outcome of implementing a FFP project.I would argue that new research paths in algae culture for bio-fuel production need to be followed in order to achieve results for application in the near-term. Maximum solar conversion efficiency are likely not necessary for commercial success with algae culture for bio-fuels— particularly in the case of food and fuel poly culture systems. A focus on simple easily applied culture techniques may yield a similar cost/benefit ratio per unit invested compared to employing the latest intensive technology to achieve maximum yield. Research and development efforts should be refocused toward small to medium scale ideas and projects with an emphasis on applied, relatively low-tech systems. The overwhelming majority of past and current work pertaining to algae culture for bio-fuel production is concentrated on microalgae. Microalgae holds great promises and this work should continue, but active work with macroalgae for bio-fuel production should be initiated and receive at minimum a similar level of effort. Screening of potential macroalgae candidate species for bio-fuel production, and developing technology transfer from current commercial algae farming operations for use in FFP systems are strongly recommended starting points. The concept of food and fuel poly culture is in its infancy, but with moderate investments in research to further develop the system’s components,greenhouse vertical farming the concept can play a role in providing food and energy to the world’s population while at the same time helping to conserve valuable natural resources— particularly those in at risk aquatic ecosystems.The rotation of the Earth acts to confer regular environmental changes in light availability and temperature and plants alter their physiology, biochemistry, and metabolism in response to these abiotic cues over the course of a day .

In addition, the inherent regularity of the transition between day and night also allows alterations in temperature and light to be predictive of subsequent abiotic stresses. For example, dusk is typically accompanied by a decrease in temperature and possible frost. It is therefore unsurprising that plants have developed an internal timing mechanism that allows prescient alterations in gene expression and biochemistry. Indeed, the circadian clock causes the regular oscillation of between 30% and 40% of genes in the model plant Arabidopsis thaliana , even when grown under constant light and temperature . These broad changes in gene expression precipitate a range of physiological responses including the regulation of hypocotyl growth , alterations to plant hormone production and sensitivity and time of flowering . In concert, such changes promote the fitness of plants grown in synchrony between endogenous and environmental cues . Circadian clocks are conceptually thought to be comprised of three parts: a central oscillator typically consisting of a negative feedback loop, input pathways to allow entrainment to local environmental conditions, and output pathways which act to modulate responses dependent on these endogenous cues. Although at its most basic level a circadian clock can consist of a single negative feedback loop with input and output pathways , evolution has typically led to the development of multiple interconnected molecular oscillators with varied levels of redundancy . The inclusion of partially redundant interlocking components likely allows greater flexibility in the modulation of clock inputs during evolution while also allowing greater accuracy of the timing mechanism itself . In addition to these core concepts, circadian clocks are recognised as having additional properties including temperature compensation and “gating”. Temperature compensation allows a circadian clock to oscillate at approximately the same frequency over a broad range of physiological temperatures while gating refers to the regulated sensitivity of the central oscillator to input stimuli. This latter mechanism enables the circadian clock to persist in plants grown in constant experimental conditions by reducing the responsiveness of core components to light during the subjective night .The other dominant zeitgeber of the Arabidopsis circadian clock is temperature, with steps as small as 4°C being capable of entraining the clock mechanism . The majority of large-scale mutant screens to identify Arabidopsis clock genes have used light as an entrainment signal and it is therefore unsurprising that comparatively little is known about temperature-sensitive entrainment of the clock.

It does appear, however, that the circadian regulation of individual genes may differ based upon the entrainment conditions used; CAB2 and TOC1 expression are similarly modulated by light and temperature whereas the phase of CAT3 expression is more sensitive to changes in temperature than light . Temperature inputs into the clock are at least in part incorporated via loops containing PRR7 and PRR9 as a prr7 prr9 double mutant is unresponsive to circadian phase changes induced by temperature and is arrhythmic if entrained to temperature steps . In contrast, it appears that TOC1 has a minor role in this mechanism as a toc1 mutant retains a wild-type entrainment response to temperature steps . Further characterization of this sensitivity is dependent upon identification of temperature sensors in Arabidopsis.Animal circadian clocks have long been recognized to contain a master clock which synchronizes multiple “slave” clocks in other tissues . This hierarchical arrangement of the clock permits individual tissues to utilize subsets of circadian genes . In comparison, plants are thought to measure time using cell-autonomous circadian oscillators , although it has remained unclear until recently whether each of these independent plant clocks sharecommon core components across different cell types. It now appears that certain loops of the plant clock act predominantly in certain tissues. PRR3 has been shown to be predominantly expressed in Arabidopsis vasculature while recent microarray analysis has indicated that only a subset of genes known to have a circadian expression pattern in aerial tissues oscillate in hydroponically grown roots . Such data suggest that circadian rhythmicity in roots is controlled by a simplified mechanism and is dramatic evidence that plant circadian rhythms need not be controlled by a uniform set of components. In support of this concept, experiments using RNAi to reduce PRR3 mRNA levels induce a greatly pronounced shortening of the circadian clock when measured using vasculature-specific luciferase reporter constructs in comparison to those with a broader range of expression . The use of modified clock circuitry in different plant tissues likely allows altered sensitivity to environmental stimuli and stresses and it will be interesting in the future to determine the functional role of tissue-specific circadian oscillations.Our understanding of the Arabidopsis circadian clock at a transcriptional level has progressed rapidly, aided by the high-throughput capabilities of luciferase reporter mutant screens and micro-array assays. While it is clear that a large percentage of the transcriptome is regulated by the circadian clock , our understanding of the molecular processes underlying these large-scale changes in transcription remains limited. Recent work has identified a correlation between histone acetylation and transcriptional activity at the TOC1 locus and similarly, a degree of histone methylation is associated with changes in transcriptional activity at CCA1 and LHY loci . Both of these observations suggest that epigenetic marks may regulate circadian gene expression and identification of proteins responsible for these epigenetic marks will allow a more thorough understanding of transcriptional regulation by the clock. While transcriptional regulation is clearly important for Arabidopsis clock function,vertical agriculture it is also increasingly clear that the rhythms generated by the transcriptional clock are modulated by a range of post translational modifications. TOC1 and PRR proteins are differentially phosphorylated and degraded over the course of a day while ZTL protein accumulates with a circadian rhythm despite being transcribed at a regular rate .

It is equally apparent that endogenous rhythms are regulated by changes in cytosolic composition, such as the concentration of free Ca2+ . Considering these examples, it is unlikely that we have either identified all components of the Arabidopsis clock or that we yet fully understand the subtleties of action and regulation of characterized transcripts and proteins. Ultimately, it will be important to transfer our understanding of the clock to real-world applications. Given the suggested role of the circadian clock in the regulation of plant responses to abiotic stresses , it is possible that altering expression of certain clock components may confer enhanced stress tolerance. Indeed, Arabidopsis prr5 prr7 prr9 triple mutants have recently been reported to have an enhanced cold, drought, and salt tolerance, caused by increased expression of stress-responsive genes . Such data are in agreement with work demonstrating that plants are differentially responsive to temperature over the course of a day and that this gating is controlled by the circadian clock . Further work to understand the process by which stress response pathways and the circadian clock interact will likely be a fruitful course of investigation.The biological oxidation of ammonia is carried out by two groups of chemotrophic bacteria , a process termed ‘nitrification’, results in transformation of NH4 + to NO3 – , making soil nitrogen susceptible to losses through multiple pathways – leaching and gaseous losses . Due to these nitrogen losses, a major portion of the soil nitrogen and applied fertilizer nitrogen is lost, in low nitrogen-use efficiency of agricultural systems . Also, nitrogen pollution is the single most environmental concern from agricultural systems, contaminating ground and surface water . Denitrification of nitrate-N is the major source of nitrous oxide emissions from agricultural systems and contributes significantly to global warming . Blocking the function of nitrifying bacteria or slowing the nitrifiers’ function can significantly reduce nitrogen losses associated with nitrification and extend the persistence of nitrogen as ammonium in the soil for uptake by plants, lead to improved nitrogen recovery and – use efficiency in agricultural systems . Recently, it was shown that some plant species have the ability to release nitrification inhibitors from roots that suppress nitrifiers’ function, a phenomenon termed ‘biological nitrification inhibition’ . Using a luminescent recombinant Nitrosomonas, an assay has been developed to detect and quantify this inhibitory effect in plant soil systems . With this newly developed methodology, it was shown that several tropical grasses and certain field crops including sorghum possessed the ability to release nitrification inhibitors from roots, termed BNI-capacity . We showed that sorghum roots release methyl 3-propionate , one of the active constituents with BNI activity of root exudates from sorghum . Here we present our findings on further characterization of BNI function in sorghum and reveal the identity of the two nitrification inhibitors released from its roots. In addition, we present evidence to show wide genetic variability in the release capacity of one of the major nitrification inhibitors from sorghum roots and also report preliminary field-based results for the existence of BNI-capacity in wild sorghum. Root architecture influences nutrient and water uptake, anchorage, and mechanical support, interactions with microbes, and responses to various abiotic stress factors . Since water and mineral supply are often limited in the soil, a plant with a more extensive root system exhibits higher performance with regard to the tolerance of drought and poor nutrient conditions . Several factors, including root angle, root growth rate, and root types, influence root architecture . Root growth requires the successive formation of new cells from stem cells in the root apical meristem , and the progeny of such stem cells divide rapidly and enter the elongation/differentiation zone . To maintain root meristem activity, the rates of cell division and differentiation have to be coordinated . In addition, the interaction between cytokinin and auxin determines the size of the RAM through the regulation of the genes involved in auxin signaling and/or transport to ensure an appropriate auxin gradient . The rice root system consists of one seminal root, numerous adventitious roots, and lateral roots that emerge from the other two types . Lateral roots are the major components involved in the absorption of nutrients and in interactions with the surrounding soil environment . Lateral root formation represents a complex developmental process modulated by several hormones, including auxin and ethylene.Well defined and closely coordinated cell division activities give rise to lateral root primordia.While lateral roots originate from pericycle cells adjacent to xylem poles in Arabidopsis,pericycle and endodermal cells located near phloem poles are the origins of lateral roots in rice and maize . Their development is initiated by the asymmetric division of the pericycle cells, and subsequent divisions result in the formation of dome-shaped, multilayered, lateral root primordia .

The resilience of these dynamics directly depends on the globalization of food through trade, including the amount of food traded, the number of links describing trade between countries, and the topological properties of the trade network. Using reconstructions of food production and trade based on FAO data , this analysis shows that in the past few decades the system has become increasingly vulnerable to instability as an effect of demographic growth, dietary shifts, and the increasing inter connectedness of the trade network. Indeed, some nodes are starting to show the first episodic signs of instability, particularly in water-poor and trade-dependent countries . This analysis of the long-term response to shocks is in agreement with the short-term propagation of perturbations in the trade network during a food crisis ; both approaches have shown how the fragility of the coupled food-demographic system has increased as an effect of the growing globalization of food through trade. If trade and interconnectedness have the effect of reducing the resilience of the system as a whole, it is paramount to investigate to what extent it would be possible to globalize without becoming more vulnerable. A possible solution of this problem is suggested by ecological systems, which often exhibit some degree of modularity.There is evidence that systems with a modular structure—that is, with groups of countries that interact more among themselves than with countries from other modules—are able to contain the spread of perturbations within the targeted module, whereas the other modules remain only marginally impacted . In other words, the modules act directly to buffer the propagation of shocks to other communities, thereby increasing the stability and resilience of the entire system . Interestingly,tower garden the virtual water trade network exhibits a growing degree of modularity with a ratio between internal and external fluxes that is approaching 70% . To date, however, the effect of modularity still needs to be investigated in the context of the resilience of trade and food security.

An alternative approach to understand the impact of virtual water trade on population growth is through carrying capacity plots , which distinguish different strategies and their success by showing the historical evolution of a region or population’s local food supply potential and net food imports relative to their local and post trade carrying capacity. Porkka and collaborators confirm that food import is the strategy nearly universally used to overcome local limits to growth. Nevertheless, they also highlight that these strategies are implemented to varying extent and with varying success . Therefore, whether dependency on imports is necessary and desirable, a clear policy priority at both local and global scale is needed and it ideally would attempt to keep the demand of food under control .There is wide agreement that humanity’s rate of resource use exceeds what can be sustainably generated and absorbed by Earth’s systems . Substantial uncertainty persists for an apparently basic question—by how much is food demand likely to grow in the coming decades—with estimates typically ranging from a required 60% to 110% increase by the year 2050 over circa year 2005 levels . More recently, Hunter et al. estimated that an increase in cereals production of 25–75% over 2014 levels could be sufficient to satisfy projected demand in 2050. The breadth of future GHG emissions trajectories—and the magnitude of their cascading consequences for agricultural productivity—leaves considerable unknowns regarding future food production under a changing climate . A radical transformation of the global food system is likely required in order to increase production while faced with the considerable uncertainties related to demand and climate impacts. New strategies for achieving FEW security worldwide may benefit from adoption of an integrated approach aimed at an improvement in the availability, access, and nutritional properties of food while enhancing the provision of affordable, clean, and reliable energy . Moreover, a secure FEW system will incorporate a sustainable use of natural resources, maintain environmental stream flows, and restore ecosystem services.

The FEW system will need to invert the ongoing trend of increasing vulnerability and enhance its resilience with respect to climate shocks, demographic growth, and consumption trends. The previous sections have highlighted the existence of several major challenges in this multi-objective strategy to food, water, and energy security. For instance, the sustainability of energy production can be improved by increasing the reliance on renewable energy sources, which would decrease the rate of fossil fuel depletion, reduce CO2 emissions, and consequently allow societies to improve their ability to meet climate change targets. Renewable energy sources based on bio-fuels, however, would claim huge volumes of water and large expanses of land, thereby inevitably competing with the food system . Moreover, bio-fuel production often entails direct and indirect land use change and associated GHG emissions, indicating that in the short term these energy sources might have a negative impact on the environment . Nonfuel-based renewable energy production may also require substantial amounts of water and therefore compete with food crops in water-stressed regions . The increasing demand for food and energy by the growing and increasingly affluent human population can hardly be met with the limited land and water resources of the planet unless we transform the FEW system. As noted in the previous sections, approaches focusing on ways to increase food and energy production instead of curbing the demand would ignore the existence of limits to growth imposed by the natural resources the planet can provide and are likely to achieve higher production rates at huge environmental costs , resilience losses , and increased food insecurity for the poor. To be sure, there are still margins for increased production through improvements in efficiency, technological innovation, and agroecologically efficient farming systems, but these measures are likely to be insufficient to meet long-term global food and energy security needs . There is the need for a complete rethinking of the FEW system to develop a comprehensive strategy for food, water, and energy security, based on enhancing the production and moderating the demand . Although a conclusive answer to the question of how to sustainably meet the food, energy and water needs of the rising and increasingly demanding human population is still missing, here we review a number of new and old approaches and ideas that could contribute to future food, water, and energy security.

Such approaches can be, in general, technological , cultural , or institutional .Large yield gaps, or the difference, between current and attainable yields still exist in many parts of the world, particularly in sub-Saharan Africa and offer the potential to increase global production of major crops by as much as 58% under currently available technologies and management practices . There is broad consensus that efforts to enhance crop yields on currently cultivated lands are crucial for avoiding additional agricultural expansion , the consequences of which would be profound and undesirable for natural systems and functioning . Agricultural intensification, however, is not free of environmental impacts in that it contributes to GHG emissions, freshwater and coastal water pollution, depletion of freshwater resources, and consequent loss of aquatic habitat . In light of the environmental impacts of conventional intensification, some scientists are advocating for an approach to food security that relies on a “sustainable intensification” of agriculture , which aims to close the yield gap while minimizing the environmental impacts . Moreover, as with historical yield trends, stacking flower pot tower harvest frequencies have generally increased through time, but many places with the potential to transition to double-cropping systems have yet to do so . On the one hand, yield and harvest gap closure offers great promise for increasing the food self-sufficiency of many developing nations because the areas with the largest potential for production increases are those places that currently rely heavily on food imports and have some of the highest rates of projected population growth to mid century . On the other hand, these remaining yield gaps raise questions about how best to promote the diffusion of high-yielding crop varieties and agricultural technologies, given that these agricultural advancements have yet to reach many places even 50 years after the start of the green revolution . Moreover, it is unclear whether additional inputs are adequately available in low-yield areas and, if so, how to avoid their unsustainable use . Thus, particularly with regard to non-mobile resources, such as land and water, it will likely be essential to ensure that increases in production occur in places where and when natural resources can support it . There are social, economic, and institutional factors that need to be accounted for while advocating for agricultural intensification versus alternative farming approaches . Most of the existing literature on this subject has recognized the pros of yield gap closure as an alternative to agricultural expansion at the expense of natural ecosystems , particularly in the tropics . “Land sparing” can, in many contexts, minimize habitat losses, land degradation, CO2 emissions, and declines in biodiversity . This approach, however, is not a panacea because its profound social impacts have often been overlooked by focusing on agrotechnological solutions without considering their effect on production systems, such as smallholder farmers . Intensification efforts require investments that are increasingly made by large-scale agribusiness corporations, particularly in the developing world. Such investments may affect the system of production and its inputs, for example, through contract farming or out grower schemes or land use, access, and tenure rights, as occurs for LSLAs , which are discussed in section 9.3. Agricultural intensification is most effective in countries where relatively large yield gaps still exist, such as sub-Saharan Africa, while ensuring that new fertilizer and water are used in the most efficient way possible . To boost crop yields, investment in modern agricultural technology is required, which many rural communities in lower-income countries cannot afford. If neither local land users nor domestic investors are able to improve crop yields, in years of increasing crop prices, foreign corporations or foreign-domestic joint ventures are not likely to miss the profit opportunities existing in under performing agricultural land .

Indeed, recent years have seen a wave of investments in agricultural land in the developing world, with import-dependent nations seeking to increase the pool of land and water resources under their control and targeted countries pursuing avenues to promote rural development and agricultural technology transfers . However, there is a growing body of scientific and anecdotal evidence showing that the development and food security goals of these land deals are often not achieved and, instead, often bring substantial social and environmental consequences . Such land deals ultimately may result in the displacement of subsistence or small-holder farmers by large-scale commercial agriculture, as well as the development of new agricultural land at the expense of savannas, forests, or other ecosystems . Because most of the cultivated land worldwide is managed by small-scale farmers , this ongoing shift in systems of production may strongly reshape the agrarian landscape around the world with important impacts on rural livelihoods because it increases the dependence on a volatile food market. Thus, agricultural intensification may be the result of important transformations in land tenure, farming systems, and livelihoods. Developing countries may enhance crop yields by introducing modern agricultural technology while promoting greater efficiency in food production through a transition in their agricultural sector toward commercial-scale farming. Commercial agriculture lends itself better to capital inputs from investors and could result from LSLAs or other forms of investment, such as contract farming , or mixed out grower schemes . However, there could be negative impacts on rural communities and their livelihoods because LSLAs may turn farmers into employees and increase their vulnerability to food price volatility The transition to large-scale farming, however, might be unnecessary: small-scale farms, which account for most of the global calorie and nutrient production , can be very productive. There is evidence in the economic literature about the inverse relationship between farm size and productivity, meaning that smallholder farmers, when provided with adequate inputs, may often achieve yields that outperform commercial, large-scale agriculture. Identifying mechanisms that support yield enhancements, technology transfers, and secure land tenure to these critical stakeholders is a key component of advancing global food security, promoting poverty alleviation, and enhancing food system resilience. Overall, intensification typically requires the introduction of modern green revolution technology in areas of the developing world in which relatively large yield gaps exist.

Water consumption for food production, including crops and livestock, accounts for about 86% of the total societal water consumption, though, locally, household and industrial uses can be predominant, particularly in major urban areas. Thus, securing water resources for agriculture, while reconciling the competing water needs of growing cities and surrounding rural areas, is a major challenge of our time. Climate change is expected to further enhance local water scarcity, especially in the subtropics. In fact, while climate warming is slightly increasing global precipitation, the global patterns of rainfall distribution are expected to become more uneven with an intensification of aridity in the dry subtropics, and an increase in precipitation in the wet tropics and the midlatitude temperate zone . The temporal variability of precipitation will likely increase, thereby enhancing the probability of drought and flood occurrences .Despite recent developments in desalinization technology , 2016, most human activities related to food and energy production rely on the consumptive use of freshwater. Desalinization remains limited to specific uses that require relatively small amounts of water and to societies that can sustain the associated costs . The freshwater available for human activities is stored in continental land masses either in soils or in surface-water bodies and groundwater aquifers. Often referred to as “green water,” soil moisture is retained in the ground by capillary forces and can be extracted only when it is subjected to a suction that overcomes the action of capillarity. Plants exert such suction through root uptake. Although most of terrestrial vegetation in natural ecosystems relies on green water , soil moisture remains for most part unavailable to direct human use because it is difficult to extract. In contrast, water stored in surface water bodies and aquifers, referred to as “blue water,” hydroponic net pots is more mobile and contributes to surface-water and groundwater runoff. Thus, green water leaves land masses in the water vapor phase as evapotranspiration , whereas blue water flows to the ocean in the liquid phase as runoff .

Since antiquity, human societies have engineered systems to withdraw blue water from rivers, lakes, and aquifers and have transported it through channels and pipes to meet the needs of a variety of human activities. Today, the main consumptive use of blue water is for irrigation , which strongly increases green water flows at the expense of blue water flows. Irrigation is a major human disruption of the water cycle ; indeed, many rivers are so strongly depleted that they no longer reach the ocean , while lakes in basins with internal drainage are drying out . Irrigation can modify the local climate, possibly by increasing evapotranspiration and effectively cooling the near-surface atmosphere . Irrigation may also moderately enhance precipitation downwind of irrigated areas and induce mesoscale circulations driven by the contrast between irrigated areas and the surrounding dry lands . It has been estimated that globally, irrigation uses a water volume that is roughly 2.56 × 1012 m3 /year , which accounts for about 2% of the precipitation . Although water is a renewable resource that is conserved in the Earth system, freshwater stocks can be depleted when their use exceeds the rates of natural replenishment. A typical example is groundwater that is often used for agriculture andis being depleted in many regions of the world , including the North American Southwest, Northern Africa, the Arabian Peninsula, and India . In some cases, groundwater use is depleting water stocks that accumulated in epochs with a wetter climate. In these aquifers “over pumping” leads to a permanent extraction of water resources, a phenomenon that is known as “groundwater mining” to better stress its unsustainability and the irreversible loss of resources that will not be available to future generations. However, even when the depletion of water resources is reversible, its environmental impacts may not be. Excessive water withdrawals from rivers and streams destroy the aquatic habitat and lead to extinction of riparian species. Interestingly, freshwater ecosystems are particularly vulnerable because the extinction rate of freshwater aquatic species is much greater than that of terrestrial organisms . Thus, sustainable use of water resources should prevent not only their permanent depletion but also the irreversible damage of downstream ecosystems.

A rich body of literature has discussed criteria to define minimum flow requirements and minimum flow variability required to conserve the aquatic habitat . A reevaluation of those efforts within the context of water sustainability has led to the formulation of the concepts of “planetary boundaries” and “safe operating space” that define a cap for sustainable water use . Such a cap is typically expressed as a fraction of the natural river flow, ranging from 20% to 60% , though recent studies have suggested referring to season-dependent fractions . Although globally, the current use of water for irrigation is smaller than the planetary boundary for blue water and accounts for only 5.4% of the global blue water flows , in many regions of the world those boundaries are locally exceeded, thereby causing habitat loss . Overall, irrigation is critical to sustaining the present rates of agricultural production. Although only 20% of the global agricultural land is irrigated , it sustains about 40% of the global crop production owing to the typically much higher yields in irrigated systems . Collectively, irrigated and rainfed agriculture accounts for about 10% of global precipitation over land, with green water flows from agroecosystems contributing to roughly 16% of the global evapotranspiration from terrestrial ecosystems . These figures give us a sense of the proportion of the water cycle that has been appropriated by agriculture. Moreover, other economic activities, such as mining, manufacturing, and energy production further increase the human demand for freshwater.Since the onset of civilization, agriculture has claimed land from natural ecosystems, such as forests, savannas, and grasslands. By converting these landscapes into agricultural land, humankind has profoundly altered the water and bio-geochemical cycles . Decades of research on deforestation have highlighted the profound hydroclimatic impacts of land use and land cover change . Compared to forests, rainfed farmland sustains lower evapotranspiration rates because of the smaller leaf area index, surface roughness, and root depth, and the greater albedo . The infiltration rates are also smaller because agricultural soils are often more compacted, typically from leaving the land fallow for part of the year and cultivated with heavy machinery. Smaller evapotranspiration and infiltration rates are expected to lead to higher runoff . However, in areas where agriculture is irrigated, water withdrawals for crop production deplete surface-water bodies and aquifers . Land use change also has an impact on the regional climate.

Land use change alters the surface energy balance and land-atmosphere interaction; these changes modify near-surface temperature, boundary layer stability, and the triggering of convection and convective precipitation . Some of these effects can alter the rainfall regime within the same region in which land cover change occurs, though it has been suggested that the impact also can be on adjacent ecosystems . Moreover, land cover change may modify the rate of emission of biological aerosols, thereby affecting cloud microphysics and cloud processes . The reduced evapotranspiration has the effect of reducing precipitation recycling, which is the fraction of regional precipitation contributed by atmospheric moisture from regional evapotransporation , a phenomenon that is relevant to policies and therefore is receiving the attention of social scientists , despite the great uncertainties with which it can be evaluated . Overall, forest or woodland conversion to cropland over large regions is expected to reduce precipitation and increase diurnal temperatures , though these effects depend on the size of the cleared area . The direct and indirect impacts of human activities on freshwater resources may strongly affect their availability to meet the competing needs of food or energy production and the environment,blueberry grow pot raising questions on the type of institutional arrangements that could improve water governance.Water is by its own nature fluid, renewable, and difficult to quantify , and its biophysical characteristics, such as the fact that it is a key input into biological processes and that is relatively plentiful and widely distributed , make the political economy of this resource very different from other similarly important strategic natural resources . From early human history, water use has led to complex dynamics of competition and cooperation . In a world with increasing societal pressure over scarce water resources and aggravating hydro climatic change, water governance is fundamental in the policy and development dimensions of water management. Even though access to safe water and sanitation is recognized as one of the UN-SDGs , about 4 billion people face water scarcity at least 1 month per year . Water availability may be affected by water quality, particularly in the case of drinking water, as the cost of treatment may become prohibitive in some locations, creating physical water scarcity of costly water resources. The reliance on water markets historically has been, and still is, strongly influenced by neoliberal governance approaches based on privatization, liberalization, and extension of property rights. The core principle behind these approaches is that water markets provide the correct economic incentives to promote the reallocation of water to higher valued uses and improve efficiency. These approaches treat water as a commodity and thus require the recognition of property rights that define the use, management, and trade of water resources . Easter et al. describe a strong legal system as the main institutional condition necessary for water markets to function properly. The creation of water markets in the Western United States and in Chile have been used as exemplary policy and governance models that could be exported and promoted in developing countries .

Since the 1980s, the World Bank has been the main promoter of water markets in developing countries, while also supporting the development of lucrative transnational opportunities in the water sector for private investors . In the context of the FEW nexus debate, however, a water market economy may lead to water resources previously used to produce food being transferred to other uses, such as industrial production or household needs in urban areas. In fact, the economic yield of food commodities may typically be orders of magnitude lower than that of the energy and water utility sectors . Food as a basic human need means that market approaches to water governance also can be evaluated in the context of their impacts on food security, particularly for the poor. For example, water markets could be structured with special consideration for certain industries, including agriculture, to avoid losing water allocations for production of food. A counterargument in favor of water markets stresses their positive environmental outcomes such as when water is partly acquired to reestablish environmental flows and improve aquatic habitat, or if the market sets a cap on the amount of water that can be withdrawn for human uses . The contemporary neoliberal trends of water commodification, that is, the multidimensional process through which goods that traditionally are not priced enter the world of money and markets , could be in stark contrast with the principle that access to water is a fundamental human right . Ostrom describes water resources as an iconic example of common-pool resources, which often have been successfully governed through diverse community and communal-property institutional arrangements. The multiple characterizations of freshwater by different cultures and societies make it difficult for freshwater to be reduced to a monetized commodity. Water can be perceived as a sacred commodity, a human right , a political good, an ecosystem medium, and a security asset . Moreover, the water sector has intrinsic characteristics that can be associated with structural market failures, with large externalities, and interconnectedness that make the level of individual and collective interdependence particularly critical . From a social and environmental justice perspective, the idea that water is not treated as a “common good” but as a commodity has generated criticism around the perpetuation of inequality and violation of fundamental human rights . Different narratives and political perceptions about the value, the meaning, and the function of water in society make a clear and uniform definition of “good water governance” difficult. As described by Meinzen-Dick , rather than considering single solutions for water governance, it may be more productive to have multiple institutions work together in an adaptive learning process.

However, if this is indeed the case, there must be a mechanism and a third component in the system which causes the inhibition of the diffusion, and which, by extension, exerts a pressure that opposes the surface tension and the external pressure, along with the internal pressure. The third component is suspected to be the hydroxide ion, which is always present in aqueous solutions, and which is detected around collapsing microbubbles, and has already been applied to wastewater treatment. This ion tends to aggregate around the nanobubble surface, and is suspected to be present in the form of a cloud of ions around the bubble, attracted to the surface by an as yet unconfirmed force, but widely thought to be physical bonds of the nature of van der Waal’s force, and plays a part in the inhibition of gas diffusion out of the bubble. The exact mechanism, distribution of the ions, the extent to which they inhibit the diffusion, and other concerns regarding their roles in the mechanism of stabilization is not yet determined, but several theories have been proposed as to their role, and more also exist which do not take into account their role, or do not require them to play any role in the process at all. The role of the ions is suspected to be due to the repulsion of the ions toward each other, which in some way opposes the external pressure and the surface tension, but this is yet to be confirmed. Thus, while there are several approaches to the question, as of recent efforts it still remains unresolved. Several theoretical approaches have been proposed, many of which are highly specific to the circumstances for which the study was conducted, and none thus far have proposed an overarching theory as to the formation and evolution of bulk nanobubbles. As far back as 1997 Ljunggren and co-workers proposed theoretical explanations for colloid-sized gas bubbles based on diffusion of the gas into the liquid,fodder system for sale which could now be considered nanobubbles. Seddon et. al. also contributed to the emerging idea around the same time, but there have been few contributions to understanding their stable presence since then.

Explanations for specific cases of phenomena such as surface nanobubbles, nanobubbles generated electrochemically, and so forth have been offered so far. Early on, the Young Laplace equation was used to describe nanobubble stability, but the internal pressures required are far higher than would be possible at ambient temperature for the amount of gas that is contained within the bubble. Attard and co-workers analysed the thermodynamic stability of bulk nanobubbles, but it was found that the radius of nanobubbles could not be accurately predicted from thermodynamic considerations, nor was an expression offered for the rate of decrease in nanobubble size. Brenner and Lohse presented a model for predicting the radius of surface bubbles based on the dynamic equilibrium between diffusion into and out of nanobubbles situated at a surface. Further work in specific cases by Weijs and Lohse suggested the use of increased length scales to counter the problem of high internal pressures due to the relatively high surface tension of a bubble in that size range. Sverdrup and colleagues offered explanations as to the rates of decrease in size based on diffusion in all directions possible through the gas-water interface at the nanobubble surface. In their models they consider the possibility of diffusion both into and out of the nanobubble, with a sufficiently high mass transfer coefficient. Their models consist of a combination of Henry’s Law and Taylor series expansion. The equations are plotted, taking time as a function of radius and show coherence with previous models given by Ljunggren. However, no comparisons with experimental data are provided. The Young-Laplace equation seems inadequate to completely describe the phenomenon as it requires extremely high internal pressures of the gas to balance the surface tension that causes the nanobubble to shrink, as summarized by Attard and coworkers. However, the interface through which the diffusion occurs has thus far been considered to have constant properties of being composed only of water molecules and gas molecules.

Yasui and colleagues also detail several theories that attempt to explain bulk nanobubble stability, based on the armoured bubble model, a particle crevice theory, a skin theory, the dynamic equilibrium model and electrostatic repulsion. Among these theories, it appears that electrostatic repulsion has the most experimental support. Studies of interfaces between water and practically all surfaces such as glass are negatively charged, assumed to be due to the accumulation of hydroxide ions physisorbed to the monolayer as reported by Zangi and Engberts. Thus, it is reasonable to suppose that the water-gas interface is also negatively charged due to similar congregation of hydroxide ions at the bubble surface. Furthermore, studies conducted by Takahashi and others have shown that nanobubbles are indeed negatively charged, with oxygen nanobubbles having a zeta potential about -35 mV. Thus, it is evident that hydroxide ions physisorbed onto the surface of the nanobubble play a role in the interactions between the molecules present there. Jin et. al. proposed a model for bulk nanobubble stability involving the electrostatic repulsion, terming the pressure due to the electrostatic force as Maxwell pressure. One rationale involving the surface charge density of a bulk nanobubble has been proposed by Ahmed and colleagues that involves electrostatic repulsion balancing the surface tension. In the following, we consider a theory of electrostatic repulsion and what it requires of the conditions imposed for nanobubbles to have the long-term stability that has been observed experimentally. Several applications have been discovered, such as for wastewater treatment, fish farming, shrimp breeding, and hydroponics. These are further substantiated by Agarwal and coworkers, for such specific issues as the disinfection of infected surfaces, the degradation of organic compounds, and the disinfection of the water itself. The effects of increased yield of fish due to higher dissolved oxygen content are summarised by Endo et al..

The usage of hydrogen nanobubbles in gasoline to improve the calorific yield is also reported by Oha et al.. Other projected uses include the use of nanobubbles as contrast agents for the ultrasound imaging of tumours, as reported by Cai and co-workers, as well as reduction and removal of deposits of calcium oxalate, which is similar to the composition of kidney stones in rat kidneys, as presented by Hirose et al. Another application of the nanobubble’s ability to permit salts to crystallize is the design of self-cleaning membranes for desalination of water,fodder growing system which use nanobubbles as electrically conductive spacers and pass current through them to force the salts to crystallize on the nanobubble surface, which will permit easy removal of the accumulated salts. This was demonstrated and presented by Abida et al. The pressure balance of the nanobubble is considered to be given by the Young-Laplace equation, which, as explained above, equates the internal pressure, external pressure and the surface tension. The first of the four forces that we consider in the Young-Laplace equation is internal pressure. It is proportional to the surface area of the nanobubble, and is assigned a positive sign since it acts to increase surface area. The second is the external pressure, given by the hydrostatic pressure acting on the surface of the bubble, which also decreases the surface area and is negative. The third is the surface tension, which acts along the surface area at the molecular level. The surface tension acts to decrease the surface area, hence the radius and size, and can also be assigned the negative sign. However, a fourth force which is thought to be integral to the stability is the electrostatic repulsion between hydroxide ions adsorbed to the surface of the nanobubble, or, possibly in the cloud surrounding the surface. This repulsion seeks to reduce the contact between the ions on the surface of the bubbles, which also acts to increase the distance between the ions, thus increasing the surface area, and therefore results in a positive pressure. The nature of the interaction between ions can be characterized by the expression for Coulombic repulsion. Since one hydroxide ion is of the order of 1 nanometre in diameter, and most nanobubbles are two orders of magnitude greater in size, we can ignore the curvature of the distance between them and take it to be linear. The repulsion should, in theory, affect all neighbouring hydroxide ions, but is assumed to be insignificant beyond the nearest neighbours. We also assume the spatial arrangement of these ions over the surface to be close-packed in nature, since the repulsion is equal in all directions, and they would ideally assume a close-packed formation. This arrangement of ions is shown schematically below, in Fig. 1a, and as shown in Fig 1b it is assumed, due to close-packing, that they assume the formation of a rhomboidal unit cell, of side and diagonal length denoted by x, which will be referred to subsequently as the inter-ionic distance. That the nanobubble shrinks due to outward diffusion of the gas contained within is, of course, undisputed, but the precise methods and the rate of diffusion are highly debated. Previous theoretical studies have always assumed a model with a higher mass transfer coefficient, or longer time scales for the process to account for the reduced rate and the high lifetime of the nanobubble.

However, it is reasonable to suggest that the change in the rate of diffusion can be attributed to two things: the velocity due to the Brownian motion of the nanobubble, and the inhibition of the diffusion due to the adsorbed hydroxide ions on the surface. In this chapter, the possible effects of Brownian motion are examined for the effect on the rate of diffusion that they may possess. Earlier studies have shown that nanobubbles can be formed by supersaturation, where the solubility limit of the gas, when surpassed will permit the gas to precipitate and form bulk nanobubbles as reported by Matsuki and co-workers . The shrinkage of nanobubbles has so far been thought to be governed by Fick’s Laws, since it is a case of how fast the gas can dissolve into the surrounding fluid. Thus, according to the first law, it must be directly proportional to the outward gas flux, but the constant is still the diffusion constant D0 for the diffusion of the gas into water. However, this only holds true where the surface area of the nanobubble remains constant. It is, however, possible, that the outward diffusion is a case of Fick’s second law, since the surface area that is available to the gas to diffuse outward also changes according to size, and that this surface area determines the rate of shrinkage and thus the lifetime of the bulk nanobubble. It is then reasonable to suppose that the cause of the change of surface area available for diffusion is the change in the surface area occupied by hydroxide ions combined with the decreasing radius of the bulk nanobubble. The rationale for the assumption that the hydroxide ions adhere to and are released the nanobubble surface is based on two observations, as mentioned before. Firstly, the observation that all interfaces formed by water are negatively charged, and we consider nanobubbles to be a special case of a gas-water interface which may be charged in the same way. Secondly, the zeta potentials measured for nanobubbles are all negative, indicating that a negative ion present in pure water is responsible for the negative charge, which by elimination is the hydroxide ion. Further observations also indicate higher negativepotentials for more electronegative gases, such as oxygen and nitrogen, than for other reported gases such as argon and xenon as reported by Ushikubo et. al.. That nano- and microbubbles release hydroxide ions as they shrink is a well-known phenomenon. The stabilization and the shrinkage can be considered to be related to the same phenomenon; thus, the ideal case can be taken to be a nanobubble that is newly formed with no hydroxide ions at the surface at the instant of its formation of an interface. Here, the hydroxide ions present in the water immediately surrounding the bubble, in the hydrodynamic layer, adhere almost instantaneously, the time taken for the adsorption to occur being too small in comparison to the overall timescale to be important.