Tag: hydroponic

The stakeholders consisted primarily of the Preserve Partners—a consortium of federal, state, and local agencies—in addition to non-profits such as TNC and Ducks Unlimited. The management scenarios were developed with reference to the Preserve Management Plan , created by the Preserve Partners through a 2-year planning process . The management plan gathered information from the public , the Preserve Partners, local municipalities, and other groups. We used a time-frame of ~30 years to 2050 . We considered each of the scenarios in isolation; for example, landscape-wide restoration does not account for development, nor urbanization for set-asides for wildlife. For parcels not affected by the management scenario, we assumed a static landscape with no change of land use occurring, as much of the land has remained pastoral for ~150 years before present. The objective of this scenario was to maximize restoration of agricultural lands to natural riparian habitat, focusing on areas of specific soil type and proximity to the river, based on an analysis in the Management Plan. In addition, Preserve goals, as set forth in the Management Plan, support maximizing the restoration of riparian habitat in the Cosumnes corridor. We applied six decision rules that relate the location of each parcel to existing landscape features. A parcel received a high likelihood of being restored if: the parcel was currently within the Cosumnes River Preserve lands; was managed for other conservation purposes ; was within a historical riparian corridor; was within 1km of standing water; was within 1km of grassland, shrubland, or wetland; or was within 1km of riparian forest.

The 1-km distance threshold was set to be inclusive of remnant riparian forest within the Preserve,flood table coupled with the assumption that areas within this threshold are practical targets for restoration. If a parcel fell within any one of these six categories, it was given a score of one. Scores were summed for each of the six rules, and a composite score was given to each parcel. Using the composite score, the upper quartile of all parcels with a score greater than one, weighted by area, were designated to be restored. We tested for multicollinearity using the variance inflation factor , which did not indicate substantial multicollinearity of the six decision rule variables . We filtered the final layer of restorable parcels to exclude existing urban areas. The restoration of these parcels was to grassland or riparian forest, which was assigned based on the potential natural vegetation . We did not consider areas defined by Kuchler as subtidal marsh within the study area for future restoration because of the current lack of feasibility; consequently, parcels remained under current land cover . The objective of this scenario was to represent a realistic growth outcome for the area projected to 2050. We assigned parcels as urban in 2050 based on whether they were currently urban or projected to become urban using the Preferred Blueprint Scenario for 2050 . SACOG generated the blueprint to help guide local government in growth and transportation planning through 2050 throughout the six-county region. This Preferred Blueprint Scenario promotes compact, mixed-use development and more transit choices as an alternative to low-density development . We filtered the final layer of potentially urbanized parcels by extracting current protected areas and areas of riparian forest, which are unlikely to be developed because they are within the 100-year floodplain. This provided the land cover data necessary to make projections in ecosystem services and disservices for 2050. The objective of this scenario was to maximize high-quality foraging habitat for the Swainson’s Hawk, and was developed based upon the literature and experience of the authors. A parcel received a high likelihood of being Swainson’s Hawk-friendly agriculture if it was designated as or within 1.25km of alfalfa, grain, pasture, or row crop . We selected all non-protected parcels of natural vegetation, such as grasslands, within 1.25km of existing fields under these four agricultural types to identify parcels available for conversion to agricultural types favorable to Swainson’s Hawk.

For practical reasons, certain agricultural types were not considered for conversion. For example, vineyards, given their high economic value , are unlikely to be converted; vineyard expansion remains a dominant trend for the region . We filtered the final layer of potentially enhanced parcels to exclude riparian forest and existing urban areas. Using the composite score, we converted the upper quartile of all parcels for these rules from existing land use to the four agriculture types more favorable for Swainson’s Hawk foraging. These types were randomly allocated in proportion to the number of parcels in which they currently occur: alfalfa 5%, grain 20%, pasture 45%, and row crop 30%. As with the restoration scenario, we tested for multicollinearity using the VIF, which indicated the model did not have substantial multicollinearity of the variables.We quantified the amount of carbon stored within three different land-cover types based on readily available data and literature: agricultural crops ; natural, non-forest vegetation types ; and forest types . We did not include soil carbon storage in this analysis, nor do we account for carbon storage associated with natural habitat within urban areas. We assumed that these steady-state estimates apply to all locations, and that changes in land cover would increase or decrease carbon storage to a new steady state.Above- and below-ground carbon storage for standing agricultural crops was based on a study by Kroodsma and Field . We divided the yield by the harvest index for each crop, and then multiplied the result by 0.45 as the proportion of biomass assumed to be carbon to estimate Mg C ha-1. For row crops not included in Kroodsma and Field , we estimated it using the National Agricultural Statistics Service yield data from the year 2000 and the average harvest index for all row crops . We estimated carbon storage for orchards by assuming the mid-point of the crop’s lifespan, multiplying this by wood accumulated , and again multiplying by 0.45 to provide Mg C ha-1.

For perennial crops not listed in Kroodsma and Field , we used the mid-point of the average lifespan and the average wood-accumulation estimates for crops within the same category as defined by the CDWR. For our broad categories of grains, orchards and row crops, we calculated an average value of Mg C ha-1 based on data available for each crop type within the category. We multiplied the area of each of the seven agricultural classes in the study area by the estimated carbon to provide total Mg C ha-1 . Carbon storage for non-forest natural vegetation types used estimates from the literature: pasture, grassland, and shrubland , and freshwater emergent wetlands . We divided forested vegetation types into three main types of riparian forest: valley oak , Fremont cottonwood , and willow . We improved the estimates of carbon storage associated with these types of riparian forest with plot data collected on the Cosumnes Preserve. Plot data included the measurement of all trees >10cm diameter at breast height within a 0.04-ha plot , applied allometric equations to calculate the amount of above-ground carbon , and summed these amounts to report a total Mg C ha-1 for riparian forests . These are in line with other estimates of live biomass from riparian studies in California . Using this variety of techniques,rolling benches we assigneda coarse estimate of total Mg C ha-1 for each parcel in the study area . Since this compilation of varied data includes a mix of both above- and below-ground carbon estimates for different classes , our estimates need to be considered conservative. We assessed the effect of different landscape management scenarios on the Swainson’s Hawk and also on a suite of 15 focal bird species. First, we used Boosted Regression Trees modeling techniques to fit the baseline land-cover data to presence and absence points of Swainson’s Hawk nest locations. We used known nest locations, identified using comprehensive field surveys of the area , to generate presence points . We generated absence points by randomly placing pseudo-absence points within the study area. We used 75% of the points to train the landscape suitability model, and 25% to test the predictive ability of those points.

We generated models by calculating the proportion of each land-use type contained within a 25-ha square that surrounded each presence and absence point. This threshold utilized research which found that 50% of Swainson’s Hawk foraging occurs within 25 to 86 ha of nest sites . We also tested model sensitivity using a 100-ha core area, and noted no significant changes n model results. Once we fitted the current land-cover type to the Swainson’s Hawk nest presence and absence data, we used the BRT to spatially project the probability of landscape suitability onto each of the three future scenarios. We assessed model performance using area under curve of the receiver operating characteristic curve scores . We converted model results to raster grids and assigned each parcel a landscape suitability score based on the average score of all grid cells contained within a parcel . Second, we assessed the effects of the three management scenarios relative to baseline on a suite of 15 other focal bird species identified as indicator species for natural habitats in the Central Valley . In contrast to the Swainson’s Hawk approach, we used existing suitability models developed for each of these focal species in the Central Valley, with suitability scores ranging from zero to one . We assigned suitability values to our baseline and three alternative scenario parcels using two steps. First, we estimated the average suitability for each bird species within each of our land-cover types by overlaying the spatial suitability surfaces onto our land-cover data and calculating the area weighted average suitability of each land-cover type for each bird species . Second, we assigned an area-weighted suitability value for each of the 15 species to each parcel in our baseline and future management scenarios, according to the parcel’s land-cover type. Based on these scores, we calculated the average suitability score for each landscape scenario across all 15 focal bird species, using a 5% increase or decrease as the threshold for meaningful change.We calculated nitrous oxide emissions for the agricultural land-use types in a manner consistent with International Panel on Climate Change Tier-1 guidelines . The key input parameter was nitrogen fertilizer use. We acquired estimates of nitrogen fertilizer application rates from a compilation of California data and summarized these by our seven agricultural types . For grain, orchard, pasture, and row crops, which contain multiple types of crops, we averaged emission rates of these individual crops to provide a single figure for the class. We used IPCC emissions factors to convert nitrogen fertilizer application to nitrous oxide emissions. This was 1% of nitrogen fertilizer applied for all crop groups except rice, for which we used an emissions factor of 0.3% . We excluded estimates for alfalfa since this crop rarely receives inorganic nitrogen fertilizer application and only accounts for a small proportion of the study area . For each parcel, we estimated the amount of nitrous oxide emissions per year under baseline and the three alternative management scenarios based on the land use type within each parcel. We calculated the amount of nitrate–nitrogen leaching for the agricultural land types based on the difference between nutrient inputs and nutrient losses. We compiled nutrient inputs from crop specific fertilization rates and based nutrient losses on the amount of nitrogen harvested in crops . We assumed atmospheric losses to be 10% of the fertilization rate, which is a conservative estimate developed to reflect the total N gaseous emissions . We assumed all surplus nitrogen was leached from soil into the groundwater in the form of nitrate , and for crops where the nitrogen harvested exceeds the nitrogen inputs, we assumed leaching loss was zero. As with emissions, we estimated the amount of nitrogen currently leached per year for each parcel and for the three alternative scenarios.Natural vegetation increased slightly in the restoration scenario from a baseline of 44% to 46% of the study area, and decreased in the urban and enhanced agriculture scenarios to 40% and 21%, respectively . Under baseline conditions, natural vegetation consisted primarily of grassland in the eastern portion of the study area , and riparian forest along the Cosumnes River accounts for 4%. Cover of riparian forest increased to 12% under the restoration scenario .

To investigate whether Tic20 can indeed form an ion channel, Tic20-proteoliposomes were subjected to swelling assays . Changes in the size of liposomes in the presence of high salt concentrations, as revealed by changes in the optical density, can be used to detect the presence of a poreforming protein. After addition of 300 mM KCl to liposomes and Tic20-proteoliposomes, their optical densities dropped initially, due to shrinkage caused by the increased salt concentration. However, the optical density of protein-free liposomes remained at this low level, showing no change in their size; whereas in the case of Tic20-proteoliposomes the optical density increased constantly with time. The increase in optical density strongly supports the presence of a channel in Tic20-proteoliposomes that is permeable for ions, thereby creating an equilibrium between the inner compartment of the proteoliposomes and the surrounding buffer. To exclude the possible effects of contaminating channel-forming proteins derived from the bacterial membrane and a protein inserted into the liposomes , a further negative control was set up: Tic110 containing only the first three transmembrane helices was purified similarly to Tic20 and reconstituted into liposomes. We chose this construct, since NtTic110 inserts into the membrane during in vitro protein import experiments. Furthermore, as the full length and N-terminally truncated Tic110 possess very similar channel activities, it is unlikely that the N-terminal part alone forms a channel. The insertion of NtTic110 into liposomes was confirmed by incubation under different buffer conditions followed by flotation experiments, similarly to Tic20 . However,hydroponic dutch buckets these NtTic110-proteoliposomes behaved similarly to the empty liposomes during swelling assays: after addition of salt, the optical density decreased, and except for a small initial increase, it remained at a constant level.

This makes it unlikely that a contamination from E. coli or simply the insertion of a protein into the liposomes caused the observed effect in the optical density of Tic20- proteoliposomes. To further characterize the channel activity of Tic20, electrophysiological measurements were performed. After the fusion of Tic20-proteoliposomes with a lipid bilayer, ion channel activity was observed . The total conductance under symmetrical buffer conditions , 250 mM KCl was dependent on the direction of the applied potential: 1260 pS and 1010 pS under negative and positive voltage values, respectively. The channel was mostly in the completely open state, however, individual single gating events were also frequently observed, varying in a broad range between 25 pS to 600 pS. All detected gating events were depicted in two histograms.Two conductance classes were defined both at negative and positive voltage values with thresholds of 220 pS and 180 pS, respectively.The observed pore seems to be asymmetric, since higher conductance classes notably differ under positive and negative voltages. This is probably due to interactions of the permeating ions with the channel, which presumably exhibits an asymmetric potential profile along the pore. Since small and large opening events were simultaneously observed in all experiments, it is very unlikely that they belong to two different pores. The selectivity of Tic20 was investigated under asymmetric salt conditions , 250/20 mM KCl. Similarly to the conductance values, the channel is intrinsically rectifying ,supporting asymmetric channel properties. The obtained reverse potential is 37.0 ± 1.4 mV . According to the Goldman-Hodgkin-Katz approach, this corresponds to a selectivity of 6.5:1 for K+ :Cl- -ions, thus indicating cation selectivity similar to Tic110.

To determine the channel’s orientation within the bilayer, two side-specific characteristics were taken into account: the highest total conductance under symmetrical buffer conditions was measured under negative voltage values, and the channel rectifies in the same direction under asymmetrical buffer conditions . Therefore, it seems that the protein is randomly inserted into the bilayer. The pore size was roughly estimated according to Hille et al.. Considering the highest conductance class , a channel length of 1-5 nm and a resistivity of 247.5 Ω cm for a solution containing 250 mM KCl, taking into account that the conductivity of the electrolyte solution within the pore is ~5 times lower than in the bulk solution, the pore size was estimated to vary between 7.8-14.1 Å. This is in good agreement with the size of protein translocation channels such as Toc75 in the outer envelope membrane and Tic110 in the IE. Thus, the size of the Tic20 pore would be sufficient for the translocation of precursor proteins through the membrane. NtTic110, as a negative control, did not show any channel activity during electrophysiological measurements, indicating that the measured channel is not the result of a possible bacterial contamination . Considering our data presented here and those published in previous studies, we can conclude that the Tic translocon consists of distinct translocation channels: On the one hand, Tic110 forms the main translocation pore and therefore facilitates import of most of the chloroplast-targeted preproteins; on the other hand, Tic20 might facilitate the translocation of a subset of proteins. This scenario would match the one found in the inner mitochondrial membrane, where specific translocases exist for defined groups of precursor proteins: the import pathway of mitochondrial carrier proteins being clearly separated from that of matrix targeted preproteins. The situation in chloroplasts does not seem as clear-cut, but an analogous separation determined by the final destination and/or intrinsic properties of translocated proteins is feasible. The severe phenotype of attic20-I mutants prompts us to hypothesize that Tic20 might be specifically required for the translocation of some essential proteins. According to cross-linking results, Tic20 is connected to Toc translocon components. Therefore, after entering the intermembrane space via the Toc complex, some preproteins might be transported through the IE via Tic20.

On the contrary, Kikuchi et al. presented that Tic20 migrates on BN-PAGE at the same molecular weight as the imported precursor of the small subunit of Rubisco and that tic20-I mutants display a reduced rate of the artificial precursor protein RbcS-nt: GFP. The authors interpreted these results in a way that Tic20 might function at an intermediate step between the Toc translocon and the channel of Tic110. However,bato bucket being a substantial part of the general import pathway seems unlikely due to the very low abundance of Tic20. It is feasible to speculate that such abundant proteins as pSSU, which are imported at a very high rate, may interact incidentally with nearby proteins or indifferently use all available import channels. To clarify this question, substrate proteins and interaction partners of Tic20 should be a matter of further investigation. Additionally, a very recent study suggested AtTic20-IV as an import channel working side by side with AtTic20-I. However, detailed characterization of the protein and experimental evidence for channel activity are still missing.Cerium oxide nanoparticles are widely used in applications such as catalyst automotive industry, glass mirrors, plate glass, and ophthalmic lenses . These NPs are among the 13 engineered nanomaterials in the list of priority for immediate testing by the Organization for Economic Cooperation and Development . However, the environmental release of CeO2 NPs from factories or applications, and their behavior and effects in the environment are not well known yet . Previous studies have shown that CeO2 NPs are stable in soil at pH values of 7 to 9 . This suggests CeO2 NPs will remain in soil for a long time. In addition, reports from recent investigations have shown a wide variety of plant responses after exposure toCeO2 NPs. For instance, Schwabe found that CeO2 NP treatments did not reduced the growth in pumpkin and wheat. However, Ma et al. reported that, at 2000 mg/L, nano-CeO2 reduced root elongation in lettuce . Van Hoecke et al. found that CeO2 NPs, at concentrations as low as 2.6 and 5.4 mg/L, produced chronic toxicity to the unicellular alga Pseudokirchneriella subcapitata. Previous results from our research group have shown that CeO2 NPs at 2000 mg/L reduced corn and tomato germination by 30% and cucumber germination by 20% . In a more recent study, we demonstrated that CeO2 NPs are taken up and stored without change in maize roots . This previous study also revealed that the uptake of CeO2 NPs by corn plants was affected by soil organic matter content and alginate surface coating . Alginates are naturally occurring polysaccharides that have been used to stabilize NPs for several applications . This suggests that excess of alginate can be released into the environment together with NPs, with unknown consequences for edible plants. Thus, more studies are needed to better understand the impact of CeO2 NPs in plants, in environments where excess alginates could be present. On the other hand, studies have shown that carbon-based nanoparticles such as single walled carbon nanotubes triggered reactive oxygen species generation in Arabidopsis and rice .

In addition, multi-wall carbon nanotubes have been found to induce gene expression of heat shock protein 90 in tomato leaves and roots . However, there are no reports on the effect of CeO2 NPs on heat shock protein expression in plants. A few studies have described the physiological impacts of rare earth elements in plants. For example, at concentration higher than 89 µmol/L, cerium affected the foliar chlorophyll content, nitrate reductase activity, shoot root length and relative yield in cowpea plants . The authors suggested the effects could be produced by the substitution of Mg2+ by Ce in chlorophyll synthesis. It has also been suggested that, due to their similar chemical characteristics, Eu, a REE, may compete with Ca for organic ligands . These studies suggest that REE elements can have serious impacts on the uptake of nutritional elements in food crops. However, to the authors’ knowledge the impact of REE NPs on the uptake of nutritional elements by plants has yet to be reported. The purposes of this work were to determine the effects of alginate on: the transport of Ce within corn plants treated with CeO2 NPs, the uptake and transport of micro and macro nutrients, the chlorophyll content, and the expression of stress related heat shock protein 70. Maize was selected for this study because it is a crop widely cultivated throughout the world for direct and indirect consumption. In addition, 40% of the corn world’s harvest is produced in the United States . In this study, corn plants were grown in soil spiked with CeO2 NPs with various alginate concentrations for one month. After harvest, the concentration of Ce and many nutrient elements were determined by ICP-OES in the root and shoots tissues. This suggests that in an eventual release of CeO2 NPs, the higher risk of food contamination would occur in organic matter enriched soil. The mechanism involved in the increase of Ce uptake and translocation by alginate is still unknown. However, our previous work showed that alginate surface coating increased the Ce translocation to shoots in corn plants grown in a soil with low organic matter content and treated with 400 mg/kg CeO2 NPs. Sodium alginate has been associated with seed germination, shoot elongation, root growth, and flower production, among others in Foeniculum vulgare Mill . However, the mechanisms of these effects are still unknown. The presence of CeO2 NPs with/without alginate did not alter the uptake of macro-nutrients Mg, K, Ca, S, and P in one-month old corn roots. However, the uptake of Al and the micro-nutrients Fe, Mn, and Zn was increased . Compared to control , the concentrations of Fe and Al were significantly higher in all NP treatments. For Al, the difference was significant at p ≤ 0.023, but for Fe, the significance was only at p ≤ 0.09. The accumulation of both Fe and Al in roots was similar in all treatments. Moreover, compared to NPs alone and NPs-low alginate, the concentrations of both Fe an Al were significantly higher at medium and high alginate concentrations. It is very likely that the CeO2 NPs were bound with Fe and Al oxides, which are widespread soil colloids. Previous results showed that Fe and Al are co-released from the soil column with ZnO NPs . Manganese accumulation pattern was different. The addition of CeO2 NPs without alginate increased Mn accumulation in roots by 34% compared to control ; but NPs-low alginate and NPs-medium alginate treatments increased the accumulation of Mn by 92% and 90% respect to NPs without alginate and 158% and 155% respect to control. These differences were significant at p ≤ 0.005.

Since thaumatin is a 22 kDa protein, a membrane with MWCO of 5 kDa is used per working process knowledge. Assuming a conservative flux of 30 L/, the inlet stream is concentrated using a concentration factor of 5, diafiltered 10 times against reverse osmosis water, then re-concentrated using a CF of 5 over 20.6 h, resulting in a 75% pure thaumatin and nicotine content of 1.08 mg/kg thaumatin. A retention coefficient of 0.9993 was assumed for thaumatin, resulting in 5.8% thaumatin loss in UF/DF . The retentate is then sent to five CEX chromatography columns operating in parallel which was modeled based on unpublished data from Nomad Bioscience GmbH . GE Healthcare Capto S resin with an assumed binding capacity of 150 g/L was used in this analysis. Table S2 shows the downstream losses breakdown per unit operation. Spray drying is used as a final formulation step over other means of industrial drying due to the heat sensitivity of thaumatin. The simulated facility consists of three sections—Virion production laboratory , spinach field growth, and DSP. A list of base case design parameters and assumptions is shown in Table S3. The VPL process is adopted from a recent article entailing the production of RNA viral particles from agrobacteria carrying a PVX construct. The laboratory is sized to produce 7900 L of spray solution per batch for application in the field. Nicotiana benthamiana plants are used as the host to produce the viral particles to inoculate spinach. N. benthamiana seeds are germinated in soilless plant substrate at a density of 94 plants per tray.

Seedlings are grown hydroponically , under LEDs, until reaching manufacturing maturity at day 35. Agrobacterium tumefaciens is grown for 24 h, before being left in a 4 L flask overnight, and the A. tumefaciens suspension is added to MES buffer in V-101. N. benthamiana infiltration takes place in a vacuum agroinfiltration chamber for 24 h followed by incubation for 7 days in . N. benthamiana biomass production,ebb flow tray agrobacterium growth, agroinfiltration, and incubation parameters are adapted from. After the incubation period, 41.5 kg of N. benthamiana fresh weight are ground and mixed with PBS buffer in a 5:1 buffer:biomass ratio. The extract is then sent to a decanter centrifuge to separate plant dry matter from the liquid phase which is clarified by dead-end filtration , followed by mixing the permeate with 35.9 kg of diatomaceous earth and 7780 L of water to reach a final concentration of 1014 viral particles/L and 4.55 g diatomaceous earth/L. Diatomaceous earth is used as an abrasive to mechanically wound plant cell walls allowing the virions to enter the cytoplasm of the cell. The final spray is stored in for 13 h before field application. Field operation starts at the beginning of each batch with the direct seeding of 28.3 million Spinacia oleracea seeds over 22.6 acres. Spinach is planted over 80-inch beds with an assumed 3 ft spacing between beds, resulting in 14,520 linear bed feet per acre. Seeds are germinated and grown in the field for 44.5 days, during which time a drip irrigation system delivers irrigation water and soluble fertilizer to the soil.A tractor on which multiple high-pressure spray devices are mounted is used to deliver the viral particle solution at a rate of 2 acres/h. This method of delivery has shown high effectiveness. Spinach plants are incubated in the field for 15 days post-infection. During that period, thaumatin starts to accumulate in the crop at an average expression level of 1 g/kg FW after 15 days post-spraying. At day 60, two mechanical harvests collect a total of 344 MT spinach biomass, carrying 344 kg thaumatin, with the aid of four hopper trucks, which is transferred to a 500-m-long conveyor belt that extends from the field collection site to the DSP section of the facility. Harvesting occurs at an average rate of 17,000 kg FW/h, which is estimated based on a harvester speed of 5 km/h and 14,520 linear bed feet per acre.

A more simplified downstream processing, enabled by the use of spinach as a host, starts with mixing plant material with 65 ◦C water before extracting the green juice through a screw press . The resulting GJ is heated for 1 h at 65 ◦C in ten jacketed tanks , then concentrated by evaporation to reduce product stream volume for further purification steps. Since thaumatin is not stable at temperatures above 70 ◦C at neutral pH, evaporation is performed at a low temperature of 40 ◦C and 0.074 bar vacuum pressure. Thermally degraded host cell proteins and impurities are eliminated in a P&F filtration unit designed to include 10 filter sheets with decreasing particle retention size from 25 to 0.1 µm. Smaller impurities are removed using a diafiltration unit with 5 kDa molecular weight cut off cassettes in a similar process as described in Section 3.3, the retentate is spray dried in to obtain a final product which has 5% water content, and 348 kg of solid material containing 94% pure thaumatin and 6% spinach impurities. These impurities are expected to be water soluble, heat stable molecules in the range of 5–100 kDa, according to the theoretical design of the filtration scheme. As shown in Figure 3a, field labor is the highest contributor to the upstream field facility followed by consumables. Detailed labor requirement and cost estimation calculations can be found in Tables S7 and S8. Consumables include mechanical harvester and tractor’s fuel, lubrication, and repair costs and other field equipment repair costs. Upstream indoor facility AOC breakdown elucidates a high cost of consumables due to the cost of soilless plant substrate, followed by high energy consumption from the LED lighting system used for plant growth. The labor category does not appear clearly on the chart because of the low need for labor hours since the indoor facility is highly automated. In both DSP scenarios, facility-dependent costs have the highest cost impact. Insurance, local taxes, and other overhead expenses are estimated to be 1%, 2%, and 5% of the section’s DFC, respectively. Maintenance costs are also included in this category and estimated to be 10% of equipment purchase prices. Facility dependent cost estimation parameters are shown in Tables S9 and S10. Consumables account for 38% of the DSP facility with chromatography due to the high cost of Capto S resin that is changed every 100 cycles. The effect of varying resin binding capacity to the product on the DSP AOC and COGS is shown in Figure 3d.

Transgenic production models were resized based on scenario design requirement for production levels ranging from 10–150 MT and expression levels ranging from 0.5–2.5 g/kg, while keeping the scheduling parameters the same from base case models. The significant impact of expression level on CAPEX and COGS is elucidated in Figure 4a–c. Production level shows a very small decline in COGS for indoor upstream facility and a linear increase in CAPEX with increasing production level. On the other hand, the field upstream facility showed a significant increase in COGS at lower production levels due to the minimum ownership costs of field equipment regardless of the small acreage size. DSP followed the expected behavior that economy of scale dictates,flood and drain tray with sharp decrease in COGS at lower production levels and diminishing returns at higher production levels. The deviation from linear trend at 150 MT/year in field upstream and DSP is likely due to the model’s specified equipment maximum rating, which allows for the inclusion of a new equipment in parallel beyond this rating. As shown in Table 2, the DSP section of the facility accounts for 79% of the project’s CAPEX and 63% of AOC. This is justified by the high equipment purchase prices, piping, instrumentation, buildings, engineering, and construction costs for a plant of this size. Figure 5a shows field labor as the highest cost contributor to the spinach field growth section due to the high direct demand of 48,800 labor-h/year, followed by the cost of spinach seeds, which is estimated to be $23.68/kg for the leafy Bloomsdale variety. Mechanical harvester and tractor’s fuel, lubrication, and repair costs are in included as consumables as well as other field machinery repair costs. Due to the small-scale scope of the VPL, labor is the highest contributor of the section’s operating cost.The impact of varying the highest cost drivers in each of the facility’s category by 25% on COGS is portrayed as a tornado diagram in Figure 5c. Field labor was the most sensitive cost variable, having the highest impact on the COGS, followed by the ultrafiltration membrane, which is replaced every 30 cycles. In this model, we assume a relatively high downstream recovery of the protein from harvest to formulation. The reason for this assumption is that spinach, being edible crop, allows for a lower target product purity and a consequently fewer DSP steps. It is particularly important to focus resources on maximizing downstream recovery during process development because it ultimately affects plant biomass and spray volume requirement upstream to appropriately compensate for these losses, which in turn affects equipment sizing in DSP based on the amount of plant material to be processed. The unit operations were resized according to the scenario design requirement for downstream recovery ranging from 50 to 95% while scheduling parameters were left unchanged. This effect of downstream recovery on the facility’s AOC and COGS is shown in Figure 5d and shows a 1.5× increase in AOC and COGS as downstream recovery decreases from 95% to 50%. Although our analysis indicates a relatively high COGS range for a sugar substitute, there are unrealized costs savings from thaumatin use due to its unique sweetness intensity. Thaumatin’s use in extremely small quantities is essentially why it is considered a noncaloric sweetener, as it provides only 4 calories per gram. Sensory evaluation studies have found that a sample with 5% sucrose +4.6 ppm thaumatin II had similar sweetness as a 10% sucrose control with minimal lingering aftertaste, suggesting that up to one-half of the sugar could be replaced by thaumatin II . SSBs including sodas, fruit drinks, and sport drinks account for 50% of the total added sugar in Western diets, and therefore provide an attractive avenue for thaumatin emergence as a sugar substitute.

The incorporation of thaumatin by the industry not only offers a tool to help decelerate the obesity epidemic caused by increased childhood sugar intake decades ago, but also provides itself with a more economically viable solution. Firstly, as sugar taxations emerge, sugar reduction becomes a financial incentive. Secondly, the reduction of sugar and the addition of thaumatin to retain the same level of sweetness has the potential to save millions of dollars per day on the cost of sweetening beverages. Assuming that the average “standard” sucrose concentration in SSBs is 35.5 g per 12 floz. drink ~10%, and a $0.30/kg sugar price, Figure 6 shows the potential savings from using thaumatin to reduce sugar content by 20%, 30%, and 50%, while maintaining the same sweetness as the standard for a range of thaumatin purchase prices. The amount of thaumatin needed to obtain the same sweetness as a 10% solution in each sugar reduction scenario was calculated using the sensory regression analysis included in a published GRAS notice . Table 3 shows the daily and annual amount of thaumatin needed for each sugar reductions scenario, assuming that one billion 12 fl oz drinks are to be sweetened per day. Successful implementation of thaumatin in this avenue can liberate R&D resources to improve expression levels and increase production volumes, both of which have a substantial impact on COGS reduction, as we have demonstrated.Our preliminary engineering facility design indicates the feasibility of thaumatin manufacturing by various molecular farming platforms. The most economic method is the field grown ethanol-inducible, transgenic N. tabacum, assuming a downstream facility without chromatography . It remains unclear whether heat incubation is sufficient to achieve the desired purity for a safe product without the inclusion of chromatography on a large-scale. In a previous plant-made food safety product techno-economic analysis, a chromatography unit was included for protein purification from N. benthamiana; however, heat precipitation of host cell proteins was not included as a purification step.

Relying on Halomonas spp. in combination with acetate as substrate may allow very rapid production of the required bio-plastic, but substrate availability constraints are higher than for CH4 or CO2/H2. A terminal electron acceptor is required in all cases, which will almost certainly be O2. Supplying O2 safely without risking explosive gas mixtures, or wasting the precious resource, is again a question of reactor design and operation. Certain purple non-sulfur alphaproteobacteria and Rhodopseudomonas palustris also feature remarkable substrate flexibility and can produce PHAs . Bioplastic recovery and purification is a major challenge. To release the intracellular compound, an osmolysis process may be employed with the halophile . However, the transfer of cells into purified water and separation of the polyesters from the cell debris, potentially through several washing steps, may require substantial amounts of water. An alternative and/or complement to the common process for extraction of PHAs with halogenated organic solvents, is to use acetate or methanol as solvents . This is applicable independent of the organism and the inputs can be provided from other bio-manufactory modules. The high crystallinity of pure PHB makes it brittle and causes it to have a narrow melting range, resulting in warp during extrusion and 3D-printing. Such behavior places operational constraints on processing and hampers applications to precision manufacturing . Workarounds may be through additives, bio-composite synthesis, and copolymerization. However, this ultimately depends on what biology can provide . There is a need to advance space bio-platforms to produce more diverse PHAs through synthetic biology. ISM of bio-materials can reduce the mission cost, increase modularity,growing hydroponically and improve system recyclability compared to abiotic approaches. In an abiotic approach, plastics will be included in the payload, thereby penalizing up-mass at launch.

As with elements of FPS and ISRU, ISM increases flexibility and can create contingencies during surface operations, therefore reducing mission risk. The high modularity of independent plastic production, filament formation, and 3D-printing allows for a versatile process, at the cost of greater resources required for systems operations. Overall, this maximizes resource use and recyclability, by utilizing mission waste streams and byproducts for circular resource management.Biomanufacturing on Mars can be supported by flexible biocatalysts that extract resources from the environment and transform them into the complex products needed to sustain human life. The Martian atmosphere contains CO2 and N2 .It is very likely that the expensive and energy-intensive Sabatier plants for CH4 production will be available per Design Reference Architecture . While a HaberBosch plant could be set up for ammonia production, this is neither part of the current DRA nor exceptionally efficient. Thus, for a biomanufactory, we must have carbon fixation reactors to fix CO2 into feed stocks for nonmethanotrophs, and have nitrogen fixation reactors to fifix N2 to fulfill nitrogen requirements for non-diazotrophs. Trace elements and small-usage compounds can be transported from Earth, or in some cases extracted from the Martian regolith. In the case where power is provided from photocollection or photovoltaics, light energy will vary with location and season, and may be critical to power our bioreactors. Although photosynthetic organisms are attractive for FPS, a higher demand for carbon-rich feed stocks and other chemicals necessitates a more rapid and efficient CO2 fixation strategy. Physicochemical conversion is inefficient due to high temperature and pressure requirements. Microbial electrosynthesis , whereby reducing power is passed from abiotic electrodes to microbes to power CO2 reduction, can offer rapid and efficient CO2 fixation at ambient temperature and pressure . MES can produce a variety of chemicals including acetate , isobutanol , PHB , and sucrose , and therefore represents a flexible and highly promising ISRU platform technology . Biological N2-fifixation offers power- and resource-efficient ammonium production. Although photoautotrophic N2 fixation with, for example, purple non-sulfur bacteria, is possible, slow growth rates due to the high energetic demand of nitrogenase limit throughput .

Therefore, heterotrophic production with similar bacteria using acetate or sucrose as a feed stock sourced from electromicrobial CO2-fifixation represents the most promising production scheme, and additionally benefits from a high degree of process redundancy with heterotrophic bioplastic production. Regolith provides a significant inventory for trace elements and, when mixed with the substantial cellulosic biomass waste from FPS processes, can facilitate recycling organic matter into fertilizer to support crop growth. However, regolith use is hampered by widespread perchlorate , indicating that decontamination is necessary prior to enrichment or use. Dechlorination can be achieved via biological perchlorate reduction using one of many dissimilatory perchlorate reducing organisms . Efforts to reduce perchlorate biologically have been explored independently and in combination with a more wholistic biological platform . Such efforts to integrate synthetic biology into human exploration missions suggest that a number of approaches should be considered within a surface biomanufactory.A biomanufactory must be able to produce and utilize feed stocks along three axes as depicted in Figure 5: CO2-fifixation to supply a carbon and energy source for downstream heterotrophic organisms or to generate commodity chemicals directly, N2-fifixation to provide ammonium and nitrate for plants and non-diazotrophic microbes, and regolith decontamination and enrichment for soil-based agriculture and trace nutrient provision. ISRU inputs are sub-module and organism dependent, with all sub-modules requiring water and power. For the carbon fixation sub-module , CO2 is supplied as the carbon source, and electrons are supplied as H2 or directly via a cathode. Our proposed biocatalysts are the lithoautotrophic Cupriavidus necator for longer-chain carbon production [e.g., sucrose ] and the acetogen Sporomusa ovata for acetate production. C. necator is a promising chassis for metabolic engineering and scale-up , with S. ovata having one of the highest current consumptions for acetogens characterized to date . The fixed-carbon outputs of this sub-module are then used as inputs for the other ISRU sub-modules in addition to the ISM module . The inputs to the nitrogen fixation sub-module include fixed carbon feedstocks, N2, and light. The diazotrophic purple-non sulfur bacterium Rhodopseudomonas palustris is the proposed biocatalyst, as this bacterium is capable of anaerobic, light-driven N2 fixation utilizing acetate as the carbon source, and has a robust genetic system allowing for rapid manipulation . The output product is fixed nitrogen in the form of ammonium, which is used as a feed stock for the carbon-fixation sub-module of ISRU along with the FPS and ISM modules.

The inputs for the regolith enrichment sub-module include regolith, fixed carbon feedstocks, and N2. Azospira suillum is a possible biocatalyst of choice due to its dual use in perchlorate reduction and nitrogen fixation . Regolith enrichment outputs include soil for the FPS module , H2 that can be fed back into the carbon fixation sub-module and the ISM module, chlorine gas from perchlorate reduction, and waste products. Replicate ISRU bioreactors operating continuously in parallel with back-up operations lines can ensure a constant supply of the chemical feed stocks, commodity chemicals, and biomass for downstream processing in ISM and FPS operations. Integration of ISRU technologies with other biomanufactory elements, especially anaerobic digestion reactors,grow strawberries hydroponically may enable complete recyclability of raw materials, minimizing resource consumption and impact on the Martian environment .Waste stream processing to recycle essential elements will reduce material requirements in the biomanufactory. Typical feed stocks include inedible crop mass, human excreta, and other mission wastes. Space mission waste management traditionally focuses on water recovery and efficient waste storage through warm air drying and lyophilization . Mission trash can be incinerated to produce CO2, CO, and H2O . Pyrolysis, another abiotic technique, yields CO and H2 alongside CH4 . The Sabatier process converts CO2 and CO to CH4 by reacting with H2. An alternate thermal degradation reactor , operating under varying conditions that promote pyrolysis, gasification, or incineration, yields various liquid and gaseous products. The fact remains however, that abiotic carbon recycling is inefficient with respect to desired product CH4, and is highly energy-intensive. Microbes that recover resources from mission wastes are a viable option to facilitate loop closure. Aerobic composting produces CO2 and a nutrient-rich extract for plant and microbial growth . However, this process requires O2, which will likely be a limited resource. Hence, anaerobic digestion, a multi-step microbial process that can produce a suite of end products at lower temperature than abiotic techniques , is the most promising approach for a Mars biomanufactory to recycle streams for the ISM and FPS processes. Digestion products CH4 and volatile fatty acids can be substrates for polymer-producing microbes . Digestate, with nutrients of N, P, and K, can be ideal for plant and microbial growth , as shown in Figure 6. Additionally, a CH4 and CO2 mixture serves as a biogas energy source, and byproduct H2 is also an energy source . Because additional infrastructure and utilities are necessary for waste processing, the extent of loop closure that is obtainable from a treatment route must be analyzed to balance yield with its infrastructure and logistic costs. Anaerobic digestion performance is a function of the composition and pretreatment of input waste streams , as well as reaction strategies like batch or continuous, number of stages, and operation conditions such as organic loading rate, solids retention time, operating temperature, pH, toxic levels of inhibitors and trace metal requirements . Many of these process parameters exhibit trade-offs between product yield and necessary resources. For example, a higher waste loading reduces water demand, albeit at the cost of process efficiency. There is also a potential for multiple co-benefits of anaerobic digestion within the biomanufactory. Anaerobic biodegradation of nitrogen-rich protein feed stocks, for example, releases free NH3 by ammonification. While NH3 is toxic to anaerobic digestion and must thus be managed , it reacts with carbonic acid to produce bicarbonate buffer and ammonium, decreasing CO2 levels in the biogas and buffering against low pH.

The resulting digestate ammonium can serve as a fertilizer for crops and nutrient for microbial cultures.FPS and ISM waste as well as human waste are inputs for an anaerobic digester, with output recycled products supplementing the ISRU unit. Depending on the configuration of the waste streams from the biomanufactory and other mission elements, the operating conditions of the process can be varied to alter the efficiency and output profile. Open problems include the design and optimization of waste processing configurations and operations, and the identification of optimal end-product distributions based on a loop closure metric against mission production profiles, mission horizon, biomanufacturing feed stock needs, and the possible use of leftover products by other mission elements beyond the biomanufactory. A comparison with abiotic waste treatment strategies is also needed, checking power demand, risk, autonomy, and modularity benefits.Biomanufactory development must be done in concert with planned NASA missions that can provide critical opportunities to test subsystems and models necessary to evaluate efficacy and technology readiness levels . Figure 7 is our attempt to place critical elements of a biomanufactory road map into this context. We label critical mission stages using Reference Mission Architecture -S and RMA-L, which refer to Mars surface missions with short and long durations, respectively. Reliance on biotechnology can increase the risk of forward biological contamination . Beyond contamination, there are ethical issues that concern both the act of colonizing a new land and justifying the cost and benefits of a mission given needs of the many here on earth. Our road map begins with the call for an extensive and ongoing discussion of ethics . Planetary protection policies can provide answers or frameworks to address extant ethical questions surrounding deep-space exploration, especially on Mars . Critically, scientists and engineers developing these technologies cannot be separate or immune to such policy development.The upcoming lunar exploration missions, Artemis and Gateway , provide additional opportunities for integration with Earth-based biomanufactory development. Early support missions will provide valuable experience in cargo predeployment for crewed operations, and is likely to help shape logistics development for short-term as well as long-term Mars exploration missions when a biomanufactory can be deployed. Here we present a subset of Artemis efforts as they relate to mission elements with opportunities for testing and maturing biomanufacturing technology. Although ISRU technologies for the Moon and Mars will be sufficiently distinct due to different resource availabilities, crewed Artemis missions provide a testing ground for crewed Mars bio-process infrastructure. Later Artemis missions also provide a suitable environment to test modular, interlocked, scalable reactor design, as well as the design of compact molecular biology labs for DNA synthesis and transformation.

In comparison with pesticides, only sporadic research has shown detoxification of PPCPs by POD and GST in plants. In the present study, activities of POD and GST increased in a dose-dependent manner in both roots and leaves after exposure to PPCPs . This observation was consistent with Bartha et al. and Huber et al. , who observed oxidation of diclofenac by plant peroxidases, and also glutathione conjugation in Typha latifolia. These mechanistic studies, together with our results, clearly show that the POD and GST enzyme families may play an important role in transformation and conjugation of PPCPs in plants. Glutathione is one of the major soluble low molecular weight antioxidants, and also the major non-protein thiol in plant cells , contributing to maintain the cellular redox homeostasis and signaling. Moreover, conjugation with xenobiotics by GSH may be a common pathway for plant metabolism of various man-made chemicals . Glutathione conjugation with diclofenac , 8:2 fluorotelomer alcohol and chlortetracycline have been previously observed in plants. It is well known that these processes require extensive utilization of reduced GSH as an electron donor and subsequently produce oxidized glutathione . The changes in the cellular glutathione pool, specially the associated ratio of reduced to oxidized glutathione, play a central role in plant defense responses . Glutathione homeostasis after exposure to trace levels of PPCPs, however,growing hydroponically has not been well documented so far. In this study, the glutathione content increased at low PPCP doses, while decreased to normal levels at the highest PPCP treatment level . The GSSG content was unchanged when the PPCP concentrations were low , but showed a significant increase when the PPCP concentration was increased to 50 mg L! 1 . The depletion observed in cellular GSH could contribute to the PPCP-induced oxidative stress and detoxification of xenobiotics.

Meanwhile, the different responses of GSH in root and leaves indicated that roots may be the main site to express PPCP toxicity and induce PPCP detoxification. Given the dominance of GSH conjugates as observed for pesticides, it is possible that trace contaminants such as PPCPs may be removed similarly by GSH conjugation. It is therefore imperative to conduct further research to explore the mechanisms and pathways of PPCP phytotransformation by GSTs after uptake by plants.On the whole, the present study provided evidence that some PPCPs may be translocated systemically, and ultimately posed toxicity effects in higher plants. Oxidative stress response may reflect the intensity of PPCP treatment and sensitivity of plant species to PPCPs, and may be used as indicators for early plant response to trace organics introduced into agroecosystems. Furthermore, plants may detoxify PPCPs through different mechanisms, including enhanced antioxidant defense systems to prevent oxidative damage and increased activities of xenobioticmetabolizing enzymes. These mechanisms help maintain plant physiological, biochemical and molecular functional integrity, offering the possibility to use some of these endpoints as biomarkers for predicting phytotoxicity induced by PPCPs and likely other man-made chemicals. Further research is needed to evaluate the physiological and biological responses of plants in realistic field practices, such as irrigation with treated wastewater or fertilization with biosolids and animal wastes in agriculture.The Paharpur Business Centre and Software Technology Incubator Park is a 7 story, 50,400 ft2 office building located near Nehru Place in New Delhi India. The occupancy of the building at full normal operations is about 500 people. The building management philosophy embodies innovation in energy efficiency while providing full service and a comfortable, safe, healthy environment to the occupants. Provision of excellent Indoor Air Quality is an expressed goal of the facility, and the management has gone to great lengths to achieve it. This is particularly challenging in New Delhi, where ambient urban pollution levels rank among the worst on the planet.

The approach to provide good IAQ in the building includes a range of technical elements: air washing and filtration of ventilation intake air from rooftop air handler, the use of an enclosed rooftop greenhouse with a high density of potted plants as a bio-filtration system, dedicated secondary HVAC/air handling units on each floor with re-circulating high efficiency filtration and UVC treatment of the heat exchanger coils, additional potted plants for bio-filtration on each floor, and a final exhaust via the restrooms located at each floor. The conditioned building exhaust air is passed through an energy recovery wheel and chemisorbent cartridge, transferring some heat to the incoming air to increase the HVAC energy efficiency. Flooring is a combination of stone, tile and “zero VOC” carpeting. Wood trim and finish appears to be primarily of solid sawn materials, with very little evidence of composite wood products. Furniture is likewise in large proportion constructed from solid wood materials. The overall impression is that of a very clean and well-kept facility. Surfaces are polished to a high sheen, probably with wax products. There was an odor of urinal cake in the restrooms. Smoking is not allowed in the building. The plants used in the rooftop greenhouse and on the floors were made up of a number of species selected for the following functions: daytime metabolic carbon dioxide absorption, nighttime metabolic CO2 absorption, and volatile organic compound and inorganic gas absorption/removal for air cleaning. The building contains a reported 910 indoor plants. Daytime metabolic species reported by the PBC include Areca Palm, Oxycardium, Rubber Plant, and Ficus alii totaling 188 plants . The single nighttime metabolic species is the Sansevieria with a total of 28 plants . The “air cleaning” plant species reported by the PBC include the Money Plant, Aglaonema, Dracaena Warneckii, Bamboo Palm, and Raphis Palm with a total of 694 plants .

The plants in the greenhouse numbering 161 of those in the building are grown hydroponically, with the room air blown by fan across the plant root zones. The plants on the building floors are grown in pots and are located on floors 1-6. We conducted a one-day monitoring session in the PBC on January 1, 2010. The date of the study was based on availability of the measurement equipment that the researchers had shipped from Lawrence Berkeley National Lab in the U.S.A. The study date was not optimal because a large proportion of the regular building occupants were not present being New Year’s Day. An estimated 40 people were present in the building all day during January 1. This being said, the building systems were in normal operations, including the air handlers and other HVAC components. The study was focused primarily on measurements in the Greenhouse and 3rd and 5th floor environments as well as rooftop outdoors. Measurements included a set of volatile organic compounds and aldehydes, with a more limited set of observations of indoor and outdoor particulate and carbon dioxide concentrations. Continuous measurements of Temperature and relative humidity were made selected indoor and outdoor locations. Air sampling stations were set up in the Greenhouse, Room 510, Room 311, the 5th and 3rd floor air handler intakes,growing strawberries hydroponically the building rooftop HVAC exhaust, and an ambient location on the roof near the HVAC intake. VOC and aldehyde samples were collected at least once at all of these locations. Both supply and return registers were sampled in rooms 510 and 311. As were a greenhouse inlet register from the air washer and outlet register ducted to the building’s floor level. Air samples for VOCs were collected and analyzed following the U.S. Environmental Protection Agency Method TO-17 . Integrated air samples with a total volume of approximately 2 L were collected at the sites, at a flow rate of <70 cc/min onto preconditioned multibed sorbent tubes containing Tenax-TA backed with a section of Carbosieve. The VOCs were desorbed and analyzed by thermodesorption into a cooled injection system and resolved by gas chromatography. The target chemicals, listed in Table 1, were qualitatively identified on the basis of the mass spectral library search, followed by comparison to reference standards. Target chemicals were quantified using multi-point calibrations developed with pure standards and referenced to an internal standard. Sampling was conducted using Masterflex L/S HV-07553-80 peristaltic pumps assembled with quad Masterflex L/S Standard HV-07017-20 pump heads. Concentrations of formaldehyde, acetaldehyde, and acetone were determined following U.S. Environmental Protection Agency Method TO-11a . Integrated samples were collected by drawing air through silica gel cartridges coated with 2,4-dinitrophenylhydrazine at a flow rate of 1 Lpm. Samples utilized an ozone scrubber cartridge installed upstream from the sample cartridge. Sample cartridges were eluted with 2 mL of high purity acetonitrile and analyzed by high-performance liquid chromatography with UV detection and quantified with a multi-point calibration for each derivitized target aldehyde. Sampling was conducted using Masterflex L/S HV-07553-71 peristaltic pumps assembled with dual Masterflex L/S Standard HV-07016-20 pump heads. Continuous measurements of PM2.5 using TSI Dustrak model 8520 monitors were made in Room 510 and at the rooftop-sampling site from about 13:30 to 16:30 of the sampling day. The indoor particle monitor was located on a desk in room 510 and the outdoor monitor was located on a surface elevated above the roof deck. Carbon dioxide spot measurements of about 10-minute duration were made throughout the building during the afternoon using a portable data logging real-time infrared monitor . Temperature and RH were monitored in the Greenhouse, room 510 and room 311 using Onset model HOBO U12-011 data loggers at one-minute recording rates. Outdoor T and RH were not monitored. The measured VOC concentrations as well as their limits of quantitation by the measurement methods are shown in Table 2. Figures 1-6 show bar graphs of these VOCs. Unless otherwise shown, all measured compounds were above the minimum detection level, but not all measurements were above the LOQ.

Those measurements with concentrations below the LOQ should be considered approximations. These air contaminants are organized by possible source categories including: carbonyl compounds that can be odorous or irritating; compounds that are often emitted by building cleaning products; those associated with bathroom products; those often found emitted from office products, supplies, materials, occupants, and in ambient air; those found from plant and wood materials as well as some cleaning products; and finally plasticizers commonly emitted from vinyl and other flexible or resilient plastic products. The groupings in this table are for convenience; many of the listed compounds have multiple sources so the attribution provided may be erroneous. The carbonyl compounds include formaldehyde that can be emitted from composite wood materials, adhesives, and indoor chemical reactions; acetaldehyde from building materials and indoor chemistry; acetone from cleaners and other solvents. Benzaldehyde sources can include plastics, dyes, fruits, and perfumes. Hexanal, nonanal, and octanal can be emitted from engineered wood products. For many of these compounds, outdoor air can also be a major source. Formaldehyde and acetone were the most abundant carbonyl compounds observed in the PBC. For context, the California 8-h and chronic non-cancer reference exposure level for formaldehyde is 9 µg m-3 and the acute REL is 55 µg m-3 . The 60 minute average formaldehyde concentrations observed in the PBC exceeded the REL by up to a factor of three. Acetone has low toxicity and the observed levels were orders of magnitude lower than concentrations of health concern. Hexanal, nonanal, and octanal are odorous compounds at low concentrations; odor thresholds established for them are 0.33 ppb, 0.53 ppb, and 0.17 ppb, respectively . Average concentrations observed within the PBC building were 3.8±0.8 ppb, 3.5±0.6 ppb, and 1.4±0.2 ppb, for these compounds, respectively, roughly ten times higher than the odor thresholds. Concentrations of these compounds in the supply air from the greenhouse were substantially lower, although stillin excess of the odor thresholds. The concentration of hexanal and nonanal roughly doubled the ambient concentrations as the outside air passed through the greenhouse. Octanal concentrations were roughly similar in the ambient air and in the air exiting the greenhouse. Concentrations of benzene, d-limonene, n-hexane, naphthalene and toluene all increased in the greenhouse air in either the AM or PM measurements. The measured levels of these compounds were far below any health relevant standards, although naphthalene concentrations reached close to 50% of the California REL of 9 µg m-3 . The concentrations of these compounds were generally somewhat higher indoors relative to the greenhouse concentrations.

Maximum concentrations of N2O were observed in the soil at 10–15 cm, shallow depths where lateral diffusion away from the dripper could only account for a small loss of N2O compared with losses to the atmosphere. It was also observed that water tended to flow laterally, past the surface wetting front, through a sandy horizon above a clayey horizon which begins at around 50 cm depth, also diminishing net lateral gas diffusion. The model is highly sensitive to variability in N2O concentration data, and at the plot level the calculations showed fluctuations between production and consumption by depth which were not plausible or consistent.Where possible, curves were fit to the concentration data of the form N2O conc = ae, which provided the dc/dz terms. Furthermore, concentrations of N2O measured at 5 cm were generally much lower than at 10 cm and led to frequent estimates of net consumption near surface. It was deemed likely that these samples had been contaminated by atmospheric air. Production of N2O at 10 cm was therefore calculated with reference to ambient N2O concentrations at 0 cm instead of the 5 cm concentration data. Measurements spaced several hours apart determined that an average 4.4% of total production was accounted for by change in concentration over time during the measured days. Ultimately no treatment statistics could be reported with the profile production data, but the model revealed general patterns and treatment effects on the depths of N2O production. The highest emissions, which were seen in summer and fall,hydroponics growing system were associated with the most consistent patterns of N2O distribution in the soil profile. Results from Days 2, 3 and 4 after fertigation, not shown, had similar distributions to those measured on Day 1, although with a slightly higher fraction of N2O concentrated in the deeper soil, 40–60 cm.

The relative stability in depths of production was seemingly contradictory to the changes in N distribution seen in soil solution ; but it was notable that distribution of extractable N , showed less change at the same points . N2O concentration patterns under most days and treatments were bimodal, with a shallow peak at 10–15 cm and a deeper peak around 45–60 cm, in the zone of higher clay content. The deeper peaks were sometimes strong 3–4 days after fertigation, especially in the Standard UAN treatment, illustrating the deeper distribution of N under higher rates of UAN application. Calculations in UAN treatments after winter typically showed points of highest production at 10–15 cm depth, usually underlain directly by the points of greatest consumption, at 15–20 cm . The calculations for 20 and 30 cm might underestimate production, because of more significant lateral diffusion of N2O around 30 cm, where WFPS generally declined . Production was seen at the lower peaks around 45 cm, but calculations suggested that N2O produced in these lower peaks was generally consumed before reaching surface , consistent with the findings of Neftel et al. . This helps to explain why emitted N2O was less per unit applied in Standard UAN than in HF UAN. Calculations in HF NO3 profiles generally showed much lower net N2O production/consumption than the UAN treatments. This is credited to the more even distribution throughout the soil of applied NO3, vertically and laterally, which led to low concentrations. Production profiles further suggest that a high proportion of the N2O produced in this treatment was consumed before it could be emitted from the surface. Overall, surface emissions of N2O decreased more quickly over the days following fertigations than did soil gas concentrations and calculated in-soil production rates, suggesting greater importance of production near surface during the first and second days. Under the driest conditions, seen on Day 3 after fertigation in late summer, increased N2O concentrations at 60 and 80 cm were concurrent with the lowest post-fertigation surface emissions. Calculations of N2O production for that date showed consumption at 45 cm in both HF treatments , supporting the conclusion that N2O produced deeper was being consumed at points immediately above, as well as possibly diffusing downwards.

Although the averaging of soil gas profiles by treatment limited the options for statistical analysis, the factors driving N2O production in the soil could still be assessed. Multiple linear regressions of surface emissions and of production at 15, 30 and 60 cm were carried out using calculated N2O production per treatment per day at those depths, and the corresponding averaged NH4 + in solution, NH4 + in soil extracts, NO3 in solution, NO3 in soil extracts, WFPS and temperature. Treatments were pooled because the dataset was limited within each treatment and the differences seen when HF NO3 was separated were minor. Regressions had little predictive capability at 30 and 60 cm depth. Nevertheless, it was notable that WFPS had negative coefficients at both depths, indicative of more complete denitrification with greater soil moisture. At 15 cm, the Adjusted R2 was only 0.14 but several alternative analyses gave better predictions. When excluding negative production values, an Adj. R2 of 0.58 was seen, which rose to 0.68 when reduced to extractable NH4 + , WFPS and temperature. If production at 10 cm was averaged with that at 15 cm, most negative values were eliminated, and using all data and variables the Adj. R2 was 0.21, or 0.26 with extractable NH4 + , NO3 in solution, WFPS and temperature. These results caused some questioning of the calculations of N2O production and consumption, which were volatile even in averaged forms, so regressions were carried out with soil gas concentrations as well. At 15 cm, all variables regressed to Adj. R2 of 0.26; reduced to NH4 + , WFPS and Temperature, the Adj. R2 was 0.32. Concentration averaged between 10 and 15 cm had an Adj. R2 of 0.41, while reduced to NH4 + , WFPS and Temperature, the Adj. R2 was 0.49. Regression of surface emissions followed the same pattern, being compared to NH4 + and NO3 in soil extracts at 2.5 cm depth, WFPS and temperature, where NO3 was found insignificant. The adjusted R2 of this regression is not reported because it is less complete than the analyses above. The superior predictive capability of extractable NH4 + at 15 cm and near surface was unexpected, since it is usually assumed that only the NH4 + in solution is available for microbial consumption .

However, little relevant investigation has been done in soils and the question can be raised whether microbial foraging on clays can desorb ammonium .The persistence of input effects on the functioning of the soil microbial community is an important agro-ecological concern. Here several assays of nitrification and denitrification capacity tested for persistent treatment effects which could influence N2O emissions. Soils were collected in late August after a month of irrigations without fertilizer. Treatment differences were of interest, not the comparison of assay results to field rates. The most ready metric of a soil’s denitrification response to NO3 amendments is its denitrification enzyme activity ,hydroponic equipment designed to assess soil process rates before they are affected by the synthesis of additional enzymes. Since fertigation applications make a large amount of NO3 available in a short time, the preevent DEA of a soil may play a significant role in denitrification derived N2O emissions. Results showed very similar N2O production by the two HF treatments in a DEA assay, which were significantly higher than Standard UAN . Over 24 h, characterized as Denitrification Potential , this initial difference was persistent, although it lost statistical significance. Given that drip fertigation saturated zones are not entirely dissimilar from the conditions of these assays, it was expected that DEA and DP modified with acetylene might also suggest differences in the product ratio of denitrification in the field treatments. Results were inconclusive, with widely dispersed values. Rates of ammonium oxidation to nitrite, as an index of nitrification potential, supported the importance of frequency and rate of NH4 + application, HF UAN > Standard UAN > HF NO3, but differences were only significant between the HF UAN and HF NO3 treatments . Strict chemoautotrophs typically dominate nitrification in cropped soils , and their numbers are more likely to be affected by availability of NH4 + than are the heterotrophs largely responsible for N2O emission through denitrification. Higher amounts of available nitrite are known to stimulate nitrifier denitrification and associated N2O losses , so a persistent effect of NH4 + application on ammonium oxidation to nitrite could increase N2O emissions under HF fertigation. Ammonium oxidation and DEA assays are predicated upon standard conditions, the former being oxic, open, shaken slurry, and the latter completely anoxic.

Actual oxygen availability in drip zones may cover a wide range between those points, but is expected to be limited. Little data is available, but Gil et al. found 4.97% O2 in the sampled soil air of a clay loam in an avocado orchard under drip. It can be assumed that many surfaces within larger aggregates would have lower O2 , being well suited for nitrifier denitrification, which takes place at <5% O2, while denitrification requires <0.05% O2 . It was therefore deemed useful to test the persistent effects of HF fertigation on potential soil production of N2O at 3% O2 . The only treatment differences were in microcosms with NO3 amendments , where N2O was presumably derived mainly from denitrification inside aggregates, supporting DEA results . The lack of HF treatment effects with NH4 + may be due to high rates of adsorption on soil surfaces expected with this N source , leading to gradual liberation. Nevertheless, emissions of N2O with NH4 + amendments were higher than those with NO3, confirming the large potential contribution of nitrifier denitrification from drip zones. The alternative explanation, being a general, rapid turnover from nitrifier-produced NO2 and NO3 to denitrifier produced N2O, has not consistently been supported by isotopic studies in laboratory . Assessments of N2O/ product ratio using acetylene in DEA, DP, and 3% O2 incubation assays did not give robust support to the hypothesis that greater microbial capacity for nitrification and/ or denitrification should correlate to a higher portion of complete reduction of N to N2 . It must be noted that N2O is more likely to be reduced to N2 when NO3 is limited , which it was not in the DEA test and DP tests. Further, the reduction of N2O to N2 dominates under anoxic conditions , which were not prevalent in the 3% O2 test. The factors affecting the “completeness” of nitrifier denitrification to N2 have been little studied and may be distinct from those affecting denitrifier denitrification. Lastly, tests of residual NO3
suggested that acetylene may have slightly inhibited NO3
reduction. The comparison of N2O from HF UAN with a HFNO3
-based treatment including Ca2 raises the question of whether differences may be ascribable to the opposite pH effects of the fertilizers. HF Ca2 + KNO3 did produce a significantly higher pH than HF UAN within 6 months of the treatment’s inception . This could partly explain lower N2O emissions from the HF NO3 treatment , but the effectis likely not a strong one because all were in neutral range . Our observation of 2.0 greater N2O emissions from HF UAN than from HF NO3 agrees well with Abalos et al. , who saw 2.4 greater N2O emissions from urea than from calcium nitrate in a drip-fertigated melon field in Spain. The greater predictive capacity of extractable NH4 + over NO3
provided evidence of a high contribution of nitrifier denitrification to N2O emitted in the field. This was supported by laboratory tests of our field soils at 3% O2, and concurred with findings by Vallejo et al. , as well as by Sanchez-Martin et al. , who calculated that with dripfertigated ammonium sulfate, 45% of N2O came from nitrification. Considering both field and laboratory data, frequency effects in the application of UAN were only seen in nitrate denitrification rates and in N present at 60 cm depth. Nitrifier capacities do not seem to have been affected, due perhaps to the adsorption of fertilizer NH4 + and its gradual release over time. Still, rates of nitrifier denitrification in the field may have seen concentration effects, as a corollary of frequency differences.

Based on the results, we propose the presence of quantitative trait locus or loci for root traits in the distal 15% of the physical length of 1RS arm. In cereals, most of the gene rich regions for agronomic traits are concentrated in the distal ends of the chromosomes . Kim et al. conducted Weld studies for the agronomic performance of 1R from different sources of origin. They found 1RS increases the grain yield significantly, and interestingly, all the lines with 1RS did not show significant differences for shoot biomass. They did not look at the root traits, which could have also been useful. In a similar study, Waines et al. compared 1RS from different sources to study root biomass in hexaploid as well as tetraploid wheats. The translocated hexaploid wheats with 1RSAmigo and 1RSKavkaz showed 9 and 31% increase in root biomass than Pavon 76, respectively. Similar results were reported for the durum wheat ‘Aconchi’ versus Aconchi with the 1RS arm. These studies point toward the definite presence of gene for greater rooting ability on 1RS, and also the differential expression of alleles from different sources of 1RS in root traits. In a recent study on rice root anatomy, Uga et al. identified a QTL for metaxylem anatomy on the distal end of the long arm of chromosome 10. In another comparative study of rye DNA sequences with rice genome, the distal end of the long arm of chromosome 10 of rice was syntenic to 1RS . Both these studies provide evidence to support the general applicability of our mapping method to locate the probable region on 1RS,hydroponic grow system carrying gene/QTL for root traits. Our present finding on root studies prepares a platform to find gene/QTL for root traits on 1RS.

Future work will focus on use of a larger number of recombinant lines to narrow down the QTL region of 1RS responsible for increased root traits and find the molecular markers linked to these QTL. Ultimately, this would lead to our goal of physical mapping and then positional cloning of the root QTL.The increasing use of NPs in commercial products has led to NP-accumulation in the environment and within the food chain.Chronic exposure to NPs can lead to health issues as some inorganic NPs have biological activity at the cellular and sub-cellular level with an unknown cytotoxicity and genotoxicity.In particular, metal oxide NPs are the most abundant form of NP in the environment with the most potential toxic risks.Locating, quantifying, and imaging NPs in vivo can provide information on bio-distribution and fate of NPs in living systems.However, many challenges to quantitatively assess their bio-distributions under realistic environmental exposure concentrations remain.To date, the visualization of most NPs within plants has relied on the use of micro-X-ray fluorescence spectrometry , confocal microscopy, TEM, SEM, scanning transmission electron microscopy , or scanning transmission ion microscopy .The most prominent quantification techniques have been inductively coupled plasma spectroscopy utilizing either optically emission spectroscopy or mass spectrometry .These techniques required mineralization of the plant material generally with hydrogen peroxide and nitric acid, and may not be sufficiently sensitive enough to quantify small changes in the amount of a metal ion.Notably, high background concentrations of essential nutrients make detecting and quantifying the small variation in NP-related metal ion content an analytical challenge as the measurement is often of the same magnitude as noise or at the detection limit.

Optically tagged NPs have also been investigated, but the challenge of overcoming the plants’ own bioluminescence can make quantification difficult.Although used in medical imaging, radio labeled NPs for noninvasive tracking and quantification in plants have not been significantly explored.Prior radio labeling of NPs for medical imaging has utilized three main approaches: post radio labeling via attachment of chelator to NPs first, then reaction with the a metal radio nuclide; preradiolabeling, where a radioactive prosthetic group, a small molecule that the radioisotope is attached, followed by attachment to the NP; and direct radio labeling.Of these three main radio labeling approaches, the third approach has been the only method used to study NP distribution in plants where Zhang et al.reported the use of [141Ce]CeO2-NPs produced via neutron bombardment of CeO2-NPs to study distribution in cucumbers. The produced radionuclide 141Ce has a 32.51 day half-life, and the specific activity of the synthesized [141Ce]CeO2-NPs was 2.7 μCi/mg of NP.Despite numerous approaches to analyze NPs, a combination of technologies is required due to the low detection limits and high resolution needed to address the intact nature of NP-transport into plants. Thus, multiple tools must be utilized to quantify and determine the intact nature of NPs at a given location within biological environments .Previous studies have provided conflicting evidence about the intact nature of NP uptake and transport into plants: some studies indicated intact NP uptake and vascular transport,some observed NPs in plants due to dissolution events and reformation within the plant tissue,and still others have indicated that NPs cannot be transported into plants.These varied observations could be linked to inadequate techniques available to track NP movement in vivo. The main challenge in determining intact NP transport into plants is ruling out NP dissolution, as reductive precipitation and formation of NPs within plant tissue has been documented.Even natural formation of NPs within plants and fungi is known.

Further complicating the picture is the fact that many studies assessing NP uptake exposed plants over long periods of time from 2 to 130 days, with very large amounts of NPs per plant, which could make dissolution events more prevalent.Avoiding excessive exposure to the NPs and carefully analyzing the stability of the administered NPs are key to avoid erroneous conclusions due to NP-dissolution and subsequent reformation. In this study we evaluated an analytical method using a radioactive label to non-invasively track and quantify transport and accumulation of NPs in lettuce seedlings in vivo. This method studied NP-size dependent transport immediately upon exposure , an early time frame that has rarely been explored in plants.The visualization of NP transport and accumulation in lettuce seedlings was done by autoradiography and PET/CT imaging and further confirmed by gamma-counting, SEM, and TEM. Our study was designed to use highly uniform NPs of two size sets , which were theoretically too large for passive transport across plant tissues.To ensure a narrow size distribution with a uniform geometric shape and the ability to thoroughly investigate stability , a preradiolabeling method with the PET-radioisotope copper-64, the “clickable” chelator ADIBO-NOTA, and commercially available spherical Fe3O4- NPs containing azides was explored. This radio labeling approach yielded a high specific activity and allowed for size characterization of the NPs after the plant accumulation and imaging period, and avoided complication from fabricating radioactive NPs and production of less stable NP material with a larger size distribution. Rigorous stability studies were carried out at a variety of pHs to investigate possible dissolution of the 64Cu-radiolabeled NPs and substantiate the intact nature of NP-transport into lettuce seedlings.To date the uptake, bio-accumulation, bio-transformation, and risks of NPs in food crops is not well understood.Most studies on NP uptake in plants have focused on the effects of NPs on plants, and have not focused on the transport or entry of intact NPs. Several studies concluded that NPs do not gain entry into plants,vertical grow rack while those that do show NP uptake in plants have found the NP amounts to vary widely between 0.05 μg/g and 38983 μg/g of plant.In vivo tracking of NP transport in plants has traditionally relied on destructive analytical techniques to quantify NP-uptake and accumulation, requiring mineralization for metal quantification mostly by ICP.These analytical techniques face the challenge of being sensitive enough to reliably measure the small changes in metal concentrations caused by NP-uptake and accumulation within the plant. The extensive range of NP accumulation reported in plants suggests that NP-uptake is dependent on several parameters: quantity of NP administered per plant, plant species, NP-size, NP-composition, and duration of exposure.Further complicating the understanding of NP transport and accumulation in plants are the studies that have observed NP-uptake due to dissolution.Collectively the variations of parameters in every study on NP transport in plants have made direct comparison and accurate conclusions challenging. Thus, it was our goal to develop a noninvasive visualization approach to track and quantify the distribution of intact radiolabeled [ 64Cu]-NPs in lettuce seedlings. Using 64Curadioactively tagged NPs, we employed a range of complementary noninvasive analytical tools including autoradiography and PET/CT imaging to spatially and temporally visualize and quantify intact-NP uptake and accumulation in plants. The stability study described demonstrates dissolution of the [ 64Cu]-NPs did not occur. Ligand effects on NP mobility within the lettuce was minimized by modification of ≤5% of the NP surface with [64Cu]-ADIBO-NOTA as to negligibly change the NP surface properties.

No ligand detachment or leaching of 64Cu-ion from the NPs within the imaging time frame and at various pHs was observed, by both HPLC and gamma counting analysis, indicating that the radioactive signal in the lettuce seedlings was due to intact [64Cu]-NPs. Additionally, indirect evidence further supported that the observed uptake was from intact [64Cu]-NPs as control lettuce seedlings given only [64Cu]CuCl2 had much higher radioactivity with 4-fold higher concentrations in the cotyledons and 10-fold higher concentrations in the root . These control plants also had visibly higher amounts of radioactivity in each part of the plant by autoradiography images suggesting that if the observed radioactivity was due to [64Cu]CuCl2, then the uptake should be much higher. Furthermore, the use of covalently bound optical-tagged NPs also exhibiting high stability and illustrated the same NP movement from the root to the cotyledon . Thus, helping to substantiate that NP-uptake and transport to the cotyledon was from intact NPs. TEM-sectioning of the plant tissue also helped to corroborate the presence of NPs within the plant tissue . It should be noted that detection of NPs within plant tissue via TEM is challenging,but based on our stability studies of the 64Cu-radiolabeled NPs along with the short exposure time that the observed uptake was attributed to intact [64Cu]-NP transport through the roots and into the cotyledons. This study has shown that NPs were transported intact into plants and can be tracked non-invasively using a radioactive tag for in vivo imaging by autoradiography and PET/CT and quantification using a gamma counter. This method allows for a highly sensitive method capable of quantifying NP amounts in an individual seedling, a level that would be challenging by the traditional ICP quantification.However, different accumulation patterns for the cotyledons were observed for the two different sized [64Cu]- NPs , while the root and whole plant were similar . Most of the accumulation for the larger [ 64Cu]-NPs was within the first hour, where cotyledon NP amounts were ∼0.35 ± 0.15 μg/g with the only significant increase after 1 h between the 12 and 24 h time point in which accumulation plateaued at around ∼0.7 μg/g . The larger [64Cu]-NPs also had higher accumulation than the [64Cu]-NPs at the early time points up to the 4 h time period. The smaller [64Cu]-NPs had ∼8.8 fold increase in cotyledon accumulation from the 4 h time point to the 24 h time point with an increase of ∼1.6 fold between 12 and 24 h time period appearing to have a linear increase in absorption over time. The differences in cotyledon accumulation between the two sized [ 64Cu]-NPs maybe linked to NP size effects on the lettuce hydraulic conductivity. Our work suggests that [64Cu]-NPs around 20 nm in size appear to clog root cortical cell walls, or pit membrane preventing further uptake, explaining why 11 reaches a plateau, while the smaller [64Cu]- NPs continued to increase in amount over time. Initial studies with duckweed also illustrated [64Cu]-NP accumulation in regions of growth and at the node and apex of the cotyledons , suggesting that [64Cu]-NP transport to the cotyledons could occur via the phloem. The TEM images further shows the appearance of intact NPs in the lettuce tissue within the expected size range for the [64Cu]-NPs , but [64Cu]-NPs had a size that appeared smaller than those administered; suggesting that the plant may filter larger NPs and has a size-threshold for uptake , which may also explain the clogging phenomenon.In summary, the combined analysis of the imaging by autoradiography and PET/CT and TEM suggested that both sized [64Cu]-NPs are transported intact from the root to the cotyledons.

Antisense suppression of NtAQP1 in tobacco lowered the level of expression of several PIP1 homologues and resulted in a significant decrease in protoplast membrane water permeability, reduced root hydraulic conductivity and decreased transpiration. The results of heterologous expression in Xenopus oocytes suggest that, in addition to functioning as a water channel, NtAQP1 is also a membrane CO2 pore that facilitates the transport of CO2 across membranes. The movement of CO2 between the substomatal cavities and the sites of carboxylation within chloroplasts, through plasma and chloroplast membranes, is generally termed leaf mesophyll conductance. The ability of NtAQP1 and its Arabidopsis homolog AtPIP1,2 to function as CO2 membrane transport facilitators has been demonstrated in in vivo experiments. Increased expression of NtAQP1 in tobacco plants enhanced CO2 incorporation and stomatal conductance; whereas antisense suppression of NtAQP1 had the opposite effect. In other studies, over expression of AtPIP1,2 or NtAQP1 in tobacco plants significantly enhanced the rates of growth, transpiration and photosynthesis; whereas antisense suppression of NtAQP1 in tobacco plants and T-DNA insertion Arabidopsis mutants in AtPIP1,2 reduced gm and led to lower rates of photosynthesis. Unlike NtAQP1, over expression of Arabidopsis hexokinase in Arabidopsis and tomato plants decreased photosynthesis, transpiration and growth. AtHXK1 is a sugar sensing enzyme that monitors glucose levels, hydroponics growing system most likely in mesophyll cells of photosynthetic tissues.

When glucose levels are sufficiently high, this enzyme inhibits the expression of photosynthetic genes, decreases chlorophyll levels and reduces the rate of photosynthesis. In addition, AtHXK1 also stimulates stomatal closure and decreases transpiration in response to increasing sugar levels. In light of the opposite effects of AtHXK1 and NtAQP1 on photosynthesis and growth, we examined the relationship between AtHXK1 and NtAQP1 using double transgenic plants that express AtHXK1 and NtAQP1 simultaneously. We found that NtAQP1 significantly compensated for the growth inhibition imposed by AtHXK1, primarily by enhancing mesophyll CO2 conductance and the rate of photosynthesis, while the hydraulic conductivity in those plants remained unchanged.The hydraulic conductance of the tomato root system was assessed using plants grown hydroponically and was determined by measuring the flow induced in response to 1 bar of applied pressure. De-topped root systems were fitted with a plastic tube filled with deionized water and connected to a beaker located on a balance . The root system was sealed in a chamber containing the hydroponic solution in which the plants had been grown. The pressure in the chamber was regulated using a needle valve, which was adjusted to allow a small leak into the chamber, so that the air used to pressurize the chamber also served to aerate the medium. Water flow through the root system was automatically recorded by a computer at 30 s intervals. At the end of each experiment, the roots were dried in an oven for 72 h at 90uC and the dry weight of the root system was then measured.Whole-plant transpiration rates and relative daily transpiration were determined using lysimeters, as described in detail by Sade et al.. WT, AQP1, HK4, AQP1xHK4 and grafted plants were planted in 3.9-L pots and grown under controlled conditions. Each pot was placed on a temperature-compensated load cell with digital output and was sealed to prevent evaporation from the surface of the growth medium. A wet, vertical wick made of 0.14 m2 cotton fibers partially submerged in a 1-L water tank was placed on a similar load cell and used as a reference for the temporal variations in the potential transpiration rate. The output of the load cells was monitored every 10 s and the average readings over 3-min intervals were logged in a data logger for further analysis.

The whole-plant transpiration rate was calculated as a numerical derivative of the load cell output following a data smoothing process.Over expression of NtAQP1 in tobacco plants enhanced leaf mesophyll CO2 conductance , hydraulic conductivity, stomatal conductance , transpiration and photosynthesis. Expression of NtAQP1 in tomato plants also enhanced photosynthesis, stomatal conductance and transpiration. However, in our study, NtAQP1 did not enhance photosynthesis, stomatal conductance or hydraulic conductivity relative to WT plants and enhanced transpiration only slightly . These differences may be due to the different tomato genotype used in our study or to different expression levels of NtAQP1. Nevertheless, photosynthesis, stomatal conductance and transpiration were elevated by NtAQP1 in the double-transgenic plants , as compared to the HK4 parental line. Yet, the hydraulic conductivity of AQP1xHK4 remained low as in the HK4 plants, implying that the increased transpiration that was observed is not directly related to hydraulic characteristics. Rather, the increased transpiration is most likely due to high gm values in the mesophyll, which opens stomata and increases the influx of CO2 to help maintain constant levels of Ci in the sub-stomatal cavity. High levels of AN, gs and gm, accompanied by constant Ci , were also reported in previous studies of tobacco plants over expressing NtAQP1. AtHXK1 is a sugar-sensing enzyme that inhibits the expression of photosynthetic genes, decreases chlorophyll levels and reduces the rate of photosynthesis in response to increasing sugar levels. As a result, tomato and Arabidopsis plants with high levels of AtHXK1 expression display severe growth inhibition directly correlated to AtHXK1 expression and activity levels. It is likely that part of the growth inhibition imposed by AtHXK1 is the result of insufficient photosynthesis, since the increased photosynthesis rate observed in AQP1xHK4 plants partially eliminated this growth inhibition. The increased rate of photosynthesis observed in AQP1xHK4 plants, despite the low level of expression of the photosynthetic gene CAB1 in those plants, can probably be attributed to NtAQP1, which accelerates CO2 mesophyll conductance.

The CO2 mesophyll conductance of HK4 plants is significantly lower than that of WT plants and is enhanced by simultaneous expression of NtAQP1, indicating that CO2 mesophyll conductance significantly affects growth. It appears that, in addition to its known sugar-sensing effect , AtHXK1 also reduces gm, perhaps by reducing the expression of TRAMP , the tomato homolog of NtAQP1. Indeed, lower gm levels have been observed in tobacco NtAQP1 antisense lines and Arabidopsis pip1;2 mutants . In those studies, the decrease in gm was accompanied by lower Cc. In agreement with the findings of those studies, the HK4 plants in our study exhibited lower Cc than the WT plants and the expression of NtAQP1in the double-transgenic plants led to full complementation of Cc . Interestingly, the HK4 plants had lower electron transport rates than the WT plants, while a clear recovery was observed in the AQP1xHK4 plants despite the low level of expression of the photosynthetic gene CAB1 in the AQP1xHK4 plants . It has previously been shown that expression level of NtAQP1 which affects gm levels also affects electron transport rates. Flexas et al. hypothesized that modified intercellular CO2 concentrations may trigger differences in the leaf photosynthetic capacity, so that the photosynthetic machinery can adjust to the change in mesophyll conductance. This would also explain why gm usually scales with photosynthetic capacity, as has been observed in broad comparisons of different species. The effect of AtHXK1 on gm suggests that HXK might coordinate photosynthesis with sugar levels by several mechanisms in different cell types. It inhibits expression of photosynthetic genes and reduces gm most likely in mesophyll photosynthetic cells. In guard cells HXK mediates stomatal closure in response to sugars and reduces stomatal conductance. These findings support the existence of a multilevel feedback-inhibition mechanism that is mediated by HXK in response to sugars. When sugar levels are high,hydroponic grow systems likely when the rate of photosynthesis exceeds the rate at which the sugar is loaded and carried by the phloem, the surplus of sugar is sensed by HXK in mesophyll and guard cells, which respond in concert to reduce both unnecessary investments in photosynthetic capacity and water loss. This response includes reducing the expression of photosynthetic genes, slowing chlorophyll production, diminishing mesophyll CO2 conductance and closing the stomata. In addition to these effects in shoots, HXK reduces the hydraulic conductivity of stem and roots via an as yet unknown mechanism. This reduction in hydraulic conductivity occurs independently of stomatal conductance, as it also happens in the double-transgenic plants that have WT levels of stomatal conductance . Nevertheless, grafting experiments indicate that neither over expression of AtHXK1 in roots nor expression of AtHXK1 in the stem has any visible physiological effects. Rather, over expression of AtHXK1 in shoots is necessary and sufficient to obtain a photosynthesis effect and growth inhibition. The dominant effect of AtHXK1, lowering hydraulic conductance in AQP1xHK4, might be the reason for the intermediate transpiration rate of AQP1xHK4 plants, which is lower than that of WT plants , despite the increase in stomatal conductance to levels similar to that of WT plants . It has been suggested that NtAQP1 might play independent roles in leaves and roots, a hydraulic role in roots and a membrane CO2 permeability role in shoots. The improved gm observed in the double-transgenic plants supports the notion that, in leaves, NtAQP1 functions as a CO2 transmembrane facilitator and that the complementation effect of NtAQP1 may be primarily attributed to its affect on CO2 conductance in leaf mesophyll. The roles of HXK and PIP1 in the regulation of photosynthesis, stomatal conductance and transpiration are well established. This study suggests that HXK and PIP1 together may influence these central properties of plant physiology and, eventually, plant growth.

Monoclonal antibodies represent the fastest growing class of therapeutics and have been especially beneficial in the treatment of cancer. Since the approval of the first anti-cancer monoclonal antibody in 1986, several innovations have improved the potency of monoclonal antibodies used in immunotherapies that offer increased drug efficiency and/or lower drug dosage for a specific treatment. Among them, glycan engineering of the oligosaccharides attached to Asn297 of the Fc region of the heavy chain has been shown to affect antibody-dependent cell-mediated cytotoxicity , complement dependent cytotoxicity , and binding to the neonatal Fc receptor, FcRn. specific oligosaccharides influence the affinity of the antibody Fc domain to Fc receptor present on effector cells resulting in altered biological functions. For example, the removal of terminal galactose residues on mammalian cell-derived antibodies lowered C1q binding, while ADCC activity is almost completely dependent on the presence or absence of fucose residues bound to the glycosylation core. Several approaches have been employed to manufacture a monoclonal antibody with a decreased or absent core fucosylation. One strategy is to use cell lines or organisms with modified glycosylation pathways. The alteration of the expression of key enzymes in the host glycosylation pathway such as the mammalian α1,6-fucosyltransferase, the plant α1,3-fucosyltransferase, the GDP -mannose 4,6-dehydratase, or the β1,4-N-Acetylglucosaminyltransferase III led to afucosylated antibodies with improved anti-tumor activity. This led to the approval of mogamulizumab and obinutuzumab in 2012 and 2013, respectively, both produced in glycoengineered mammalian cell lines. Another approach to alter the antibody glycosylation profile is to modify the culture conditions of the host cells by adjusting the growth environment or supplementing the media with inhibitors of enzymes in the glycosylation pathway such as N-butyldeoxynojirimycin , mannostatin A, swainsonine, or kifunensine. Kifunensine from the actinomycete Kitasatosporia kifunense 9482 inhibits class I α-mannosidases and blocks N-glycan synthesis at the Man8GlcNAc2 or Man9GlcNAc2 stage before the core fucose is added. In mammalian cell culture, kifunensine was successfully employed to produce protein with >90% high-mannose content. This effect was similar across many different proteins including antibodies, suggesting that this simple treatment could be applied broadly. Compared to other α-mannosidase I inhibitors, kifunensine is highly effective on mammalian cell culture without significantly affecting cell growth or protein yield, even at concentrations as low as 100 ng/mL culture. Similar to mammalian cell studies, kifunensine was used in conjunction with the Nicotiana benthamiana transient protein expression systems to produce proteins with >98% afucosylated high-mannose glycans. In plants, the non-human α1,3-fucose and β1,2-xylose residues are commonly added in the Golgi apparatus after mannose trimming by mannosidases in the endoplasmic reticulum. Upon kifunensine treatment, addition of α1,3-fucose and β1,2-xylose residues were not observed on the Man3 to Man9 structures. However, the amount of kifunensine used in these studies was at or above 1.16 µg/mL, which significantly increases production costs at the manufacturing scale. Kifunensine is currently being used to manufacture a recombinant glucocerebrosidase in HT1080 fibrosarcoma cells to treat type 1 Gaucher disease.

The productivity of rainfed farming is also affected by the selection of planting date, which often depends on the timing of the first effective rainfall events. For this joint soil-terrain-climate analysis, all regions with a growing season of two months or shorter were assigned a suitability value of zero and thus classified as unsuitable for agriculture. We then evaluated the capacity of land for rainfed farming by using a precipitation cut-of of 250 mm year−1 , which is often regarded as the minimum threshold for the rainfed farming . As shown in Table 1, the inclusion of the length of growing period and precipitation threshold into the analysis only slightly reduced the total area of high-quality lands from 5.8 to 5.4 million ha. This implies that most lands with suitable soil and terrain conditions also receive sufficient amount of moisture to sustain rainfed agriculture. On the contrary, the area of unsuitable lands increased from 39.7 to 112.9 million ha when precipitation and duration of growing season thresholds were superimposed on the soil and topographic constraints. This increase in unsuitable acreage was mainly driven by the demotion of lands from the very poor class to the unsuitable class . The addition of moisture constraints also reduced the area of medium suitability lands by 4.8 million ha. In summary, for the rainfed farming suitability analysis, 125 million ha of Iran’s land might be classified as poor or lower ranks whilst only 18 million ha meet the required conditions for the medium or higher suitability classes . The geographical distribution of these land classes is mapped in Fig. 4. Almost the entire central Iran ,growing strawberries vertical system and the vast majority of land area in the eastern , southeastern and southern provinces were found to be unsuitable for rainfed farming. Almost half the area of Khuzestan and three-quarters of Fars provinces were also characterized unsuitable. Over the entire east, only in the northern part of Khorasan Razavi province, is there a belt of marginally suitable lands satisfying the requirements of a potentially prosperous rainfed agriculture .

In the next step of the analysis, the suitability of land was scaled with the annual precipitation over the range of 100 to 500 mm year−1 . The lower limit is deemed to exclude the desert areas for agricultural use whilst the upper limit represents a benign moisture environment for the growth of many crops. This last analysis, here after referred to as precipitation scaling method, makes no assumption as to whether the cropping practices rely on rainfall or irrigation to satisfy crop water requirement and may thus represent a more comprehensive approach for agricultural suitability assessment. The same minimum length of growing period and soil/topographic constraints as with the two previous methods were used in this analysis. Compared to the rainfed agriculture analysis, the precipitation scaling method mainly changed the distribution of lands within the lower suitability classes . For example, a great proportion of lands within the unsuitable class was shifted up to the very poor and poor classes. This implies that, to a limited extent, irrigation can compensate for the below threshold precipitation . Nevertheless, water availability cannot necessarily justify agriculture in areas with low soil and topographic suitability. This has an important implication for water management in Iran that has a proven record of strong desire for making water available to drier areas through groundwater pumping, water transfer, and dam construction. The majority of high-quality lands , which also retains sufficient levels of moisture are located in the western and northern provinces of Iran . Kermanshah province accommodates the largest area of such lands followed by Kurdistan . High-quality lands were estimated to cover 33% and 21% of these two provinces, respectively. Other provinces with high percentages of high quality lands were Gilan , Mazandaran , West Azerbaijan , and Lorestan . For 17 provinces, however, high-quality lands covered less than 1% of their total area .To estimate the total area of croplands within each suitability class, we visually inspected 1.2 million ha of Iran’s land by randomly sampling images from Google Earth . The proportion of land used for cropping increased almost linearly with the suitability values obtained from the precipitation scaling method . Total cropping area in Iran was estimated to be about 24.6 million ha, which is greater than the reported value by the Iran’s Ministry of Agriculture. This authority reports the harvested area; hence, the fallow or abandoned lands are not included in their calculation of active agricultural area.

Our visual method, however, captures all lands that are currently under cultivation or had been used for cropping in the near past that are now in fallow or set-aside . The relative distribution of croplands amongst the suitability classes shows that about 52% of the croplands in Iran are located in areas with poor suitability or lower ranks as identified by the precipitation scaling method. Particularly concerning are the 4.2 million ha of lands that fall within the unsuitable class. Approximately 3.4 million ha of cropping areas occur in good and very good lands . However, no agricultural expansion can be practiced in these areas as all available lands in these suitability classes have already been fully exploited. Medium quality lands comprise 12.8 million ha of Iran’s land surface area , of which about 8.6 million ha have been already allocated to agriculture . Nevertheless, due to their sparse spatial distribution and lack of proper access, only a small portion of the unused lands with medium suitability can be practically deployed for agriculture. Using FAO’s spatial data on rainfed wheat yield in Iran, we estimated the mean yield for wheat cropping areas located within each of the six suitability classes. As shown in Fig. 7, the yield of the rainfed wheat increased proportionally with improving suitability index, showing that our suitability index adequately translates to crop yield. Using the observed yield-suitability relationship , we estimated that 0.8 million ton of wheat grain might be produced per year by allocating 1 million ha of the unused lands from the medium suitability class to rainfed wheat cropping.Whilst the insufciency of water resources has long been realized as a major impediment to developing a productive agriculture in Iran, our study highlights the additional limitations caused by the paucity of suitable land resources.Tat is, Iran as a member of Convention on Biological Diversity is obliged to fulfil Aichi Biodiversity Targets whose Target 11 requires Iran to expand its protected area to 17% by 2020, which is almost double the size of the current protected areas in Iran .

Agriculture also needs to compete with other types of land uses with urbanization being an important driver of agricultural land loss. By converting arable lands to a barren desert, desertification is a growing global concern, particularly in the MENA region and Iran. The redistribution of croplands from the low-quality lands to more suitable ones has potentials to improve crop yields and the sustainability of agriculture in Iran. A recent global-scale study concluded that by reallocating croplands to suitable environmental conditions, the global biomass production could increase by 30% even without any land expansion. However, reallocation planning requires accurate mapping of croplands, which is not currently available for Iran. Inefficient agricultural practices in unsuitable lands need to be avoided as they produce little yields at the cost of exacerbating land degradation and water scarcity problem. Our estimations shows that rainfed wheat production from a small acreage of 1.0 million ha in the medium suitability class can equal that from 5.5 million ha of lands in unsuitable or very poor areas . Although this conclusion may not hold for other crops grown in Iran,growing vegetables in vertical pvc pipe the wheat crop could be a good candidate to make such a generalization as wheat is the most widely cultivated crop in the country and is considered as a very low demanding plant, which has adapted to a broad range of contrasting environments. Redistribution of croplands, however, will not be a trivial task for both the Iranian decision makers and stakeholders due to various sociol-economic and logistic barriers. Lands found suitable for agriculture may not be easily accessible if scattered sparsely or occur in remote areas. Given the land and water limitations, increasing the crop production in Iran needs to be achieved through sustainable intensification, which has been found a promising approach for ensuring food security in several global-scale studies. As such, it is of vital importance for Iran to properly use its limited agricultural lands, improve water use efficiency, optimize crop pattern distribution, and adopt modern cultivation techniques. Practicing certain industrial agriculture methods in the unsuitable lands might be a viable strategy to sustainably maintain these lands in the agricultural sector while avoiding the potential socio-economic and political costs associated with redistribution of agricultural lands and farming populations. For example, protected agriculture can be established at some of these locations to cope with both land suitability and water availability constraints. While water insufficiency is a major limiting factor for both field and protected farming, the latter will be affected to a lesser extent. Our suitability assessment is based on a general set of requirements known to affect the productivity of a large number of crops, but there would exist crops with exceptional adaptive traits that can grow under less favourable conditions.

Although we used the most updated geospatial data at the finest available resolution, the result of our suitability analysis should be interpreted in commensuration with the reliability and quality of the original data. For example, whereas the GlobCover database reliably maps the distribution of forests and rangelands in Iran, our visual inspection of satellite images showed that sometimes their utilized method lacks the required precision to distinguish cultivated from uncultivated croplands. Although soil erosion was not directly incorporated into our analysis, the use of slope at the very high resolution implicitly accounts for this effect. The interaction between variables and the quality of subsoil are among other factors that can be considered in the future studies. This study used precipitation as the only water availability factor. Including surface water and groundwater availability can further improve the adequacy of the land evaluation analysis. Given the good correlation between water availability and land suitability for agriculture, the general findings of this study are not expected to change significantly by the inclusion of water availability conditions. Nevertheless, due to the current water shortage constraints across the country, the potential agricultural capacity of the country is likely to decrease when water availability is added to the analysis. Although global projections suggest that the suitable lands may expand with climate changes, how these changes, particularly in precipitation pattern, would affect the suitability of Iran’s land for crop production in the future is subject to high degree of uncertainty and needs further work.Atmospheric carbon dioxide has increased about 35% since 1800 , and computer models predict that it will reach between 530 and 970 ppm by the end of the century . This rise in carbon dioxide could potentially be mitigated by crop plants, in which photosynthesis converts atmospheric carbon dioxide into carbohydrates and other organic compounds. The extent of this mitigation remains uncertain, however, due to the complex relationship between carbon and nitrogen metabolism in plants . Carbon metabolism provides the energy and carbon molecules to synthesize organic nitrogen compounds in plants, whereas nitrogen metabolism provides the amino groups for all proteins . Proteins include all enzymes that catalyze biochemical reactions in plants, including carbon metabolism. Any environmental perturbation that interferes with nitrogen metabolism sooner or later inhibits carbon metabolism.The focal point of crop responses to rising carbon dioxide levels is the enzyme rubisco . Rubisco is the most prevalent protein on Earth and contains as much as half of the nitrogen in plant leaves. It catalyzes two different chemical reactions: one reaction combines a 5-carbon sugar RuBP with carbon dioxide, and the other reaction combines this same sugar with oxygen. The reaction of RuBP with carbon dioxide produces a 6-carbon compound that immediately divides into two molecules of a 3-carbon compound , hence the name C3 carbon fi xation .

During the cultivation, four independent samples were taken at a given time point for fresh and dry weight analysis, sugar analysis, and quantification of rrBChE and total soluble protein . The bioreactor run was terminated at day 5 following induction and the rice cell aggregates were allowed time to sediment . The culture medium was withdrawn from the bioreactor through a sampling port using a peristaltic pump and stored at 4 ◦C The bioreactor head plate was then opened. Rice cell biomass was collected and vacuum-filtered on Whatman Grade 1 . Fresh biomass was weighed and stored at −20 ◦C. Performing a media exchange using 1.25-times-concentrated sugar-free medium together with 1.25-times-reduced culture volume and addition of kifunensine prior to and after the media exchange resulted in increased total production levels of active rrBChE, volumetric productivity, and specific productivity by 1.5 times, 3.4 times, and 1.5 times, respectively, compared with a bioreactor run with same operating conditions but no kifunensine treatment. Moreover, kifunensine enhanced the excretion of recombinant rrBChE glycoprotein through the secretory pathway, leading to 44% of total rrBChE in the culture medium at day 5 following induction and increasing extracellular rrBChE purity to 1.6% rrBChE/TSP compared with 0.8% cell-associated rrBChE/TSP. Coomassie-stained SDS-PAGE and Western blot analyses showed different migration bands of rrBChE with and without kifunensine treatment due to different N-glycan structures. N-Glycosylation site-specific analysis revealed increased oligomannose glycans at site N57, N246, N341, and N455 in both purified cell-associated and culture medium-derived rrBChE in the presence of kifunensine, while the mass transfer limitation of kifunensine was thought to be the main reason for the weak inhibition of α-mannosidase I in this bioreactor study.

At the laboratory scale, we produced ~16 mg of rrBChE in a 2 L working volume during a 12-day batch run,vertical farming supplies corresponding to a volumetric productivity of 0.680 mg L−1 day−1 . A technoeconomic model developed for semicontinuous large-scale production of rrBChE at a higher volumetric productivity showed that the process could be cost-effective with a cost-of-goods sold of ~$660/gram, less than 3% of the estimated cost of plasma-derived hBChE at ~$25,000/gram. The addition of compounds to the culture medium to alter the function of glycan-modifying enzymes is the simplest method to modify N-glycan structure of a target glycoprotein compared to other methods. As a bio-processing approach, it does not require alteration of the primary amino acid sequence of the target protein , or time-consuming glycoengineering of the host that could impact cell growth or viability, yet still allows secretion of the product into the culture medium. For example, adding kifunensine for N-glycan modification is a simple and effective way of obtaining oligomannose glycoproteins with reduced plant-specific xylose and fucose moieties. However, this method may not be cost-effective in large-scale production depending on the production level and market price of the glycoprotein product, the amount and frequency of kifunensine addition , and the price of kifunensine in bulk quantities. Currently, at our laboratory-scale pricing for kifunensine , the addition of 5 µM kifunensine in NB-S increases induction medium costs by ~14-fold and contributes ~$225 in reagent costs for the 5 L bioreactor run. Although the cost of the growth and induction medium is still significantly lower than mammalian cell culture medium, and the price of kifunensine is likely to decrease more than 10-fold with larger demand and bulk pricing, it may be advantageous to reduce bioreactor working volume during the induction phase to minimize kifunensine cost, enhance mass transfer, and concentrate extracellular rrBChE. Employing current genomic editing techniques such as CRISPR/Cas9 to knock out XylT and FucT genes to remove plant-specific α-1,3 fucose and β-1,2 xylose in host rice lines, similar to what was done in N. tabacum BY-2 cell lines without negative impacts in terms of cell growth rate, would be worth investigating as an alternative to modify N-glycans of secreted glycoproteins, such as rrBChE, for large-scale operations.

Graminaceous plants, like other so-called metal-tolerant plants, mostly sequester metals in roots to protect reproductive and photosynthetic tissues . The ability to store metals in underground tissues is used in phytoremediation to reinstall a vegetation cover on heavily contaminated areas and limit the propagation of metals into the food chain . Panfili et al. showed that the grass species Festuca rubra and Agrostis tenuis accelerate the weathering of zinc sulfide when grown on contaminated dredged sediment, thus increasing Zn bio-availability in the rhizosphere. After two years of plant growth, micrometersized Mn-Zn black precipitates were observed at the surface of Festuca rubra roots, but not characterized . Zinc precipitation may be a bio-active tolerance mechanism in response to metal toxicity, or a passive mineralization at the soil-root interface . Clarifying this issue and determining the mineralogy and structure of this natural precipitate is important to enhance the effectiveness of using graminaceous plants in phytoremediation. These questions are addressed here with electron microscopy and synchrotron-based microanalytical tools, including X-ray fluorescence , extended Xray absorption fine structure spectroscopy and X-ray diffraction . Micro-XRD was employed to determine the nanocrystalline structure of the Mn-Zn precipitates and the nature of defects through modeling of their scattering properties . We show that the root precipitates are present in the root epidermis and consist of a poorly crystallized phyllomanganate with a constant Zn:Mn ratio higher than reported so far for any natural and synthetic manganate.The composition in major elements of the dredged sediment was 68.3 % SiO2, 6.9 % CaO, 4.8 % Al2O3, 2.4 % Fe2O3, 0.7 % P2O5, and 7.2% organic carbon, and the composition in a trace metals was 4700 mg.kg-1 Zn, 700 mg.kg-1 Pb, and ~270 mg.kg-1 Mn. Seeds of F. rubra were sown in plastic pots filled with 40 kg of either the untreated sediment, the sediment amended with 3 wt. % hydroxylapatite, or the sediment amended with 5 wt. % Thomas basic slag.

The pots were placed in a greenhouse without artificial lighting and daily irrigated with tap water in an amount similar to the mean rainfall in northern France. After two years of culture, the pots were dismantled to collect samples. The texture and color of the sediment in areas colonized by the roots were similar to a brown silty soil, whereas the initial sediment was black and compact. Roots of F. rubra from the treated and untreated pots were washed meticulously with distilled water to remove soil particles from the surface and then freeze-dried. The speciation of zinc in the initial sediment and in the rhizosphere of F. rubra after the two years of vegetation was described previously . Briefly, in the untreated and unvegetated sediment, Zn was distributed as ~50% sphalerite, ~40% Zn-ferrihydrite, and ~10 to 20% -hydrotalcite plus Zn-phyllosilicate. In the presence of plants, ZnS was almost completely dissolved, and the released Zn bound to phosphate and to Zn phyllosilicate plus -hydrotalcite . The coaddition of mineral amendment did not affect the Zn speciation in the vegetated sediment. The Zn:Mn and Ca:Mn ratios were measured with an Eagle III µ-XRF spectrometer equipped with a Rh anode and a 40 µm poly-capillary. The spectrometer was operated under vacuum at 20 kV and 400 µA,vertical lettuce tower and fluorescence was measured for 300 s per point. Micro XRF, XRD and EXAFS data were collected on beamline 10.3.2 at the Advanced Light Source . Short root fragments were attached to the tips of glass capillaries and cooled down to 110-150 K to minimize radiation damage . X-ray fluorescence maps were taken at 10 keV incident energy, with a beam size ranging from 5×5 µm to 16×7 µm . Fluorescence counts were collected for K, Ca, Mn, Fe and Zn with a seven-element Ge solid-state detector and a counting time of 100 ms per pixel. For µ-EXAFS measurements, the vertical beam size ranged from 5 to 7 µm. A maximum of two spectra per precipitate were taken at either the Mn or the Zn K-edge to prevent the reduction of tetravalent to divalent Mn and the increase of structural disorder under the beam . Diffraction data were collected with a CCD camera at 17 keV and exposure times of 120-240 seconds. At this energy, the incident flux and absorption cross-sections are low enough to make radiation damage during an exposure negligible even at room temperature. A background pattern was recorded next to each precipitate to subtract the scattering contribution from the root so as to obtain the precipitate pattern. Diffraction patterns collected on different precipitates were all statistically identical, and thus summed up to optimize data quality. Calibration of the energy and camera distance were obtained using an Al2O3 standard and Fit2D software . This software was also used to calculate the one-dimensional XRD traces from the radial integration of the two-dimensional patterns. Under the optical microscope, the Mn-Zn precipitates appear as black stains about ten to several tens of micrometers in size on the root surface . They are also observed in back scattered electron microscopy due to the presence of high-Z elements , but always are hardly noticeable in secondary electronimaging mode . This suggests that the precipitates are engulfed in the root epidermis and do not coat the root surface as iron and manganese plaques do . No differences were observed among precipitates from plants grown in the untreated and mineral amended sediments. This result, together with the compartmentation of the precipitates inside the roots, suggests a biological origin. This interpretation is supported also by the absence of Zn-rich phyllomanganates in the surrounding soil matrix . Elemental mapping of F. rubra roots shows that Zn is associated with Mn in localized spots, and uniformly distributed without manganese in the vascular cylinder as expected for this nutritive element .

All roots have Zn in their central stele, but not all are speckled with Mn-Zn precipitates. Some root fragments are partly covered by Zn-free Fe-rich plaques . These plaques are made of ferric oxyhydroxides, as indicated by their optical rusty color . In Zn-Mn-Ca tricolor representation all Mn-Zn precipitates generally have the same color , even among different roots , meaning that the relative proportions of Zn, Mn and Ca are about the same. The correlation coefficient between Zn and Mn counts for the precipitates is 0.8, with P-value < 0.0001 for the Anova F-test . The Zn:Mn atomic ratio was calculated from the relative absorption jumps measured at the Mn and Zn K-edges on four particles. For each particle, a pre-absorption edge background was removed first, and then a linear fit to the post-edge region was extrapolated back to the edge to measure edge jumps. The ratio of the Zn to Mn edge jumps is 0.310, which translates into a Zn:Mn ratio of 0.46 when taking into account the atomic absorptions of the two elements. A consistent 0.44 value was obtained independently with the Eagle III spectrometer. This analysis also confirmed that root precipitates have a constant Ca:Mn ratio. An atomic ratio of 0.41 was calculated after correction of the Ca-fluorescence from the root. Micro-EXAFS spectra were recorded in the vascular cylinder of four distinct roots, at spots containing little Mn. All spectra were indistinguishable, indicating that Zn speciation is uniform, and thus averaged. The resulting Root spectrum has the same frequency as the MnZn precipitate spectrum, which suggests that Zn is also mostly tetrahedral in the roots . However, in contrast to Mn-Zn precipitate, the second and third oscillations of the Root spectrum are not split, indicative of “light” back scatters from second-shell contributions. Consistently, the best spectral match to our organic and inorganic database of the Root spectrum was provided with Zn in a biofilm . This reference has 80 ± 10% Zn complexed to phosphoryl groups and 20 ±10% to carboxyl groups . Consistent with this other study, consideration of carboxyl and phosphate ligands alone, did not yield an optimal fit to the data. Zinc preferential binding to phosphate groups has been reported also in the roots of Arabidopsis halleri and A. lyrata grown hydroponically, on bacterial and fungi cells, and in biofilms . These studies have shown that Zn has a higher affinity for phosphate than for carboxyl groups, which is consistent with the predominance of the phosphate species in F. rubra roots. As the optimal fit to data was obtained using a trial-and-error approach, the sensitivity of the XRD simulations to key structural parameters needs to be assessed. A key parameter for birnessite’s ability to sorb trace metals is the origin of the layer charge.